Genotypic analysis of genes associated with transmission and drug resistance in the Beijing lineage of Mycobacterium tuberculosis Genotypic analysis of genes associated with transmission and drug resistance in the Beijing lineage of Mycobacterium tuberculosis J-R. Chang 1 *, C-H. Lin 2 *, S-F. Tsai 2 , I-J. Su 1 , F-C. Tseng 1 , Y-T. Chen 2 , T-S. Chiueh 3 , J-R. Sun 3 , T-S. Huang 4 , Y-S. Chen 4 and H-Y. Dou 1 1) Division of Infectious Diseases, 2) Division of Molecular and Genomic Medicine, National Health Research Institutes, Zhunan, Miaoli, 3) Department of Clinical Pathology, Tri-Service General Hospital, Taipei and 4) Department of Microbiology, Kaohsiung Veterans General Hospital, Kaohsiung, Taiwan Abstract 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 muta- tions 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. Keywords: Beijing lineage, drug resistance, Mycobacterium tuberculosis Original Submission: 20 July 2010; Revised Submission: 1 November 2010; Accepted: 17 November 2010 Editor: M. Drancourt Article published online: 23 November 2010 Clin Microbiol Infect 2011; 17: 1391–1396 10.1111/j.1469-0691.2010.03436.x Corresponding author: H-Y. Dou, Division of Infectious Diseases, National Health Research Institutes, No. 35 Keyan Road, Zhunan Town, Miaoli County 350, Taiwan E-mail: hydou@nhri.org.tw *These authors contributed equally to this work. Introduction Tuberculosis (TB) remains a worldwide healthcare concern and has been characterized as an epidemic by the World Health Organisation (WHO). It is estimated that one-third of the world’s population has been infected with Mycobacte- rium tuberculosis (MTB) and that 3 million people die of TB annually. Although both the incidence and mortality rates of TB in Taiwan have shown a steady decline since 1950, TB remains a leading notifiable infectious disease on the island. In 2001, 14 486 cases were reported, with a notification rate of 64.9 per 100 000 people [1]. Amongst the most prevalent MTB strains, the Beijing genotype is a major concern on a global basis. Beijing strains are rapidly spreading worldwide and are most often associ- ated with major TB outbreaks [2–4]. At present, Beijing strains account for >10% of TB cases worldwide and are highly endemic throughout much of east and southeast Asia, where they account for >50% of TB cases [3,5]. The impor- tance of this genotype is further highlighted by the fact that the recent epidemic spread of Beijing strains is frequently associated with multiple drug resistance (MDR) [4–6]. Recent studies have demonstrated worldwide dissemination of modern strains of the Beijing family (characterized chromo- somally as having an IS6110 insertion in the NTF region of the genome) [2,7–9] and have led to speculation about the hypervirulent features of this sublineage [10–13]. In contrast to the worldwide prevalence of modern Beijing strains, the ancient Beijing strains (having an intact NTF region) are highly diverse and dominant in Japan [14,15]. In a previous study we reported that the Beijing family strains in Taipei mainly belong to the modern subfamily; this suggested that they became endemic after originating from the evolutionary stream that led to the dominant modern Beijing subfamily [16,17]. Therefore, phylogenetic investigation of ª2010 The Authors Clinical Microbiology and Infection ª2010 European Society of Clinical Microbiology and Infectious Diseases ORIGINAL ARTICLE BACTERIOLOGY Beijing family strains in Taiwan will be useful for understand- ing how they acquired hypervirulent phenotypes and adapted to the Taiwanese human host population. It was suggested that variable number of tandem repeats (VNTR) alleles are phylogenetically informative for the M. tuberculosis Beijing family. In the present study, sublineages of the Beijing family were classified by using ten synonymous single nucleotide polymorphisms (SNPs) [14,15]; in addition, the IS6110 inser- tion in the NTF region, the presence or absence of five large sequence polymorphisms (RD105, RD207, RD181, RD142 and RD150) [18,19], and non-synonymous SNPs in putative DNA repair genes (mutT2, mutT4, and ogt) [10] were analy- sed. All strains were subjected to 24 mycobacterial inter- spersed repetitive units (MIRU)-VNTR analyses [20] to detect probable epidemiological linkage amongst patients. Our analyses will promote a better understanding of the pop- ulation genetic structure of the Beijing family and extend the information obtained from the VNTR genotyping method. Materials and Methods Study population and bacterial isolates Isolates were collected between 2004 and 2007 from the mycobacteriology laboratories of five general hospitals located in four geographical regions in Taiwan, namely, Taipei Tri-Service General Hospital (northern region), Mennonite Christian Hospital (eastern region), Wan-Ciao Veterans Hos- pital (central region), Tainan Chest Hospital (southern region), and Kaohsiung Veterans General Hospital (southern region). All of the patients were sputum microscopy positive and culture positive. Mycobacterial genomic DNA was extracted from cultured cells as described previously [21]. Briefly, mycobacterial colonies were resuspended in 100– 200 lL of distilled H2O and incubated at 85�C for 30 min to obtain genomic DNA. After centrifugation of the suspension, the supernatant containing the DNA was removed and stored at )20�C until further use. NTF locus analysis A multiplex PCR approach was used to determine possible IS6110 insertion(s) in the NTF region of M. tuberculosis strains. The method, including primer choices within the NTF region, the IS6110 sequence and PCR parameters, was adapted from a paper by Plikaytis et al. [18]. Detection of RD deletions Previous studies have shown that the type of RD deletion correlates with the relative evolutionary age of the MTB strain [19]. Beijing strains in our collection were further classified by using PCR amplification to determine the pres- ence of an RD deletion. A primer set was used to check for the presence or absence of RD105, RD181, RD150, RD142 and RD207. The PCR mixture consisted of 0.2 lg DNA tem- plate, 13.9 lL Q buffer, 5 lL 5· buffer, 4 lL 10 mM deoxy- nucleoside triphosphates, 1 lL of each primer (10 pmol/lL), 1 lL DMSO, and 0.6 lL Herculase II Fusion DNA polymer- ase (Stratagene, La Jolla, CA, USA). Sterile water was used to dilute the mixture up to 25 lL. A detailed explanation of this methodology has been described [19]. SNP typing PCR and extension primers were designed using MassArray Assay Design 3.1 software (Sequenom, San Diego, CA, USA). PCRs contained, in a volume of 5 lL, 1 pmol of the corre- sponding primers, 10 ng genomic DNA and HotStar reaction Mix (Qiagen, Valenca, CA, USA) in 384-well plates. PCR condi- tions were as follows: 94�C for 15 min, followed by 40 cycles of 94�C (20 s), 56�C (30 s) and 72�C (60 s), and a final exten- sion of 72�C for 3 min. In the primer extension procedure, each sample was denatured at 94�C, followed by 40 cycles of 94�C (5 s), 52�C (5 s) and 72�C (5 s). The mass spectrum from time-resolved spectra was retrieved by using a Mass- Array mass spectrometer (Sequenom), and each spectrum was then analysed using SpectroTyper software (Sequenom) to perform the genotype calling. Although the application of mass spectrometry-based genotyping of the M. tuberculosis genome had been demonstrated in previous studies [22,23], the Sanger sequencing method was used to validate 600 ran- domly-selected SNP calls. The false-positive and false-negative rates of mass spectrometry-based genotyping in this study are both equal to 0%, showing that the results of mass spectrome- try-based genotyping are highly accurate and sensitive. Results Molecular polymorphisms and drug resistance of M. tuberculosis Beijing isolates From a collection of 338 Beijing strains a total of nine inde- pendently evolving Beijing sublineages were identified (Table 1). Two of these, ST11 and ST26, each possessing intact RD181 and NTF regions, correspond to ancient sublin- eages whilst the rest correspond to modern sublineages: ST3, ST10, ST19, ST22, ST25, STK, and newly assigned STN in this study; all possess an RD181 region deletion and some of them have an IS6110 insertion on the right side of the NTF region [14,16,17,19]. The most frequent strain found was ST10 (53.3%), followed by ST19 (14.8%) and ST22 (14.5%), which all belong to modern sublineages. 1392 Clinical Microbiology and Infection, Volume 17 Number 9, September 2011 CMI ª2010 The Authors Clinical Microbiology and Infection ª2010 European Society of Clinical Microbiology and Infectious Diseases, CMI, 17, 1391–1396 All 338 clinical isolates were tested for drug resistance (isoniazid, rifampicin, ethambutol and streptomycin) and clas- sified as either sensitive (S) to all four drugs, resistant to any one of the drugs (R), or multi-drug resistant (MDR; resistant to isoniazid and rifampicin) (Table 1). Of the nine lineages, ST26 is associated with a higher percentage of MDR (18.5%) as compared with strains of all other eight lineages (6.43%), with the difference being statistically significant (p 0.038 by exact test; Table 1). Chuang et al. [24] reported that modern Beijing sublineag- es can be distinguished from ancient lineages based on SNPs in the antigen 85 (Ag85) complex genes fbpA and fbpB. In our analysis, none of the ancient Beijing sublineages (RD type 1–2) were found to have an SNP in the fbpA or fbpB gene; however, the modern Beijing sublineages (RD type 3–6) were found to carry an SNP in fbpB codon 238 (Table 2). We also tested the isolates for the presence of missense mutations in mutT2, mutT4, ogt12 and ogt37, which are genes encoding putative DNA repair enzymes. Mutations in the four putative repair genes were detected with variable per- centages in all of the modern (ST3, ST10, ST19, ST22, ST25, STK and STN) sublineages. However, mutations were not observed in the ST11 and ST26 ancient sublineages, the lat- ter of which had the highest rate of MDR. Thus, our data suggest that mutations in these DNA repair enzymes may more likely be present in the modern Beijing sublineages, but they were not associated with the presence of MDR (Table 1). The relatively high rate of cluster formation that was found in the ST10 strains, as observed by 24-MIRU-VNTR analysis (Table 2), suggests the occurrence of recent trans- mission at a significant frequency. Phylogenetic classification of Beijing family isolates based on MIRU-VNTR typing and RD typing The phylogenetic classification was projected into a cluster analysis of 24-MIRU-VNTR (Fig. 1). The majority of the mod- ern Beijing sublineages were not clustered according to their ST classifications. Amongst these, the isolates ST22 and ST10 were actually merged together in the tree. Similar results were reported in a recent study in Japan, in which the phylo- genetic interpretation based on MIRU-VNTR genotyping also TABLE 1. Distribution of drug resistance and various genetic characteristics of each sublineage Beijing sublineage a Allele in indicated SNP position of H37Rv Age No. (%) of isolates of the indicated strain 797 736 909 166 147 759 154 814 169 206 189 201 237 613 253 261 282 558 413 782 S b R c MDR d ST11(4) C C C G A T A G T C 57.5 4 (100) 0 0 ST26(27) C T C G A T A G T C 53.5 18 (66.7) 4 (14.8) 5 (18.5) ST3(13) T C C G A T A G G C 61.6 8 (61.5) 5 (38.5) 0 (0) STK(4) T T C G A T A G G C 69 4 (100) 0 (0) 0 (0) ST19(50) T T C G A C A G G T 64.1 41 (82) 6 (12) 3 (6) ST25(7) T C C G A C A G G T 52.2 6 (85.7) 1 (14.3) 0 (0) ST22(49) T T T G A C G A G T 65.3 41 (83.7) 8 (16.3) 0 (0) ST10(180) T T T G A C A G G T 58.4 142 (78.9) 24 (13.3) 14 (7.8) STN(4) e T C T G A C A G G T 63 3 (75) 1 (25) 0 (0) Total (338) 267 (79) 49 (14.5) 22 (6.5) a Types of sequence of Beijing sublineages; designations from Filliol et al. [5] and Iwamoto et al. [14]. b Sensitive to any one of the four anti-TB drugs. c Resistant to any one of the anti-TB drugs. d Resistant to isoniazid and rifampicin. e New sublineage found in this study. TABLE 2. Genotypic characteristics of the Beijing lineage of M. tuberculosis isolates Beijing sublineage %% of N branch a Point mutations in indicated putative repair genes (%%) SNP in fbpA 156 and fbpB 238 (%%) No. of cluster No. of cases in cluster No. of VNTR patternMut2 Mut4 Ogt12 Ogt37 fbpA 156 fbpB 238 ST11(4) 100 0 0 0 0 0 0 0 0 4 ST26(27) 100 0 0 0 0 30 0 5 13 19 ST3(13) 77 0 15 0 0 8 78 2 4 11 STK(4) 75 0 25 0 0 0 50 0 0 4 ST19(50) 71 12 98 0 58 0 98 5 10 45 ST25(7) 57 0 86 0 43 0 100 1 2 6 ST22(49) 4 100 100 100 100 100 98 8 32 25 ST10(180) 2 99 99 99 1 0 99 25 71 134 STN(4) 0 100 100 100 0 0 50 1 2 3 a Defined by IS6110 insertion in the NTF region. CMI Chang et al. Genotypic analysis of the Beijing lineage of M. tuberculosis 1393 ª2010 The Authors Clinical Microbiology and Infection ª2010 European Society of Clinical Microbiology and Infectious Diseases, CMI, 17, 1391–1396 resulted in poor resolution of modern Beijing strains [25]. The ancient Beijing strains ST11 and ST26 were positioned away from the major MIRU-VNTR genotypes, indicating their more distant genetic relationship to the modern sublineages. To validate the phylogenetic reconstruction by RD typing, we analysed STs of our isolates using ten SNPs, as previously described [11,14]. Nine STs were identified in our popula- tion. RD group 1 sublineage: 1 isolate of ST11; this isolate shows a deletion of the RD105 region. RD group 2 sublin- eages: 30 isolates of ST11 and ST26; these isolates show deletion of the RD105 and RD207 regions. RD group 3 sub- lineages: 225 isolates of ST3, ST10, ST19, ST22, STK and STN; these isolates show deletion of the RD105, RD207 and RD181 regions. RD group 4 sublineages: 43 isolates of ST10 and ST19; these isolates show deletion of the RD105, RD207, RD181 and RD150 regions. RD group 5 sublineages: 32 isolates of ST3, ST10, ST19, ST22 and STK; these isolates show deletion of the RD105, RD207, RD181 and RD142 regions. RD group 6 sublineages: seven isolates of ST10 and ST19; these isolates show deletion of the RD105, RD207, RD181, RD142 and RD150 regions. Strains in groups 1 and 2 have neither a deletion of RD181 nor an insertion of IS6110 in the NTF region, and can be thought of as being ancestral to the modern Beijing lineage (Fig. 2). Discussion The reasons for the apparent global success of the Beijing strains are not yet understood, but could include a variety of host-related factors, such as human population movements [9], selective pressure due to increases in worldwide BCG vaccine coverage [12], and ineffective treatment of drug- resistant strains, leading to increased transmission periods. The high adaptability of these bacteria to stress conditions, such as the host’s immune response and/or exposure to anti-tuberculosis drugs, has been hypothetically attributed to defects in DNA repair systems, which would confer selective advantages due to an increased mutation rate [10]. A num- ber of genes coding for presumptive DNA repair enzymes, such as ogt, mutM, mutT and mutY, have been detected in the M. tuberculosis genome [10], and analysis of strains represent- ing different branches of the Beijing genotype has shown that Beijing strains display unique missense alterations in putative mut genes, designated ogt, mutT2 and mutT4 [10]. Although it FIG. 1. A minimum spanning tree based on 24-MIRU-VNTR genotyping of 338 Mycobacte- rium tuberculosis Beijing isolates. The circles represent different types classified by 24- MIRU-VNTR genotypes and were coloured according to the ST classification described in the text. The sizes of circles represent the number of isolates with a particular genotype. RD type (No.) N branch (%) ST 105 207 181 150 142 150 142 1 (1) 1 (100) 2 (30) 30 (100) ST11 ST26, ST11 Insertion of IS6110 in NTF 3 (225) 4 (43) 43 (19.1) 1 (2.3) 5 (32) 6 (21.9) 6 (7) 2 (28.6) ST3, ST10, ST19, ST22, ST25, STK, STN ST10, ST19 ST3, ST10, ST19, ST22, STK ST10, ST19 FIG. 2. Scheme of the proposed evolution of Beijing lineages. The scheme is based on the deletion of genomic regions (RD, region of difference, shown in grey rectangles), the NTF region with IS6110 insertions, and types of sequence designations from the studies of Filliol et al. [5] and Iwamoto et al. [14]. 1394 Clinical Microbiology and Infection, Volume 17 Number 9, September 2011 CMI ª2010 The Authors Clinical Microbiology and Infection ª2010 European Society of Clinical Microbiology and Infectious Diseases, CMI, 17, 1391–1396 has been recently shown that the product of mutT2 is unlikely to function as a DNA repair enzyme, its role in regu- lating the availability of cellular cytidine-triphosphate may help explain the success of Beijing strains expressing the mutated mutT2 [26]. Mutations in the mutT2 and mutT4 genes occur in the most recently evolved Beijing lineages, which are associ- ated with an increased ability to spread and cause disease [27]. In the present study, mutations were observed in most of the Beijing sublineages, except ST11 and ST26. Thus, our data do not demonstrate an association between the pres- ence of mutations in these genes and MDR, but an association between the SNP subclassification and polymorphism of the genes. We have also demonstrated that sublineage ST26 occurs at a higher frequency in the MDR population. To our surprise, our findings are similar to those of Wada et al. [25]. Our 24-loci MIRU-VNTR data also support the hypothesis that parts of the ancestral Beijing lineage may come from two different branches (Fig. 1). This is contrary to colonal evolution of MTB [28]. However, further investi- gation of this dual-origin possibility is required. Furthermore, the 12-loci MIRU pattern of one of the strains from the present investigation was determined to be 223325173533, which is the most predominant pattern for the classical Beijing type (ST1) in Taiwan [16]. Interestingly, this strain was also found in other countries, including Russia, China, Japan and Vietnam [29]. Sequence typing per- mitted us to further subdivide this major group of strains into ST3, ST10, ST19, ST22 and ST25 in our studies. We believe that these modern Beijing strains, with their high degree of transmissibility, are currently spreading throughout the world. It was previously reported that the BCG vaccina- tion favours the positive selection of modern Beijing strains [12]. Our results support this finding. In a previous study we established that modern Beijing sublineages are the most predominant strains in Taiwan [16,17]. Moreover, a recent study showed that 80% of the strains from modern Beijing sublineages, but not from ancient sublineages, synthesize relatively high quantities of phenolic glycolipid (PGL), which suppresses proinflammatory cytokines. These findings suggest that modern sublineages may be more pathogenic [30]. In addition, studies have shown that the Ag85 complex plays a role in survival of tuberculosis bacilli and their ability to escape the human immune response [31]. Although Chuang et al. [24] reported that modern Beijing sublineages can be distinguished based on SNPs in the Ag85 complex genes fbpA and fbpB, in the present study we found that ancestral and modern Beijing sublineages can be distinguished by an SNP only in fbpB. We also demonstrated that most modern Beijing sublineages carry a mutated mutT gene and deletion RD181, which may facilitate their spreading ability. ST26 should be assigned to the early ancestral Beijing sublineage, and did not have an SNP in mut4, mut2 or ogt, although it occurred at a signifi- cantly high frequency in the MDR population. This finding suggests that different sublineages of the Beijing family may differ in their mechanisms of adaptation to the host’s immune response and drug selection pressure. Acknowledgements We thank the mycobacteriology laboratories of Mennonite Christian Hospital, Tri-Service General Hospital, Tainan Chest Hospital, Wan-Ciao Veterans Hospital and Kaohsiung Veterans General Hospital for providing bacterial isolates. We also thank Dr Daryl Henderson for his kind help in improving the English of this manuscript. All participants of this consortium are acknowledged for valuable discussions. Transparency Declaration This project was supported by grants from the National Health Research Institutes and National Science Council (NSC97-3112-B-400-012), Taiwan. Other authors had no conflicts of interest. References 1. Chen ZC. Tuberculosis annual report. Center for Disease Control DoH, 2002; 34–39. 2. Bifani PJ, Mathema B, Kurepina NE et al. Global dissemination of the Mycobacterium tuberculosis W-Beijing family strains. Trends Microbiol 2002; 10: 45–52. 3. Brudey K, Driscoll JR, Rigouts L et al. 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Infect Immun 2000; 68: 767–778. 1396 Clinical Microbiology and Infection, Volume 17 Number 9, September 2011 CMI ª2010 The Authors Clinical Microbiology and Infection ª2010 European Society of Clinical Microbiology and Infectious Diseases, CMI, 17, 1391–1396 Genotypic analysis of genes associated with transmission and drug resistance in the Beijing lineage of Mycobacterium tuberculosis Introduction Materials and Methods Study population and bacterial isolates NTF locus analysis Detection of RD deletions SNP typing Results Molecular polymorphisms and drug resistance of M. tuberculosis Beijing isolates Phylogenetic classification of Beijing family isolates based on MIRU-VNTR typing and RD typing Discussion Acknowledgements Transparency Declaration References