Kanglin Wan

Genome sequencing of 161 Mycobacterium tuberculosis isolates from China identifies genes and intergenic regions associated with drug resistance.

The worldwide emergence of multidrug-resistant (MDR) and extensively drug-resistant (XDR) tuberculosis threatens to make this disease incurable. Drug resistance mechanisms are only partially understood, and whether the current understanding of the genetic basis of drug resistance in M. tuberculosis is sufficiently comprehensive remains unclear. Here we sequenced and analyzed 161 isolates with a range of drug resistance profiles, discovering 72 new genes, 28 intergenic regions (IGRs), 11 nonsynonymous SNPs and 10 IGR SNPs with strong, consistent associations with drug resistance. On the basis of our examination of the dN/dS ratios of nonsynonymous to synonymous SNPs among the isolates, we suggest that the drug resistance–associated genes identified here likely contain essentially all the nonsynonymous SNPs that have arisen as a result of drug pressure in these isolates and should thus represent a near-complete set of drug resistance–associated genes for these isolates and antibiotics. Our work indicates that the genetic basis of drug resistance is more complex than previously anticipated and provides a strong foundation for elucidating unknown drug resistance mechanisms.

Southern East Asian origin and coexpansion of Mycobacterium tuberculosis Beijing family with Han Chinese

Significance Mycobacterium tuberculosis Beijing family is a group of globally emerging bacterial strains that are responsible for more than a quarter of the global tuberculosis epidemic. Here, we combine whole-genome sequencing and large-scale genotyping to map the temporal and spatial changes of the genetic diversity within this strain family. We reveal a southern East Asia origin and a parallel evolution of this bacterial genotype with modern humans in East Asia during the last 30,000 years. The recently globally emerged Beijing strains mainly belong to a hypervirulent subtype that most likely has initially been selected for adaption to increased population densities during the agricultural transition in northern China. The Beijing family is the most successful genotype of Mycobacterium tuberculosis and responsible for more than a quarter of the global tuberculosis epidemic. As the predominant genotype in East Asia, the Beijing family has been emerging in various areas of the world and is often associated with disease outbreaks and antibiotic resistance. Revealing the origin and historical dissemination of this strain family is important for understanding its current global success. Here we characterized the global diversity of this family based on whole-genome sequences of 358 Beijing strains. We show that the Beijing strains endemic in East Asia are genetically diverse, whereas the globally emerging strains mostly belong to a more homogenous subtype known as “modern” Beijing. Phylogeographic and coalescent analyses indicate that the Beijing family most likely emerged around 30,000 y ago in southern East Asia, and accompanied the early colonization by modern humans in this area. By combining the genomic data and genotyping result of 1,793 strains from across China, we found the “modern” Beijing sublineage experienced massive expansions in northern China during the Neolithic era and subsequently spread to other regions following the migration of Han Chinese. Our results support a parallel evolution of the Beijing family and modern humans in East Asia. The dominance of the “modern” Beijing sublineage in East Asia and its recent global emergence are most likely driven by its hypervirulence, which might reflect adaption to increased human population densities linked to the agricultural transition in northern China.

Spoligotypes of Mycobacterium tuberculosis from Different Provinces of China

ABSTRACT A total of 2,346 Mycobacterium tuberculosis isolates from 13 provinces in China were genotyped by spoligotyping. Two hundred seventy-eight spoligotypes were identified: 2,153 isolates were grouped into 85 clusters, and the remaining 193 isolates were orphans. Comparison with the SpolDB4.0 database revealed that 118 spoligotypes had shared international type numbers in the database and the other 160 were novel. These 160 novel spoligotypes were assigned to families and subfamilies using the SpotClust program. The most prevalent family was the Beijing family (74.08%), followed by the T family (14.11%). CAS family strains were found only in the Xinjiang and Tibet regions, while EAI family strains were found only in Fujian Province. In conclusion, the present study of the M. tuberculosis population in China demonstrated that Beijing family isolates are the most prevalent strains in China and that they exhibit geographical variation. Furthermore, many new spoligotypes were found in this study.

Distribution of Borrelia burgdorferi Sensu Lato in China

ABSTRACT We genotyped 102 Borrelia burgdorferi sensu lato strains isolated from ticks, animals, and patients in 11 provinces in China by PCR–restriction fragment length polymorphism (PCR-RFLP) amplification of 5S (rrf)-23S (rrl) rRNA gene spacer amplicons and multilocus sequence analysis (MLSA). The results showed that Borrelia garinii was the main genotype in China (65/102) and that it was distributed mainly in northern China. Borrelia afzelii was the second most frequently found species (22/102), and it was distributed in both northern and southern China. All Borrelia valaisiana strains were isolated from Guizhou Province. Additionally, one B. burgdorferi strain was isolated from Hunan Province. Our results show the diversity and wide distribution of B. burgdorferi sensu lato in China.

Genome sequencing and analysis of BCG vaccine strains.

Background Although the Bacillus Calmette-Guérin (BCG) vaccine against tuberculosis (TB) has been available for more than 75 years, one third of the worlds population is still infected with Mycobacterium tuberculosis and approximately 2 million people die of TB every year. To reduce this immense TB burden, a clearer understanding of the functional genes underlying the action of BCG and the development of new vaccines are urgently needed. Methods and Findings Comparative genomic analysis of 19 M. tuberculosis complex strains showed that BCG strains underwent repeated human manipulation, had higher region of deletion rates than those of natural M. tuberculosis strains, and lost several essential components such as T-cell epitopes. A total of 188 BCG strain T-cell epitopes were lost to various degrees. The non-virulent BCG Tokyo strain, which has the largest number of T-cell epitopes (359), lost 124. Here we propose that BCG strain protection variability results from different epitopes. This study is the first to present BCG as a model organism for genetics research. BCG strains have a very well-documented history and now detailed genome information. Genome comparison revealed the selection process of BCG strains under human manipulation (1908–1966). Conclusions Our results revealed the cause of BCG vaccine strain protection variability at the genome level and supported the hypothesis that the restoration of lost BCG Tokyo epitopes is a useful future vaccine development strategy. Furthermore, these detailed BCG vaccine genome investigation results will be useful in microbial genetics, microbial engineering and other research fields.

Efflux Pump Gene Expression in Multidrug-Resistant Mycobacterium tuberculosis Clinical Isolates

Isoniazid (INH) and rifampicin (RIF) are the two most effective drugs in tuberculosis therapy. Understanding the molecular mechanisms of resistance to these two drugs is essential to quickly diagnose multidrug-resistant (MDR) tuberculosis and extensive drug-resistant tuberculosis. Nine clinical Mycobacterium tuberculosis isolates resistant to only INH and RIF and 10 clinical pan-sensitive isolates were included to evaluate the expression of 20 putative drug efflux pump genes and sequence mutations in rpoB (RIF), katG (INH), the inhA promoter (INH), and oxyR-ahpC (INH). Nine and three MDR isolates were induced to overexpress efflux pump genes by INH and RIF, respectively. Eight and two efflux pump genes were induced to overexpress by INH and RIF in MDR isolates, respectively. drrA, drrB, efpA, jefA (Rv2459), mmr, Rv0849, Rv1634, and Rv1250 were overexpressed under INH or RIF stress. Most efflux pump genes were overexpressed under INH stress in a MDR isolates that carried the wild-type katG, inhA, and oxyR-ahpC associated with INH resistance than in those that carried mutations. The expression levels of 11 genes (efpA, Rv0849, Rv1250, P55 (Rv1410c), Rv1634, Rv2994, stp, Rv2459, pstB, drrA, and drrB) without drug inducement were significantly higher (P < 0.05) in nine MDR isolates than in 10 pan-sensitive isolates. In conclusion, efflux pumps may play an important role in INH acquired resistance in MDR M. tuberculosis, especially in those strains having no mutations in genes associated with INH resistance; basal expression levels of some efflux pump genes are higher in MDR isolates than in pan-sensitive isolates and the basal expressional differences may be helpful to diagnose and treat resistant tuberculosis.

Complete Genome Sequences of Mycobacterium tuberculosis Strains CCDC5079 and CCDC5080, Which Belong to the Beijing Family

Mycobacterium tuberculosis is one of most prevalent pathogens in the world. Drug-resistant strains of this pathogen caused by the excessive use of antibiotics have long posed serious threats to public health worldwide. A broader picture of drug resistance mechanisms at the genomic level can be obtained only with large-scale comparative genomic methodology. Two closely related Beijing family isolates, one resistant to four first-line drugs (CCDC5180) and one sensitive to them (CCDC5079), were completely sequenced. These sequences will serve as valuable references for further drug resistance site identification studies and could be of great importance for developing drugs targeting these sites.

Molecular typing of mycobacterium tuberculosis isolates circulating in Jiangsu Province, China

BackgroundGlobally, China is the second place with high burden of tuberculosis (TB). To explore the characteristics of the pathogens of Mycobacterium tuberculosis (MTB) circulating in this area is helpful for understanding and controlling the spread of the strains. Recent developments in molecular biology have allowed prompt identification and tracking specific strains of MTB spreading through the population.MethodsSpacer-oligonucleotide typing (spoligotyping) and mycobacterial interspersed repetitive units variable number tandem repeat (MIRU-VNTR) were performed in combination to yield specific genetic profiles of 260 MTB strains isolated from 30 counties of Jiangsu province in China between June and July 2010. The spoligotyping results were in comparison to the world Spoligotyping Database of Institute Pasteur de Guadeloupe (SpolDB4). Drug susceptibility test (DST) was performed on all strains by proportion method on Lowenstein-Jensen (LJ) culture media.ResultsBased on the spoligotyping method, 246 strains displayed known patterns and 14 were absent in the database. Predominant spoligotypes belonged to the Beijing family (80.4%). By using the 24-loci VNTR typing scheme, 224 different patterns were identified, including 20 clusters and 204 unique patterns. The largest clade comprised 195 strains belonging to the Beijing family. The combination of spoligotyping and 24-loci MIRU-VNTR demonstrated maximal discriminatory power. Furthermore, we observed a significant association between Beijing family strains and drug-resistant phenotypes. The Beijing family strains presented increased risks for developing multi-drug resistant TB, with the OR (95% CI) of 11.07(1.45-84.50).ConclusionsThe present study demonstrated that Beijing family isolates were the most prevalent strains circulating in Jiangsu province of China. The utility of spoligotyping in combination with 24-loci MIRU-VNTR might be a useful tool for epidemiological analysis of MTB transmission.

Genetic Diversity of Mycobacterium tuberculosis Isolates from Tibetans in Tibet, China

Background Tuberculosis (TB) is a serious health problem in Tibet where Tibetans are the major ethnic group. Although genotyping of Mycobacterium tuberculosis (M. tuberculosis) isolates is a valuable tool for TB control, our knowledge of population structure of M. tuberculosis circulating in Tibet is limited. Methodology/Principal Findings In our study, a total of 576 M. tuberculosis isolates from Tibetans in Tibet, China, were analyzed via spoligotyping and 24-locus MIRU-VNTR. The Beijing genotype was the most prevalent family (90.63%, nu200a=u200a522). Shared-type (ST) 1 was the most dominant genotype (88.89%, nu200a=u200a512). We found that there was no association between the Beijing genotype and sex, age and treatment status. In this sample collection, 7 of the 24 MIRU-VNTR loci were highly or moderately discriminative according to their Hunter-Gaston discriminatory index. An informative set of 12 loci had similar discriminatory power with 24 loci set. Conclusions/Significance The population structure of M. tuberculosis isolates in Tibetans is homogeneous and dominated by Beijing genotype. The analysis of 24-locus MIRU-VNTR data might be useful to select appropriate VNTR loci for the genotyping of M. tuberculosis.

Analysis of embCAB Mutations Associated with Ethambutol Resistance in Multidrug-Resistant Mycobacterium tuberculosis Isolates from China

ABSTRACT Ethambutol (EMB) plays a pivotal role in the chemotherapy of drug-resistant tuberculosis (TB), including multidrug-resistant tuberculosis (MDR-TB). Resistance to EMB is considered to be caused by mutations in the embCAB operon (embC, embA, and embB). In this study, we analyzed the embCAB mutations among 139 MDR-TB isolates from China and found a possible association between embCAB operon mutation and EMB resistance. Our data indicate that 56.8% of MDR-TB isolates are resistant to EMB, and 82.2% of EMB-resistant isolates belong to the Beijing family. Overall, 110 (79.1%) MDR-TB isolates had at least one mutation in the embCAB operon. The majority of mutations were present in the embB gene and the embA upstream region, which also displayed significant correlations with EMB resistance. The most common mutations occurred at codon 306 in embB (embB306), followed by embB406, embA(−16), and embB497. Mutations at embB306 were associated with EMB resistance. DNA sequencing of embB306–497 was the best strategy for detecting EMB resistance, with 89.9% sensitivity, 58.3% specificity, and 76.3% accuracy. Additionally, embB306 had limited value as a candidate predictor for EMB resistance among MDR-TB infections in China.