# Molecular Identification of Aminoglycoside-Modifying Enzymes and Plasmid-Mediated Quinolone Resistance Genes amongKlebsiella pneumoniae Clinical Isolates Recovered from Egyptian Patients

Authors: Mohamed F. El-Badawy; Wael M. Tawakol; Shaymaa W. El-Far; Ibrahim A. Maghrabi; Saleh A. Al-Ghamdi; Moselhy S. Mansy; Mohammed S. Ashour; Mohamed M. Shohayeb
Journal: International Journal of Microbiology
 (2017)

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## Abstract

Inappropriate use of antibiotics in clinical settings is thought to have led to the global emergence and spread of multidrug-resistant pathogens. The goal of this study was to investigate the prevalence of genes encoding aminoglycoside resistance and plasmid-mediated quinolone resistance among clinical isolates ofKlebsiella pneumoniae. AllK. pneumoniae isolates were phenotypically identified using API 20E and then confirmed genotypically through amplification of the specificK. pneumoniae phoE gene. All isolates were genotyped by the enterobacterial repetitive intergenic consensus polymerase chain reaction technique (ERIC-PCR). Antibiotic susceptibility testing was done by a modified Kirby-Bauer method and broth microdilution. All resistant or intermediate-resistant isolates to either gentamicin or amikacin were screened for 7 different genes encoding aminoglycoside-modifying enzymes (AMEs). In addition, all resistant or intermediate-resistant isolates to either ciprofloxacin or levofloxacin were screened for 5 genes encoding the quinolone resistance protein (Qnr), 1 gene encoding quinolone-modifying enzyme, and 3 genes encoding quinolone efflux pumps. Biotyping using API 20E revealed 13 different biotypes. Genotyping demonstrated that all isolates were related to 2 main phylogenetic groups. Susceptibility testing revealed that carbapenems and tigecycline were the most effective agents. Investigation of genes encoding AMEs revealed thatacc(6′)-Ib was the most prevalent, followed byacc(3′)-II,aph(3′)-IV, andant(3′′)-I. Examination of genes encoding Qnr proteins demonstrated thatqnrB was the most prevalent, followed byqnrS,qnrD, andqnrC. It was found that 61%, 26%, and 12% of quinolone-resistantK. pneumoniaeisolates harboredacc(6′)-Ib-cr,oqxAB, andqebA, respectively. The current study demonstrated a high prevalence of aminoglycoside and quinolone resistance genes among clinical isolates ofK. pneumoniae.


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## Body

## 1. Introduction

Few studies have been performed in Egypt concerning the coexistence of genes encoding aminoglycoside-modifying enzymes (AMEs) and plasmid-mediated quinolone resistance (PMQR) among isolates ofKlebsiella pneumoniae. The present study investigated the prevalence and coexistence of seven genes encoding AMEs and nine genes encoding PMQR. Also, clonal relatedness betweenK. pneumoniae isolates was determined by the enterobacterial repetitive intergenic consensus polymerase chain reaction technique (ERIC-PCR). We found a high prevalence and coexistence of genes encoding quinolone and aminoglycosides resistance that were heterogenous and mostly clonally unrelated.The most common mechanism of aminoglycoside resistance arises from enzymatic modification rendering aminoglycosides unable to bind with the aminoacyl site of 16S rRNA with a subsequent failure to inhibit protein synthesis [1]. Modification of aminoglycosides is mediated by AMEs, which catalyze the modification at -OH or -NH2 groups of the 2-deoxystreptamine nucleus or of the sugar moieties of aminoglycoside molecules [2, 3], resulting in reduced or abolished binding of the aminoglycoside molecule to the ribosome. AMEs can be divided into three families: (i) aminoglycoside N-acetyltransferases (AACs), (ii) aminoglycoside O-phosphotransferases (APHs), and (iii) aminoglycoside O-nucleotidyltransferases (ANTs) [4]. Many of the AMEs result in clinically relevant resistance, but only APHs produce high-level resistance [2].Regarding the quinolones, there are four known mechanisms of resistance that work separately or in combination, resulting in varying degrees of resistance that range from reduced susceptibility to clinically relevant resistance. These mechanisms may be chromosomal or plasmid-mediated [5]. The term “resistance” in the setting of PMQR refers to any increase in minimum inhibitory concentration (MIC) rather than to an increase above a susceptibility breakpoint [6]. Three mechanisms are responsible for PMQR: (i) target alteration by Qnr, (ii) drug modification by the aminoglycoside acetyltransferase AAC(6′)-Ib-cr, which can reduce ciprofloxacin activity, and (iii) efflux pump activation by two quinolone efflux pumps, which are known as OqxAB and QepA [6, 7].Qnr proteins protect DNA gyrase and topoisomerase IV from the inhibitory activity of quinolones [8]. Currently, there are six differentqnr genes:qnrA,qnrB,qnrC,qnrD,qnrS, and the most recently reported,qnrVC [6]. The sequences ofqnr genes generally differ from each other and fromqnrA by 35% [9]. Enzymatic inactivation of quinolones arises from aminoglycoside acetyltransferase [AAC (6′)-Ib-cr], which is a bifunctional variant of a common AAC(6′)-Ib. AAC(6′)-Ib-cr acetylates fluoroquinolones, such as ciprofloxacin and norfloxacin, that have an amino nitrogen on the C7 of piperazinyl ring [10]. Finally, we consider PMQR attributed to efflux pumps that specifically extrude quinolones from bacterial cells. Plasmid-mediated quinolone efflux involves two types of pumps, the quinolone efflux pump (QepA) and the olaquindox (OqxAB) efflux pump. QepA belongs to the major facilitator (MFS) family that decreases susceptibility to hydrophilic fluoroquinolones, especially ciprofloxacin and norfloxacin [7]. TheqepA gene is often located on plasmids that encode aminoglycoside ribosomal methylase(rmtB) [6]. The OqxAB pump belongs to the resistance-nodulation-division (RND) family. The OqxAB pump was first detected on a conjugative plasmid (pOLA52) that was harbored byEscherichia coli strains isolated from swine manure [7, 11, 12]. The QqxAB efflux pump has wide substrate specificity that includes chloramphenicol, trimethoprim, and quinolones (ciprofloxacin, norfloxacin, and nalidixic acid [13]).oqxAB genes are located on plasmids in clinical isolates ofE. coli and on both chromosomes and plasmids ofSalmonella spp. andK. pneumoniae. We found thatoqxAB genes are commonly located on the chromosome ofK. pneumoniae[6, 14].


## 2. Materials and Methods

### 2.1. Bacterial Strains

A total of 114 nonduplicate clinical isolates ofK. pneumoniaewere selected from 301 randomly collected isolates of Gram-negative bacilli. The isolates ofK. pneumoniae were collected from 84 cases (39 females and 45 males, age between 2 months and 85 years) who were admitted to or attended medical departments at Ain Shams University Hospital, Cairo, Egypt, over a period of one year (May 2012 to April 2013). Isolates ofK. pneumoniaewere recovered from ascitic fluid (n=5), pus (n=2), blood (n=8), throat swab (n=4), endotracheal tube (n=1), sputum (n=25), urinary catheter (n=1), urine (n=22), wound (n=38), cerebrospinal fluid (n=3), central line catheter (n=3), surgical drain (n=1), and nasal swab (n=1).


### 2.2. Isolation and Identification ofK. pneumoniaeIsolates

All isolates ofK. pneumoniaewere initially isolated on MacConkey’s agar (Oxoid, UK) and then subcultured on eosin methylene blue (EMB) agar (Scharlau, Spain). The isolated strains were identified phenotypically using API 20E (Biomerieux, France) and then confirmed genotypically through amplification of the specificphoEgene using primers and cycling conditions listed in Table 1.Table 1
Primer sets and PCR cycling conditions used for genotyping and amplification of genes encoding AMES.
Primer
Sequence
Gene
Reference
Amplification conditions
Amplicon size (bp)
aac(3′)-II
F: ATATCGCGATGCATACGCGGR: GACGGCCTCTAACCGGAAGG
aac(3′)-II
[20]
Initial denaturation at 95°C for 15 min, then 30 cycles of 95°C for 1 min, 55°C for 1 min, and 72°C for 5 minutes, and one cycle of final elongation at 72°C.
877
aac(6′)-Ib
F: TTGCGATGCTCTATGAGTGGCTAR: CTCGAATGCCTGGCGTGTTT
aac(6′)-Ib
[20]
Initial denaturation at 95°C for 15 min, then 30 cycles of 95°C for 1 min, 55°C for 1 min, and 72°C for 5 minutes, and one cycle of final elongation at 72°C.
472
aac(6′)-II
F: CGACCATTTCATGTCCR: GAAGGCTTGTCGTGTTT
aac(6′)-II
[20]
542
ant(3′′)-I
F: CATCATGAGGGAAGCGGTGR: GACTACCTTGGTGATCTCG
ant(3′′)-I
[20]
Initial denaturation at 95°C for 15 min, then 30 cycles of 95°C for 1 min, 55°C for 1 min, and 72°C for 5 minutes, and one cycle of final elongation at 72°C.
787
aph(3′)-VI
F: ATGGAATTGCCCAATATTATTR: TCAATTCAATTCATCAAGTTT
aph(3′)-VI
[20]
Initial denaturation at 95°C for 15 min, then 30 cycles of 95°C for 1 min, 55°C for 1 min, and 72°C for 5 minutes, and one cycle of final elongation at 72°C.
780
armA
F: CCGAAATGACAGTTCCTATCR: GAAAATGAGTGCCTTGGAGG
armA
[20]
846
rmtB
F: ATGAACATCAACGATGCCCTCR: CCTTCTGATTGGCTTATCCA
rmtB
[20]
Initial denaturation at 95°C for 15 min, then 30 cycles of 95°C for 1 min, 60°C for 1 min, and 72°C for 5 minutes, and one cycle of final elongation at 72°C.
769
phoE
F: TGGCCCGCGCCCAGGGTTCGAAAR: GATGTCGTCATCGTTGATGCCGAG
phoE
[21]
Initial denaturation at 95°C for 15 min, then 35 cycles of 95°C for 1 min, 40°C for 1 min, and 72°C for 5 minutes, and one cycle of final elongation at 72°C.
368
ERIC-1R
R: AACCCACGATGTGGGTAGC
—
[22]
—
acc: aminoglycoside acetyl transferase, ant: aminoglycoside nucleotidyl transferase, aph: aminoglycoside phosphor transferase, arm: aminoglycoside ribosomal methylase, and rmt: ribosomal methylase, phoE; phosphoporin E, ERIC, enterobacterial repetitive intergenic consensus.


### 2.3. Genotyping of Clinical Isolates

Clonal relatedness between clinical isolates ofK. pneumoniaewas determined by ERIC-PCR. The primer was obtained from Macrogen (Korea, Geumcheon-gu, Seoul). Gene amplification was carried out according to cycling conditions as described in Table 1 using Mastercycler® personal (Eppendorf, California, USA).


### 2.4. Fingerprint Pattern Analysis

The banding pattern generated by ERIC-PCR was analyzed using BioNumerics 7.5 software (Applied Maths, Kortrijk, Belgium). The PCR fingerprint profile was analyzed using Dice (similarity) coefficient. Cluster analysis was performed based on the unweighted pair group method with arithmetic averages (UPGMA) at position tolerance at 0.15, as previously described [15].


### 2.5. Antimicrobial Susceptibility Testing

AllK. pneumoniaeisolates were tested for susceptibility to 23 different antibiotics of several classes. Antimicrobial susceptibility testing was by Kirby-Bauer disc diffusion using Mueller-Hinton agar (MHA) (Oxoid, UK) [16]. Broth microdilution [17] was performed using cation-modified Mueller-Hinton broth (Oxoid, UK) to determine the MIC for the tested antibiotics by the Kirby-Bauer disc-diffusion method. Results were interpreted according to guidelines of the Clinical Laboratory Standards Institute (CLSI) [18]. BothE. coli ATCC 25922 andK. pneumoniae ATCC 700603 were used as quality-control strains.


### 2.6. Genotypic Detection of Genes Encoding Aminoglycoside and Quinolone Resistance

AllK. pneumoniae isolates that were resistant to amikacin and/or gentamicin were screened for 7 genes encoding AMEs, namely,aac(3)-II,aac(6′)-Ib,aac(6′)-II,ant(3′′)-I,aph(3′)-VI,armA,andrmtB, using primers and cycling conditions listed in Table 1.


### 2.7. Genotypic Detection of Genes Encoding Quinolone Resistance

AllK. pneumoniae isolates that were resistant to ciprofloxacin and/or levofloxacin were screened for 5 quinolone resistance proteins (qnrA,qnrB,qnrC,qnrD,andqnrS) and one quinolone-modifying enzyme,acc(6′)-Ib-cr. Also, 3 genes (oqxA,oqxB, andqebA) encoding quinolone efflux pump proteins were screened using primers and cycling conditions listed in Table 2.Table 2
Primer sets and PCR cycling conditions used for amplification of genes encoding quinolone resistance.
Prime
Sequence
Gene
Reference
Amplification conditions
Amplicon size (bp)
QnrA
F: AGAGGATTTCTCACGCCAGGR: TGCCAGGCACAGATCTTGAC
qnrA
[23]
Initial denaturation at 95°C for 15 min, then 30 cycles of 95°C for 1 min, 55°C for 1 min, and 72°C for 5 minutes, and one cycle of final elongation at 72°C.
580
QnrB
F: GGMATHGAAATTCGCCACTGR: TTTGCYGYYCGCCAGTCGAA
qnrB
[23]
264
QnrC
F: GGGTTGTACATTTATTGAATCGR: CACCTACCCATTTATTTTCA
qnrC
[24]
307
QnrD
F: CGAGATCAATTTACGGGGAATAR: AACAAGCTGAAGCGCCTG
qnrD
[24]
Initial denaturation at 95°C for 15 min, then 30 cycles of 95°C for 1 min, 56°C for 1 min, and 72°C for 5 minutes, and one cycle of final elongation at 72°C.
533
QnrS
F: GCAAGTTCATTGAACAGGGTR: TCTAAACCGTCGAGTTCGGCG
qnrS
[23]
428
ACC (6′)-Ib-cr
F: TTGCGATGCTCTATGAGTGGCTAR: CTCGAATGC-CTGGCGTGTTT
acc (6′)-Ib-cr
[25]
482
OqxA
F: CTCGGCGCGATGATGCTR: CCACTCTTCACGGGAGACGA
oqxA
[26]
Initial denaturation at 95°C for 15 min, then 30 cycles of 95°C for 1 min, 60°C for 1 min, and 72°C for 5 minutes, and one cycle of final elongation at 72°C.
392
OqxB
F: TTCTCCCCCGGCGGGAAGTACR: CTCGGCCATTTTGGCGCGTA
oqxB
[26]
Initial denaturation at 95°C for 15 min, then 35 cycles of 95°C for 1 min, 58°C for 1 min, and 72°C for 5 minutes, and one cycle of final elongation at 72°C.
512
Qep
F: AACTGCTTGAGCCCGTAGATR: GTCTACGCCATGGACCTCAC
qepA
[27]
Initial denaturation at 95°C for 15 min, then 30 cycles of 95°C for 1 min, 55°C for 1 min, and 72°C for 5 minutes, and one cycle of final elongation at 72°C.
596
qnr: quinolone resistance protein, acc(6′)-Ib-cr: aminoglycoside acetyl transferase-ciprofloxacin variant, Oqx: olaquindox, and Qep: quinolone efflux pump.


### 2.8. Preparation of DNA Templates

DNA was extracted as previously described by Englen and Kelley [19]. Briefly, three to six colonies of bacterial isolates (depending on colony size) were picked from a nutrient agar plate and suspended in 100 μl of DNase-free water in a sterile 1.5 ml microfuge tube to obtain a bacterial suspension equivalent to 1-2 × 109 CFU/ml. The bacterial suspension was placed in a boiling water bath for 10 min to lyse the bacterial cells. The lysed bacterial suspension was centrifuged at maximum speed (13,000 ×g) for 3 min. The supernatant, which contains total genomic DNA, was transferred to a new sterile tube using DNase-free tips. DNA was stored in −20°C.


### 2.9. PCR Setup

The PCR reaction was performed at a final reaction volume of 20μl. The reaction mixture contained 4 μl of extracted DNA, 4 μl of 5x master mix (HOT FIREPol® Blend Master Mix, Solis BioDyne, Tartu, Estonia), 0.6 μl of forward primer (10 pmol/μl), 0.6 μl of reverse primer (10 pmol/μl), and 10.8 μl distilled water.


## 2.1. Bacterial Strains

A total of 114 nonduplicate clinical isolates ofK. pneumoniaewere selected from 301 randomly collected isolates of Gram-negative bacilli. The isolates ofK. pneumoniae were collected from 84 cases (39 females and 45 males, age between 2 months and 85 years) who were admitted to or attended medical departments at Ain Shams University Hospital, Cairo, Egypt, over a period of one year (May 2012 to April 2013). Isolates ofK. pneumoniaewere recovered from ascitic fluid (n=5), pus (n=2), blood (n=8), throat swab (n=4), endotracheal tube (n=1), sputum (n=25), urinary catheter (n=1), urine (n=22), wound (n=38), cerebrospinal fluid (n=3), central line catheter (n=3), surgical drain (n=1), and nasal swab (n=1).


## 2.2. Isolation and Identification ofK. pneumoniaeIsolates

All isolates ofK. pneumoniaewere initially isolated on MacConkey’s agar (Oxoid, UK) and then subcultured on eosin methylene blue (EMB) agar (Scharlau, Spain). The isolated strains were identified phenotypically using API 20E (Biomerieux, France) and then confirmed genotypically through amplification of the specificphoEgene using primers and cycling conditions listed in Table 1.Table 1
Primer sets and PCR cycling conditions used for genotyping and amplification of genes encoding AMES.
Primer
Sequence
Gene
Reference
Amplification conditions
Amplicon size (bp)
aac(3′)-II
F: ATATCGCGATGCATACGCGGR: GACGGCCTCTAACCGGAAGG
aac(3′)-II
[20]
Initial denaturation at 95°C for 15 min, then 30 cycles of 95°C for 1 min, 55°C for 1 min, and 72°C for 5 minutes, and one cycle of final elongation at 72°C.
877
aac(6′)-Ib
F: TTGCGATGCTCTATGAGTGGCTAR: CTCGAATGCCTGGCGTGTTT
aac(6′)-Ib
[20]
Initial denaturation at 95°C for 15 min, then 30 cycles of 95°C for 1 min, 55°C for 1 min, and 72°C for 5 minutes, and one cycle of final elongation at 72°C.
472
aac(6′)-II
F: CGACCATTTCATGTCCR: GAAGGCTTGTCGTGTTT
aac(6′)-II
[20]
542
ant(3′′)-I
F: CATCATGAGGGAAGCGGTGR: GACTACCTTGGTGATCTCG
ant(3′′)-I
[20]
Initial denaturation at 95°C for 15 min, then 30 cycles of 95°C for 1 min, 55°C for 1 min, and 72°C for 5 minutes, and one cycle of final elongation at 72°C.
787
aph(3′)-VI
F: ATGGAATTGCCCAATATTATTR: TCAATTCAATTCATCAAGTTT
aph(3′)-VI
[20]
Initial denaturation at 95°C for 15 min, then 30 cycles of 95°C for 1 min, 55°C for 1 min, and 72°C for 5 minutes, and one cycle of final elongation at 72°C.
780
armA
F: CCGAAATGACAGTTCCTATCR: GAAAATGAGTGCCTTGGAGG
armA
[20]
846
rmtB
F: ATGAACATCAACGATGCCCTCR: CCTTCTGATTGGCTTATCCA
rmtB
[20]
Initial denaturation at 95°C for 15 min, then 30 cycles of 95°C for 1 min, 60°C for 1 min, and 72°C for 5 minutes, and one cycle of final elongation at 72°C.
769
phoE
F: TGGCCCGCGCCCAGGGTTCGAAAR: GATGTCGTCATCGTTGATGCCGAG
phoE
[21]
Initial denaturation at 95°C for 15 min, then 35 cycles of 95°C for 1 min, 40°C for 1 min, and 72°C for 5 minutes, and one cycle of final elongation at 72°C.
368
ERIC-1R
R: AACCCACGATGTGGGTAGC
—
[22]
—
acc: aminoglycoside acetyl transferase, ant: aminoglycoside nucleotidyl transferase, aph: aminoglycoside phosphor transferase, arm: aminoglycoside ribosomal methylase, and rmt: ribosomal methylase, phoE; phosphoporin E, ERIC, enterobacterial repetitive intergenic consensus.


## 2.3. Genotyping of Clinical Isolates

Clonal relatedness between clinical isolates ofK. pneumoniaewas determined by ERIC-PCR. The primer was obtained from Macrogen (Korea, Geumcheon-gu, Seoul). Gene amplification was carried out according to cycling conditions as described in Table 1 using Mastercycler® personal (Eppendorf, California, USA).


## 2.4. Fingerprint Pattern Analysis

The banding pattern generated by ERIC-PCR was analyzed using BioNumerics 7.5 software (Applied Maths, Kortrijk, Belgium). The PCR fingerprint profile was analyzed using Dice (similarity) coefficient. Cluster analysis was performed based on the unweighted pair group method with arithmetic averages (UPGMA) at position tolerance at 0.15, as previously described [15].


## 2.5. Antimicrobial Susceptibility Testing

AllK. pneumoniaeisolates were tested for susceptibility to 23 different antibiotics of several classes. Antimicrobial susceptibility testing was by Kirby-Bauer disc diffusion using Mueller-Hinton agar (MHA) (Oxoid, UK) [16]. Broth microdilution [17] was performed using cation-modified Mueller-Hinton broth (Oxoid, UK) to determine the MIC for the tested antibiotics by the Kirby-Bauer disc-diffusion method. Results were interpreted according to guidelines of the Clinical Laboratory Standards Institute (CLSI) [18]. BothE. coli ATCC 25922 andK. pneumoniae ATCC 700603 were used as quality-control strains.


## 2.6. Genotypic Detection of Genes Encoding Aminoglycoside and Quinolone Resistance

AllK. pneumoniae isolates that were resistant to amikacin and/or gentamicin were screened for 7 genes encoding AMEs, namely,aac(3)-II,aac(6′)-Ib,aac(6′)-II,ant(3′′)-I,aph(3′)-VI,armA,andrmtB, using primers and cycling conditions listed in Table 1.


## 2.7. Genotypic Detection of Genes Encoding Quinolone Resistance

AllK. pneumoniae isolates that were resistant to ciprofloxacin and/or levofloxacin were screened for 5 quinolone resistance proteins (qnrA,qnrB,qnrC,qnrD,andqnrS) and one quinolone-modifying enzyme,acc(6′)-Ib-cr. Also, 3 genes (oqxA,oqxB, andqebA) encoding quinolone efflux pump proteins were screened using primers and cycling conditions listed in Table 2.Table 2
Primer sets and PCR cycling conditions used for amplification of genes encoding quinolone resistance.
Prime
Sequence
Gene
Reference
Amplification conditions
Amplicon size (bp)
QnrA
F: AGAGGATTTCTCACGCCAGGR: TGCCAGGCACAGATCTTGAC
qnrA
[23]
Initial denaturation at 95°C for 15 min, then 30 cycles of 95°C for 1 min, 55°C for 1 min, and 72°C for 5 minutes, and one cycle of final elongation at 72°C.
580
QnrB
F: GGMATHGAAATTCGCCACTGR: TTTGCYGYYCGCCAGTCGAA
qnrB
[23]
264
QnrC
F: GGGTTGTACATTTATTGAATCGR: CACCTACCCATTTATTTTCA
qnrC
[24]
307
QnrD
F: CGAGATCAATTTACGGGGAATAR: AACAAGCTGAAGCGCCTG
qnrD
[24]
Initial denaturation at 95°C for 15 min, then 30 cycles of 95°C for 1 min, 56°C for 1 min, and 72°C for 5 minutes, and one cycle of final elongation at 72°C.
533
QnrS
F: GCAAGTTCATTGAACAGGGTR: TCTAAACCGTCGAGTTCGGCG
qnrS
[23]
428
ACC (6′)-Ib-cr
F: TTGCGATGCTCTATGAGTGGCTAR: CTCGAATGC-CTGGCGTGTTT
acc (6′)-Ib-cr
[25]
482
OqxA
F: CTCGGCGCGATGATGCTR: CCACTCTTCACGGGAGACGA
oqxA
[26]
Initial denaturation at 95°C for 15 min, then 30 cycles of 95°C for 1 min, 60°C for 1 min, and 72°C for 5 minutes, and one cycle of final elongation at 72°C.
392
OqxB
F: TTCTCCCCCGGCGGGAAGTACR: CTCGGCCATTTTGGCGCGTA
oqxB
[26]
Initial denaturation at 95°C for 15 min, then 35 cycles of 95°C for 1 min, 58°C for 1 min, and 72°C for 5 minutes, and one cycle of final elongation at 72°C.
512
Qep
F: AACTGCTTGAGCCCGTAGATR: GTCTACGCCATGGACCTCAC
qepA
[27]
Initial denaturation at 95°C for 15 min, then 30 cycles of 95°C for 1 min, 55°C for 1 min, and 72°C for 5 minutes, and one cycle of final elongation at 72°C.
596
qnr: quinolone resistance protein, acc(6′)-Ib-cr: aminoglycoside acetyl transferase-ciprofloxacin variant, Oqx: olaquindox, and Qep: quinolone efflux pump.


## 2.8. Preparation of DNA Templates

DNA was extracted as previously described by Englen and Kelley [19]. Briefly, three to six colonies of bacterial isolates (depending on colony size) were picked from a nutrient agar plate and suspended in 100 μl of DNase-free water in a sterile 1.5 ml microfuge tube to obtain a bacterial suspension equivalent to 1-2 × 109 CFU/ml. The bacterial suspension was placed in a boiling water bath for 10 min to lyse the bacterial cells. The lysed bacterial suspension was centrifuged at maximum speed (13,000 ×g) for 3 min. The supernatant, which contains total genomic DNA, was transferred to a new sterile tube using DNase-free tips. DNA was stored in −20°C.


## 2.9. PCR Setup

The PCR reaction was performed at a final reaction volume of 20μl. The reaction mixture contained 4 μl of extracted DNA, 4 μl of 5x master mix (HOT FIREPol® Blend Master Mix, Solis BioDyne, Tartu, Estonia), 0.6 μl of forward primer (10 pmol/μl), 0.6 μl of reverse primer (10 pmol/μl), and 10.8 μl distilled water.


## 3. Result

### 3.1. Isolation and Identification

From 301 recovered isolates, 114 (37%) wereK. pneumoniae, 3 (1%) wereKlebsiella oxytoca, 61 (20%) wereE. coli, 48 (16%) wereProteusspp., 38 (13%) werePseudomonas aeruginosa, 12 (4%) wereAcinetobacter baumannii, 9 (3%) wereSerratia marcescens, 6 (2%) wereEnterobacter cloacae,and 3 (1%) were single isolates for each ofProvidencia stuartii,Burkholderia cepacia, andAeromonas hydrophilia.


### 3.2. Phenotypic and Genotypic Identification ofK. pneumoniae Isolates

Biotyping ofK. pneumoniae clinical isolates using API 20E revealed 13 different biotypes. The most prevalent were 5215773 and 5205773, which occurred at a prevalence of 56% (64/114) and 29% (34/114), respectively. Other detected biotypes were 5005573, 5215573, and 5205573, which occurred at a prevalence of 3.5% (4/114), 1.8% (2/114), and 1.8% (2/114), respectively. The lowest detected biotypes were 1205773, 1215773, 5004573, 5204773, 5204553, 5215763, 5217773, and 5214773, which each occurred at a prevalence of 0.88% (1/114).Genotypic confirmation of phenotypically identified isolates through amplification of theK. pneumoniae phoEgene revealed that these isolates were related toK. pneumoniae.ERIC-PCR-based DNA fingerprinting identified only 95.6% (109/114) of theK. pneumoniaeisolates, as 5 isolates gave no band following agarose gel electrophoresis. The 109 genotypedK. pneumoniae isolates displayed 85 different fingerprint patterns, as shown in Figure 1.Figure 1
Representative DNA fingerprint pattern ofK. pneumoniaeclinical isolates genotyped by ERIC-PCR.


### 3.3. Fingerprint Pattern Analysis

A UPGMA dendrogram generated according to Dice (similarity) coefficient revealed that the 85 fingerprint profiles were related to 67 different profiles, including 67 isolates with 18 different combined profiles that included 42 isolates. All genotypedK. pneumoniae were classified into 2 major phylogenetic groups (group A and group B), as shown in Figure 2. Phylogenetic group A included 4 isolates (K184, K109, K162, and K161). Two isolates (K161 and K162) within phylogenetic group A had the same fingerprint pattern. Phylogenetic group B contained the remaining 105 isolates.Figure 2
Generated UPGMA dendrogram based on Dice similarity coefficient for clustering ofK. pneumoniaeisolates using ERIC-PCR as DNA fingerprinting method. KL:Klebsiella pneumoniae, PGA: phylogenetic group A, and PGB: phylogenetic group B.


### 3.4. Correlation between Phenotyping by API 20E and Genotyping by ERIC-PCR

No relationship was found between the phenotypes detected by API 20E and the genotypes detected by ERIC-PCR, as identical clones, such as KL169 and KL174, showed different biotypes (5005773 and 5215773). Other identical genotypes also revealed different biotypes, as shown in Figure2.


### 3.5. Antimicrobial Susceptibility

Antimicrobial susceptibility tests revealed that carbapenems (imipenem and meropenem) were more effective than 3rd- and 4th-generation cephalosporins for which 75% (86/114) and 75% (85/114) of the isolates were susceptible to imipenem and meropenem, respectively, as shown in Table3. Regarding non-β-lactam antibiotics, tigecycline showed the lowest resistance, as 97% (111/114) of isolates were susceptible. As regarding quinolones and aminoglycosides, we found that 60% (68/114), 26% (30/114), 47% (54/114), and 43% (49/114) of isolates were resistant to gentamicin, amikacin, ciprofloxacin, and levofloxacin, respectively.Table 3
Antibiotic susceptibility ofK. pneumoniae clinical isolates.
Antibiotic
K. pneumoniae susceptibility pattern (n=114)
MIC50 and MIC90
Sensitive
Intermediate
Resistant
MIC50
MIC90
Number (%)
Number (%)
Number (%)
Cefotaxime
15(13)
1(1)
98(86)
256
>1024
Ceftazidime
26(23)
14(12)
74(65)
32
>1024
Cefoperazone
17(15)
3(2)
94(82)
512
>1024
Cefoperazone/sulbactam
51(45)
12(10)
51(45)
32
1024
Ceftriaxone
18(16)
—
96(84)
256
>1024
Cefepime
21(18)
24(21)
69(61)
32
256
Imipenem
86(75)
5(4)
23(20)
≤0.5
4
Meropenem
85(75)
2(2)
27(24)
≤0.5
16
Tetracycline
32(28)
4(3)
78(68)
128
512
Tigecycline
111(97)
—
3(3.0)
≤0.5
≤0.5
Gentamicin
46(40)
12(11)
56(49)
8
>1024
Amikacin
84(74)
1(1)
29(25)
≤0.5
>1024
Thiamphenicol
7(6.0)
2(2)
105(92)
1024
>1024
Ciprofloxacin
60(53)
3(2)
51(45)
1
128
Levofloxacin
65(57)
5(4)
44(39)
≤0.5
64Apart from imipenem and meropenem, the MIC50 and MIC90 values for other tested β-lactams ranged between 32 to 512 μg/ml and 256 to >1024 μg/ml, respectively. On the other hand, the MIC50 and MIC90 for gentamicin and amikacin ranged from ≤0.5 to 8 μg/ml and >1024 μg/ml, respectively, while MIC50 and MIC90 of ciprofloxacin and levofloxacin ranged from ≤0.5 to 1 μg/ml and 64 to 128 μg/ml, respectively.


### 3.6. Detection of Genes Encoding AMEs

Genotypic results for AMEs among the aminoglycoside-resistant isolates, as shown in Figure3, revealed that acetyltransferases were the most prevalent type of AME. Theacc(6′)-Ib andacc(3′)-II genes were detected among 88% (58/66) and 58% (38/66) of the investigated isolates (Table 4). In contrast, theacc(6′)-II variant was not detected.Table 4
Resistance pattern and genetic profile of quinolone and aminoglycoside resistantK. pneumoniaeisolates.
Isolate number
Quinolone and aminoglycoside resistance profile
Quinolone resistance genetic profile
Number of quinolone resistance genes
Aminoglycoside resistance genetic profile
Number of aminoglycoside resistance genes
Total number of detected genes
KLP2
CN, AK, CIP
qnrB,qnrD, oqxA, oqxB
4
ant(3′′)-I,aac(6′)-Ib
2
6
KLP3
CN, CP, LEV
qnrB,qnrD,oqxA
3
aac(3)-II,aac(6′)-Ib
2
5
KLP5
CN
NA
NA
aac(3)-II,aac(6′)-Ib
2
2
KLP6
CN
NA
NA
aac(3)-II,aac(6′)-Ib
2
5
KLP13
CN, AK, CIP, LEV
qnrB,oqxA, oqxB
3
—
—
3
KLP14
CN, AK, CIP, LEV
qnrB
1
aph(3′)-VI, ant(3′′)-I
2
3
KLP15
CN, AK, CIP
qnrB,oqxA, acc(6′)-Ib-cr
3
ant(3′′)-I,aac(3)-II,aac(6′)-Ib,aph(3′)-VI,armA
5
8
KLP19
CN, AK, CIP, LEV
qnrB,oqxA, oqxB
3
ant(3′′)-I,aac(6′)-Ib
2
5
KLP20
CN, AK, CIP, LEV
qnrB,oqxA
2
aac(3)-II,aac(6′)-Ib
2
4
KLP23
CN, AK, CIP, LEV
qnrB,qnrD, qnrS,oqxA
4
aac(3)-II,aac(6′)-Ib,aph(3′)-VI
3
7
KLP27
CN, CIP, LEV
qnrB,qnrD,oqxA
3
aac(3)-II,aac(6′)-Ib
2
5
KLP28
CN, CIP, LEV
qnrB,oqxA
2
aac(6′)-Ib, aph(3′)-VI,armA
3
5
KLP29
CN, AK, CIP
qnrB,oqxA
2
ant(3′′)-I, aac(3)-II,aac(6′)-Ib,aph(3′)-VI
4
6
KLP30
CN, AK, CIP, LEV
qnrB, qnrS,oqxA, oqxB, acc(6′)-Ib-cr
5
ant(3′′)-I,aac(3)-II,aac(6′)-Ib,aph(3′)-VI
4
9
KLP33
CN, CIP, LEV
qnrB, qnrS,oqxA, acc(6′)-Ib-cr
4
ant(3′′)-I,aac(3)-II,aac(6′)-Ib,aph(3′)-VI
4
8
KLP34
CN
oqxA
1
aac(3)-II,aac(6′)-Ib,aph(3′)-VI
3
4
KLP35
CN, CIP
acc(6′)-Ib-cr
1
aac(6′)-Ib, aac(6′)-Ib,aph(3′)-VI
3
4
KLP36
CN, AK, CIP, LEV
oqxA
1
aac(3)-II,aac(6′)-Ib
2
3
KLP42
CN
NA
NA
aac(3)-II,aac(6′)-Ib
2
2
KLP43
CN
NA
NA
aac(3)-II, aac(6′)-Ib, aph(3′)-VI
3
3
KLP50
CN, LEV
qnrS,oqxA, acc(6′)-Ib-cr
3
ant(3′′)-I,aac(3)-II,aac(6′)-Ib
3
6
KLP58A
CIP, LEV
qnrB, qnrS,oqxA, acc(6′)-Ib-cr
4
NA
NA
4
KLP58B
CN, CIP, LEV
qnrB, qnrS,oqxA, acc(6′)-Ib-cr
4
ant(3′′)-I,aac(3)-II,aac(6′)-Ib, aph(3′)-VI
4
8
KLP63
CN, AK, CIP, LEV
qnrB,oqxA, acc(6′)-Ib-cr
3
ant(3′′)-I,aac(3)-II,aac(6′)-Ib,aph(3′)-VI
4
7
KLP65
CN
NA
NA
aac(6′)-Ib
1
1
KLP69
CN, AK, CIP, LEV
qnrB,qnrD,oqxA, oqxB, acc(6′)-Ib-cr
5
—
—
5
KLP72
CN, AK, CIP, LEV
qnrB,qnrD, qnrS,oqxA, acc(6′)-Ib-cr
5
ant(3′′)-I,aac(6′)-Ib,aph(3′)-VI,armA
4
9
KLP73
CN
NA
NA
aac(6′)-Ib
1
1
KLP78
CN, CIP, LEV
qnrB,qnrD, oqxA
3
aac(3)-II,aac(6′)-Ib
2
5
KLP80
CN, AK, CIP, LEV
qnrD, qnrS,oqxA, acc(6′)-Ib-cr
4
ant(3′′)-I,aac(6′)-Ib,aph(3′)-VI,armA
4
8
KLP81
CN, CIP
qnrB,oqxA, acc(6′)-Ib-cr
3
aac(3)-II,aac(6′)-Ib,aph(3′)-VI
3
6
KLP88
CN
NA
NA
aac(3)-II, aac(6′)-Ib,aph(3′)-VI
3
3
KLP89
CN, AK
NA
NA
ant(3′′)-I,aac(3)-II,aac(6′)-Ib, aph(3′)-VI
4
4
KLP93
CN
NA
NA
ant(3′′)-I,aac(3)-II,aac(6′)-Ib,aph(3′)-VI
4
4
KLP96
CN, AK, CIP, LEV
qnrB,qnrD, qnrS,qebA,oqxA, oqxB, acc(6′)-Ib-cr
7
ant(3′′)-I,aac(3)-II,aac(6′)-Ib,aph(3′)-VI,armA
5
12
KLP99
CN, AK, CIP, LEV
qnrB, oqxA, oqxB, acc(6′)-Ib-cr
4
aac(6′)-Ib,aph(3′)-VI
2
6
KLP100
CN, AK, CIP, LEV
qnrB, oqxA, oqxB, acc(6′)-Ib-cr
4
aac(3)-II,aac(6′)-Ib
2
6
KLP101
CN, CIP, LEV
qnrB, oqxA, acc(6′)-Ib-cr
3
aac(6′)-Ib, aph(3′)-VI
2
5
KLP102
CIP, LEV
qnrD,oqxA
2
NA
NA
2
KLP103
CN, AK, CIP, LEV
qnrB, oqxA, oqxB
3
aac(6′)-Ib,aph(3′)-VI
2
5
KLP104
CN, CIP, LEV
qnrS,oqxA, acc(6′)-Ib-cr
3
ant(3′′)-I,aac(6′)-Ib
2
5
KLP107
CN
NA
NA
aac(6′)-Ib
1
1
KLP108
CN, LEV
qnrB,qnrS, acc(6′)-Ib-cr
3
aac(3)-II, aac(6′)-Ib
2
5
KLP110
CN, CIP, LEV
qnrB,qnrD, qnrS,oqxA, acc(6′)-Ib-cr
5
ant(3′′)-I,aac(3)-II,aac(6′)-Ib, aph(3′)-VI
4
9
KLP111
CN
NA
NA
ant(3′′)-I,aac(3)-II,aph(3′)-VI
3
3
KLP112
CN, CIP, LEV
qnrB,qnrD,qnrS,oqxA, acc(6′)-Ib-cr
5
ant(3′′)-I,aac(6′)-Ib,aph(3′)-VI
3
8
KLP115
CN, CIP, LEV
qnrB,qnrD,qnrS,oqxA, acc(6′)-Ib-cr
5
aac(6′)-Ib
1
6
KLP117
CIP, LEV
qnrB,qnrD,qnrS,oqxA, acc(6′)-Ib-cr
5
NA
NA
5
KLP119
CN
NA
NA
aac(3)-II
1
1
KLP121
CIP, LEV
qnrB,qnrD,qnrS,oqxA, oqxB
5
NA
NA
5
KLP125
CN, AK, CIP, LEV
qnrB,qebA,oqxA, oqxB, acc(6′)-Ib-cr
5
aac(6′)-Ib
1
6
KLP127
CN, AK, CIP, LEV
qnrB,qnrD,qebA,oqxA, oqxB, acc(6′)-Ib-cr
6
ant(3′′)-I,aac(6′)-Ib
2
8
KLP128
CN
NA
NA
—
—
—
KLP129
CN, AK, CIP, LEV
qnrB,qnrS, qebA,oqxA, acc(6′)-Ib-cr
5
ant(3′′)-I,aac(6′)-Ib,aph(3′)-VI,armA
4
9
KLP134
CN, AK, CIP, LEV
qnrB,qnrS,oqxA, oqxB, acc(6′)-Ib-cr
5
ant(3′′)-I,aac(3)-II,aac(6′)-Ib,aph(3′)-VI
4
9
KLP136
CIP, LEV
qnrS, oqxB
2
aac(6′)-Ib
1
3
KLP137
CIP, LEV
qnrS,oqxA, acc(6′)-Ib-cr
3
NA
NA
3
KLP140
CN
NA
NA
aac(3)-II
1
1
KLP141
CN
NA
NA
aac(3)-II
1
1
KLP144
CN, AK, CIP, LEV
acc(6′)-Ib-cr
1
ant(3′′)-I,aac(6′)-Ib,aph(3′)-VI
3
4
KLP147
CN, LEV
qnrB,qnrD,qnrS,oqxA, acc(6′)-Ib-cr
5
aac(3)-II,aac(6′)-Ib
2
7
KLP148
CN
NA
NA
—
—
—
KLP154
CIP
qnrB,qnrD,qnrS,oqxA
4
NA
NA
4
KLP157
CN
NA
NA
aac(3)-II, aac(6′)-Ib,aph(3′)-VI
3
3
KLP169
CN, AK, CIP, LEV
qnrB, oqxA, acc(6′)-Ib-cr
3
ant(3′′)-I,aac(3)-II,aac(6′)-Ib,aph(3′)-VI
4
7
KLP170
CN, AK, CIP, LEV
qnrB, oqxA,oqxB, acc(6′)-Ib-cr
4
ant(3′′)-I,aac(6′)-Ib,aph(3′)-VI
3
7
KLP177
CN, CIP, LEV
—
—
—
—
—
KLP180
CN, AK, CIP, LEV
qnrB,qnrD,qnrS,oqxA,oqxB, acc(6′)-Ib-cr
6
ant(3′′)-I,aac(6′)-Ib
2
8
KLP184
CIP, LEV
qnrS,oqxA,oqxB
3
NA
NA
3
KLP186
CN, CIP
qnrC,qnrD, qnrS,oqxA
4
aac(6′)-Ib
1
5
KLP187
CIP
qnrS,qnrD
2
NA
NA
2
KLP197
CN, AK, CIP, LEV
qnrS,qebA,oqxA, acc(6′)-Ib-cr
4
ant(3′′)-I,aac(6′)-Ib,aph(3′)-VI,armA
4
8
KLP198
CN, AK, CIP, LEV
qnrB,qnrD,qebA,oqxA, acc(6′)-Ib-cr
5
aac(3)-II,aac(6′)-Ib
2
7
KLP199
CN
NA
NA
aac(3)-II,aac(6′)-Ib
2
2
KLP201
CN, CIP, LEV
qnrB,qnrD,oqxA
3
ant(3′′)-I,aac(3)-II,aac(6′)-Ib,aph(3′)-VI,armA
5
8
KLP207
CN, AK, CIP, LEV
qnrB,qnrS, qebA,oqxA, acc(6′)-Ib-cr
5
ant(3′′)-I,aac(6′)-Ib, armA
3
8
KL:Klebsiella pneumoniae, CN: gentamicin, AK: amikacin, CIP: ciprofloxacin, LEV: levofloxacin, and NA: not applicable.Figure 3
Representative PCR products of detected genes encoding AMEs. Lanes:(1)ant (3′′)-I;(L) 100 bp ladder;(2)armA;(3)acc(6′)-Ib;(4)aph(3′)-VI;(5)acc(3-)-II.The second most common types of AME were phosphotransferases, followed by nucleotidyltransferases in whichaph(3′)-IV andant(3′)-I were detected among 50% (33/66) and 44% (29/66) of isolates, respectively. The lowest detected types of AMEs were ribosomal methylases in whicharmAwas detected among 14% (9/66) of tested isolates. ArmtB variant was not detected.


### 3.7. Detection of Genes Encoding Qnr Proteins

Screening of quinolone-resistant isolates for genes encoding Qnr proteins (Figure4) revealed thatqnrBwas most prevalent (74% (42/57) tested positive). Other detected genes wereqnrSandqnrD,which occurred at a prevalence of 49% (28/57) and 40% (23/57), respectively (Table 4). The gene encoding Qnr protein detected least often wasqnrC: only one isolate tested positive;qnrAwas not detected.Figure 4
Representative PCR products of detected quinolone resistance genes. Lanes:(1)qnrB;(2)qnrC;(3)qnrD;(4)qnrS;(L) 100 bp ladder;(5)acc(6′)-Ib-cr;(6)oqxA;(7)oqxb;(8)qebA.


### 3.8. Detection of Genes Encoding Quinolone Efflux Pumps

The current study revealed that genes encodingqebA, oqxA, andoqxB efflux pumps were detected at a prevalence of 12% (7/57), 88% (50/58), and 30% (17/57), respectively, among the quinolone-resistant isolates. Only 26% (15/57) of the isolates harbored bothoqxAandoqxB.


### 3.9. Detection of Gene Encoding Quinolone-Modifying Enzyme

Screening for a quinolone-modifying enzyme among the quinolone-resistant isolates revealed that 61% (35/57) of the tested isolates harboredacc(6′)-Ib-cr.


### 3.10. Correlation between MIC for Quinolones and Aminoglycosides and Genetic Determinants of Resistance

K. pneumoniaeisolates that showed elevated MIC (16 to 256 μg/ml) for ciprofloxacin and levofloxacin mainly harboredqnrB andacc(6′)-Ib-cr. Isolates that showed high MIC values (64 to ≥ 1024 μg/ml) for gentamicin and amikacin harboredaph(3′)-VIandant(3′′)-I.


## 3.1. Isolation and Identification

From 301 recovered isolates, 114 (37%) wereK. pneumoniae, 3 (1%) wereKlebsiella oxytoca, 61 (20%) wereE. coli, 48 (16%) wereProteusspp., 38 (13%) werePseudomonas aeruginosa, 12 (4%) wereAcinetobacter baumannii, 9 (3%) wereSerratia marcescens, 6 (2%) wereEnterobacter cloacae,and 3 (1%) were single isolates for each ofProvidencia stuartii,Burkholderia cepacia, andAeromonas hydrophilia.


## 3.2. Phenotypic and Genotypic Identification ofK. pneumoniae Isolates

Biotyping ofK. pneumoniae clinical isolates using API 20E revealed 13 different biotypes. The most prevalent were 5215773 and 5205773, which occurred at a prevalence of 56% (64/114) and 29% (34/114), respectively. Other detected biotypes were 5005573, 5215573, and 5205573, which occurred at a prevalence of 3.5% (4/114), 1.8% (2/114), and 1.8% (2/114), respectively. The lowest detected biotypes were 1205773, 1215773, 5004573, 5204773, 5204553, 5215763, 5217773, and 5214773, which each occurred at a prevalence of 0.88% (1/114).Genotypic confirmation of phenotypically identified isolates through amplification of theK. pneumoniae phoEgene revealed that these isolates were related toK. pneumoniae.ERIC-PCR-based DNA fingerprinting identified only 95.6% (109/114) of theK. pneumoniaeisolates, as 5 isolates gave no band following agarose gel electrophoresis. The 109 genotypedK. pneumoniae isolates displayed 85 different fingerprint patterns, as shown in Figure 1.Figure 1
Representative DNA fingerprint pattern ofK. pneumoniaeclinical isolates genotyped by ERIC-PCR.


## 3.3. Fingerprint Pattern Analysis

A UPGMA dendrogram generated according to Dice (similarity) coefficient revealed that the 85 fingerprint profiles were related to 67 different profiles, including 67 isolates with 18 different combined profiles that included 42 isolates. All genotypedK. pneumoniae were classified into 2 major phylogenetic groups (group A and group B), as shown in Figure 2. Phylogenetic group A included 4 isolates (K184, K109, K162, and K161). Two isolates (K161 and K162) within phylogenetic group A had the same fingerprint pattern. Phylogenetic group B contained the remaining 105 isolates.Figure 2
Generated UPGMA dendrogram based on Dice similarity coefficient for clustering ofK. pneumoniaeisolates using ERIC-PCR as DNA fingerprinting method. KL:Klebsiella pneumoniae, PGA: phylogenetic group A, and PGB: phylogenetic group B.


## 3.4. Correlation between Phenotyping by API 20E and Genotyping by ERIC-PCR

No relationship was found between the phenotypes detected by API 20E and the genotypes detected by ERIC-PCR, as identical clones, such as KL169 and KL174, showed different biotypes (5005773 and 5215773). Other identical genotypes also revealed different biotypes, as shown in Figure2.


## 3.5. Antimicrobial Susceptibility

Antimicrobial susceptibility tests revealed that carbapenems (imipenem and meropenem) were more effective than 3rd- and 4th-generation cephalosporins for which 75% (86/114) and 75% (85/114) of the isolates were susceptible to imipenem and meropenem, respectively, as shown in Table3. Regarding non-β-lactam antibiotics, tigecycline showed the lowest resistance, as 97% (111/114) of isolates were susceptible. As regarding quinolones and aminoglycosides, we found that 60% (68/114), 26% (30/114), 47% (54/114), and 43% (49/114) of isolates were resistant to gentamicin, amikacin, ciprofloxacin, and levofloxacin, respectively.Table 3
Antibiotic susceptibility ofK. pneumoniae clinical isolates.
Antibiotic
K. pneumoniae susceptibility pattern (n=114)
MIC50 and MIC90
Sensitive
Intermediate
Resistant
MIC50
MIC90
Number (%)
Number (%)
Number (%)
Cefotaxime
15(13)
1(1)
98(86)
256
>1024
Ceftazidime
26(23)
14(12)
74(65)
32
>1024
Cefoperazone
17(15)
3(2)
94(82)
512
>1024
Cefoperazone/sulbactam
51(45)
12(10)
51(45)
32
1024
Ceftriaxone
18(16)
—
96(84)
256
>1024
Cefepime
21(18)
24(21)
69(61)
32
256
Imipenem
86(75)
5(4)
23(20)
≤0.5
4
Meropenem
85(75)
2(2)
27(24)
≤0.5
16
Tetracycline
32(28)
4(3)
78(68)
128
512
Tigecycline
111(97)
—
3(3.0)
≤0.5
≤0.5
Gentamicin
46(40)
12(11)
56(49)
8
>1024
Amikacin
84(74)
1(1)
29(25)
≤0.5
>1024
Thiamphenicol
7(6.0)
2(2)
105(92)
1024
>1024
Ciprofloxacin
60(53)
3(2)
51(45)
1
128
Levofloxacin
65(57)
5(4)
44(39)
≤0.5
64Apart from imipenem and meropenem, the MIC50 and MIC90 values for other tested β-lactams ranged between 32 to 512 μg/ml and 256 to >1024 μg/ml, respectively. On the other hand, the MIC50 and MIC90 for gentamicin and amikacin ranged from ≤0.5 to 8 μg/ml and >1024 μg/ml, respectively, while MIC50 and MIC90 of ciprofloxacin and levofloxacin ranged from ≤0.5 to 1 μg/ml and 64 to 128 μg/ml, respectively.


## 3.6. Detection of Genes Encoding AMEs

Genotypic results for AMEs among the aminoglycoside-resistant isolates, as shown in Figure3, revealed that acetyltransferases were the most prevalent type of AME. Theacc(6′)-Ib andacc(3′)-II genes were detected among 88% (58/66) and 58% (38/66) of the investigated isolates (Table 4). In contrast, theacc(6′)-II variant was not detected.Table 4
Resistance pattern and genetic profile of quinolone and aminoglycoside resistantK. pneumoniaeisolates.
Isolate number
Quinolone and aminoglycoside resistance profile
Quinolone resistance genetic profile
Number of quinolone resistance genes
Aminoglycoside resistance genetic profile
Number of aminoglycoside resistance genes
Total number of detected genes
KLP2
CN, AK, CIP
qnrB,qnrD, oqxA, oqxB
4
ant(3′′)-I,aac(6′)-Ib
2
6
KLP3
CN, CP, LEV
qnrB,qnrD,oqxA
3
aac(3)-II,aac(6′)-Ib
2
5
KLP5
CN
NA
NA
aac(3)-II,aac(6′)-Ib
2
2
KLP6
CN
NA
NA
aac(3)-II,aac(6′)-Ib
2
5
KLP13
CN, AK, CIP, LEV
qnrB,oqxA, oqxB
3
—
—
3
KLP14
CN, AK, CIP, LEV
qnrB
1
aph(3′)-VI, ant(3′′)-I
2
3
KLP15
CN, AK, CIP
qnrB,oqxA, acc(6′)-Ib-cr
3
ant(3′′)-I,aac(3)-II,aac(6′)-Ib,aph(3′)-VI,armA
5
8
KLP19
CN, AK, CIP, LEV
qnrB,oqxA, oqxB
3
ant(3′′)-I,aac(6′)-Ib
2
5
KLP20
CN, AK, CIP, LEV
qnrB,oqxA
2
aac(3)-II,aac(6′)-Ib
2
4
KLP23
CN, AK, CIP, LEV
qnrB,qnrD, qnrS,oqxA
4
aac(3)-II,aac(6′)-Ib,aph(3′)-VI
3
7
KLP27
CN, CIP, LEV
qnrB,qnrD,oqxA
3
aac(3)-II,aac(6′)-Ib
2
5
KLP28
CN, CIP, LEV
qnrB,oqxA
2
aac(6′)-Ib, aph(3′)-VI,armA
3
5
KLP29
CN, AK, CIP
qnrB,oqxA
2
ant(3′′)-I, aac(3)-II,aac(6′)-Ib,aph(3′)-VI
4
6
KLP30
CN, AK, CIP, LEV
qnrB, qnrS,oqxA, oqxB, acc(6′)-Ib-cr
5
ant(3′′)-I,aac(3)-II,aac(6′)-Ib,aph(3′)-VI
4
9
KLP33
CN, CIP, LEV
qnrB, qnrS,oqxA, acc(6′)-Ib-cr
4
ant(3′′)-I,aac(3)-II,aac(6′)-Ib,aph(3′)-VI
4
8
KLP34
CN
oqxA
1
aac(3)-II,aac(6′)-Ib,aph(3′)-VI
3
4
KLP35
CN, CIP
acc(6′)-Ib-cr
1
aac(6′)-Ib, aac(6′)-Ib,aph(3′)-VI
3
4
KLP36
CN, AK, CIP, LEV
oqxA
1
aac(3)-II,aac(6′)-Ib
2
3
KLP42
CN
NA
NA
aac(3)-II,aac(6′)-Ib
2
2
KLP43
CN
NA
NA
aac(3)-II, aac(6′)-Ib, aph(3′)-VI
3
3
KLP50
CN, LEV
qnrS,oqxA, acc(6′)-Ib-cr
3
ant(3′′)-I,aac(3)-II,aac(6′)-Ib
3
6
KLP58A
CIP, LEV
qnrB, qnrS,oqxA, acc(6′)-Ib-cr
4
NA
NA
4
KLP58B
CN, CIP, LEV
qnrB, qnrS,oqxA, acc(6′)-Ib-cr
4
ant(3′′)-I,aac(3)-II,aac(6′)-Ib, aph(3′)-VI
4
8
KLP63
CN, AK, CIP, LEV
qnrB,oqxA, acc(6′)-Ib-cr
3
ant(3′′)-I,aac(3)-II,aac(6′)-Ib,aph(3′)-VI
4
7
KLP65
CN
NA
NA
aac(6′)-Ib
1
1
KLP69
CN, AK, CIP, LEV
qnrB,qnrD,oqxA, oqxB, acc(6′)-Ib-cr
5
—
—
5
KLP72
CN, AK, CIP, LEV
qnrB,qnrD, qnrS,oqxA, acc(6′)-Ib-cr
5
ant(3′′)-I,aac(6′)-Ib,aph(3′)-VI,armA
4
9
KLP73
CN
NA
NA
aac(6′)-Ib
1
1
KLP78
CN, CIP, LEV
qnrB,qnrD, oqxA
3
aac(3)-II,aac(6′)-Ib
2
5
KLP80
CN, AK, CIP, LEV
qnrD, qnrS,oqxA, acc(6′)-Ib-cr
4
ant(3′′)-I,aac(6′)-Ib,aph(3′)-VI,armA
4
8
KLP81
CN, CIP
qnrB,oqxA, acc(6′)-Ib-cr
3
aac(3)-II,aac(6′)-Ib,aph(3′)-VI
3
6
KLP88
CN
NA
NA
aac(3)-II, aac(6′)-Ib,aph(3′)-VI
3
3
KLP89
CN, AK
NA
NA
ant(3′′)-I,aac(3)-II,aac(6′)-Ib, aph(3′)-VI
4
4
KLP93
CN
NA
NA
ant(3′′)-I,aac(3)-II,aac(6′)-Ib,aph(3′)-VI
4
4
KLP96
CN, AK, CIP, LEV
qnrB,qnrD, qnrS,qebA,oqxA, oqxB, acc(6′)-Ib-cr
7
ant(3′′)-I,aac(3)-II,aac(6′)-Ib,aph(3′)-VI,armA
5
12
KLP99
CN, AK, CIP, LEV
qnrB, oqxA, oqxB, acc(6′)-Ib-cr
4
aac(6′)-Ib,aph(3′)-VI
2
6
KLP100
CN, AK, CIP, LEV
qnrB, oqxA, oqxB, acc(6′)-Ib-cr
4
aac(3)-II,aac(6′)-Ib
2
6
KLP101
CN, CIP, LEV
qnrB, oqxA, acc(6′)-Ib-cr
3
aac(6′)-Ib, aph(3′)-VI
2
5
KLP102
CIP, LEV
qnrD,oqxA
2
NA
NA
2
KLP103
CN, AK, CIP, LEV
qnrB, oqxA, oqxB
3
aac(6′)-Ib,aph(3′)-VI
2
5
KLP104
CN, CIP, LEV
qnrS,oqxA, acc(6′)-Ib-cr
3
ant(3′′)-I,aac(6′)-Ib
2
5
KLP107
CN
NA
NA
aac(6′)-Ib
1
1
KLP108
CN, LEV
qnrB,qnrS, acc(6′)-Ib-cr
3
aac(3)-II, aac(6′)-Ib
2
5
KLP110
CN, CIP, LEV
qnrB,qnrD, qnrS,oqxA, acc(6′)-Ib-cr
5
ant(3′′)-I,aac(3)-II,aac(6′)-Ib, aph(3′)-VI
4
9
KLP111
CN
NA
NA
ant(3′′)-I,aac(3)-II,aph(3′)-VI
3
3
KLP112
CN, CIP, LEV
qnrB,qnrD,qnrS,oqxA, acc(6′)-Ib-cr
5
ant(3′′)-I,aac(6′)-Ib,aph(3′)-VI
3
8
KLP115
CN, CIP, LEV
qnrB,qnrD,qnrS,oqxA, acc(6′)-Ib-cr
5
aac(6′)-Ib
1
6
KLP117
CIP, LEV
qnrB,qnrD,qnrS,oqxA, acc(6′)-Ib-cr
5
NA
NA
5
KLP119
CN
NA
NA
aac(3)-II
1
1
KLP121
CIP, LEV
qnrB,qnrD,qnrS,oqxA, oqxB
5
NA
NA
5
KLP125
CN, AK, CIP, LEV
qnrB,qebA,oqxA, oqxB, acc(6′)-Ib-cr
5
aac(6′)-Ib
1
6
KLP127
CN, AK, CIP, LEV
qnrB,qnrD,qebA,oqxA, oqxB, acc(6′)-Ib-cr
6
ant(3′′)-I,aac(6′)-Ib
2
8
KLP128
CN
NA
NA
—
—
—
KLP129
CN, AK, CIP, LEV
qnrB,qnrS, qebA,oqxA, acc(6′)-Ib-cr
5
ant(3′′)-I,aac(6′)-Ib,aph(3′)-VI,armA
4
9
KLP134
CN, AK, CIP, LEV
qnrB,qnrS,oqxA, oqxB, acc(6′)-Ib-cr
5
ant(3′′)-I,aac(3)-II,aac(6′)-Ib,aph(3′)-VI
4
9
KLP136
CIP, LEV
qnrS, oqxB
2
aac(6′)-Ib
1
3
KLP137
CIP, LEV
qnrS,oqxA, acc(6′)-Ib-cr
3
NA
NA
3
KLP140
CN
NA
NA
aac(3)-II
1
1
KLP141
CN
NA
NA
aac(3)-II
1
1
KLP144
CN, AK, CIP, LEV
acc(6′)-Ib-cr
1
ant(3′′)-I,aac(6′)-Ib,aph(3′)-VI
3
4
KLP147
CN, LEV
qnrB,qnrD,qnrS,oqxA, acc(6′)-Ib-cr
5
aac(3)-II,aac(6′)-Ib
2
7
KLP148
CN
NA
NA
—
—
—
KLP154
CIP
qnrB,qnrD,qnrS,oqxA
4
NA
NA
4
KLP157
CN
NA
NA
aac(3)-II, aac(6′)-Ib,aph(3′)-VI
3
3
KLP169
CN, AK, CIP, LEV
qnrB, oqxA, acc(6′)-Ib-cr
3
ant(3′′)-I,aac(3)-II,aac(6′)-Ib,aph(3′)-VI
4
7
KLP170
CN, AK, CIP, LEV
qnrB, oqxA,oqxB, acc(6′)-Ib-cr
4
ant(3′′)-I,aac(6′)-Ib,aph(3′)-VI
3
7
KLP177
CN, CIP, LEV
—
—
—
—
—
KLP180
CN, AK, CIP, LEV
qnrB,qnrD,qnrS,oqxA,oqxB, acc(6′)-Ib-cr
6
ant(3′′)-I,aac(6′)-Ib
2
8
KLP184
CIP, LEV
qnrS,oqxA,oqxB
3
NA
NA
3
KLP186
CN, CIP
qnrC,qnrD, qnrS,oqxA
4
aac(6′)-Ib
1
5
KLP187
CIP
qnrS,qnrD
2
NA
NA
2
KLP197
CN, AK, CIP, LEV
qnrS,qebA,oqxA, acc(6′)-Ib-cr
4
ant(3′′)-I,aac(6′)-Ib,aph(3′)-VI,armA
4
8
KLP198
CN, AK, CIP, LEV
qnrB,qnrD,qebA,oqxA, acc(6′)-Ib-cr
5
aac(3)-II,aac(6′)-Ib
2
7
KLP199
CN
NA
NA
aac(3)-II,aac(6′)-Ib
2
2
KLP201
CN, CIP, LEV
qnrB,qnrD,oqxA
3
ant(3′′)-I,aac(3)-II,aac(6′)-Ib,aph(3′)-VI,armA
5
8
KLP207
CN, AK, CIP, LEV
qnrB,qnrS, qebA,oqxA, acc(6′)-Ib-cr
5
ant(3′′)-I,aac(6′)-Ib, armA
3
8
KL:Klebsiella pneumoniae, CN: gentamicin, AK: amikacin, CIP: ciprofloxacin, LEV: levofloxacin, and NA: not applicable.Figure 3
Representative PCR products of detected genes encoding AMEs. Lanes:(1)ant (3′′)-I;(L) 100 bp ladder;(2)armA;(3)acc(6′)-Ib;(4)aph(3′)-VI;(5)acc(3-)-II.The second most common types of AME were phosphotransferases, followed by nucleotidyltransferases in whichaph(3′)-IV andant(3′)-I were detected among 50% (33/66) and 44% (29/66) of isolates, respectively. The lowest detected types of AMEs were ribosomal methylases in whicharmAwas detected among 14% (9/66) of tested isolates. ArmtB variant was not detected.


## 3.7. Detection of Genes Encoding Qnr Proteins

Screening of quinolone-resistant isolates for genes encoding Qnr proteins (Figure4) revealed thatqnrBwas most prevalent (74% (42/57) tested positive). Other detected genes wereqnrSandqnrD,which occurred at a prevalence of 49% (28/57) and 40% (23/57), respectively (Table 4). The gene encoding Qnr protein detected least often wasqnrC: only one isolate tested positive;qnrAwas not detected.Figure 4
Representative PCR products of detected quinolone resistance genes. Lanes:(1)qnrB;(2)qnrC;(3)qnrD;(4)qnrS;(L) 100 bp ladder;(5)acc(6′)-Ib-cr;(6)oqxA;(7)oqxb;(8)qebA.


## 3.8. Detection of Genes Encoding Quinolone Efflux Pumps

The current study revealed that genes encodingqebA, oqxA, andoqxB efflux pumps were detected at a prevalence of 12% (7/57), 88% (50/58), and 30% (17/57), respectively, among the quinolone-resistant isolates. Only 26% (15/57) of the isolates harbored bothoqxAandoqxB.


## 3.9. Detection of Gene Encoding Quinolone-Modifying Enzyme

Screening for a quinolone-modifying enzyme among the quinolone-resistant isolates revealed that 61% (35/57) of the tested isolates harboredacc(6′)-Ib-cr.


## 3.10. Correlation between MIC for Quinolones and Aminoglycosides and Genetic Determinants of Resistance

K. pneumoniaeisolates that showed elevated MIC (16 to 256 μg/ml) for ciprofloxacin and levofloxacin mainly harboredqnrB andacc(6′)-Ib-cr. Isolates that showed high MIC values (64 to ≥ 1024 μg/ml) for gentamicin and amikacin harboredaph(3′)-VIandant(3′′)-I.


## 4. Discussion

Aminoglycoside-modifying enzymes are the most important determinants of aminoglycoside resistance amongK. pneumoniae isolates [28]. The current study revealed that 88% (58/66) and 58% (38/66) of aminoglycoside-resistantK. pneumoniae isolates tested positive foracc(6′)-Ib andacc(3′)-I,I, respectively. Similar high prevalence rates foracc(6′)-Ib andacc(3′)-II amongK. pneumoniae isolates were reported by Lotfollahi et al. (74% (63/85) and 73% (62/85) foracc(6′)Ibandacc(3′)-II, resp., [29]). But lower rates have also been reported (20% (32/162) and 30% (49/162) foracc(6′)-Ib andacc (3′)-II, resp., [30]). The present study also found that 44% (29/66) of aminoglycoside-resistantK. pneumoniae isolates tested positive forant(3′′)-1, which is three times that seen previously (14% (22/162) [30]). Foraph(3′)-IV we found that 50% (33/66) of the aminoglycoside-resistantK. pneumoniae isolates harbored this gene, which was similar to the findings of Almaghrabi et al. and Gad et al. (56% (28/50) and 50% (4/8), resp., [28, 31]).The Qnr proteins are considered as one of the three reported mechanisms of PMQR. Theqnr genes encode proteins that protect DNA gyrase and topoisomerase IV from inhibition by quinolones and have recently been found worldwide [32]. The current study examined the prevalence of Qnr proteins amongK. pneumoniae isolates that showed full or intermediate resistance to quinolones. TheqnrA gene was not detected, which was consistent with Yang et al. [32] but differed from a Portuguese study (19% (4/21) of MDRK. pneumoniae isolates [33] and another Egyptian report (12% (14/121) of ESBL-producingK. pneumoniae [34]). RegardingqnrB, we found that 74% (42/57) of the isolates tested positive, which was slightly more than the 50% (11/22) seen in a Korean study [32]. In contrast, a relatively low prevalence rate was reported by Tunisian study (13% (21/165) [35]). We found thatqnrCwas represented by only a single isolate, which was consistent with findings from the recent Turkish and Tunisian studies that failed to detectqnrCamong quinolone-resistantK. pneumoniae isolates [35, 36]. The current study detectedqnrD at a prevalence of 40% (23/57); previous work failed to detect this gene [35, 37]. TheqnrS gene was seen in 49% (28/57) of the investigatedK. pneumoniae isolates. Lower incidences (9% (2/22) and 2% (3/165)) had been reported in Korean [37] and Tunisian [35] studies, respectively. A much higher incidence (64%, 28/44) was reported in China [38]. We conclude that plasmids carryingqnr genes were highly spread in Egypt and China, probably due to misuse of quinolones in clinical settings.The current work is the first Egyptian study to investigate the QepA and OqxAB efflux pumps amongK. pneumoniae isolates. We found that 12% (7/57) tested positive forqepA, which is far higher than the 2% (5/247) reported previously [39] or the absence ofqebA amongK. pneumoniae isolates [40]. The prevalence ofoqxAandoqxB was higher, 88% (50/57) and 30% (17/57), respectively. Previously Rodríguez-Martínez et al. reported values of 76% (87/114) and 75% (86/114), respectively [41]. Only 26% (15/57) of quinolone-resistantK. pneumoniae isolates were positive for bothoqxAandoqxB, double that reported earlier (11% (11/102) [32]). Interestingly, Yuan et al. reported that 100% (154/154) of theirK. pneumoniae isolates tested positive for bothoqxA andoqxB, suggesting that in that case the genes encoding the OqxAB protein were located on the chromosome ofK. pneumoniae, perhaps as a reservoir for these genes [42]. Thus, high resistance rates to quinolones may be expected amongK. pneumoniaeisolates recovered from clinical settings that frequently prescribe quinolones, since the chromosomal genes coding for OqxAB efflux pump proteins will be overexpressed.Enzymatic modification of quinolones by the AAC(6′)-Ib-cr enzyme is a third reported mechanism underlying PMQR [32]. The current study revealed that 61% (35/57) of quinolone-resistantK. pneumoniae isolates tested positive foraac(6′)-Ib-cr, which is several times higher than seen by Jlili et al. and by Kim et al. (19% (8/42) and 13% (21/165), resp., [35, 43]).Genotypic identification ofK. pneumoniae isolates via amplification ofphoE identified 81% (92/114) of theK. pneumoniae isolates; this finding contradicted Sun et al., who reported 100% [21].This difference may be due to a mutation in thephoEgene of our isolates. Genotyping ofK. pneumoniae isolates using the ERIC-PCR technique revealed that the majority of isolates had different origins; 32 isolates were related to 18 different single origins, indicating that the spread ofK. pneumoniae among different hospital departments was due to poor infection control.


## 5. Conclusion

The current study demonstrated that ACCs were the most prevalent AMEs, followed by APHs and then ANTs. Screening ofqnr genes revealed thatqnrB was the most prevalent, followed byqnrS. This is the first Egyptian study to detectqnrCandacc(6′)-Ib-cr among quinolone-resistantK. pneumoniaeisolates. Genotypic identification ofK. pneumoniae through amplification of thephoE gene was not 100%. MostK. pneumoniaeisolates included in this study displayed different genetic and phenotypic profiles, indicating different origins of dissemination.



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*Source: 8050432-2017-05-30.xml*