Mycobacterium tuberculosis drug susceptibility and drug resistance mechanism to cycloserine
NIU Jin-xia1,2, CUI Zhen-ling3, PANG Wen-hui4, ZHU Chang-tai2, FAN Lin3
1. Shanghai Ocean University, College of Fisheries and Life Sciences, Shanghai 201306, China; 2. Department of Transfusion, Shanghai Jiao Tong University Affiliated Sixth People’s Hospital, Shanghai 200233, China; 3. Shanghai Pulmonary Hospital, Tongji University School of Medicine,Shanghai Key Laboratory of Tuberculosis Shanghai 200433, China; 4. China Ocean University, College of Environmental Science and Engineering, Qingdao 266100, China
Abstract:To detect the minimum inhibitory concentration of (MIC) circulating serine in clinical isolates of multidrug-resistant tuberculosis (MDR-TB), we studied the resistance mechanism of the genotypes of cycloserine-resistance Mycobacterium tuberculosis (M. tuberculosis), and provide the scientific evidence for the rapid resistance determination of cycloserine. The 140 clinical strains of drug-resistant M. tuberculosis and 37 clinical strains of drug-susceptible M. tuberculosis were isolated and tested by Middlebrook 7H9 liquid medium in 96-well plate. The Ald, Alr and ddlA mutations and gene expression levels were analysed to define the mechanism of cycloserine-resistant M. tuberculosis. The results showed cycloserine-resistance rates were as low as 4.28% in 140 strains of drug-resistant M. tuberculosis, we preliminarily defined the MICs of M. tuberculosis ≥32 μg/mL as cycloserine-resistance. There were no significant correlations between Ald, Alr, ddlA gene mutations and cycloserine-resistant M. tuberculosis. The Alr gene expression levels in cycloserine-resistant M. tuberculosis were significantly higher than those in cycloserine-susceptible M. tuberculosis. It is indicated that the drug resistance rate of cycloserine is low in MDR-TB, suggesting that cycloserine can be an effective and reliable clinical choice for treatment on MDR-TB. The cycloserine-resistance mutation in MTB has not been found, yet the overexpression of Alr gene found to be highly related to the cycloserine-resistance of MTB, providing a possible explanation of MTB resistance mechanism.
牛金霞, 崔振玲, 逄文慧, 朱长太, 范琳. 结核分枝杆菌环丝氨酸药物敏感性及耐药机制研究[J]. 中国人兽共患病学报, 2019, 35(1): 39-44.
NIU Jin-xia, CUI Zhen-ling, PANG Wen-hui, ZHU Chang-tai, FAN Lin. Mycobacterium tuberculosis drug susceptibility and drug resistance mechanism to cycloserine. Chinese Journal of Zoonoses, 2019, 35(1): 39-44.
[1] Falzon D, Jaramillo E, Schunemann HJ, et al.WHO guidelines for the programmatic management of drug-resistant tuberculosis: 2011 update[J]. Eur Respir J,2011,38(3):516-528. [2] Janmeja AK, Raj B.Acquired drug resistance in tuberculosis in Harayana, India[J]. J Assoc Physicians India,1998,46(2):194-198. DOI:10.4103/0971-5916.155593 [3] Balabanova Y, Ruddy M, Hubb J, et al.Multidrug-resistant tuberculosis in Russia: clinical characteristics, analysis of second-line drug resistance and development of standardized therapy[J]. Eur J Clin Microbiol Infect Dis,2005,24(2):136-139. DOI:10.1007/s10096-004-1268-4 [4] Otto-Knapp R, Bos L, Schonfeld N, et al.Resistance to second-line drugs in migrants with multidrug-resistant tuberculosis in the Berlin region[J]. Pneumologie,2014,68(7):496-500. DOI:10.1055/s-0034-1377226 [5] Mok JH, Kang BH, Lee T, et al.Additional drug resistance patterns among multidrug-resistant tuberculosis patients in Korea: implications for regimen design[J]. J Korean Med Sci,2017,32(4):636-641. DOI:10.3346/jkms.2017.32.4.636 [6] 中国防痨协会基础专业委员会. 结核病诊断实验室检验规程[M]. 中国教育文化出版社,2006.46-51. [7] Baisa G, Stabo NJ, Welch RA.Characterization of Escherichia coli D-cycloserine transport and resistant mutants[J]. J Bacteriol,2013,195(7):1389-1399. DOI:10.1128/JB.01598-12 [8] Feng Z, Barletta RG.Roles of Mycobacterium smegmatis D-alanine:D-alanine ligase and D-alanine racemase in the mechanisms of action of and resistance to the peptidoglycan inhibitor D-cycloserine[J]. Antimicrob Agents Chemother,2003,47(1):283-291. DOI:10.1128/AAC.47.1.283-291.2003 [9] 唐神结,徐绍发,李亮,等.耐药结核病学[M]. 北京:人民日报出版社,2014.63-64. [10] World Health Organization. Global tuberculosis report2017[R]. Geneva, Switzerland. [11] 陈松华,王晓萌,柳正卫,等. 浙江省耐多药结核病例中二线耐药状况分析[J]. 中国预防医学杂志,2011,12(9):761-764. DOI:10.16506/j.1009-6639.2011.09.013 [12] 赵兵,宋媛媛,逄宇,等.中国耐多药结核分枝杆菌二线抗结核药物敏感性分析[J]. 中国防痨杂志,2013,10(35):831-834. DOI:10.3969/j.issn.2095-1752.2017.01.028 [13] Tb-Alliance.Handbook of anti-tuberculosis agents. Introduction[J]. Tuberculosis (Edinb),2008,88(2):85-86. DOI:10.1016/S1472-9792(08)70002-7 [14] Bruning JB, Murillo AC, Chacon O, et al.Structure of the Mycobacterium tuberculosis D-alanine:D-alanine ligase, a target of the antituberculosis drug D-cycloserine[J]. Antimicrob Agents Chemother,2011,55(1):291-301. DOI:10.1128/AAC.00558-10 [15] Halouska S, Chacon O, Fenton RJ, et al.Use of NMR metabolomics to analyze the targets of D-cycloserine in mycobacteria: role of D-alanine racemase[J]. J Proteome Res,2007,6(12):4608-4614. DOI:10.1021/pr0704332 [16] Caceres NE, Harris NB, Wellehan JF, et al.Overexpression of the D-alanine racemase gene confers resistance to D-cycloserine in Mycobacterium smegmatis[J]. J Bacteriol,1997,179(16):5046-5055. DOI:10.1128/jb.179.16.5046-5055.1997 [17] Belanger AE, Porter JC, Hatfull GF.Genetic analysis of peptidoglycan biosynthesis in mycobacteria: characterization of a ddlA mutant of Mycobacterium smegmatis[J]. J Bacteriol,2000,182(23):6854-6856. DOI:10.1128/JB.182.23.6854-6856.2000 [18] 于霞. 环丝氨酸对结核分枝杆菌的耐药性及耐药的分子机制研究[C]. 2011年中国防痨协会全国学术会议论文集, 2011. [19] Desjardins CA, Cohen KA, Munsamy V, et al.Genomic and functional analyses of Mycobacterium tuberculosis strains implicate ald in D-cycloserine resistance[J]. Nature Genetics,2016,48(5):544-551. DOI:10.1038/ng.3548 [20] Prosser GA, de Carvalho LP. Metabolomics reveald D-alanine:D-alanine ligase as the target of D-cycloserine in Mycobacterium tuberculosis[J]. ACS Medicinal Chemistry Letters,2013,4(12):1233-1237. DOI:10.1021/ml400349n [21] Saier MJ, Yen MR, Noto K, et al.The transporter classification database: recent advances[J]. Nucleic Acids Res,2009,37(Database issue):D274-D278. DOI:10.1093/nar/gkn862 [22] Chen JM, Uplekar S, Gordon SV, et al.A point mutation in cycA partially contributes to the D-cycloserine resistance trait of Mycobacterium bovis BCG vaccine Strains[J]. PLOS One,2012,7(8). DOI:10.1371/journal.pone.0043467