Genotyping and molecular characteristics of multidrug-resistant Mycobacterium tuberculosis isolates from China

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.

336

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 <0.05 was considered statistically significant. The statistical analysis was performed in the SPSS 15.0 (SPSS Inc., Chicago, Illinois, USA).

Results A total of 401 MDR isolated were enrolled in the national drug-resistant survey. Out of 401 MDR strains, 376 could be sub-cultured successfully for subsequent genotyping analysis and DNA sequencing.

Beijing genotype Among the 376 MDR isolates, 261 strains (69.4%) belonged to Beijing genotype, while the other 115 (30.6%) to nonBeijing genotype. By detecting the IS6110 in the NTF region, the 261 Beijing genotype strains were further identified as 177 modern Beijing genotype (67.8%) and 84 ancient Beijing genotype (32.2%) strains. We firstly compared the percentages of drug-resistance and socialdemographic characteristics of patients identified as infected with ancient Beijing genotype and modern Beijing genotype. As shown in Table 1, the percentages of SMresistant, KAN-resistant, pre-XDR and XDR TB in modern Beijing genotype were significantly lower than ancient genotype (69.5% vs. 82.1% for SM; 7.9% vs. 21.4% for KAN, 69.5% vs. 85.1% for pre-XDR; 5.6% vs. 15.5% for XDR, P < 0.05), while the percentages of the other drug susceptibility profiles and several social characteristics showed no difference between ancient Beijing genotype and modern Beijing genotype. Furthermore, we analyzed the distribution of drug-resistance and social-demographic characteristics among the Beijing genotype and non-Beijing genotype. Our data demonstrated that the proportions of OFLXresistance and pre-XDR was significantly lower in the nonBeijing genotype than in the Beijing genotype group (17.4% vs. 31.8% for OFLX; 15.7%vs. 30.7% for pre-XDR, P < 0.01). In addition, statistical analysis revealed that no difference was observed among other characteristics between Beijing genotype and non-Beijing genotype (Table 2).

MIRU-VNTR The 376 MDR isolates were classified into 307 genotypes by 15-loci MIRU-VNTR. Of 307 genotypes, 278 isolates harbored a unique pattern, while the other 98 isolates belonged to 29 clusters (2e16 isolates per cluster), and the cumulative clustering rate was 26.1% and the HGDI was 0.996 (Table 3 & Table S1). By different genotypes, ancient Beijing had 10 clustered strains, modern Beijing had 82, Beijing had 92 and non-Beijing had 6. Statistical analysis revealed that the clustering rate of modern Beijing genotype strains was significantly higher than ancient Beijing genotype strains (P < 0.01). Similarly, we also observed the higher clustering rate of Beijing genotype strains when comparing with that of non-Beijing genotype (P < 0.01) (Fig. 1 & Table 3). The discriminatory power of each MIRU-VNTR locus was evaluated by using the HGDI. As shown in Table S2, the HGDI

338 Table 1

Z. Zhang et al. Differences of characteristics between ancient and modern Beijing genotype strains.

Characteristics

Resistance to: SM EMB OFLX KAN Pre-XDRa XDR Sex Men Women Age group (years) <25 25e44 45e64 >64 Treatment History New case Re-treated

No. (%) of Beijing genotype isolates (n Z 261)

No. (%) of isolates Ancient Beijing (n Z 84)

Modern Beijing (n Z 177)

OR 95%CI

P value

192(73.6) 136(52.1) 83(31.8) 32(12.3) 80(30.7) 23(8.4)

69(82.1) 41(48.8) 32(38.1) 18(21.4) 34(40.5) 13(15.5)

123(69.5) 95(53.7) 51(28.8) 14(7.9) 46(26.0) 10(5.6)

0.50(0.26e0.94) 1.22(0.72e2.04) 0.66(0.38e1.14) 0.31(0.15e0.65) 0.52(0.30e0.89) 0.33(0.14e0.67)

0.03 0.46 0.13 <0.01 0.02 <0.01

174(66.7) 87(33.3)

61(72.6) 23(27.4)

113(63.8) 64(36.2)

1.0(Ref.) 0.67(0.38e1.17)

e

42(16.1) 118(45.2) 78(29.9) 23(8.8)

10(11.9) 43(51.2) 22(26.2) 9(10.7)

32(18.1) 75(42.4) 56(31.6) 14(7.9)

0.55(0.25e1.21) 1.0(Ref.) 0.69(0.37e1.27) 1.12(0.45e2.81)

e

115(44.1) 146(55.9)

36(42.9) 48(57.1)

79(44.6) 98(55.4)

1.0(Ref.) 1.07(0.64e1.82)

e

0.16 0.14 0.23 0.81

0.79

a

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.

of 3 loci exceeded 0.6, classified as highly discriminating, and the 8 loci showed moderate discrimination (0.3  HGDI  0.6). The other 4 loci were found to be less polymorphic (HGDI < 0.3). In comparison with Beijing genotype strains, most of the loci in non-Beijing genotype

Table 2

Differences of characteristics between Beijing and non-Beijing genotype strains.

Characteristics

Resistance to: SM EMB OFLX KAN Pre-XDRa XDR Sex Men Women Age group (years) <25 25e44 45e64 >64 Treatment History New case Re-treated a

strains showed more allelic diversity. In addition, our data revealed that the discriminatory power of most loci among ancient Beijing genotype strains was higher than that among modern Beijing genotype strains. Interestingly, the locus Qub4156 showed negligible diversity (HGDI < 0.1) for

No. (%) of isolates (n Z 376)

No. (%) of isolates Beijing (n Z 261)

Non-Beijing (n Z 115)

OR 95%CI

P value

269(71.5) 199(52.9) 103(27.4) 43(11.4) 98(26.1) 29(7.7)

192(73.6) 136(52.1) 83(31.8) 32(12.3) 80(30.7) 23(8.4)

77(67.0) 63(54.8) 20(17.4) 11(9.6) 18(15.7) 6(5.2)

0.73(0.45e1.71) 1.11(0.72e1.73) 0.45(0.26e0.77) 0.76(0.37e1.56) 0.42(0.24e0.73) 0.57(0.23e1.42)

0.19 0.63 <0.01 0.45 <0.01 0.23

252(67.0) 124(33.0)

174(66.7) 87(33.3)

78(67.8) 37(32.2)

1.0(Ref.) 1.05(0.66e1.83)

e

68(18.1) 159(42.3) 115(30.6) 34(9.0)

42(16.1) 118(45.2) 78(29.9) 23(8.8)

26(22.6) 41(35.7) 37(32.2) 11(9.6)

0.56(0.31e1.02) 1.0(Ref.) 0.73(0.43e1.24) 0.73(0.33e1.62)

e

164(43.6) 212(56.4)

115(44.1) 146(55.9)

49(42.6) 66(57.4)

1.0(Ref.) 0.94(0.61e1.47)

e

0.83 0.06 0.25 0.43

0.79

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.

Genotyping of MDR-TB isolates from China

339

Table 3 Discriminatory index and clustering rate of 15-locus set applied to Mycobacterium tuberculosis strains in various MDRTB groups. Group Genotype Beijing Ancient Modern Non-Beijing Population New case Re-treated case Total a

Total no. of isolates

No. of clustered isolates

No. of isolates in each cluster

Clustering rate (%)a

HGDI

261 84 177 115

92 10 82 6

2e16 ‘2 2e16 2

35.2% 11.9% 46.3% 5.2%

0.993 0.999 0.984 0.999

164 212 376

46 52 98

2e8 2e8 2e16

28.0% 24.5% 26.1%

0.994 0.992 0.996

P < 0.01 (Ancient Beijing vs. Modern Beijing); P < 0.01 (Beijing vs. non-Beijing); P Z 0.44 (New case vs. re-treated case).

modern Beijing genotype strains (HGDI Z 0.017), while the discriminating ability of this locus was significantly higher for ancient Beijing genotype (HGDI Z 0.516) and nonBeijing genotype strains (HGDI Z 0.414).

Association between rpoB mutations and genotypes In total, 355 out of 376 (94.7%) MDR isolates harbored a mutation located in the rpoB genes. The most prevalent mutation conferring RIF resistance was codon 531 (63.3%), resulting in the amino acid substitution of Ser to Leu (60.4%), Trp (2.7%) or Tyr (0.3%). The second most affected codon was 526, conferring RIF resistance of 69 (18.4%) MDRTB isolates, and had five types of amino acid substitutions. 25 isolates had a mutation in codon 516, 8 in codon 511, 3 in codon 513, 9 in codon 515, 2 in codon 522 and 2 in codon 533 (Table 4). We also analyzed distribution of mutation types among different genotype strains. As shown in Table 4, there was no significant difference of mutation distribution between modern and ancient Beijing genotype strains (P > 0.05). When compared with Beijing genotype strains (67.4%), the proportion of MDR isolates harboring codon 531 mutation among non-Beijing genotype strains (54.1%) was significantly lower (P Z 0.01). In contrast, statistical analysis indicated that the non-Beijing genotype strains (14.8%) had significantly higher percentage of MDR isolates with no mutation located in the RRDR of rpoB gene than Beijing genotype (1.1%, P < 0.01).

Association between INH resistance-related mutations and genotypes Out of 376 MDR isolates, 231 (61.4%) carried katG mutations, the vast majority of which was the commonly described substitution of codon 315 from Ser to Thr (55.3%, 208/376). In addition, we also observed that sixteen isolates harbored substitution from Ser to Asn, and four from Ser to Ile. In addition to codon 315, substitution SNP was identified at codon 299. We also screened for mutations in inhA promoter and oxyR-ahpC intergenic region. Mutations in the inhA promoter region were observed in 67 (17.8%) MDR isolates; 48 (12.8%) of which had a mutation

at 15 of the inhA regulatory gene region. In addition, 48 isolates (12.8%) had mutations in the intergenic region of oxyR-ahpC, where nine different types of nucleotide substitution were detected. Of these isolates, 13(3.5%) and 9 (2.4%) isolates had 12C/T and 10C/T substitution, respectively. In addition, we also identified 78 (20.7%) MDR isolates harboring no mutation in the three genes (Table 5). We next evaluated for the potential relationship between mutations and strain families. Statistical analysis demonstrated that Beijing genotype strains (66.3%) showed high frequency of katG mutation than non-Beijing genotype (50.4%, P < 0.01). On the contrary, Beijing genotype was not associated with mutations in the inhA promoter (P Z 0.66) and intergenic region of oxyR-ahpC (P Z 0.66) (Table 5).

Discussion In this study, we analyzed the genotypes and molecular characteristics of MDR-TB strains from China by using a combination of DTM-PCR and 15-loci MIRU-VNTR typing. Beijing genotype is one of the most successful clades in the present worldwide tuberculosis epidemic,25,26 especially in Eastern Asia, South Africa, and Northern Eurasia.27,28 Similar with the results of different local regions in China, our data confirm that Beijing genotype remains the predominant lineage among MDR isolates in China. However, the rate of Beijing genotype among MDR isolates is unequal among the different regions of China, ranging from 73.3% to 93.3% (Table 6).7e9,29e31 Numerous studies have demonstrated that Beijing genotype is associated with drug resistance, suggesting increased bacterial fitness.32e35 In contrast, several epidemiological studies from various geographic setting suggested that Beijing strains were no more likely to acquire drug resistance than non-Beijing strains.21,36 Our data are in agreement with former findings. Among the MDR isolates, Beijing genotype strains present higher rate of OFLX and pre-XDR resistance than nonBeijing strains. In addition to potential association with drug resistance, Beijing genotype strains were more likely to cause transmission than non-Beijing strains in several molecular epidemiological studies.36,37 Similarly, we also

340

Figure 1 profiles.

Z. Zhang et al.

Dendrogram of 376 MDR M. tuberculosis isolates from China. The phylogenetic tree was genetated from the MIRU-VNTR

observed that Beijing genotype showed higher genotypic clustering rate than non-Beijing genotype, reflecting recent transmission. One hypothesis for the emergency of Beijing genotype is that Beijing genotype strains can escape from the protective effect of the BCG vaccine.26,36 Therefore,

the widely vaccinated BCG in China might be a selective force favoring the transmission of the Beijing genotype, especially MDR Beijing strains. The Beijing linage consists of two major groups, modern and ancient Beijing strains.26 The modern Beijing strains

Genotyping of MDR-TB isolates from China Table 4

341

Distribution of different mutations located in RDRR of rpoB among Beijing and non-Beijing genotypes.

Mutation type

No. of isolates with different mutations (%) Beijing (n Z 261)

rpoB511 Leu511Pro rpoB513 Gln513Pro rpoB515 Met515Ile ins515Phe rpoB516 Asp516Ala Asp516Asn Asp516Gly Asp516Tyr Asp516Val rpoB522 Ser522Leu rpoB526 His526Arg His526Asn His526Asp His526Leu His526Tyr rpoB531 Ser531Leu Ser531Trp Ser531Tyr rpoB533 Leu533Pro No mutation

Ancient (n Z 84)

Modern (n Z 177)

P value

Total

Non-Beijing (n Z 115)

1(1.2%) 1(1.2%) 2(2.4%) 2(2.4%) 2(2.4%) 1(1.2%) 1(1.2%) 11(13.1%) 5(6.0%) 2(2.4%) 0(0.0%) 3(3.6%) 1(1.2%) 0(0.0%) 0(0.0%) 9(10.7%) 4(4.8%) 3(3.6%) 0(0.0%) 0(0.0%) 2(2.4%) 59(70.2%) 57(67.9%) 2(2.4%) 0(0.0%) 0(0.0%) 0(0.0%) 0(0.0%)

5(2.8%) 5(2.8%) 1(0.6%) 0(0.0%) 2(1.1%) 2(1.1%) 0(0.0%) 14(7.9%) 0(0.0%) 3(1.7%) 5(2.8%) 0(0.0%) 6(3.4%) 0(0.0%) 0(0.0%) 33(18.6%) 4(2.3%) 0(0.0%) 15(8.5%) 1(0.6%) 13(7.3%) 117(66.1%) 112(63.3%) 5(2.8%) 0(0.0%) 2(1.1%) 2(1.1%) 3(1.7%)

e

6(2.3%) 6(2.3%) 3(1.1%) 3(1.1%) 4(1.5%) 3(1.1%) 1(0.4%) 25(9.6%) 5(1.9%) 5(1.9%) 5(1.9%) 3(1.1%) 7(2.7%) 0(0.0%) 0(0.0%) 42(16.1%) 8(3.1%) 3(1.1%) 15(5.7%) 1(0.4%) 15(5.7%) 176(67.4%) 169(64.8%) 7(2.7%) 0(0.0%) 2(0.8%) 2(0.8%) 3(1.1%)

2(1.7%) 2(1.7%) 0(0.0%) 0(0.0%) 5(4.3%) 3(2.6%) 2(1.7%) 0(0.0%) 0(0.0%) 0(0.0%) 0(0.0%) 0(0.0%) 0(0.0%) 2(1.7%) 2(1.7%) 27(23.5%) 5(4.3%) 1(0.9%) 6(5.2%) 2(1.7%) 13(11.3%) 62(54.1%) 58(50.4%) 3(2.6%) 1(0.9%) 0(0.0%) 0(0.0%) 17(14.8%)

e e

0.18

e 0.10

0.51

e e

are the most widely disseminated Beijing strains, accounting for 95% of Beijing strains in Russian, 86% in Hong Kong, 76% in Beijing and 75% in Vietnam.27,36,38 Consistent with previous reports, we observed that the frequency of modern Beijing strains was 67.8% among MDR Beijing strains in China. Several publications indicated that these two major evolutionary lineages of Beijing genotype displayed different pathogenic and drug-resistant features.27 Mokrousov et al. revealed that ancient Beijing genotype was significantly associated with RIF, pyrazinamide and MDR resistance.27 In agreement to previous report, we also found that the ancient Beijing strains were more likely to acquire antituberculosis drug resistance, although only the proportions of KAN resistant and XDR strains were statistically different due to sample size. Our findings indicate that the different ratios of modern to ancient Beijing genotype subpopulations from different studies may be responsible for the discrepancy of the association between Beijing genotype and drug resistance profiles. Interestingly, we also detected an association of the modern sublineage with genotypic clustering in the present study, which was consistent to previous studies in China, South Africa, Japan and Taiwan.36,39e41 A recent research indicated that BCG vaccination is less protective against

P value

Total (n Z 376)

0.73

8(2.1%) 8(2.1%) 3(0.8%) 3(0.8%) 9(2.4%) 6(1.6%) 3(0.8%) 25(6.6%) 5(1.3%) 5(1.3%) 5(1.3%) 3(0.8%) 7(1.9%) 2(0.5%) 2(0.5%) 69(18.4%) 13(3.5%) 4(1.1%) 21(5.6%) 3(0.8%) 28(7.4%) 238(63.3%) 227(60.4%) 10(2.7%) 1(0.3%) 2(0.5%) 2(0.5%) 20(5.3%)

e e

e

e 0.09

0.01

e <0.01

modern Beijing strains.42 Hence, the higher proportion of clustered modern Beijing MDR strains may reflect the increased transmissibility of modern Beijing sublineage escaping from effective BCG. Based on the current study, our evidence supports the previous findings that the ancient and modern Beijing MDR strains undergo different mechanisms of co-existence with human populations.43e45 Modern Beijing MDR strains adapt to the increasing availability of susceptible hosts, while the emergency of ancient Beijing MDR strains depends on the higher proportion of drug resistance. Resistance to RIF in clinical M. tuberculosis isolates occurs primarily due to mutations in the RRDR of rpoB.13 Consistent with previous reports, we observed that 94.7% of MDR isolates harbored mutations in the RRDR of rpoB.13,15,46 The most frequently mutated codon in our study was codon 531 (63.3%), similar to those in India (59.0%), Nepal (58.7%) and various part of China (58.3%e 61.2%).46e48 In accordance with previous studies in Germany and Russia, we found that Beijing genotype strains had a substantially higher proportion of the rpoB531 mutation than non-Beijing strains.49,50 Besides, non-Beijing genotype showed stronger association with isolates lacking mutation in RRDR of rpoB.

342 Table 5

Z. Zhang et al. Distribution of different mutations conferring INH resistance among Beijing and non-Beijing genotypes.

Mutation type

No. of isolates with different mutations (%) Beijing (n Z 261)

KatG Gly299Ser Ser315Asn Ser315Ile Ser315Thr InhA 8T/C 15C/T 17G/T oxyR-ahpC 4insA 6G/A 8A/G 9G/A 10C/T 10C/A 12C/T 32G/A 39C/T Single Mutation KatG InhA oxyR-ahpC Double Mutations KatG þ InhA KatG þ oxyR-ahpC InhA þ oxyR-ahpC No mutation

Ancient (n Z 84)

Modern (n Z 177)

P value

Total

Non-Beijing (n Z 115)

61(72.6%) 1(1.2%) 6(7.1%) 3(3.6%) 51(60.7%) 12(14.3%) 5(6.0%) 4(4.8%) 3(3.6%) 8(9.5%) 0(0.0%) 1(1.2%) 0(0.0%) 2(2.4%) 1(1.2%) 1(1.2%) 2(2.4%) 1(1.2%) 0(0.0%) 59(70.2%) 51(60.7%) 5(6.0%) 3(3.6%) 11(13.1%) 6(7.1%) 4(4.8%) 1(1.2%) 14(16.7%)

112(63.3%) 1(0.6%) 5(2.8%) 0(0.0%) 106(59.9%) 33(18.6%) 4(2.3%) 27(15.3%) 2(1.1%) 24(13.6%) 2(1.1%) 5(2.8%) 2(1.1%) 1(0.6%) 7(4.0%) 1(0.6%) 5(2.8%) 0(0.0%) 1(0.6%) 123(69.5%) 96(54.2%) 19(10.7%) 18(10.2%) 18(10.2%) 12(4.5%) 4(2.3%) 2(1.1%) 36(20.3%)

0.14

173(66.3%) 2(0.8%) 11(4.2%) 3(1.1%) 157(60.2%) 45(17.2%) 9(3.4%) 31(11.9%) 5(1.9%) 32(12.3%) 2(0.8%) 6(2.3%) 2(0.8%) 3(1.1%) 8(3.1%) 2(0.8%) 7(2.7%) 1(0.4%) 1(0.4%) 182(69.7%) 147(56.3%) 24(9.2%) 21(8.0%) 29(11.1%) 18(6.9%) 8(3.1%) 3(1.1%) 50(19.2%)

58(50.4%) 1(0.9%) 5(4.3%) 1(0.9%) 51(44.3%) 22(19.1%) 2(1.7%) 17(14.8%) 3(2.6%) 16(13.9%) 3(2.6%) 1(0.9%) 2(1.7%) 0(0.0%) 1(0.9%) 1(0.9%) 6(5.2%) 1(0.9%) 1(0.9%) 78(67.8%) 51(44.3%) 17(14.8%) 10(8.7%) 9(7.8%) 3(2.6%) 4(3.5%) 2(1.7%) 28(24.3%)

0.38

0.35

0.90

0.48

0.48

Previous studies revealed that INH resistance-conferring mutations were most frequently detected at katG, especially in codon 315, and the promoter region of inhA.13,24 As expected, the primary mutation conferring INH resistance in this study was the katG315 substitution (60.6%), the frequency of which is similar to those in Jiangxi (65.8%) and Shanghai (72.7%), although it is lower than those in Russia (93.6%) and Fujian (82.7%).24,51,52 We also found that Beijing genotype exhibited high rate of

Table 6

P value

Total (n Z 376)

<0.01

231(61.4%) 3(0.8%) 16(4.3%) 4(1.1%) 208(55.3%) 67(17.8%) 11(2.9%) 48(12.8%) 8(2.1%) 48(12.8%) 5(1.3%) 7(1.9%) 4(1.1%) 3(0.8%) 9(2.4%) 3(0.8%) 13(3.5%) 2(0.5%) 2(0.5%) 260(69.1%) 198(52.6%) 41(10.9%) 31(8.2%) 38(10.1%) 21(5.6%) 12(3.2%) 5(1.3%) 78(20.7%)

0.66

0.66

0.71

0.33

0.25

mutations in katG, which corresponds well with previously published results.49,50 The mutations in intergenic region of oxyR-ahpC, compensate for the loss of catalase activity in M. tuberculosis, have been proven as an indicator of INH resistance.13 Nevertheless, many reports have revealed that the frequency of mutation in the oxyR-ahpC region is rare; otherwise, it always appears along with the mutations at katG315 in the INH-resistant strains.53,54 In line with a previous study by Chen et al., our data

Distribution of Beijing genotype strains among MDR TB isolates from different regions of China.

Region

No. (%)

Reference

Total

Beijing genotype

Non-Beijing genotype

Shanghai Fujian Jiangxi Hubei Chongqing Five provinces China

189 75 110 60 208 128 376

165(87.3) 55(73.3) 85(77.3) 56(93.3) 156(75.0) 108(84.4) 261(69.4)

24(12.7) 20(26.7) 25(22.7) 4(6.7) 52(25.0) 20(15.6) 115(30.6)

9 8 7 29 30 31 This study

Genotyping of MDR-TB isolates from China demonstrated that 5.1% of MDR isolates only harbored point mutations in the oxyR-ahpC region.24 Recently, several commercial kits have been developed for rapidly detecting INH-resistance by detecting the frequent mutations in katG and the promoter region of inhA.55,56 Based on this study, the combination mutations in katG gene and the promoter of inhA gene can only identify less than 75% of INH-resistant isolates in the MDR population from China. Our findings were supported by a recent multi-center evaluation of Genechip, a molecular DST diagnosis tool, which demonstrated that Genechip could only detect 80.34% INH-resistant TB patients.55 Twenty percent of MDR isolates with no resistance-associated alterations in this study may be due to mutations present outside of the sequenced region or in other genes, and the efflux-related mechanisms may also contribute to INH resistance in those M. tuberculosis isolates.57 Hence, further molecular analysis among INH-resistant strains without the known mutation will expand our current knowledge of the mechanism conferring INH resistance in the MDR isolates circulating in China. In conclusion, Beijing genotype MDR strains revealed a significant association with OFLX-resistance, and also showed a higher proportion of clustered strains, reflecting possible recent transmission. In addition, the rpoB531 and katG mutation was more frequently observed among Beijing genotype strains than non-Beijing strains. Our finding demonstrated that ancient Beijing MDR strains were associated with drug resistance, while modern Beijing MDR strains were more likely to be clustered. The unsatisfactory relationship between the phenotypic resistance and genetic mutation for INH indicates that it is necessary to investigate the potential molecular mechanism conferring for INH resistance among the MDR isolates circulating in China.

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10.

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12.

13.

14.

Acknowledgments 15.

This work was supported by the National Basic Research Program of China (2014CB744403). We are grateful to members of the National Tuberculosis Reference Laboratory at the Chinese Center for Disease Control and Prevention for their cooperation and technical help.

16.

Appendix A. Supplementary data Supplementary data related to this article can be found at http://dx.doi.org/10.1016/j.jinf.2014.11.008

17.

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