Genotypic analysis of genes associated with transmission and drug resistance in the Beijing lineage of Mycobacterium tuberculosis

The Beijing genotype of Mycobacterium tuberculosis is an endemic lineage in East Asia that has disseminated worldwide. It is a major health concern, as it is geographically widespread and is considered to be hypervirulent. To elucidate its genetic diversity in Taiwan, phylogenetic reconstruction was performed using 338 M. tuberculosis Beijing family clinical isolates. Region-of-difference analysis revealed the strains from Taiwan to be distributed among six subgroups of a phylogenetic tree. Synonymous single nucleotide polymorphisms at 10 chromosomal positions were also analysed. Among the 338 isolates analysed for single-nucleotide polymorphisms by using mass spectrometry, the most frequent strain found was ST10 (53.3%), followed by ST19 (14.8%) and ST22 (14.5%). Tests of drug resistance showed that the sublineages ST10, ST19 and ST26 were over-represented in the multidrug-resistant population. The presence of mutations in putative genes coding for DNA repair enzymes, which could confer a mutator phenotype to facilitate spreading of the pathogen, did not demonstrate an association with multidrug resistance. Therefore, the DNA repair genes may be involved in transmission but not in drug resistance.     

Genotypic diversity of Mycobacterium tuberculosis in the Guiana-Antilles region

This investigation dealt with 226 strains (1 isolate/patient) of Mycobacterium tuberculosis isolated in the French West Indies and French Guiana over a three-year period (1994-1996). The genotypic diversity of the isolates was investigated using various molecular markers; essentially two PCR-based rapid methods, namely spoligotyping and double-repetitive-element (DRE)-PCR, as well as three restriction fragment length polymorphism (RFLP)-based methods, namely IS6110-RFLP, DR-RFLP and PGRS-RFLP. Out of 226 isolates investigated, a total of 166 isolates were distributed in 31 spoligotype-defined clusters containing 2-31 strains, which corresponded to a rate of 73% of primary clustering. After secondary typing with DRE-PCR, IS6110-RFLP, DR-RFLP and/or PGRS-RFLP, molecular clonality was established for 73 isolates organised in 25 clusters (32% of clustered isolates). Considering one reactivation case per cluster, the rate of recent transmission was estimated to a minimal rate of 21%, however the available epidemiologic information led to the positive conclusion for only 14% of cases. The data obtained demonstrated the presence of common genotypes of M. tuberculosis among the three overseas French territories, i.e. Guadeloupe, Martinique and French Guiana. The results obtained during this retrospective study clearly indicate the importance of future prospective epidemiological investigations around the clustered cases of tuberculosis, so as to detect the persisting foci of endemic disease and characterize the chain of transmission as well as the subpopulations which are at an increased risk of contracting and/or propagating the disease. Last but not least, the present study also deals with a first phylogenetic approach of M. tuberculosis based on a comparison of the spoligotyping results obtained locally with those reported elsewhere in the world.     

Genotypic and phenotypic heterogeneity among Mycobacterium tuberculosis isolates from pulmonary tuberculosis patients

Although the heterogeneity of Mycobacterium tuberculosis populations and the existence of mixed infections are now generally accepted, systematic studies on their relative importance are rare. In the present study, 10 individual colonies of each M. tuberculosis isolate (primary isolate) from 97 tuberculosis patients in a primarily human immunodeficiency virus-negative population were screened for heterogeneity and detectable mixed infections by spoligotyping, IS6110-based restriction fragment length polymorphism analysis, and mycobacterial interspersed repetitive unit-variable number of tandem repeat typing. The MICs of antituberculosis drugs for colonies with divergent fingerprints were determined. Infections with different bacterial subpopulations were detected in the samples from eight patients (8.2%), and the frequency of detectable mixed infections in the study population was estimated to be 2.1%. Genotypic variations were found to be independent of the drug susceptibilities, and the various molecular markers evolved independently in most cases. The predominant strains and the primary isolates always had concordant drug susceptibility and MIC testing results. These findings have implications on the interpretation of molecular epidemiology results for patient follow-up and in transmission studies.     

一种鉴定结核分枝杆菌"北京家族"菌株的新方法

目的 研究结核分枝杆菌"北京家族"菌株鉴定的新方法——RD105缺失检测的可行性.方法 分别应用间隙寡核苷酸分型(Spoligotyping)和RD105缺失检测两种方法对结核分枝杆菌进行"北京家族"的鉴定,比较分析两种方法进行菌株鉴定结果的差异.结果 共对来自北京、福建、新疆和吉林的342株结核分枝杆菌临床分离菌株进行了鉴定,"北京家族"菌株261株,占76.32%.非"北京家族"菌株81株,占23.68%.两种方法鉴定结果的符合度达100%.结论 简单、快速的新方法——RD105缺失检测可替代原始经典方法Spoligotyping用于结核分枝杆菌"北京家族"菌株的鉴定.     

Rapid test for identification of a highly transmissible Mycobacterium tuberculosis Beijing strain of sub-Saharan origin

The development of a rapid test to identify Mycobacterium tuberculosis Beijing isolates and specifically strain GC1237, coming from a sub-Saharan country, is needed due to its alarming wide spread on Gran Canaria Island (Spain). A rapid test that detects IS6110 present between dnaA and dnaN in the Beijing strains and in a specific site for GC1237 (Rv2180c) has been developed. This test would be a useful tool in the surveillance of subsequent cases.     

Spread of a low-fitness drug-resistant Mycobacterium tuberculosis strain in a setting of high human immunodeficiency virus prevalence

The fitness cost associated with the evolution of resistance to rifampin in Mycobacterium tuberculosis may be different in clinical isolates compared to in vitro-generated mutants. An atypical Beijing strain (attenuated phenotype) demonstrated the ability to spread despite acquiring resistance to rifampin. Transmission was linked to human immunodeficiency virus coinfection (P = 0.029), raising concern for the spread of drug resistance in vulnerable populations.     

Distribution of the Beijing family genotypes of Mycobacterium tuberculosis in Taiwan

To investigate the distribution of the Beijing family genotypes of Mycobacterium tuberculosis in Taiwan, we collected 421 M. tuberculosis complex clinical isolates at random from four geographic regions of Taiwan and analyzed them by spacer oligonucleotide typing (spoligotyping) in 2003. We found 113 resolved spoligotypes, among which we identified 28 (24.8%) clusters. One hundred eighty-seven (44.4%) isolates were Beijing family genotypes and consisted of 172 (40.9%) characteristic Beijing genotypes and 15 (3.6%) Beijing-like ones. We also found that substantially larger proportions of tuberculosis patients were infected with Beijing family genotypes in the northern (51.6%) and eastern (46.2%) regions of Taiwan, while 31.6 and 28.0% of the tuberculosis patients in the central and southern regions, respectively, were infected with these genotypes. The proportion of Beijing genotype isolates was the highest in patients below the age of 24 years (61.5%), the second highest in elderly patients over age 65 years (46.8%), and the lowest in middle-age patients between the ages of 45 and 54 years (34.0%). Geographic location and age were found by multivariate analysis to be associated with Beijing family genotypes. Antituberculosis drug resistance was found more often in Beijing family genotype strains (46.4%) than in non-Beijing family genotype strains (34.3%), with more Beijing family genotype strains being resistant to ethambutol and isoniazid. These findings suggest that M. tuberculosis Beijing family genotypes have been dominant for several decades and that they are the cause of a significant proportion of the recent transmissions of tuberculosis in Taiwan.     

Subpopulation analysis of heteroresistance to fluoroquinolone in Mycobacterium tuberculosis isolates from Beijing, China

The presence of heteroresistance was represented by 23% of 235 fluoroquinolone (FQ)-resistant Mycobacterium tuberculosis isolates in Beijing, China, from 2008 to 2010. The main mechanism of FQ heteroresistance is due to the segregation of a single M. tuberculosis strain in patients; the majority of isolates with multidrug-resistant tuberculosis contained a mixture of bacterial subpopulations consisting of various mutant types, suggesting that the improper use of FQ is the major cause of FQ resistance.     

Antigenic evidence of prevalence and diversity of Mycobacterium tuberculosis arabinomannan

Arabinomannan (AM) is a polysaccharide of the mycobacterial capsule. The capsular polysaccharides of various microorganisms are diverse, and this diversity is important for classification of organisms into serotypes and vaccine development. In the present study we examined the prevalence and diversity of AM among Mycobacterium tuberculosis strains using four AM-binding monoclonal antibodies (MAbs). One of these MAbs, MAb 9d8, is known to bind to AM specifically. By whole-cell enzyme-linked immunosorbent assay (ELISA), the AM recognized by MAb 9d8 was detected on the surfaces of 9 of 11 strains, while 2 strains showed no reactivity with MAb 9d8. However, the AM recognized by MAb 9d8 was found in the culture supernatants of all 11 M. tuberculosis strains tested, as demonstrated by capture ELISA. Other AM-binding MAbs reacted both with the surfaces and with the culture supernatants of all 11 strains. Mice immunized with an experimental AM-recombinant Pseudomonas aeruginosa exoprotein A (rEPA) conjugate vaccine had an increased antibody response to AM and a moderate reduction in the numbers of CFU in their organs 7 days after challenge. Our results indicate that AM was detected in all M. tuberculosis strains tested, with differences in epitope distributions of certain strains. In addition, our results suggest that an experimental AM-rEPA vaccine has a moderate effect on the numbers of CFU in organs early after infection.     

Immunological consequences of strain variation within the Mycobacterium tuberculosis complex

In 2015, there were an estimated 10.4 million new cases of tuberculosis (TB) globally, making it one of the leading causes of death due to an infectious disease. TB is caused by members of the Mycobacterium tuberculosis complex (MTBC), with human disease resulting from infection by M. tuberculosis sensu stricto and M. africanum. Recent progress in genotyping techniques, in particular the increasing availability of whole genome sequence data, has revealed previously under appreciated levels of genetic diversity within the MTBC. Several studies have shown that this genetic diversity may translate into differences in TB transmission, clinical manifestations of disease, and host immune responses. This suggests the existence of MTBC genotype‐dependent host–pathogen interactions which may influence the outcome of infection and progression of disease. In this review, we highlight the studies demonstrating differences in innate and adaptive immunological outcomes consequent on MTBC genetic diversity, and discuss how these differences in immune response might influence the development of TB vaccines, diagnostics and new therapies.     

Beijing Sublineages of Mycobacterium tuberculosis Differ in Pathogenicity in the Guinea Pig

The Beijing family of Mycobacterium tuberculosis strains is part of lineage 2 (also known as the East Asian lineage). In clinical studies, we have observed that isolates from the sublineage RD207 of lineage 2 were more readily transmitted among humans. To investigate the basis for this difference, we tested representative strains with the characteristic Beijing spoligotype from four of the five sublineages of lineage 2 in the guinea pig model and subjected these strains to comparative whole-genome sequencing. The results of these studies showed that all of the clinical strains were capable of growing and causing lung pathology in guinea pigs after low-dose aerosol exposure. Differences between the abilities of the four sublineages to grow in the lungs of these animals were not overt, but members of RD207 were significantly more pathogenic, resulting in severe lung damage. The RD207 strains also induced much higher levels of markers associated with regulatory T cells and showed a significant loss of activated T cells in the lungs over the course of the infections. Whole-genome sequencing of the strains revealed mutations specific for RD207 which may explain this difference. Based on these data, we hypothesize that the sublineages of M. tuberculosis are associated with distinct pathological and clinical phenotypes and that these differences influence the transmissibility of particular M. tuberculosis strains in human populations.     

异烟肼耐药和敏感结核分枝杆菌的比较蛋白质组学研究

目的　研究结核分枝杆菌异烟肼耐药菌株与敏感菌株的蛋白质表达差异 ,鉴定结核分枝杆菌中与异烟肼耐药相关的菌体蛋白。方法　应用双向电泳技术比较 2株异烟肼耐药菌株与 2株敏感菌株的全菌蛋白表达图谱差异 ,发现异烟肼耐药结核分枝杆菌表达量显著增加 2 6个蛋白质斑点 ,通过基质辅助激光解吸 /电离飞行时间质谱仪进行质谱分析 ,获得 6个明确的肽质量指纹谱 ,通过蛋白质数据库进行检索分析。结果　 6个蛋白分别为海藻糖磷酸磷酸酶、铁钼蛋白A、莽草酸 5脱氢酶、葡萄糖胺果糖 6磷酸氨基转移酶、吲哚 3甘油磷酸合成酶、TetR家族转录调控因子。结论　上述蛋白的鉴定将对发现新型抗结核药物靶位和异烟肼耐药结核病诊断的分子标志物可能有所裨益     

结核分枝杆菌异烟肼耐药株和敏感株亚细胞蛋白质组差异研究

目的 研究结核分枝杆菌异烟肼耐药株和敏感株亚细胞蛋白质组差异,鉴定菌体细胞壁蛋白和细胞膜蛋白中与异烟肼耐药相关的蛋白,并初步探讨其在临床检验中的应用价值。方法 应用密度梯度离心法分离5株异烟肼耐药株和5株敏感株的细胞壁和细胞膜蛋白,利用二维液相色谱分离技术进一步获得耐药株和敏感株的亚细胞蛋白表达差异图谱。运用基质辅助激光解析/电离飞行时间质谱技术对获得的1280个细胞壁和细胞膜组分进行质谱鉴定,并运用DAVID数据库中GO注释功能对鉴定到的蛋白进行细胞成分和生物过程的注释和分类。运用NASF技术对蛋白表达进行定量,报告阈值为1.5。选取5个在耐药株中表达上调、2个在敏感株中表达上调的蛋白分别与菌株来源患者的血清进行ELISA检测,检测上述组分与血清样品发生免疫应答的强度,组间比较采用t检验。结果 共鉴定到347个蛋白。蛋白定位分析表明58％蛋白定位于细胞膜或跨膜。蛋白功能聚类分析表明31％蛋白参与三羧酸循环,26％、15％蛋白分别参与脂类、脂肪酸生物合成和代谢,28％蛋白参与脂质体的生物合成、代谢、转运和定位。其中琥珀酰氯化胆碱合酶、单加氧酶、假想蛋白Rv2255c、烟碱核苷二甲基联苯酰磷酸核酮糖激酶、膜磷脂胞嘧啶转移酶在耐药株中表达上调,含有上述蛋白的组分与感染耐药株患者的血清免疫学反应的吸光度（A450值）明显高于与感染敏感株患者的血清免疫学反应强度,差异有统计学意义(t值分别为0.028、0.044、0.066、0.064、0.083,P均＜0.01)。Rv2002蛋白A链、Rv2632c蛋白A链在敏感株中表达上调,含有上述蛋白的组分与感染敏感株患者的血清免疫学反应的A450值明显高于与感染耐药株患者的血清免疫学反应强度,差异有统计学意义（t值分别为0.053、0.073,P均＜0.05）。结论 运用密度梯度离心法和二维液相色谱-质谱技术能在亚细胞水平上富集并鉴定结核分枝杆菌异烟肼耐药株和敏感株中表达有差异的蛋白。有助于寻找异烟肼耐药株感染相关的抗原蛋白,为进一步探讨结核分枝杆菌耐异烟肼机制、研究耐药株-宿主相互作用提供了有价值的线索。     

Assessment of population structure and major circulating phylogeographical clades of Mycobacterium tuberculosis complex in Bangladesh suggests a high prevalence of a specific subclade of ancient M. tuberculosis genotypes

Spoligotyping was performed to study the population structure of Mycobacterium tuberculosis complex strains (n = 224) from Bangladesh. Strains were split into principal genetic group 1 (PGG 1 [75.0%]) and PGG 2 and 3 (25%). Forty-nine strains with a new spoligotype signature and considered as south or southeast Asian-linked emerging clones were designated as "Matlab type."     

Molecular strain identification of the Mycobacterium tuberculosis complex in archival tissue samples

AIMS: To investigate the use of different molecular analyses that can identify distinct strains of human pathogenic mycobacteria in formalin fixed and paraffin wax embedded archival tissue samples to see whether it is possible to differentiate between the members of the Mycobacterium tuberculosis complex (M tuberculosis, M bovis, M africanum, M microti, or M canettii) and/or substrains in a high number of samples. This would be of interest for identifying individual infection traits and superinfection by different mycobacterial strains. METHODS: Forty nine archival tissue samples with clinically and/or histologically suspected tuberculosis infection were subjected to molecular DNA analysis. RESULTS: The molecular analysis revealed the presence of M tuberculosis complex DNA in 20 samples, whereas acid fast bacilli could be detected by Ziehl-Neelsen staining in only eight samples. All IS6110 positive samples were further characterised by spoligotyping and seven cases provided M tuberculosis specific signatures, whereas M bovis specific signatures were obtained in four cases. The analysis of mtp40, oxyR, and pncA partial gene sequences confirmed the presence of M tuberculosis in six cases and M bovis in one case. The amplification and sequencing of four further genetic regions (katG, gyrA, TbD1, RD9) characterised six "modern" M tuberculosis strains belonging to genetic groups 2 or 3. CONCLUSION: This study provides clear evidence that archival paraffin wax embedded material can be used for further studies on the strain identification of M tuberculosis complex strains and can therefore unequivocally be used for the study of the epidemiology and evolution of tuberculosis pathogens.     

Genotypic analysis of the earliest known prehistoric case of tuberculosis in Britain

The earliest known case of human tuberculosis in Britain dates to the middle period of the Iron Age, approximately 2,200 years before present. Bone lesions on the spine of a male skeleton excavated at Tarrant Hinton in Dorset, United Kingdom, show evidence of Pott's disease and are supported by molecular evidence of Mycobacterium tuberculosis complex DNA amplified by IS6110 PCR (19). In the present study, we used a further series of sensitive PCR methods to confirm the diagnosis of tuberculosis and to determine the genotype of the infecting strain. These tests demonstrated that this individual was infected with a strain of M. tuberculosis rather than Mycobacterium bovis. The strain had undergone the tuberculosis D1 deletion affecting the mmpS6 and mmpL6 genes and can therefore be identified as a member of the family of "modern" M. tuberculosis isolates. All evidence obtained was consistent with surviving mycobacterial DNA being highly fragmented in this case.