# Drug Susceptibility Testing of 31 Antimicrobial Agents on Rapidly Growing Mycobacteria Isolates from China

Authors: Hui Pang; Guilian Li; Xiuqin Zhao; Haican Liu; Kanglin Wan; Ping Yu
Journal: BioMed Research International
 (2015)

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## Abstract

Objectives. Several species of rapidly growing mycobacteria (RGM) are now recognized as human pathogens. However, limited data on effective drug treatments against these organisms exists. Here, we describe the species distribution and drug susceptibility profiles of RGM clinical isolates collected from four southern Chinese provinces from January 2005 to December 2012. Methods. Clinical isolates (73) were subjected to in vitro testing with 31 antimicrobial agents using the cation-adjusted Mueller-Hinton broth microdilution method. The isolates included 55 M. abscessus, 11 M. fortuitum, 3 M. chelonae, 2 M. neoaurum, and 2 M. septicum isolates. Results. M. abscessus (75.34%) and M. fortuitum (15.07%), the most common species, exhibited greater antibiotic resistance than the other three species. The isolates had low resistance to amikacin, linezolid, and tigecycline, and high resistance to first-line antituberculous agents, amoxicillin-clavulanic acid, rifapentine, dapsone, thioacetazone, and pasiniazid. M. abscessus and M. fortuitum were highly resistant to ofloxacin and rifabutin, respectively. The isolates showed moderate resistance to the other antimicrobial agents. Conclusions. Our results suggest that tigecycline, linezolid, clofazimine, and cefmetazole are appropriate choices for M. abscessus infections. Capreomycin, sulfamethoxazole, tigecycline, clofazimine, and cefmetazole are potentially good choices for M. fortuitum infections. Our drug susceptibility data should be useful to clinicians.


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## Body

## 1. Introduction

Nontuberculous mycobacteria (NTM) form a large class within the Mycobacteriaceae family. More than 100 NTM species are found in soil, potable water, food, and animals [1]. In China, the proportion of NTM among all mycobacterial isolates has increased from 11.1% to 22.9% according to National surveys conducted in 1990 and 2010 [2]. Thus, the rising percentage of NTM in China is now an important public health concern [1, 2].NTM can be classified into rapidly growing mycobacteria (RGM) and slowly growing mycobacteria (SGM). More than 50 RGM species are able to produce mature colonies on agar plates within 7 days [3]. Many of these are important human pathogens that cause pulmonary and soft tissue infections and various other infections [4, 5]. RGM comprise a diverse group of species, includingM. abscessus,M. fortuitum,M. chelonae, and various rare species. Most studies have shown thatM. abscessus accounts for 80% of the lung disease caused by RGM, and afterM. fortuitum,M abscessus is the second most common RGM to cause extrapulmonary disease [3, 6].Diagnosing and treating RGM diseases is challenging for clinicians [6, 7]. Over the past few decades, RGM infections have been diagnosed based on a patient’s clinical characteristics, risk factors, and the results of antimicrobial susceptibility testing [3]. However, drug susceptibility patterns vary greatly between RGM species and optimally therapeutic regimens have not been established [3, 6].In this study, the antimicrobial susceptibility of 73 clinical RGM isolates and their corresponding standard strains were tested with 31 antibiotics. The tests were based on the recommendations of the Clinical and Laboratory Standards Institute (CLSI) [8] for determining the clinical criteria for therapeutic treatment of RGM infections.


## 2. Materials and Methods

### 2.1. Clinical Isolates and Reference Strains

During the period from January 2005 to December 2012, RGM clinical strains were isolated from the sputum specimens of suspected tuberculosis patients in Fujian, Hunan, Gansu, and Sichuan Provinces of China. Eighteen clinical strains were isolated in 2005, 6 in 2006, 8 in 2009, 25 in 2010, 8 in 2011, and 8 in 2012. International-standard RGM species were used as the corresponding quality control strains for the clinical isolates tested herein (i.e.,M. abscessusATCC19977,M. chelonae ATCC35752,M. fortuitum DSM44220,M. neoaurum ATCC25795, andM. septicum DSM44393).


### 2.2. Species Identification

Species identification of the isolates was conducted by sequence analysis of thehsp65 gene. When anhsp65 sequence match was less than 97%, therpoB gene and the 16S–23S internal transcribed spacer region were also sequenced [9, 10]. PCR products were sequenced by the Beijing Tsingke Bio Tech Co. Ltd. (Beijing, China). The sequences obtained were compared with those in the GenBank (National Center for Biotechnology Information: http://www.ncbi.nlm.nih.gov/) DNA sequence database; species identification was confirmed if a 97% match was achieved [9, 10].


### 2.3. RGM Growth Medium

The strains tested were cultured using Difco Middlebrook 7H10 Agar (BD company). The medium was prepared as follows. First, 19 g of 7H10 (powder) was suspended in 900 mL of purified water containing 5 mL of glycerol and then mixed thoroughly. Next, the powder was completely dissolved by heating with frequent agitation for 1 min and then sterilized at 121°C for 10 min. Last, 100 mL of Middlebrook OADC enrichment solution (BD, Franklin Lakes, NJ, USA) was added aseptically to the medium after cooling to 50–55°C.


### 2.4. Medium for Drug Susceptibility Testing of RGM

The medium used for antimicrobial susceptibility testing of RGM was BBL cation-adjusted Mueller-Hinton (CAMH) Broth (BD, Franklin Lakes, NJ, USA). The medium was prepared by suspending 22 g of powder in 1 L of purified water and autoclaving the bottle at 121°C for 10 min, followed by supplementation with 20–25 mg/L of calcium and 10–12.5 mg/L of magnesium.


### 2.5. Drug Susceptibility Tests

The strains were grown on 7H10 agar and incubated at 37°C in ambient air. Drug susceptibility tests were performed using the broth microdilution method according to CLSI recommendations [6]. Tests on the strains were repeated at least twice using 96-well microplates. The final minimum inhibitory concentration (MIC) of each drug used for each strain was the average value of the two tests. Bacterial inocula were adjusted with normal saline to a density of a 0.5 McFarland standard with an organism density of approximately 1 × 107 colony forming units (CFU)/mL. Fifty microliters of the suspension, added to 10 mL of CAMH broth, was vortexed thoroughly to make a 1 : 200 bacterial dilution. First, 100 μL of CAMH medium was added to each well of a 96-well microplate, except for the first well of the each row. CAMH medium (180 μL) was added to the first well of every row, followed by a 20 μL aliquot of a drug solution. The thoroughly mixed solution in the first well was serially diluted into the next well, and so on up to the 11th well. The 12th well in every row was a blank control. Second, 100 μL of the bacterial dilution was added to the wells of the 96-well microplate. The final volume in each row was 200 μL. Finally, the 96-well microplate was sealed in a plastic bag and incubated at 37°C. The concentration ranges for rifampicin, isoniazid, ethambutol, streptomycin, tobramycin, sulfamethoxazole, dapsone, amoxicillin-clavulanic acid, cefoxitin, cefmetazole, thioacetazone, pasiniazid, minocycline, doxycycline, tigecycline, and meropenem were all 0.25–256 μg/mL, those of amikacin, kanamycin, capreomycin, ofloxacin, ciprofloxacin, levofloxacin, moxifloxacin, sparfloxacin, clarithromycin, azithromycin, roxithromycin, clofazimine, rifapentine, and rifabutin were 0.03–32 μg/mL, and the concentration of linezolid was 0.06–64 μg/mL. All the drugs were purchased from Sigma-Aldrich (St. Louis, MO). The following two negative controls were used: CAMH broth plus inoculum (drug-free control), which was used to decide the optimal time to add Alamar blue to the assay; the other was only CAMH broth, which was used to decide the interference level of CAMH to Alamar blue. The plates were checked after 72 h. If the drug-free growth control showed sufficient bacterial growth, the indicator (20 μL of Alamar Blue and 50 μL of sterile 5% Tween-80) turned pink. Generally, the minimal inhibitory concentration (MIC) value was read on day 3 or 4 after addition of the inoculum. If bacterial growth in the drug-free control was insufficient on day 5, the test was repeated. The MIC values for clarithromycin were evaluated 3 to 5 days after inoculation and were incubated for a further 14 days at 37°C for the final reading.MIC values were defined as the lowest concentration of drug that inhibited the visible growth of the isolates tested. MIC50 and MIC90 values were defined as the drug concentrations at which 50% and 90% of the isolates tested showed no visible growth, respectively. The MIC breakpoints of antibiotics displaying susceptibility, intermediate susceptibility, and resistance were interpreted by the World Health Organization (WHO) [11] and CLSI guidelines [8], except for sparfloxacin [12], clofazimine [13], azithromycin [14], roxithromycin [14], amoxicillin-clavulanic acid [15], cefmetazole [16], rifapentine [17], rifabutin [17], dapsone [18], and thioacetazone [19] (Table 1).Table 1
MIC (μg/mL) breakpoints of 31 antimicrobial agents.
Antibacterial agents
MIC breakpoints
Susceptibility
Intermediate susceptibility
Resistance
Rifampicin
—
—
⩾1  [8]
Isoniazid
—
—
⩾1  [8]
Ethambutol
—
—
⩾4  [8]
Streptomycin
—
—
⩾5  [8]
Amikacin
⩽16
32
⩾64  [8]
Kanamycin
—
—
⩾4  [11]
Capreomycin
—
—
⩾2.5  [11]
Tobramycin
⩽2
4
⩾8  [8]
Ofloxacin
—
—
⩾2  [11]
Ciprofloxacin
⩽1
2
⩾4  [8]
Levofloxacin
⩽2
4
⩾8  [8]
Sparfloxacin
⩽1
2
⩾4  [12]
Moxifloxacin
⩽1
2
⩾4  [8]
Linezolid
⩽8
16
⩾32  [8]
Clofazimine
—
—
⩾1  [13]
Sulfamethoxazole
⩽38
—
⩾76  [8]
Minocycline
⩽1
2–4
⩾8  [8]
Doxycycline
⩽1
2–4
⩾8  [8]
Tigecycline
⩽1
2–4
⩾8  [8]
Clarithromycin
⩽2
4
⩾8  [8]
Azithromycin
⩽2
4
⩾8  [14]
Roxithromycin
⩽2
4
⩾8  [14]
Amoxicillin-clavulanicacid
⩽8/4
16/8
⩾32/16  [15]
Cefoxitin
⩽16
32–64
⩾128  [8]
Cefmetazole
⩽16
32
⩾64  [16]
Meropenem
⩽4
8–16
⩾32  [8]
Rifapentine
—
—
⩾1  [17]
Rifabutin
—
—
⩾1  [17]
Dapsone
—
—
⩾4  [18]
Thioacetazone
—
—
⩾8  [19]
Pasiniazid
—
—
⩾2  [11]


### 2.6. Statistical Analysis

The data were analyzed by SPSS17.0 software. The drug susceptibility percentages and the MIC50 and MIC90 among the antimicrobial agents tested were determined for the five species of RGM isolates (M. abscessus,M. chelonae,M. fortuitum,M. neoaurum, andM. septicum).


## 2.1. Clinical Isolates and Reference Strains

During the period from January 2005 to December 2012, RGM clinical strains were isolated from the sputum specimens of suspected tuberculosis patients in Fujian, Hunan, Gansu, and Sichuan Provinces of China. Eighteen clinical strains were isolated in 2005, 6 in 2006, 8 in 2009, 25 in 2010, 8 in 2011, and 8 in 2012. International-standard RGM species were used as the corresponding quality control strains for the clinical isolates tested herein (i.e.,M. abscessusATCC19977,M. chelonae ATCC35752,M. fortuitum DSM44220,M. neoaurum ATCC25795, andM. septicum DSM44393).


## 2.2. Species Identification

Species identification of the isolates was conducted by sequence analysis of thehsp65 gene. When anhsp65 sequence match was less than 97%, therpoB gene and the 16S–23S internal transcribed spacer region were also sequenced [9, 10]. PCR products were sequenced by the Beijing Tsingke Bio Tech Co. Ltd. (Beijing, China). The sequences obtained were compared with those in the GenBank (National Center for Biotechnology Information: http://www.ncbi.nlm.nih.gov/) DNA sequence database; species identification was confirmed if a 97% match was achieved [9, 10].


## 2.3. RGM Growth Medium

The strains tested were cultured using Difco Middlebrook 7H10 Agar (BD company). The medium was prepared as follows. First, 19 g of 7H10 (powder) was suspended in 900 mL of purified water containing 5 mL of glycerol and then mixed thoroughly. Next, the powder was completely dissolved by heating with frequent agitation for 1 min and then sterilized at 121°C for 10 min. Last, 100 mL of Middlebrook OADC enrichment solution (BD, Franklin Lakes, NJ, USA) was added aseptically to the medium after cooling to 50–55°C.


## 2.4. Medium for Drug Susceptibility Testing of RGM

The medium used for antimicrobial susceptibility testing of RGM was BBL cation-adjusted Mueller-Hinton (CAMH) Broth (BD, Franklin Lakes, NJ, USA). The medium was prepared by suspending 22 g of powder in 1 L of purified water and autoclaving the bottle at 121°C for 10 min, followed by supplementation with 20–25 mg/L of calcium and 10–12.5 mg/L of magnesium.


## 2.5. Drug Susceptibility Tests

The strains were grown on 7H10 agar and incubated at 37°C in ambient air. Drug susceptibility tests were performed using the broth microdilution method according to CLSI recommendations [6]. Tests on the strains were repeated at least twice using 96-well microplates. The final minimum inhibitory concentration (MIC) of each drug used for each strain was the average value of the two tests. Bacterial inocula were adjusted with normal saline to a density of a 0.5 McFarland standard with an organism density of approximately 1 × 107 colony forming units (CFU)/mL. Fifty microliters of the suspension, added to 10 mL of CAMH broth, was vortexed thoroughly to make a 1 : 200 bacterial dilution. First, 100 μL of CAMH medium was added to each well of a 96-well microplate, except for the first well of the each row. CAMH medium (180 μL) was added to the first well of every row, followed by a 20 μL aliquot of a drug solution. The thoroughly mixed solution in the first well was serially diluted into the next well, and so on up to the 11th well. The 12th well in every row was a blank control. Second, 100 μL of the bacterial dilution was added to the wells of the 96-well microplate. The final volume in each row was 200 μL. Finally, the 96-well microplate was sealed in a plastic bag and incubated at 37°C. The concentration ranges for rifampicin, isoniazid, ethambutol, streptomycin, tobramycin, sulfamethoxazole, dapsone, amoxicillin-clavulanic acid, cefoxitin, cefmetazole, thioacetazone, pasiniazid, minocycline, doxycycline, tigecycline, and meropenem were all 0.25–256 μg/mL, those of amikacin, kanamycin, capreomycin, ofloxacin, ciprofloxacin, levofloxacin, moxifloxacin, sparfloxacin, clarithromycin, azithromycin, roxithromycin, clofazimine, rifapentine, and rifabutin were 0.03–32 μg/mL, and the concentration of linezolid was 0.06–64 μg/mL. All the drugs were purchased from Sigma-Aldrich (St. Louis, MO). The following two negative controls were used: CAMH broth plus inoculum (drug-free control), which was used to decide the optimal time to add Alamar blue to the assay; the other was only CAMH broth, which was used to decide the interference level of CAMH to Alamar blue. The plates were checked after 72 h. If the drug-free growth control showed sufficient bacterial growth, the indicator (20 μL of Alamar Blue and 50 μL of sterile 5% Tween-80) turned pink. Generally, the minimal inhibitory concentration (MIC) value was read on day 3 or 4 after addition of the inoculum. If bacterial growth in the drug-free control was insufficient on day 5, the test was repeated. The MIC values for clarithromycin were evaluated 3 to 5 days after inoculation and were incubated for a further 14 days at 37°C for the final reading.MIC values were defined as the lowest concentration of drug that inhibited the visible growth of the isolates tested. MIC50 and MIC90 values were defined as the drug concentrations at which 50% and 90% of the isolates tested showed no visible growth, respectively. The MIC breakpoints of antibiotics displaying susceptibility, intermediate susceptibility, and resistance were interpreted by the World Health Organization (WHO) [11] and CLSI guidelines [8], except for sparfloxacin [12], clofazimine [13], azithromycin [14], roxithromycin [14], amoxicillin-clavulanic acid [15], cefmetazole [16], rifapentine [17], rifabutin [17], dapsone [18], and thioacetazone [19] (Table 1).Table 1
MIC (μg/mL) breakpoints of 31 antimicrobial agents.
Antibacterial agents
MIC breakpoints
Susceptibility
Intermediate susceptibility
Resistance
Rifampicin
—
—
⩾1  [8]
Isoniazid
—
—
⩾1  [8]
Ethambutol
—
—
⩾4  [8]
Streptomycin
—
—
⩾5  [8]
Amikacin
⩽16
32
⩾64  [8]
Kanamycin
—
—
⩾4  [11]
Capreomycin
—
—
⩾2.5  [11]
Tobramycin
⩽2
4
⩾8  [8]
Ofloxacin
—
—
⩾2  [11]
Ciprofloxacin
⩽1
2
⩾4  [8]
Levofloxacin
⩽2
4
⩾8  [8]
Sparfloxacin
⩽1
2
⩾4  [12]
Moxifloxacin
⩽1
2
⩾4  [8]
Linezolid
⩽8
16
⩾32  [8]
Clofazimine
—
—
⩾1  [13]
Sulfamethoxazole
⩽38
—
⩾76  [8]
Minocycline
⩽1
2–4
⩾8  [8]
Doxycycline
⩽1
2–4
⩾8  [8]
Tigecycline
⩽1
2–4
⩾8  [8]
Clarithromycin
⩽2
4
⩾8  [8]
Azithromycin
⩽2
4
⩾8  [14]
Roxithromycin
⩽2
4
⩾8  [14]
Amoxicillin-clavulanicacid
⩽8/4
16/8
⩾32/16  [15]
Cefoxitin
⩽16
32–64
⩾128  [8]
Cefmetazole
⩽16
32
⩾64  [16]
Meropenem
⩽4
8–16
⩾32  [8]
Rifapentine
—
—
⩾1  [17]
Rifabutin
—
—
⩾1  [17]
Dapsone
—
—
⩾4  [18]
Thioacetazone
—
—
⩾8  [19]
Pasiniazid
—
—
⩾2  [11]


## 2.6. Statistical Analysis

The data were analyzed by SPSS17.0 software. The drug susceptibility percentages and the MIC50 and MIC90 among the antimicrobial agents tested were determined for the five species of RGM isolates (M. abscessus,M. chelonae,M. fortuitum,M. neoaurum, andM. septicum).


## 3. Results

Among the 73 clinical isolates, 55 (75.34%) wereM. abscessus, 11 (15.07%) wereM. fortuitum, 3 (4.11%) wereM. chelonae, 2 (2.74%) wereM. neoaurum, and 2 (2.74%) wereM. septicum. Of the isolates, 63 were from Fujian, 7 were from Hunan, 2 were from Gansu, and 1 was from Sichuan.The antimicrobial susceptibility profiles of the five reference RGM strains are shown in Table2. The strains were highly resistant to the four first-line antituberculous agents tested on them, especiallyM. chelonae,M. abscessus, andM. fortuitum. We found that aminoglycoside antibiotics including amikacin, kanamycin, capreomycin, and tobramycin, were effective antimicrobials for the RGM species. However,M. abscessus was resistant to tobramycin andM. chelonae was resistant to kanamycin and capreomycin. Fluoroquinolones (including ofloxacin, ciprofloxacin, levofloxacin, sparfloxacin, and moxifloxacin) also exhibited favorablein vitro activities against the standard RGM strains. However,M. chelonae was resistant to all four of the fluoroquinolones we tested.M. chelonae andM. abscessus were not susceptible to minocycline and doxycycline, but were susceptible to tigecycline. Clarithromycin, azithromycin, and roxithromycin all exhibited favorablein vitro activities (except for azithromycin) againstM. septicum. The reference species were not susceptible to amoxicillin-clavulanic acid but were susceptible or moderately susceptible to cefoxitin and cefmetazole, apart fromM. chelonae andM. septicum, which were not susceptible to cefoxitin. UnlikeM. chelonae, the RGM species were susceptible to meropenem. Rifapentine had a more favorable MIC than rifabutin againstM. chelonae,M. abscessus, andM. fortuitum. Linezolid, clofazimine, and sulfamethoxazole were highly active against the standard RGM organisms. However, dapsone, thioacetazone, and pasiniazid displayed poor activities against the reference species.Table 2
MIC (μg/mL) results of the antimicrobial susceptibility tests for five rapidly growing reference mycobacteria.
Sp.
RFP
INH
EMB
SM
AM
KN
CPM
TOB
OF
CIP
LEV
SPA
MXF
LNZ
CLO
SMZ
M. abscessus
64
>256
32
16
1
2
0.5
8
4
2
1
2
1
4
0.06
32
M. chelonae
>256
>256
128
32
4
16
16
2
32
4
16
16
4
8
0.25
64
M. fortuitum
128
64
256
32
0.25
4
<0.03
16
0.13
0.03
0.03
<0.03
<0.03
8
0.03
2
M. septicum
16
256
4
16
0.13
0.5
<0.03
1
0.25
0.06
0.13
0.03
0.06
1
0.13
1
M. neoaurum
1
>256
8
0.5
0.5
0.06
<0.03
0.25
0.06
0.06
0.03
<0.03
<0.03
0.5
0.03
0.5
Sp.
MIN
DOX
TIG
CLR
AZM
ROX
AMC
FOX
CMZ
MEM
RFT
RFB
DAP
THI
PASI
M. abscessus
8
32
4
<0.03
<0.03
0.03
>256
32
32
64
0.13
32
64
16
32
M. chelonae
64
>32
1
0.06
0.5
2
>256
128
<0.25
>256
0.13
32
32
>256
>256
M. fortuitum
8
0.03
0.5
1
2
4
>256
32
4
4
1
16
8
256
2
M. septicum
32
8
1
1
16
4
>256
128
1
16
0.13
<0.03
32
256
4
M. neoaurum
0.03
0.06
0.03
1
1
2
32
8
1
0.5
0.06
0.13
4
>256
64
Note1: M. abscessus: ATCC19977;M. chelonae: ATCC35752; M. fortuitum: DSM44220; M. neoaurum: ATCC25795; and M. septicum: DSM44393.
Note2: INH: isoniazid; RFP: rifampicin; EMB: ethambutol; SM: streptomycin; AM: amikacin; KN: kanamycin; CPM: capreomycin; TOB: tobramycin; OF: ofloxacin; CIP: ciprofloxacin; LEV: levofloxacin; SPA: sparfloxacin; MXF: moxifloxacin; LNZ: linezolid; CLO: clofazimine; SMZ: sulfamethoxazole; CLR: clarithromycin; AZM: azithromycin; ROX: roxithromycin; MIN: minocycline; DOX: doxycycline; TIG: tigecycline; AMC: Amoxicillin-clavulanic Acid; FOX: cefoxitin; CMZ: cefmetazole; MEM: meropenem; RPT: rifapentine; RBT: rifabutin; DAP: dapsone; THI: thioacetazone; PASI: pasiniazid.
Note3: Bold, italic values indicate drug susceptibility. Values shown in bold indicate moderate drug susceptibility.The percentage ofin vitro drug susceptibility values of the 31 antibacterial agents against the 73 clinical RGM isolates is shown in Table 3. Among the four first-line antituberculous drugs, no (0/73) strains were susceptible to isoniazid and 3 (4.11%), 2 (2.74%), and 4 (5.48%) strains were susceptible to rifampicin, ethambutol, and streptomycin, respectively. However, theM. chelonae isolates were less resistant to rifampicin thanM. abscessus andM. fortuitum.Aminoglycosides and fluoroquinolones displayed a range of activities against the RGM isolates. Amikacin displayed the highest activity (72/73, 98.63%), while moxifloxacin displayed a range of activities (57/73, 78.08%). Tigecycline (70/73, 95.89%) had much higher activity against the isolates than minocycline (30/73, 41.10%) and doxycycline (25/73, 34.25%). Clarithromycin (48/73, 65.75%), azithromycin (53/73, 72.60%), and roxithromycin (48/73, 65.75%) showed variousin vitro activities against the RGM isolates. Meropenem (52/73, 71.23%) exhibited good activity against the strains tested. Cefmetazole (62/73, 84.93%) was more active than cefoxitin (49/73, 67.12%) and amoxicillin-clavulanic acid (11/73, 15.07%). In contrast, linezolid (71/73, 97.26%) was highly active against the majority of the RGM isolates. Clofazimine (47/73, 64.38%) and sulfamethoxazole (56/73, 76.71%) inhibited the majority of isolates. Rifapentine (5/73, 6.85%) and rifabutin (26/73, 35.62%), which are rifamycin derivatives, were better than rifampicin. However, dapsone (5/73, 6.85%), thioacetazone (0/73, 0%), and pasiniazid (0/73, 0%) displayed poor activities against the RGM isolates.Table 3
In vitro drug susceptibility percentage per species for rapidly growing mycobacteria isolates.
Drugs
Species
Total (n=73)  (%)
M. abscessus (n=55) (%)
M. fortuitum (n=11) (%)
M. chelonae (n=3) (%)
M. septicum (n=2) (%)
M. neoaurum (n=2) (%)
INH
0 (0)
0 (0)
0 (0)
0 (0)
0 (0)
0 (0)
RFP
3 (5.46)
0 (0)
0 (0)
0 (0)
0 (0)
3 (4.11)
EMB
2 (3.64)
0 (0)
0 (0)
0 (0)
0 (0)
2 (2.74)
SM
3 (5.46)
0 (0)
0 (0)
0 (0)
1 (50.00)
4 (5.48)
AM
55 (100.00)
11 (100.00)
3 (100.00)
2 (100.00)
1 (50.00)
72 (98.63)
KN
27 (49.09)
5 (45.45)
1 (33.33)
1 (50.00)
0 (0)
34 (46.58)
CPM
41 (74.55)
11 (100.00)
1 (33.33)
2 (100.00)
0 (0)
55 (75.34)
TOB
25 (45.45)
6 (54.55)
3 (100.00)
1 (50.00)
0 (0)
35 (47.94)
OF
9 (16.36)
5 (45.45)
0 (0)
2 (100.00)
2 (100.00)
18 (24.66)
CIP
24 (43.64)
5 (45.45)
1 (33.33)
2 (100.00)
1 (50.00)
33 (45.21)
LEV
26 (47.27)
8 (72.73)
2 (66.67)
2 (100.00)
1 (50.00)
39 (53.42)
SPA
23 (41.82)
6 (54.55)
3 (100.00)
2 (100.00)
1 (50.00)
35 (47.94)
MXF
43 (78.18)
8 (72.73)
3 (100.00)
2 (100.00)
1 (50.00)
57 (78.08)
LNZ
53 (96.36)
11 (100.00)
3 (100.00)
2 (100.00)
2 (100.00)
71 (97.26)
CLO
35 (63.64)
8 (72.73)
1 (33.33)
2 (100.00)
1 (50.00)
47 (64.38)
SMZ
41 (74.55)
10 (90.91)
2 (66.67)
2 (100.00)
1 (50.00)
56 (76.71)
MIN
24 (43.64)
3 (27.27)
2 (66.67)
1 (50.00)
0 (0)
30 (41.10)
DOX
19 (34.55)
3 (27.27)
2 (66.67)
0 (0)
1 (50.00)
25 (34.25)
TIG
53 (96.36)
10 (90.91)
3 (100.00)
2 (100.00)
2 (100.00)
70 (95.89)
CLR
37 (67.27)
7 (63.64)
2 (66.67)
1 (50.00)
1 (50.00)
48 (65.75)
AZM
43 (78.18)
6 (54.55)
2 (66.67)
0 (0)
2 (100.00)
53 (72.60)
ROX
38 (69.09)
7 (63.64)
2 (66.67)
0 (0)
1 (50.00)
48 (65.75)
AMC
10 (18.18)
0 (0)
1 (33.33)
0 (0)
0 (0)
11 (15.07)
FOX
38 (69.09)
9 (81.82)
1 (33.33)
0 (0)
1 (50.00)
49 (67.12)
CMZ
48 (87.27)
9 (81.82)
2 (66.67)
1 (50.00)
2 (100.00)
62 (84.93)
MEM
39 (70.91)
8 (72.73)
1 (33.33)
2 (100.00)
2 (100.00)
52 (71.23)
RPT
4 (7.27)
0 (0)
1 (33.33)
0 (0)
0 (0)
5 (6.85)
RBT
22 (40.00)
1 (9.09)
2 (66.67)
0 (0)
1 (50.00)
26 (35.62)
DAP
4 (7.27)
1 (9.09)
0 (0)
0 (0)
0 (0)
5 (6.85)
THI
0 (0)
0 (0)
0 (0)
0 (0)
0 (0)
0 (0)
PASI
0 (0)
0 (0)
0 (0)
0 (0)
0 (0)
0 (0)
Note:n: number of strains tested.The MICs, MIC50, and MIC90 ranges for each antimicrobial agent tested against each RGM species are shown in Table 4. Based on the MIC90 values of the isolates, capreomycin was 8 μg/mL forM. abscessus and 2 μg/mL forM. fortuitum. The MIC50 value for levofloxacin was 8 μg/mL forM. abscessus and 2 μg/mL forM. fortuitum. Clofazimine had very high activity, with MIC90 values of 8 μg/mL forM. abscessus and 2 μg/mL forM. fortuitum. The MIC50 and MIC90 values of doxycycline were 16 μg/mL and 32 μg/mL, respectively, forM. abscessus, and 64 μg/mL and 256 μg/mL, respectively, forM. fortuitum. Azithromycin had better activity againstM. abscessus thanM. fortuitum, with MIC50 values of 0.5 μg/mL and 2 μg/mL, respectively.Table 4
The MIC range, MIC50, and MIC90 (μg/mL) per species of rapidly growing mycobacterial isolates for all the antibacterial agents tested.
Drugs
M. abscessus (n=55)
M. fortuitum (n=11)
Other species (n=7)
MIC range
MIC50
MIC90
MIC range
MIC50
MIC90
MIC range
MIC50
MIC90
RFP
0.5–>256
16
128
1–>256
128
>256
2–8
2
8
INH
2–>256
>256
>256
2–>256
>256
>256
16–>256
>256
>256
EMB
1–>256
>256
>256
32–>256
256
>256
128–>256
>256
>256
SM
<0.25–>256
32
256
8–>256
32
128
4–64
32
64
AM
0.06–16
4
8
0.06–16
2
4
0.25–>32
4
8
KN
0.25–>32
4
32
0.5–>32
8
16
2–>32
16
>32
CPM
0.03–16
1
8
0.03–2
0.5
2
0.03–32
4
16
TOB
<0.25–64
8
32
0.5–32
4
32
0.5–32
4
16
OF
0.06–32
8
16
0.06–32
4
16
0.03–8
0.5
8
CIP
0.03–32
4
4
0.06–8
4
8
0.13–8
2
4
LEV
0.03–32
8
8
0.06–8
2
8
0.13–16
4
8
SPA
0.03–32
4
16
0.03–16
2
16
0.06–16
0.25
2
MXF
0.03–16
2
4
0.03–8
1
4
0.06–8
0.5
2
LNZ
0.5–>64
4
16
0.5–16
4
16
0.25–16
4
8
CLO
0.06–>32
0.25
8
0.03–16
0.25
2
0.06–>32
0.5
4
SMZ
0.5–>256
32
>256
4–128
16
64
4–>256
16
256
MIN
<0.25–>256
16
32
1–256
16
64
2–32
16
32
DOX
<0.25–>256
16
32
<0.25–256
64
256
1–64
8
32
TIG
<0.25–8
2
2
<0.25–8
4
4
<0.25–1
0.5
1
CLR
0.03–>32
0.13
>32
0.06–>32
0.25
16
0.03–>32
1
8
AZM
0.03–>32
0.5
>32
0.13–>32
2
>32
0.13–>32
1
32
ROX
0.03–>32
0.25
>32
0.13–32
1
32
0.03–16
8
16
AMC
0.5–>256
>256
>256
256–>256
>256
>256
1–>256
>256
>256
FOX
4–>256
64
256
16–>256
32
128
32–>256
128
256
CMZ
1–128
16
64
8–64
16
64
8–>256
32
64
MEM
<0.25–>256
8
128
<0.25–>256
8
256
0.5–32
4
32
RPT
0.03–>32
8
32
1–>32
8
>32
0.06–32
2
16
RBT
0.03–>32
1
16
0.5–>32
2
16
0.03–4
1
4
DAP
1–>256
16
128
2–>256
64
128
4–256
16
64
THI
8–>256
>256
>256
128–>256
>256
>256
128–>256
>256
>256
PASI
2–>256
256
>256
8–>256
>256
>256
16–>256
>256
>256
Note:n: number of strains tested.


## 4. Discussion

With the development of improved microbiological and laboratory techniques, more RGM have been identified [20]. Effective treatment of RGM-related diseases is challenging to physicians because it is not obvious which drugs should be selected. In this study, the susceptibilities of 73 clinical RGM isolates and their corresponding reference RGM strains were examined for 31 antimicrobial agents using CAMH broth microdilution methodology.Some studies have shown thatM. abscessus,M. fortuitum, andM. chelonaeare important human pathogens among RGM isolates [21–24]. Here, we showed thatM. abscessus(75.34%) is the predominant RGM species in the 73 clinical isolates, followed byM. fortuitum(15.07%). Both organisms were susceptible to amikacin, linezolid, tigecycline, cefmetazole, capreomycin, moxifloxacin, macrolides, and carbapenems, but were highly resistant to the first-line antituberculous drugs, dapsone, thioacetazone, and pasiniazid. The percentage of resistance to numerous drugs was higher inM. abscessus than inM. fortuitum, except for moxifloxacin, minocycline, doxycycline, roxithromycin, cefmetazole, and rifabutin. In a recent report, amikacin and clarithromycin were the optimal choices against infection withM. abscessus [25]. Additionally, quinolones and trimethoprim-sulfamethoxazole were effective againstM. fortuitum [25]. In the present study, Amikacin was the most active drug againstM. abscessus. Furthermore, we found that amikacin, capreomycin, and linezolid had the highest antibacterial activities againstM. fortuitum.Previous studies have reported that numerous RGM strains were highly resistant to the first-line antituberculous agents [26, 27]. Our data confirms this finding. Elsewhere, researchers have shown that dapsone had little activity against RGM isolates [18]. Thioacetazone is used mainly as an antituberculous agent but has variable activity, and the drug was formerly used in conjunction with isoniazid [19]. RGM strains have been shown to be highly resistant to pasiniazid [28]. Our data shows that dapsone had little activity againstM. abscessus andM. fortuitum isolates, while thioacetazone and pasiniazid had no activity against any of the RGM organisms.Aminoglycosides and quinolones, which are second-line antituberculous drugs, have good activities against RGM strains [28–30]. In our study, amikacin was found to have potential to be effective for treatment of RGM diseases and showed higher activity than the other aminoglycoside antibiotics we tested. However, a higher percentage ofM. chelonae isolates were sensitive to tobramycin thanM. abscessus, although the sample size of the latter was smaller. The third generation fluoroquinolone drugs levofloxacin and sparfloxacin displayed higher activities than ofloxacin. Moxifloxacin, a fourth generation fluoroquinolone, displayed higher activity than the third generation ones [30]. Quinolones exhibited better activity againstM. fortuitum thanM. abscessus, especially levofloxacin, against whichM. fortuitum was more susceptible thanM. abscessus.Minocycline, doxycycline, and tigecycline represent the newest tetracycline derivatives [31]. A recent study [26] showed that NTM displayed ~50% susceptibility to doxycycline and minocycline, but in our research susceptibility to these two drugs was more than 20% (Table 3). This finding may reflect the small sample number ofM. fortuitum in this study. Tigecycline displayed activity against RGM organisms [26]. We found that tigecycline had more activity than minocycline and doxycycline, with lower MIC50 and MIC90 values.RGM strains have been shown to be susceptible to the newer generation of macrolide antibiotics (i.e., clarithromycin, azithromycin, and roxithromycin) [25, 26, 30]. This drug class is a good alternative for treating RGM species because of its high activity and oral formulations. Clinical experience shows that azithromycin toxicity is dose dependent and most adult patients withM. avium complex (MAC) lung disease do not tolerate azithromycin doses greater than 300 mg/day because of frequent of adverse events, including gastrointestinal symptoms (primarily diarrhea) and reversible hearing impairment [26]. In our research, the isolates were less susceptible to clarithromycin than to azithromycin, making the latter more applicable in the future. Some reports suggest that clarithromycin resistance can be induced inM. abscessus andM. fortuitum, and the resistance is associated with erm (41) and erm (39) genes, respectively [26, 32]. TheM. abscessus complex is subclassified into three closely related subspecies (M. abscessus,M. massiliense, andM. bolletii) [33]. Historically, theM. fortuitum group has included three species:M. fortuitum,M. peregrinum, and an unnamed third biovariant complex [34]. The different subspecies potentially exhibit different drug susceptibilities. Macrolide resistance inM. abscessus andM. fortuitum will be the subject of our future research.Cefoxitin and cefmetazole are second and third generation cephalosporin antibiotics and in previous studies cefoxitin was frequently found to have good mycobacterial activity [25, 26, 28, 30]. The isolates were more susceptible to cefmetazole than cefoxitin, so cefmetazole can be used where cefoxitin is ineffective. In addition, the antibacterial mechanism for meropenem, a carbapenem antibiotic, occurs via inhibition of bacterial cell wall synthesis [35]. Carbapenems include imipenem, meropenem, and ertapenem, among others. In previous studies, imipenem was widely used in experiments [25, 26, 30]. Here, among the RGM strains, meropenem was found to have good activity and a lower MIC value than cefoxitin. In clinical work, rifabutin has been used mainly to target SGM, and toxicity to this drug was dose related. Clarithromycin has been shown to increase rifabutin serum levels and this effect was likely to be related to the hepatic metabolism of rifabutin [26]. Our data indicates that rifabutin and rifapentine can be used to treat rifampicin-resistant strains; the RGM isolates have better susceptibility to rifabutin than rifapentine, but rifapentine was more active againstM. chelonae,M. abscessus, andM. fortuitum. Otherwise, the rifabutin dose should be reduced when used in combination with clarithromycin to treat infections with RGM strains.Several studies have shown that linezolid and clofazimine have potent activities against NTM [25, 26, 29, 30, 36]. In our experiments, linezolid had >95% activity against the strains tested and clofazimine > 60% susceptibility for the RGM isolates (with the exception ofM. chelonae). The smallM. chelonae sample size probably affected this result. In the future, a larger sample size should be used to determine the MICs of the 31 drugs used in this study.The RGM isolates used in our research were from four different Chinese provinces. Most of them (86.30%) were from Fujian Province, making it important to obtain more samples from different geographical areas in the future.The data presented here suggest that tigecycline or linezolid combined with clofazimine or cefmetazole should be the most efficacious drug combination for treatingM. abscessus infections. ForM. fortuitum, capreomycin, sulfamethoxazole, tigecycline, clofazimine, and cefmetazole in combination may be a good choice.


## 5. Conclusions

In summary, our data provide useful information on antibiotics that are effective against RGM and this information may help to identify suitable therapy for patients infected with such organisms. Future studies should address whether combining two or more antimicrobial agents for treatment of RGM infections is better than treatment with a single drug alone.



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*Source: 419392-2015-08-13.xml*