Molecular Characterization of Isoniazid-Resistant Mycobacterium tuberculosis Clinical Isolates in Lithuania

Abstract

Mutations at codon 315 of the katG gene were detected in 312 of 364 (85.7%) isoniazid-resistant Mycobacterium tuberculosis isolates. Seven of 52 (13.5%) isoniazid-resistant isolates with the wild-type Ser315 codon and 10 of 52 (19.2%) isoniazid-resistant isolates with a mutated katG allele had mutation ؊15C3T in the promoter of the mabA-inhA operon.Isoniazid (INH) together with rifampin (RIF) form the cornerstone of a short chemotherapy course for tuberculosis, and resistance to either drug hampers seriously the complete cure of patients (17). Mutations at several chromosome loci of Mycobacterium tuberculosis (fur-katG, mabA-inhA, oxyR-aphC, kasA, and ndh) are associated with resistance to INH (6,7,11,13). Mutations both at the Ser315 codon of katG, encoding catalase-peroxidase, the enzyme oxidizing INH (4), and at the regulatory region of the mabA-inhA operon, encoding a target of activated prodrug, enoyl-acyl carrier protein reductase (2), occur most frequently in INH-resistant isolates (9,11,13). In this study we searched for the most common mutations in katG and the mabA-inhA regulon of INH-resistant isolates to evaluate their significance for rapid drug resistance prediction in Lithuania.In total, 364 INH-resistant and 78 INH-susceptible M. tuberculosis isolates collected from different patients (93 female, 349 male; age range, 19 to 94 years) in the National Tuberculosis Reference Laboratory from 1998 to May 2002 were studied. New and previously treated tuberculosis cases were defined according to World Health Organization recommendations (19). Among INH-resistant isolates 157 were from new cases and 207 were from previously treated cases. Among INH-susceptible isolates 66 and 12 were from new and previously treated cases, respectively. Analysis of M. tuberculosis was carried out by standard procedures (5, 18). Drug susceptibility testing was done either by the proportion method on Löwenstein-Jensen medium or with BACTEC 460 (Becton Dickinson, Sparks, Md.). Critical drug concentrations were 0.2 g/ml for INH, 4 g/ml for streptomycin (STR), 40 g/ml for RIF, and 2 g/ml for ethambutol (EMB) in Löwenstein-Jensen medium and 0.1, 2, 2, and 2.5 g/ml for INH, STR, RIF, and EMB, respectively, with the BACTEC 460. DNA from M. tuberculosis was extracted as described elsewhere (15). The 264-bp katG fragment targeting codon 315 and the 248-bp fragment spanning the mabA-inhA regulon were amplified by using primers 5Ј-TGGAGCAGATGGGCTTGG and 5Ј-CAG TGGCCAGCATCGTCG (12) and primers 5Ј-CCTCGCTGC CCAGAAAGGGA and 5Ј-ATCCCCCGGTTTCCTCCGG (14), respectively. PCR products purified with silica powder were subjected to manual sequencing by using the CycleReader DNA sequencing kit (Fermentas, Vilnius, Lithuania). For the restriction fragment length polymorphism (RFLP) assay, katG-specific PCR products were digested with TauI or SatI (Fermentas) and analyzed in a 1.5% agarose gel. Both endonucleases possess the overlapping GC sequence at a single recognition site of the 315 triplet AGC, and mutations at these positions eliminate cle...

High level isoniazid resistance correlates with multiple mutation in the katG encoding catalase proxidase of pulmonary tuberculosis isolates from the frontier localities of Iran

“…Our findings are in agreement with similar data reported in Lithuania where 95% of strains displayed mutation in codon 315 of the katG gene with the predominance of codon substitution AGC→ACC (Ser→Thr) (90%) (Bakonyte et al 2003). However, we could not identify the mutation of AGC→ACA (Ser→Thr) which has been reported in Lithuania (5).…”

“…Mutations at the Ser315 codon of katG have been reported to be associated with high-level of isoniazid resistance (10). Resistance to isoniazid has a second degree of magnitude in Iran, and combinations of mutations conferring M. tuberculosis resistance to isoniazid have been reported to be more common in the multidrug resistance tuberculosis (MDR-TB) than in monoresistant M. tuberculosis isolates, suggesting that isolates develop resistance to isoniazid by a stepwise accumulation of mutations (5,11).…”

“…Most isoniazid-resistant M. tuberculosis strains have not been reported to have a high proportion of katG deletions suggesting the need to more precisely analyze the structure of the katG gene in the resistant organisms. Further studies have revealed that katG gene deletions are very rare and this requires more detailed analysis of the katG its structure (4,5,9). Several groups have recently reported that many isoniazid-resistant strains contain missense and other types of mutations (4).…”

“…Resistance to isoniazid is associated with a variety of mutations affecting one or more genes such as that encoding catalase peroxidase (katG) (4,5). The katG gene is the most commonly targeted region of the Mycobacterium tuberculosis genome with the majority of mutations occurring in codon 315 in 30-90% of isoniazid-resistant strains depending on geographical distribution (6,7).…”

Isoniazid Resistance and the Future of Drug-Resistant Tuberculosis

“…Other mutations observed at this site have led to Ser-Asn, Ser-Ile, Ser-Arg, and Ser-Leu substitutions, although these have been observed less frequently than S315T. 4,6,12,15,22,26,28 Because these mutations are relatively rare, they may indeed be less fit than their Ser-Thr counterpart. If this is the case, their inclusion would most likely diminish any association detected between 315 mutations and transmissibility.…”