Genetic diversity and molecular basis of antimicrobial resistance of mycobacterium tuberculosis strains from Pakistan

Pakistan ranks 8th amongst the 22 high burden tuberculosis (TB) disease countries with an estimated incidence rate of 181/100,000 population. The high incidence of tuberculosis in Pakistan is further compounded by the increasing emergence of drug resistant strains including multi-drug resistant (MDR). Prevalence of MDR-TB in Pakistan has been shown to be between2-4%o in the untreated patients, while the global prevalence of MDR is estimated at 3%. Molecular epidemiological studies, based on the assumption that patients infected with clustered strains are epidemiologically linked, have helped understand the transmission dynamics of disease. It has also helped to investigate the basis of variation in Mycobacterium tuberculosis (MTB) strains, differences in transmission, severity of disease or drug resistance mechanisms in a defined geographical location. This is in turn helpful in developing strategies for the treatment and prevention of the disease including MDR. Molecular epidemiological data using spoligotyping available from Pakistan shows a predominance of the Central Asian Strain 1 (CAS1) (39%), and Beijing strains (6%). Beijing strains have been shown an association with MDR. Although CASI strains have not shown an association with MDR, about forty percent of total CAS1 strains were comprised of MDR strains. The data about the types and frequency of drug resistance gene mutations within these strains is limited. The overall aims of this study were to explore genetic diversity amongst the predominant genogroup of Mycobacterium tuberculosis from the country as well as to investigate genetic basis of drug resistance amongst these predominant MTB strains. In lv I this study variable number of tandem repeat mycobacterial interspersed repetitive unit (VNTR-MIRU) and 1S6110 based restriction fragment length polymorphism (1S6110- RFLP) was used to study the relationship within CAS1 strains. Knowledge of type and frequency of mutations amongst prevalent genogroup has been shown to be essential for the development of appropriate tools for early diagnosis and control of MDR-TB strains. There is limited information on mutations leading to drug resistance within MTB strains from the country. Therefore in this study prevalent mutation in the rpoB, katG and inhA genes for rifampicin (RMP) and isoniazid (INH) resistance were also investigated in MDR strains of predominant genogroups. Twelve loci based VNTR-MIRU typing showed highly diverse profile of 367 MTB strains. Of the 178 CAS1 strains studied only 34 (19 %) clustered into groups based on MIRU profiles, while all 189 \u27unique\u27 spoligotypes studied had non-matching MIRU profiles and therefore remained un-clustered. The MIRU-VNTR data shows a close relationship (70%) within the prevalent CASI strains. Therefore it is proposed that in a region where CASI family strains are prevalent most discriminatory MIRU loci 26, 31, 16, 10, 27, 39 and 40, could be used for differentiation and estimation of the phylogenetic relatedness of Mycobacterium tuberculosis. IS6110-RFLP typing of 78 strains (43 CAS1 and 35 \u27unique\u27 spoligotypes strains) resulted in 73 different RFLP types. One cluster of two unique strains, with single copy of IS6110 was identified; the remaining seventy-two strains revealed unique RFLP patterns. The most common mutations determined in MDR strains were in codons 531 (60%),526 (23%) and 516 (5%) of rpoB gene by sequencing, while probe based assay detected 44%o and 2l%o mutations at codon 531 and 526 respectively. Occurrence of mutation at codon 526 as well as concurrent mutation in rpoB gene was significantly higher in CAS1 than Beijing and other un-clustered strains tested. Sixty three percent of 62 MDR isolates showed mutation at codon 315 by sequencing while by probe based assay sixty percent of resistance was detected. CAS1 family strains exhibited higher rate of mutation at codon 315 as compared with Beijing. Multi-drug resistant CAS1 strains are more prone to develop resistance against RMP and INH through mutations at codon 526 of rpoB gene and codon 315 of katG gene respectively than non-CASI MDR strains. 67% of RMP and INH resistance within MDR CASI strains could be determined by detecting mutations at only three codons 526 and 531 of rpoB and 315 of katG gene