# In Vitro Susceptibility ofMycobacterium abscessus andMycobacterium fortuitum Isolates to 30 Antibiotics

Authors: Yaojie Shen; Xuyang Wang; Jialin Jin; Jing Wu; Xuelian Zhang; Jiazhen Chen; Wenhong Zhang
Journal: BioMed Research International
 (2018)

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## Abstract

Objective. Nontuberculous mycobacteria (NTM) cause various diseases in humans and animals. Recently, the prevalence of NTM-related disease has been on the rise, becoming an emerging public health problem. The aim of this study was to determine the antibiotic susceptibility profiles of clinical isolates ofMycobacterium abscessus and Mycobacterium fortuitum. Methods. We performed susceptibility tests on 37 clinical NTM isolates to 30 antibiotics with the microdilution method recommended by the Clinical and Laboratory Standards Institute.Results. BothM. abscessus andM. fortuitum were highly resistant to antitubercular drugs such as isoniazid, rifampin, ethambutol, clofazimine, ethionamide, and rifabutin.M. abscessus showed the lowest resistant rates to cefoxitin (10%), azithromycin (10%), amikacin (10%), and clarithromycin (20%) and very high resistant to sulfamethoxazole, vancomycin, oxacillin, clindamycin, and all fluoroquinolones.M. fortuitum showed low resistance to tigecycline (0%), tetracycline (0%), cefmetazole (12%), imipenem (12%), linezolid (18%), and the aminoglycosides amikacin (0%), tobramycin (0%), neomycin (0%), and gentamycin (24%).Conclusion.Amikacin, cefoxitin, and azithromycin have the highestin vitro activity againstM. abscessus. Isolates ofM. fortuitum need to be individually evaluated for drug susceptibility before choosing an effective antimicrobial regimen for treatment of infections.


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## Body

## 1. Introduction

Nontuberculous mycobacteria (NTM) are widely distributed in nature [1] and are opportunistic pathogens that can cause various diseases in multiple organs in humans and animals. Recently, the prevalence of NTM diseases has been on the rise [2, 3], and they are now recognized as representing an emerging public health problem [4].Mycobacterium abscessus andMycobacterium fortuitum are the most important RGMs (rapidly growing mycobacteria), with the former accounting for 80% of chronic pulmonary diseases caused by all RGM [5] and the latter being the main RGM responsible for extra-pulmonary disease, especially in cutaneous and plastic surgery-related infections [6].M. abscessusis an opportunistic pathogen, which can cause human to human infection [7] and nosocomial infection [8]. It was reported to cause multiple community outbreaks of cutaneous infection by means of wading pool or swimming pool [9–11]. In addition,M. abscessus is one of the most severe drug resistant bacteria among the RGM [6, 12] and is therefore very difficult to treat [5, 13]. In order to achieve the goal of 12-month sputum conversion on medication, it is essential to guide treatment regimens based on drug susceptibility results [14].M. fortuitum can often cause soft tissue infection during trauma and surgery. It had also been reported in many implant-associated infections [15] and in endocarditis infections [15, 16]. It was reported susceptible to multiple drugs except for macrolides [17]. However, the antibiotics resistance spectrum varies with different geographic locations or different hospital administration situation.In this study, we investigated the drug susceptibility status of 30 commonly used antibiotics among 37 clinical isolates ofM. abscessus andM. fortuitum. In addition, we compared the drug susceptibility results with those of other studies worldwide to provide a clearer picture of the current antibiotic resistance levels for these two common RGM species, which could serve as valuable reference data to guide treatment.


## 2. Methods

### 2.1. Strains and Antibiotics

In total, 20M. abscessus and 17M. fortuitum isolates were collected from various clinical specimens, including 23 sputum isolates, 5 bronchoalveolar lavage isolates, 2 puncture fluid isolates, 2 tissue isolates, 2 urine isolates, 1 wound isolate, 1 cerebrospinal fluid isolate, and 1 exudate isolate, from patients at the Huashan Hospital affiliated to Fudan University between January 2009 and December 2013. All isolates were from unique patients, except that one isolate ofM. abcessus andM. fortuitum were cultured from two specimens of the same patient. All 37 isolates were recovered in Mueller-Hinton Broth (Oxoid, Hampshire, UK).M. abscessus ATCC19977 andM. fortuitum ATCC6841 were used as the reference strains.Mycobacterium peregrinum ATCC700686 was used as the quality control strain in drug susceptibility tests.Rifampin, ethambutol, streptomycin, kanamycin, amikacin, ethionamide, clarithromycin, doxycycline, imipenem, linezolid, tobramycin, clindamycin, sulfamethoxazole, clofazimine, minocycline, neomycin, tetracycline, gentamycin, and vancomycin were purchased from Sigma-Aldrich (St. Louis, MO, USA). Isoniazid, levofloxacin, moxifloxacin, rifabutin, cefoxitin, ciprofloxacin, and oxacillin were purchased from Fluka/Sigma-Aldrich (St. Louis, MO, USA). Azithromycin, tigecycline, teicoplanin, and cefmetazole were purchased from Aladdin (Shanghai, China), Calbiochem/Sigma-Aldrich (St. Louis, MO, USA), Bio Vision (CA, USA), and Meilunbio (Dalian, China), respectively.


### 2.2. Subspecies Identification among M. abscessus Complex Isolates

Isolates were thawed and recovered on Lowenstein-Jensen medium at 37°C or on BACTER MGIT 960 medium for 4–7 days. DNA was extracted from cultured colonies using DNeasy Blood & Tissue Kit (Qiagen, Hilden, Germany) in accordance with the manufacturer’s protocol and used as templates for polymerase chain reaction (PCR). Therrs gene [38] was amplified with primersrrs-F (5′-AGTTTGATCCTGGCTCAG) andrrs-R (5′-GGTTACCTTGTTACGACTT) andhsp65[18, 19, 39, 40] was amplified with primershsp-F (5′-CGATGCGGTAAAGGTGACATTG) andhsp-R (5′-CCTTGACAGTGGACACCTTGGA). PCR was carried out in a final volume of 50 μl with 1 μl of DNA supernatant containing approximately 10 ng of genomic DNA, 5 μl of 10× ExTaq PCR Buffer, 4 μl of dNTPs (2.5 mM each), 0.4 μM of each primer, 0.5 μl of ExTaq DNA Polymerase (5 U/μl) (Takara, Japan), and 37.5 μl of distilled water. DNA samples were first denatured completely by incubation at 95°C for 5 min and then amplified using 35 cycles of (i) denaturation at 95°C for 40 s, (ii) primer annealing at 58°C for 40 s, and (iii) elongation at 72°C for 1 min in a thermocycler. The PCR products were sequenced by the Sanger method. Consensus sequences for each isolate were assembled using Lasergene SeqMan II software (DNAStar, Inc., Madison, WI, USA).


### 2.3. Drug Susceptibility Testing

The susceptibility tests of all 37 isolates and the referenceM. peregrinum ATCC700686 against 30 antibiotics were carried out by the broth microdilution method in 96-well plates (Nunc, Denmark) according to the Clinical and Laboratory Standards Institute (CLSI) guidelines M24- A2. [41]. The final drug concentrations tested are shown in Table 1. The minimum inhibitory concentrations (MICs) of all antibiotics except for clarithromycin were determined after 3 days of incubation at 37°C. For clarithromycin, the incubation process lasted for 14 days. The MIC was determined as the lowest concentration of the drug that resulted in no visible bacterial growth. MIC90 values were defined as drug concentrations that inhibited 90% of the isolates. The susceptibility was determined based on CLSI breakpoint recommendations and published studies (Table 2). The resistance rates comparison betweenM. abscessussubsp.abscessus andM. abscessus subsp.massiliense was statistically analyzed by Fisher’s exact chi-square test. A p-value of < 0.05 indicated statistical significance.Table 1
The drug susceptibility results of 37 RGM isolates.
Antimicrobial Agent MIC(μg/ml) M. abscessus (n=20) MIC(μg/ml) M. fortuitum (n=17) range MIC 90 R I S range MIC 90 R I S Macrolides CLR 0.0625-64 32 4 (20%)/7(35%) 0 (0%)/0 (0%) 16 (80%)/13(65%) 32-64 32 16 (94%)/17(100%) 0 (0%)/0 (0%) 1 (6%)/0(0%) AZM 0.125-128 2 2 (10%) 0 (0%) 18 (90%) 8-128 32 17 (100%) 0 (0%) 0 (0%) Rifamycins RFB 0.5-8 4 15 (75%) - 5 (25%) 1-8 4 16 (94%) - 1 (6%) RIF 64-256 128 20 (100%) - 0 (0%) 32-256 128 17 (100%) - 0 (0%) Aminoglycosides STR 32-128 64 20 (100%) - 0 (0%) 16-128 32 17 (100%) - 0 (0%) GEN 0.5-64 32 14 (70%) 1 (5%) 5 (25%) 4-64 32 4 (24%) 9 (53%) 4 (24%) KAN 4-16 8 16 (80%) - 4 (20%) 4-32 16 16 (94%) - 1 (6%) TOB 2-32 8 12 (60%) 3 (15%) 5 (25%) 16-64 16 0 (0%) 0 (0%) 17 (100%) NEO 0.5-64 16 13 (65%) - 7 (35%) 2-8 2 0 (0%) - 17 (100%) AMK 0.5-64 32 2 (10%) 9 (45%) 9 (45%) 2-8 4 0 (0%) 0 (0%) 17 (100%) Fluoroquinolones MXF 0.0625-16 8 19 (95%) 0 (0%) 1 (5%) 0.0625-8 2 10 (59%) 5 (29%) 2 (12%) CIP 0.125-128 64 19 (95%) 0 (0%) 1 (5%) 0.125-256 128 8 (47%) 0 (0%) 9 (53%) LVX 0.125-32 16 19 (95%) 0 (0%) 1 (5%) 0.125-32 16 7 (41%) 0 (0%) 10 (59%) Cephalosporins FOX 16-256 64 2 (10%) 10 (50%) 8 (40%) 16-128 32 15 (88%) 0 (0%) 2 (12%) CMZ 2-256 128 11 (55%) 5 (25%) 4 (20%) 2-64 8 2 (12%) 0 (0%) 15 (88%) Tetracyclines TCY 2-256 64 9 (45%) 3 (15%) 8 (40%) 1-8 4 0 (0%) 12 (70%) 5 (30%) DOX 0.5-256 128 14 (70%) 2 (10%) 4 (20%) 0.5-256 128 15 (88%) 0 (0%) 2 (12%) MNO 0.125-128 64 11 (55%) 3 (15%) 6 (30%) 0.25-32 16 14 (82%) 1 (6%) 2 (12%) Glycylcycline TGC 0.0625-16 8 8 (40%) 6 (30%) 6 (30%) 0.0625-4 0.5 0 (0%) 1 (6%) 16 (94%) Sulfonamides SOX 2-256 128 18 (90%) - 2 (10%) 16-256 128 14 (82%) - 3 (18%) Carbapenems IMP 1-256 64 13 (65%) 2 (10%) 5 (25%) 2-32 16 2 (12%) 12 (70%) 3 (18%) Oxazolidinones LNZ 2-128 16 3 (15%) 1 (5%) 16 (80%) 8-32 16 3 (18%) 12 (70%) 2 (12%) Lincosamides CLI 8-256 128 20 (100%) 0 (0%) 0 (0%) 256 256 17 (100%) 0 (0%) 0 (0%) Penicillins OXA 256 256 20 (100%) - 0 (0%) 256 256 17 (100%) - 0 (0%) Polypeptides TEC 0.5-256 128 17 (85%) 1 (5%) 2 (10%) 128-256 128 17 (100%) 0 (0%) 0 (0%) VAN 256 256 20 (100%) 0 (0%) 0 (0%) 256 256 17 (100%) 0 (0%) 0 (0%) Others CFZ 0.25-128 32 19 (95%) - 1 (5%) 0.125-128 0.25 10 (59%) - 7 (41%) EMB 256 256 20 (100%) - 0 (0%) 256 256 17 (100%) - 0 (0%) INH 8-256 16 20 (100%) - 0 (0%) 8-16 8 17 (100%) - 0 (0%) ETH 16-256 64 20 (100%) - 0 (0%) 4-256 32 16 (94%) - 1 (6%) (a) CLR= clarithromycin, AZM= azithromycin, RFB= rifabutin, RIF= rifampin, STR= streptomycin, GEN= gentamycin, KAN= kanamycin, TOB= tobramycin, NEO= neomycin, AMK= amikacin, MXF= moxifloxacin, CIP= ciprofloxacin, LVX= levofloxacin, FOX= cefoxitin, CMZ= cefmetazole, TCY= tetracycline, DOX= doxycycline, MNO= minocycline, TGC= tigecycline, SOX= sulfamethoxazole, IMP= imipenem, LNZ= linezolid, CLI= clindamycin, OXA= oxacillin, TEC= teicoplanin, VAN= vancomycin, CFZ= clofazimine, EMB= ethambutol, INH= isoniazid, and ETH= ethionamide. (b) “-“ indicates data not available. (c) For CLR, each resistance and susceptible rate has two data. The first is the MIC result for 3 days and the second is the MIC result for 14 days.Table 2
Breakpoints of 30 antibiotics.
Antimicrobial Agent MIC breakpoints (μg/ml) Susceptibility Intermediate Resistance Macrolides CLRa ⩽2 4 ⩾8 AZMc ⩽2 4 ⩾8 Rifamycins RIFa - - >1 RFBa - - >2 Aminoglycosides STRa - - ⩾5 GENb <=4 8 >=16 KANa - - ⩾4 TOBa ⩽2 4 ⩾8 NEOb - - >=10 AMKa ⩽16 32 ⩾64 Fluoroquinolones MXFa ⩽1 2 ⩾4 CIPa ⩽1 2 ⩾4 LVXa ⩽2 4 ⩾8 Cephalosporin FOXa ⩽16 32-64 ⩾128 CMZd ⩽16 32 ⩾64 Tetracyclines TCYb <=4 8 >=16 DOXa ⩽1 2-4 ⩾8 MNOa ⩽1 2-4 ⩾8 Glycylcycline TGCa ⩽1 2-4 ⩾8 Sulfonamides SOXa ⩽38 - ⩾76 Carbapenems IMPa ⩽4 8-16 ⩾32 Oxazolidinones LNZa ⩽8 16 ⩾32 Lincosamides CLIb <=0.5 1-2 >=4 Penicillins OXAb <=2 - >=4 Polypeptides TECb <=8 16 >=32 VANb <=2 4-8 >=16 Others CFZe - - >1 INHa - - ⩾1 EMBa - - ⩾4 ETHa - - >5 a denotes the breakpoints coming fromSusceptibility Testing of Mycobacteria, Nocardia, and Other Aerobic Actinomycetes; Approved Standard–Second Edition. CLSI document M24-A2. b denotes the breakpoints coming from Performance Standards Antimicrobial Susceptibility Testing-27th Edition.CLSI document M100. c, d, e denote the breakpoints coming from [18–20], respectively. d, S, susceptible, I, intermediate susceptible, and R, resistant.


## 2.1. Strains and Antibiotics

In total, 20M. abscessus and 17M. fortuitum isolates were collected from various clinical specimens, including 23 sputum isolates, 5 bronchoalveolar lavage isolates, 2 puncture fluid isolates, 2 tissue isolates, 2 urine isolates, 1 wound isolate, 1 cerebrospinal fluid isolate, and 1 exudate isolate, from patients at the Huashan Hospital affiliated to Fudan University between January 2009 and December 2013. All isolates were from unique patients, except that one isolate ofM. abcessus andM. fortuitum were cultured from two specimens of the same patient. All 37 isolates were recovered in Mueller-Hinton Broth (Oxoid, Hampshire, UK).M. abscessus ATCC19977 andM. fortuitum ATCC6841 were used as the reference strains.Mycobacterium peregrinum ATCC700686 was used as the quality control strain in drug susceptibility tests.Rifampin, ethambutol, streptomycin, kanamycin, amikacin, ethionamide, clarithromycin, doxycycline, imipenem, linezolid, tobramycin, clindamycin, sulfamethoxazole, clofazimine, minocycline, neomycin, tetracycline, gentamycin, and vancomycin were purchased from Sigma-Aldrich (St. Louis, MO, USA). Isoniazid, levofloxacin, moxifloxacin, rifabutin, cefoxitin, ciprofloxacin, and oxacillin were purchased from Fluka/Sigma-Aldrich (St. Louis, MO, USA). Azithromycin, tigecycline, teicoplanin, and cefmetazole were purchased from Aladdin (Shanghai, China), Calbiochem/Sigma-Aldrich (St. Louis, MO, USA), Bio Vision (CA, USA), and Meilunbio (Dalian, China), respectively.


## 2.2. Subspecies Identification among M. abscessus Complex Isolates

Isolates were thawed and recovered on Lowenstein-Jensen medium at 37°C or on BACTER MGIT 960 medium for 4–7 days. DNA was extracted from cultured colonies using DNeasy Blood & Tissue Kit (Qiagen, Hilden, Germany) in accordance with the manufacturer’s protocol and used as templates for polymerase chain reaction (PCR). Therrs gene [38] was amplified with primersrrs-F (5′-AGTTTGATCCTGGCTCAG) andrrs-R (5′-GGTTACCTTGTTACGACTT) andhsp65[18, 19, 39, 40] was amplified with primershsp-F (5′-CGATGCGGTAAAGGTGACATTG) andhsp-R (5′-CCTTGACAGTGGACACCTTGGA). PCR was carried out in a final volume of 50 μl with 1 μl of DNA supernatant containing approximately 10 ng of genomic DNA, 5 μl of 10× ExTaq PCR Buffer, 4 μl of dNTPs (2.5 mM each), 0.4 μM of each primer, 0.5 μl of ExTaq DNA Polymerase (5 U/μl) (Takara, Japan), and 37.5 μl of distilled water. DNA samples were first denatured completely by incubation at 95°C for 5 min and then amplified using 35 cycles of (i) denaturation at 95°C for 40 s, (ii) primer annealing at 58°C for 40 s, and (iii) elongation at 72°C for 1 min in a thermocycler. The PCR products were sequenced by the Sanger method. Consensus sequences for each isolate were assembled using Lasergene SeqMan II software (DNAStar, Inc., Madison, WI, USA).


## 2.3. Drug Susceptibility Testing

The susceptibility tests of all 37 isolates and the referenceM. peregrinum ATCC700686 against 30 antibiotics were carried out by the broth microdilution method in 96-well plates (Nunc, Denmark) according to the Clinical and Laboratory Standards Institute (CLSI) guidelines M24- A2. [41]. The final drug concentrations tested are shown in Table 1. The minimum inhibitory concentrations (MICs) of all antibiotics except for clarithromycin were determined after 3 days of incubation at 37°C. For clarithromycin, the incubation process lasted for 14 days. The MIC was determined as the lowest concentration of the drug that resulted in no visible bacterial growth. MIC90 values were defined as drug concentrations that inhibited 90% of the isolates. The susceptibility was determined based on CLSI breakpoint recommendations and published studies (Table 2). The resistance rates comparison betweenM. abscessussubsp.abscessus andM. abscessus subsp.massiliense was statistically analyzed by Fisher’s exact chi-square test. A p-value of < 0.05 indicated statistical significance.Table 1
The drug susceptibility results of 37 RGM isolates.
Antimicrobial Agent MIC(μg/ml) M. abscessus (n=20) MIC(μg/ml) M. fortuitum (n=17) range MIC 90 R I S range MIC 90 R I S Macrolides CLR 0.0625-64 32 4 (20%)/7(35%) 0 (0%)/0 (0%) 16 (80%)/13(65%) 32-64 32 16 (94%)/17(100%) 0 (0%)/0 (0%) 1 (6%)/0(0%) AZM 0.125-128 2 2 (10%) 0 (0%) 18 (90%) 8-128 32 17 (100%) 0 (0%) 0 (0%) Rifamycins RFB 0.5-8 4 15 (75%) - 5 (25%) 1-8 4 16 (94%) - 1 (6%) RIF 64-256 128 20 (100%) - 0 (0%) 32-256 128 17 (100%) - 0 (0%) Aminoglycosides STR 32-128 64 20 (100%) - 0 (0%) 16-128 32 17 (100%) - 0 (0%) GEN 0.5-64 32 14 (70%) 1 (5%) 5 (25%) 4-64 32 4 (24%) 9 (53%) 4 (24%) KAN 4-16 8 16 (80%) - 4 (20%) 4-32 16 16 (94%) - 1 (6%) TOB 2-32 8 12 (60%) 3 (15%) 5 (25%) 16-64 16 0 (0%) 0 (0%) 17 (100%) NEO 0.5-64 16 13 (65%) - 7 (35%) 2-8 2 0 (0%) - 17 (100%) AMK 0.5-64 32 2 (10%) 9 (45%) 9 (45%) 2-8 4 0 (0%) 0 (0%) 17 (100%) Fluoroquinolones MXF 0.0625-16 8 19 (95%) 0 (0%) 1 (5%) 0.0625-8 2 10 (59%) 5 (29%) 2 (12%) CIP 0.125-128 64 19 (95%) 0 (0%) 1 (5%) 0.125-256 128 8 (47%) 0 (0%) 9 (53%) LVX 0.125-32 16 19 (95%) 0 (0%) 1 (5%) 0.125-32 16 7 (41%) 0 (0%) 10 (59%) Cephalosporins FOX 16-256 64 2 (10%) 10 (50%) 8 (40%) 16-128 32 15 (88%) 0 (0%) 2 (12%) CMZ 2-256 128 11 (55%) 5 (25%) 4 (20%) 2-64 8 2 (12%) 0 (0%) 15 (88%) Tetracyclines TCY 2-256 64 9 (45%) 3 (15%) 8 (40%) 1-8 4 0 (0%) 12 (70%) 5 (30%) DOX 0.5-256 128 14 (70%) 2 (10%) 4 (20%) 0.5-256 128 15 (88%) 0 (0%) 2 (12%) MNO 0.125-128 64 11 (55%) 3 (15%) 6 (30%) 0.25-32 16 14 (82%) 1 (6%) 2 (12%) Glycylcycline TGC 0.0625-16 8 8 (40%) 6 (30%) 6 (30%) 0.0625-4 0.5 0 (0%) 1 (6%) 16 (94%) Sulfonamides SOX 2-256 128 18 (90%) - 2 (10%) 16-256 128 14 (82%) - 3 (18%) Carbapenems IMP 1-256 64 13 (65%) 2 (10%) 5 (25%) 2-32 16 2 (12%) 12 (70%) 3 (18%) Oxazolidinones LNZ 2-128 16 3 (15%) 1 (5%) 16 (80%) 8-32 16 3 (18%) 12 (70%) 2 (12%) Lincosamides CLI 8-256 128 20 (100%) 0 (0%) 0 (0%) 256 256 17 (100%) 0 (0%) 0 (0%) Penicillins OXA 256 256 20 (100%) - 0 (0%) 256 256 17 (100%) - 0 (0%) Polypeptides TEC 0.5-256 128 17 (85%) 1 (5%) 2 (10%) 128-256 128 17 (100%) 0 (0%) 0 (0%) VAN 256 256 20 (100%) 0 (0%) 0 (0%) 256 256 17 (100%) 0 (0%) 0 (0%) Others CFZ 0.25-128 32 19 (95%) - 1 (5%) 0.125-128 0.25 10 (59%) - 7 (41%) EMB 256 256 20 (100%) - 0 (0%) 256 256 17 (100%) - 0 (0%) INH 8-256 16 20 (100%) - 0 (0%) 8-16 8 17 (100%) - 0 (0%) ETH 16-256 64 20 (100%) - 0 (0%) 4-256 32 16 (94%) - 1 (6%) (a) CLR= clarithromycin, AZM= azithromycin, RFB= rifabutin, RIF= rifampin, STR= streptomycin, GEN= gentamycin, KAN= kanamycin, TOB= tobramycin, NEO= neomycin, AMK= amikacin, MXF= moxifloxacin, CIP= ciprofloxacin, LVX= levofloxacin, FOX= cefoxitin, CMZ= cefmetazole, TCY= tetracycline, DOX= doxycycline, MNO= minocycline, TGC= tigecycline, SOX= sulfamethoxazole, IMP= imipenem, LNZ= linezolid, CLI= clindamycin, OXA= oxacillin, TEC= teicoplanin, VAN= vancomycin, CFZ= clofazimine, EMB= ethambutol, INH= isoniazid, and ETH= ethionamide. (b) “-“ indicates data not available. (c) For CLR, each resistance and susceptible rate has two data. The first is the MIC result for 3 days and the second is the MIC result for 14 days.Table 2
Breakpoints of 30 antibiotics.
Antimicrobial Agent MIC breakpoints (μg/ml) Susceptibility Intermediate Resistance Macrolides CLRa ⩽2 4 ⩾8 AZMc ⩽2 4 ⩾8 Rifamycins RIFa - - >1 RFBa - - >2 Aminoglycosides STRa - - ⩾5 GENb <=4 8 >=16 KANa - - ⩾4 TOBa ⩽2 4 ⩾8 NEOb - - >=10 AMKa ⩽16 32 ⩾64 Fluoroquinolones MXFa ⩽1 2 ⩾4 CIPa ⩽1 2 ⩾4 LVXa ⩽2 4 ⩾8 Cephalosporin FOXa ⩽16 32-64 ⩾128 CMZd ⩽16 32 ⩾64 Tetracyclines TCYb <=4 8 >=16 DOXa ⩽1 2-4 ⩾8 MNOa ⩽1 2-4 ⩾8 Glycylcycline TGCa ⩽1 2-4 ⩾8 Sulfonamides SOXa ⩽38 - ⩾76 Carbapenems IMPa ⩽4 8-16 ⩾32 Oxazolidinones LNZa ⩽8 16 ⩾32 Lincosamides CLIb <=0.5 1-2 >=4 Penicillins OXAb <=2 - >=4 Polypeptides TECb <=8 16 >=32 VANb <=2 4-8 >=16 Others CFZe - - >1 INHa - - ⩾1 EMBa - - ⩾4 ETHa - - >5 a denotes the breakpoints coming fromSusceptibility Testing of Mycobacteria, Nocardia, and Other Aerobic Actinomycetes; Approved Standard–Second Edition. CLSI document M24-A2. b denotes the breakpoints coming from Performance Standards Antimicrobial Susceptibility Testing-27th Edition.CLSI document M100. c, d, e denote the breakpoints coming from [18–20], respectively. d, S, susceptible, I, intermediate susceptible, and R, resistant.


## 3. Results

### 3.1. Species Identification

There were 17 isolates identified asM. fortuitum and 20 isolates identified asM. abscessus. Among 20M. abscessusisolates, 12 isolates were classified asM. abscessussubsp.abscessus, 7 isolates were classified asM. abscessus subsp.massiliense, and one isolate was classified asM. abscessus subsp.bolletii based on theirhsp65 gene.


### 3.2. Resistance to Antitubercular Drugs

BothM. abscessus andM. fortuitum were highly resistant to antitubercular drugs such as isoniazid, rifampin, ethambutol, clofazimine, ethionamide, and rifabutin. Specifically, all isolates (100%) were resistant to isoniazid, ethambutol, and rifampin (Table 1). All of theM. abscessus isolates (100%) and 16 of the 17 (94%)M. fortuitum isolates were resistant to both ethionamide and rifabutin. Only oneM. abscessus subsp.abscessus isolate and 7 of 17 (41%)M. fortuitum isolates were susceptible to clofazimine. This result reconfirmed that first-line antitubercular drugs are not useful for treating infections byM. abscessus orM. fortuitum. In addition, in order to better investigate the susceptibility of second-line antitubercular drugs to these strains, such as clofazimine, linezolid, and kanamycin, the first-line antitubercular drugs were partially used as a control in the study.


### 3.3. Resistance of M. abscessus to Non-Antitubercular Antibiotics

In general, the antibiotic resistance rates of the 20M. abscessus isolates were very high. All or almost all isolates were resistant to sulfamethoxazole, vancomycin, oxacillin, clindamycin, and all fluoroquinolones, and more than 50% of the isolates were resistant to tetracyclines, carbapenems, and aminoglycosides except for amikacin (Table 1).M. abscessus showed the lowest resistance rates to cefoxitin (10%), azithromycin (10%), amikacin (10%), and clarithromycin (20%). For clarithromycin, the 14-day inducible resistance rate is 15% (3/20). There was no statistically difference in the resistance rates betweenM. abscessus subsp.abscessus and subsp.massiliense isolates except to minocycline, in which the latter subspecies showed less resistance (Table 1).


### 3.4. Resistance of M. fortuitum to Non-Antitubercular Antibiotics

Although macrolides and cefoxitin are important component of the treatment regimen for RGM, the results showed that 16 (94%), 17 (100%), and 15 (88%)M. fortuitum isolates were resistant to clarithromycin, azithromycin, and cefoxitin, respectively. Besides macrolides, all or almost all of theM. fortuitum isolates were resistant to kanamycin (94%), doxycycline (82%), minocycline (82%), vancomycin (100%), teicoplanin (100%), oxacillin (100%), and clindamycin (100%).M. fortuitum showed the lowest levels of resistance to tigecycline (0%), tetracycline (0%), cefmetazole (12%), imipenem (12%), linezolid (18%), and the aminoglycosides, including amikacin (0%), tobramycin (0%), neomycin (0%), and gentamycin (24%) (Table 1). Isolates ofM. fortuitum showed intermediate resistance rates to fluoroquinolones, which are also commonly considered in RGM treatment regimens. The resistance rates to moxifloxacin, ciprofloxacin, and levofloxacin were 59%, 47%, and 41%, respectively (Table 1). Importantly, the isolates showed variable resistance to three tetracyclines, tigecycline, and two cephalosporins. They were mostly resistant to doxycycline, minocycline, and cefoxitin but were susceptible to tigecycline and cefmetazole.


## 3.1. Species Identification

There were 17 isolates identified asM. fortuitum and 20 isolates identified asM. abscessus. Among 20M. abscessusisolates, 12 isolates were classified asM. abscessussubsp.abscessus, 7 isolates were classified asM. abscessus subsp.massiliense, and one isolate was classified asM. abscessus subsp.bolletii based on theirhsp65 gene.


## 3.2. Resistance to Antitubercular Drugs

BothM. abscessus andM. fortuitum were highly resistant to antitubercular drugs such as isoniazid, rifampin, ethambutol, clofazimine, ethionamide, and rifabutin. Specifically, all isolates (100%) were resistant to isoniazid, ethambutol, and rifampin (Table 1). All of theM. abscessus isolates (100%) and 16 of the 17 (94%)M. fortuitum isolates were resistant to both ethionamide and rifabutin. Only oneM. abscessus subsp.abscessus isolate and 7 of 17 (41%)M. fortuitum isolates were susceptible to clofazimine. This result reconfirmed that first-line antitubercular drugs are not useful for treating infections byM. abscessus orM. fortuitum. In addition, in order to better investigate the susceptibility of second-line antitubercular drugs to these strains, such as clofazimine, linezolid, and kanamycin, the first-line antitubercular drugs were partially used as a control in the study.


## 3.3. Resistance of M. abscessus to Non-Antitubercular Antibiotics

In general, the antibiotic resistance rates of the 20M. abscessus isolates were very high. All or almost all isolates were resistant to sulfamethoxazole, vancomycin, oxacillin, clindamycin, and all fluoroquinolones, and more than 50% of the isolates were resistant to tetracyclines, carbapenems, and aminoglycosides except for amikacin (Table 1).M. abscessus showed the lowest resistance rates to cefoxitin (10%), azithromycin (10%), amikacin (10%), and clarithromycin (20%). For clarithromycin, the 14-day inducible resistance rate is 15% (3/20). There was no statistically difference in the resistance rates betweenM. abscessus subsp.abscessus and subsp.massiliense isolates except to minocycline, in which the latter subspecies showed less resistance (Table 1).


## 3.4. Resistance of M. fortuitum to Non-Antitubercular Antibiotics

Although macrolides and cefoxitin are important component of the treatment regimen for RGM, the results showed that 16 (94%), 17 (100%), and 15 (88%)M. fortuitum isolates were resistant to clarithromycin, azithromycin, and cefoxitin, respectively. Besides macrolides, all or almost all of theM. fortuitum isolates were resistant to kanamycin (94%), doxycycline (82%), minocycline (82%), vancomycin (100%), teicoplanin (100%), oxacillin (100%), and clindamycin (100%).M. fortuitum showed the lowest levels of resistance to tigecycline (0%), tetracycline (0%), cefmetazole (12%), imipenem (12%), linezolid (18%), and the aminoglycosides, including amikacin (0%), tobramycin (0%), neomycin (0%), and gentamycin (24%) (Table 1). Isolates ofM. fortuitum showed intermediate resistance rates to fluoroquinolones, which are also commonly considered in RGM treatment regimens. The resistance rates to moxifloxacin, ciprofloxacin, and levofloxacin were 59%, 47%, and 41%, respectively (Table 1). Importantly, the isolates showed variable resistance to three tetracyclines, tigecycline, and two cephalosporins. They were mostly resistant to doxycycline, minocycline, and cefoxitin but were susceptible to tigecycline and cefmetazole.


## 4. Discussion

Although the role ofin vitro drug susceptibility testing has not been validated for most NTM species, these tests may nevertheless be important in the management of NTM-related diseases [19]. The need for long-term antibiotic treatment and associated toxicities contribute to the frequent unsatisfactory treatment outcomes in patients, thereby posing a great challenge to physicians in choosing the optimal regimen for patients infected with RGM. The present results of antibiotic susceptibility tests of the 30 most commonly used antibiotics against 37 RGM isolates, including subspecies of theM. abscessus complex isolates, supported the current recommendation of using amikacin, cefoxitin, and macrolides to treatM. abscessus infections [14]. Among the macrolides, azithromycin appears to be a better choice for treatingM. abscessus infections than clarithromycin, since the susceptibility rate was lower for clarithromycin and the inducible resistance rate is 15%(3/20).AllM. abscessus isolates were resistant to streptomycin in our study (100%), which was in agreement with previous study [26]. However, in a study in Japan [30], the resistance rate to streptomycin was only 61%, which indicated the variance of the resistance in different healthcare background. For other rarely used aminoglycosides like tobramycin, neomycin, kanamycin, and gentamycin, the resistance rates ofM. abscessus were also very high. The resistance rate to tobramycin was 60% in this study, which is higher than those of studies in Taiwan province of China [33], Korea [32], and Japan [30], in which the resistance rate varies from 30% to 32%. Broad spectrum antibiotics like minocycline, doxycycline, and sulfamethoxazole were ineffective againstM. abscessus isolates; for their resistance rates reached 70%, 80%, and 90%, respectively. A recent study by Ruth et al. [42] proposed that minocycline has no clear roles in the treatment ofM. abscessus disease, because of their high MICs against minocycline, rapid emergence of drug resistance, and no synergy effect with other antibiotics used to treatM. abscessus. Therefore, these antibiotics should not be used in clinics againstM. abscessus infections.Treatment with linezolid also appears to be a potentially good choice for this bacillus. The major difference betweenM. abscessus subsp.abscessus andM. abscessus subsp.massiliense is that the former has an innateerm(41) gene that confers the ability for inducible macrolide resistance; therefore, precise differentiation between these subspecies has been proposed to be important for clinical purposes [40, 43]. Our study showed that the macrolides resistance rate ofM. abscessus subsp.abscessus was higher than that ofM. abscessus subsp.massiliense, but the difference was not statistically significant due to the limitation of isolate numbers, which was consistent with the results conducted by Nie et al. [40]. However, except for macrolides and minocycline, differentiation between these two subspecies could not provide additional drug resistance information, as their resistance profiles were largely similar. In addition, the results of our study indicate that two commonly used drugs, macrolides and cefoxitin, should not be used in the treatment ofM. fortuitum infection, since the isolates showed high resistance rates to both drugs. Given the lower resistance rates, aminoglycosides, cefmetazole, tigecycline, imipenem, and linezolid are potentially good choices for treatment regimens againstM. fortuitum infection.When the drug resistance rates obtained in the present study were compared with those reported previously, we found that high resistance to multiple antimicrobials was mostly prevalent inM. abscessus. Almost all studies indicated thatM. abscessus isolates are highly susceptible to amikacin, cefoxitin, and macrolides and low resistance to linezolid (Table 3). By contrast, the imipenem data varied markedly among studies. The present study and those conducted in 2016 in Shanghai [24], 2017 in Taiwan [21], and 2015 in Australia [28] showed high resistance ofM. abscessusto imipenem, whereas studies conducted in 2017 in Korea [22], 2013 in Guangdong [31], 2014 in Beijing [4], 2008 in Korea [32], and 2003 in Taiwan [33] showed relatively lower resistance rates to imipenem. Most studies did not support the use of fluoroquinolones and doxycycline, except for tigecycline (Table 3).Table 3
The comparison of drug resistance rate ofM. abscessusisolates from various studies.
district/nation year Test method Tetracyclines Glycylcycline Carbapenems    Aminoglycosides Fluoroquinolones Macrolides source DOX MNO TGC IMP MEM LNZ SXT/SOX3 FOX AMK TOB STR CIP MXF LVX CLR-ERT CLR-LRT AZM Shanghai 2017 1 14 (70%) 11 (55%) 8 (40%) 13 (65%) - 3 (15%) 18 (90%) 2 (10%) 2 (10%) 12 (60%) 20 (100%) 19 (95%) 19 (95%) 19 (95%) - 9 (45%) 2 (10%) This study Taiwan 2017 1 66 (99%) 66 (99%) - 41 (61%) - 47 (70%) 60 (90%) 14 (21%) 4 (6%) - - 60 (90%) 63 (94%) - 5 (7%) 19 (28%) - [21] Korea 2017 1 - - - 17 (18%) - 5 (5%) - 10 (10%) 6 (6%) - - 94 (99%) 88 (93%) - 9 (9%) 39 (41%) - [22] Beijing 2016 1 - - 4 (18%) 9 (41%) - 2 (9%) 10 (45%) 7 (32%) 1 (5%) 8 (36%) - - 6 (27%) 17 (77%) 3 (14%) - 17 (77%) [23] Shanghai 2016 1 52 (98%) - - 52 (98%) - 11 (21%) - 15 (28%) 1 (2%) 44 (83%) - - 51 (96%) - 15 (28%) 36 (68%) - [24] UK 2015 2 - - 3 (1%) - 534 (99%) 311 (96%) - 531 (96%) 74 (14%) - - 495 (99%) 257 (98%) - 33 (6%) - 191 (38%) [25] Fujian et al. 2015 1 36 (65%) 31 (56%) 2 (4%) - 16 (29%) 2 (4%) - 17 (31%) 0 (0%) 30 (55%) 52 (95%) 31 (56%) 12 (22%) 29 (53%) 18 (33%) - 12 (22%) [26] Singapore 2015 1 235 (80%) - - 50 (20%) - 50 (16%) 95 (94%) 8 (3%) 1 (0.9%) 282 (98%) - 293 (94%) 115 (93%) - 11 (3%) - - [27] Australia 2015 1 32 (84%) - - 26 (68%) - 7 (18%) 35 (92%) 7 (18%) 2 (5%) 22 (58%) - 36 (95%) 35 (92%) - - 18 (47%) - [28] Beijing 2014 1 - - - 7 (10%) - 2 (3%) - 1 (1%) 1 (1%) - - - 10 (14%) 68 (97%) 22 (31%) 38 (54%) 3 (4%) [4] Korea 2014 1 - - - - - - - - 40 (10%) - - 361 (89%) 319 (79%) - 64 (16%) 186 (46%) - [29] Japan 2013 1 139 (97%) 119 (83%) 73 (51%) 34 (24%) - 16 (11%) 143(100%) - 7 (5%) 44 (31%) 87 (61%) 137 (96%) 135 (94%) - 18 (13%) - - [30] Guangdong 2013 1 - - - 15 (21%) - - - 3 (4%) 0 (0%) - - 56 (80%) - - 10 (14%) - - [31] Korea 2008 1 60 (81%) - - 8 (11%) - - - 0 (0%) 0 (0%) 22 (30%) - 13 (18%) 5 (7%) - 2 (3%) - - [32] Taiwan 2003 1 85 (92%) - - 17 (10%) 91 (99%) 39 (42%) 91 (99%) 4 (4%) 4 (4%) 28 (32%) - 87 (96%) 75 (82%) 88 (96%) 10 (11%) - 44 (48%) [33] ( 1 ) 1 means broth microdilution method and 2 means disc diffusion method. ( 2 ) “-“ means the results are not available. ( 3 ) SXT=trimethoprim-sulfamethoxazole. SXT and SOX have the same mainly effective ingredient sulfamethoxazole, so we compare the results together.Although the drug resistance ofM. fortuitum does not appear to be as prevalent as that ofM. abscessus, the resistance rates to multiple antibiotics, including sulfamethoxazole, linezolid, clarithromycin, cefoxitin, and tobramycin, vary extensively among studies (Table 4). In addition, except for the present study, amikacin was reported to show the most effectivein vitro activity in all previous studies. Most studies recommended the use of imipenem, except for the study conducted in 2017 in Guangzhou [34], in the treatment ofM. fortuitum infection (Table 4). Low to medium degrees of fluoroquinolones resistance were observed inM. fortuitumin the majority of studies, in which moxifloxacin resistance varied from 0% to 59% and ciprofloxacin resistance varied from 3% to 47% (Table 4). The variability of susceptibility to different antimicrobials among studies emphasizes the importance of drug susceptibility testing in cases ofM. fortuitum infection. The antibiotic susceptibility variance may be due to the difference in the choices of DST methods and breakpoints.Table 4
The comparison of drug resistance rate ofM. fortuitum isolates from various studies.
district/nation year Test method Aminoglycosides  Fluoroquinolones   Carbapenems  Sulfonamides source AMK TOB FOX MXF CIP CLR DOX IMP MEM LNZ SXT/SOX3 Shanghai 2017 1 0 (0%) 0 (0%) 15 (88%) 10 (59%) 8 (47%) 17 (100%) 15 (88%) 2 (12%) - 3 (18%) 14 (82%) This study Guangzhou 2017 1 7 (14%) 51 (100%) - 2 (4%) 21 (41%) 39 (76%) - 29 (57%) 13 (25%) 43 (84%) - [34] Mumbai 2016 2 0 (0%) 0 (0%) 22 (100%) - 5 (23%) 0 (0%) - 4 (18%) - 0 (0%) 21 (95%) [35] India 2016 1 0 (0%) - - - 5 (24%) 3 (14%) - - - - - [36] Iran 2016 1 1 (2%) 0 (0%) 8 (14%) 25 (29%) 12(14%) - 36 (42%) 8 (9%) 42 (49%) 6 (7%) 0 (0%) [37] Singapore 2015 1 3 (3%) 73 (92%) 7 (8%) 0 (0%) 3 (3%) 44 (47%) - 3 (3%)  6 (7%) 1 (3%) [27] Taiwan 2003 1 0 (0%) 32 (46%) 1 (1%) 17 (25%) 23(33%) 14 (20%) 47 (68%) 5 (7%) 28 (41%) 17 (25%) 35 (51%) [33] ( 1 ) 1 means broth microdilution method and 2 means disc diffusion method. ( 2 ) “-“ means the results are not available. ( 3 ) SXT=trimethoprim-sulfamethoxazole. SXT and SOX have the same mainly effective ingredient sulfamethoxazole, so we compare the results together.For some rarely used antibiotics applied inM. fortuitum treatment like neomycin and tigecycline, the susceptibility rates were 100% and 94% separately, suggesting they are very prospective antibiotics in the treatment. For gentamycin, although the susceptibility was only 24%, the intermediate rate was as high as 52%, which should be used in cautions in high doses.Among the 36 patients, 29 were hospitalized and had detailed record of hospitalization. Among them, 27 have empirical antibiotic treatment, including meropenem, levofloxacin, clarithromycin, and rifampin, before the diagnosis of NTM associated diseases. Only 2 patients had no history of antibiotics treatment. Mostly patients had empirical anti-infection therapy in the hospital which could be the reason of the generally higher resistance rates in these isolates.Nevertheless, this study has several limitations that should be mentioned. First, the isolates were geographically limited to a single province. Second, there was a limited number of RGM isolates collected, because of the relatively low incidence of RGM infections in Shanghai. Consequently, the power for detecting resistance to multiple antibiotics could be reduced in terms of generalizability to other RGM isolates worldwide.In conclusion, the present results showed that amikacin, cefoxitin, and azithromycin had the highestin vitro activity againstM. abscessus, which is in line with current recommendations. However, in contrast to previous studies,M. fortuitum was found to be mostly resistant to macrolides and cefoxitin. Therefore, isolates ofM. fortuitum should be individually evaluated with the drug susceptibility test in deciding the most effective antimicrobials for treatment of infections.



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