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庞慧, 李桂莲, 万康林, 余平. 快速生长分枝杆菌标准株对10种新型抗菌药物的敏感性[J]. 中国人兽共患病学报, 2015,31(9): 783-788
PANG Hui, LI Gui-lian, WAN Kang-lin, YU Ping. Drug susceptibility of reference species of rapidly growing mycobacteria against 10 new antimicrobial agents[J]. CHINESE JOURNAL OF ZOONOSES, 2015,31(9): 783-788  
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Copyright©2015, 中国人兽共患病学报
中国人兽共患病学报
快速生长分枝杆菌标准株对10种新型抗菌药物的敏感性
庞慧1,2,3, 李桂莲3,4, 万康林3,4, 余平1
1.中南大学湘雅医学院基础医学院免疫学系,长沙 410078
2.长治医学院免疫学教研室,长治 046000
3.中国疾病预防控制中心传染病预防控制所国家重点实验室,北京 102206
4.杭州感染性疾病诊断和治疗协同中心,杭州 310000
余平,Email: yuping1953@sina.com; 万康林,Email: wankanglin@icdc.cn
收稿日期:2015-08-11
基金:国家重点感染性疾病项目(2013ZX10004-101, 2013ZX10003002-001); 中国疾病预防控制中心国家重点实验室项目(2014SKLID104); 长治医学院科技创新团队支持项目(CX201412)资助
摘要

目的 越来越多的快速生长分枝杆菌对于常规抗结核药物耐药,并且可以引起种类繁多的疾病。本研究主要聚焦在快速生长分枝杆菌的标准菌株对新型抗菌药物的敏感性实验方面。方法 在体外,应用阳离子调节的MH微量肉汤稀释法,针对40种快速生长分枝杆菌进行10种新型抗菌药物的敏感性实验。结果 结果显示氯法齐明(39/40)具有最好的抗菌活性。司帕沙星(37/40)和头孢美唑(36/40)具有强大的抗菌活性。罗红霉素(33/40)、阿奇霉素(31/40)和利福喷丁(29/40)具有较好的抗菌活性。利福布丁(16/40)对于实验菌株具有中等程度的抗菌活性。氨苯砜(4/40)和帕司烟肼(9/40)具有很低的抗菌活性。氨硫脲(1/40)的抗菌活性最低。在40株快速生长分枝杆菌的标准株当中, Mycobacterium aurum, Mycobacterium duvalii和 Mycobacterium parafortuitum对于测试药物的敏感性最高,可达80%的敏感性。 Mycobacterium massiliense, Mycobacterium boenickei和 Mycobacterium porcinum的药物敏感性最低,只有30%的敏感性。其它物种有40%~70%的敏感性。结论 通过对40株快速生长分枝杆菌标准株的药物敏感性测试,可以为临床医生选择和优化抗菌药物治疗方案提供一定的帮助和依据。

关键词: 药物敏感性; 快速生长分枝杆菌; 抗菌药物; 最小抑菌浓度; 阳离子调节的MH肉汤
中图分类号:R378.91 文献标志码:A 文章编号:1002-2694(2015)09-0783-06
Drug susceptibility of reference species of rapidly growing mycobacteria against 10 new antimicrobial agents
PANG Hui1,2,3, LI Gui-lian3,4, WAN Kang-lin3,4, YU Ping1
1.Department of Immunology, Xiangya School of Medicine, Central South University, Changsha 410078, China
2.Department of Immunology, Changzhi Medical College, Changzhi 046000, China
3.State Key Laboratory for Infectious Disease Prevention and Control, National Institute for Communicable Disease Control and Prevention, Chinese Center for Disease Control and Prevention, Beijing 102206, China
4.Collaborative Innovation Center for Diagnosis and Treatment of Infectious Diseases, Hangzhou 310000, China
Corresponding authors: Yu Ping, Email: yuping1953@sina.com; Wan Kang-lin, Email: wankanglin@icdc.cn
Fund:Financially supported by the Project of the National Key Programme of Mega Infectious Diseases (Nos.2013ZX10004-101 and 2013ZX10003002-001), the Key Project (No.2014SKLID104) of the State Key Laboratory for Infectious Disease Prevention and Control, and a Science and Technology Innovation Team Support Project (No.CX201412) from Changzhi Medical College
Abstract

An increasing number of rapidly growing mycobacteria (RGM) are being found to be resistant to conventional antituberculous agents and have been associated with a broad spectrum of diseases. In this study, a cation-adjusted Mueller-Hinton broth microdilution assay was employed to assess the susceptibility of 40 reference species of RGM to 10 new antimicrobial agents. Clofazimine (39/40) was found to be the most effective antimicrobial agent tested against the RGM species, closely followed by sparfloxacin (37/40) and cefmetazole (36/40). Roxithromycin (33/40), azithromycin (31/40) and rifapentine (29/40) displayed a good level of activity, and rifabutin (16/40) showed moderate activity. Dapsone (4/40) and pasiniazid (9/40) displayed little antimicrobial activity against RGM species, and thioacetazone (1/40) was the least effective agent. Among the 40 reference RGM species, Mycobacterium aurum, Mycobacterium duvalii and Mycobacterium parafortuitum exhibited 80% susceptibility to the tested drugs, and Mycobacterium massiliense, Mycobacterium boenickei and Mycobacterium porcinum exhibited 30% susceptibility to these agents. All other species displayed 40-70% susceptibility. These data will be valuable for physicians when formulating and optimizing drug treatment programs for RGM.

Keywords: drug susceptibility; rapidly growing mycobacteria; antimicrobial agents; minimum inhibitory concentration; cation-adjusted Mueller-Hinton broth

Rapidly growing mycobacteria (RGM) are hardy microbes that are ubiquitous, being found in municipal tap water and biofilms in piped water systems[1]. Infections caused by RGM are becoming an increasing threat to human health worldwide[2]. RGM are Gram-positive bacilli that usually produce visible colonies within 7 days when cultured at room temperature[3]. RGM organisms cause a variety of clinical manifestations including skin and soft tissue infections, osteomyelitis, respiratory tract infections and disseminated infections[4]. More than 50 species of RGM have been described[1, 3] including Mycobacterium abscessus, Mycobacterium chelonae, the Mycobacterium fortuitum group, Mycobacterium peregrinum, Mycobacterium immunogenum and the Mycobacterium smegmatis group[1].

However, the drug susceptibility profiles of these different RGM species are lacking. In this study, an in vitro microdilution assay was employed to assess the susceptibility of 40 reference species of RGM to 10 new antimicrobial agents according to the criteria of the Clinical and Laboratory Standards Institute[5].

Materials and methods
Strains and culture conditions

In total, 40 international standard RGM strains from Deutsche Sammlung Von Mikroorganismen and Zellkulturen (DSMZ) and the American Type Culture Collection (ATCC) were used in this study. The strains were grown in DifcoTM Middlebrook 7H10 agar (BD Biosciences, San Jose, CA, USA) at 37 ℃, with the exception of Mycobacterium chelonae and Mycobacterium pseudoshottsii that were cultured at 32 ℃ and 25 ℃ respectively. Drug susceptibility testing was performed using BBLTM cation-adjusted Mueller-Hinton (CAMH) broth (BD Biosciences).

Drug susceptibility testing

Drug susceptibility testing was performed for 10 antimicrobial agents using 96-well microplates by the broth microdilution method as recommended by the Clinical and Laboratory Standards Institute[5]. All of the experiments were carried out in duplicate. The final minimum inhibitory concentration (MIC) of each antibacterial agent for each bacterium was the mean value derived from these two tests. The inocula were adjusted with saline to a density of 0.5 McFarland standard, with a density of approximately 1× 107 colony forming units/mL. Then, 50 μ L of the suspension were added to 10 mL of CAMH broth and mixed thoroughly. Antibiotics were serially diluted two-fold in 100 μ L of CAMH broth. The final reaction volume was 200 μ L. The range of concentrations used for sparfloxacin, clofazimine, roxithromycin, azithromycin, rifapentine and rifabutin was 0.03-32 μ g/mL, while that for dapsone, thioacetazone, pasiniazid and cefmetazole was 0.25-256 μ g/mL. Two negative controls were included: a no-drug control (used to determine the time of alamar blue addition) and a CAMH broth-only control (used to analyze the interference of CAMH with alamar blue). The 96-well microplates were placed in plastic bags and cultured at the appropriate temperature. Plates were examined after 3 days. In the case of the no-drug control, when the indicator (alamar blue and sterile 5% Tween-80) turned the suspension pink, this indicated that the MIC value should be determined. If this had not occurred by day 5, the experiment was repeated. The MIC breakpoints of the antibacterial agents exhibiting sensitivity, moderate sensitivity and resistance were defined according to the guidelines of the World Health Organization[6] or published literature: sparfloxacin[7], clofazimine[8], roxithromycin[9], azithromycin[9], dapsone[10], thioacetazone[11], rifapentine[12], rifabutin[12] and cefmetazole[13] (Table 1).

Tab.1 Minimum inhibitory concentration (MIC) (μ g/mL) breakpoints of 31 antimicrobial agents
Results

Of the 10 antimicrobial agents tested against the 40 reference RGM species, clofazimine (39/40) showed excellent antimicrobial activity, followed closely by sparfloxacin (37/40) and cefmetazole (36/40). Roxithromycin (33/40), azithromycin (31/40) and rifapentine showed a good level of antimicrobial activity, rifabutin (16/40) showed moderate activity, whereas dapsone (4/40), thioacetazone (1/40) and pasiniazid (9/40) showed little activity (Table 2). Clofazimine therefore showed the highest activity and thioacetazone showed the lowest activity against the reference RGM species (Figure 1).

Fig.1 Susceptibility distribution of the 40 reference species of rapidly growing mycobacteria against 10 antimicrobial agents (SPA: sparfloxacin; CLO: clofazimine; ROX: roxithromycin; AZM: azithromycin; DAP: dapsone; THI: thioacetazone; PASI: pasiniazid; RPT: rifapentine; RBT: rifabutin; CMZ: cefmetazole.)

Tab.2 MIC values (μ g/mL) for the antimicrobial susceptibility of reference species of rapidly growing mycobacteria

Among the 40 reference RGM species, Mycobacterium aurum, Mycobacterium duvalii and Mycobacterium parafortuitum displayed 80% susceptibility to the tested drugs, and Mycobacterium massiliense, Mycobacterium boenickei and Mycobacterium porcinum displayed 30% susceptibility to the antibacterial agents. All other species showed 40-70% susceptibility (Table 3).

Tab.3 Susceptibility rates of the 40 reference species of rapidly growing mycobacteria against 10 antimicrobial agents
Discussion

RGM pose a threat to human health worldwide. However, data are lacking regarding the drug susceptibility of RGM species. In this study, the susceptibility profiles of 10 drugs were evaluated for 40 reference RGM species. Clofazimine, sparfloxacin and cefmetazole displayed excellent activity against the RGM species, while Mycobacterium massiliense, Mycobacterium boenickei and Mycobacterium porcinum showed resistance to multiple drugs.

A previous study reported that sparfloxacin was a new generation fluoroquinolone with good activity against Mycobacterium tuberculosis[14]. In our study, sparfloxacin also showed strong activity against RGM organisms, with the exception of Mycobacterium chelonae, Mycobacterium bolletii and Mycobacterium pseudoshottsii. Clofazimine, a drug designed for tuberculosis therapy[15], has also been reported to be effective against RGM strains[15, 16]. In our study, clofazimine showed excellent antimicrobial activity against all species, with the exception of Mycobacterium massiliense. Roxithromycin and azithromycin are macrolide antibiotics with wide ranging antimicrobial activities[9]. These antibiotics were also found to be effective in our study. Rifapentine and rifabutin were derivatives of rifampin, showing higher activity and, on occasion, cross resistance with rifampin[12, 17]. In our study, compared with rifabutin, rifapentine showed increased activity against Mycobacterium chelonae, Mycobacterium abscessus, Mycobacterium senegalense, Mycobacterium flavescens, Mycobacterium brumae, Mycobacterium canariasense, Mycobacterium confluentis, Mycobacterium fluoranthenivorans, Mycobacterium poriferae and Mycobacterium vanbaalenii. Rifabutin in turn showed higher activity against Mycobacterium bolletii and Mycobacterium wolinskyi. Cefmetazole is one of the cephalosporin antibiotics reported to have powerful activity against Gram-negative bacilli[13]. In our study, it was effective against most RGM species. Dapsone, thioacetazone and pasiniazid have been widely employed to combat Mycobacterium tuberculosis infections[6, 10, 11]. Our research demonstrated that these three drugs showed little activity against RGM organisms.

In summary, most of the antibacterial agents tested in our study have activity against RGM species and could be used therapeutically, especially clofazimine, sparfloxacin and cefmetazole. In the future, other cephalosporin antibiotics should be investigated for their effectiveness against RGM species, with the aim of further expanding the repertoire of therapeutic agents to treat RGM pathogens.

The authors have declared that no competing interests exist.

Acknowledgments

We thank the staffs of the State Key Laboratory for Infectious Disease Prevention and Control, National Institute for Communicable Disease Control and Prevention, Chinese Center for Disease Control and Prevention.

The authors have declared that no competing interests exist.

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