Elsevier

Gene

Volume 582, Issue 2, 15 May 2016, Pages 128-136
Gene

Research paper
Whole genome analysis of an MDR Beijing/W strain of Mycobacterium tuberculosis with large genomic deletions associated with resistance to isoniazid

https://doi.org/10.1016/j.gene.2016.02.003Get rights and content

Highlights

  • An MDR strain of M. tb W146 was analyzed by the whole genome sequencing.

  • Three large genomic deletions were identified in W146.

  • The missing of furA–katG operon of LGD2 in W146 conferred resistance to isoniazid.

Abstract

Mycobacterium tuberculosis (M.tb) is one of the most prevalent bacterial pathogens in the world. With geographical wide spread and hypervirulence, Beijing/W family is the most successful M.tb lineage. China is a country of high tuberculosis (TB) and high multiple drug-resistant TB (MDR-TB) burden, and the Beijing/W family strains take the largest share of MDR strains. To study the genetic basis of Beijing/W family strains' virulence and drug resistance, we performed the whole genome sequencing of M.tb strain W146, a clinical Beijing/W genotype MDR isolated from Wuxi, Jiangsu province, China. Compared with genome sequence of M.tb strain H37Rv, we found that strain W146 lacks three large fragments and the missing of furA-katG operon confers isoniazid resistance. Besides the missing of furA-katG operon, strain W146 harbored almost all known drug resistance-associated mutations. Comparison analysis of single nucleotide polymorphisms (SNPs) and indels between strain W146 and Beijing/W genotype strains and non-Beijing/W genotype strains revealed that strain W146 possessed some unique mutations, which may be related to drug resistance, transmission and pathogenicity. These findings will help to understand the large sequence polymorphisms (LSPs) and the transmission and drug resistance related genetic characteristics of the Beijing/W genotype of M.tb.

Introduction

Drug-resistant strains of Mycobacterium tuberculosis (M.tb) pose a major threat to the control of TB worldwide (Tomioka, H. and Namba, K., 2006, Nodieva, A., et al., 2010). Although the curative anti-tuberculosis therapy has been established a half century ago, M.tb resistance to the most important anti-tuberculosis drugs emerged quickly due to inappropriate treatment (van der Werf, M.J., et al., 2012, Langendam, M.W., et al., 2012). The drug-resistant tuberculosis spread quickly owing to the convergence of resistant strains of M.tb especially in high-risk patients such as those with HIV/AIDS and in high-risk environments such as hospitals (Moro, M.L., et al., 1998, Pedersen, C., et al., 1997). Meanwhile, compared with patients with drug-susceptible tuberculosis, patients with drug-resistant tuberculosis were more likely to generate new secondary cases (Prasad, 2010).

The molecular mechanisms of drug resistance have been elucidated in several studies. Spontaneous chromosomal mutations account for drug resistance in M.tb and mutations in a few genes of M.tb result in the occurrence of resistance to rifampin (RIF), isoniazid (INH), streptomycin (SM), and ethambutol (EMB), the main components of the first-line multidrug therapy for tuberculosis (Sintchenko, V., et al., 1999, Kozhamkulov, U., et al., 2011, Ramaswamy, S.V., et al., 2003, Tracevska, T., et al., 2004, Wong, S.Y., et al., 2011). More than 97% of RIF-resistant cases is due to mutations within the 81 bp “core region” of the rpoB gene, called the RIF resistance-determining region (RRDR) (Cole, 1996). As a prodrug, INH inhibits mycolic acid synthesis and its primary target is believed to be the enoyl-acyl carrier protein InhA. Mutations in the promoter and ORF region of inhA lead to a low level of INH resistance. In contrast, mutations in katG, which encodes the catalase–peroxidase enzyme KatG, result in a high level resistance (Oliveira, J.S., et al., 2006, O'Brien, K.L., et al., 1996). Nearly 70% of the streptomycin-resistant clinical isolates present mutations associated with rpsL and rrs genes, which encode the ribosomal protein S12 and 16S rRNA respectively (Tracevska et al., 2004). Mutations in codon 43 in the rpsL gene are the most common case. 47–65% mutations associated with resistance to EMB are found in the embB gene (Cuevas-Cordoba et al., 2015).

The World Health Organization (WHO) estimates that there were 480,000 new cases of MDR-TB worldwide in 2014, defined as strains that are resistant to at least the two most powerful first-line anti-TB drugs (isoniazid and rifampicin) (Zumla et al., 2015). Among patients with pulmonary TB who were notified in 2013, an estimated 300,000 (range: 230,000–380,000) had MDR-TB. More than half of these patients were in China, India and the Russian Federation (Zumla et al., 2015).The Beijing/W genotype of M.tb is one of the most successful M.tb lineages in China even in the world (Zumla et al., 2015). It has been reported that several sublineages of the Beijing/W genotype are more frequently associated with MDR-TB (Iwamoto et al., 2008).

Gao and his colleagues reported that Beijing/W genotype strains have an increased ability to transmit infection, with Beijing/W genotype strains progressing more rapidly to active tuberculosis (Yang et al., 2012). However, the mechanisms underlying these epidemiological findings remain to be clearly elucidated. It is important to search for molecular basis for transmission, pathogenicity warrant and drug resistance in Beijing/W genotype. The whole genome sequencing is an efficient method for acquiring the genetic information of M.tb. Sequencing of the whole genomic DNA of M.tb strain H37Rv was completed in 1998 and it opened the way to understand the biology, metabolism and evolution of this pathogen in the genomic level (Brosch et al., 1998). In recent years, some Beijing/W genotype strains including drug-sensitive and multidrug-resistant strains' complete genome sequences have been determined (Wu, W., et al., 2013, Zhang, Y., et al., 2011). However, the whole genome sequence of MDR-M.tb with large deletions is still lacking.

In this study, we sequenced the genome of one clinical MDR isolate-strain W146 isolated from Wuxi, Jiangsu province, China. We found W146 missing three large sequences and the missing of furA-katG operon conferred isoniazid resistance. We also analyzed the SNPs and indels of strain W146. We performed the comparative genome analysis of W146 with other Beijing/W genotype strains and non-Beijing/W genotype strains. We hope our report could promote the understanding of LSPs (large sequence polymorphisms) of M.tb and the genome polymorphism of the Beijing/W family.

Section snippets

Ethics statement

The study was reviewed and approved by the local ethics committee (The ethics committee of Wuxi People's Hospital). Written informed consent was obtained from participants prior to their enrollment in the study.

Strain isolation and drug susceptibility testing

The clinical isolate of M.tb strain W146 was obtained from patient with pulmonary tuberculosis in Wuxi. The isolate was grown in Lowenstein–Jenson (L–J) at 37 °C for approximately 3–4 weeks. The isolate was tested for drug susceptibility according to the Clinical and Laboratory Standards

Genotyping and phenotypic drug resistance of strain W146

Strain W146 was isolated from a young male patient in Wuxi who had a history of previous TB. W146 was an MDR isolate and had resistance to five first-line anti-TB drugs (isoniazid, rifampin, streptomycin, pyrazinamide, and ethambutol). The genomic deletion RD105 phylogenetically defines the Beijing/W family as a separate lineage within M.tb. By targeting RD105, we used Beijing P1, Beijing P2 and Beijing P3 primers in a multiplex PCR and obtained a 761 bp product for W146. These results indicated

Discussion

Beijing/W family has a higher ability to transmit infection compared to non-Beijing/W family. It has been reported that the Beijing/W genotype MDR strains have successfully disseminated in the world (Yang, C., et al., 2012, Mariam, D.H., et al., 2004). However, the genetic basis for the robust transmitting ability of Beijing/W genotype MDR strains remains unclear. In our study, we performed the whole-genome sequence for the strain W146 – a Beijing/W genotype MDR strain. Most interestingly, we

Transparency declarations

The authors declare no conflicts of interest.

Acknowledgements

This work was supported by grants from the National Natural Science Foundation of China (No. 81361120383 and No. 31270173), the State Key Development Programs for Basic Research of China (973 Program No. 2015CB554203), the Program for Professor of Special Appointment (Eastern Scholar) at Shanghai Institutions of Higher Learning.

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