Patent Publication Number: US-10760110-B2

Title: Antibiotic testing and screening system

Description:
RELATED APPLICATION 
     This application claims priority to U.S. Provisional Application No. 62/463,168, filed on Feb. 24, 2017, the content of which is hereby incorporated by reference in its entirety. 
    
    
     FIELD OF THE INVENTION 
     The present invention relates to a platform technology for evaluating the antimicrobial activity of a known antibiotic against specified resistance mechanisms and also for screening for new antimicrobial agents. 
     BACKGROUND OF THE INVENTION 
     The emergence of antibiotic resistance has become a major clinical and public health threat worldwide. 1.2  According to a recent report from the United Kingdom, if antimicrobial resistance continues to increase, 10 million people will die due to it annually by 2050, a mortality rate greater than that of cancer. 1  Correct use of antibiotics and the development of novel antibiotics are both necessary to overcome this crisis. 
     World Health Organization (WHO) reported an extremely high resistant rates in  Klebsiella pneumoniae, Escherichia coli  and  Staphylococcus aureus , which are common causes of hospital and community infections. 4    K. pneumoniae  and  E. coli  both belong to Enterobacteriaceae and have similar antibiotic resistance mechanisms. 5  Understanding the antibiotic effectiveness against each resistant mechanism can help us to develop and use antibiotics. Traditionally, a large number of clinical drug-resistance isolates are collected and used for testing and screening antibiotics before going into clinical trials. 6  However, the resistance mechanisms in clinical isolates usually are complex and not fully identified. It is difficult to verify which mechanism is actually involved or plays the major role in conferring the drug resistance. Without the certain information, it is also difficult to modify the antibiotic candidates for improving its antimicrobial activity in an efficient manner. 
     SUMMARY OF THE INVENTION 
     The present design provides a platform technology for testing, screening, selecting and evaluating antibiotics by using genetically engineered strains with identified, individual or combined, resistance mechanisms, prepared from a multidrug (or preferably fully) susceptible clinical isolate. 
     Specifically, the present invention provides a platform technology for screening for antimicrobial agents by using genetic engineering bacterial strains, wherein these bacterial strains before genetic engineering are in nature highly susceptible to antibiotic treatment (preferably susceptible to multiple antibiotic treatment) and after genetic engineering it is given one or more mutations or antibiotic resistance genes based on the major types of drug-resistant mechanisms, selected form the group consisting of (i) decrease antibiotic permeability by loss of outer membrane proteins, (ii) pump out the antibiotics by overexpression of efflux pumps, (iii) eliminates or reduces binding of antibiotic by modification of antibiotic target or by acquirement of antibiotic-resistant target, and (iv) inactivate antibiotic by enzymatic cleavage or modification, and any combinations thereof. Since the drug-resistant mechanism given in the genetic engineering bacterial strains is known and identified, once an agent is cultured with the genetic engineering bacterial strain and determined to have an antimicrobial activity against it, we can thus figure out that the agent is specifically active against the drug-resistant mechanism given in the genetic engineering bacterial strains. Accordingly, the platform technology of the invention is feasible and efficient to identify an antimicrobial agent targeting a particular drug-resistant mechanism as desired. In addition, the platform technology of the invention, as compared with a conventional screening method using clinical resistant isolates, is less labor intensive and also is workable for carrying out a large-scale screening for an antimicrobial agent, especially for an antimicrobial agent active against multiple drug-resistant mechanisms, and moreover is meaningful to apply the selected antimicrobial agent to subsequent drug modification. 
     In particular, the present invention provides a method for screening for an antimicrobial agent, comprising: 
     (1) providing a wild-type (WT) susceptible bacterial strain which has a WT-referenced resistance level to a reference antibiotic; 
     (2) providing a first genetic engineering bacterial strain generated from the WT susceptible bacterial strain which is given a first mutation or antibiotic resistance gene (ET1) via a genetic engineering manner to confer drug resistance to the reference antibiotic, exhibiting a ET1-referenced resistance level to the reference antibiotic, wherein the ET1-referenced resistance level is higher than the WT-referenced resistance level; 
     (3) culturing the wild-type susceptible bacterial strain in the presence of a first test agent and analyzing the first test agent for its activity against the wild-type susceptible bacterial strain to obtain a WT-Test1 resistance level; 
     (4) culturing the first engineered bacterial strain in the presence of the first test agent and analyzing the first test agent for its activity against the first engineered bacterial strain to obtain a ET1-Test1 resistance level; 
     (5) obtaining a ET1-Test1 ratio of the ET1-Test1 resistance level to the WT-Test1 resistance level; and 
     (6) determining if the first test agent is a potentially effective antimicrobial agent against the drug resistance associated with ET1 in the first engineered bacterial strain based on the ET1-Test1 ratio. 
     In some embodiments, the ET1-referenced resistance level is higher than the WT-referenced resistance level by 1-fold, 2-fold, 3-fold, or 4-fold or more. 
     In some embodiments, the wild-type susceptible bacterial strain is susceptible to multiple antibiotics, selected from the group consisting of β-lactams, quinolones, aminoglycosides, tetracyclines, folate pathway inhibitors, polymyxins, phenicols, fosomycins, nitrofurans and any combinations thereof. 
     In some embodiments, the first test agent is determined as a potentially effective antimicrobial agent against the drug resistance associated with ET1 if the ET1-Test1 ratio is no more than 4 (e.g. 4 or less, 3 or less, 2 or less, 1 or less). 
     In more particular, the method of the invention further comprises: 
     (7) (17) culturing the wild-type susceptible strain in the presence of a second test agent and analyzing the second test agent for its activity against the wild-type susceptible strain to obtain a WT-Test2 resistance level; 
     (8) (18) culturing the first engineered bacterial strain in the presence of the second test agent and analyzing the second test agent for its activity against the first engineered bacterial strain to obtain a ET1-Test2 resistance level; 
     (9) (19) obtaining a ET1-Test2 ratio of the ET1-Test2 resistance level to the WT-Test2 resistance level; and 
     (10) (20) determining if the second test agent is a potentially effective antimicrobial agent against the drug resistance associated with ET1 in the first engineered bacterial strain based on the ET1-Test2 ratio. 
     In some embodiments, the second test agent is determined as a potentially effective antimicrobial agent against the drug resistance associated with ET1 if the ET1-Test2 ratio is no more than 4 (e.g. 4 or less, 3 or less, 2 or less, 1 or less). 
     In some embodiments, the second test agent is determined as a potentially effective antimicrobial agent against the drug resistance associated with ET1 if the ET1-Test2 resistance level, which is expressed by minimum inhibitory concentration (MIC), is no more than 10 μg/ml, 5 μg/ml, 2.5 μg/ml, 1 μg/ml or 0.5 μg/ml. 
     In more particular, the method of the invention further comprises: 
     (11) ranking the first test agent and the second test agent for a preferable antimicrobial activity according to the ET1-Test1 ratio and the ET1-Test2 ratio, the lower the ratio, the higher the ranking. 
     In more particular, the method of the invention further comprises: 
     (12) providing a second engineered bacterial strain generated from the WT susceptible bacterial strain which is given a second mutation or antibiotic resistance gene (ET2) in a genetic engineering manner to confer drug resistance to the reference antibiotic, exhibiting a ET2-referenced resistance level to the reference antibiotic, wherein the ET2-referenced resistance level is higher than the WT-referenced resistance level; 
     (13) culturing the second engineered bacterial strain in the presence of the first test agent and analyzing the first test agent for its activity against the second engineered bacterial strain to obtain a ET2-Test1 resistance level; 
     (14) obtaining a ET2-Test1 ratio of the ET2-Test1 resistance level to the WT-Test1 resistance level; 
     (15) determining if the first test agent is a potentially effective antimicrobial agent against the drug resistance associated with ET2 in the second engineered bacterial strain based on the ET2-Test1 ratio. 
     In some embodiments, the first test agent is determined as a potentially effective antimicrobial agent against the drug resistance associated with ET2 if the ET2-Test1 ratio is no more than 4 (e.g. 4 or less, 3 or less, 2 or less, 1 or less). 
     In some embodiments, the first test agent is determined as a potentially effective antimicrobial agent against the drug resistance associated with ET2 if the ET2-Test1 resistance level, which is expressed by minimum inhibitory concentration (MIC), is no more than 10 μg/ml, 5 μg/ml, 2.5 μg/ml, 1 μg/ml or 0.5 μg/ml. 
     In more particular, the method of the invention further comprises: (16) obtaining a first sum ratio value with respect to the first test agent by adding the ET1-Test1 ratio to the ET2-Test1 ratio. 
     In more particular, the method of the invention further comprises: 
     (17) culturing the wild-type susceptible strain in the presence of a second test agent and analyzing the second test agent for its activity against the wild-type susceptible strain to obtain a WT-Test2 resistance level; 
     (18) culturing the first genetic engineering bacterial strain in the presence of the second test agent and analyzing the second test agent for its activity against the first genetic engineering bacterial strain to obtain a ET1-Test2 resistance level; 
     (19) obtaining a ET1-Test2 ratio of the ET1-Test2 resistance level to the WT-Test2 resistance level; and 
     (20) determining if the second test agent is a potentially effective antimicrobial agent against the drug resistance associated with ET1 in the first genetic engineering bacterial strain based on the ET1-Test2 ratio. 
     In some embodiments, the second test agent is determined as a potentially effective antimicrobial agent against the drug resistance associated with ET1 if the ET1-Test2 ratio is no more than 4 (e.g. 4 or less, 3 or less, 2 or less, 1 or less). 
     In some embodiments, the second test agent is determined as a potentially effective antimicrobial agent against the drug resistance associated with ET1 if the ET1-Test2 resistance level, which is expressed by minimum inhibitory concentration (MIC), is no more than 10 μg/ml, 5 μg/ml, 2.5 μg/ml, 1 μg/ml or 0.5 μg/ml. 
     In more particular, the method of the invention further comprises: 
     (21) culturing the second engineered bacterial strain in the presence of the second test agent and analyzing the second test agent for its activity against the second engineered bacterial strain to obtain a ET2-Test2 resistance level; 
     (22) obtaining a ET2-Test2 ratio of the ET2-Test2 resistance level to the WT-Test2 resistance level; 
     (23) determining if the second test agent is a potentially effective antimicrobial agent against the drug resistance associated with ET2 in the second engineered bacterial strain based on the ET2-Test2 ratio. 
     In some embodiments, the second test agent is determined as a potentially effective antimicrobial agent against the drug resistance associated with ET2 if the ET2-Test2 ratio is no more than 4 (e.g. 4 or less, 3 or less, 2 or less, 1 or less). 
     In some embodiments, the second test agent is determined as a potentially effective antimicrobial agent against the drug resistance associated with ET2 if the ET2-Test2 resistance level, which is expressed by minimum inhibitory concentration (MIC), is no more than 10 μg/ml, 5 μg/ml, 2.5 μg/ml, 1 μg/ml or 0.5 μg/ml. 
     In more particular, the method of the invention further comprises: (24) obtaining a second sum ratio value with respect to the second test agent by adding the ET1-Test2 ratio to the ET2-Test2 ratio. 
     In more particular, the method of the invention further comprises: (25) ranking the first test agent and the second test agent for a preferable antimicrobial activity according to the first sum ratio value and the second sum ratio value, the lower the value, the higher the ranking. 
     In more particular, the method of the invention further comprises: (26) ranking the first test agent and the second test agent for a preferable antimicrobial activity according to the ET2-Test1 ratio and the ET2-Test2 ratio, the lower the ratio, the higher the ranking. 
     In some embodiments, the preferable antimicrobial activity includes an antimicrobial activity against multiple drug-resistance mechanisms. 
     In some embodiments, the first mutation or antibiotic resistance gene, or the second mutation or antibiotic resistance gene is given based on one or more drug-resistant mechanisms selected form the group consisting of (i) decrease antibiotic permeability by loss of outer membrane proteins, (ii) pump out the antibiotics by overexpression of efflux pumps, (iii) eliminates or reduces binding of antibiotic by modification of antibiotic target or by acquirement of antibiotic-resistant target, and (iv) inactivate antibiotic by enzymatic cleavage or modification, and any combinations thereof. 
     In some embodiments, the first mutation or antibiotic resistance gene, or the second mutation or antibiotic resistance gene is selected from the group consisting of: ΔompK35, ΔompK36, ΔramR, GyrA S83I, GyrA S83L, GyrA S83F, GyrA S83Y, GyrA D87N, ParC S80I , CTX-M-14, CTX-M-15, SHV-12, CMY-2, DHA-1-AmpR, KPC-2, KPC-3, IMP-8, NDM-1, VIM-1, OXA-48, QnrB, QnrS, AAC(6′)-Ib-cr, AAC(6′)-Ib-cr, AAC(3)-IId, AAC(3)-IVa, ANT(2″)-Ia, ANT(3″)-Ia, APH(3′)-Ia, APH(3′)-IIa, StrA-StrB, ArmA, RmtB, Tet(A), Tet(B), Tet(C), Tet(D), Tet(M), Sul1, Sul2, DfrA1, DfrA16, AdeR D20N, AdeR A91V, AdeR P116L, AdeS G30D, AdeS A94V, AdeS R152K, AdeS T153M, ParC G78C, ParC S80L, ParC S80W, ParC S80Y, ParC E84K, VEB-3, ADC-30, IMP-1, OXA-23, OXA-58, OXA-66, OXA-72, AAC(3)-IIa, APH(3′)-VIa, and any combinations thereof. 
     In some embodiments, the first mutation or antibiotic resistance gene, or the second mutation or antibiotic resistance gene is given by a chromosome-mediated approach or a plasmid-mediated approach. 
    
    
     
       BRIEF DESCRIPTION OF THE DRAWINGS 
       The foregoing summary, as well as the following detailed description of the invention, will be better understood when read in conjunction with the appended drawings. For the purpose of illustrating the invention, there are shown in the drawings embodiments which are presently preferred. It should be understood, however, that the invention is not limited to the precise arrangements and instrumentalities shown. 
       In the drawings: 
         FIGS. 1A and 1B  show in vivo efficacy of ceftazidime ( FIG. 1A ) and cefotaxime ( FIG. 1B ) against  K. pneumoniae  NVT1001 harboring the pACYC177 plasmid with bla OXA-48  in a mouse peritonitis-sepsis model. 
     
    
    
     DETAILED DESCRIPTION OF THE INVENTION 
     Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by a person skilled in the art to which this invention belongs. 
     As used herein, the singular forms “a”, “an”, and “the” include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to “a sample” includes a plurality of such samples and equivalents thereof known to those skilled in the art. 
     As used herein, the term “drug resistance” can refer to a cell&#39;s increasing resistance to drug treatment, such as antibiotic treatment. 
     As used herein, the terms “susceptible” and “antibiotic susceptibility” can indicate that the growth of a microorganism is inhibited by a normal achievable concentration of an antimicrobial agent when the recommended dosage is used. 
     As used herein, the terms “resistant” and “antibiotic resistance” can indicate that microorganism growth is not inhibited by a normal achievable concentration of the agent and clinical efficacy of the agent against the microorganism has not been shown in treatment studies. 
     As used herein, a resistance level with reference to a bacterial strain for a particular agent can indicate a minimal amount of the particular agent that will inhibit the visible growth of a microorganism, such as a minimum inhibitory concentration (MIC). A bacterial strain with a lower resistance level to an agent can indicate that the bacterial strain is relatively susceptible to the agent (reflecting a relatively lower MIC), while a bacterial strain with a higher resistance level to an agent can indicate that the bacterial strain is relatively resistant to the agent (reflecting a relatively higher MIC). 
     According to the present invention, a bacterial strain can be from any bacterial species, particularly a species leading to common infectious diseases in human, including but not limited to, Gram-negative bacteria, such as  K. pneumonia, Escherichia coli, Salmonella  spp.,  Acinetobacter baumannii  and  Pseudomonas aeruginosa , and Gram-positive bacteria, such as  Staphylococcus aureus  and  Enterococcus  spp. 
     As used herein, a wild-type susceptible bacterial strain can indicate a bacterial strain, the genome of which is not modified by a genetic engineering manner and which is susceptible to antibiotic treatment. Preferably, a wild-type susceptible bacterial strain as used herein is susceptible to multiple antibiotics, selected from the group consisting of β-lactams, quinolones, aminoglycosides, tetracyclines, folate pathway inhibitors, polymyxins, phenicols, fosomycins, nitrofurans and any combinations thereof. A particular example of a wild-type susceptible bacterial strain as used herein includes  K. pneumoniae  strain NVT1001, which can be obtained for example from Division of Infectious Diseases and Tropical Medicine, Department of Internal Medicine, Tri-Service General Hospital, Taipei 114, Taiwan. 
     As used herein, a genetically engineered bacterial strain can be generated from a wild-type susceptible bacterial strain via a genetic engineering manner, which after the genetic engineering, is given mutation or antibiotic resistance gene to confer drug resistance. The genetic engineering can be conducted by a conventional method as known in the art, such as by a chromosome-mediated approach (e.g. mutating a gene in the chromosome of the strain to confer drug resistance) or a plasmid-mediated approach (e.g. adding a foreign gene to confer drug resistance in the strain). The genetically engineered bacterial strain can include one or more genetic changes based on one or more drug-resistant mechanisms selected form the group consisting of (i) decrease antibiotic permeability by loss of outer membrane proteins, (ii) pump out the antibiotics by overexpression of efflux pumps, (iii) eliminates or reduces binding of antibiotic by modification of antibiotic target or by acquirement of antibiotic-resistant target, and (iv) inactivate antibiotic by enzymatic cleavage or modification, and any combinations thereof. In some embodiments, the mutation or antibiotic resistance gene is selected from the group consisting of: ΔompK35, ΔompK36, ΔramR, GyrA S83I, GyrA S83L, GyrA S83F, GyrA S83Y, GyrA D87N, GyrA S80I, CTX-M-14, CTX-M-15, SHV-12, CMY-2, DHA-1-AmpR, KPC-2, KPC-3, IMP-8, NDM-1, VIM-1, OXA-48, QnrB, QnrS, AAC(6′)-Ib-cr, AAC(6′)-Ib-cr, AAC(3)-IId, AAC(3)-IVa, ANT(2″)-Ia, ANT(3″)-Ia, APH(3′)-Ia, APH(3′)-IIa, StrA-StrB, ArmA, RmtB, Tet(A), Tet(B), Tet(C), Tet(D), Tet(M), Sul1, Sul2, DfrA1, DfrA16, AdeR D20N, AdeR A91V, AdeR P116L, AdeS G30D, AdeS A94V, AdeS R152K, AdeS T153M, ParC G78C, ParC S80L, ParC S80W, ParC S80Y, ParC E84K, VEB-3, ADC-30, IMP-1, OXA-23, OXA-58, OXA-66, OXA-72, AAC(3)-IIa, APH(3′)-VIa, and any combinations thereof. In some embodiments, the mutation or antibiotic resistance gene is preferably include genetic changes as many as possible which can be used to screen for a strong antimicrobial agent against multiple drug-resistant mechanisms. 
     The present invention provides a method for screening for an antibiotic agent. In particular, the method of the invention creates a genetically engineered bacterial strain from a wild-type susceptible bacterial strain via a genetic engineering manner, which after the genetic engineering, is given mutation or antibiotic resistance gene to confer drug resistance to a reference antibiotic based on one or more drug-resistant mechanisms; a test agent is then added to a culture of the wild-type susceptible bacterial strain and a culture of the genetically engineered bacterial strain to analyze for its activity against the wild-type susceptible bacterial strain and the genetically engineered bacterial strain, respectively, wherein the activity of the test agent against the bacterial strains can be expressed by a minimum inhibitory concentration (MIC) (also referring to a resistance level as described herein; typically, a higher MIC indicates a higher resistance level and a lower antimicrobial activity, and a lower MIC indicates a lower resistance level and a higher antimicrobial activity); and if the activity of the test agent against the genetically engineered bacterial strain is similar to that against the wild-type susceptible bacterial strain, the test agent is selected as a potential antimicrobial agent against the same species of the bacterial strain. In general, the antimicrobial activity of a test agent is expressed by MIC, which is preferably no more than 10 μg/ml, 5 μg/ml, 2.5 μg/ml, 1 μg/ml or 0.5 μg/ml. 
     Table A shows an embodiment of the method of the present invention. 
     
       
         
           
               
               
               
               
             
               
                   
               
             
            
               
                   
                 (1) Providing a wild- 
                 (2) Creating a first 
                 (12) Creating a second 
               
               
                   
                 type (WT) 
                 genetically engineered 
                 genetically engineered 
               
               
                   
                 susceptible bacterial 
                 bacterial strain having 
                 bacterial strain having 
               
               
                   
                 strain 
                 mutation 1 or antibiotic 
                 mutation 2 or antibiotic 
               
               
                   
                 (Preferably, the WT 
                 resistance gene 1 (ET1), 
                 resistance gene 2 (ET2), 
               
               
                   
                 susceptible bacterial 
                 from the wild-type (WT) 
                 from the wild-type (WT) 
               
               
                   
                 strain is susceptible 
                 susceptible bacterial 
                 susceptible bacterial strain 
               
               
                   
                 to multiple antibiotics.) 
                 strain via a genetically 
                 via a genetically 
               
               
                   
                   
                 engineering technology to 
                 engineering technology to 
               
               
                   
                   
                 confer drug resistance. 
                 confer drug resistance. 
               
               
                 A reference 
                 (1) the WT 
                 (2) the first genetically 
                 (12) the second genetically 
               
               
                 antibiotic 
                 susceptible bacterial 
                 engineered bacterial 
                 engineered bacterial strain 
               
               
                   
                 strain has a WT 
                 strain exhibits a ET1- 
                 exhibits a ET2-referenced 
               
               
                   
                 referenced resistance 
                 referenced resistance 
                 resistance level, which is 
               
               
                   
                 level to a reference 
                 level, which is higher 
                 higher than the WT- 
               
               
                   
                 antibiotic 
                 than the WT-referenced 
                 referenced resistance level, 
               
               
                   
                   
                 resistance level, 
                 preferably by 1-fold, 2- 
               
               
                   
                   
                 preferably by 1-fold, 2- 
                 fold, 3-fold, or 4-fold or 
               
               
                   
                   
                 fold, 3-fold, or 4-fold or 
                 more. 
               
               
                   
                   
                 more. 
               
               
                 Test 1 agent 
                 (3) Culturing the WT 
                 (4) Culturing the first 
                 (13) Culturing the second 
               
               
                   
                 susceptible bacterial 
                 genetically engineered 
                 genetically engineered 
               
               
                   
                 strain in the presence 
                 bacterial strain with ET1 
                 bacterial strain with ET2 in 
               
               
                   
                 of the Test 1 agent to 
                 in the presence of the 
                 the presence of the Test 1 
               
               
                   
                 obtain a WT-Test1 
                 Test 1 agent to obtain a 
                 agent to obtain a ET2-Test1 
               
               
                   
                 resistance level. 
                 ET1-Test1 resistance 
                 resistance level. 
               
               
                   
                   
                 level. 
               
               
                   
                   
                 (5) Obtaining a ET1- 
                 (14) Obtaining a ET2-Test1 
               
               
                   
                   
                 Test1 ratio of the ET1- 
                 ratio of the ET2-Test1 
               
               
                   
                   
                 Test1 resistance level to 
                 resistance level to the WT- 
               
               
                   
                   
                 the WT-Test1 resistance 
                 Test1 resistance level is 
               
               
                   
                   
                 level. 
                 obtained. 
               
               
                   
                   
                 (a ET1-Test1 ratio = the 
                 (a ET2-Test1 ratio = the 
               
               
                   
                   
                 ET1-Test1 resistance 
                 ET2-Test1 resistance level ÷ 
               
               
                   
                   
                 level ÷ the WT-Test1 
                 the WT-Test1 resistance 
               
               
                   
                   
                 resistance level) 
                 level) 
               
               
                   
                   
                 (6) Determining if Test 1 
                 (15) Determining if Test 1 
               
               
                   
                   
                 agent is a potentially 
                 agent is an effective 
               
               
                   
                   
                 effective antimicrobial 
                 antimicrobial agent against 
               
               
                   
                   
                 agent against the drug 
                 the drug resistance 
               
               
                   
                   
                 resistance associated with 
                 associated with ET2 in the 
               
               
                   
                   
                 ET1 in the first 
                 second genetically 
               
               
                   
                   
                 genetically engineered 
                 engineered bacterial strain 
               
               
                   
                   
                 bacterial strain based on 
                 based on the ET2-Test1 
               
               
                   
                   
                 the ET1-Test1 ratio. 
                 ratio. 
               
               
                   
                   
                 (Particularly, the Test 1 
                 (Particularly, the Test 1 
               
               
                   
                   
                 agent is determined as a 
                 agent is determined as a 
               
               
                   
                   
                 potentially effective 
                 potentially effective 
               
               
                   
                   
                 antimicrobial agent 
                 antimicrobial agent against 
               
               
                   
                   
                 against the drug 
                 the drug resistance 
               
               
                   
                   
                 resistance associated with 
                 associated with ET2 if the 
               
               
                   
                   
                 ET1 if the ET1-Test1 
                 ET2-Test1 ratio is no more 
               
               
                   
                   
                 ratio is no more than 4) 
                 than 4) 
               
               
                   
                   
                 (Particularly, the first test 
                 (Particularly, the first test 
               
               
                   
                   
                 agent is determined as a 
                 agent is determined as a 
               
               
                   
                   
                 potentially effective 
                 potentially effective 
               
               
                   
                   
                 antimicrobial agent 
                 antimicrobial agent against 
               
               
                   
                   
                 against the drug 
                 the drug resistance 
               
               
                   
                   
                 resistance associated with 
                 associated with ET2 if the 
               
               
                   
                   
                 ET1 if the ET1-Test1 
                 ET2-Test1 resistance level, 
               
               
                   
                   
                 resistance level, 
                 expressed by minimum 
               
               
                   
                   
                 expressed by minimum 
                 inhibitory concentration 
               
               
                   
                   
                 inhibitory concentration 
                 (MIC), is no more than 10 μg/ 
               
               
                   
                   
                 (MIC), is no more than 
                 ml, 5 μg/ml, 2.5 μg/ml, 
               
               
                   
                   
                 10 μg/ml, 5 μg/ml, 2.5 μg/ 
                 1 μg/ml or 0.5 μg/ml) 
               
               
                   
                   
                 ml, 1 μg/ml or 0.5 μg/ml) 
               
            
           
           
               
               
            
               
                   
                 (16) Obtaining a first sum ratio value with respect to Test 1 agent by adding the 
               
               
                   
                 ET1-Test1 ratio to the ET2-Test1 ratio 
               
            
           
           
               
               
               
               
            
               
                 Test 2 agent 
                 (7) (17) Culturing 
                 (8) (18) Culturing the 
                 (21) Culturing the second 
               
               
                   
                 the WT susceptible 
                 first genetically 
                 genetically engineered 
               
               
                   
                 bacterial strain in the 
                 engineered bacterial 
                 bacterial strain with ET2 in 
               
               
                   
                 presence of the Test 
                 strain with ET1 in the 
                 the presence of the Test 2 
               
               
                   
                 2 agent to obtain a 
                 presence of the Test 2 
                 agent to obtain a ET2-Test2 
               
               
                   
                 WT-Test2 resistance 
                 agent to obtain a ET1- 
                 resistance level. 
               
               
                   
                 level. 
                 Test2 resistance level. 
               
               
                   
                   
                 (9) (19) Obtaining a ET1- 
                 (22) Obtaining a ET2-Test2 
               
               
                   
                   
                 Test2 ratio of the ET1- 
                 ratio of the ET2-Test2 
               
               
                   
                   
                 Test2 resistance level to 
                 resistance level to the WT- 
               
               
                   
                   
                 the WT-Test2 resistance 
                 Test2 resistance level is 
               
               
                   
                   
                 level. 
                 obtained. 
               
               
                   
                   
                 (a ET1-Test2 ratio = the 
                 (a ET2-Test2 ratio = the 
               
               
                   
                   
                 ET1-Test2 resistance 
                 ET2-Test2 resistance level ÷ 
               
               
                   
                   
                 level ÷ the WT-Test2 
                 the WT-Test2 resistance 
               
               
                   
                   
                 resistance level) 
                 level) 
               
               
                   
                   
                 (10) (20) Determining if 
                 (23) Determining if Test 2 
               
               
                   
                   
                 Test 2 agent is an 
                 agent is an effective 
               
               
                   
                   
                 effective antimicrobial 
                 antimicrobial agent against 
               
               
                   
                   
                 agent against the drug 
                 the drug resistance 
               
               
                   
                   
                 resistance associated with 
                 associated with ET2 in the 
               
               
                   
                   
                 ET1 in the first 
                 second genetically 
               
               
                   
                   
                 genetically engineered 
                 engineered bacterial strain 
               
               
                   
                   
                 bacterial strain based on 
                 based on the ET2-Test2 
               
               
                   
                   
                 the ET1-Test2 ratio. 
                 ratio. 
               
               
                   
                   
                 (Particularly, the Test 2 
                 (Particularly the Test 2 
               
               
                   
                   
                 agent is determined as an 
                 agent is determined as an 
               
               
                   
                   
                 effective antimicrobial 
                 effective antimicrobial 
               
               
                   
                   
                 agent against the drug 
                 agent against the drug 
               
               
                   
                   
                 resistance associated with 
                 resistance associated with 
               
               
                   
                   
                 ET1 if the ET1-Test2 
                 ET2 if the ET2-Test2 ratio 
               
               
                   
                   
                 ratio is no more than 4) 
                 is no more than 4) 
               
               
                   
                   
                 (Particularly, the second 
                 (Particularly, the second 
               
               
                   
                   
                 test agent is determined 
                 test agent is determined as 
               
               
                   
                   
                 as a potentially effective 
                 a potentially effective 
               
               
                   
                   
                 antimicrobial agent 
                 antimicrobial agent against 
               
               
                   
                   
                 against the drug 
                 the drug resistance 
               
               
                   
                   
                 resistance associated with 
                 associated with ET2 if the 
               
               
                   
                   
                 ET1 if the ET1-Test2 
                 ET2-Test2 resistance level, 
               
               
                   
                   
                 resistance level, 
                 expressed by minimum 
               
               
                   
                   
                 expressed by minimum 
                 inhibitory concentration 
               
               
                   
                   
                 inhibitory concentration 
                 (MIC), is no more than 10 μg/ 
               
               
                   
                   
                 (MIC), is no more than 
                 ml, 5 μg/ml, 2.5 μg/ml, 
               
               
                   
                   
                 10 μg/ml, 5 μg/ml, 2.5 μg/ 
                 1 μg/ml or 0.5 μg/ml) 
               
               
                   
                   
                 ml, 1 μg/ml or 0.5 μg/ml) 
               
            
           
           
               
               
            
               
                   
                 (24) Obtaining a second sum ratio value with respect to Test 2 agent by adding 
               
               
                   
                 the ET1-Test2 ratio to the ET2-Test2 ratio. 
               
               
                   
                 (25) Ranking Test 1 agent and Test 2 agent for a preferable antimicrobial 
               
               
                   
                 activity according to the first sum ratio value and the second sum ratio value, 
               
               
                   
                 the lower the value, the higher the ranking. 
               
               
                   
                 (11) Ranking Test 1 agent and Test 2 agent for a preferable antimicrobial 
               
               
                   
                 activity according to the ET1-Test1 ratio and the ET1-Test2 ratio, the lower the 
               
               
                   
                 ratio, the higher the ranking. 
               
               
                   
                 (26) Ranking Test 1 agent and Test 2 agent for a preferable antimicrobial 
               
               
                   
                 activity according to the ET2-Test1 ratio and the ET2-Test2 ratio, the lower the 
               
               
                   
                 ratio, the higher the ranking. 
               
               
                   
                   
               
            
           
         
       
     
     In certain embodiments, the method of the invention is to identify a potential agent with antimicrobial activity against multiple drug-resistance mechanisms. 
     In particular, the method of the present invention is advantageous to develop a platform technology to screen for a large number of agents for their antimicrobial activity. In addition, once a test agent is determined as a potentially antimicrobial agent in a first run of the process, it can be subjected to a subsequent run of the process by creating another genetically engineered bacterial strain with a new mutation or antibiotic resistance gene and repeating the steps of culturing, obtaining a resistance level and analyzing for its activity against the wild-type susceptible bacterial strain and the genetically engineered bacterial strain. The more times the process is conducted, the more accurate the results, and an agent thus selected by more runs of the process is more effective against various types of drug-resistant mechanisms. 
     The present invention will now be described more specifically with reference to the following examples, which are provided for the purpose of demonstration rather than limitation. 
     EXAMPLES 
     The system developed in the present study provides a platform technology for testing, screening, selecting and evaluating antibiotics using genetically engineered bacterial strains with individual or combined resistance mechanisms that were constructed from the fully susceptible clinical  K. pneumoniae  NVT1001 isolate. The chromosome-mediated resistance mechanisms that were constructed in this study are decreased antibiotic permeability, generated by deletion of the genes encoding the outer-membrane proteins OmpK35 and/or OmpK36; 7  active antibiotic export, generated by deletion of the ramR regulatory gene, which leads to overexpression of the AcrAB-TolC efflux pump (Tables 1); 8-11  and modification of quinolone target sites via gyrA and/or parC mutations. 12-15  Most plasmid-mediated mechanisms are specific to one kind of antibiotic group, 16  with this study focusing on β-lactams, quinolones, aminoglycosides, tetracyclines and folate pathway inhibitors. Because many kinds of resistance are conferred by combined resistance mechanisms in the clinical setting, mechanisms related to the same antibiotic group were combined. Testing antibiotics against these combined mechanisms can be used to further evaluate their antimicrobial activities. 
     
       
         
           
               
             
               
                 TABLE 1 
               
             
            
               
                   
               
               
                 Relative transcription levels of the acrA, tolC, 
               
               
                 ramA and ramR in the genetically engineered strains 
               
               
                 in comparison with  K. pneumoniae  NVT1001 a   
               
            
           
           
               
               
               
            
               
                   
                 Gene transcription level (fold change) b   
                   
               
            
           
           
               
               
               
               
               
               
            
               
                   
                 Strain 
                 acrA 
                 tolC 
                 ramA 
                 ramR 
               
               
                   
                   
               
            
           
           
               
               
               
               
               
               
            
               
                   
                 NVT1001 
                 1 
                 1 
                 1 
                 1 
               
               
                   
                 ΔramR c   
                 
                   3.18 
                 
                 
                   3.12 
                 
                 
                   13.37 
                 
                 
                   0 
                 
               
               
                   
                 ΔramR::ramR 
                 1.02 
                 0.95 
                 0.95 
                 1 
               
               
                   
                   
               
               
                   
                   a The expression of acrAB and tolC is positively regulated by RamA, while RamR is a repressor that regulates ramA expression. 1  Previous study has found that after ramR disruption, the expression of acrA, tolC and ramA was increased 3.36-, 3.35- and 25.04-fold respectively, 2  while similar results can also obtained in this study. 
               
               
                   
                   b Boldface letter indicates at least a 2-fold change compared with that of  K. pneumoniae  NVT1001 and the results are the mean of three different experiments. 
               
               
                   
                   c ΔramR, ramR deletion strain of NVT1001; ΔramR::ramR, ramR-complemented strain of ΔramR strain. 
               
            
           
         
       
     
     In addition, Enterobacteriaceae,  Acinetobacter baumannii  and  Pseudomonas aeruginosa  have been identified by the WHO as three critical priority pathogens for the research and development of new antibiotics in 2017. 3  Given that the genetic background of  A. baumannii  is quite different from that of  K. pneumoniae , specific resistance genes can be found in this pathogen. Therefore, the strategic system has been constructed by using  A. baumannii.    
     This study includes generation of genetically engineered strains from a fully susceptible clinical isolate of  A. baumannii . The chromosome-mediated resistance mechanisms that were constructed in this study are active antibiotic export, generated by mutation of the adeR or adeS regulatory gene, which leads to overexpression of the AdeABC efflux pump;41-46 and modification of quinolone target sites via gyrA and/or parC mutations. 47-54  Most plasmid-mediated mechanisms are specific to one kind of antibiotic group, 55  with this study focusing on β-lactams, aminoglycosides and tetracyclines. All of these genetically engineered  A. baumannii  strains are with clear and simple resistance mechanisms and their usability was evaluated via antimicrobial susceptibility testing in this study. 
     Prior to the present invention, no complete system is available for testing antibiotics against specific resistance mechanisms. This study generated genetically engineered strains with clear and simple resistance mechanisms and evaluated their usability via both in vitro and in vivo assays. 
     1. Materials and Methods 
     1.1 Concept and Approach to Engineer the Resistant Strains for Antibiotic Screening System. 
     To create the bacterial strains for antibiotic screening system, four approaches with different target related antibiotic resistance were used. 
     For construction of chromosomal mediated resistance, (i) decreasing penetration of antibiotic (knockout outer membrane protein genes ompK35 and ompK36 in  K. pneumoniae ), 7  (ii) increasing the expression of AcrAB-TolC (knockout regulatory gene ramR 8-11  or overexpression of the AdeABC efflux pump (mutation of the adeR or adeS regulatory gen) 41-46  and (iii) antibiotic target gene mutation were performed. 12-15,47-54  For plasmid mediated resistance, different class of plasmid mediated resistant genes including (i) β-lactam resistant genes, (ii) quinolones resistant genes, (iii) aminoglycosides resistant genes, (iv) tetracyclines resistant genes and (v) folate pathway inhibitor resistant genes 16  were individually transferred into susceptible strain. See, Tables 2 and 4. 
     1.2 Bacterial Strains and Plasmids 
     1.2.1  K. pneumoniae  NVT1001 
       K. pneumoniae  NVT1001, capsular serotype 1, is an isolate from a liver-abscess patient in Taiwan 17  that was fully susceptible to all antibiotics tested, except ampicillin. The NVT1001 strain was used to construct genetically engineered strains with the antibiotic resistance mechanisms that are shown in Table 2, and the revertants of the NVT1001 mutants constructed in this study are shown in Table 3. 
     
       
         
           
               
             
               
                 TABLE 2 
               
             
            
               
                   
               
               
                 Antibiotic resistance mechanisms in fully susceptible clinical  K. pneumoniae  NVT1001 
               
            
           
           
               
               
               
            
               
                 Strain or plasmid 
                 Genotype or description a   
                 Major relevant antibiotic b   
               
               
                   
               
            
           
           
               
            
               
                 Strain with chromosome-mediated resistance mechanism 
               
            
           
           
               
               
               
            
               
                 ΔompK35 mutant 
                 ΔompK35 
                 β-lactams 
               
               
                 ΔompK36 mutant 
                 ΔompK36 
                 β-lactams, Folate pathway inhibitors, 
               
               
                   
                   
                 Nitrofurans 
               
               
                 ΔompK35/36 mutant 
                 ΔompK35/36 
                 β-lactams, Folate pathway inhibitors, 
               
               
                   
                   
                 Fosfomycins, Nitrofurans 
               
               
                 ΔramR mutant 
                 ΔramR 
                 β-lactams, Quinolones, Tetracyclines, 
               
               
                   
                   
                 Folate pathway inhibitors, Phenicols, 
               
               
                   
                   
                 Nitrofurans 
               
               
                 ΔramRΔompK35 mutant 
                 ΔramRΔompK35 
                 β-lactams, Quinolones, Tetracyclines, 
               
               
                   
                   
                 Folate pathway inhibitors, Phenicols, 
               
               
                   
                   
                 Nitrofurans 
               
               
                 ΔramRΔompK36 mutant 
                 ΔramRΔompK36 
                 β-lactams, Quinolones, Tetracyclines, 
               
               
                   
                   
                 Folate pathway inhibitors, Phenicols, 
               
               
                   
                   
                 Fosfomycins, Nitrofurans 
               
               
                 ΔramRΔompK35/36 mutant 
                 ΔramRΔompK35/36 
                 β-lactams, Quinolones, Tetracyclines, 
               
               
                   
                   
                 Folate pathway inhibitors, Phenicols, 
               
               
                   
                   
                 Fosfomycins, Nitrofurans 
               
               
                 S83I mutant 
                 GyrA S83I 
                 Quinolones 
               
               
                 S83L mutant 
                 GyrA S83L 
                 Quinolones 
               
               
                 S83F mutant 
                 GyrA S83F 
                 Quinolones 
               
               
                 S83Y mutant 
                 GyrA S83Y 
                 Quinolones 
               
               
                 D87N mutant 
                 GyrA D87N 
                 Quinolones 
               
               
                 S80I mutant 
                 ParC S80I 
                 None 
               
               
                 D87N/S80I mutant 
                 GyrA D87N; ParC S80I 
                 Quinolones 
               
               
                 S83I/D87N mutant 
                 GyrA S83I/D87N 
                 Quinolones 
               
               
                 S83L/D87N mutant 
                 GyrA S83L/D87N 
                 Quinolones 
               
               
                 S83F/D87N mutant 
                 GyrA S83F/D87N 
                 Quinolones 
               
               
                 S83Y/D87N mutant 
                 GyrA S83Y/D87N 
                 Quinolones 
               
               
                 S83I/S80I mutant 
                 GyrA S83I; ParC S80I 
                 Quinolones 
               
               
                 S83L/S80I mutant 
                 GyrA S83L; ParC S80I 
                 Quinolones 
               
               
                 S83F/S80I mutant 
                 GyrA S83F; ParC S80I 
                 Quinolones 
               
               
                 S83Y/S80I mutant 
                 GyrA S83Y; ParC S80I 
                 Quinolones 
               
               
                 S83I/D87N/S80I mutant 
                 GyrA S83I/D87N; ParC S80I 
                 Quinolones 
               
               
                 S83L/D87N/S80I mutant 
                 GyrA S83L/D87N; ParC S80I 
                 Quinolones 
               
               
                 S83F/D87N/S80I mutant 
                 GyrA S83F/D87N; ParC S80I 
                 Quinolones 
               
               
                 S83Y/D87N/S80I mutant 
                 GyrA S83Y/D87N; ParC S80I 
                 Quinolones 
               
               
                 ΔramR/S83I mutant 
                 ΔramR; GyrA S83I 
                 β-lactams, Quinolones, Tetracyclines, 
               
               
                   
                   
                 Folate pathway inhibitors, Phenicols, 
               
               
                   
                   
                 Nitrofurans 
               
               
                 ΔramR/S83I/S80I mutant 
                 ΔramR; GyrA S83I; ParC S80I 
                 β-lactams, Quinolones, Tetracyclines, 
               
               
                   
                   
                 Folate pathway inhibitors, Phenicols, 
               
               
                   
                   
                 Nitrofurans 
               
               
                 ΔramR/S83I/D87N/S80I mutant 
                 ΔramR; GyrA S83I/D87N; ParC 
                 β-lactams, Quinolones, Tetracyclines, 
               
               
                   
                 S80I 
                 Folate pathway inhibitors, Phenicols, 
               
               
                   
                   
                 Nitrofurans 
               
            
           
           
               
            
               
                 Plasmid with plasmid-mediated resistance mechanism 
               
            
           
           
               
               
               
            
               
                 p177/CTX-M-14 
                 bla CTX-M-14  cloned into 
                 β-lactams 
               
               
                   
                 pACYC177 
               
               
                 p177/CTX-M-15 
                 bla CTX-M-15  cloned into 
                 β-lactams 
               
               
                   
                 pACYC177 
               
               
                 p177/SHV-12 
                 bla SHV-12  cloned into pACYC177 
                 β-lactams 
               
               
                 p1771/CMY-2 
                 bla CMY-2  cloned into pACYC177 
                 β-lactams 
               
               
                 p177/DHA-1-AmpR 
                 bla DHA-1-AmpR  cloned into 
                 β-lactams 
               
               
                   
                 pACYC177 
               
               
                 p177/KPC-2 
                 bla KPC-2  cloned into pACYC177 
                 β-lactams 
               
               
                 p177/KPC-3 
                 bla KPC-3  cloned into pACYC177 
                 β-lactams 
               
               
                 p177/IMP-8 
                 bla IMP-8  cloned into pACYC177 
                 β-lactams 
               
               
                 p177/NDM-1 
                 bla NDN-1  cloned into pACYC177 
                 β-lactams 
               
               
                 p177/VIM-1 
                 bla VIM-1  cloned into pACYC177 
                 β-lactams 
               
               
                 p177/OXA-48 
                 bla OXA-48  cloned into pACYC177 
                 β-lactams 
               
               
                 p177/QnrB 
                 qnrB cloned into pACYC177 
                 Quinolones 
               
               
                 p177/QnrS 
                 qnrS cloned into pACYC177 
                 Quinolones 
               
               
                 p177/AAC(6′)-Ib-cr 
                 aac(6′)-Ib-cr cloned into 
                 Aminoglycosides, Quinolones 
               
               
                   
                 pACYC177 
               
               
                 p184/AAC(6′)-Ib-cr 
                 aac(6′)-Ib-cr cloned into 
                 Aminoglycosides, Quinolones 
               
               
                   
                 pACYC184 
               
               
                 p184/AAC(3)-IId 
                 aac(3)-IId cloned into 
                 Aminoglycosides 
               
               
                   
                 pACYC184 
               
               
                 p184/AAC(3)-IVa 
                 aac(3)-IVa cloned into 
                 Aminoglycosides 
               
               
                   
                 pACYC184 
               
               
                 p184/ANT(2″)-Ia 
                 ant(2″)-Ia cloned into 
                 Aminoglycosides 
               
               
                   
                 pACYC184 
               
               
                 p184/ANT(3″)-Ia 
                 ant(3″)-Ia cloned into 
                 Aminoglycosides 
               
               
                   
                 pACYC184 
               
               
                 p184/APH(3′)-Ia 
                 aph(3′)-Ia cloned into 
                 Aminoglycosides 
               
               
                   
                 pACYC184 
               
               
                 p184/APH(3′)-IIa 
                 aph(3′)-IIa cloned into 
                 Aminoglycosides 
               
               
                   
                 pACYC184 
               
               
                 p184/StrA-StrB 
                 strA-strB cloned into 
                 Aminoglycosides 
               
               
                   
                 pACYC184 
               
               
                 p184/ArmA 
                 armA cloned into pACYC184 
                 Aminoglycosides 
               
               
                 p184/RmtB 
                 rmtB cloned into pACYC184 
                 Aminoglycosides 
               
               
                 p177/Tet(A) 
                 tet(A) cloned into pACYC177 
                 Tetracyclines 
               
               
                 p177/Tet(B) 
                 tet(B) cloned into pACYC177 
                 Tetracyclines 
               
               
                 p177/Tet(C) 
                 tet(C) cloned into pACYC177 
                 Tetracyclines 
               
               
                 p177/Tet(D) 
                 tet(D) cloned into pACYC177 
                 Tetracyclines 
               
               
                 p177/Tet(M) 
                 tet(M) cloned into pACYC177 
                 Tetracyclines 
               
               
                 p177/Sul1 
                 sul1 cloned into pACYC177 
                 Folate pathway inhibitors 
               
               
                 p177/Sul2 
                 sul2 cloned into pACYC177 
                 Folate pathway inhibitors 
               
               
                 p177/DfrA1 
                 dfrA1 cloned into pACYC177 
                 Folate pathway inhibitors 
               
               
                 p177/DfrA16 
                 dfrA16 cloned into pACYC177 
                 Folate pathway inhibitors 
               
               
                   
               
               
                   a Amino acid replacements of GyrA or ParC are listed; Resistance genes of plasmid-mediated mechanisms were cloned into the low-copy-number plasmid pACYC177 or pACYC184. 
               
               
                   b The antibiotics listed indicate a significant (≥4-fold) difference in the MICs of the NVT1001 strain and its genetically engineered strains in this study. 
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 3 
               
             
            
               
                   
               
               
                 Revertants of the NVT1001&#39;s mutants that were constructed in this study 
               
            
           
           
               
               
               
            
               
                 Strain 
                 Genotype 
                 Description 
               
               
                   
               
               
                 ΔramRΔompK35/36::ramR::ompK35/36 revertant 
                 NVT1001 
                 ramR, ompK35 and/or ompK36 
               
               
                 ΔramRΔompK35/36::ramR::ompK36 revertant 
                 ΔompK35 
                 restored strains of the 
               
               
                 ΔramRΔompK35/36::ramR::ompK35 revertant 
                 ΔompK36 
                 ΔramRΔompK35/36 mutant 
               
               
                 ΔramRΔompK35/36::ramR revertant 
                 ΔompK35/36 
               
               
                 ΔramRΔompK35/36::ompK35/36 revertant 
                 ΔramR 
               
               
                 ΔramRΔompK35/36::ompK36 revertant 
                 ΔramRΔompK35 
               
               
                 ΔramRΔompK35/36::ompK35 revertant 
                 ΔramRΔompK36 
               
               
                 S83I/D87N/S80I::I83S/N87D/I80S revertant 
                 NVT1001 
                 GyrA S83/D87 and ParC S80 
               
               
                 S83L/D87N/S80I::L83S/N87D/I80S revertant 
                 NVT1001 
                 restored strains of the 
               
               
                 S83F/D87N/S80I::F83S/N87D/I80S revertant 
                 NVT1001 
                 S83I/D87N/S80I, 
               
               
                 S83Y/D87N/S80I::Y83S/N87D/I80S revertant 
                 NVT1001 
                 S83L/D87N/S80I, 
               
               
                   
                   
                 S83F/D87N/S80I or 
               
               
                   
                   
                 S83Y/D87N/S80I mutant 
               
               
                 S83I/D87N/S80I::N87D/I80S revertant 
                 GyrA S83I 
                 GyrA D87 and ParC S80 
               
               
                 S83L/D87N/S80I::N87D/I80S revertant 
                 GyrA S83L 
                 restored strains of the 
               
               
                 S83F/D87N/S80I::N87D/I80S revertant 
                 GyrA S83F 
                 S83I/D87N/S80I, 
               
               
                 S83Y/D87N/S80I::N87D/I80S revertant 
                 GyrA S83Y 
                 S83L/D87N/S80I, 
               
               
                   
                   
                 S83F/D87N/S80I or 
               
               
                   
                   
                 S83Y/D87N/S80I mutant 
               
               
                 ΔramR/S83I/D87N/S80I::ramR/I83S/I80S revertant 
                 GyrA D87N 
                 ramR, GyrA S83, GyrA D87, 
               
               
                 ΔramR/S83I/D87N/S80I::ramR/I83S/N87D revertant 
                 ParC S80I 
                 and/or ParC S80 restored 
               
               
                 ΔramR/S83I/D87N/S80I::I83S/N87D/I80S revertant 
                 ΔramR 
                 strains of the 
               
               
                 ΔramR/S83I/D87N/S80I::ramR/I83S revertant 
                 GyrA D87N; ParC S80I 
                 ΔramR/S83I/D87N/S80I 
               
               
                   
                   
                 mutant 
               
               
                 S83I/D87N/S80I::I80S revertant 
                 GyrA S83I/D87N 
                 ParC S80 restored strains of 
               
               
                 S83L/D87N/S80I::I80S revertant 
                 GyrA S83L/D87N 
                 the S83I/D87N/S80I, 
               
               
                 S83F/D87N/S80I::I80S revertant 
                 GyrA S83F/D87N 
                 S83L/D87N/S80I, 
               
               
                 S83Y/D87N/S80I::I80S revertant 
                 GyrA S83Y/D87N 
                 S83F/D87N/S80I or 
               
               
                   
                   
                 S83Y/D87N/S80I mutant 
               
               
                 S83I/D87N/S80I::N87D revertant 
                 GyrA S83I; ParC S80I 
                 GyrA D87 restored strains of 
               
               
                 S83L/D87N/S80I::N87D revertant 
                 GyrA S83L; ParC S80I 
                 the S83I/D87N/S80I, 
               
               
                 S83F/D87N/S80I::N87D revertant 
                 GyrA S83F; ParC S80I 
                 S83L/D87N/S80I, 
               
               
                 S83Y/D87N/S80I::N87D revertant 
                 GyrA S83Y; ParC S80I 
                 S83F/D87N/S80I or 
               
               
                   
                   
                 S83Y/D87N/S80I mutant 
               
               
                 ΔramR/S83I/D87N/S80I::ramR/I83S/N87D/I80S revertant 
                 NVT1001 
                 ramR, GyrA S83, GyrA D87 
               
               
                 ΔramR/S83I/D87N/S80I::N87D/I80S revertant 
                 ΔramR; GyrA S83I 
                 and/or ParC S80, restored 
               
               
                 ΔramR/S83I/D87N/S80I::N87D revertant 
                 ΔramR; GyrA S83I; ParC 
                 strains of the 
               
               
                   
                 S80I 
                 ΔramR/S83I/D87N/S80I 
               
               
                   
                   
                 mutant 
               
               
                   
               
            
           
         
       
     
     1.2.2  A. baumannii  KW1 
       A. baumannii  KW1 is a clinical isolate that was fully susceptible to antibiotics tested. The KW1 strain was used to construct genetically engineered strains with the antibiotic resistance mechanisms that are shown in Table 4. The plasmids pYMAb5 and pYMab5Tc are shuttle vectors able to replicate in  A. baumannii  and  Escherichia coli , which carry a resistant determinant of kanamycin or tetracycline respectively. The two shuttle vectors were used to clone the resistance genes of the plasmid-mediated resistance mechanisms in this study (Table 4). 
     
       
         
           
               
             
               
                 TABLE 4 
               
             
            
               
                   
               
               
                 Antibiotic resistance mechanisms in fully susceptible clinical  A. baumannii  KW1 
               
            
           
           
               
               
               
            
               
                 Strain or plasmid 
                 Genotype or description a   
                 Major relevant antibiotic b   
               
               
                   
               
            
           
           
               
            
               
                 Strain with chromosome-mediated resistance mechanism 
               
            
           
           
               
               
               
            
               
                 D20N mutant 
                 AdeR D20N 
                 β-lactams, quinolones, aminoglycosides, 
               
               
                   
                   
                 tetracyclines 
               
               
                 A91V mutant 
                 AdeR A91V 
                 β-lactams, quinolones, aminoglycosides, 
               
               
                   
                   
                 tetracyclines 
               
               
                 P116L mutant 
                 AdeR P116L 
                 β-lactams, quinolones, aminoglycosides, 
               
               
                   
                   
                 tetracyclines 
               
               
                 G30D mutant 
                 AdeS G30D 
                 β-lactams, quinolones, aminoglycosides, 
               
               
                   
                   
                 tetracyclines 
               
               
                 A94V mutant 
                 AdeS A94V 
                 β-lactams, quinolones, aminoglycosides, 
               
               
                   
                   
                 tetracyclines 
               
               
                 R152K mutant 
                 AdeS R152K 
                 β-lactams, quinolones, aminoglycosides, 
               
               
                   
                   
                 tetracyclines 
               
               
                 T153M mutant 
                 AdeS T153M 
                 β-lactams, quinolones, aminoglycosides, 
               
               
                   
                   
                 tetracyclines 
               
               
                 S83L mutant 
                 GyrA S83L 
                 quinolones 
               
               
                 G78C mutant 
                 ParC G78C 
                 none 
               
               
                 S80L mutant 
                 ParC S80L 
                 none 
               
               
                 S80W mutant 
                 ParC S80W 
                 none 
               
               
                 S80Y mutant 
                 ParC S80Y 
                 none 
               
               
                 E84K mutant 
                 ParC E84K 
                 none 
               
               
                 S83L/G78C mutant 
                 GyrA S83L; ParC G78C 
                 quinolones 
               
               
                 S83L/S80L mutant 
                 GyrA S83L; ParC S80L 
                 quinolones 
               
               
                 S83L/S80W mutant 
                 GyrA S83L; ParC S80W 
                 quinolones 
               
               
                 S83L/S80Y mutant 
                 GyrA S83L; ParC S80Y 
                 quinolones 
               
               
                 S83L/E84K mutant 
                 GyrA S83L; ParC E84K 
                 quinolones 
               
            
           
           
               
            
               
                 Plasmid with plasmid-mediated resistance mechanism 
               
            
           
           
               
               
               
            
               
                 pB5/CTX-M-15 
                 bla CTX-M-15  cloned into 
                 β-lactams 
               
               
                   
                 pYMAb5 
               
               
                 pB5/VEB-3 
                 bla VEB-3  cloned into 
                 β-lactams 
               
               
                   
                 pYMAb5 
               
               
                 pB5/ADC-30 
                 bla ADC-30  cloned into 
                 β-lactams 
               
               
                   
                 pYMAb5 
               
               
                 pB5/IMP-1 
                 bla IMP-1  cloned into 
                 β-lactams 
               
               
                   
                 pYMAb5 
               
               
                 pB5/NDM-1 
                 bla NDM-1  cloned into 
                 β-lactams 
               
               
                   
                 pYMAb5 
               
               
                 pB5/VIM-1 
                 bla VIM-1  cloned into 
                 β-lactams 
               
               
                   
                 pYMAb5 
               
               
                 pB5/OXA-23 
                 bla OXA-23  cloned into 
                 β-lactams 
               
               
                   
                 pYMAb5 
               
               
                 pB5/OXA-58 
                 bla OXA-58  cloned into 
                 β-lactams 
               
               
                   
                 pYMAb5 
               
               
                 pB5/OXA-66 
                 bla OXA-66  cloned into 
                 β-lactams 
               
               
                   
                 pYMAb5 
               
               
                 pB5/OXA-72 
                 bla OXA-72  cloned into 
                 β-lactams 
               
               
                   
                 pYMAb5 
               
               
                 pB5Tc/AAC(3)-IIa 
                 aac(3)-IIa cloned into 
                 aminoglycosides 
               
               
                   
                 pYMAb5Tc 
               
               
                 pB5Tc/ANT(2″)-Ia 
                 ant(2″)-Ia cloned into 
                 aminoglycosides 
               
               
                   
                 pYMAb5Tc 
               
               
                 pB5Tc/ANT(3″)-Ia 
                 ant(3″)-Ia cloned into 
                 aminoglycosides 
               
               
                   
                 pYMAb5Tc 
               
               
                 pB5Tc/APH(3′)-Ia 
                 aph(3′)-Ia cloned into 
                 aminoglycosides 
               
               
                   
                 pYMAb5Tc 
               
               
                 pB5Tc/APH(3′)-VIa 
                 aph(3′)-VIa cloned into 
                 aminoglycosides 
               
               
                   
                 pYMAb5Tc 
               
               
                 pB5Tc/ArmA 
                 armA cloned into 
                 aminoglycosides 
               
               
                   
                 pYMAb5Tc 
               
               
                 pB5/Tet(A) 
                 tet(A) cloned into 
                 tetracyclines 
               
               
                   
                 pYMAb5 
               
               
                 pB5/Tet(B) 
                 tet(B) cloned into 
                 tetracyclines 
               
               
                   
                 pYMAb5 
               
               
                 pB5/Tet(M) 
                 tet(M) cloned into 
                 tetracyclines 
               
               
                   
                 pYMAb5 
               
               
                   
               
               
                   a Amino acid replacement of AdeR, AdeS, GyrA or ParC are listed; resistance genes of plasmid-mediated mechanisms were clone into the shuttle vector pYMAb5 or pYMAb5Tc, which carries a resistant determinant of kanamycin or tetracycline respectively. 
               
               
                   b The antibiotics listed indicate a ≥2-fold difference in the MICs for the KW1 strain and its genetically engineered strains in this study. 
               
            
           
         
       
     
     1.3 In-Frame Deletion Mutagenesis 
     The plasmid pUT-kmy, which consists of an R6K origin of replication, an mobRP4 origin of transfer, and a kanamycin resistance cassette, 18  was ligated with the sacB gene to generate the plasmid pUT-KB, which was then used to construct the mutants. 19  The plasmid pUT-KB is a suicide vector containing the counter-selection marker sacB, which originates from  Bacillus subtilis.   20  When this gene is expressed via the integrated pUT-KB, it confers a sucrose-susceptibility phenotype, which enables positive selection with sucrose to detect loss of the vector. 
     Gene deletions in  K. pneumoniae  strain NVT1001 were constructed via in-frame deletion mutagenesis. 7  Briefly, two DNA fragments (approximately 1 kb in size) that flanked the regions to be deleted were amplified by PCR using specific primer pairs. The two gel-purified PCR products, containing complementary ends, were then mixed and amplified via overlap PCR. 21,22  The resulting PCR fragment (approximately 2 kb in size) was digested with restriction enzymes and then cloned into pUT-KB that had been similarly digested. For homologous recombination, each of the gene-deletion constructs in pUT-KB was transformed into  E. coli  S17-1 λpir 23  via electroporation and then mobilized into  K. pneumoniae  strain NVT1001 via conjugation. Single-crossover strains were selected from BIND (Brilliant green containing Inositol-Nitrate-Deoxycholate) plates supplemented with kanamycin (50 mg/L), as the growth of non- K. pneumoniae  strains is effectively suppressed on BIND plates. 24  After the kanamycin-resistant transconjugants were selected, the insertions of the plasmids were verified via PCR using primer pairs that flanked the target genes. After incubation in 20 mL brain-heart infusion (BHI) broth for 6 hours at 37° C. in the absence of kanamycin, the fully grown cultures were spread onto LB agar plates supplemented with 10% sucrose. After the double crossover occurred, the sucrose-resistant and kanamycin-susceptible colonies were selected, and the gene deletions were confirmed via PCR. 
     1.4 Site-Directed Mutagenesis 
     DNA fragments of the entire gyrA and parC sequences along with their flanking regions were amplified from  K. pneumoniae  strain NVT1001 using PCR with specific primer pairs and then cloned into the plasmid pUT-KB. The QuikChange site-directed mutagenesis kit (Stratagene) was used to generate mutations in the gyrA and parC genes in the plasmid using the methods described by the manufacturer. For homologous recombination, plasmids containing mutations in the gyrA or parC gene were transformed into  E. coli  S17-1 λpir 23  via electroporation and mobilized into  K. pneumoniae  strain NVT1001 via conjugation. Single-crossover strains were selected from BIND plates supplemented with kanamycin (50 mg/L), as the growth of non- K. pneumoniae  contaminants is suppressed on BIND plates. 24  After the kanamycin-resistant transconjugants were selected, the insertion of plasmids was verified via PCR. After incubation in 20 mL BHI broth for 6 hours at 37° C. in the absence of kanamycin, the fully grown cultures were spread onto LB agar plates supplemented with 10% sucrose. After the double crossover occurred, the sucrose-resistant and kanamycin-susceptible colonies were selected, and the gene mutations were confirmed via PCR. 
     Plasmid pUT-kmy, which consists of an R6K origin of replication, a mobRP4 origin of transfer, and a kanamycin resistant determinant, 56  was ligated with a sacB gene to generate plasmid pUT-KB for constructing mutants. 57  Plasmid pUT-KB is a suicide vector containing a counter-selection marker, sacB, which originates from  Bacillus subtilis.   58  When this gene is expressed on the integrated pUT-KB, it confers a sucrose-sensitivity phenotype, which enables positive selection with sucrose to detect the loss of the vector. 
     DNA fragments of the entire adeR, adeS, gyrA and parC along with their flanking regions were amplified from  A. baumannii  KW1 using PCR with specific primer pairs and then cloned into plasmid pUT-KB. The QuikChange site-directed mutagenesis kit (Stratagene) was used to generate mutations in the adeR, adeS, gyrA and parC genes in the plasmids using the methods described by the manufacturer. For homologous recombination, plasmids containing mutations in the adeR, adeS, gyrA or parC gene were transformed into  E. coli  S17-1 λpir 23  via electroporation and mobilized into  A. baumannii  KW1 via conjugation. Single-crossover strains were selected from Luria-Bertani (LB) plates supplemented with cefotaxime (1 μg/ml) and kanamycin (50 m/ml), as the growth of non- A. baumannii  contaminants is suppressed by cefotaxime (1 μg/ml). The kanamycin-resistant transconjugants were selected and the insertion of plasmids was verified via PCR. After incubation in 20 ml brain heart infusion (BHI) broth for 6 hours in the absence of kanamycin at 37° C., the fully grown cultures were spread onto LB plates supplemented with 10% sucrose. After the double crossover occurred, the sucrose-resistant and kanamycin-susceptible colonies were selected, and the gene mutations were confirmed via PCR. 
     1.5 Construction of Revertants 
     The allelic-exchange method was used to restore the wild-type gene in the  K. pneumoniae  NVT1001 mutants with constructed chromosome-mediated resistance mechanisms, as described by Tsai et al. 19  Briefly, a DNA fragment of the entire wild-type gene sequence along with flanking regions was amplified from  K. pneumoniae  NVT1001 using PCR with specific primers. The resulting PCR fragment was digested and then cloned into pUT-KB. For homologous recombination, this plasmid was transformed into  E. coli  S17-1 λpir 23  via electroporation and mobilized into the  K. pneumoniae  NVT1001 mutant via conjugation. Single-crossover strains were selected from BIND plates supplemented with kanamycin (50 mg/L), as the growth of non- K. pneumoniae  strains is effectively suppressed on the BIND plates. 24  After the kanamycin-resistant transconjugants were selected, the insertion of the plasmid was verified via PCR. After incubation in 20 mL BHI broth for 6 hours at 37° C. in the absence of kanamycin, the fully grown cultures were spread onto LB agar plates supplemented with 10% sucrose. After the double crossover occurred, the sucrose-resistant and kanamycin-susceptible colonies were selected, and the restoration of the wild-type gene was confirmed via DNA sequencing. 
     1.6 Plasmid Construction and Transformation 
     DNA fragments of the resistance genes along with their flanking regions were amplified from clinical plasmids via PCR with specific primer pairs. The resulting PCR fragments were digested and then cloned into the plasmid pACYC177 or pACYC184. The resulting plasmids were then transformed into  K. pneumoniae  strain NVT1001 via electroporation. The recombinant bacteria were plated onto LB agar plates containing kanamycin (50 mg/L) or chloramphenicol (50 mg/L), and the presence of the cloned gene was confirmed via PCR and DNA sequencing. 
     DNA fragments of the resistance genes along with their flanking regions were amplified from clinical plasmids via PCR with specific primer pairs. The resulting PCR fragments were digested and then cloned into the plasmid pYMAb5 or pYMAb5Tc. The resulting plasmids were then transformed into  A. baumannii  KW1 via electroporation. The recombinant bacteria were plated onto LB agar plates containing kanamycin (50 μg/ml) or tetracycline (50 μg/ml), and the presence of the cloned gene was confirmed via PCR and DNA sequencing. 
     1.7 Antimicrobial Susceptibility Testing 
     The MICs of antibiotics were determined using the Etest (Biodisk AB, Sweden) according to the manufacturer&#39;s instructions. The MICs from the Etest, which fall between standard two-fold dilutions, were rounded up to the next highest two-fold value. The MICs of ceftazidime and cefotaxime against  K. pneumoniae  NVT1001 harbouring the pACYC177 plasmid with bla OXA-48  were further determined using a broth microdilution test according to the recommendations of the CLSI, 25  and the results were interpreted according to the breakpoints established by the CLSI in 2017. 25  Meanwhile, due to the lack of CLSI breakpoints in 2016 and 2017, the results for cephalothin were interpreted according to the breakpoints established by the CLSI in 2015, 26  and the results for moxifloxacin were interpreted according to the breakpoints established by the EUCAST in 2017. 27    
     1.8 Mouse Infection Model 
     Pathogen-free 6- to 8-week-old male BALB/c mice were obtained from the National Laboratory Animal Center (Taiwan) and maintained in the pathogen-free vivarium of the Laboratory Animal Center of National Defense Medical Center (Taiwan).  K. pneumoniae  NVT1001 harbouring the pACYC177 plasmid with bla OXA-48  was cultured overnight at 37° C. in BHI broth and then diluted (1:100) in fresh BHI broth. The culture was incubated until the mid-exponential growth phase, and the cells were then washed once, resuspended in 0.85% sterile saline, and adjusted to the desired concentrations according to the OD 600  value. The concentrations were verified by plating the cells to determine viable counts. Six mice from each group were then injected intraperitoneally with 0.1 mL of the cell suspension containing twice the 90-100% lethal dose (LD90-100); this inoculum of 4×10 3  cfu/mouse was known to be 100% lethal within 7 days (data not shown). Meanwhile, antibiotic doses were prepared in 0.85% sterile saline. At 1 hour post-inoculation, antibiotic or 0.85% sterile saline alone was administered as a single subcutaneous injection with a volume of 0.3 mL per dose. The mice were then monitored daily for 7 days to measure survival. The ED50 was defined as the single dose giving protection to 50% of the test mice. 
     1.9 Quantitative real-time PCR (qRT-PCR) 
       K. pneumoniae  strains were cultured in Mueller-Hinton broth (MHB) at 37° C. overnight, diluted (1:100) in fresh MHB, and then incubated at 37° C. until an OD600 of 0.8 (the mid-exponential growth phase) is reached. RNA was extracted using the RNeasy kit and treated with RNase-free DNase I according to the manufacturer&#39;s instructions (Qiagen). RNA yield and quality was measured using the NanoVue spectrophotometer (GE Healthcare Life Sciences). cDNAs were synthesized from 1 μg of RNA template using the Omniscript Reverse Transcriptase according to the manufacturer&#39;s instructions (Qiagen). Relative quantification of gene expression was performed using ABI PRISM 7900HT real-time PCR System (Applied Biosystems) with the Maxima SYBR Green qPCR Master Mix (Thermo Scientific) and primers listed in Table 5. Fold-change values were calculated after normalization of each gene to the 23S rRNA internal control, 3  using the comparative threshold method with the NVT1001 strain as the reference strain. 
     
       
         
           
               
             
               
                 TABLE 5 
               
             
            
               
                   
               
               
                 Oligonucleotide primers for qRT-PCR in this study 
               
            
           
           
               
               
               
               
            
               
                 Primer 
                 Sequence (5′-3′) 
                 Target 
                 Reference 
               
               
                   
               
               
                 acrA-qF 
                 ATGTGACGATAAACCGGCTC (SEQ ID NO: 1) 
                 acrA 
                 This study 
               
               
                   
               
               
                 acrA-qR 
                 CTGGCAGTTCGGTGGTTATT (SEQ ID NO: 2) 
                   
                   
               
               
                   
               
               
                 tolC-qF 
                 AACGGGCAGAACCAAATCGGC (SEQ ID NO: 3) 
                 tolC 
                 This study 
               
               
                   
               
               
                 tolC-qR 
                 CGTTGATGCTGCTGATGGAGGC (SEQ ID NO: 4) 
                   
                   
               
               
                   
               
               
                 ramA-qF 
                 TGATTCGCAACAGACTTTTACCCG (SEQ ID NO: 5) 
                 ramA 
                 This study 
               
               
                   
               
               
                 ramA-qR 
                 GCGACTGTGGTTCTCTTTGCGGT (SEQ ID NO: 6) 
                   
                   
               
               
                   
               
               
                 ramR-qF 
                 AGGATGAGTTGCTCAACGAG (SEQ ID NO: 7) 
                 ramR 
                 This study 
               
               
                   
               
               
                 ramR-qR 
                 CCAGTCGATATAGCTGTTCCAG (SEQ ID NO: 8) 
                   
                   
               
               
                   
               
               
                 23S-qF 
                 GGTAGGGGAGCGTTCTGTAA (SEQ ID NO: 9) 
                 23S 
                 40 
               
               
                   
                   
                 rRNA 
                   
               
               
                   
               
               
                 23S-qR 
                 TCAGCATTCGCACTTCTGAT (SEQ ID NO: 10) 
               
               
                   
               
            
           
         
       
     
     2. Results 
     2.1  K. pneumoniae  NVT1001 
     2.1.1 Porin Loss and Efflux-Pump Overexpression in Antibiotic Resistance 
     The MICs of antibiotics against chromosome-mediated resistance mechanisms, namely, OmpK35/36 loss and AcrAB-TolC overexpression (ΔramR), are shown in Table 6. These results demonstrate that OmpK35/36 loss is related to increased resistance to β-lactams, folate pathway inhibitors, fosfomycins and nitrofurans, whereas AcrAB-To1C overexpression (ΔramR) is associated with increased resistance to β-lactams, quinolones, tetracyclines, folate pathway inhibitors, phenicols and nitrofurans. The two resistance mechanisms alone or in combination had no significant (≥4-fold) effects on the MIC(s) of imipenem, aminoglycosides or polymyxins. The results shown above were further validated by testing the antimicrobial susceptibility of the revertant strains, and similar results were found when comparing the MIC of the wild-type strain NVT1001 or its mutant to the MIC of the revertant with the same genotype (Table 7). 
     
       
         
           
               
             
               
                 TABLE 6 
               
             
            
               
                   
               
               
                 MICs of antibiotics against the chromosome-mediated resistance mechanisms, OmpK35/36 loss 
               
               
                 and AcrAB-TolC overexpression (ΔramR), in  K. pneumoniae  NVT1001 
               
            
           
           
               
               
               
            
               
                   
                   
                 MIC (mg/L) b   
               
            
           
           
               
               
               
               
               
               
               
               
               
               
            
               
                   
                   
                   
                   
                   
                   
                   
                 ΔramR 
                 ΔramR 
                 ΔramR 
               
               
                 Antibiotic group 
                 Antibiotic a   
                 WT c   
                 Δ35 
                 Δ36 
                 Δ35/36 
                 ΔramR 
                 Δ35 
                 Δ36 
                 Δ35/36 
               
               
                   
               
            
           
           
               
               
               
               
               
               
               
               
               
               
            
               
                 β-lactams 
                 Aztreonam 
                 0.03 
                 
                   0.25 
                 
                 0.06 
                 
                   0.5 
                 
                 
                   0.5 
                 
                 
                   0.5 
                 
                 
                   1 
                 
                 
                   1 
                 
               
               
                   
                 Piperacillin/TZB 
                 4 
                 4 
                 4 
                 8 
                 
                   32 
                 
                 
                   32 
                 
                 
                   32 
                 
                 
                   32 
                 
               
               
                   
                 Ticarcillin/CLA 
                 4 
                 4 
                 4 
                 
                   16 
                 
                 8 
                 
                   16 
                 
                 
                   16 
                 
                 8 
               
               
                   
                 Cephalothin 
                 8 
                 8 
                 16 
                 
                   256 
                 
                 
                   64 
                 
                 
                   64 
                 
                 
                   &gt;256 
                 
                 
                   &gt;256 
                 
               
               
                   
                 Cefuroxime 
                 4 
                 8 
                 
                   16 
                 
                 
                   32 
                 
                 
                   32 
                 
                 
                   64 
                 
                 
                   &gt;256 
                 
                 
                   128 
                 
               
               
                   
                 Cefoxitin 
                 4 
                 4 
                 
                   16 
                 
                 
                   128 
                 
                 
                   64 
                 
                 
                   64 
                 
                 
                   &gt;256 
                 
                 
                   &gt;256 
                 
               
               
                   
                 Ceftazidime 
                 0.25 
                 0.5 
                 0.5 
                 
                   1 
                 
                 
                   2 
                 
                 
                   2 
                 
                 
                   2 
                 
                 
                   2 
                 
               
               
                   
                 Cefotaxime 
                 0.06 
                 0.125 
                 
                   0.25 
                 
                 
                   2 
                 
                 
                   1 
                 
                 
                   1 
                 
                 
                   4 
                 
                 
                   4 
                 
               
               
                   
                 Cefepime 
                 0.06 
                 0.125 
                 0.125 
                 
                   1 
                 
                 
                   0.5 
                 
                 
                   0.5 
                 
                 
                   4 
                 
                 
                   2 
                 
               
               
                   
                 Ceftaroline 
                 0.25 
                 0.25 
                 0.5 
                 
                   1 
                 
                 
                   1 
                 
                 
                   1 
                 
                 
                   4 
                 
                 
                   4 
                 
               
               
                   
                 Ertapenem 
                 0.015 
                 0.03 
                 0.03 
                 
                   2 
                 
                 
                   0.125 
                 
                 
                   0.125 
                 
                 
                   4 
                 
                 
                   4 
                 
               
               
                   
                 Imipenem 
                 0.5 
                 0.5 
                 0.5 
                 0.5 
                 0.25 
                 0.25 
                 0.5 
                 0.5 
               
               
                   
                 Meropenem 
                 0.06 
                 0.06 
                 0.06 
                 
                   0.5 
                 
                 0.06 
                 0.06 
                 
                   2 
                 
                 
                   2 
                 
               
               
                   
                 Doripenem 
                 0.06 
                 0.06 
                 0.06 
                 0.125 
                 0.03 
                 0.03 
                 
                   0.25 
                 
                 
                   0.5 
                 
               
               
                 Quinolones 
                 Nalidixic acid 
                 4 
                 8 
                 4 
                 8 
                 
                   32 
                 
                 
                   64 
                 
                 
                   32 
                 
                 
                   32 
                 
               
               
                   
                 Ciprofloxacin 
                 0.06 
                 0.06 
                 0.06 
                 0.125 
                 
                   0.5 
                 
                 
                   0.5 
                 
                 
                   0.5 
                 
                 
                   0.5 
                 
               
               
                   
                 Norfloxacin 
                 0.25 
                 0.25 
                 0.25 
                 0.5 
                 
                   2 
                 
                 
                   2 
                 
                 
                   2 
                 
                 
                   2 
                 
               
               
                   
                 Ofloxacin 
                 0.25 
                 0.25 
                 0.25 
                 0.25 
                 
                   2 
                 
                 
                   2 
                 
                 
                   2 
                 
                 
                   2 
                 
               
               
                   
                 Levofloxacin 
                 0.06 
                 0.125 
                 0.125 
                 0.125 
                 
                   1 
                 
                 
                   1 
                 
                 
                   1 
                 
                 
                   1 
                 
               
               
                   
                 Moxifloxacin 
                 0.125 
                 0.125 
                 0.06 
                 0.125 
                 
                   1 
                 
                 
                   1 
                 
                 
                   1 
                 
                 
                   1 
                 
               
               
                 Aminoglycosides 
                 Amikacin 
                 2 
                 2 
                 2 
                 2 
                 2 
                 2 
                 2 
                 2 
               
               
                   
                 Gentamicin 
                 0.5 
                 0.5 
                 0.5 
                 0.5 
                 0.5 
                 0.25 
                 0.5 
                 0.5 
               
               
                   
                 Kanamycin 
                 2 
                 2 
                 2 
                 4 
                 2 
                 2 
                 2 
                 2 
               
               
                   
                 Netilmicin 
                 0.5 
                 0.5 
                 0.5 
                 0.5 
                 0.5 
                 0.25 
                 0.5 
                 0.5 
               
               
                   
                 Spectinomycin 
                 16 
                 16 
                 16 
                 16 
                 16 
                 16 
                 16 
                 16 
               
               
                   
                 Streptomycin 
                 2 
                 4 
                 4 
                 4 
                 2 
                 2 
                 4 
                 4 
               
               
                   
                 Tobramycin 
                 0.5 
                 0.5 
                 0.5 
                 0.5 
                 0.5 
                 0.5 
                 0.5 
                 1 
               
               
                 Tetracyclines 
                 Tetracycline 
                 4 
                 4 
                 2 
                 8 
                 
                   32 
                 
                 
                   32 
                 
                 
                   32 
                 
                 
                   64 
                 
               
               
                   
                 Doxycycline 
                 4 
                 4 
                 4 
                 4 
                 
                   64 
                 
                 
                   64 
                 
                 
                   64 
                 
                 
                   128 
                 
               
               
                   
                 Minocycline 
                 4 
                 4 
                 4 
                 4 
                 
                   64 
                 
                 
                   128 
                 
                 
                   128 
                 
                 
                   128 
                 
               
               
                   
                 Tigecycline 
                 1 
                 1 
                 1 
                 1 
                 
                   8 
                 
                 
                   8 
                 
                 
                   8 
                 
                 
                   8 
                 
               
               
                 Folate pathway 
                 Trimethoprim 
                 2 
                 2 
                 2 
                 2 
                 
                   8 
                 
                 
                   8 
                 
                 
                   16 
                 
                 
                   &gt;32 
                 
               
               
                 inhibitors 
                 Sulfamethoxazol 
                 256 
                 256 
                 
                   &gt;1024 
                 
                 
                   &gt;1024 
                 
                 
                   &gt;1024 
                 
                 
                   &gt;1024 
                 
                 
                   &gt;1024 
                 
                 
                   &gt;1024 
                 
               
               
                   
                 SXT 
                 0.25 
                 0.25 
                 0.5 
                 0.5 
                 0.5 
                 0.5 
                 
                   1 
                 
                 
                   8 
                 
               
               
                 Polymyxins 
                 Polymyxin B 
                 2 
                 2 
                 2 
                 2 
                 1 
                 1 
                 2 
                 1 
               
               
                   
                 Colistin 
                 1 
                 1 
                 1 
                 1 
                 1 
                 1 
                 1 
                 1 
               
               
                 Phenicols 
                 Chloramphenico 
                 8 
                 8 
                 8 
                 8 
                 
                   128 
                 
                 
                   128 
                 
                 
                   128 
                 
                 
                   128 
                 
               
               
                 Fosfomycins 
                 Fosfomycin 
                 128 
                 128 
                 256 
                 
                   512 
                 
                 64 
                 128 
                 
                   512 
                 
                 
                   &gt;1024 
                 
               
               
                 Nitrofurans 
                 Nitrofurantoin 
                 64 
                 64 
                 
                   &gt;512 
                 
                 
                   &gt;512 
                 
                 
                   256 
                 
                 
                   256 
                 
                 
                   &gt;512 
                 
                 
                   &gt;512 
                 
               
               
                   
               
               
                   a TZB, tazobactam with a fixed concentration of 4 mg/L; CLA, clavulanic acid with a fixed concentration of 2 mg/L; SXT, trimethoprim/sulfamethoxazole (only the trimethoprim portion of the 1/19 drug ratio is displayed). 
               
               
                   b Boldface numbers indicate a significant (≥4-fold) difference in the MICs of the NVT1001 strain and its mutants. 
               
               
                   c WT, NVT1001; Δ35, ΔompK35 mutant; Δ36, ΔompK36 mutant; Δ35/36, ΔompK35/36 mutant; ΔramR, ΔramR mutant; ΔramRΔ35, ΔramRΔompK35 mutant; ΔramRΔ36, ΔramRΔompK36 mutant; ΔramRΔ35/36, ΔramRΔompK35/36 mutant. 
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 7 
               
             
            
               
                   
               
               
                 MICs of antibiotics against the ΔramRΔompK35/36 mutant and its revertants 
               
            
           
           
               
               
               
            
               
                   
                   
                 MIC (mg/L) b   
               
            
           
           
               
               
               
               
               
               
               
               
               
               
            
               
                   
                   
                 ΔramR 
                 ΔramR 
                 ΔramR 
                 ΔramR 
                 ΔramR 
                 ΔramR 
                 ΔramR 
                   
               
               
                 Antibiotic 
                   
                 Δ35/36::ramR:: 
                 Δ35/36:: 
                 Δ35/36:: 
                 Δ35/36:: 
                 Δ35/36:: 
                 Δ35/ 
                 Δ35/ 
                 ΔramR 
               
               
                 group 
                 Antibiotic a   
                 35/36 c   
                 ramR::36 
                 ramR::35 
                 ramR 
                 35/36 
                 36::36 
                 36::35 
                 Δ35/36 
               
               
                   
               
            
           
           
               
               
               
               
               
               
               
               
               
               
            
               
                 β-lactams 
                 Aztreonam 
                 0.03 
                 
                   0.25 
                 
                 0.06 
                 
                   0.5 
                 
                 
                   0.5 
                 
                 
                   0.5 
                 
                 
                   1 
                 
                 
                   1 
                 
               
               
                   
                 Piperacillin/TZB 
                 4 
                 4 
                 4 
                 8 
                 
                   32 
                 
                 
                   32 
                 
                 
                   32 
                 
                 
                   32 
                 
               
               
                   
                 Ticarcillin/CLA 
                 4 
                 4 
                 4 
                 
                   16 
                 
                 
                   16 
                 
                 
                   16 
                 
                 
                   16 
                 
                 8 
               
               
                   
                 Cephalothin 
                 8 
                 8 
                 
                   32 
                 
                 
                   &gt;256 
                 
                 
                   64 
                 
                 
                   128 
                 
                 
                   &gt;256 
                 
                 
                   &gt;256 
                 
               
               
                   
                 Cefuroxime 
                 4 
                 8 
                 
                   16 
                 
                 
                   32 
                 
                 
                   32 
                 
                 
                   32 
                 
                 
                   &gt;256 
                 
                 
                   128 
                 
               
               
                   
                 Cefoxitin 
                 4 
                 8 
                 
                   16 
                 
                 
                   64 
                 
                 
                   64 
                 
                 
                   64 
                 
                 
                   &gt;256 
                 
                 
                   &gt;256 
                 
               
               
                   
                 Ceftazidime 
                 0.25 
                 
                   1 
                 
                 0.25 
                 
                   1 
                 
                 
                   2 
                 
                 
                   2 
                 
                 
                   2 
                 
                 
                   2 
                 
               
               
                   
                 Cefotaxime 
                 0.06 
                 0.125 
                 
                   0.25 
                 
                 
                   2 
                 
                 
                   1 
                 
                 
                   1 
                 
                 
                   4 
                 
                 
                   4 
                 
               
               
                   
                 Cefepime 
                 0.06 
                 0.125 
                 0.125 
                 
                   1 
                 
                 
                   0.5 
                 
                 
                   0.5 
                 
                 
                   2 
                 
                 
                   2 
                 
               
               
                   
                 Ceftaroline 
                 0.25 
                 0.5 
                 0.5 
                 
                   1 
                 
                 
                   1 
                 
                 
                   1 
                 
                 
                   4 
                 
                 
                   4 
                 
               
               
                   
                 Ertapenem 
                 0.015 
                 0.03 
                 0.03 
                 
                   1 
                 
                 
                   0.125 
                 
                 
                   0.125 
                 
                 
                   4 
                 
                 
                   4 
                 
               
               
                   
                 Imipenem 
                 0.5 
                 0.25 
                 0.25 
                 0.5 
                 0.25 
                 0.25 
                 0.25 
                 0.5 
               
               
                   
                 Meropenem 
                 0.125 
                 0.125 
                 0.125 
                 
                   0.5 
                 
                 0.06 
                 0.06 
                 
                   2 
                 
                 
                   2 
                 
               
               
                   
                 Doripenem 
                 0.06 
                 0.125 
                 0.125 
                 
                   0.25 
                 
                 0.06 
                 0.06 
                 
                   0.25 
                 
                 
                   0.5 
                 
               
               
                 Quinolones 
                 Nalidixic acid 
                 8 
                 8 
                 8 
                 8 
                 
                   64 
                 
                 
                   64 
                 
                 
                   32 
                 
                 
                   32 
                 
               
               
                   
                 Ciprofloxacin 
                 0.06 
                 0.06 
                 0.06 
                 0.125 
                 
                   0.25 
                 
                 
                   0.25 
                 
                 
                   0.5 
                 
                 
                   0.5 
                 
               
               
                   
                 Norfloxacin 
                 0.25 
                 0.25 
                 0.25 
                 0.5 
                 
                   2 
                 
                 
                   2 
                 
                 
                   2 
                 
                 
                   2 
                 
               
               
                   
                 Ofloxacin 
                 0.25 
                 0.25 
                 0.25 
                 0.25 
                 
                   2 
                 
                 
                   2 
                 
                 
                   2 
                 
                 
                   2 
                 
               
               
                   
                 Levofloxacin 
                 0.06 
                 0.125 
                 0.06 
                 0.125 
                 
                   0.5 
                 
                 
                   0.5 
                 
                 
                   1 
                 
                 
                   1 
                 
               
               
                   
                 Moxifloxacin 
                 0.125 
                 0.125 
                 0.06 
                 0.06 
                 
                   0.5 
                 
                 
                   0.5 
                 
                 
                   1 
                 
                 
                   1 
                 
               
               
                 Tetracyclines 
                 Tetracycline 
                 4 
                 4 
                 4 
                 8 
                 
                   32 
                 
                 
                   32 
                 
                 
                   32 
                 
                 
                   64 
                 
               
               
                   
                 Doxycycline 
                 4 
                 4 
                 4 
                 4 
                 
                   64 
                 
                 
                   64 
                 
                 
                   64 
                 
                 
                   128 
                 
               
               
                   
                 Minocycline 
                 4 
                 4 
                 4 
                 4 
                 
                   128 
                 
                 
                   128 
                 
                 
                   128 
                 
                 
                   128 
                 
               
               
                   
                 Tigecycline 
                 1 
                 1 
                 1 
                 1 
                 
                   8 
                 
                 
                   8 
                 
                 
                   8 
                 
                 
                   8 
                 
               
               
                 Folate pathway 
                 Trimethoprim 
                 2 
                 2 
                 2 
                 2 
                 
                   8 
                 
                 
                   8 
                 
                 
                   8 
                 
                 
                   &gt;32 
                 
               
               
                 inhibitors 
                 Sulfamethoxazole 
                 256 
                 256 
                 
                   &gt;1024 
                 
                 
                   &gt;1024 
                 
                 
                   &gt;1024 
                 
                 
                   &gt;1024 
                 
                 
                   &gt;1024 
                 
                 
                   &gt;1024 
                 
               
               
                   
                 SXT 
                 0.25 
                 0.25 
                 0.5 
                 0.5 
                 0.5 
                 0.5 
                 
                   1 
                 
                 
                   8 
                 
               
               
                 Phenicols 
                 Chloramphenicol 
                 4 
                 8 
                 4 
                 8 
                 
                   64 
                 
                 
                   64 
                 
                 
                   128 
                 
                 
                   128 
                 
               
               
                 Fosomycins 
                 Fosfomycin 
                 128 
                 128 
                 256 
                 
                   512 
                 
                 64 
                 128 
                 
                   512 
                 
                 
                   &gt;1024 
                 
               
               
                 Nitrofurans 
                 Nitrofurantoin 
                 64 
                 64 
                 
                   &gt;512 
                 
                 
                   &gt;512 
                 
                 
                   256 
                 
                 
                   256 
                 
                 
                   &gt;512 
                 
                 
                   &gt;512 
                 
               
               
                   
               
               
                   a TZB, tazobactam with a fixed concentration of 4 mg/L; CLA, clavulanic acid with a fixed concentration of 2 mg/L; SXT, trimethoprim/sulfamethoxazole (only the trimethoprim portion of the 1/19 drug ratio is displayed). 
               
               
                   b Boldface numbers indicate a significant (≥4-fold) difference in the MICs of the NVT1001 strain and its derived strains. 
               
               
                   c ΔramRΔ35/36::ramR::35/36, ΔramRΔompK35/36::ramR::ompK35/36 revertant; ΔramRΔ35/36::ramR::36, ΔramRΔompK35/36::ramR::ompK36 revertant; ΔramRΔ35/36::ramR::35, ΔramRΔompK35/36::ramR::ompK35 revertant; ΔramRΔ35/36::ramR, ΔramRΔompK35/36::ramR revertant; ΔramRΔ35/36::35/36, ΔramRΔompK35/36::ompK35/36 revertant; ΔramRΔ35/36::36, ΔramRΔompK35/36::ompK36 revertant; ΔramRΔ35/36::35, ΔramRΔompK35/36::ompK35 revertant; ΔramR435/36, ΔramRΔompK35/36 mutant. 
               
            
           
         
       
     
     2.1.2 β-Lactams 
     The MICs of β-lactams against associated resistance mechanisms are shown in Tables 8 and 9. In particular, the MICs for extended-spectrum β-lactamases (ESBLs) and AmpC β-lactamases are shown in Table 8, while those for carbapenemases are shown in Table 9. The production of ESBLs when tested alone showed no significant (≥4-fold) effects on the MIC of piperacillin/tazobactam, cefoxitin, imipenem or doripenem, and the production of AmpC β-lactamases when tested alone showed no significant (≥4-fold) effects on the MIC of imipenem (Table 8). With or without ESBLs or AmpC β-lactamases, the strains with AcrAB-TolC overexpression (ΔramR) all showed no significant (≥4-fold) effects on the MIC of imipenem or doripenem (Table 8). The production of KPC-2 or KPC-3 alone could confer intermediate resistance to cefoxitin as well as resistance to the other tested β-lactams (Table 9). In addition, with or without OmpK35/36 loss and/or AcrAB-TolC overexpression (ΔramR), the IMP-8, NDM-1, VIM-1 and OXA-48 strains were susceptible to aztreonam (Table 9). 
     
       
         
           
               
             
               
                 TABLE 8 
               
             
            
               
                   
               
               
                 MICs of β-lactams against the chromosome-mediated resistance mechanisms, OmpK35/36 loss and AcrAB-TolC 
               
               
                 overexpression (ΔramR), and/or the plasmid-mediated resistance mechanisms, extended-spectrum β-lactamase  
               
               
                 and AmpC β-lactamase, in  K. pneumoniae  NVT1001 
               
            
           
           
               
               
            
               
                 Supplemental 
                 MIC (mg/L) b   
               
            
           
           
               
               
               
               
               
               
               
               
               
            
               
                 plasmid 
                   
                   
                   
                   
                   
                 ΔramR 
                 ΔramR 
                 ΔramR 
               
               
                 and antibiotic a   
                 WT c   
                 Δ35 
                 Δ36 
                 Δ35/36 
                 ΔramR 
                 Δ35 
                 Δ36 
                 Δ35/36 
               
               
                   
               
            
           
           
               
               
               
               
               
               
               
               
               
            
               
                 No 
                   
                   
                   
                   
                   
                   
                   
                   
               
               
                 Aztreonam 
                 0.03 
                 
                   0.25 
                 
                 0.06 
                 
                   0.5 
                 
                 
                   0.5 
                 
                 
                   0.5 
                 
                 
                   1 
                 
                 
                   1 
                 
               
               
                 Piperacillin/TZB 
                 4 
                 4 
                 4 
                 8 
                 
                   32 
                 
                 
                   32 
                 
                 
                   32 
                 
                 
                   32 
                 
               
               
                 Ticarcillin/CLA 
                 4 
                 4 
                 4 
                 
                   16 
                 
                 8 
                 
                   16 
                 
                 
                   16 
                 
                 8 
               
               
                 Cephalothin 
                 8 
                 8 
                 16 
                 
                   256 
                 
                 
                   64 
                 
                 
                   64 
                 
                 
                   &gt;256 
                 
                 
                   &gt;256 
                 
               
               
                 Cefuroxime 
                 4 
                 8 
                 
                   16 
                 
                 
                   32 
                 
                 
                   32 
                 
                 
                   64 
                 
                 
                   &gt;256 
                 
                 
                   128 
                 
               
               
                 Cefoxitin 
                 4 
                 4 
                 
                   16 
                 
                 
                   128 
                 
                 
                   64 
                 
                 
                   64 
                 
                 
                   &gt;256 
                 
                 
                   &gt;256 
                 
               
               
                 Ceftazidime 
                 0.25 
                 0.5 
                 0.5 
                 
                   1 
                 
                 
                   2 
                 
                 
                   2 
                 
                 
                   2 
                 
                 
                   2 
                 
               
               
                 Cefotaxime 
                 0.06 
                 0.125 
                 
                   0.25 
                 
                 
                   2 
                 
                 
                   1 
                 
                 
                   1 
                 
                 
                   4 
                 
                 
                   4 
                 
               
               
                 Cefepime 
                 0.06 
                 0.125 
                 0.125 
                 
                   1 
                 
                 
                   0.5 
                 
                 
                   0.5 
                 
                 
                   4 
                 
                 
                   2 
                 
               
               
                 Ceftaroline 
                 0.25 
                 0.25 
                 0.5 
                 
                   1 
                 
                 
                   1 
                 
                 
                   1 
                 
                 
                   4 
                 
                 
                   4 
                 
               
               
                 Ertapenem 
                 0.015 
                 0.03 
                 0.03 
                 
                   2 
                 
                 
                   0.125 
                 
                 
                   0.125 
                 
                 
                   4 
                 
                 
                   4 
                 
               
               
                 Imipenem 
                 0.5 
                 0.5 
                 0.5 
                 0.5 
                 0.25 
                 0.25 
                 0.5 
                 0.5 
               
               
                 Meropenem 
                 0.06 
                 0.06 
                 0.06 
                 
                   0.5 
                 
                 0.06 
                 0.06 
                 
                   2 
                 
                 
                   2 
                 
               
               
                 Doripenem 
                 0.06 
                 0.06 
                 0.06 
                 0.125 
                 0.03 
                 0.03 
                 
                   0.25 
                 
                 
                   0.5 
                 
               
               
                 CTX-M-14 
                   
                   
                   
                   
                   
                   
                   
                   
               
               
                 Aztreonam 
                 
                   2 
                 
                 
                   16 
                 
                 
                   2 
                 
                 
                   32 
                 
                 
                   16 
                 
                 
                   16 
                 
                 
                   16 
                 
                 
                   32 
                 
               
               
                 Piperacillin/TZB 
                 4 
                 8 
                 8 
                 
                   16 
                 
                 
                   32 
                 
                 
                   32 
                 
                 
                   32 
                 
                 
                   64 
                 
               
               
                 Ticarcillin/CLA 
                 
                   32 
                 
                 
                   64 
                 
                 
                   64 
                 
                 
                   &gt;256 
                 
                 
                   64 
                 
                 
                   64 
                 
                 
                   &gt;256 
                 
                 
                   &gt;256 
                 
               
               
                 Cephalothin 
                 
                   &gt;256 
                 
                 
                   &gt;256 
                 
                 
                   &gt;256 
                 
                 
                   &gt;256 
                 
                 
                   &gt;256 
                 
                 
                   &gt;256 
                 
                 
                   &gt;256 
                 
                 
                   &gt;256 
                 
               
               
                 Cefuroxime 
                 
                   &gt;256 
                 
                 
                   &gt;256 
                 
                 
                   &gt;256 
                 
                 
                   &gt;256 
                 
                 
                   &gt;256 
                 
                 
                   &gt;256 
                 
                 
                   &gt;256 
                 
                 
                   &gt;256 
                 
               
               
                 Cefoxitin 
                 4 
                 8 
                 
                   16 
                 
                 
                   64 
                 
                 
                   64 
                 
                 
                   64 
                 
                 
                   &gt;256 
                 
                 
                   &gt;256 
                 
               
               
                 Ceftazidime 
                 
                   1 
                 
                 
                   4 
                 
                 
                   2 
                 
                 
                   8 
                 
                 
                   8 
                 
                 
                   8 
                 
                 
                   8 
                 
                 
                   16 
                 
               
               
                 Cefotaxime 
                 
                   64 
                 
                 
                   128 
                 
                 
                   &gt;256 
                 
                 
                   &gt;256 
                 
                 
                   128 
                 
                 
                   128 
                 
                 
                   &gt;256 
                 
                 
                   &gt;256 
                 
               
               
                 Cefepime 
                 
                   8 
                 
                 
                   16 
                 
                 
                   16 
                 
                 
                   128 
                 
                 
                   16 
                 
                 
                   16 
                 
                 
                   256 
                 
                 
                   256 
                 
               
               
                 Ceftaroline 
                 
                   128 
                 
                 
                   &gt;256 
                 
                 
                   &gt;256 
                 
                 
                   &gt;256 
                 
                 
                   &gt;256 
                 
                 
                   &gt;256 
                 
                 
                   &gt;256 
                 
                 
                   &gt;256 
                 
               
               
                 Ertapenem 
                 
                   0.125 
                 
                 
                   0.25 
                 
                 
                   0.25 
                 
                 
                   8 
                 
                 
                   0.25 
                 
                 
                   0.25 
                 
                 
                   16 
                 
                 
                   16 
                 
               
               
                 Imipenem 
                 0.5 
                 0.5 
                 0.5 
                 1 
                 0.25 
                 0.25 
                 0.5 
                 0.5 
               
               
                 Meropenem 
                 0.06 
                 0.125 
                 0.125 
                 
                   2 
                 
                 0.125 
                 0.125 
                 
                   2 
                 
                 
                   2 
                 
               
               
                 Doripenem 
                 0.125 
                 0.125 
                 0.125 
                 
                   1 
                 
                 0.06 
                 0.06 
                 
                   0.5 
                 
                 
                   0.5 
                 
               
               
                 CTX-M-15 
                   
                   
                   
                   
                   
                   
                   
                   
               
               
                 Aztreonam 
                 
                   256 
                 
                 
                   &gt;256 
                 
                 
                   &gt;256 
                 
                 
                   &gt;256 
                 
                 
                   &gt;256 
                 
                 
                   &gt;256 
                 
                 
                   &gt;256 
                 
                 
                   &gt;256 
                 
               
               
                 Piperacillin/TZB 
                 4 
                 8 
                 8 
                 
                   &gt;256 
                 
                 
                   64 
                 
                 
                   64 
                 
                 
                   &gt;256 
                 
                 
                   &gt;256 
                 
               
               
                 Ticarcillin/CLA 
                 
                   32 
                 
                 
                   128 
                 
                 
                   64 
                 
                 
                   &gt;256 
                 
                 
                   128 
                 
                 
                   &gt;256 
                 
                 
                   &gt;256 
                 
                 
                   &gt;256 
                 
               
               
                 Cephalothin 
                 
                   &gt;256 
                 
                 
                   &gt;256 
                 
                 
                   &gt;256 
                 
                 
                   &gt;256 
                 
                 
                   &gt;256 
                 
                 
                   &gt;256 
                 
                 
                   &gt;256 
                 
                 
                   &gt;256 
                 
               
               
                 Cefuroxime 
                 
                   &gt;256 
                 
                 
                   &gt;256 
                 
                 
                   &gt;256 
                 
                 
                   &gt;256 
                 
                 
                   &gt;256 
                 
                 
                   &gt;256 
                 
                 
                   &gt;256 
                 
                 
                   &gt;256 
                 
               
               
                 Cefoxitin 
                 4 
                 8 
                 
                   16 
                 
                 
                   64 
                 
                 
                   64 
                 
                 
                   64 
                 
                 
                   &gt;256 
                 
                 
                   &gt;256 
                 
               
               
                 Ceftazidime 
                 
                   64 
                 
                 
                   &gt;256 
                 
                 
                   64 
                 
                 
                   &gt;256 
                 
                 
                   256 
                 
                 
                   &gt;256 
                 
                 
                   &gt;256 
                 
                 
                   &gt;256 
                 
               
               
                 Cefotaxime 
                 
                   &gt;256 
                 
                 
                   &gt;256 
                 
                 
                   &gt;256 
                 
                 
                   &gt;256 
                 
                 
                   &gt;256 
                 
                 
                   &gt;256 
                 
                 
                   &gt;256 
                 
                 
                   &gt;256 
                 
               
               
                 Cefepime 
                 
                   64 
                 
                 
                   &gt;256 
                 
                 
                   &gt;256 
                 
                 
                   &gt;256 
                 
                 
                   256 
                 
                 
                   &gt;256 
                 
                 
                   &gt;256 
                 
                 
                   &gt;256 
                 
               
               
                 Ceftaroline 
                 
                   &gt;256 
                 
                 
                   &gt;256 
                 
                 
                   &gt;256 
                 
                 
                   &gt;256 
                 
                 
                   &gt;256 
                 
                 
                   &gt;256 
                 
                 
                   &gt;256 
                 
                 
                   &gt;256 
                 
               
               
                 Ertapenem 
                 
                   0.5 
                 
                 
                   0.5 
                 
                 
                   0.5 
                 
                 
                   &gt;32 
                 
                 
                   0.5 
                 
                 
                   0.5 
                 
                 
                   &gt;32 
                 
                 
                   &gt;32 
                 
               
               
                 Imipenem 
                 0.5 
                 0.5 
                 0.5 
                 
                   4 
                 
                 0.5 
                 0.25 
                 
                   2 
                 
                 
                   2 
                 
               
               
                 Meropenem 
                 
                   0.25 
                 
                 
                   0.25 
                 
                 
                   0.25 
                 
                 
                   8 
                 
                 
                   0.25 
                 
                 
                   0.25 
                 
                 
                   4 
                 
                 
                   4 
                 
               
               
                 Doripenem 
                 0.125 
                 0.125 
                 
                   0.25 
                 
                 
                   4 
                 
                 0.125 
                 0.125 
                 
                   2 
                 
                 
                   2 
                 
               
               
                 SHV-12 
                   
                   
                   
                   
                   
                   
                   
                   
               
               
                 Aztreonam 
                 
                   128 
                 
                 
                   &gt;256 
                 
                 
                   128 
                 
                 
                   &gt;256 
                 
                 
                   &gt;256 
                 
                 
                   &gt;256 
                 
                 
                   &gt;256 
                 
                 
                   &gt;256 
                 
               
               
                 Piperacillin/TZB 
                 4 
                 4 
                 4 
                 
                   &gt;256 
                 
                 
                   64 
                 
                 
                   32 
                 
                 
                   &gt;256 
                 
                 
                   &gt;256 
                 
               
               
                 Ticarcillin/CLA 
                 
                   32 
                 
                 
                   128 
                 
                 
                   64 
                 
                 
                   &gt;256 
                 
                 
                   256 
                 
                 
                   256 
                 
                 
                   &gt;256 
                 
                 
                   &gt;256 
                 
               
               
                 Cephalothin 
                 
                   &gt;256 
                 
                 
                   &gt;256 
                 
                 
                   &gt;256 
                 
                 
                   &gt;256 
                 
                 
                   &gt;256 
                 
                 
                   &gt;256 
                 
                 
                   &gt;256 
                 
                 
                   &gt;256 
                 
               
               
                 Cefuroxime 
                 
                   64 
                 
                 
                   64 
                 
                 
                   128 
                 
                 
                   &gt;256 
                 
                 
                   256 
                 
                 
                   128 
                 
                 
                   &gt;256 
                 
                 
                   &gt;256 
                 
               
               
                 Cefoxitin 
                 4 
                 8 
                 
                   16 
                 
                 
                   128 
                 
                 
                   64 
                 
                 
                   64 
                 
                 
                   &gt;256 
                 
                 
                   &gt;256 
                 
               
               
                 Ceftazidime 
                 
                   64 
                 
                 
                   &gt;256 
                 
                 
                   128 
                 
                 
                   &gt;256 
                 
                 
                   &gt;256 
                 
                 
                   &gt;256 
                 
                 
                   &gt;256 
                 
                 
                   &gt;256 
                 
               
               
                 Cefotaxime 
                 
                   32 
                 
                 
                   32 
                 
                 
                   32 
                 
                 
                   &gt;256 
                 
                 
                   64 
                 
                 
                   64 
                 
                 
                   256 
                 
                 
                   256 
                 
               
               
                 Cefepime 
                 
                   4 
                 
                 
                   4 
                 
                 
                   4 
                 
                 
                   128 
                 
                 
                   8 
                 
                 
                   8 
                 
                 
                   64 
                 
                 
                   128 
                 
               
               
                 Ceftaroline 
                 
                   16 
                 
                 
                   16 
                 
                 
                   16 
                 
                 
                   64 
                 
                 
                   16 
                 
                 
                   16 
                 
                 
                   32 
                 
                 
                   32 
                 
               
               
                 Ertapenem 
                 
                   0.06 
                 
                 
                   0.06 
                 
                 
                   0.06 
                 
                 
                   4 
                 
                 
                   0.125 
                 
                 
                   0.125 
                 
                 
                   4 
                 
                 
                   4 
                 
               
               
                 Imipenem 
                 0.5 
                 0.5 
                 0.5 
                 1 
                 0.25 
                 0.25 
                 0.5 
                 1 
               
               
                 Meropenem 
                 0.125 
                 0.125 
                 0.125 
                 
                   1 
                 
                 0.06 
                 0.06 
                 
                   1 
                 
                 
                   2 
                 
               
               
                 Doripenem 
                 0.125 
                 0.125 
                 0.125 
                 
                   1 
                 
                 0.06 
                 0.06 
                 
                   0.5 
                 
                 
                   0.5 
                 
               
               
                 CMY-2 
                   
                   
                   
                   
                   
                   
                   
                   
               
               
                 Aztreonam 
                 
                   64 
                 
                 
                   128 
                 
                 
                   64 
                 
                 
                   256 
                 
                 
                   64 
                 
                 
                   64 
                 
                 
                   64 
                 
                 
                   64 
                 
               
               
                 Piperacillin/TZB 
                 
                   &gt;256 
                 
                 
                   &gt;256 
                 
                 
                   &gt;256 
                 
                 
                   &gt;256 
                 
                 
                   256 
                 
                 
                   256 
                 
                 
                   &gt;256 
                 
                 
                   &gt;256 
                 
               
               
                 Ticarcillin/CLA 
                 
                   &gt;256 
                 
                 
                   &gt;256 
                 
                 
                   &gt;256 
                 
                 
                   &gt;256 
                 
                 
                   &gt;256 
                 
                 
                   &gt;256 
                 
                 
                   &gt;256 
                 
                 
                   &gt;256 
                 
               
               
                 Cephalothin 
                 
                   &gt;256 
                 
                 
                   &gt;256 
                 
                 
                   &gt;256 
                 
                 
                   &gt;256 
                 
                 
                   &gt;256 
                 
                 
                   &gt;256 
                 
                 
                   &gt;256 
                 
                 
                   &gt;256 
                 
               
               
                 Cefuroxime 
                 
                   256 
                 
                 
                   256 
                 
                 
                   &gt;256 
                 
                 
                   &gt;256 
                 
                 
                   &gt;256 
                 
                 
                   &gt;256 
                 
                 
                   &gt;256 
                 
                 
                   &gt;256 
                 
               
               
                 Cefoxitin 
                 
                   &gt;256 
                 
                 
                   &gt;256 
                 
                 
                   &gt;256 
                 
                 
                   &gt;256 
                 
                 
                   &gt;256 
                 
                 
                   &gt;256 
                 
                 
                   &gt;256 
                 
                 
                   &gt;256 
                 
               
               
                 Ceftazidime 
                 
                   &gt;256 
                 
                 
                   &gt;256 
                 
                 
                   &gt;256 
                 
                 
                   &gt;256 
                 
                 
                   &gt;256 
                 
                 
                   &gt;256 
                 
                 
                   &gt;256 
                 
                 
                   &gt;256 
                 
               
               
                 Cefotaxime 
                 
                   64 
                 
                 
                   128 
                 
                 
                   &gt;256 
                 
                 
                   &gt;256 
                 
                 
                   128 
                 
                 
                   128 
                 
                 
                   &gt;256 
                 
                 
                   &gt;256 
                 
               
               
                 Cefepime 
                 
                   2 
                 
                 
                   2 
                 
                 
                   16 
                 
                 
                   32 
                 
                 
                   4 
                 
                 
                   4 
                 
                 
                   16 
                 
                 
                   16 
                 
               
               
                 Ceftaroline 
                 
                   64 
                 
                 
                   64 
                 
                 
                   256 
                 
                 
                   &gt;256 
                 
                 
                   64 
                 
                 
                   64 
                 
                 
                   256 
                 
                 
                   128 
                 
               
               
                 Ertapenem 
                 
                   1 
                 
                 
                   1 
                 
                 
                   4 
                 
                 
                   &gt;32 
                 
                 
                   1 
                 
                 
                   1 
                 
                 
                   &gt;32 
                 
                 
                   &gt;32 
                 
               
               
                 Imipenem 
                 1 
                 1 
                 
                   4 
                 
                 
                   &gt;32 
                 
                 1 
                 1 
                 
                   32 
                 
                 
                   32 
                 
               
               
                 Meropenem 
                 
                   0.25 
                 
                 
                   0.25 
                 
                 
                   2 
                 
                 
                   16 
                 
                 
                   0.25 
                 
                 
                   0.25 
                 
                 
                   8 
                 
                 
                   8 
                 
               
               
                 Doripenem 
                 
                   0.25 
                 
                 
                   0.25 
                 
                 
                   1 
                 
                 
                   8 
                 
                 0.125 
                 0.125 
                 
                   4 
                 
                 
                   4 
                 
               
               
                 DHA-1-AmpR 
                   
                   
                   
                   
                   
                   
                   
                   
               
               
                 Aztreonam 
                 
                   1 
                 
                 
                   8 
                 
                 
                   1 
                 
                 
                   16 
                 
                 
                   4 
                 
                 
                   4 
                 
                 
                   8 
                 
                 
                   8 
                 
               
               
                 Piperacillin/TZB 
                 4 
                 8 
                 8 
                 
                   &gt;256 
                 
                 
                   32 
                 
                 
                   32 
                 
                 
                   64 
                 
                 
                   64 
                 
               
               
                 Ticarcillin/CLA 
                 
                   256 
                 
                 
                   &gt;256 
                 
                 
                   &gt;256 
                 
                 
                   &gt;256 
                 
                 
                   &gt;256 
                 
                 
                   &gt;256 
                 
                 
                   &gt;256 
                 
                 
                   &gt;256 
                 
               
               
                 Cephalothin 
                 
                   &gt;256 
                 
                 
                   &gt;256 
                 
                 
                   &gt;256 
                 
                 
                   &gt;256 
                 
                 
                   &gt;256 
                 
                 
                   &gt;256 
                 
                 
                   &gt;256 
                 
                 
                   &gt;256 
                 
               
               
                 Cefuroxime 
                 
                   &gt;256 
                 
                 
                   &gt;256 
                 
                 
                   &gt;256 
                 
                 
                   &gt;256 
                 
                 
                   &gt;256 
                 
                 
                   &gt;256 
                 
                 
                   &gt;256 
                 
                 
                   &gt;256 
                 
               
               
                 Cefoxitin 
                 
                   &gt;256 
                 
                 
                   &gt;256 
                 
                 
                   &gt;256 
                 
                 
                   &gt;256 
                 
                 
                   &gt;256 
                 
                 
                   &gt;256 
                 
                 
                   &gt;256 
                 
                 
                   &gt;256 
                 
               
               
                 Ceftazidime 
                 
                   16 
                 
                 
                   128 
                 
                 
                   32 
                 
                 
                   &gt;256 
                 
                 
                   64 
                 
                 
                   64 
                 
                 
                   64 
                 
                 
                   128 
                 
               
               
                 Cefotaxime 
                 
                   8 
                 
                 
                   16 
                 
                 
                   16 
                 
                 
                   64 
                 
                 
                   16 
                 
                 
                   16 
                 
                 
                   64 
                 
                 
                   64 
                 
               
               
                 Cefepime 
                 0.125 
                 
                   0.25 
                 
                 
                   0.25 
                 
                 
                   1 
                 
                 
                   0.5 
                 
                 
                   0.5 
                 
                 
                   4 
                 
                 
                   4 
                 
               
               
                 Ceftaroline 
                 
                   8 
                 
                 
                   8 
                 
                 
                   8 
                 
                 
                   16 
                 
                 
                   8 
                 
                 
                   8 
                 
                 
                   16 
                 
                 
                   16 
                 
               
               
                 Ertapenem 
                 
                   1 
                 
                 
                   1 
                 
                 
                   1 
                 
                 
                   &gt;32 
                 
                 
                   1 
                 
                 
                   1 
                 
                 
                   &gt;32 
                 
                 
                   &gt;32 
                 
               
               
                 Imipenem 
                 0.5 
                 0.5 
                 0.5 
                 
                   32 
                 
                 0.25 
                 0.25 
                 
                   16 
                 
                 
                   16 
                 
               
               
                 Meropenem 
                 
                   0.25 
                 
                 
                   0.25 
                 
                 
                   0.25 
                 
                 
                   8 
                 
                 0.125 
                 
                   0.25 
                 
                 
                   8 
                 
                 
                   4 
                 
               
               
                 Doripenem 
                 0.125 
                 0.125 
                 
                   0.25 
                 
                 
                   8 
                 
                 0.125 
                 0.125 
                 
                   4 
                 
                 
                   4 
                 
               
               
                   
               
               
                   a No, no supplemental plasmid. The β-lactamase on the low-copy-number plasmid pACYC177 is shown, and the plasmid was transferred into  K. pneumoniae  NVT1001 and its mutants. The DHA-1 was cloned with its regulator AmpR. TZB, tazobactam with a fixed concentration of 4 mg/L; CLA, clavulanic acid with a fixed concentration of 2 mg/L. 
               
               
                   b Boldface numbers indicate a significant (≥4-fold) difference in the MICs of the NVT1001 strain and its derived strains, while no significant (≥4-fold) differences in the MICs of the NVT1001 with or without plasmid pACYC177 alone (data not shown). 
               
               
                   C WT, NVT1001; Δ35, ΔompK35 mutant; Δ36, ΔompK36 mutant; Δ35/36, ΔompK35/36 mutant; ΔramR, ΔramR mutant; ΔramRΔ35, ΔramRΔompK35 mutant; ΔramRΔ36, ΔramRΔompK36 mutant; ΔramRΔ35/36, ΔramRΔompK35/36 mutant. 
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 9 
               
             
            
               
                   
               
               
                 MICs of β-lactams against the chromosome-mediated resistance mechanisms, OmpK35/36 loss and AcrAB- 
               
               
                 TolC overexpression (ΔramR), and/or the plasmid-mediated resistance mechanism, carbapenemase, in  K. pneumoniae   
               
               
                 NVT1001 
               
            
           
           
               
               
            
               
                 Supplemental 
                 MIC (mg/L) b   
               
            
           
           
               
               
               
               
               
               
               
               
               
            
               
                 plasmid and 
                   
                   
                   
                   
                   
                 ΔramR 
                 ΔramR 
                 ΔramR 
               
               
                 antibiotic a   
                 WT c   
                 Δ35 
                 Δ36 
                 Δ35/36 
                 ΔramR 
                 Δ35 
                 Δ36 
                 Δ35/36 
               
               
                   
               
            
           
           
               
               
               
               
               
               
               
               
               
            
               
                 No 
                   
                   
                   
                   
                   
                   
                   
                   
               
               
                 Aztreonam 
                 0.03 
                 
                   0.25 
                 
                 0.06 
                 
                   0.5 
                 
                 
                   0.5 
                 
                 
                   0.5 
                 
                 
                   1 
                 
                 
                   1 
                 
               
               
                 Piperacillin/TZB  
                 4 
                 4 
                 4 
                 8 
                 
                   32 
                 
                 
                   32 
                 
                 
                   32 
                 
                 
                   32 
                 
               
               
                 Ticarcillin/CLA 
                 4 
                 4 
                 4 
                 
                   16 
                 
                 8 
                 
                   16 
                 
                 
                   16 
                 
                 8 
               
               
                 Cephalothin 
                 8 
                 8 
                 16 
                 
                   256 
                 
                 
                   64 
                 
                 
                   64 
                 
                 
                   &gt;256 
                 
                 
                   &gt;256 
                 
               
               
                 Cefuroxime 
                 4 
                 8 
                 
                   16 
                 
                 
                   32 
                 
                 
                   32 
                 
                 
                   64 
                 
                 
                   &gt;256 
                 
                 
                   128 
                 
               
               
                 Cefoxitin 
                 4 
                 4 
                 
                   16 
                 
                 
                   128 
                 
                 
                   64 
                 
                 
                   64 
                 
                 
                   &gt;256 
                 
                 
                   &gt;256 
                 
               
               
                 Ceftazidime 
                 0.25 
                 0.5 
                 0.5 
                 
                   1 
                 
                 
                   2 
                 
                 
                   2 
                 
                 
                   2 
                 
                 
                   2 
                 
               
               
                 Cefotaxime 
                 0.06 
                 0.125 
                 
                   0.25 
                 
                 
                   2 
                 
                 
                   1 
                 
                 
                   1 
                 
                 
                   4 
                 
                 
                   4 
                 
               
               
                 Cefepime 
                 0.06 
                 0.125 
                 0.125 
                 
                   1 
                 
                 
                   0.5 
                 
                 
                   0.5 
                 
                 
                   4 
                 
                 
                   2 
                 
               
               
                 Ceftaroline 
                 0.25 
                 0.25 
                 0.5 
                 
                   1 
                 
                 
                   1 
                 
                 
                   1 
                 
                 
                   4 
                 
                 
                   4 
                 
               
               
                 Ertapenem 
                 0.015 
                 0.03 
                 0.03 
                 
                   2 
                 
                 
                   0.125 
                 
                 
                   0.125 
                 
                 
                   4 
                 
                 
                   4 
                 
               
               
                 Imipenem 
                 0.5 
                 0.5 
                 0.5 
                 0.5 
                 0.25 
                 0.25 
                 0.5 
                 0.5 
               
               
                 Meropenem 
                 0.06 
                 0.06 
                 0.06 
                 
                   0.5 
                 
                 0.06 
                 0.06 
                 
                   2 
                 
                 
                   2 
                 
               
               
                 Doripenem 
                 0.06 
                 0.06 
                 0.06 
                 0.125 
                 0.03 
                 0.03 
                 
                   0.25 
                 
                 
                   0.5 
                 
               
               
                 KPC-2 
                   
                   
                   
                   
                   
                   
                   
                   
               
               
                 Aztreonam 
                 
                   &gt;256 
                 
                 
                   &gt;256 
                 
                 
                   256 
                 
                 
                   &gt;256 
                 
                 
                   &gt;256 
                 
                 
                   &gt;256 
                 
                 
                   &gt;256 
                 
                 
                   &gt;256 
                 
               
               
                 Piperacillin/TZB &gt;256 
                 
                   &gt;256 
                 
                 
                   &gt;256 
                 
                 
                   &gt;256 
                 
                 
                   &gt;256 
                 
                 
                   &gt;256 
                 
                 
                   &gt;256 
                 
                 
                   &gt;256 
                 
                   
               
               
                 Ticarcillin/CLA 
                 
                   &gt;256 
                 
                 
                   &gt;256 
                 
                 
                   &gt;256 
                 
                 
                   &gt;256 
                 
                 
                   &gt;256 
                 
                 
                   &gt;256 
                 
                 
                   &gt;256 
                 
                 
                   &gt;256 
                 
               
               
                 Cephalothin 
                 
                   &gt;256 
                 
                 
                   &gt;256 
                 
                 
                   &gt;256 
                 
                 
                   &gt;256 
                 
                 
                   &gt;256 
                 
                 
                   &gt;256 
                 
                 
                   &gt;256 
                 
                 
                   &gt;256 
                 
               
               
                 Cefuroxime 
                 
                   &gt;256 
                 
                 
                   &gt;256 
                 
                 
                   &gt;256 
                 
                 
                   &gt;256 
                 
                 
                   &gt;256 
                 
                 
                   &gt;256 
                 
                 
                   &gt;256 
                 
                 
                   &gt;256 
                 
               
               
                 Cefoxitin 
                 
                   16 
                 
                 
                   32 
                 
                 
                   64 
                 
                 
                   &gt;256 
                 
                 
                   128 
                 
                 
                   128 
                 
                 
                   &gt;256 
                 
                 
                   &gt;256 
                 
               
               
                 Ceftazidime 
                 
                   16 
                 
                 
                   64 
                 
                 
                   32 
                 
                 
                   256 
                 
                 
                   128 
                 
                 
                   128 
                 
                 
                   128 
                 
                 
                   128 
                 
               
               
                 Cefotaxime 
                 
                   128 
                 
                 
                   256 
                 
                 
                   256 
                 
                 
                   &gt;256 
                 
                 
                   256 
                 
                 
                   256 
                 
                 
                   &gt;256 
                 
                 
                   &gt;256 
                 
               
               
                 Cefepime 
                 
                   64 
                 
                 
                   64 
                 
                 
                   64 
                 
                 
                   &gt;256 
                 
                 
                   64 
                 
                 
                   64 
                 
                 
                   &gt;256 
                 
                 
                   &gt;256 
                 
               
               
                 Ceftaroline 
                 
                   256 
                 
                 
                   &gt;256 
                 
                 
                   &gt;256 
                 
                 
                   &gt;256 
                 
                 
                   &gt;256 
                 
                 
                   &gt;256 
                 
                 
                   &gt;256 
                 
                 
                   &gt;256 
                 
               
               
                 Ertapenem 
                 
                   32 
                 
                 
                   32 
                 
                 
                   32 
                 
                 
                   &gt;32 
                 
                 
                   &gt;32 
                 
                 
                   &gt;32 
                 
                 
                   &gt;32 
                 
                 
                   &gt;32 
                 
               
               
                 Imipenem 
                 
                   &gt;32 
                 
                 
                   &gt;32 
                 
                 
                   &gt;32 
                 
                 
                   &gt;32 
                 
                 
                   &gt;32 
                 
                 
                   &gt;32 
                 
                 
                   &gt;32 
                 
                 
                   &gt;32 
                 
               
               
                 Meropenem 
                 
                   32 
                 
                 
                   &gt;32 
                 
                 
                   &gt;32 
                 
                 
                   &gt;32 
                 
                 
                   32 
                 
                 
                   &gt;32 
                 
                 
                   &gt;32 
                 
                 
                   &gt;32 
                 
               
               
                 Doripenem 
                 
                   16 
                 
                 
                   32 
                 
                 
                   &gt;32 
                 
                 
                   &gt;32 
                 
                 
                   16 
                 
                 
                   32 
                 
                 
                   &gt;32 
                 
                 
                   &gt;32 
                 
               
               
                 KPC-3 
                   
                   
                   
                   
                   
                   
                   
                   
               
               
                 Aztreonam 
                 
                   &gt;256 
                 
                 
                   &gt;256 
                 
                 
                   &gt;256 
                 
                 
                   &gt;256 
                 
                 
                   &gt;256 
                 
                 
                   &gt;256 
                 
                 
                   &gt;256 
                 
                 
                   &gt;256 
                 
               
               
                 Piperacillin/TZB  
                 
                   &gt;256 
                 
                 
                   &gt;256 
                 
                 
                   &gt;256 
                 
                 
                   &gt;256 
                 
                 
                   &gt;256 
                 
                 
                   &gt;256 
                 
                 
                   &gt;256 
                 
                 
                   &gt;256 
                 
               
               
                 Ticarcillin/CLA 
                 
                   &gt;256 
                 
                 
                   &gt;256 
                 
                 
                   &gt;256 
                 
                 
                   &gt;256 
                 
                 
                   &gt;256 
                 
                 
                   &gt;256 
                 
                 
                   &gt;256 
                 
                 
                   &gt;256 
                 
               
               
                 Cephalothin 
                 
                   &gt;256 
                 
                 
                   &gt;256 
                 
                 
                   &gt;256 
                 
                 
                   &gt;256 
                 
                 
                   &gt;256 
                 
                 
                   &gt;256 
                 
                 
                   &gt;256 
                 
                 
                   &gt;256 
                 
               
               
                 Cefuroxime 
                 
                   &gt;256 
                 
                 
                   &gt;256 
                 
                 
                   &gt;256 
                 
                 
                   &gt;256 
                 
                 
                   &gt;256 
                 
                 
                   &gt;256 
                 
                 
                   &gt;256 
                 
                 
                   &gt;256 
                 
               
               
                 Cefoxitin 
                 
                   16 
                 
                 
                   32 
                 
                 
                   64 
                 
                 
                   &gt;256 
                 
                 
                   128 
                 
                 
                   128 
                 
                 
                   &gt;256 
                 
                 
                   &gt;256 
                 
               
               
                 Ceftazidime 
                 
                   128 
                 
                 
                   &gt;256 
                 
                 
                   128 
                 
                 
                   &gt;256 
                 
                 
                   &gt;256 
                 
                 
                   &gt;256 
                 
                 
                   &gt;256 
                 
                 
                   &gt;256 
                 
               
               
                 Cefotaxime 
                 
                   128 
                 
                 
                   256 
                 
                 
                   256 
                 
                 
                   &gt;256 
                 
                 
                   256 
                 
                 
                   256 
                 
                 
                   &gt;256 
                 
                 
                   &gt;256 
                 
               
               
                 Cefepime 
                 
                   128 
                 
                 
                   128 
                 
                 
                   256 
                 
                 
                   &gt;256 
                 
                 
                   128 
                 
                 
                   128 
                 
                 
                   &gt;256 
                 
                 
                   &gt;256 
                 
               
               
                 Ceftaroline 
                 
                   256 
                 
                 
                   256 
                 
                 
                   &gt;256 
                 
                 
                   &gt;256 
                 
                 
                   256 
                 
                 
                   256 
                 
                 
                   &gt;256 
                 
                 
                   &gt;256 
                 
               
               
                 Ertapenem 
                 
                   32 
                 
                 
                   32 
                 
                 
                   32 
                 
                 
                   &gt;32 
                 
                 
                   32 
                 
                 
                   32 
                 
                 
                   &gt;32 
                 
                 
                   &gt;32 
                 
               
               
                 Imipenem 
                 
                   32 
                 
                 
                   &gt;32 
                 
                 
                   &gt;32 
                 
                 
                   &gt;32 
                 
                 
                   32 
                 
                 
                   32 
                 
                 
                   &gt;32 
                 
                 
                   &gt;32 
                 
               
               
                 Meropenem 
                 
                   16 
                 
                 
                   32 
                 
                 
                   &gt;32 
                 
                 
                   &gt;32 
                 
                 
                   32 
                 
                 
                   &gt;32 
                 
                 
                   &gt;32 
                 
                 
                   &gt;32 
                 
               
               
                 Doripenem 
                 
                   8 
                 
                 
                   32 
                 
                 
                   &gt;32 
                 
                 
                   &gt;32 
                 
                 
                   16 
                 
                 
                   32 
                 
                 
                   &gt;32 
                 
                 
                   &gt;32 
                 
               
               
                 IMP-8 
                   
                   
                   
                   
                   
                   
                   
                   
               
               
                 Aztreonam 
                 0.06 
                 
                   0.25 
                 
                 0.06 
                 
                   0.5 
                 
                 
                   0.5 
                 
                 
                   0.5 
                 
                 
                   0.5 
                 
                 
                   0.5 
                 
               
               
                 Piperacillin/TZB  
                 
                   16 
                 
                 
                   16 
                 
                 
                   16 
                 
                 
                   64 
                 
                 
                   32 
                 
                 
                   64 
                 
                 
                   64 
                 
                 
                   64 
                 
               
               
                 Ticarcillin/CLA 
                 
                   &gt;256 
                 
                 
                   &gt;256 
                 
                 
                   &gt;256 
                 
                 
                   &gt;256 
                 
                 
                   &gt;256 
                 
                 
                   &gt;256 
                 
                 
                   &gt;256 
                 
                 
                   &gt;256 
                 
               
               
                 Cephalothin 
                 
                   &gt;256 
                 
                 
                   &gt;256 
                 
                 
                   &gt;256 
                 
                 
                   &gt;256 
                 
                 
                   &gt;256 
                 
                 
                   &gt;256 
                 
                 
                   &gt;256 
                 
                 
                   &gt;256 
                 
               
               
                 Cefuroxime 
                 
                   &gt;256 
                 
                 
                   &gt;256 
                 
                 
                   &gt;256 
                 
                 
                   &gt;256 
                 
                 
                   &gt;256 
                 
                 
                   &gt;256 
                 
                 
                   &gt;256 
                 
                 
                   &gt;256 
                 
               
               
                 Cefoxitin 
                 
                   &gt;256 
                 
                 
                   &gt;256 
                 
                 
                   &gt;256 
                 
                 
                   &gt;256 
                 
                 
                   &gt;256 
                 
                 
                   &gt;256 
                 
                 
                   &gt;256 
                 
                 
                   &gt;256 
                 
               
               
                 Ceftazidime 
                 
                   &gt;256 
                 
                 
                   &gt;256 
                 
                 
                   &gt;256 
                 
                 
                   &gt;256 
                 
                 
                   &gt;256 
                 
                 
                   &gt;256 
                 
                 
                   &gt;256 
                 
                 
                   &gt;256 
                 
               
               
                 Cefotaxime 
                 
                   64 
                 
                 
                   128 
                 
                 
                   256 
                 
                 
                   &gt;256 
                 
                 
                   128 
                 
                 
                   128 
                 
                 
                   256 
                 
                 
                   256 
                 
               
               
                 Cefepime 
                 
                   16 
                 
                 
                   32 
                 
                 
                   64 
                 
                 
                   &gt;256 
                 
                 
                   32 
                 
                 
                   64 
                 
                 
                   128 
                 
                 
                   128 
                 
               
               
                 Ceftaroline 
                 
                   64 
                 
                 
                   64 
                 
                 
                   64 
                 
                 
                   &gt;256 
                 
                 
                   32 
                 
                 
                   64 
                 
                 
                   64 
                 
                 
                   128 
                 
               
               
                 Ertapenem 
                 
                   2 
                 
                 
                   4 
                 
                 
                   2 
                 
                 
                   &gt;32 
                 
                 
                   4 
                 
                 
                   4 
                 
                 
                   &gt;32 
                 
                 
                   &gt;32 
                 
               
               
                 Imipenem 
                 
                   4 
                 
                 
                   4 
                 
                 
                   8 
                 
                 
                   &gt;32 
                 
                 
                   4 
                 
                 
                   4 
                 
                 
                   &gt;32 
                 
                 
                   &gt;32 
                 
               
               
                 Meropenem 
                 
                   2 
                 
                 
                   1 
                 
                 
                   2 
                 
                 
                   &gt;32 
                 
                 
                   2 
                 
                 
                   2 
                 
                 
                   &gt;32 
                 
                 
                   &gt;32 
                 
               
               
                 Doripenem 
                 
                   1 
                 
                 
                   2 
                 
                 
                   2 
                 
                 
                   &gt;32 
                 
                 
                   1 
                 
                 
                   2 
                 
                 
                   32 
                 
                 
                   32 
                 
               
               
                 NDM-1 
                   
                   
                   
                   
                   
                   
                   
                   
               
               
                 Aztreonam 
                 0.06 
                 
                   0.25 
                 
                 0.06 
                 
                   0.5 
                 
                 
                   0.5 
                 
                 
                   0.5 
                 
                 
                   0.5 
                 
                 
                   0.5 
                 
               
               
                 Piperacillin/TZB  
                 
                   &gt;256 
                 
                 
                   &gt;256 
                 
                 
                   &gt;256 
                 
                 
                   &gt;256 
                 
                 
                   &gt;256 
                 
                 
                   &gt;256 
                 
                 
                   &gt;256 
                 
                 
                   &gt;256 
                 
               
               
                 Ticarcillin/CLA 
                 
                   &gt;256 
                 
                 
                   &gt;256 
                 
                 
                   &gt;256 
                 
                 
                   &gt;256 
                 
                 
                   &gt;256 
                 
                 
                   &gt;256 
                 
                 
                   &gt;256 
                 
                 
                   &gt;256 
                 
               
               
                 Cephalothin 
                 
                   &gt;256 
                 
                 
                   &gt;256 
                 
                 
                   &gt;256 
                 
                 
                   &gt;256 
                 
                 
                   &gt;256 
                 
                 
                   &gt;256 
                 
                 
                   &gt;256 
                 
                 
                   &gt;256 
                 
               
               
                 Cefuroxime 
                 
                   &gt;256 
                 
                 
                   &gt;256 
                 
                 
                   &gt;256 
                 
                 
                   &gt;256 
                 
                 
                   &gt;256 
                 
                 
                   &gt;256 
                 
                 
                   &gt;256 
                 
                 
                   &gt;256 
                 
               
               
                 Cefoxitin 
                 
                   &gt;256 
                 
                 
                   &gt;256 
                 
                 
                   &gt;256 
                 
                 
                   &gt;256 
                 
                 
                   &gt;256 
                 
                 
                   &gt;256 
                 
                 
                   &gt;256 
                 
                 
                   &gt;256 
                 
               
               
                 Ceftazidime 
                 
                   &gt;256 
                 
                 
                   &gt;256 
                 
                 
                   &gt;256 
                 
                 
                   &gt;256 
                 
                 
                   &gt;256 
                 
                 
                   &gt;256 
                 
                 
                   &gt;256 
                 
                 
                   &gt;256 
                 
               
               
                 Cefotaxime 
                 
                   256 
                 
                 
                   256 
                 
                 
                   &gt;256 
                 
                 
                   &gt;256 
                 
                 
                   &gt;256 
                 
                 
                   &gt;256 
                 
                 
                   &gt;256 
                 
                 
                   &gt;256 
                 
               
               
                 Cefepime 
                 
                   32 
                 
                 
                   128 
                 
                 
                   &gt;256 
                 
                 
                   &gt;256 
                 
                 
                   &gt;256 
                 
                 
                   256 
                 
                 
                   &gt;256 
                 
                 
                   &gt;256 
                 
               
               
                 Ceftaroline 
                 
                   &gt;256 
                 
                 
                   &gt;256 
                 
                 
                   &gt;256 
                 
                 
                   &gt;256 
                 
                 
                   &gt;256 
                 
                 
                   &gt;256 
                 
                 
                   &gt;256 
                 
                 
                   &gt;256 
                 
               
               
                 Ertapenem 
                 
                   32 
                 
                 
                   &gt;32 
                 
                 
                   &gt;32 
                 
                 
                   &gt;32 
                 
                 
                   &gt;32 
                 
                 
                   &gt;32 
                 
                 
                   &gt;32 
                 
                 
                   &gt;32 
                 
               
               
                 Imipenem 
                 
                   &gt;32 
                 
                 
                   &gt;32 
                 
                 
                   &gt;32 
                 
                 
                   &gt;32 
                 
                 
                   &gt;32 
                 
                 
                   &gt;32 
                 
                 
                   &gt;32 
                 
                 
                   &gt;32 
                 
               
               
                 Meropenem 
                 
                   32 
                 
                 
                   32 
                 
                 
                   &gt;32 
                 
                 
                   &gt;32 
                 
                 
                   32 
                 
                 
                   32 
                 
                 
                   &gt;32 
                 
                 
                   &gt;32 
                 
               
               
                 Doripenem 
                 
                   16 
                 
                 
                   32 
                 
                 
                   &gt;32 
                 
                 
                   &gt;32 
                 
                 
                   32 
                 
                 
                   32 
                 
                 
                   &gt;32 
                 
                 
                   &gt;32 
                 
               
               
                 VIM-1 
                   
                   
                   
                   
                   
                   
                   
                   
               
               
                 Aztreonam 
                 0.06 
                 
                   0.25 
                 
                 0.06 
                 
                   0.5 
                 
                 
                   0.5 
                 
                 
                   0.5 
                 
                 
                   1 
                 
                 
                   1 
                 
               
               
                 Piperacillin/TZB  
                 
                   &gt;256 
                 
                 
                   &gt;256 
                 
                 
                   &gt;256 
                 
                 
                   &gt;256 
                 
                 
                   &gt;256 
                 
                 
                   &gt;256 
                 
                 
                   &gt;256 
                 
                 
                   &gt;256 
                 
               
               
                 Ticarcillin/CLA 
                 
                   &gt;256 
                 
                 
                   &gt;256 
                 
                 
                   &gt;256 
                 
                 
                   &gt;256 
                 
                 
                   &gt;256 
                 
                 
                   &gt;256 
                 
                 
                   &gt;256 
                 
                 
                   &gt;256 
                 
               
               
                 Cephalothin 
                 
                   &gt;256 
                 
                 
                   &gt;256 
                 
                 
                   &gt;256 
                 
                 
                   &gt;256 
                 
                 
                   &gt;256 
                 
                 
                   &gt;256 
                 
                 
                   &gt;256 
                 
                 
                   &gt;256 
                 
               
               
                 Cefuroxime 
                 
                   &gt;256 
                 
                 
                   &gt;256 
                 
                 
                   &gt;256 
                 
                 
                   &gt;256 
                 
                 
                   &gt;256 
                 
                 
                   &gt;256 
                 
                 
                   &gt;256 
                 
                 
                   &gt;256 
                 
               
               
                 Cefoxitin 
                 
                   &gt;256 
                 
                 
                   &gt;256 
                 
                 
                   &gt;256 
                 
                 
                   &gt;256 
                 
                 
                   &gt;256 
                 
                 
                   &gt;256 
                 
                 
                   &gt;256 
                 
                 
                   &gt;256 
                 
               
               
                 Ceftazidime 
                 
                   &gt;256 
                 
                 
                   &gt;256 
                 
                 
                   &gt;256 
                 
                 
                   &gt;256 
                 
                 
                   &gt;256 
                 
                 
                   &gt;256 
                 
                 
                   &gt;256 
                 
                 
                   &gt;256 
                 
               
               
                 Cefotaxime 
                 
                   &gt;256 
                 
                 
                   &gt;256 
                 
                 
                   &gt;256 
                 
                 
                   &gt;256 
                 
                 
                   &gt;256 
                 
                 
                   &gt;256 
                 
                 
                   &gt;256 
                 
                 
                   &gt;256 
                 
               
               
                 Cefepime 
                 
                   128 
                 
                 
                   256 
                 
                 
                   &gt;256 
                 
                 
                   &gt;256 
                 
                 
                   &gt;256 
                 
                 
                   &gt;256 
                 
                 
                   &gt;256 
                 
                 
                   &gt;256 
                 
               
               
                 Ceftaroline 
                 
                   256 
                 
                 
                   &gt;256 
                 
                 
                   &gt;256 
                 
                 
                   &gt;256 
                 
                 
                   &gt;256 
                 
                 
                   &gt;256 
                 
                 
                   &gt;256 
                 
                 
                   &gt;256 
                 
               
               
                 Ertapenem 
                 
                   2 
                 
                 
                   4 
                 
                 
                   2 
                 
                 
                   &gt;32 
                 
                 
                   4 
                 
                 
                   4 
                 
                 
                   &gt;32 
                 
                 
                   &gt;32 
                 
               
               
                 Imipenem 
                 
                   16 
                 
                 
                   32 
                 
                 
                   &gt;32 
                 
                 
                   &gt;32 
                 
                 
                   16 
                 
                 
                   16 
                 
                 
                   &gt;32 
                 
                 
                   &gt;32 
                 
               
               
                 Meropenem 
                 
                   16 
                 
                 
                   &gt;32 
                 
                 
                   &gt;32 
                 
                 
                   &gt;32 
                 
                 
                   &gt;32 
                 
                 
                   32 
                 
                 
                   &gt;32 
                 
                 
                   &gt;32 
                 
               
               
                 Doripenem 
                 
                   16 
                 
                 
                   32 
                 
                 
                   &gt;32 
                 
                 
                   &gt;32 
                 
                 
                   32 
                 
                 
                   32 
                 
                 
                   &gt;32 
                 
                 
                   &gt;32 
                 
               
               
                 OXA-48 
                   
                   
                   
                   
                   
                   
                   
                   
               
               
                 Aztreonam 
                 0.06 
                 
                   0.25 
                 
                 
                   0.125 
                 
                 
                   0.5 
                 
                 
                   0.5 
                 
                 
                   0.5 
                 
                 
                   0.5 
                 
                 
                   0.5 
                 
               
               
                 Piperacillin/TZB  
                 
                   &gt;256 
                 
                 
                   &gt;256 
                 
                 
                   &gt;256 
                 
                 
                   &gt;256 
                 
                 
                   &gt;256 
                 
                 
                   &gt;256 
                 
                 
                   &gt;256 
                 
                 
                   &gt;256 
                 
               
               
                 Ticarcillin/CLA 
                 
                   &gt;256 
                 
                 
                   &gt;256 
                 
                 
                   &gt;256 
                 
                 
                   &gt;256 
                 
                 
                   &gt;256 
                 
                 
                   &gt;256 
                 
                 
                   &gt;256 
                 
                 
                   &gt;256 
                 
               
               
                 Cephalothin 
                 
                   256 
                 
                 
                   256 
                 
                 
                   &gt;256 
                 
                 
                   &gt;256 
                 
                 
                   &gt;256 
                 
                 
                   &gt;256 
                 
                 
                   &gt;256 
                 
                 
                   &gt;256 
                 
               
               
                 Cefuroxime 
                 8 
                 
                   16 
                 
                 
                   32 
                 
                 
                   128 
                 
                 
                   64 
                 
                 
                   64 
                 
                 
                   &gt;256 
                 
                 
                   &gt;256 
                 
               
               
                 Cefoxitin 
                 4 
                 8 
                 
                   16 
                 
                 
                   128 
                 
                 
                   128 
                 
                 
                   64 
                 
                 
                   &gt;256 
                 
                 
                   &gt;256 
                 
               
               
                 Ceftazidime 
                 0.5 
                 
                   1 
                 
                 0.5 
                 
                   2 
                 
                 
                   2 
                 
                 
                   2 
                 
                 
                   2 
                 
                 
                   2 
                 
               
               
                 Cefotaxime 
                 
                   2 
                 
                 
                   4 
                 
                 
                   16 
                 
                 
                   32 
                 
                 
                   4 
                 
                 
                   4 
                 
                 
                   32 
                 
                 
                   32 
                 
               
               
                 Cefepime 
                 
                   0.5 
                 
                 
                   1 
                 
                 
                   1 
                 
                 
                   8 
                 
                 
                   1 
                 
                 
                   1 
                 
                 
                   8 
                 
                 
                   8 
                 
               
               
                 Ceftaroline 
                 
                   8 
                 
                 
                   8 
                 
                 
                   8 
                 
                 
                   32 
                 
                 
                   8 
                 
                 
                   8 
                 
                 
                   8 
                 
                 
                   16 
                 
               
               
                 Ertapenem 
                 
                   4 
                 
                 
                   8 
                 
                 
                   8 
                 
                 
                   &gt;32 
                 
                 
                   8 
                 
                 
                   8 
                 
                 
                   &gt;32 
                 
                 
                   &gt;32 
                 
               
               
                 Imipenem 
                 
                   4 
                 
                 
                   4 
                 
                 
                   8 
                 
                 
                   &gt;32 
                 
                 
                   4 
                 
                 
                   4 
                 
                 
                   &gt;32 
                 
                 
                   &gt;32 
                 
               
               
                 Meropenem 
                 
                   2 
                 
                 
                   2 
                 
                 
                   8 
                 
                 
                   &gt;32 
                 
                 
                   2 
                 
                 
                   2 
                 
                 
                   &gt;32 
                 
                 
                   &gt;32 
                 
               
               
                 Doripenem 
                 
                   2 
                 
                 
                   2 
                 
                 
                   8 
                 
                 
                   &gt;32 
                 
                 
                   2 
                 
                 
                   1 
                 
                 
                   &gt;32 
                 
                 
                   &gt;32 
                 
               
               
                   
               
               
                   a No, no supplemental plasmid. The carbapenemase on the low-copy-number plasmid pACYC177 is shown, and the plasmid was transferred into  K. pneumoniae  NVT1001 and its mutants. TZB, tazobactam with a fixed concentration of 4 mg/L; CLA, clavulanic acid with a fixed concentration of 2 mg/L. 
               
               
                   b Boldface numbers indicate a significant (≥4-fold) difference in the MICs of the NVT1001 strain and its derived strains, while no significant (≥4-fold) differences in the MICs of the NVT1001 with or without plasmid pACYC177 alone (data not shown). 
               
               
                   c WT, NVT1001; Δ35, ΔompK35 mutant; Δ36, ΔompK36 mutant; Δ35/36, ΔompK35/36 mutant; ΔramR, ΔramR mutant; ΔramRΔ35, ΔramRΔompK35 mutant; ΔramRΔ36, ΔramRΔompK36 mutant; ΔramRΔ35/36, ΔramRAΔompK35/36 mutant. 
               
            
           
         
       
     
     2.1.3 Quinolones 
     The MICs of quinolones against chromosome-mediated resistance mechanisms are shown in Table 10. Strains with a single mutation at GyrA (S83I, S83L, S83F, S83Y or D87N) or with AcrAB-TolC overexpression (ΔramR) showed significant (≥4-fold) increases in the MICs of all quinolones tested (Table 5). In contrast, the single mutation at ParC (S80I) showed no significant (≥4-fold) effects on the MIC of any of the quinolones tested, whereas significant (≥4-fold) increases in the MICs of quinolones were observed for the S83I, S83L, S83F, S83Y, S83I/D87N, S83L/D87N, S83F/D87N, S83Y/D87N and ΔramR/S83I mutants (Table 10). The results shown above were further validated by testing the antimicrobial susceptibility of the revertant strains, and similar results were found when comparing the MIC of the wild-type strain NVT1001 or its mutant to the MIC of the revertant with the same genotype (Table 11). 
     The MICs of quinolones against plasmid-mediated resistance mechanisms with or without combination with chromosome-mediated resistance mechanisms are shown in Table 6. In contrast to QnrB or QnrS, strains with AAC(6′)-Ib-cr exhibited a significant (≥4-fold) increase in the MIC of only ciprofloxacin or norfloxacin (Table 12). Even without plasmid-mediated resistance mechanisms, strains with the GyrA (S83I) mutation and AcrAB-TolC overexpression (ΔramR) could resist all tested quinolones (Table 12). 
     
       
         
           
               
             
               
                 TABLE 10 
               
             
            
               
                   
               
               
                 MICs of quinolones against the chromosome-mediated resistance  
               
               
                 mechanisms, GyrA/ParC mutations and AcrAB-TolC  
               
               
                 overexpression (ΔramR), in  K. pneumoniae  NVT1001 
               
            
           
           
               
               
            
               
                   
                 MIC (mg/L) a   
               
            
           
           
               
               
               
               
               
               
               
            
               
                 Strain 
                 NAL b   
                 CIP 
                 NOR 
                 OFX 
                 LVX 
                 MXF 
               
               
                   
               
            
           
           
               
               
               
               
               
               
               
            
               
                 NVT1001 
                 4 
                 0.06 
                 0.25 
                 0.25 
                 0.06 
                 0.125 
               
               
                 S83I mutant 
                 
                   &gt;256 
                 
                 
                   0.5 
                 
                 
                   2 
                 
                 
                   2 
                 
                 
                   0.5 
                 
                 
                   1 
                 
               
               
                 S83L mutant 
                 
                   &gt;256 
                 
                 
                   0.5 
                 
                 
                   2 
                 
                 
                   2 
                 
                 
                   0.5 
                 
                 
                   0.5 
                 
               
               
                 S83F mutant 
                 
                   &gt;256 
                 
                 
                   0.5 
                 
                 
                   2 
                 
                 
                   2 
                 
                 
                   0.5 
                 
                 
                   0.5 
                 
               
               
                 S83Y mutant 
                 
                   &gt;256 
                 
                 
                   0.5 
                 
                 
                   2 
                 
                 
                   2 
                 
                 
                   0.5 
                 
                 
                   0.5 
                 
               
               
                 D87N mutant 
                 
                   &gt;256 
                 
                 
                   0.25 
                 
                 
                   2 
                 
                 
                   1 
                 
                 
                   0.5 
                 
                 
                   0.5 
                 
               
               
                 S80I mutant 
                 8 
                 0.03 
                 0.25 
                 0.25 
                 0.125 
                 0.25 
               
               
                 ΔramR mutant 
                 
                   32 
                 
                 
                   0.5 
                 
                 
                   2 
                 
                 
                   2 
                 
                 
                   1 
                 
                 
                   1 
                 
               
               
                 D87N/S80I mutant 
                 
                   &gt;256 
                 
                 
                   0.5 
                 
                 
                   2 
                 
                 
                   1 
                 
                 
                   0.5 
                 
                 
                   1 
                 
               
               
                 S83I/D87N mutant 
                 
                   &gt;256 
                 
                 
                   0.5 
                 
                 
                   2 
                 
                 
                   2 
                 
                 
                   1 
                 
                 
                   1 
                 
               
               
                 S83L/D87N mutant 
                 
                   &gt;256 
                 
                 
                   0.5 
                 
                 
                   2 
                 
                 
                   2 
                 
                 
                   0.5 
                 
                 
                   0.5 
                 
               
               
                 S83F/D87N mutant 
                 
                   &gt;256 
                 
                 
                   0.5 
                 
                 
                   2 
                 
                 
                   2 
                 
                 
                   0.5 
                 
                 
                   0.5 
                 
               
               
                 S83Y/D87N mutant 
                 
                   &gt;256 
                 
                 
                   0.5 
                 
                 
                   2 
                 
                 
                   2 
                 
                 
                   0.5 
                 
                 
                   0.5 
                 
               
               
                 S83I/S80I mutant 
                 
                   &gt;256 
                 
                 
                   4 
                 
                 
                   16 
                 
                 
                   4 
                 
                 
                   2 
                 
                 
                   8 
                 
               
               
                 S83L/S80I mutant 
                 
                   &gt;256 
                 
                 
                   1 
                 
                 
                   8 
                 
                 
                   4 
                 
                 
                   1 
                 
                 
                   2 
                 
               
               
                 S83F/S80I mutant 
                 
                   &gt;256 
                 
                 
                   0.5 
                 
                 
                   8 
                 
                 
                   2 
                 
                 
                   0.5 
                 
                 
                   1 
                 
               
               
                 S83Y/S80I mutant 
                 
                   &gt;256 
                 
                 
                   0.5 
                 
                 
                   8 
                 
                 
                   2 
                 
                 
                   1 
                 
                 
                   1 
                 
               
               
                 S83I/D87N/S80I mutant 
                 
                   &gt;256 
                 
                 
                   32 
                 
                 
                   64 
                 
                 
                   32 
                 
                 
                   8 
                 
                 
                   &gt;32 
                 
               
               
                 S83L/D87N/S80I mutant 
                 
                   &gt;256 
                 
                 
                   32 
                 
                 
                   64 
                 
                 
                   32 
                 
                 
                   8 
                 
                 
                   32 
                 
               
               
                 S83F/D87N/S80I mutant 
                 
                   &gt;256 
                 
                 
                   32 
                 
                 
                   32 
                 
                 
                   16 
                 
                 
                   8 
                 
                 
                   32 
                 
               
               
                 S83Y/D87N/S80I  
                 
                   &gt;256 
                 
                 
                   16 
                 
                 
                   32 
                 
                 
                   16 
                 
                 
                   4 
                 
                 
                   32 
                 
               
               
                 mutant 
                   
                   
                   
                   
                   
                   
               
               
                 ΔramR/S83I mutant 
                 
                   &gt;256 
                 
                 
                   4 
                 
                 
                   8 
                 
                 
                   &gt;32 
                 
                 
                   16 
                 
                 
                   8 
                 
               
               
                 ΔramR/S83I/S801  
                 
                   &gt;256 
                 
                 
                   &gt;32 
                 
                 
                   128 
                 
                 
                   &gt;32 
                 
                 
                   &gt;32 
                 
                 
                   &gt;32 
                 
               
               
                 mutant 
                   
                   
                   
                   
                   
                   
               
               
                 ΔramR/S83I/ 
                 
                   &gt;256 
                 
                 
                   &gt;32 
                 
                 
                   &gt;256 
                 
                 
                   &gt;32 
                 
                 
                   &gt;32 
                 
                 
                   &gt;32 
                 
               
               
                 D87N/S80I mutant 
               
               
                   
               
               
                   a Boldface numbers indicate a significant (≥4-fold) difference in the MICs of the NVT1001 strain and its mutants. 
               
               
                   b NAL, nalidixic acid; CIP, ciprofloxacin; NOR, norfloxacin; OFX, ofloxacin; LVX, levofloxacin; MXF, moxifloxacin. 
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 11 
               
             
            
               
                   
               
               
                 MICs of quinolones against the gyrA/parC and gyrA/parC/ramR mutants, and their revertants 
               
            
           
           
               
               
            
               
                   
                 MIC (mg/L) a    
               
            
           
           
               
               
               
               
               
               
               
            
               
                 Strain 
                 NAL b   
                 CIP 
                 NOR 
                 OFX 
                 LVX 
                 MXF 
               
               
                   
               
            
           
           
               
               
               
               
               
               
               
            
               
                 S83I/D87N/S80I::I83S/N87D/I80S revertant 
                 4 
                 0.06 
                 0.25 
                 0.25 
                 0.06 
                 0.125 
               
               
                 S83L/D87N/S80I::L83S/N87D/I80S revertant 
                 4 
                 0.06 
                 0.25 
                 0.25 
                 0.06 
                 0.125 
               
               
                 S83F/D87N/S80I::F83S/N87D/I80S revertant 
                 4 
                 0.03 
                 0.25 
                 0.25 
                 0.06 
                 0.125 
               
               
                 S83Y/D87N/S80I::Y83S/N87D/I80S revertant 
                 8 
                 0.03 
                 0.25 
                 0.25 
                 0.06 
                 0.125 
               
               
                 S83I/D87N/S80I::N87D/I80S revertant 
                 
                   &gt;256 
                 
                 
                   0.5 
                 
                 
                   2 
                 
                 
                   2 
                 
                 
                   1 
                 
                 
                   1 
                 
               
               
                 S83L/D87N/S80I::N87D/I80S revertant 
                 
                   &gt;256 
                 
                 
                   0.5 
                 
                 
                   2 
                 
                 
                   2 
                 
                 
                   1 
                 
                 
                   0.5 
                 
               
               
                 S83F/D87N/S80I::N87D/I80S revertant 
                 
                   &gt;256 
                 
                 
                   0.5 
                 
                 
                   2 
                 
                 
                   2 
                 
                 
                   0.5 
                 
                 
                   0.5 
                 
               
               
                 S83Y/D87N/S80I::N87D/I80S revertant 
                 
                   &gt;256 
                 
                 
                   0.5 
                 
                 
                   2 
                 
                 
                   2 
                 
                 
                   0.5 
                 
                 
                   0.5 
                 
               
               
                 ΔramR/S83I/D87N/S80I:: ramR/I83S/I80S revertant 
                 
                   &gt;256 
                 
                 
                   0.25 
                 
                 
                   1 
                 
                 
                   1 
                 
                 
                   0.5 
                 
                 
                   0.5 
                 
               
               
                 ΔramR/S83I/D87N/S80I:: ramR/I83S/N87D revertant 
                 8 
                 0.06 
                 0.25 
                 0.25 
                 0.06 
                 0.25 
               
               
                 ΔramR/S83I/D87N/S80I::I83S/N87D/I80S revertant 
                 
                   32 
                 
                 
                   0.25 
                 
                 
                   2 
                 
                 
                   2 
                 
                 
                   1 
                 
                 
                   1 
                 
               
               
                 ΔramR/S83I/D87N/S80I:: ramR/I83S revertant 
                 
                   &gt;256 
                 
                 
                   0.5 
                 
                 
                   2 
                 
                 
                   1 
                 
                 
                   0.5 
                 
                 
                   1 
                 
               
               
                 S83I/D87N/S80I::I80S revertant 
                 
                   &gt;256 
                 
                 
                   0.5 
                 
                 
                   2 
                 
                 
                   2 
                 
                 
                   1 
                 
                 
                   1 
                 
               
               
                 S83L/D87N/S80I::I80S revertant 
                 
                   &gt;256 
                 
                 
                   0.5 
                 
                 
                   2 
                 
                 
                   2 
                 
                 
                   1 
                 
                 
                   1 
                 
               
               
                 S83F/D87N/S80I::I80S revertant 
                 
                   &gt;256 
                 
                 
                   0.5 
                 
                 
                   2 
                 
                 
                   2 
                 
                 
                   0.5 
                 
                 
                   1 
                 
               
               
                 S83Y/D87N/S80I::I80S revertant 
                 
                   &gt;256 
                 
                 
                   0.5 
                 
                 
                   2 
                 
                 
                   2 
                 
                 
                   0.5 
                 
                 
                   0.5 
                 
               
               
                 S83I/D87N/S80I::N87D revertant 
                 
                   &gt;256 
                 
                 
                   4 
                 
                 
                   16 
                 
                 
                   8 
                 
                 
                   4 
                 
                 
                   4 
                 
               
               
                 S83L/D87N/S80I::N87D revertant 
                 
                   &gt;256 
                 
                 
                   2 
                 
                 
                   8 
                 
                 
                   4 
                 
                 
                   2 
                 
                 
                   1 
                 
               
               
                 S83F/D87N/S80I::N87D revertant 
                 
                   &gt;256 
                 
                 
                   1 
                 
                 
                   4 
                 
                 
                   4 
                 
                 
                   1 
                 
                 
                   0.5 
                 
               
               
                 S83Y/D87N/S80I::N87D revertant 
                 
                   &gt;256 
                 
                 
                   1 
                 
                 
                   4 
                 
                 
                   4 
                 
                 
                   1 
                 
                 
                   0.5 
                 
               
               
                 S83I/D87N/S80I mutant 
                 
                   &gt;256 
                 
                 
                   32 
                 
                 
                   64 
                 
                 
                   32 
                 
                 
                   8 
                 
                 
                   &gt;32 
                 
               
               
                 S83L/D87N/S80I mutant 
                 
                   &gt;256 
                 
                 
                   32 
                 
                 
                   64 
                 
                 
                   32 
                 
                 
                   8 
                 
                 
                   32 
                 
               
               
                 S83F/D87N/S80I mutant 
                 
                   &gt;256 
                 
                 
                   32 
                 
                 
                   32 
                 
                 
                   16 
                 
                 
                   8 
                 
                 
                   32 
                 
               
               
                 S83Y/D87N/S80I mutant 
                 
                   &gt;256 
                 
                 
                   16 
                 
                 
                   32 
                 
                 
                   16 
                 
                 
                   4 
                 
                 
                   32 
                 
               
               
                 ΔramR/S83I/D87N/S80I::ramR/I83S/N87D/I80S 
                 8 
                 0.06 
                 0.25 
                 0.25 
                 0.06 
                 0.125 
               
               
                 ΔramR/S83I/D87N/S80I::N87D/I80S revertant 
                 
                   &gt;256 
                 
                 
                   4 
                 
                 
                   8 
                 
                 
                   &gt;32 
                 
                 
                   8 
                 
                 
                   8 
                 
               
               
                 ΔramR/S83I/D87N/S80I::N87D revertant 
                 
                   &gt;256 
                 
                 
                   &gt;32 
                 
                 
                   128 
                 
                 
                   &gt;32 
                 
                 
                   &gt;32 
                 
                 
                   &gt;32 
                 
               
               
                 ΔramR/S83I/D87N/S80I mutant 
                 
                   &gt;256 
                 
                 
                   &gt;32 
                 
                 
                   &gt;256 
                 
                 
                   &gt;32 
                 
                 
                   &gt;32 
                 
                 
                   &gt;32 
                 
               
               
                   
               
               
                   a Boldface numbers indicate a significant (≥4-fold) difference in the MICs of the NVT1001 strain and its derived strains. 
               
               
                   b NAL, nalidixic acid; CIP, ciprofloxacin; NOR, norfloxacin; OFX, ofloxacin; LVX, levofloxacin; MXF, moxifloxacin. 
               
            
           
         
       
     
     
       
         
           
               
             
               
                 TABLE 12 
               
             
            
               
                   
               
               
                 MICs of quinolones against the chromosome-mediated resistance mechanisms, GyrA/ParC mutations and AcrAB- 
               
               
                 TolC overexpression (ΔramR) and/or the plasmid-mediated resistance mechanisms, QnrB, QnrS or AAC(6′)-Ib-cr, in  
               
               
                   K. pneumoniae  NVT1001 
               
            
           
           
               
               
            
               
                   
                 MIC (mg/L) b   
               
            
           
           
               
               
               
               
               
               
               
               
               
            
               
                 Supplemental 
                   
                   
                   
                   
                   
                   
                   
                 ΔramR/S8 
               
               
                 plasmid and 
                   
                   
                   
                 S83I/D8 
                   
                 ΔramR/ 
                 ΔramR/ 
                 3I/D87N/ 
               
               
                 antibiotic a   
                 WT c   
                 S83I 
                 S83I/S80I 
                 7N/S80I 
                 ΔramR 
                 S83I 
                 S83I/S80I 
                 S80I 
               
               
                   
               
            
           
           
               
               
               
               
               
               
               
               
               
            
               
                 No 
                   
                   
                   
                   
                   
                   
                   
                   
               
               
                 Nalidixic acid 
                 4 
                 
                   &gt;256 
                 
                 
                   &gt;256 
                 
                 
                   &gt;256 
                 
                 
                   32 
                 
                 
                   &gt;256 
                 
                 
                   &gt;256 
                 
                 
                   &gt;256 
                 
               
               
                 Ciprofloxacin 
                 0.06 
                 
                   0.5 
                 
                 
                   4 
                 
                 
                   32 
                 
                 
                   0.5 
                 
                 
                   4 
                 
                 
                   &gt;32 
                 
                 
                   &gt;32 
                 
               
               
                 Norfloxacin 
                 0.25 
                 
                   2 
                 
                 
                   16 
                 
                 
                   64 
                 
                 
                   2 
                 
                 
                   8 
                 
                 
                   128 
                 
                 
                   &gt;256 
                 
               
               
                 Ofloxacin 
                 0.25 
                 
                   2 
                 
                 
                   4 
                 
                 
                   32 
                 
                 
                   2 
                 
                 
                   &gt;32 
                 
                 
                   &gt;32 
                 
                 
                   &gt;32 
                 
               
               
                 Levofloxacin 
                 0.06 
                 
                   0.5 
                 
                 
                   2 
                 
                 
                   8 
                 
                 
                   1 
                 
                 
                   16 
                 
                 
                   &gt;32 
                 
                 
                   &gt;32 
                 
               
               
                 Moxifloxacin 
                 0.125 
                 
                   1 
                 
                 
                   8 
                 
                 
                   &gt;32 
                 
                 
                   1 
                 
                 
                   8 
                 
                 
                   &gt;32 
                 
                 
                   &gt;32 
                 
               
               
                 QnrB 
                   
                   
                   
                   
                   
                   
                   
                   
               
               
                 Nalidixic acid 
                 
                   32 
                 
                 
                   &gt;256 
                 
                 
                   &gt;256 
                 
                 
                   &gt;256 
                 
                 
                   256 
                 
                 
                   &gt;256 
                 
                 
                   &gt;256 
                 
                 
                   &gt;256 
                 
               
               
                 Ciprofloxacin 
                 
                   0.5 
                 
                 
                   2 
                 
                 
                   &gt;32 
                 
                 
                   &gt;32 
                 
                 
                   4 
                 
                 
                   &gt;32 
                 
                 
                   &gt;32 
                 
                 
                   &gt;32 
                 
               
               
                 Norfloxacin 
                 
                   2 
                 
                 
                   8 
                 
                 
                   &gt;256 
                 
                 
                   &gt;256 
                 
                 
                   16 
                 
                 
                   64 
                 
                 
                   &gt;256 
                 
                 
                   &gt;256 
                 
               
               
                 Ofloxacin 
                 
                   4 
                 
                 
                   8 
                 
                 
                   &gt;32 
                 
                 
                   &gt;32 
                 
                 
                   32 
                 
                 
                   &gt;32 
                 
                 
                   &gt;32 
                 
                 
                   &gt;32 
                 
               
               
                 Levofloxacin 
                 
                   1 
                 
                 
                   2 
                 
                 
                   16 
                 
                 
                   &gt;32 
                 
                 
                   8 
                 
                 
                   &gt;32 
                 
                 
                   &gt;32 
                 
                 
                   &gt;32 
                 
               
               
                 Moxifloxacin 
                 
                   2 
                 
                 
                   8 
                 
                 
                   &gt;32 
                 
                 
                   &gt;32 
                 
                 
                   &gt;32 
                 
                 
                   &gt;32 
                 
                 
                   &gt;32 
                 
                 
                   &gt;32 
                 
               
               
                 QnrS 
                   
                   
                   
                   
                   
                   
                   
                   
               
               
                 Nalidixic acid 
                 
                   16 
                 
                 
                   &gt;256 
                 
                 
                   &gt;256 
                 
                 
                   &gt;256 
                 
                 
                   64 
                 
                 
                   &gt;256 
                 
                 
                   &gt;256 
                 
                 
                   &gt;256 
                 
               
               
                 Ciprofloxacin 
                 
                   1 
                 
                 
                   2 
                 
                 
                   &gt;32 
                 
                 
                   &gt;32 
                 
                 
                   4 
                 
                 
                   32 
                 
                 
                   &gt;32 
                 
                 
                   &gt;32 
                 
               
               
                 Norfloxacin 
                 
                   2 
                 
                 
                   8 
                 
                 
                   64 
                 
                 
                   128 
                 
                 
                   32 
                 
                 
                   64 
                 
                 
                   &gt;256 
                 
                 
                   &gt;256 
                 
               
               
                 Ofloxacin 
                 
                   4 
                 
                 
                   8 
                 
                 
                   &gt;32 
                 
                 
                   &gt;32 
                 
                 
                   16 
                 
                 
                   &gt;32 
                 
                 
                   &gt;32 
                 
                 
                   &gt;32 
                 
               
               
                 Levofloxacin 
                 
                   1 
                 
                 
                   4 
                 
                 
                   32 
                 
                 
                   &gt;32 
                 
                 
                   8 
                 
                 
                   &gt;32 
                 
                 
                   &gt;32 
                 
                 
                   &gt;32 
                 
               
               
                 Moxifloxacin 
                 
                   1 
                 
                 
                   8 
                 
                 
                   &gt;32 
                 
                 
                   &gt;32 
                 
                 
                   &gt;32 
                 
                 
                   &gt;32 
                 
                 
                   &gt;32 
                 
                 
                   &gt;32 
                 
               
               
                 AAC(6′)-Ib-cr 
                   
                   
                   
                   
                   
                   
                   
                   
               
               
                 Nalidixic acid 
                 4 
                 
                   &gt;256 
                 
                 
                   &gt;256 
                 
                 
                   &gt;256 
                 
                 
                   64 
                 
                 
                   &gt;256 
                 
                 
                   &gt;256 
                 
                 
                   &gt;256 
                 
               
               
                 Ciprofloxacin 
                 0.125 
                 
                   2 
                 
                 
                   16 
                 
                 
                   &gt;32 
                 
                 
                   0.5 
                 
                 
                   16 
                 
                 
                   &gt;32 
                 
                 
                   &gt;32 
                 
               
               
                 Norfloxacin 
                 
                   1 
                 
                 
                   8 
                 
                 
                   128 
                 
                 
                   &gt;256 
                 
                 
                   8 
                 
                 
                   128 
                 
                 
                   &gt;256 
                 
                 
                   &gt;256 
                 
               
               
                 Ofloxacin 
                 0.25 
                 
                   4 
                 
                 
                   8 
                 
                 
                   32 
                 
                 
                   2 
                 
                 
                   &gt;32 
                 
                 
                   &gt;32 
                 
                 
                   &gt;32 
                 
               
               
                 Levofloxacin 
                 0.06 
                 
                   0.5 
                 
                 
                   2 
                 
                 
                   8 
                 
                 
                   1 
                 
                 
                   8 
                 
                 
                   &gt;32 
                 
                 
                   &gt;32 
                 
               
               
                 Moxifloxacin 
                 0.125 
                 
                   1 
                 
                 
                   8 
                 
                 
                   &gt;32 
                 
                 
                   1 
                 
                 
                   16 
                 
                 
                   &gt;32 
                 
                 
                   &gt;32 
                 
               
               
                   
               
               
                   a No, no supplemental plasmid. The Qnr protein or the aminoglycoside modifying enzyme on the low-copy-number plasmid pACYC177 is shown, and the plasmid was transferred into  K. pneumoniae  NVT1001 and its mutants. 
               
               
                   b Boldface numbers indicate a significant (≥4-fold) difference in the MICs of the NVT1001 strain and its derived strains, while no significant (≥4-fold) differences in the MICs of the NVT1001 with or without plasmid pACYC177 alone (data not shown). 
               
               
                   c WT, NVT1001; S83I, S83I mutant; S83I/S80I, S83I/S80I mutant; S83I/D87N/S80I, S83I/D87N/S80I mutant; ΔramR, ΔramR mutant; ΔramR/S83I, ΔramR/S83I mutant; ΔramR/S83I/S80I, ΔramR/S83I/S80I mutant; ΔramR/S83I/D87N/S80I, ΔramR/S83I/D87N/S80I mutant. 
               
            
           
         
       
     
     2.1.4 Aminoglycosides 
     The MICs of aminoglycosides for each resistance mechanism are shown in Table 13. Several different resistance profiles were found among the strains with different aminoglycoside-modifying enzymes, though all were susceptible to amikacin (Table 13). The production of 16S rRNA methylase, ArmA and RmtB was not associated with observable effects on the MIC of spectinomycin or streptomycin, whereas both conferred strong resistance to all other aminoglycosides tested (Table 13). 
     
       
         
           
               
             
               
                 TABLE 13 
               
             
            
               
                   
               
               
                 MICs of aminoglycosides against the plasmid-mediated  
               
               
                 resistance mechanism, aminoglycoside-modifying 
               
               
                 enzyme or 16S rRNA methylase, in  K. pneumoniae  NVT1001 
               
            
           
           
               
               
            
               
                 Supplemental  
                 MIC (mg/L)b 
               
            
           
           
               
               
               
               
               
               
               
               
            
               
                 plasmid a   
                 AMK c   
                 GEN 
                 KAN 
                 NET 
                 SPT 
                 STR 
                 TOB 
               
               
                   
               
            
           
           
               
               
               
               
               
               
               
               
            
               
                 No 
                 2 
                 0.5 
                 2 
                 0.5 
                 16 
                 2 
                 0.5 
               
               
                 AAC(3)-IId 
                 2 
                 
                   32 
                 
                 4 
                 
                   4 
                 
                 16 
                 2 
                 4 
               
               
                 AAC(3)-IVa 
                 2 
                 
                   32 
                 
                 
                   8 
                 
                 
                   64 
                 
                 16 
                 2 
                 
                   &gt;256 
                 
               
               
                 AAC(6′)-Ib-cr 
                 
                   8 
                 
                 0.25 
                 
                   32 
                 
                 
                   8 
                 
                 16 
                 2 
                 
                   8 
                 
               
               
                 ANT(2″)-Ia 
                 2 
                 
                   64 
                 
                 
                   &gt;256 
                 
                 1 
                 16 
                 2 
                 
                   64 
                 
               
               
                 ANT(3″)-Ia 
                 2 
                 0.25 
                 2 
                 0.5 
                 
                   &gt;1024 
                 
                 
                   16 
                 
                 0.5 
               
               
                 APH(3′)-Ia 
                 2 
                 0.5 
                 
                   &gt;256 
                 
                 0.5 
                 16 
                 2 
                 1 
               
               
                 APH(3′)-IIa 
                 2 
                 0.5 
                 
                   &gt;256 
                 
                 0.5 
                 16 
                 2 
                 0.5 
               
               
                 StrA-StrB 
                 2 
                 0.25 
                 2 
                 0.5 
                 16 
                 
                   1024 
                 
                 0.5 
               
               
                 ArmA 
                 
                   &gt;256 
                 
                 
                   &gt;256 
                 
                 
                   &gt;256 
                 
                 
                   &gt;256 
                 
                 16 
                 2 
                 
                   &gt;256 
                 
               
               
                 RmtB 
                 
                   &gt;256 
                 
                 
                   &gt;256 
                 
                 
                   &gt;256 
                 
                 
                   &gt;256 
                 
                 16 
                 2 
                 
                   &gt;256 
                 
               
               
                   
               
               
                   a No, no supplemental plasmid. The aminoglycoside modifying enzyme or 16S rRNA methylases on the low-copy-number plasmid pACYC184 is shown, and the plasmid was transferred into  K. pneumoniae  NVT1001. 
               
               
                   b Boldface numbers indicate a significant (≥4-fold) difference in the MICs of the NVT1001 strain and its derived strains, while no significant (≥4-fold) differences in the MICs of the NVT1001 with or without plasmid pACYC184 alone (data not shown). 
               
               
                   c AMK, amikacin; GEN, gentamicin; KAN, kanamycin; NET, netilmicin; SPT, spectinomycin; STR, streptomycin; TOB, tobramycin. 
               
            
           
         
       
     
     2.1.5 Tetracyclines 
     The MICs of tetracyclines for various resistance mechanisms are shown in Table 14. With or without AcrAB-To1C overexpression (ΔramR), the production of Tet(B), Tet(C), Tet(D), or Tet(M) showed no observable effects on the MIC of tigecycline (Table 8). AcrAB-TolC overexpression (ΔramR) and Tet(A) production both conferred an 8-fold increase in the MIC of tigecycline, and a 32-fold increase was found when the two mechanisms were combined (Table 14). 
     
       
         
           
               
             
               
                 TABLE 14 
               
             
            
               
                   
               
               
                 MICs of tetracyclines against the chromosome-mediated  
               
               
                 resistance mechanism, AcrAB- 
               
               
                 TolC overexpression (ΔramR), and/or the  
               
               
                 plasmid-mediated resistance mechanism, the tet 
               
               
                 resistance gene, in  K. pneumoniae  NVT1001 
               
            
           
           
               
               
            
               
                 Strain and  
                 MIC (mg/L) a   
               
            
           
           
               
               
               
               
               
               
               
            
               
                 antibiotic 
                 No b   
                 Tet(A) 
                 Tet(B) 
                 Tet(C) 
                 Tet(D) 
                 Tet(M) 
               
               
                   
               
            
           
           
               
               
               
               
               
               
               
            
               
                 NVT1001 
                   
                   
                   
                   
                   
                   
               
               
                 Tetracycline 
                 4 
                 
                   &gt;256 
                 
                 
                   &gt;256 
                 
                 
                   16 
                 
                 
                   &gt;256 
                 
                 
                   16 
                 
               
               
                 Doxycycline 
                 4 
                 
                   &gt;256 
                 
                 
                   &gt;256 
                 
                 
                   16 
                 
                 
                   128 
                 
                 
                   32 
                 
               
               
                 Minocycline 
                 4 
                 
                   32 
                 
                 
                   32 
                 
                 
                   4 
                 
                 
                   128 
                 
                 
                   32 
                 
               
               
                 Tigecycline 
                 1 
                 
                   8 
                 
                 1 
                 1 
                 1 
                 1 
               
               
                 ΔramR 
                   
                   
                   
                   
                   
                   
               
               
                 Tetracycline 
                 
                   32 
                 
                 
                   &gt;256 
                 
                 
                   &gt;256 
                 
                 
                   256 
                 
                 
                   &gt;256 
                 
                 
                   128 
                 
               
               
                 Doxycycline 
                 
                   64 
                 
                 
                   &gt;256 
                 
                 
                   &gt;256 
                 
                 
                   128 
                 
                 
                   256 
                 
                 
                   128 
                 
               
               
                 Minocycline 
                 
                   64 
                 
                 
                   &gt;256 
                 
                 
                   &gt;256 
                 
                 
                   64 
                 
                 
                   &gt;256 
                 
                 
                   &gt;256 
                 
               
               
                 Tigecycline 
                 
                   8 
                 
                 
                   32 
                 
                 
                   8 
                 
                 
                   8 
                 
                 
                   8 
                 
                 
                   8 
                 
               
               
                   
               
               
                   a Boldface numbers indicate a significant (≥4-fold) difference in the MICs of the NVT1001 strain and its derived strains, while no significant (≥4-fold) differences in the MICs of the NVT1001 with or without plasmid pACYC177 alone (data not shown). 
               
               
                   b No, no supplemental plasmid. The tet resistance gene on the low-copy-number plasmid pACYC177 is shown, and the plasmid was transferred into  K. pneumoniae  NVT1001. 
               
            
           
         
       
     
     2.1.6 Folate Pathway Inhibitors 
     The MICs of folate pathway inhibitors against associated resistance mechanisms are shown in Table15. Strains with single resistance mechanisms were all susceptible (≤2/38 mg/L) to trimethoprim/sulfamethoxazole, and strains with certain combinations of mechanisms became resistant to trimethoprim/sulfamethoxazole (Table 15). Strains with porin loss and/or Sul production all showed no significant (≥4-fold) effects on the MIC of trimethoprim, whereas most of the other strains with combined mechanisms were associated with significant (≥4-fold) increases in the MICs of all folate pathway inhibitors tested (Table 15). 
     
       
         
           
               
             
               
                 TABLE 15 
               
             
            
               
                   
               
               
                 MICs of folate pathway inhibitors against the chromosome-mediated resistance mechanism, OmpK35/36 loss and 
               
               
                 AcrAB-TolC overexpression (ΔramR), and/or the plasmid-mediated resistance mechanism, drug-resistant target enzyme, in  
               
               
                   K. pneumoniae  NVT1001 
               
            
           
           
               
               
            
               
                   
                 MIC (mg/L) b   
               
            
           
           
               
               
               
               
               
               
               
               
               
            
               
                 Supplemental plasmid and 
                   
                   
                   
                   
                   
                 ΔramR 
                 ΔramR 
                 ΔramR 
               
               
                 antibiotic a   
                 WT c   
                 Δ35 
                 Δ36 
                 Δ35/36 
                 ΔramR 
                 Δ35 
                 Δ36 
                 Δ35/36 
               
               
                   
               
            
           
           
               
               
               
               
               
               
               
               
               
            
               
                 No 
                   
                   
                   
                   
                   
                   
                   
                   
               
               
                 Trimethoprim 
                 2 
                 2 
                 2 
                 2 
                 
                   8 
                 
                 
                   8 
                 
                 
                   16 
                 
                 
                   &gt;32 
                 
               
               
                 Sulfamethoxazole 
                 256 
                 256 
                 
                   &gt;1024 
                 
                 
                   &gt;1024 
                 
                 
                   &gt;1024 
                 
                 
                   &gt;1024 
                 
                 
                   &gt;1024 
                 
                 
                   &gt;1024 
                 
               
               
                 SXT 
                 0.25 
                 0.25 
                 0.5 
                 0.5 
                 0.5 
                 0.5 
                 
                   1 
                 
                 
                   8 
                 
               
               
                 Sul1 
                   
                   
                   
                   
                   
                   
                   
                   
               
               
                 Trimethoprim 
                 2 
                 2 
                 2 
                 4 
                 
                   16 
                 
                 
                   32 
                 
                 
                   &gt;32 
                 
                 
                   &gt;32 
                 
               
               
                 Sulfamethoxazole 
                 
                   &gt;1024 
                 
                 
                   &gt;1024 
                 
                 
                   &gt;1024 
                 
                 
                   &gt;1024 
                 
                 
                   &gt;1024 
                 
                 
                   &gt;1024 
                 
                 
                   &gt;1024 
                 
                 
                   &gt;1024 
                 
               
               
                 SXT 
                 0.25 
                 
                   2 
                 
                 
                   2 
                 
                 
                   4 
                 
                 
                   16 
                 
                 
                   16 
                 
                 
                   &gt;32 
                 
                 
                   &gt;32 
                 
               
               
                 Sul2 
                   
                   
                   
                   
                   
                   
                   
                   
               
               
                 Trimethoprim 
                 2 
                 4 
                 4 
                 4 
                 
                   16 
                 
                 
                   32 
                 
                 
                   32 
                 
                 
                   &gt;32 
                 
               
               
                 Sulfamethoxazole 
                 
                   &gt;1024 
                 
                 
                   &gt;1024 
                 
                 
                   &gt;1024 
                 
                 
                   &gt;1024 
                 
                 
                   &gt;1024 
                 
                 
                   &gt;1024 
                 
                 
                   &gt;1024 
                 
                 
                   &gt;1024 
                 
               
               
                 SXT 
                 
                   1 
                 
                 
                   2 
                 
                 
                   2 
                 
                 
                   2 
                 
                 
                   8 
                 
                 
                   8 
                 
                 
                   16 
                 
                 
                   &gt;32 
                 
               
               
                 DfrA1 
                   
                   
                   
                   
                   
                   
                   
                   
               
               
                 Trimethoprim 
                 
                   &gt;32 
                 
                 
                   &gt;32 
                 
                 
                   &gt;32 
                 
                 
                   &gt;32 
                 
                 
                   &gt;32 
                 
                 
                   &gt;32 
                 
                 
                   &gt;32 
                 
                 
                   &gt;32 
                 
               
               
                 Sulfamethoxazole 
                 256 
                 
                   &gt;1024 
                 
                 
                   &gt;1024 
                 
                 
                   &gt;1024 
                 
                 
                   &gt;1024 
                 
                 
                   &gt;1024 
                 
                 
                   &gt;1024 
                 
                 
                   &gt;1024 
                 
               
               
                 SXT 
                 
                   2 
                 
                 
                   8 
                 
                 
                   &gt;32 
                 
                 
                   &gt;32 
                 
                 
                   &gt;32 
                 
                 
                   &gt;32 
                 
                 
                   &gt;32 
                 
                 
                   &gt;32 
                 
               
               
                 DfrA16 
                   
                   
                   
                   
                   
                   
                   
                   
               
               
                 Trimethoprim 
                 
                   &gt;32 
                 
                 
                   &gt;32 
                 
                 
                   &gt;32 
                 
                 
                   &gt;32 
                 
                 
                   &gt;32 
                 
                 
                   &gt;32 
                 
                 
                   &gt;32 
                 
                 
                   &gt;32 
                 
               
               
                 Sulfamethoxazole 
                 256 
                 
                   &gt;1024 
                 
                 
                   &gt;1024 
                 
                 
                   &gt;1024 
                 
                 
                   &gt;1024 
                 
                 
                   &gt;1024 
                 
                 
                   &gt;1024 
                 
                 
                   &gt;1024 
                 
               
               
                 SXT 
                 
                   1 
                 
                 
                   4 
                 
                 
                   8 
                 
                 
                   16 
                 
                 
                   4 
                 
                 
                   4 
                 
                 
                   &gt;32 
                 
                 
                   &gt;32 
                 
               
               
                   
               
               
                   a No, no supplemental plasmid. The drug-resistant target enzyme on the low-copy-number plasmid pACYC177 is shown, and the plasmid was transferred into  K. pneumoniae  NVT1001. SXT, trimethoprim/sulfamethoxazole (only the trimethoprim portion of the 1/19 drug ratio is displayed). 
               
               
                   b Boldface numbers indicate a significant (≥4-fold) difference in the MICs of the NVT1001 strain and its derived strains, while no significant (≥4-fold) differences in the MICs of the NVT1001 with or without plasmid pACYC177 alone (data not shown). 
               
               
                   c WT, NVT1001; Δ35, ΔompK35 mutant; Δ36, ΔompK36 mutant; Δ35/36, ΔompK35/36 mutant; ΔramR, ΔramR mutant; ΔramRΔ35, ΔramRΔompK35 mutant; ΔramRΔ6, ΔramRΔompK36 mutant; ΔramRΔ35/36, ΔramRΔompK35/36 mutant. 
               
            
           
         
       
     
     2.1.7 In Vivo Investigation 
     To further demonstrate that this system can also be used to test the efficacy of antibiotics in vivo, a mouse infection model was used. The 50% effective doses (ED50 values) of ceftazidime and cefotaxime were specifically determined using  K. pneumoniae  NVT1001 harbouring the pACYC177 plasmid with bla OXA-48  ( FIG. 1 ). The in vivo efficacy of ceftazidime was higher than that of cefotaxime, which had an estimated ED50 of 30 mg/kg, whereas no mice survived treatment with 30 mg/kg cefotaxime ( FIG. 1 ). The higher efficacy of ceftazidime was also observed in the in vitro MIC assays, in which the MICs of ceftazidime and cefotaxime were 0.5 mg/L and 2 mg/L, respectively, using the broth microdilution test. 
     2.2  A. baumannii  KW1 
     2.2.1 Efflux Pump Overexpression 
     The single mutation in the regulator genes of AdeABC efflux pump could significantly (≥4-fold) increase the MICs of ceftazidime, gentamicin, tetracycline and/or tigecycline, including D20N, A91V or P116L mutation in AdeR and G30D, A94V, R152K or T153M mutation in AdeS (Table 16). These mutations could also confer a 2 or 3-fold increase in the MIC of ciprofloxacin (Table 16). 
     2.2.2 Quinolones Target Site Mutation 
     The single mutation S83L in GyrA could significantly (4-fold) increase the MICs of all quinolones tested (Table 17). With the further mutation in ParC, including G78C, S80L, 
                     TABLE 16                  MICs of antibiotics against the chromosome-mediated       resistance mechanism, AdeABC overexpression via AdeR       or AdeS mutation, in  A. baumannii  KW1                         MIC (μg/ml) a                                               Strain   CAZ b     CIP   GM   TC   TGC                                                         KW1   4   0.25   0.75   3   0.25           D20N mutant   12   0.75     3     8     2             A91V mutant     16     0.75     4       12       3             P116L mutant   8   0.5     3     6     1.5             G30D mutant   8   0.75     4     8     3             A94V mutant   8   0.75     4     6     2             R152K mutant   8   0.75     4     6     2             T153M mutant   8   0.75     4     8     3                           a Boldface numbers indicate a significant (≥4-fold) difference in the MICs of the KW1 strain and its mutants.             b CAZ, ceftazidime; CIP, ciprofloxacin; GM, gentamicin; TC, tetracycline; TGC, tigecycline.            
S80W, S80Y or E84K mutation, could increase these MICs to high-level resistance (Table 17), whereas the single mutation of all these ParC mutations showed no significant (≥4-fold) effects on the MICs of any of the quinolones tested (Table 17).
 
     
       
         
           
               
             
               
                 TABLE 17 
               
             
            
               
                   
               
               
                 MICs of quinolones against the chromosome-mediated  
               
               
                 resistance mechanisms, GyrA and/or ParC 
               
               
                 mutations, in  A. baumannii  KW1 
               
            
           
           
               
               
            
               
                   
                 MIC (μg/ml) a   
               
            
           
           
               
               
               
               
               
               
               
            
               
                 Strain 
                 NAL b   
                 CIP 
                 NOR 
                 OFX 
                 LVX 
                 MXF 
               
               
                   
               
            
           
           
               
               
               
               
               
               
               
            
               
                 KW1 
                 12 
                 0.25 
                 4 
                 0.38 
                 0.19 
                 0.19 
               
               
                 S83L mutant 
                 
                   &gt;256 
                 
                 
                   2 
                 
                 
                   24 
                 
                 
                   6 
                 
                 
                   1 
                 
                 
                   0.75 
                 
               
               
                 G78C mutant 
                 12 
                 0.25 
                 4 
                 0.38 
                 0.19 
                 0.19 
               
               
                 S80L mutant 
                 12 
                 0.25 
                 4 
                 0.5 
                 0.19 
                 0.25 
               
               
                 S80W mutant 
                 12 
                 0.25 
                 6 
                 0.5 
                 0.19 
                 0.19 
               
               
                 S80Y mutant 
                 12 
                 0.25 
                 4 
                 0.38 
                 0.19 
                 0.19 
               
               
                 E84K mutant 
                 12 
                 0.25 
                 4 
                 0.38 
                 0.19 
                 0.19 
               
               
                 S83L/G78C mutant 
                 
                   &gt;256 
                 
                 
                   32 
                 
                 
                   &gt;256 
                 
                 
                   32 
                 
                 
                   32 
                 
                 
                   4 
                 
               
               
                 S83L/S80L mutant 
                 
                   &gt;256 
                 
                 
                   &gt;32 
                 
                 
                   &gt;256 
                 
                 
                   &gt;32 
                 
                 
                   &gt;32 
                 
                 
                   6 
                 
               
               
                 S83L/S80W mutant 
                 
                   &gt;256 
                 
                 
                   &gt;32 
                 
                 
                   &gt;256 
                 
                 
                   &gt;32 
                 
                 
                   &gt;32 
                 
                 
                   8 
                 
               
               
                 S83L/S80Y mutant 
                 
                   &gt;256 
                 
                 
                   &gt;32 
                 
                 
                   &gt;256 
                 
                 
                   &gt;32 
                 
                 
                   &gt;32 
                 
                 
                   6 
                 
               
               
                 S83L/E84K mutant 
                 
                   &gt;256 
                 
                 
                   &gt;32 
                 
                 
                   &gt;256 
                 
                 
                   &gt;32 
                 
                 
                   &gt;32 
                 
                 
                   6 
                 
               
               
                   
               
               
                   a Boldface numbers indicate a significant (≥4-fold) difference in the MICs of the KW1 strain and its mutants. 
               
               
                   b NAL, nalidixic acid; CIP, ciprofloxacin; NOR, norfloxacin; OFX, ofloxacin; LVX, levofloxacin; MXF, moxifloxacin. 
               
            
           
         
       
     
     2.2.3 Plasmid-Mediated Resistance Mechanism of β-Lactams 
     The MICs of β-lactams against associated resistance mechanisms are shown in Table 18. The production of CTX-M-15 or ADC-30 could significantly (≥4-fold) increase the MICs of all β-lactams tested. The production of each carbapenemase tested all showed no increase effects on the MIC of aztreonam, and no increase effects on the MIC of ceftazidime could also be found on the production of OXA-type carbapenemases tested in this study. 
     
       
         
           
               
             
               
                 TABLE 18 
               
             
            
               
                   
               
               
                 MICs of β-lactams against the plasmid-mediated  
               
               
                 resistance mechanism, extended-spectrum β-lactamase, 
               
               
                 AmpC β-lactamase or carbapenemase, in  A. baumannii  KW1 
               
            
           
           
               
               
            
               
                 Supplemental  
                 MIC (μg/ml) b   
               
            
           
           
               
               
               
               
               
               
               
            
               
                 plasmid a   
                 ATM c   
                 PP 
                 CAZ 
                 ETP 
                 MP 
                 DOR 
               
               
                   
               
            
           
           
               
               
               
               
               
               
               
            
               
                 No 
                 16 
                 12 
                 4 
                 3 
                 0.5 
                 0.38 
               
               
                 pYMAb5 
                 12 
                 12 
                 3 
                 3 
                 0.5 
                 0.38 
               
               
                 CTX-M-15 
                 
                   &gt;256 
                 
                 
                   &gt;256 
                 
                 
                   &gt;256 
                 
                 
                   12 
                 
                 
                   2 
                 
                 
                   1.5 
                 
               
               
                 VEB-3 
                 
                   &gt;256 
                 
                 12 
                 
                   &gt;256 
                 
                 3 
                 0.5 
                 0.38 
               
               
                 ADC-30 
                 
                   64 
                 
                 
                   &gt;256 
                 
                 
                   &gt;256 
                 
                 
                   &gt;32 
                 
                 
                   3 
                 
                 
                   4 
                 
               
               
                 IMP-1 
                 16 
                 16 
                 
                   48 
                 
                 
                   &gt;32 
                 
                 
                   12 
                 
                 
                   16 
                 
               
               
                 NDM-1 
                 12 
                 
                   &gt;256 
                 
                 
                   &gt;256 
                 
                 
                   &gt;32 
                 
                 
                   &gt;32 
                 
                 
                   &gt;32 
                 
               
               
                 VIM-1 
                 12 
                 
                   &gt;256 
                 
                 
                   &gt;256 
                 
                 
                   &gt;32 
                 
                 
                   &gt;32 
                 
                 
                   &gt;32 
                 
               
               
                 OXA-23 
                 16 
                 
                   &gt;256 
                 
                 3 
                 
                   &gt;32 
                 
                 
                   &gt;32 
                 
                 
                   &gt;32 
                 
               
               
                 OXA-58 
                 16 
                 
                   128 
                 
                 3 
                 
                   &gt;32 
                 
                 
                   6 
                 
                 
                   6 
                 
               
               
                 OXA-66 
                 12 
                 
                   64 
                 
                 3 
                 
                   &gt;32 
                 
                 
                   8 
                 
                 
                   8 
                 
               
               
                 OXA-72 
                 16 
                 
                   &gt;256 
                 
                 3 
                 
                   &gt;32 
                 
                 
                   &gt;32 
                 
                 
                   &gt;32 
                 
               
               
                   
               
               
                   a No, no supplemental plasmid. The extended-spectrum β-lactamase, AmpC β-lactamase or carbapenemase on the shuttle vector pYMAb5 is shown, and the plasmid was transferred into  A. baumannii  KW1. 
               
               
                   b Boldface numbers indicate a significant (≥4-fold) difference in the MICs of the KW1 strain and its derived strains. 
               
               
                   c ATM, aztreonam; PP, piperacillin; PTc, piperacillin- tazobactam, tazobactam with a fixed concentration of 4 μg/ml; CAZ, ceftazidime; ETP, ertapenem; IP, imipenem; MP, meropenem; DOR, doripenem. 
               
            
           
         
       
     
     2.2.4 Plasmid-Mediated Resistance Mechanism of Aminoglycosides 
     The MICs of aminoglycosides for each resistance mechanism are shown in Table 19. Different resistance effects could be found on the production of these aminoglycoside-modifying enzymes, whereas all showed no increase effects on the MICs of amikacin, netilmicin and streptomycin. The production of 16S rRNA methylase, ArmA, was not associated with increase effects on the MICs of spectinomycin and streptomycin, whereas conferred strong resistance to all other aminoglycosides tested. 
     
       
         
           
               
             
               
                 TABLE 19 
               
             
            
               
                   
               
               
                 MICs of aminoglycosides against the plasmid-mediated  
               
               
                 resistance mechanism, aminoglycoside-modifying 
               
               
                 enzyme or 16S rRNA methylase, in  A. baumannii  KW1 
               
            
           
           
               
               
            
               
                 Supplemental  
                 MIC (μg/ml) b   
               
            
           
           
               
               
               
               
               
               
               
               
            
               
                 plasmid a   
                 AMK c   
                 GEN 
                 KAN 
                 NET 
                 SPT 
                 STR 
                 TOB 
               
               
                   
               
            
           
           
               
               
               
               
               
               
               
               
            
               
                 No 
                 2 
                 0.5 
                 2 
                 1.5 
                 24 
                 12 
                 0.5 
               
               
                 pYMAb5Tc 
                 2 
                 0.5 
                 2 
                 1.5 
                 24 
                 12 
                 0.5 
               
               
                 AAC(3)-IIa 
                 2 
                 
                   3 
                 
                 2 
                 1.5 
                 24 
                 8 
                 0.5 
               
               
                 ANT(2”)-Ia 
                 2 
                 
                   3 
                 
                 4 
                 1.5 
                 24 
                 8 
                 
                   2 
                 
               
               
                 ANT(3”)-Ia 
                 2 
                 0.5 
                 2 
                 1.5 
                 
                   192 
                 
                 8 
                 0.5 
               
               
                 APH(3&#39;)-Ia 
                 2 
                 0.5 
                 
                   16 
                 
                 1.5 
                 24 
                 12 
                 0.5 
               
               
                 APH(3&#39;)-VIa 
                 2 
                 0.5 
                 
                   16 
                 
                 1.5 
                 24 
                 12 
                 0.5 
               
               
                 ArmA 
                 
                   &gt;256 
                 
                 
                   &gt;256 
                 
                 
                   &gt;256 
                 
                 
                   &gt;256 
                 
                 24 
                 8 
                 
                   &gt;256 
                 
               
               
                   
               
               
                   a No, no supplemental plasmid. The aminoglycoside modifying enzyme or 16S rRNA methylase on the shuttle vector pYMAb5Tc is shown, and the plasmid was transferred into  A. baumannii  KW1. 
               
               
                   b Boldface numbers indicate a significant (≥4-fold) difference in the MICs of the KW1 strain and its derived strains. 
               
               
                   c AMK, amikacin; GEN, gentamicin; KAN, kanamycin; NET, netilmicin; SPT, spectinomycin; STR, streptomycin; TOB, tobramycin. 
               
            
           
         
       
     
     2.2.5 Plasmid-Mediated Resistance Mechanism of Tetracyclines 
     The MICs of tetracyclines for its resistance mechanisms are shown in Table 20. The production of Tet(A), Tet(B) or Tet(C) could significantly (4-fold) increase the MICs of tetracycline, doxycycline and minocycline. In the MICs of tigecycline, the production of Tet(B) or Tet(M) showed no observable effects, whereas the production of Tet(A) could confer a 2 to 3-fold increase. 
     
       
         
           
               
             
               
                 TABLE 20 
               
             
            
               
                   
               
               
                 MICs of tetracyclines against the plasmid-mediated resistance 
               
               
                 mechanism, tet resistance gene, in  A. baumannii  KW1 
               
            
           
           
               
               
               
            
               
                   
                 MIC (μg/ml) b   
                   
               
            
           
           
               
               
               
               
               
               
            
               
                   
                 Supplemental plasmid a   
                 TC c   
                 DC 
                 MC 
                 TGC 
               
               
                   
                   
               
            
           
           
               
               
               
               
               
               
            
               
                   
                 No 
                 3 
                 0.75 
                 0.38 
                 0.38 
               
               
                   
                 pYMAb5 
                 3 
                 0.75 
                 0.25 
                 0.38 
               
               
                   
                 Tet(A) 
                 
                   128 
                 
                 
                   12 
                 
                 
                   1.5 
                 
                 1 
               
               
                   
                 Tet(B) 
                 
                   &gt;256 
                 
                 
                   32 
                 
                 
                   1.5 
                 
                 0.38 
               
               
                   
                 Tet(M) 
                 
                   48 
                 
                 
                   192 
                 
                 
                   2 
                 
                 0.38 
               
               
                   
                   
               
               
                   
                   a No, no supplemental plasmid. The tet resistance genes on the shuttle vector pYMAb5 is shown, and the plasmid was transferred into  A. baumannii  KW1. 
               
               
                   
                   b Boldface numbers indicate a significant (≥4-fold) difference in the MICs of the KW1 strain and its derived strains. 
               
               
                   
                   c TC, tetracycline; DC, doxycycline; MC, Minocycline; TGC, tigecycline. 
               
            
           
         
       
     
     3. Discussion 
     In this study, 193 genetically engineered strains with different resistance mechanisms were constructed from  K. pneumoniae  NVT1001, a fully susceptible clinical isolate, including 29 strains with chromosome-mediated resistance, 33 strains with plasmid-mediated resistance and 131 strains with a combination of the two resistance mechanisms. In addition, 37 genetically engineered strains with different resistance mechanisms were constructed from  A. baumannii  KW1, a fully susceptible clinical isolate, including 18 strains with chromosome-mediated resistance and 19 strains with plasmid-mediated resistance. Because the resistance mechanisms of these strains were constructed, all are known to be resistant to specific antibiotics. In addition, the plasmid-mediated resistance mechanisms were constructed using non-conjugative plasmids without transposons, so these mechanisms are not easy to spread. These features suggest that using these strains is safer than using drug-resistant clinical isolates. 
     Also in contrast to clinical drug-resistant isolates, these genetically engineered strains have clear and simple antibiotic resistance mechanisms. In in vitro MIC assays, the resistance profiles of these strains were confirmed by testing the MICs against several well-known antibiotics. Investigators can estimate the effectiveness of their antibiotics by comparing their MIC results to those of well-known antibiotics. The MIC assay is usually the starting point for assessing antibiotics, and our results suggest that these strains were ready for testing antibiotic activities in vitro. 
     In vivo mouse infection models are commonly used to demonstrate the efficacy of antibiotics in protecting against lethal infection. 28-30  Using a clinical  K. pneumoniae  isolate producing the carbapenemase OXA-48, a previous study found that the MIC of ceftazidime was 4-fold lower than that of cefotaxime and that the ED50 of ceftazidime was lower than that of cefotaxime. 28  A similar result was obtained in the present study using  K. pneumoniae  NVT1001 harbouring the pACYC177 plasmid with bla OXA-48 . In contrast to many other clinical isolates, this genetically engineered strain can effectively infect BALB/c mice without weakening their immune system, and its 100% lethal dose (LD100) is 4×10 3  cfu in the mouse infection model, similar to its parental strain NVT1001 (data not shown). Obtained without changing the immune system of mice, the in vivo results should be closer to the true antibiotic efficacy during clinical use. The results thus suggest that these genetically engineered strains are adequate for testing antibiotic activities in vivo. 
     For the development of antibiotics in the face of multidrug resistance, in vitro selection and earlier evaluation are important steps before in vivo studies and further clinical evaluation. In contrast to the use of clinical isolates, the use of the platform technology described here to evaluate antibiotics may elucidate the exact reason for and the level of resistance both in vitro and in vivo. This information may in turn help in estimation of the efficacy of and potential resistance to antibiotics. The modification of antibiotics&#39; chemical structure and discontinuities in development can lower the costs associated with the development process. Furthermore, the global prevalence and distribution of antibiotic resistance genes are already available from various studies and monitoring groups. 31-36  In combination with these data, the data obtained from this platform technology may also help in estimation of the rates of resistance to newly developed drugs across regions. Additionally, antibiotics should be used more carefully in regions where specific resistance mechanisms are epidemic. 
     The set of resistant strains included in this platform technology can be expanded. Specifically, through genetic construction, other resistance mechanisms or different combinations of mechanisms can also be constructed in the same parental strain, including newly identified mechanisms. Given that the genetic background of several important bacterial pathogens with high resistance rates is quite different from that of  K. pneumoniae , specific resistance genes can be found in these pathogens. 37,38  For example, the overexpression of the AdeABC efflux pump can confer antibiotic resistance in  A. baumannii , whereas this efflux pump does not exist in  K. pneumoniae.   37  To completely evaluate the activities of antibiotics, these specific resistance mechanisms should also be constructed in their original pathogens; in fact, several of them have been successfully constructed by our group (data not shown). 
     In summary, various chromosomal and plasmid resistance mechanisms were constructed in this study, and especially mechanisms conferring resistance to β-lactams, quinolones, aminoglycosides, tetracyclines or folate pathway inhibitors, in fully susceptible bacterial strains. Our results demonstrate that this platform technology can be used to efficiently and effectively evaluate antibiotics targeting specific resistance mechanisms both in vitro and in vivo. The system can also be used to screen new antibiotics against multidrug-resistant bacteria. 
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