Effect of mutation and genetic background on drug resistance in Mycobacterium tuberculosis

Bacterial factors may contribute to the global emergence and spread of drug-resistant tuberculosis (TB). Only a few studies have reported on the interactions between different bacterial factors. We studied drug-resistant Mycobacterium tuberculosis isolates from a nationwide study conducted from 2000 to 2008 in Switzerland. We determined quantitative drug resistance levels of first-line drugs by using Bactec MGIT-960 and drug resistance genotypes by sequencing the hot-spot regions of the relevant genes. We determined recent transmission by molecular methods and collected clinical data. Overall, we analyzed 158 isolates that were resistant to isoniazid, rifampin, or ethambutol, 48 (30.4%) of which were multidrug resistant. Among 154 isoniazid-resistant strains, katG mutations were associated with high-level and inhA promoter mutations with low-level drug resistance. Only katG(S315T) (65.6% of all isoniazid-resistant strains) and inhA promoter -15C/T (22.7%) were found in molecular clusters. M. tuberculosis lineage 2 (includes Beijing genotype) was associated with any drug resistance (adjusted odds ratio [OR], 3.0; 95% confidence interval [CI], 1.7 to 5.6; P < 0.0001). Lineage 1 was associated with inhA promoter -15C/T mutations (OR, 6.4; 95% CI, 2.0 to 20.7; P = 0.002). We found that the genetic strain background influences the level of isoniazid resistance conveyed by particular mutations (interaction tests of drug resistance mutations across all lineages; P < 0.0001). In conclusion, M. tuberculosis drug resistance mutations were associated with various levels of drug resistance and transmission, and M. tuberculosis lineages were associated with particular drug resistance-conferring mutations and phenotypic drug resistance. Our study also supports a role for epistatic interactions between different drug resistance mutations and strain genetic backgrounds in M. tuberculosis drug resistance.     

Lack of correlation between embB mutation and ethambutol MIC in Mycobacterium tuberculosis clinical isolates from China

Seventy-four Mycobacterium tuberculosis clinical isolates from China were subjected to drug susceptibility testing using ethambutol, isoniazid, rifampin, and ofloxacin. The results revealed that the presence of embB mutations did not correlate with ethambutol resistance but was associated with multiple-drug resistance, especially resistance to both ethambutol and rifampin.     

结核分支杆菌临床分离株耐药性调查与耐多药结核病流行特点探讨

目的调查结核临床分离株的耐药性,探讨多耐药结核病（MDR-TB）流行特点。方法采用匡氏琼脂培养基按比例法测定287株结核菌（56株来自初治病例）对8种抗痨药物的耐药性。结果初治病例菌株和复治病例菌株对异烟肼、利福平、链霉素、吡嗪酰胺高度耐药率分别为1．8％、3．6％、17．9％、0％和74．9％、58．0％、68．4％、32．0％,差异均极显著（P〈0．001）。复治病例MDR株占63．2％,广泛耐药（XDR）株占10％,15．8％的MDR株为XDR株;初治病例无XDR株感染,MDR株占1.8％,极显著低于复治病例MDR株比率（P〈0．001）。结论住院结核病患者MDR株和XDR株感染率高,且主要由获得性耐药产生。DOTS策略仍是我国耐药结核病防治重点。     

上海地区肺结核病患者氧氟沙星耐药情况分析

目的 了解上海地区肺结核病患者氧氟沙星耐药的分布情况及可能的危险因素.方法 收集2009-2010年上海市各结核病定点医院的447株对任意一种一线抗结核药物(异烟肼、利福平、链霉素和乙胺丁醇)耐药的菌株,同期随机选取151株对上述4种一线抗结核药物全敏感菌株,对这598株结核分枝杆菌菌株进行氧氟沙星敏感性检测,分析氧氟沙星耐药的分布情况.收集肺结核患者的年龄、性别、抗结核治疗史和户籍资料,采用多因素分析研究氧氟沙星耐药可能的危险因素.用DNA测序分析氧氟沙星耐药菌株gyrA、gyrB基因耐药突变的特征.结果 447株耐药菌株中,72株(16.1%)对氧氟沙星耐药,MDR(至少同时对异烟肼、利福平耐药)结核分枝杆菌中氧氟沙星耐药44株(39.6%).在151株一线抗结核药物全敏感菌株中,4株(2.6%)耐氧氟沙星.多因素分析结果显示MDR和多耐药(对一种以上抗结核药物耐药,但不对异烟肼和利福平同时耐药)等与氧氟沙星耐药有关(OR分别为19.5、5.6,95% CI 分别为6.4～59.4、1.7～18.1,P均＜0.05).患者为复治病例与氧氟沙星耐药相关(OR=2.3,95%CI:1.2～4.0,P＜0.05).氧氟沙星耐药与年龄偏大有关(OR=1.03,95%CI:1.01～1.05,P＜0.05).76株氧氟沙星耐药菌株的gyrA、gyrB基因序列分析显示62株(81.6%)发生耐药突变.结论 上海地区MDR结核病患者中氧氟沙星耐药率高于对一线抗结核药物全敏感的肺结核病患者.MDR、多耐药、复治、年龄为氧氟沙星耐药可能的危险因素,其中以MDR与氧氟沙星耐药的关联强度最大.     

Evaluation of the CombiChip Mycobacteria Drug-Resistance detection DNA chip for identifying mutations associated with resistance to isoniazid and rifampin in Mycobacterium tuberculosis

The CombiChip Mycobacteriatrade mark Drug-Resistance Detection DNA chip, recently developed by GeneIn (Pusan, South Korea), is an oligonucleotide microchip coupled with polymerase chain reaction for the detection of mutations associated with resistance to isoniazid (INH) and rifampin (RIF). This oligonucleotide chip was compared with DNA sequencing and phenotypic drug susceptibility testing with 69 INH- and/or RIF-resistant and 27 all tested drug-susceptible Mycobacterium tuberculosis isolates. Two selected codons (the katG codon 315 and inhA15) allowed identification of 84.1% of INH-resistant isolates and 100% of RIF resistance were detected by screening for 7 codons: rpoB511, rpoB513, rpoB516, rpoB522, rpoB526, rpoB531, and rpoB533. The overall specificity of this oligonucleotide chip for detecting INH and RIF resistance were 100 and 95.3%, respectively. This level of sensitivity and specificity is concordant with that from the determination of M. tuberculosis drug resistance by DNA sequencing. This oligonucleotide chip is a rapid and reliable genotypic method capable of detecting multiple mutations associated with INH and RIF resistance simultaneously in a single microchip slide.     

焦磷酸测序技术检测结核分枝杆菌四种药物耐药性

目的 利用焦磷酸测序技术检测结核分枝杆菌对利福平、异烟肼、氧氟沙星、阿米卡星的耐药性,并对其临床应用进行评价.方法 收集上海市肺科医院2008-2009年临床确诊的肺结核患者的痰标本培养阳性结核分枝杆菌89株.所有菌株按<结核病诊断实验室检验规程>进行分枝杆菌培养、菌种鉴定.另外10株已知药敏结果的结核分枝杆菌来自上海市肺科医院菌株库.利用焦磷酸测序技术,以10株已知药敏结果和经直接测序已知耐药基因突变情况的结核分枝杆菌为试验菌株,探索焦磷酸测序检测结核分枝杆菌rpoB、katG、gyrA、rrs耐药基因的最佳模式,并用该条件检测89株结核分枝杆菌临床分离株,检测结果与Bactec 960药敏法进行比较.结果 rpoB、gyrA基因检测宜采用焦磷酸测序序列分析模式,katG、rrs基因检测宜采用焦磷酸测序单核苷酸多态性模式.以Bactec 960药敏法测定结果为判断标准,则焦磷酸测序法检测89株结核分枝杆菌临床分离株对利福平、异烟肼、氧氟沙星、阿米卡星耐药性的敏感度分别为98.0%、64.1%、79.5%、78.3%,特异度分别为97.5%、100.0%、90.0%、100.0%,准确性分别为97.8%、77.5%、85.4%、94.4%,该法检测利福平、氧氟沙星、阿米卡星的检测结果与Bactec 960药敏检测结果一致性较好,Kappa值均≥0.7.结论 焦磷酸测序技术检测结核分枝杆菌对利福平、异烟肼、氧氟沙星、阿米卡星耐药性具有快速、准确、高通量的优点,是一种可对结核分枝杆菌多种药物耐药性进行快速检测的方法.

Abstract：

Objective To develop an assay to determine Mycobacterium tuberculosis resistance to rifampin, isoniazid, ofloxacin and amikacin by pyrosequencing and evaluate the value of this method in clinical application. Methods Eighty-nine clinical isolates of Mycobacterium tuberculosis from tuberculosis patients were collected from Shanghai Pulmonary Hospital during 2008 to 2009. All strains were isolated from decontaminated sputum, cultured on Lowenstein-Jensen media and identified by traditional biochemical tests with standard methods. Ten Mycobacterium tuberculosis were selected from the strain bank of Shanghai Pulmonary Hospital. The optimal conditions of detection of rpoB, katG, gyrA and rrs gene by pyroseuencing were determined, using the 10 Mycobacterium tuberculosis strains whose drug susceptibility of Bactec 960 and sequence of rpoB, katG, gyrA, rrs gene were known. Then the drug susceptibility of 89 Mycobacterium tuberculosis clinical isolate strains were detected by pyrosequencing using this conditions and the results were compared with that of the Bactec 960 methods. Results The pyrosequencing program of sequence analysis was suitable for the detection of the mutations of rpoB and gyrA genes, and the program of single nucleotide polymorphism was suitable for katG and rrs genes. Among the 89 Mycobacterium tuberculosis clinical isolates,when using the drug susceptibility of Bactec 960 as the standard, the sensitivity of rifampin, isoniazid,ofloxacin and amikacin is 98.0%, 64. 1%, 79.5%, 78. 3% respectively, the specificity is 97.5%,100. 0%, 90. 0%, 100. 0% respectively, the accuracy is 97.8%, 77. 5%, 85.4%, 94. 4% respectively,tested by pyrosequencing. The results of the detection of resistance to rifampin, isoniazid, ofloxacin and amikacin in Mycobacterium tuberculosis using pyrosequencing technique were almost the same with that of Bactec 960, and Kappa ≥0. 7 in each detection. Conclusion Pyrosequencing is thus a rapid, accurate and high throughput method for detecting Mycobacterium tuberculosis resistance to these four drugs.     

Molecular basis of rifampin and isoniazid resistance in Mycobacterium bovis strains isolated in Sardinia, Italy

Fourteen of 22 (68%) Mycobacterium bovis strains isolated from cattle in Sardinia were found to be resistant to rifampin and isoniazid. Analysis of the rpoB and the katG, oxyR-ahpC, and inhA gene regions of these strains was performed in order to investigate the molecular basis of rifampin and isoniazid resistance, respectively. The most frequent mutation, encountered in 6 of 10 strains (60%), was in the rpoB gene; it occurred, at codon position 521 and resulted in leucine changed to proline. This suggests that codon 521 may be important for the development of rifampin resistance in M. bovis. Resistance to isoniazid is associated in Mycobacterium tuberculosis with a variety of mutations affecting one or more genes. Our results confirm the difficulty of interpreting the sequence variations observed in clinical strains of M. bovis. M. bovis strains isolated from the same geographic area showed similar mutations within the genes responsible for rifampin and isoniazid resistance. Our results represent the first study to elucidate the molecular genetic basis of drug resistance in M. bovis isolated from cattle.     

Population genetics study of isoniazid resistance mutations and evolution of multidrug-resistant Mycobacterium tuberculosis

The molecular basis for isoniazid resistance in Mycobacterium tuberculosis is complex. Putative isoniazid resistance mutations have been identified in katG, ahpC, inhA, kasA, and ndh. However, small sample sizes and related potential biases in sample selection have precluded the development of statistically valid and significant population genetic analyses of clinical isoniazid resistance. We present the first large-scale analysis of 240 alleles previously associated with isoniazid resistance in a diverse set of 608 isoniazid-susceptible and 403 isoniazid-resistant clinical M. tuberculosis isolates. We detected 12 mutant alleles in isoniazid-susceptible isolates, suggesting that these alleles are not involved in isoniazid resistance. However, mutations in katG, ahpC, and inhA were strongly associated with isoniazid resistance, while kasA mutations were associated with isoniazid susceptibility. Remarkably, the distribution of isoniazid resistance-associated mutations was different in isoniazid-monoresistant isolates from that in multidrug-resistant isolates, with significantly fewer isoniazid resistance mutations in the isoniazid-monoresistant group. Mutations in katG315 were significantly more common in the multidrug-resistant isolates. Conversely, mutations in the inhA promoter were significantly more common in isoniazid-monoresistant isolates. We tested for interactions among mutations and resistance to different drugs. Mutations in katG, ahpC, and inhA were associated with rifampin resistance, but only katG315 mutations were associated with ethambutol resistance. There was also a significant inverse association between katG315 mutations and mutations in ahpC or inhA and between mutations in kasA and mutations in ahpC. Our results suggest that isoniazid resistance and the evolution of multidrug-resistant strains are complex dynamic processes that may be influenced by interactions between genes and drug-resistant phenotypes.     

Rapid detection of rifampicin-resistant Mycobacterium tuberculosis by in-house, reverse line blot assay

Drug resistance in tuberculosis is a significant problem in countries endemic for tuberculosis. A sensitive, specific, and high-throughput reverse line blot assay (RLBA) for the detection of genotypic resistance to rifampicin (RIF) was designed and evaluated. DNA sequencing done for 72 resistant isolates from Delhi, for baseline data, showed mutations within the rpoB core region in all RIF-resistant strains. The RLBA includes oligonucleotide probes specific for wild-type and mutant sequences, allowing sensitive detection of both genotypes in a single assay. The assay based on reverse hybridization principle simultaneously detects 13 different mutations affecting 6 independent codons, including the most prevalent mutations at positions 531 and 526. Application of the method to a panel of 292 MDR TB isolates and susceptible strains from 5 different cities in India showed 98% concordance with the sequencing results. This rapid, simple, economical, and highly sensitive assay provides a practical alternative to sequencing for genotypic evaluation of RIF resistance in Mycobacterium tuberculosis.     

应用多重PCR-单链构象多态性分析同时检测耐异烟肼和利福平的结核分枝杆菌

目的 探讨应用多重PCR-单链构象多态性分析(multiplexpulymerase chain reaction-single strand conformation polymorphism,multi-PCR-SSCP)方法快速、特异地同时快速检测结核分枝杆菌对异烟肼和利福平耐药性的效能.方法 根据结核分枝杆菌的inhA序列、katG序列、rpoB序列,分别设计出3对特异性寡聚核苷酸引物.采用multi-PCR-SSCP技术,一次性检出耐异烟肼和利福平的结核分枝杆菌.新方法的有效性通过116株临床分离株(70株耐异烟肼,66株耐利福平)的验证.结果 名 Multi-PCR-SSCP方法检测临床分离株基因突变的有效性,以细菌培养和药敏试验结果为金标准.116株临床分离株和H37Rv标准株中除了4株katG缺失突变,其余菌株3个基因katG、inhA和rpoB在单基因PCR中都扩增成功.与H37Rv标准株相比,46株katG基因突变,14株inhA基因突变,58株rpoB基因突变.38株katG和rpoB,4株inhA和rpoB,4株inhA和katG同时突变,还有2株3个基因都有突变.multi-PCR-SSCP对于耐异烟肼和利福平的结核分枝杆菌检出的敏感度分别为80%、82%,特异度分别为100%和92%.结论 multi-PCR-SSCP方法敏感、特异,能同时快速有效地检测耐多药结核分枝杆菌,有望成为临床指导用药的好方法,为深入研究耐药基凶检测奠定了良好的基础.     

Prevalence of and molecular basis for tuberculosis drug resistance in the Republic of Georgia: validation of a QIAplex system for detection of drug resistance-related mutations

We developed a QIAplex system for the simultaneous detection of 24 Mycobacterium tuberculosis gene mutations responsible for resistance to isoniazid (INH), rifampin (RIF), streptomycin (STM), and ethambutol (EMB) in 196 M. tuberculosis isolates recovered in the Republic of Georgia. In comparison to phenotypic susceptibility tests, the QIAplex showed sensitivity and specificity of 85.4% and 96.1% for INH, 94.4% and 99.4% for RIF, 69.6% and 99.2% for STM, 50.0% and 98.8% for EBM, and 86.7% and 100.0% for multidrug resistance, respectively. The dominant resistance mutations revealed were a mutation in katG resulting in S315T (katG S315T), rpsL K43R, and rpoB S531L. Mutations katG S315G and S315T and rpoB S531L were detected with higher frequencies in pretreated patients than in naive patients (P < 0.05). Simultaneous detection of 24 common drug resistance-related mutations provides a molecular tool for studying and monitoring M. tuberculosis resistance mechanism and epidemiology.     

Mutation analysis of mycobacterial rpoB genes and rifampin resistance using recombinant Mycobacterium smegmatis

Rifampin is a major drug used to treat leprosy and tuberculosis. The rifampin resistance of Mycobacterium leprae and Mycobacterium tuberculosis results from a mutation in the rpoB gene, encoding the β subunit of RNA polymerase. A method for the molecular determination of rifampin resistance in these two mycobacteria would be clinically valuable, but the relationship between the mutations and susceptibility to rifampin must be clarified before its use. Analyses of mutations responsible for rifampin resistance using clinical isolates present some limitations. Each clinical isolate has its own genetic variations in some loci other than rpoB, which might affect rifampin susceptibility. For this study, we constructed recombinant strains of Mycobacterium smegmatis carrying the M. leprae or M. tuberculosis rpoB gene with or without mutation and disrupted their own rpoB genes on the chromosome. The rifampin and rifabutin susceptibilities of the recombinant bacteria were measured to examine the influence of the mutations. The results confirmed that several mutations detected in clinical isolates of these two pathogenic mycobacteria can confer rifampin resistance, but they also suggested that some mutations detected in M. leprae isolates or rifampin-resistant M. tuberculosis isolates are not involved in rifampin resistance.     

Selection of Mutations To Detect Multidrug-Resistant Mycobacterium tuberculosis Strains in Shanghai,China

Novel tools are urgently needed for the rapid, reliable detection of multidrug-resistant (MDR) and extensively drug-resistant (XDR) strains of Mycobacterium tuberculosis. To develop such tools, we need information about the frequency and distribution of the mycobacterial mutations and genotypes that are associated with phenotypic drug resistance. In a population-based study, we sequenced specific genes of M. tuberculosis that were associated with resistance to rifampin and isoniazid in 242 phenotypically MDR isolates and 50 phenotypically pan-susceptible isolates from tuberculosis (TB) cases in Shanghai, China. We estimated the sensitivity and specificity of the mutations, using the results of conventional, culture-based phenotypic drug susceptibility testing as the standard. We detected mutations within the 81-bp core region of rpoB in 96.3% of phenotypically MDR isolates. Mutations in two structural genes (katG and inhA) and two regulatory regions (the promoter of mabA-inhA and the intergenic region of oxyR-ahpC) were found in 89.3% of the MDR isolates. In total, 88.0% (213/242 strains) of the phenotypic MDR strains were confirmed by mutations in the sequenced regions. Mutations in embB306 were also considered a marker for MDR and significantly increased the sensitivity of the approach. Based on our findings, an approach that prospectively screens for mutations in 11 sites of the M. tuberculosis genome (rpoB531, rpoB526, rpoB516, rpoB533, and rpoB513, katG315, inhA-15, ahpC-10, ahpC-6, and ahpC-12, and embB306) could detect 86.8% of MDR strains in Shanghai. This study lays the foundation for the development of a rapid, reliable molecular genetic test to detect MDR strains of M. tuberculosis in China.Multidrug-resistant (MDR) tuberculosis (TB), defined as resistance to at least rifampin (RIF) and isoniazid (INH), and extensively drug-resistant (XDR) TB, defined as additional resistance to any fluoroquinolone and one injectable second-line drug, are among the most serious health threats of the 21st century. The epidemic of MDR TB is especially severe in China, a nation with the world''s second largest number of TB cases and the largest number of MDR TB cases (39). A recent study reported that 9.3% of all TB cases in China are MDR, almost twice the worldwide MDR prevalence (4.8%) (14). While a lot of attention has been focused on acquired drug resistance among TB patients who receive an inadequate treatment regimen or who cannot adhere to their treatment regimen, several studies also showed that a large number of MDR TB cases are likely caused by transmission of MDR strains of Mycobacterium tuberculosis (2, 19). Therefore, there is an urgent need for new tools and approaches that will provide a rapid, reliable, and cost-effective diagnosis of MDR TB, particularly in resource-limited settings. This will help to prevent transmission of MDR strains and to optimize treatment regimens for MDR cases.Drug susceptibility testing by the conventional solid medium culture method is highly sensitive and specific but extremely slow, due to the slow growth of M. tuberculosis. Liquid culture methods can reduce the turnaround time but require specialized instrumentation and reagents and are not feasible in most resource-limited settings. New molecular diagnostic methods represent a potentially rapid and sensitive alternative to conventional diagnostics. The molecular basis for phenotypic rifampin resistance is linked to mutations in the 81-bp core region of rpoB. Phenotypic isoniazid resistance has been associated with mutations in katG, particularly at codon 315, as well as with mutations in inhA, the promoter of mabA-inhA, and the intergenic region of oxyR-ahpC (3, 33, 34, 37, 43). Recently, a database of tuberculosis drug resistance mutations (TBDReaMDB) was established (27), and several genotypic diagnostic methods based on specific drug resistance-conferring mutations were developed. Two line probe assays, the INNO-LiPARif.TB assay (Innogenetics, Belgium) and the GenoType MTBDR Plus assay (Hain Lifescience, Nehren, Germany), have been approved by the World Health Organization (WHO) as tools for the rapid diagnosis of MDR TB (11, 20, 22). These tools are rapid and reproducible, but performance varies by geographic location, depending on the prevalent strains of M. tuberculosis and the type and frequency of drug resistance-conferring mutations in the population being tested (20, 22). Therefore, a thorough understanding of the diversity of the mycobacterial genetic mutations will form the foundation for new diagnostic methods.Despite the large number of MDR TB cases in China, relatively few studies have determined the prevalence of different drug resistance-conferring mutations among MDR clinical isolates. In this study, we investigated the type and frequency of drug resistance-conferring mutations that occurred among M. tuberculosis clinical isolates that were phenotypically MDR. Our goal was to identify and select a limited, parsimonious number of mutation sites that can be used to prospectively and rapidly screen isolates to detect MDR TB in Shanghai.     

Molecular characterization of multidrug-resistant Mycobacterium tuberculosis isolated in Nepal

Despite the fact that Nepal is one of the first countries globally to introduce multidrug-resistant tuberculosis (MDR-TB) case management, the number of MDR-TB cases is continuing to rise in Nepal. Rapid molecular tests applicable in this setting to identify resistant organisms would be an effective tool in reversing this trend. To develop such tools, information about the frequency and distribution of mutations that are associated with phenotypic drug resistance in Mycobacterium tuberculosis is required. In the present study, we investigated the prevalence of mutations in rpoB and katG genes and the inhA promoter region in 158 M. tuberculosis isolates (109 phenotypically MDR and 49 non-MDR isolates collected in Nepal) by DNA sequencing. Mutations affecting the 81-bp rifampin (RIF) resistance-determining region (RRDR) of rpoB were identified in 106 of 109 (97.3%) RIF-resistant isolates. Codons 531, 526, and 516 were the most commonly affected, at percentages of 58.7, 15.6, and 15.6%, respectively. Of 113 isoniazid (INH)-resistant isolates, 99 (87.6%) had mutations in the katG gene, with Ser315Thr being the most prevalent (81.4%) substitution. Mutations in the inhA promoter region were detected in 14 (12.4%) INH-resistant isolates. The results from this study provide an overview of the current situation of RIF and INH resistance in M. tuberculosis in Nepal and can serve as a basis for developing or improving rapid molecular tests to monitor drug-resistant strains in this country.     

The Combination of Sulfamethoxazole, Trimethoprim, and Isoniazid or Rifampin Is Bactericidal and Prevents the Emergence of Drug Resistance in Mycobacterium tuberculosis

The challenges of developing new drugs to treat tuberculosis (TB) are indicated by the relatively small number of candidates entering clinical trials in the past decade. To overcome these issues, we reexamined two FDA-approved antibacterial drugs, sulfamethoxazole (SMX) and trimethoprim (TMP), for use in TB treatment. SMX and TMP inhibit folic acid biosynthesis and are used in combination to treat infections of the respiratory, urinary, and gastrointestinal tracts. The MICs of SMX and TMP, alone and in combination, were determined for drug-susceptible, multidrug-resistant (MDR), and extensively drug-resistant Mycobacterium tuberculosis strains. While TMP alone was not effective against M. tuberculosis, the combination of TMP and SMX was bacteriostatic against M. tuberculosis. Surprisingly, the combination of SMX and TMP was also active against a subset of MDR M. tuberculosis strains. Treatment of M. tuberculosis with TMP-SMX and a first-line anti-TB drug, either isoniazid or rifampin, was bactericidal, demonstrating that the combination of TMP and SMX with isoniazid or rifampin was not antagonistic. Moreover, the addition of SMX-TMP in combination with either isoniazid or rifampin also prevented the emergence of drug resistance in vitro. In conclusion, this study further illustrates the opportunity to reevaluate the activity of TMP-SMX in vivo to prevent the emergence of drug-resistant M. tuberculosis.     

Molecular Characterization of Multidrug-Resistant Isolates of Mycobacterium tuberculosis from Patients in North India

The World Health Organization has identified India as a major hot-spot region for Mycobacterium tuberculosis infection. We have characterized the sequences of the loci associated with multidrug resistance in 126 clinical isolates of M. tuberculosis from India to identify the respective mutations. The loci selected were rpoB (rifampin), katG and the ribosomal binding site of inhA (isoniazid), gyrA and gyrB (ofloxacin), and rpsL and rrs (streptomycin). We found known as well as novel mutations at these loci. Few of the mutations at the rpoB locus could be correlated with the drug resistance levels exhibited by the M. tuberculosis isolates and occurred with frequencies different from those reported earlier. Missense mutations at codons 526 to 531 seemed to be crucial in conferring a high degree of resistance to rifampin. We identified a common Arg463Leu substitution in the katG locus and certain novel insertions and deletions. Mutations were also mapped in the ribosomal binding site of the inhA gene. A Ser95Thr substitution in the gyrA locus was the most common mutation observed in ofloxacin-resistant isolates. A few isolates showed other mutations in this locus. Seven streptomycin-resistant isolates had a silent mutation at the lysine residue at position 121. While certain mutations are widely present, pointing to the magnitude of the polymorphisms at these loci, others are not common, suggesting diversity in the multidrug-resistant M. tuberculosis strains prevalent in this region. Our results additionally have implications for the development of methods for multidrug resistance detection and are also relevant in the shaping of future clinical treatment regimens and drug design strategies.     

Characterization of mutations conferring extensive drug resistance to Mycobacterium tuberculosis isolates in Pakistan

The increasing incidence of extensively drug-resistant (XDR) Mycobacterium tuberculosis in high-tuberculosis-burden countries further highlights the need for improved rapid diagnostic assays. An increasing incidence of XDR M. tuberculosis strains in Pakistan has been reported, but drug resistance-associated mutations in these strains have not been evaluated previously. We sequenced the "hot-spot" regions of rpoB, katG, inhA, ahpC, gyrA, gyrB, and rrs genes in 50 XDR M. tuberculosis strains. It was observed that 2% of rifampin, 6% of isoniazid, 24% of fluoroquinolone, and 32% of aminoglycoside/capreomycin resistance in XDR M. tuberculosis strains would be undetected if only these common hot-spot regions were tested. The frequencies of resistance-conferring mutations were found to be comparable among all XDR M. tuberculosis strain families present, including the Central Asian Strain, Beijing, and East African Indian genogroups and the Unique isolates. Additional genetic loci need to be tested for detection of mutations conferring fluoroquinolone, aminoglycoside, and capreomycin resistance in order to improve molecular diagnosis of regional XDR M. tuberculosis strains.     

Molecular characterization of isoniazid-resistant Mycobacterium tuberculosis isolates collected in Australia

Elucidation of the molecular basis of isoniazid (INH) resistance in Mycobacterium tuberculosis has led to the development of different genotypic approaches for the rapid detection of INH resistance in clinical isolates. Mutations in katG, in particular the S315T substitution, are responsible for INH resistance in a large proportion of tuberculosis cases. However, the frequency of the katG S315T substitution varies with population samples. In this study, 52 epidemiologically unrelated clinical INH-resistant M. tuberculosis isolates collected in Australia were screened for mutations at katG codon 315 and the fabG1-inhA regulatory region. Importantly, 52 INH-sensitive isolates, selected to reflect the geographic and genotypic diversity of the isolates, were also included for comparison. The katG S315T substitution and fabG1-inhA -15 C-to-T mutation were identified in 34 and 13 of the 52 INH-resistant isolates, respectively, and none of the INH-sensitive isolates. Three novel katG mutations, D117A, M257I, and G491C, were identified in three INH-resistant strains with a wild-type katG codon 315, fabG1-inhA regulatory region, and inhA structural gene. When analyzed for possible associations between resistance mechanisms, resistance phenotype, and genotypic groups, it was found that neither the katG S315T nor fabG1-inhA -15 C-to-T mutation clustered with any one genotypic group, but that the -15 C-to-T substitution was associated with isolates with intermediate INH resistance and isolates coresistant to ethionamide. In total, 90.4% of unrelated INH-resistant isolates could be identified by analysis of just two loci: katG315 and the fabG1-inhA regulatory region.     

实时荧光PCR分子信标检测耐利福平结核分枝杆菌印rpoB基因

目的 应用实时荧光PCR分子信标技术,建立快速检测临床标本中结核分枝杆菌利福平rpoB相关耐药突变点方法,探讨其缩短耐药实验报告时间的临床应用价值.方法 以分枝杆菌药物敏感性实验绝对浓度法为标准,12株非结核分枝杆菌、4株非分枝杆菌作对照,对174例结核患者临床分离株应用实时荧光PCR分子信标方法,检测利福平rpoB核心区域的耐药突变点并将结果与直接测序进行比较.结果 (1)实时荧光PCR分子信标方法:82例结核分枝杆菌利福平敏感菌株中,3例发生rpoB基因突变,特异度为96.3%;92例结核分枝杆菌利福平耐药菌株中,82例检出耐药突变,敏感度为89.1%;准确性为92.5%.(2)DNA直接测序分析:82例结核分枝杆菌利福平敏感株中,1例发生rpoB基因突变,特异度为98.8%;92例结核分枝杆菌利福平耐药菌株中,83例发生:rpoB基因突变,敏感度为90.2%;准确性为94.2%.检测174株结核分枝杆菌临床分离菌株,与实时荧光PCR分子信标方法检测一致性为98.3%(171/174).结论 实时荧光PCR分子信标方法检测耐利福平结核分枝杆菌rpoB基因突变点可作为结核患者快速耐药检测的初筛方法之一.     

Profiling and identification of novel rpoB mutations in rifampicin-resistant Mycobacterium tuberculosis clinical isolates from Pakistan

IntroductionRifampicin (RIF) is one of the most effective anti-tuberculosis first-line drugs prescribed along with isoniazid. However, the emergence of RIF resistance Mycobacterium tuberculosis (MTB) isolates is a major issue towards tuberculosis (TB) control program in high MDR TB-burdened countries including Pakistan. Molecular data behind phenotypic resistance is essential for better management of RIF resistance which has been linked with mutations in rpoB gene. Since molecular studies on RIF resistance is limited in Pakistan, the current study was aimed to investigate the molecular data of mutations in rpoB gene behind phenotypic RIF resistance isolates in Pakistan.MethodA total of 322 phenotypically RIF-resistant isolates were randomly selected from National TB Reference Laboratory, Pakistan for sequencing while 380 RIF resistance whole-genome sequencing (WGS) of Pakistani isolates (BioProject PRJEB25972), were also analyzed for rpoB mutations.ResultAmong the 702 RIF resistance samples, 675 (96.1%) isolates harbored mutations in rpoB in which 663 (94.4%) were detected within the Rifampicin Resistance Determining Region (RRDR) also known as a mutation hot spot region, including three novel. Among these mutations, 657 (97.3%) were substitutions including 603 (89.3%) single nucleotide polymorphism, 49 (7.25%) double and five (0.8%) triple. About 94.4% of Phenotypic RIF resistance strains, exhibited mutations in RRDR, which were also detectable by GeneXpert.ConclusionMutations in the RRDR region of rpoB is a major mechanism of RIF resistance in MTB circulating isolates in Pakistan. Molecular detection of drug resistance is a faster and better approach than phenotypic drug susceptibility testing to reduce the time for transmission of RIF resistance strains in population. Such insights will inform the deployment of anti-TB drug regimens and disease control tools and strategies in high burden settings, such as Pakistan.