Molecular Characterization of Isoniazid-Resistant Mycobacterium tuberculosis Isolates Collected in Australia

Mycobacterial isolates.The Victorian Infectious Diseases Reference Laboratory (VIDRL) serves as the reference laboratory for all mycobacterial infections in Victoria, Australia. Six hundred forty-five new laboratory-confirmed cases of TB caused by M. tuberculosis were collected during the study period (2001 to 2003). All isolates were tested for susceptibility to first-line drugs INH (0.1 μg/ml), ethambutol (EMB) (5 μg/ml), and rifampin (RIF) (1 μg/ml), using the BACTEC MGIT 960 system. Resistance to pyrazinamide (PZA) was initially tested using Wayne's method (33), with confirmation of resistance in the BACTEC 460 (100 μg/ml). Those strains resistant to one or more first-line drugs were tested for their susceptibility to second-line drugs amikacin, ciprofloxacin, kanamycin, ethionamide (ETH) (5 μg/ml), and rifabutin in the BACTEC 460 system. All strains resistant to RIF were also resistant to rifabutin. As defined by the BACTEC MGIT 960 manual, 42 isolates were resistant to INH at 0.4 μg/ml and 10 isolates were resistant to INH at 0.1 μg/ml but sensitive at 0.4 μg/ml (defined as having intermediate resistance to INH and denoted as INH^I). For the purposes of this study, unless specified, those isolates displaying intermediate resistance have been classified as INH^r. Each matched pair of INH^r and INH^s isolates belonged to the same genotypic group, had similar MIRU-exact tandem repeat (ETR) profiles, and was isolated from patients born in the same country or region. DNA was extracted from M. tuberculosis cultures by the method described by Ross et al. (24). Patient country-of-origin data were kindly provided by the Department of Human Services, Victoria, Australia.

MIRU-ETR typing.ETR loci A, B, and C were amplified using the primers and amplification conditions described by Frothingham and Meeker-O'Connell (10). MIRU loci 2, 4, 10, 16, 20, 23, 24, 26, 27, 31, 39, and 40 were amplified using the primers and amplification conditions described by Supply et al. (30). PCRs were performed singly rather than using the multiplex format. The number of tandem repeats at each locus was determined by estimating the amplicon sizes after electrophoresis on 2% (wt/vol) agarose Tris-acetate-EDTA gels.

Classification of strains into major genetic groups and ancestral and modern strains.For the purpose of association studies, the isolates were each assigned to either genetic group 1 or groups 2/3 according to the classification scheme described by Sreevatsan et al. (28). (Genetic groups 2 and 3 were not separated due to the small number of isolates.) The presence of katG463 CTG (Leu) (group 1) or CGG (Arg) (groups 2/3) was determined by PCR-restriction fragment length polymorphism as described by Sreevatsan et al. (28). Genetic group 1 isolates were divided into “ancestral” and “modern” strains based on the number of repeats at MIRU locus 24 (29). Ancestral isolates have more than one repeat at locus 24, while modern strains have one repeat.

Identification of the Beijing and CAS genotype.Strains belonging to the Beijing family were identified based on the scheme proposed by Ferdinand et al. (8), by comparing the MIRU profiles of the study isolates with those of previously published Beijing isolates (18) or by comparison of IS6110 profiles to those described by Kremer et al. (15; data not shown). The aphC −46A polymorphism has been reported to be associated with the Delhi/CAS family of strains (2). The presence of either G or A at aphC −46A was determined by nucleotide sequencing of the 200-bp PCR product obtained using primers FSQ AphC-67 (5′-GTCGACTGGCTCATATCGAGA-3′) and R4OxyR-AhpC (5′-GGTTAGCAGTGGCATGACTCT-3′) (M. Hazbon; personal communication). PCR products were sequenced using BigDye terminator 3.1 cycle sequencing kit (Applied Biosystems) according to the manufacturer's instructions, and precipitated reaction products were run on an Applied Biosystems 3730 DNA analyzer.

Cluster analysis.The unweighted-pair group method using average linkages (UPGMA) tree was constructed using the Sequence Type Analysis and Recombinational Tests (START) program (http://outbreak.ceid.ox.ac.uk/software.htm). This algorithm, while not appropriate for the generation of a phylogenetic tree, grouped the isolates according to similarities in their profiles based on the 15 MIRU-ETR loci.

Statistical analyses.Tests for association were performed using the chi-square distribution.

katG315 MAS-PCR.Screening for mutations at katG codon 315 was carried out using the multiplex allele-specific PCR (MAS-PCR) assay developed and described by Mokrousov et al. (20). In this assay, the codon 315 region is PCR amplified with two outer primers and an inner reverse primer specific for the katG315 wild-type allele (AGC). Isolates with a wild-type codon 315 produce two PCR products, while those isolates with a mutation at codon 315 produce only one PCR product. All 52 INH^r and 52 INH^s isolates were screened using this method. Those isolates identified as not having a wild-type katG codon 315 (AGC) were then screened using a modified MAS-PCR assay, which, instead of the inner reverse primer specific for the katG315 wild-type sequence (20), used an inner reverse primer designed to detect the presence of either the katG315 ACC mutation, primer 523 (5′-ATACGACCTCGATGCCGG-3′), or the ACA mutation, primer 534 (5′-ATACGACCTCGATGCCTG-3′).

Sequencing of the fabG1-inhA regulatory region, inhA ORF, and katG ORF.The fabG1-inhA regulatory region was PCR amplified using primers 519 (5′-CCTCGCTGCCCAGAAAGGGA-3′) and 520 (5′-ATCCCCCGGTTTCCTCCGGT-3′). The inhA open reading frame (ORF) was amplified using primers 534 (5′-TCCGGTGCGGTCATCCCG-3′) and 535 (5′-AACGGCCGCACCTGCTCG-3′). The katG ORF was amplified in three overlapping segments: segment 1 was amplified using primers 527 (5′-ACACTTCGCGATCACATCCG-3′) and 528 (5′-ACCTCGATGCCGCTGGTG-3′), segment 2 with primers 540 (5′-CGGTCACACTTTCGGTAAGA-3′) and 541 (5′-GGCGAAGGACACTTTGATGT-3′), and segment 3 using primers 542 (5′-GCCAGCCTTAAGAGCCAGAT-3′) and 543 (5′-ACGCGGGGTCTGACAAAT3′). PCR products were sequenced as described previously.

Detection of mutations at katG codon 315 and in the fabG1-inhA regulatory region.The 52 INH^r and 52 matched INH^s sensitive control strains were screened for mutations at katG codon 315 and the fabG1-inhA regulatory region by MAS-PCR and DNA sequence analysis, respectively. A complete list of specific mutations identified is provided in Table 1. Thirty-four (65.4%) INH^r isolates had mutations at katG codon 315. The wild-type codon, AGC (Ser), was altered to ACC (Thr) in 31 strains and ACA (Thr) in three strains. Mutations in the fabG1-inhA regulatory region were identified in 13 (25.0%) of the 52 INH^r isolates (Table 1). All but one of these was the substitution of C for T 15 nucleotides upstream from the fabG1 start codon. The one exception was a G-to-C substitution at position −17. None of the INH^r isolates had mutations in both katG315 and the fabG1-inhA regulatory region. No mutations at either katG codon 315 or in the fabG1-inhA regulatory region were identified in any of the INH^s isolates.

Identification of mutations in the katG and inhA ORFs.Five INH^r isolates had a wild-type sequence at both katG315 and in the fabG1-inhA regulatory region. The entire katG and inhA ORFs of these isolates were sequenced together with their matched INH^s control strains. All five INH^r and five INH^s isolates had a wild-type inhA structural gene. Mutations in katG were identified in three of the five INH^r isolates (Table 1). Isolate 2285 had an Asp117Ala substitution, isolate 2562 a Gly491Cys substitution, and isolate 2938 a Met257Ile substitution. These mutations were not present in the three INH^s control strains tested; however, a more extensive survey of INH^r and INH^s isolates would need to be performed for an association between these mutations and INH resistance to be determined. No mutations in katG, the fabG1-inhA regulatory region, or the inhA structural gene were identified in isolate 2919 or 2956. Thus, resistance in these isolates is unlikely to be associated with either of these enzymes.

Drug resistance pattern, geographic origin, and strain differentiation.The drug resistance phenotype, MIRU-ETR profile, genotypic group, geographic origin, and mutation identified for each of the 52 INH^r isolates are presented in Table 2. Twenty-three isolates were INH monoresistant; 16 isolates were resistant to both INH and streptomycin; 5 isolates were multidrug resistant (resistant to INH and rifampin); 4 isolates were resistant to both INH and ETH; 3 isolates were resistant to INH, pyrazinamide, and streptomycin; and 1 isolate was resistant to INH and ethambutol. Ten isolates displayed intermediate resistance to INH.

Although the isolates were collected from patients resident in Australia, only 7.7% (n = 4) of patients were born in Australia. The majority of overseas-born patients originated from Vietnam (n = 16), India (n = 8), China (n = 4), and the Philippines (n = 4), with 11 other countries represented. To determine whether INH resistance was associated with isolates from particular geographic origins, the proportion of strains resistant to INH from each country was compared to the proportion in the rest of the population (data not shown). Patients born in Vietnam were more likely to be infected with INH^r isolates than non-Vietnamese-born patients (P = 0.001). This finding cannot be attributed to patient-to-patient transmission, as the isolates were epidemiologically unrelated.

The UPGMA tree (Fig. 1) based on MIRU-ETR patterns (Table 2) illustrates the genetic relationship, genotypic group, drug resistance profile, and mutations identified in the 52 INH^r M. tuberculosis isolates. The 16 INH^r isolates identified as ancestral genetic group 1 represented 9.5% (16 of 168) of the total ancestral strains collected during the study period. In comparison, 11.6% (25 of 215) of modern group 1 and only 4.2% (11 of 262) of group 2/3 strains were found to be resistant to INH. Group 2/3 isolates were therefore less likely to be resistant to INH than strains belonging to group 1 (P = 0.008). All five multidrug-resistant isolates were modern strains: one isolate belonged to groups 2/3, and four belonged to group 1. Beijing family strains accounted for 30.8% (n = 16) of the INH^r isolates (Fig. 1), which was slightly higher than the proportion of Beijing strains in the total collection (22.6%). With the exception of strain 2817, the remaining modern group 1 strains (n = 8) were of the Delhi/CAS genotype (Fig. 1).

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FIG. 1.

Genetic relationship, genotypic group, drug resistance profile, and mutations identified in the 52 INH-resistant M. tuberculosis isolates. This UPGMA tree was generated using the START program based on the 50 distinct MIRU-ETR patterns (Table 2) that represented the 52 isolates. I, isolate identification number; II, drug resistance phenotype; INHI, INH intermediate resistance; III, inhAP, fabG1-inhA regulatory region mutation; katG117, katG D117A substitution; katG257, katG M257I substitution; katG315, katG S315T substitution; katG491, katG G491C substitution.

Association between mutations at katG codon 315 and in the fabG1-inhA regulatory region, genotypic group, and drug resistance phenotype.The proportions of isolates within each genotypic group with the katG S315T substitution were as follows: ancestral, 12/16; group 1, 15/25; and groups 2/3, 7/11. The proportion of Beijing isolates harboring the S315T substitution (68.8%) was slightly higher than that of non-Beijing strains (63.9%). The numbers of isolates with the fabG1-inhA −15C→T mutation in each of the genotypic groups were as follows: ancestral, 4/16; group 1, 6/25; and groups 2/3, 2/11. Thus, neither the katG S315T substitution nor the fabG1-inhA −15C→T mutation clustered with any one genetic group. However, there was an association between the −15C→T substitution and those isolates with intermediate resistance to INH, with the mutation identified in eight of the 10 INH^I isolates. In addition, the −15C→T substitution was strongly associated with resistance to ETH, with four of the five ETH-resistant isolates having the mutation.