Journal of Infection (2015) 70, 335e345

www.elsevierhealth.com/journals/jinf

Genotyping and molecular characteristics of multidrug-resistant Mycobacterium tuberculosis isolates from China Zhijian Zhang a,b,e, Jie Lu c,e, Min Liu d,e, Yufeng Wang b, Geping Qu a, Hongxia Li a, Jichun Wang b, Yu Pang a,b,*, Changting Liu a,***, Yanlin Zhao b,** a

Respiratory Diseases Department of Nanlou, Chinese People’s Liberation Army General Hospital, Beijing, China b National Center for Tuberculosis Control and Prevention, Chinese Center for Disease Control and Prevention, Beijing, China c Beijing Key Laboratory for Pediatric Diseases of Otolaryngology, Head and Neck Surgery, Beijing Pediatric Research Institute, Beijing Children’s Hospital, Capital Medical University, Beijing, China d Liaoning Provincial Center for Disease Control and Prevention, Shenyang, China Accepted 25 November 2014 Available online 5 December 2014

KEYWORDS Mycobacterium tuberculosis; Multidrug-resistant; Genotyping

Summary Objectives: The aim of this study was to explore the population structure of multidrug-resistant (MDR) tuberculosis strains and distribution of resistance-associated nucleotide alteration among the different genotype MDR strains in China. Methods: The genotypes of 376 MDR strain were analyzed by 15-loci MIRU-VNTR and RD105 deletion-targeted multiplex PCR (DTM-PCR) method. In addition, all the MDR isolates were sequenced for genetic mutations conferring rifampicin (rpoB) and isonizid resistance (katG, inhA and oxyR-ahpC ). Results: Among the 376 MDR isolates, 261 (69.4%) belonged to Beijing genotype, including 177 modern Beijing strains (67.8%) and 84 ancient Beijing (32.2%) strains. The percentages of streptomycin-resistant, kanamycin-resistant, pre-XDR and XDR TB in modern Beijing genotype were significantly lower than ancient genotype (P < 0.05). The Beijing MDR strains had

* Corresponding author. National Center for Tuberculosis Control and Prevention, Chinese Center for Disease Control and Prevention, No. 155, Chang Bai Road, Changping District, Beijing 102206, China. Tel./fax: þ86 10 5890 0779. ** Corresponding author. National Center for Tuberculosis Control and Prevention, Chinese Center for Disease Control and Prevention, No. 155, Chang Bai Road, Changping District, Beijing 102206, China. Tel./fax: þ86 10 5890 0777. *** Corresponding author. Respiratory Department of Nanlou, Chinese People’s Liberation Army General Hospital, 28# Fuxing Road, Haidian District, Beijing 100853, China. Tel./fax: þ86 10 6687 6272. E-mail addresses: [email protected] (Y. Pang), [email protected] (C. Liu), [email protected] (Y. Zhao). e These authors contributed equally to this work. http://dx.doi.org/10.1016/j.jinf.2014.11.008 0163-4453/ª 2014 The British Infection Association. Published by Elsevier Ltd. All rights reserved.

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Z. Zhang et al. significantly higher proportions of ofloxacin-resistant and pre-XDR isolates than non-Beijing strains (P < 0.01). In addition, the clustering rate of modern Beijing strains was significantly higher than that of ancient Beijing strains (46.3% vs. 11.9%, P < 0.01). 94.7% and 79.3% of MDR isolates harbored genetic mutations conferring rifampicin and isonizid resistance, respectively, and the most prevalent mutation was located in codon rpoB531 and katG315. In addition, the rpoB531 and katG mutation were more frequently observed among Beijing genotype strains than non-Beijing strains, while non-Beijing genotype showed stronger association with isolates lacking mutation in rifampicin resistance determination region (P < 0.05). Conclusions: Our findings demonstrated that ancient Beijing MDR strains were associated with drug resistance, while modern Beijing MDR strains were more likely to be clustered. ª 2014 The British Infection Association. Published by Elsevier Ltd. All rights reserved.

Introduction Multidrug-resistant tuberculosis (MDR-TB), defined as the strains resistant to at least isoniazid (INH) and rifampicin (RIF), is the major threat to TB control and prevention strategy worldwide.1,2 World Health Organization (WHO) estimated that there were approximately 0.31 million cases of MDR-TB throughout the world in 2011.3 China has a serious epidemic of MDR-TB, accounting for nearly a quarter of MDR-TB burden in the world.2,3 A national drug resistance survey conducted in China in 2007 reported that 5.7% of new TB patients and 35.6% of previously treated patients had MDR-TB.2 In recently years, researchers have demonstrated the use of molecular epidemiology tools to investigate the transmission and prevalence of different Mycobacterium tuberculosis strains.4,5 Rapid and inexpensive genotyping based on PCR assays, such as mycobacterial interspersed repetitive unitevariable number tandem repeat (MIRUVNTR) method, has been proven to be useful in investigating the genetic relationships and epidemiology of MDRTB in numerous literature.6e9 Similarly, the RD105 deletion-targeted multiplex polymerase chain reaction (DTM-PCR) has also been considered as a good alternative method to spoligotyping to predict M. tuberculosis Beijing strains, as it is faster and easier to perform.10,11 In clinical MTB isolates, the resistance to anti-TB drugs can be the result of genomic mutations in genes encoding either the drug target or enzyme conferring drug activation.12,13 To date, several resistance-associated mutations have been identified for commonly used anti-TB drugs, including RIF, INH, ethambutol (EMB), fluroquinolone.13 A mutation located in the 81-bp region of the gene encoding the beta subunit of RNA polymerase (rpoB) dtermed the rifampin resistance determinant region (RRDR)dis responsible for more than 95% of RIF-resistant isolates.13,14 The most common mutations in the RRDR region are observed in codons 516, 526 and 531.13 For INH, several genes, including katG, the promoter of inhA and the intergenic region of oxyR-ahpC, are associated with INH resistance in M. tuberculosis isolates.13,15 Of those INH resistance targets, the substitution of a single nucleotide at codon 315 of the katG gene is the most frequently identified mutation type, conferring approximately 70% of INH resistant isolates.15,16 Due to the high prevalence of MDR among TB patients, China has been classified as global “hotspots” of MDR-

TB.2,17,18 Although several molecular epidemiological studies have been performed among MDR strains isolated from different regions of China, the knowledge on molecular characteristics of MDR-TB isolates representative of the whole China is still unknown now.7e9 In the present study, we sought to investigate the population structure of MDR strains in China by using standard 15-loci MIRU-VNTR method and DTM-PCR method. We also analyzed the molecular characteristics of resistance-associated mutations in four specific genes (rpoB for RIF; katG, the inhA promoter and intergenic region oxyR-ahpC for INH) by DNA sequencing. Furthermore, the data have been used to determine the distribution of resistance-associated nucleotide alteration among the strains of different genotypes.

Materials and methods Bacterial strains and culture conditions MDR M. tuberculosis strains, identified by conventional drug susceptibility testing(DST), were all obtained from national tuberculosis drug resistance survey of China conducted in 2007.2 The DST was performed by the proportion method as recommended by WHO/IUATLD in National Tuberculosis Reference Laboratory (NTRL) in China.2,19 The concentrations of anti-TB drugs in Lowenstein-Jensen (L-J) media was as follows: INH 0.2 mg/mL, RIF 40 mg/mL, EMB2 mg/ mL, streptomycin (SM) 4 mg/mL, kanamycin (KAN) 30 mg/ mL and ofloxacin (OFLX) 2 mg/mL. The strains was determined as resistant to the specific drug when the growth rate was >1% compared to the control. MDR-TB isolates were defined as the strains resistant at least to INH and RIF. In addition, Pre-XDR is defined as MDR strains resistant to either OFLX or KAN, but not both; XDR is defined as MDR strains resistant to both OFLX and KAN. The NTRL participated in the annual proficiency testing of DST organized by the Hong Kong Supranational tuberculosis Reference Laboratory and has passed each testing since 2003. All the MDR strains were recovered on L-J media for 4 weeks at 37  C.

Genomic DNA extraction Genomic DNA was extracted from freshly cultured bacteria as previously reported.20 The bacterial cells were transferred into a microcentrifuge tube containing 500 ml Tris-

Genotyping of MDR-TB isolates from China EDTA (TE) buffer, followed by centrifuging at 13,000 rpm for 2 min. Then, the supernatant was discarded, and the pellet was resuspended in 500 ml TE buffer. The suspension of bacterial cells were heated in a 95  C water bath for 1 h, and centrifuged at 13,000 rpm for 5 min. The DNA in the supernatant was used for PCR amplification.

Genotyping RD105 DTM-PCR was performed to distinguish Beijing genotype from non-Beijing genotype as previously described.11 The strains with no RD105 region amplification were classified to Beijing genotype, while the others containing RD105 region belonged to non-Beijing genotype. In order to differentiate modern Beijing and ancient Beijing strains, the IS6110 PCR in the NTF region was analyzed according to methods described as the previous study.21 The modern Beijing strains were defined as the Beijing strains showing a 1.8-kb DNA fragment after amplification, while those without the insert posed a 700-bp PCR product. In addition, all the MDR isolates were genotyped using the classical 15-loci MIRU-VNTR method described by Supply et al.22 The PCR products were analyzed with 1.5% agarose electrophoresis at 5 V/cm for 1 h using 100bp DNA ladder (Genestar, Beijing, China) as a size marker. The amplicons of H37Rv were loaded per 8 lanes as an additional control for accuracy. The corresponding number at each locus was calculated according to the repeat and flank length. In addition, the HuntereGaston discriminatory Index (HGDI) was used to evaluate the discriminatory power of the MIRU-VNTR loci. The HGDI was calculated as previous reported.23

DNA sequencing The fragments of rpoB, katG, the inhA promoter and intergenic region oxyR-ahpC were amplified by PCR, respectively. The primer pairs were synthesized as previously reported.24 The genomic DNA was used as template to perform PCR amplification as follows: each PCR mixture was prepared in a volume of 50 ml containing 25 ml 2  PCR Mixture, 2 ml of DNA template and 0.2 mM of each primer set. PCR program was performed under the following conditions: initial denaturation at 94  C for 5 min, and then 35 cycles of denaturation at 94  C for 1 min, annealing at 60  C for 1 min, and extension at 72  C for 2 min, followed by a final extension at 72  C for 10 min. PCR products were purified using a QIAquickQiagen PCR purification kit (Qiagen), and then sent to Qingke Company (Beijing, China) for sequencing service. The resulting sequences were compared to the homologous sequences of the reference M. tuberculosis H37Rv strains using BLASTn optimized for megablast in the National Center for Biotechnology Information website (www.ncbi.nlm.nih.gov/BLAST).

Statistical analysis The Pearson chi-square test or the Fisher exact test was used to compare the proportions of Beijing genotype and non-Beijing genotype M. tuberculosis isolates with

337 different characteristics. Two-sided P values of