Molecular characteristics of drug-resistance Mycobacterium tuberculosis strains isolated from extra pulmonary tuberculosis sites

ObjectivesChina is the second high tuberculosis (TB) burden country in the world. This article was to determinate the molecular characteristic of drug resistance Mycobacterium tuberculosis (DRTB) strains from extra pulmonary tuberculosis (EPTB).MethodsThe medical records of patients with EPTB were reviewed and collected from 2006 to 2016. The drug sensitivity of all samples was studied. All multiple drug resistance (MDR) and extensive drug resistance (XDR) strains were included. The detection of the deletion of region of difference 105 (RD105) and mycobacterial interspersed repetitive-unit variable-number tandem-repeat (MIRU-VNTR) were used to discriminate the molecular type of EPTB strains.Results162 DRTB isolates were from patients with EPTB including 104 male and 58 female. Beijing genotype had a significant correlation with the patterns of DR (P < 0.05), re-treatment patients (P < 0.05) and gender (P < 0.05). The history of treatment had a statistically significant correlation with patterns of DR (P < 0.05) and gender (P < 0.05). Patterns of DR had no correlation with gender (P > 0.05). Of 162 strains Beijing family strains represented 91.4%. The cluster rate was 17.9% and clustering ratio was 11.1%. Beijing family genotype is predominant in the patients with EPTB. The cluster rate and clustering ratio was low.ConclusionsBeijing family genotype is predominant and highly epidemic in the patients with drug resistance extra pulmonary tuberculosis (DR-EPTB). The cluster rate and clustering ratio was low. Genotype of re-treatment male patient with DR-EPTB is more likely Beijing family genotype.     

福建省耐多药结核分枝杆菌对氟喹诺酮类药物表型耐药与gyrA基因突变特征分析

目的 了解福建省耐多药(Multi-drug resistant, MDR)结核分枝杆菌(Mycobacterium tuberculosis, Mtb)氟喹诺酮类药物(Fluoroquinolones,FQs)的gyrA基因突变特征,为FQs耐药菌株的快速分子药敏检测提供基础科学数据。方法 收集来自福建省2010-2011年和2008-2009年耐药监测点的所有MDR Mtb临床菌株,采用常规比例法进行FQs敏感性试验。PCR扩增包含gyrA耐药决定区的基因片段,测序后比对分析。结果 共收集到MDR结核分枝杆菌临床菌株119株,经常规药敏试验,氧氟沙星(Ofloxacin,Ofx)的耐药率为26.89%,左氧氟沙星(Levofloxacin,Lfx)耐药率为25.21%,莫西沙星(Moxifloxacin,Mfx)耐药率为11.76%。FQs敏感株gyrA基因未检测到突变。gyrA基因在Ofx耐药菌株的突变率为84.38%(27/32),Lfx耐药菌株的突变率为83.33%(25/30), Mfx耐药菌株的突变率为92.86%(13/14)。gyrA基因突变为点突变,共发现有5种突变类型,以Asp94Gly,Asp94Asn和Ala90Val为主。结论 福建省MDR-Mtb对FQs耐药的主要原因是gyrA基因突变,最常见的突变位于第94位,第90位和第91位密码子。     

Molecular characterisation of streptomycin-resistant Mycobacterium tuberculosis strains isolated in Poland.

Primary drug resistance of Mycobacterium tuberculosis strains in Poland increased two-fold between 1997 and 2000. Among 3705 drug-resistant strains investigated in 2000, 169 were resistant to streptomycin alone or in combination with isoniazid, rifampicin and/or ethambutol. The molecular basis of streptomycin resistance for 88 (52%) of these strains in comparison with 15 susceptible controls was determined. The most prevalent mutation was the single substitution Lys43Arg in the rpsL gene, found in 30.7% of the strains analysed. However, as many as 51% of the strains investigated carried no mutation in the rpsL or rrs genes. The multiple mutations present in two Beijing family strains were also identified.     

Molecular characterization of rifampicin- and isoniazid-resistant Mycobacterium tuberculosis strains isolated in Kazakhstan

Kazakhstan is one of the 14 countries with a high rate of morbidity due to multidrug-resistant tuberculosis (MDR TB) in WHO European region. The aim of our study was to characterize mutations associated with drug resistance to rifampicin and isoniazid in Mycobacterium tuberculosis isolates from Kazakhstan. M. tuberculosis strains were isolated from TB patients in different regions of Kazakhstan. A drug susceptibility test was performed on Lowenstein-Jensen medium using the absolute concentration method. Sequencing analysis was performed of the rpoB rifampicin resistance-determining region and the katG gene, the oxyR-ahpC intergenic region, and the inhA promoter region in 259 MDR M. tuberculosis isolates, in 51 isoniazid-resistant isolates, and in 13 rifampicin-resistant isolates. The mutational analysis revealed that the most frequent mutations associated with rifampicin and isoniazid resistance in M. tuberculosis are the substitutions at codons 531 (82.7%) and 315 (98.4%) in the rpoB and katG genes, respectively. In addition, we have found mutations with lower frequency at codon 526 (8.4%), 533 (1.5%), and 516 (1.1%) in the rpoB gene. In 6.2% of the isolates, no mutations were found in the rpoB gene. The findings of this study provide useful data for a better understanding of the mutation spectrum of isoniazid and rifampicin resistance among strains isolated from patients in Kazakhstan. Our results are also useful for the development of diagnostic tests of MDR M. tuberculosis.     

GenoType MTBDRplus快速检测耐多药结核分枝杆菌复合群临床分离株效果评价

目的评价GenoType MTBDRplus快速检测耐多药结核分枝杆菌复合群(multi-drug resistant Mycobacterium tuberculosis Complex,MDR-MTBC)临床分离株的应用效能。方法随机抽取福建省30个耐药监测点的129株MTBC包括全敏感菌株50株和79株MDR菌株,应用MTBDRplus 检测,同时扩增MDR菌株的rpoB, katG, inhA基因进行测序比对,并将检测结果与比例法药敏结果比较分析。结果129株MTBC经MTBDRplus检测,Tub条带均阳性(100%),50株全敏感株的检测结果与比例法药敏结果完全一致。79株MDR菌株经MTBDRplus检出67株(84.81%)MDR,10株(12.66%)单耐RFP,2株(2.53%)单耐INH。经测序,77株MDR菌株的rpoB基因检测出突变,71株的katG 和/或inhA检测出突变。以比例法药敏结果为金标准,MTBDRplus检测RFP、INH耐药性及MDR的敏感度分别为97.47%(77/79), 87.34%(69/79), 84.81%(67/79),MTBDRplus检测RFP、INH耐药性及MDR的特异度均为100%(50/50),一致率分别为98.45%(127/129),92.25%(119/129),90.70%(117/129)。结论MTBDRplus是一种快速、准确、操作简便的技术,具有很好的应用前景,适用于耐多药结核分枝杆菌临床分离株的快速检测,但灵敏度有待进一步提高。     

Molecular genetic characteristics of Mycobacterium tuberculosis isolated from patients operated on for pulmonary tuberculosis

The RFLP-IS6110 assay was used to genotype 67 Mycobacterium tuberculosis isolated from different specimens (including intraoperative ones) of 24 patients operated on for chronic progressive pulmonary tuberculosis who lived in north-western Russia. More than half (53%) of 17 types of RFLP profiles of isolates are identical and typical of M. tuberculosis of the Beijing family. Comparing the results of isolate genotyping with clinical, microbiological, and pathomorphological findings allows cases of endogenous reactivation to be differentiated from exogenous (nosocomial, in particular) reinfection in recurrence. Beijing genotype strains are shown to cause more severe unarrested course of drug-resistant fibrocavernous tuberculosis. At the same time such strains are more frequently detected in relatively young persons with a shorter duration of the disease, which reflects current clonal Beijing genotype dissemination that present a severe epidemiological hazard.     

江西省结核分枝杆菌耐二线抗结核药的分子学特征

目的 初步明确江西省耐二线抗结核药相关基因突变的特征,评价等位基因特异性多重PCR（multiplex allele-specific polymerase chain reaction,MAS-PCR）检测二线抗结核药耐药性的可行性。 方法 采用DNA测序方法和MAS-PCR技术,对江西省52株耐二线抗结核药的结核分枝杆菌进行耐药相关基因突变位点检测。 结果DNA测序分析：39株耐氧氟沙星（Ofx）菌株中,32株存在gyrA基因错义突变,2株发生gyrB基因错义突变。29株耐卡那霉素（Km）或卷曲霉素（Cm）菌株中,22株为rrs1401位点A→G突变。52株耐二线抗结核药的结核分枝杆菌,40株为北京基因型（76.92%,40/52）,其中17株北京基因型菌株发生gyrA-GAC94GGC突变（42.50%,17/40）,19株北京基因型菌株发生rrs-A1401G突变（47.50%,19/40）。北京基因型与基因突变类型gyrA-GAC94GGC和rrs-A1401G无明显相关性（χ²=1.16、1.92,P值均＞0.05）。MAS-PCR检测Ofx耐药株的敏感度为61.54%（24/39）,检测Km或Cm耐药株的敏感度为79.31%（23/29）。 结论 gyrA基因94位密码子突变是江西省结核分枝杆菌Ofx耐药的主要机制; rrs-A1401G突变则是Km或Cm耐药的主要原因。MAS-PCR方法对于快速检测二线抗结核药的耐药性有一定的临床应用价值。     

Drug-resistant strains of Mycobacterium tuberculosis isolated in Russia.

SETTING: State Research Center for Applied Microbiology, Russian Research Institute of Phthisiopulmonology (Ministry of Health, Moscow). OBJECTIVE: To analyze drug-resistant clinical isolates of Mycobacterium tuberculosis obtained from patients referred to the institute from different parts of Russia, and to study the mechanisms of their rifampicin resistance. DESIGN: Fifty clinical isolates of M. tuberculosis were analysed. Polymerase chain reaction (PCR) and sequencing were used to study the mechanisms of rifampicin resistance in 25 isolates. RESULTS: Among cultures isolated from 50 patients, drug resistance was detected in 33. Most of the isolates were resistant to rifampicin (25 isolates), isoniazid (14 isolates), and streptomycin (seven isolates). Only 6% of the isolates were resistant to one drug, while 14% were resistant to two, 32% to three, 40% to four, and 8% to five drugs. Susceptible isolates were derived from 17 patients. The following point mutations and deletions in the rpoB locus, responsible for high level rifampicin resistance (more than 50 microg/ml in egg-based medium), were detected: G-->A/395 (Arg-->Gln), C-->T/232 (His-->Tyr), C-->T/221 (Ser-->Leu), G-->T/202 (Asp-->Tyr), GA-->TT/202-203 (Asp-->Phe), deltaATGGACCAG/199-207 (Met, Asp, Gin), A-->T/91 (Met-->Leu), TG-->CC/227-228 (Leu-->Ser), GAG-->AGT/349-350-351 (Gln-->Ser), deltaGGG/354(Gly). CONCLUSION: A number of previously unrecognised genetic modifications in the rpoB region were found in rifampicin-resistant strains isolated from patients from different parts of Russia.     

Change in biochemical and biological characteristics of Mycobacterium tuberculosis strains isolated in recent years

One hundred strains of Mycobacterium tuberculosis isolated in 1988-89 from patients who were newly hospitalized in the National Chubu Hospital were studied on their biochemical and biological characteristics and compared with the strains isolated previously. Recently isolated strains showed frequently a much stronger arylsulfatase activity, grew at a higher rate at 42 degrees C, and showed a little stronger niacin production.     

Molecular fingerprinting of Mycobacterium tuberculosis strains isolated in Vietnam using IS6110 as probe.

SETTING: Northern and Southern areas of Vietnam. OBJECTIVE: To study the correlation between DNA fingerprinting of 168 Mycobacterium tuberculosis strains isolated from patients with a particular historical past (political separation of Vietnam for 20 years) and data about geographical origin, drug susceptibility, HIV infection and BCG vaccination status. METHODS: Comparison of restriction fragment length polymorphism (RFLP) patterns produced by Southern hybridization of Pvull-digested chromosomal DNA. RESULTS: The number of IS6110 copies for the 168 strains ranges from 0 to 23. Strains originating from the North or the South differ strongly with respect to the number of copies of IS6110. Indeed, the strains originating from the north have predominantly from 3 to 14 IS6110 copies while the southern strains have predominantly from 15 to 23 IS6110 copies. Furthermore, strains isolated in the North are dispersed into 6 groups whereas 80% of the strains isolated in the South form a single group. Moreover, the prevalence of drug resistance is higher in strains isolated in the South than in the North. No noticeable correlation is observed between RFLP patterns, drug susceptibility, or HIV infection. CONCLUSION: The IS6110 fingerprints of 168 M. tuberculosis strains isolated in Vietnam showed a high range of polymorphism. Only a few strains have been found with no IS6110 (1.8%). The differences between the strains from the North and South, having more than six IS6110, suggests that they derived from ancestral strains that would be distinguishable by the number of IS6110 and their transposition sites throughout the genome. The genomic structure of the population of strains from South Vietnam resembles that of the Beijing strain population. This could account for a similar evolution of M. tuberculosis due to a selection by BCG-induced immunity in the two populations.     

分枝杆菌临床分离株菌种的分子鉴定

目的 建立对临床分离分枝杆菌菌种快速、精确鉴定的方法学平台,了解分枝杆菌菌种的分布,为结核分枝杆菌(MTB)和非结核分枝杆菌(NTM)病临床精确诊断和有效治疗提供依据.方法 330株分枝杆菌分离株来自于2007年5月至2008年12月在哈尔滨市胸科医院诊断为结核病的住院患者.菌株灭活后提取基因组DNA,采用PCR方法,结合PCR-限制性片段长度多态性(RFLP)和DNA序列测定方法进行菌种精确鉴定.结果 330株临床分离菌株全部精确鉴定,其中328株[占99.4％(328/330)]为结核分枝杆菌复合群(MTBC),2株[占0.6％(2/330)]为NTM.328株MTBC中,MTB 326株、非洲分枝杆菌(M.African)1株、田鼠分枝杆菌(M.microti)1株,分别占MTBC的99.4％ (326/328)、0.3％(1/328)和0.3％(1/328).经测序和基因组同源性分析发现,2株NTM为胞内分枝杆菌(MAC),与MAC的同源性分别为99％和93％,2株菌之间的同源性为93％.结论 建立了临床分离分枝杆菌菌种快速鉴定的方法学平台,临床诊断为结核病的患者MTB比例占绝对优势,也发现有M.African、M.microti和NTM感染.     

福建省布鲁氏菌分离株的分子生物学鉴定

目的利用分子生物学方法鉴定福建省布鲁氏菌分离株。方法对布鲁氏菌分离株进行AMOS-PCR和MLVA分型。结果3株福建省分离株经AMOS-PCR鉴定为羊种,与传统生物学分型一致;MLVA分型将其分为3个基因型,聚类分析鉴定为羊种。结论AMOS-PCR、MLVA分型可以作为我省布鲁氏菌分离株鉴定的辅助手段。     

波兰肺结核患者耐药结核分枝杆菌分子流行病学研究

目的：描述波兰耐药结核分枝杆菌分离株的特征，估计人群中近期传播的数量。 设计：应用间隔区寡核苷酸分型及IS6110DNA指纹图谱法对2000年波兰251名耐药肺结核患者分离株进行分析。对部分菌株进行rpoB、katG和，或inhA基因调节区测序分析。 结果：应用间隔区寡核苷酸分型BS6110-RFLP相结合的成簇定义，29％的菌株成簇，提示存在近期传播。成簇菌株中某些病例的传播联系得到了流行病学资料证实，多数分离株的突变与利福平和异烟肼耐药相关。年龄、性别、移民身份、用药史和戍簇没有关系，耐多药病例更容易成簇。在波兰，也发现了北京基因型分离株，但频率较邻国偏低。 结论：证实了耐药结核菌的传播及其对波兰发生耐药结核病的作用。     

重庆地区耐多药结核分枝杆菌三种二线注射类药物耐药相关基因特征分析

目的 分析重庆市耐多药结核病(MDR-TB)临床菌株二线注射类药物耐药相关基因突变特征,为本地区合理应用分子生物学技术进行注射类药物耐药性检测提供科学依据。方法 收集2015年1月至2017年6月重庆市39个区(县)967例MDR-TB可疑患者的所有耐多药(MDR)结核分枝杆菌临床分离株229株,通过微孔板Alamar blue显色法检测3种二线注射类药物[卡那霉素(Km)、阿米卡星(Am)、卷曲霉素(Cm)]的耐药性,用PCR测序方法对其耐药相关基因rrs、eis启动子区、tlyA进行分析,并采用荧光定量熔解曲线方法进行北京基因型鉴定。结果 在229株MDR菌株中,35株(15.3%,35/229)对三种二线注射类药物任一耐药,Km、Am和Cm耐药率分别为14.8%(34/229)、11.8%(27/229)、8.3%(19/229)。在34株Km耐药株中,21株发生rrs基因A1401G突变,1株rrs基因G1484T突变,4株eis启动子区G(-10)A突变及1株C(-14)T突变。在27株Am耐药株中,20株发生rrs基因A1401G突变,1株rrs基因G1484T突变,1株eis启动子区G(-10)A突变。在19株Cm耐药株中,13株发生rrs基因A1401G突变,1株rrs基因G1484T突变,1株tlyA基因137位碱基插入改变。北京与非北京基因型间注射类药物耐药及相关基因突变情况差异均无统计学意义。结论 重庆地区MDR结核分枝杆菌Km、Am和Cm各耐药相关基因呈现不同的突变频率特征,其中rrs基因突变频率最高,可作为三种注射类药物的耐药诊断标记物。北京基因型与二线注射类药物耐药无相关性。     

Drug resistance of Mycobacterium tuberculosis strains isolated from patients with pulmonary tuberculosis in Archangels, Russia.

SETTING: The Archangels oblast, Russia, 1998-2000. OBJECTIVE: To study Mycobacterium tuberculosis resistance to anti-tuberculosis drugs in the Archangels oblast, and to reveal risk factors for the development of drug-resistant tuberculosis. DESIGN: The drug susceptibility of strains isolated from 119 patients with pulmonary tuberculosis was studied using the BACTEC method. Medical records of the patients were reviewed, retrospectively, to identify factors associated with drug resistance. RESULTS: Sixty-seven strains (56.3%) were resistant to at least one anti-tuberculosis drug. The highest rates of resistance were observed for streptomycin and isoniazid: respectively 40.4% and 66.7% of strains isolated from newly and previously treated patients were resistant to streptomycin, and respectively 37.1% and 73.3% of strains isolated from newly and previously treated patients were resistant to isoniazid. Thirty of the 119 strains (25.2%) were multidrug-resistant. Multidrug resistance was four times higher among previously treated patients than among new patients. A history of previous or interrupted treatment for tuberculosis and being female were significantly associated with drug resistance. CONCLUSION: Drug-resistant tuberculosis is an important problem in the Archangels oblast, Russia. The spread of drug resistance is attributed to several risk factors. Being female and evidence of previous treatment for tuberculosis are risk factors for the development of drug-resistant tuberculosis in the Archangels oblast. Patients with drug-resistant tuberculosis also showed a higher risk of interrupting their treatment.