Received 2 May 2008/Returned for modification 12 June 2008/Accepted 28 July 2008

Transcription

1 ANTIMICROBIAL AGENTS AND CHEMOTHERAPY, Oct. 2008, p Vol. 52, No /08/$ doi: /aac Copyright 2008, American Society for Microbiology. All Rights Reserved. Population Structure Analysis of the Mycobacterium tuberculosis Beijing Family Indicates an Association between Certain Sublineages and Multidrug Resistance Tomotada Iwamoto, 1 * Shiomi Yoshida, 2 Katsuhiro Suzuki, 2 and Takayuki Wada 3 Department of Microbiology, Kobe Institute of Health, 4-6 Minatojima-Nakamachi, Chuo-ku, Kobe ; Clinical Research Center, National Hospital Organization Kinki-Chuo Chest Medical Center, 1180 Nagasone-cho, Sakai ; and Department of Microbiology, Osaka City Institute of Public Health and Environmental Sciences, 8-34 Tojo-cho, Tennoji-ku, Osaka , Japan 3 Received 2 May 2008/Returned for modification 12 June 2008/Accepted 28 July 2008 Our population-based study of the Mycobacterium tuberculosis Beijing family examined the frequency of occurrence of each sublineage of this family, classified by using 10 synonymous single-nucleotide polymorphisms. The results revealed the overabundance of two evolutionary sublineages in a population of multidrugresistant and extensively drug-resistant tuberculosis bacteria. Mycobacterium tuberculosis Beijing family strains are suspected to be an evolving lineage of M. tuberculosis that has acquired the advantage of drug resistance (1, 8, 13, 26). However, the association between this genotype and drug resistance varies in different countries (2, 7, 26). This may be due to heterogeneity in the fitness of the sublineages of the Beijing family and to the different proportions of these sublineages in local populations (6, 9, 15, 23). To determine whether certain sublineages are associated with multidrug-resistant (MDR) and extensively drug-resistant (XDR) tuberculosis, the population structure of the Beijing strains was analyzed, based on 10 synonymous single-nucleotide polymorphisms (ssnps) (5, 9) in pandrug-sensitive (DS), MDR, and XDR strains. Two hundred eighty-five Beijing family strains were obtained from individual patients with pulmonary tuberculosis (TB) at the Kinki-chuo Chest Medical Center. All the patients in this study were human immunodeficiency virus negative, and most of them were residents of the Kinki area of Japan. Of the 285 strains, 189 DS strains were collected between 1 January 2003 and 31 August 2003, and 96 MDR strains (including 47 XDR strains) were collected between 1 January 2001 and 31 December These strains were from all of the DS, MDR, and XDR-TB patients with Beijing strains in this hospital during the strain collection periods except for one MDR patient. The Beijing family strains were defined by spoligotyping (12). The strains were analyzed for drug susceptibility by using the simplified proportion method (22). All 96 MDR strains were analyzed for the presence of mutations in the rifampin resistance-determining region (RRDR) (25) and in katg codon 315 (29). Subdivisions within the Beijing family were characterized on the basis of ssnps at 10 chromosomal positions (5, 9). In addition, the IS6110 insertion in the NTF * Corresponding author. Mailing address: Department of Microbiology, Kobe Institute of Health, 4-6 Minatojima-Nakamachi, Chuo-ku, Kobe , Japan. Phone: Fax: kx2t-iwmt@asahi-net.or.jp. Supplemental material for this article may be found at Published ahead of print on 11 August regions (19), the presence or absence of three large sequence polymorphisms (RD181, RD150, and RD142) (27), and three nonsynonymous SNPs in putative repair genes (mutt2, mutt4, and ogt [codon 37]) (21) were analyzed. All strains were subjected to Supply s optimized 15-locus variable number of tandem repeats (15-MIRU-VNTR) analysis (24) to detect probable epidemiological linkage among the patients (see Table S1 in the supplemental material). A total of eight independently evolving Beijing sublineages that corresponded to ancient sublineages (ST11, ST26, ST3, ST25, ST19 and newly assigned STK in this study, possessing an intact NTF region) and modern sublineages (ST10 and ST22, possessing an IS6110 insertion on the right side of the NTF region) were identified in our population (17, 18) (Table 1). The population structures based on the numbers of patients demonstrated that two sublineages, ST26 (RD181 ) and ST3 (RD181 ), were significantly overrepresented in the population of MDR/XDR strains compared with their numbers in the population of DS strains; however, the ST19 sublineage was significantly underrepresented in the MDR/XDR populations (Table 2). The age, gender, and human immunodeficiency virus status of the patients did not account for such differences between the DS and MDR/XDR populations (Table 2). The MDR/XDR populations in this study mostly comprised previously treated patients (Table 2). This might imply that clonal evolution within a patient is a driving force for acquiring MDR/XDR. In order to clarify the contribution of clonal evolution, excluding clonal expansion (human-to-human transmission), on the overabundance of ST26 and ST3, the population structures based on the genotypes determined by 15-MIRU- VNTR analysis, which can discriminate epidemiologically unrelated strains as different VNTR profiles (10, 11, 28), were compared (Table 2). Genotype-based analysis demonstrated a trend similar to that observed with patient-based analysis, although there was a slight decrease in the statistical significance (Table 2). The variety of RRDR and katg 315 mutations in the strains employed in this study (Table 3; see Table S2 in the supplemental material) confirms the assumption that the strains from each sublineage evolved independently, not from 3805

2 3806 NOTES ANTIMICROB. AGENTS CHEMOTHER. TABLE 1. Distribution of drug resistance and various genetic characteristics of each sublineage Presence or absence of indicated large sequence polymorphism f : Point mutations in indicated putative repair gene e : IS6110 insertion in the NTF region No. (%) of isolates of indicated strain: Allele in indicated SNP position of H37Rv: Beijing sublineage a RD181 RD150 RD142 ogt (codon 37) mutt2 mutt4 XDR- TB MDR- TB DS-TB ST11 C C C G A T A G T C 1 (0.5) 0 (0) 0 (0) WT WT WT ST26 C T C G A T A G T C 10 (5.3) 21 (21.9) 17 (36.2) WT WT WT b T T C G A T A G G C 30 (16.0) 8 (8.2) 2 (4.3) WT WT WT - ST3 T C C G A T A G G C 44 (23.4) 34 (35.1) 20 (42.6) WT WT WT - ST25 T C C G A C A G G T 3 (1.6) 1 (1.0) 0 (0) WT MT MT - ST19 T T C G A C A G G T 63 (33.5) 14 (14.4) 3 (6.4) WT MT MT c - ST10 T T T G A C A G G T 29 (15.4) 8 (8.2) 1 (2.1) NTF::IS6110 MT MT WT - d ST22 T T T G A C G A G T 9 (4.8) 10 (10.3) 4 (8.5) NTF::IS6110 MT MT WT - a ST designations from reference 5. b New ST, STK, assigned in this study. c Nine DS-TB strains and one MDR-TB strain possessed the wild type. d Two strains exhibiting ST10 had the RD150 deletion. e WT, wild type; MT, mutation. f, presence of large sequence polymorphism;, absence of large sequence polymorphism. an endemic MDR-TB strain. Taken together, the overrepresentation of the two sublineages could be considered reflective of the actual situation in the MDR/XDR population rather than an artifact biased by the prevalence of an endemic MDR-TB strain. The relatively high rate of cluster formation by the MDR/ XDR strains, as observed by 15-MIRU-VNTR analysis (Table 2), suggests the occurrence of exogenous reinfection and/or transmission (clonal expansion) at a certain frequency. Due to the lack of information on patients initially isolated strains, the inability to discriminate between clonal evolution and reinfection/transmission is a limitation of this study. Only four cases of epidemiological links were identified by tracing the patients contacts (see Table S2 in the supplemental material). Although we cannot rule out factors of social behavior in this study, the overrepresentation of the two sublineages in the study leads to the hypothesis that there are certain bacterial factors favoring their emergence and spread. Since the relative fitness of drug-resistant M. tuberculosis strains is considered one of the key determinants of MDR-TB burden (3, 4, 6), these sublineages may be at an advantage in acquiring drug resistance via mechanisms having a low fitness cost. To examine this possibility, we analyzed the rpob S531L and katg S315T mutations, which are suspected to be mutations with low fitness costs. As expected, a high rate of the rpob S531L and katg S315T mutations was observed in ST26 (85.7% and 71.4%, respectively, in MDR-TB) (Table 3). However, in ST3, the rate of katg S315T mutations was high (50% in MDR-TB), but that of rpob S531L mutations was unexpectedly low (23.5% in MDR-TB) (Table 3). It is interesting to note that ST3 showed a variety of mutations in the RRDR regions (Table 3). Two strains demonstrated double mutations and one showed mixed peaks corresponding to wild-type and mutant genotypes in codons 516, 526, 530, and 531, which may imply the existence of subpopulations with different drug resistance alleles (20). Besides low-cost resistance mutations, compensatory mutations that ameliorate fitness costs are suggested to be important factors influencing fitness (3, 4, 6). It is possible that the ST3 sublineages are at an advantage due to the occurrence of compensatory mutations. We also examined the appearance of missense alterations in mutt2, mutt4, and ogt, which are putative genes encoding DNA repair enzymes (21). Mutations were observed in ST25, ST19, ST10, and ST22 but not in ST11, ST26, STK, and ST3 (Table 1). Thus, our data did not demonstrate an association between the presence of mutations in these genes and MDR/ XDR, while there was an association between the ssnp subclassification and the polymorphism of the genes (Table 1). Only a few studies have investigated the association of the Beijing family with drug resistance at the sublineage level (9, 15). Compared with those of previous studies, our population is advantageous for the analysis of the ancient Beijing subgroup because of its high proportion of ancient subgroup lineages versus global dissemination of modern subgroup lineages (2, 14, 16, 17). We demonstrated that two sublineages, ST26 and ST3, which occurred with a significantly higher frequency in the MDR/XDR population than in the DS population, belong to an ancient subgroup. This finding suggests that different sublineages of the Beijing family may differ in their mechanisms of adaptation to drug selection pressures. The

3 VOL. 52, 2008 NOTES 3807 TABLE 2. Epidemiological, clinical, and genotypic data from DS-TB, MDR-TB, and XDR-TB isolates Epidemiological/clinical information Genotypic information as determined by VNTR analysis Drug sensitivity group a and Beijing sublineage Total no. (%) of patients P value b No. (%) male Avg age (yrs) No. (%) of new cases Total no. (%) of genotypes by VNTR analysis P value c No. of clusters No. (%) of cases in cluster DS ST11 1 (0.5) 1 (100) 54 1 (100) 1 (0.8) 0 0 (0) ST26 10 (5.3) 6 (60.0) (80.0) 9 (7.3) 1 2 (22.2) STK 30 (15.9) 22 (73.3) (76.7) 27 (22.0) 3 6 (20.0) ST3 44 (23.3) 33 (75.0) (84.1) 26 (21.1) (63.6) ST25 3 (1.6) 3 (100) (66.7) 2 (1.6) 1 2 (66.7) ST19 63 (33.3) 48 (76.2) (82.5) 31 (25.2) 6 38 (60.3) ST10 29 (15.3) 23 (79.3) (75.9) 21 (17.1) 4 12 (41.4) ST22 9 (4.8) 5 (55.6) (100) 6 (4.9) 1 4 (44.4) MDR ST11 0 (0) ST26 21 (21.9) (85.7) (19.0) 10 (18.2) (63.6) STK 8 (8.3) (75.0) (25.0) 8 (14.6) (0) ST3 34 (35.4) (70.6) (14.7) 18 (32.8) (64.7) ST25 1 (1.0) (100) 50 1 (100) 1 (1.8) (0) ST19 14 (14.6) (71.4) (28.6) 8 (14.6) (57.1) ST10 8 (8.3) (62.5) (62.5) 6 (10.9) (50) ST22 10 (10.4) (90) (20.0) 4 (7.3) (90) XDR ST11 0 (0) ST26 17 (36.2) (82.4) (17.6) 7 (29.2) (70.6) STK 2 (4.3) (100) 68 0 (0) 2 (8.3) ST3 20 (42.6) (65.0) (15.0) 10 (41.7) (65.0) ST25 0 (0) ST19 3 (6.4) (66.7) (66.7) 2 (8.3) (66.7) ST10 1 (2.1) (100) 52 0 (0) 1 (4.2) (0) ST22 4 (8.5) (75.0) (25.0) 2 (8.3) (75.0) a There were 189 DS-TB, 96 MDR-TB, and 47 XDR-TB isolates. b P value of Z test for proportion between DS versus MDR and DS versus XDR patients. c P value of Z test for proportion between DS versus MDR and DS versus XDR genotypes.

4 3808 NOTES ANTIMICROB. AGENTS CHEMOTHER. TABLE 3. Mutations in RRDR and katg 315 rpob mutation(s) a katg 315 mutation a No. (%) of MDR-TB isolates from indicated sublineage: No. (%) of XDR-TB isolates from indicated sublineage: ST26 STK ST3 ST25 ST19 ST10 ST22 ST26 STK ST3 ST19 ST10 ST22 Leu 511 Pro 1 (12.5) (CTG 3 CCG) Ser 315 Thr Glu 513 Lys (CAA 3 AAA) Ser 315 Thr 1 (12.5) 1 (2.9) 1 (50) Asp 516 Val 1 (4.8) 3 (37.5) 1 (2.9) (GAC 3 CTC) Ser 315 Thr 1 (12.5) 11 (32.4) 10 (50) Ser 522 Leu 3 (8.8) (TCG 3 TTG) Ser 315 Thr 525 (ACG 1 (2.9) insertion) Ser 315 Thr His 526 Ser 2 (5.9) 4 (40) 3 (75) (CAC 3 AGC) Ser 315 Thr His 526 Pro 1 (2.9) 1 (5) (CAC 3 CCC) Ser 315 Thr His 526 Arg 2 (25) 1 (100) (CAC 3 CGC) Ser 315 Thr His 526 Leu 1 (4.8) (CAC 3 CTC) Ser 315 Thr His 526 Leu (CAC 3 TTG) Ser 315 Thr 1 (12.5) His 526 Asp 1 (10) (CAC 3 GAC) Ser 315 Thr 1 (100) 1 (7.1) His 526 Tyr 1 (12.5) 2 (5.9) 1 (7.1) 1 (5) (CAC 3 TAC) Ser 315 Asn 1 (2.9) 1 (5) (AGC 3 AAC) His 526 Cys (CAC 3 TGC) Ser 315 Thr 1 (7.1) Ser 531 Leu 4 (19.0) 1 (12.5) 5 (14.7) 9 (64.3) 1 (12.5) 4 (40) 3 (17.6) 1 (50) 4 (20) 2 (66.7) 1 (25) (TCG 3 TTG) Ser 315 Thr 14 (66.7) 3 (8.8) 3 (37.5) 14 (82.4) 1 (5) Leu 533 Pro (CTG 3 CCG) Ser 315 Thr 1 (10) Ser 512 Ile 1 (2.9) 1 (5) (AGC 3 ATC), His 526 Pro Ser 315 Thr (CAC 3 CCC) Asp 516 Glu (GAC 3 GAA), Ser 522 Leu Ser 315 Thr 1 (2.9) 1 (5) (TCG 3 TTG) Asp 516 Ala (GAC 3 GCC), Leu 533 Pro Ser 315 Thr 1 (7.1) 1 (33.3) (CTG 3 CCG) Mixed peak in (2.9) (CAC, GTC), Ser 315 Thr 526 (CAC, CAA), 530 (CTG, ATG), 531 (TCG, TTC) Wild-type RRDR 1 (7.1) 1 (12.5) Ser 315 Thr 1 (4.8) a The mutant codon is indicated, followed by the nucleotide change. increasing prevalence of these sublineages would make it more difficult to control the threat posed by MDR/XDR than by those currently encountered. Therefore, greater vigilance in monitoring the occurrence of these strains is indispensable for achieving better TB control in this region. This work was supported by grants from JSPS Grant-in-Aid for Scientific Research (A) ( ) and the US-Japan Cooperative Medical Science Program (TB leprosy panel). REFERENCES 1. Anh, D. D., M. W. Borgdorff, L. N. Van, N. T. Lan, T. van Gorkom, K. Kremer, and D. van Soolingen Mycobacterium tuberculosis Beijing genotype emerging in Vietnam. Emerg. Infect. Dis. 6: Bifani, P. J., B. Mathema, N. E. Kurepina, and B. N. Kreiswirth Global dissemination of the Mycobacterium tuberculosis W-Beijing family strains. Trends Microbiol. 10: Cohen, T., and M. Murray Modeling epidemics of multidrug-resistant M. tuberculosis of heterogeneous fitness. Nat. Med. 10: Cohen, T., B. Sommers, and M. Murray The effect of drug resistance on the fitness of Mycobacterium tuberculosis. Lancet Infect. Dis. 3: Filliol, I., A. S. Motiwala, M. Cavatore, W. Qi, M. H. Hazbon, M. Bobadilla del Valle, J. Fyfe, L. Garcia-Garcia, N. Rastogi, C. Sola, T. Zozio, M. I. Guerrero, C. I. Leon, J. Crabtree, S. Angiuoli, K. D. Eisenach, R. Durmaz, M. L. Joloba, A. Rendon, J. Sifuentes-Osornio, A. Ponce de Leon, M. D. Cave, R. Fleischmann, T. S. Whittam, and D. Alland Global phylogeny of Mycobacterium tuberculosis based on single nucleotide polymorphism (SNP) analysis: insights into tuberculosis evolution, phylogenetic accuracy of other DNA fingerprinting systems, and recommendations for a minimal standard SNP set. J. Bacteriol. 188: Gagneux, S., C. D. Long, P. M. Small, T. Van, G. K. Schoolnik, and B. J. Bohannan The competitive cost of antibiotic resistance in Mycobacterium tuberculosis. Science 312: Glynn, J. R., A. C. Crampin, H. Traore, M. D. Yates, F. D. Mwaungulu, B. M. Ngwira, S. D. Chaguluka, D. T. Mwafulirwa, S. Floyd, C. Murphy, F. A. Drobniewski, and P. E. Fine Mycobacterium tuberculosis Beijing genotype, northern Malawi. Emerg. Infect. Dis. 11: Glynn, J. R., J. Whiteley, P. J. Bifani, K. Kremer, and D. van Soolingen.

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