Characterization of Hypervariable Region of Gyra And Gyrb Genes of Mycobacterium Tuberculosis from Jeddah, KSA

Mycobacterium tuberculosis (MTB) is an old enemy of the human race, with evidence of infection observed as early as 5000 years ago.MTB infection is relatively high in Jeddah as compared to other cities of Saudi Arabia due to high influx of people from across the globe for pilgrimage. This study aimed to investigate the phenotypic drug resistance for the first line antituberculous drugs and to explore mutations in fluoroquinolone resistance gyrA and gyrB genes in  Mycobacterium tuberculosis (MTB) isolates from  tuberculosis patients from Jeddah, Saudi Arabia during 2015. Firstly, phenotypic drug susceptibility tests (DST) were performed for first line antituberculous drugs for all the MTB isolates. DST for rifampicin, isoniazid,streptomycin, ethambutol, and pyrazinamide were performed. Then the hypervariable regions of gyrA and gyrB genes were sequenced to identify mutations for Fluoroquinolones (FQs) resistance . Overall, all the MTB isolates were susceptible to Sterpt, RIF and Eth. Where as resistance to INH and Pyr were 2% and 14% respectively. Genotypic resistance revealed mutations in gyrA and gyrB genes among 96% (48/50) of FQ resistant isolates. 96% (48/50) of FQ resistant isolates showed mutations at codons 95 (S95T)  and three pattern of double mutations in gyrA gene ; six with E21Q & S95T, and the two with I29S & S95T, and one with A90V & S95T. The mutation in gyrB gene was identified in two of 50 clinical isolates in this study . K421R and T420. For clinical isolates, gyrB mutations appear to be of much rarer occurrence.We conclude that occurrence of mutations at only four codons in gyrA and two codon in  gyrB genes among FQ resistant isolates may assist in development of rapid molecular method for FQ resistance detection. Presence of mutations among more than fifty percent of intermediate susceptible FQ MTB isolates could also serve as a predictor for pre-resistant isolates

Mycobacterium tuberculosis Central Asian Strain (CAS) lineage strains in Pakistan reveal lower diversity of MIRU loci than other strains

Mycobacterium tuberculosis (MTB) Central Asian Strain (CAS) lineage strains are predominant in South Asia. Mycobacterial interspersed repetitive units-variable number of tandem repeats (MIRU-VNTR) typing is an effective way of determining genetic diversity of strains. A maximum of 24 loci-based MIRU-VNTR typing can be used, however, it is important to investigate the relevance of specific MIRU loci for regional strains for more cost-effective MIRU typing. MIRU-VNTR typing was performed on MTB strains from Pakistan. Strains were comprised of CAS (n=113) and non-CAS lineages (n=87) - both multi-drug resistant (MDR) and drug susceptible. Hunter Gaston Discriminatory Index (HGDI) for each MIRU loci was interpreted as poor, moderate or highly discriminatory. Results were analyzed using Bionumerics software and miru-vntrplus database link. Clustering analysis revealed 185 different MIRU types. Eight clusters of 2 strains each were present amongst MDR (3 clusters) and drug susceptible (5 clusters) isolates. MDR clusters had orphan and Haarlem strains, whereas drug susceptible strain clusters were comprised of CAS and Beijing lineage strains. The HGDI for 15 loci-based MIRU typing of all isolates was 0.620, whereas HGDI for CAS was lower than non-CAS lineage strains (p-value: 0.023). HGDI of 8 MIRU-VNTR loci (Qub 26b, 10, 26, 4156, Mtub 04, 16, 31 and ETR-A) were all highly discriminatory. The average HGDI based on these 8 loci was significantly lower for CAS than non-CAS strains (P value: 0.03). The lower discriminatory index for CAS using both 15 and 8 MIRU loci-based analysis suggests less genetic diversity in these isolates than in other lineages. The eight highly discriminatory MIRU loci for CAS may help in monitoring the transmission of MTB strains in regions with high CAS lineage prevalence

Line probe assay for detection of rifampicin and isoniazid resistant tuberculosis in Pakistan

Objective: To assess the efficacy of a line-probe assay delta (LiPA) as rapid diagnostic test for early detection of drug-resistant tuberculosis compared to conventional susceptibility methods in Pakistan.Methods: Resistance to rifampicin (RIF) and isoniazid (INH) in 108 smear-positive pulmonary tuberculosis samples was detected using a line-probe assay [GenoType MTBDRplus (Hain Lifescience, GmbH, Nehren, Germany)] at the clinical microbiology laboratory of Aga Khan University Hospital in May, 2009. Results were compared with susceptibilities performed while using agar proportion.Results: In comparison to the agar proportion method, the detection rate and specificity of resistance using MTBDR plus was 92.5% and 98.2% for rifampicin, and 76.3% and 100% for isoniazid. Mutations in codons 531 and 533 of rpoB gene (62%S531L) were responsible for 67.9% of rifampicin resistance. S315T mutation of katG gene was detected in 55.9% and inhA promoter mutation at positions -15 (C15T) in 11.9% of isoniazid resistant isolates. Four phenotypically rifampicin-resistant and 14 isoniazid-resistant strains were not detected by MTBDRplus. Sequencing these strains revealed mutations in 4 strains; 2 in rpoB gene S531W, del518 and 2 in katG genesW300L, S315N. Hence, two phenotypic rifampicin-resistant and 13 phenotypic isoniazid-resistant strains were not detected by the commercial line probe assay.Conclusion: The study showed that MTBDRplus had a high detection rate for rifampicin resistance. However, additional probes need to be included in the assay to improve the detection of isoniazid-resistant mycobacterium tuberculosis strains in Pakistan

Characterization of Mycobacterium tuberculosis Central Asian Strain1 using mycobacterial interspersed repetitive unit genotyping

<p>Abstract</p> <p>Background</p> <p>The Central Asian Strain1 (CAS1) genogroup of <it>Mycobacterium tuberculosis </it>(MTB) is the most prevalent in Pakistan, India and Bangladesh. Mycobacterial interspersed repetitive units variable number tandem repeat (MIRU-VNTR) typing is a reliable and reproducible method for differentiation of MTB isolates. However, information of its utility in determining the diversity of CAS1 strain is limited. We performed standard 12 loci based MIRU-VNTR typing on previously spoligotyped CAS1 strains and 'unique' strains in order to evaluate its discriminatory power for these isolates.</p> <p>Methods</p> <p>Twelve loci based MIRU- VNTR typing was used to type178 CAS1 and 189 'unique' MTB strains. The discriminatory index for each of the loci was calculated using the Hunter Gaston Discriminatory Index (HGDI). A subset of these strains (n = 78) were typed using IS<it>6110 </it>restriction fragment length polymorphism (RFLP). MIRU-VNTR profiles were studied together with their drug susceptibility patterns.</p> <p>Results</p> <p>A total of 349 MIRU patterns were obtained for the 367 strains tested. The CAS1 strains were subdivided into 160 distinct patterns; 15 clusters of 2 strains each, 1 cluster of four strains and 144 unique patterns. Using HGDI, seven MIRU loci, (numbers 26, 31, 27, 16, 10, 39, and 40) were found to be "highly discriminatory" (DI: ≥0.6), four MIRU loci (numbers 20, 24, 23, and 4) were "moderately discriminatory" (DI: 0.3–0.59), and one locus (number 2) was "poorly discriminatory" (DI< 0.3). Loci 26 and 31 were the most discriminatory for the CAS1 isolates. Amongst 'unique' strains in addition to loci 26, 31, 27, 16, 10, 39, and 40, locus 23 was highly discriminatory, while no locus was poorly discriminating. DI values for loci 4, 10 and 26 were significantly lower (P-value < .01) in CAS1 strains than in 'unique' strains. The association between CAS1 strains and MDR was not found to be significant (p value = 0.21).</p> <p>Conclusion</p> <p>We propose that MIRU typing could be used to estimate the phylogenetic relatedness amongst prevalent CAS1 strains, for which MIRU loci 26, 31, 16, 10, 27, 39 and 40 were found to be the most discriminatory.</p

Alternate efflux pump mechanism may contribute to drug resistance in extensively drug-resistant isolates of mycobacterium tuberculosis

INTRODUCTION: Extensively drug-resistant tuberculosis (XDR-TB) has emerged as one of the biggest threats to public health and TB control programs worldwide. XDR-TB is caused by Mycobacterium tuberculosis (MTB) strains resistant to rifampin and isoniazid, as well as to a fluoroquinolone and to at least one injectable aminoglycoside. Drug resistance in MTB has primarily been associated with single nucleotide polymorphisms (SNPs) in particular genes. However, it has also been shown that efflux pumps may play a role in resistance of MTB. Upregulation of drug efflux pumps can decrease the intracellular concentration of drugs and reduce their efficacy. METHODS: Whole genome sequencing was performed on 32 XDR-TB clinical isolates. Sequence data were used to investigate SNPs in efflux pump genes as compared with the H37Rv reference genome. RESULTS: Of the XDR MTB strains, eight (21.62%) were wild type for rpsL, rrs (500 region), and gidB genes, but had non-synonymous (ns) SNPs (aspartic acid to histidine) in the drrA efflux pump gene at position 3273138. Three of eight (37.5%) XDR MTB strains, wild type for rpsL, rrs (500 region), gidB, and gyrB genes were phenotypically streptomycin sensitive and five (62.5%) XDR MTB strains were streptomycin resistant, while all XDR MTB strains, wild type for rpsL, rrs, gidB, and gyrB genes were resistant to fluoroquinolone (ofloxacin) and ethambutol. In addition, three XDR MTB strains wild type for rpsL, rrs, gidB, and drrA genes showed nsSNPs (isoleucine to valine) in the major facilitator superfamily, Rv1634 efflux pump gene at position 1839306. CONCLUSION: Our data show an nsSNP in the drrA efflux pump gene that may result in upregulation of drug efflux mechanisms in MTB strains. It is therefore imperative to understand the mechanism of efflux and its role in drug resistance, which will enable the identification of new drug targets and development of new drug regimens to counteract the drug efflux mechanism of MTB

Characterization of genomic variations in SNPs of PE_PGRS genes reveals deletions and insertions in extensively drug resistant (XDR) M. tuberculosis strains from Pakistan.

BACKGROUND: Mycobacterium tuberculosis (MTB) PE_PGRS genes belong to the PE multigene family. Although the function of PE_PGRS genes is unknown, it is hypothesized that the PE_PGRS genes may be associated with antigenic variability in MTB. MATERIAL AND METHODS: Whole genome sequencing analysis was performed on (n=37) extensively drug-resistant (XDR) MTB strains from Pakistan, which included Lineage 1 (East African Indian, n=2); Other lineage 1 (n=3); Lineage 3 (Central Asian, n=24); Other lineage 3 (n=4); Lineage 4 (X3, n=1) and T group (n=3) MTB strains. RESULTS: There were 107 SNPs identified from the analysis of 42 PE_PGRS genes; of these, 13 were non-synonymous SNPs (nsSNPs). The nsSNPs identified in PE_PGRS genes - 6, 9 and 10 - were common in all EAI, CAS, Other lineages (1 and 3), T1 and X3. Deletions (DELs) in PE_PGRS genes - 3 and 19 - were observed in 17 (80.9%) CAS1 and 6 (85.7%) in Other lineages (1 and 3) XDR MTB strains, while DELs in the PE_PGRS49 were observed in all CAS1, CAS, CAS2 and Other lineages (1 and 3) XDR MTB strains. All CAS, EAI and Other lineages (1 and 3) strains showed insertions (INS) in PE_PGRS6 gene, while INS in the PE_PGRSgenes 19 and 33 were observed in 20 (95.2%) CAS1, all CAS, CAS2, EAI and Other lineages (1 and 3) XDR MTB strains. CONCLUSION: Genetic diversity in PE_PGRS genes contributes to antigenic variability and may result in increased immunogenicity of strains. This is the first study identifying variations in nsSNPs and INDELs in the PE_PGRS genes of XDR-TB strains from Pakistan. It highlights common genetic variations which may contribute to persistence

Single nucleotide polymorphisms in efflux pumps genes in extensively drug resistant Mycobacterium tuberculosis isolates from Pakistan

It is challenging to understand mechanisms of drug resistance in Mycobacterium tuberculosis (MTB) due to the large variability in resistance associated genes. Efflux pump genes contribute to drug resistance and thus add to this complexity. Efflux pump gene protein superfamilies have been characterized by genome analysis of drug resistant strains and through invitro transcriptional studies. However, there is limited information regarding efflux pump genes in extensively drug resistant (XDR) tuberculosis (TB) isolates. Whole genome sequencing (WGS) based analysis of 37 extensively drug resistant (XDR) and five drug sensitive (DS) MTB clinical isolates was performed. Single nucleotide polymorphisms (SNPs) in efflux pump genes Rv0194, Rv1217, Rv1218, drrA, drrB, Rv1258, Rv1634, Rv2688, Rv1273, Rv1819, Rv1458, Rv1877 and Rv1250 were determined in the clinical isolates as compared with the H37Rv reference strain. Allele frequencies of SNPs identified in XDR strains were compared with DS strains. Gene expression of Rv0194, Rv2688, Rv1634, drrA and drrB was determined in XDR -TB isolates (n=9), DS-TB strains (n=4) and H37Rv. We identified SNPs in XDR-TB isolates which were either unique or present at very low frequencies in DS strains; Rv0194 G170V; Rv1217 L151R; Rv1258 P369T and G391R; Rv1273 S118G and I175T; Rv1877 I534T; Rv1250 V318X/A and S333A, and Rv2688 P156T. The expression of Rv2688 and drrB was found to be raised in XDR-TB as compared with DS-TB strains. We identified unique SNPs in efflux pump genes which may be associated with increased drug resistance in the isolates. Increased levels of Rv2688 and drrB efflux pump gene expression observed in XDR strains even in the absence of antibiotics suggests that these clinical isolates may be more refractory to treatment. Further studies are required to directly associate these mutations with increased resistance in MTB

Extensively Drug-Resistant Tuberculosis, Pakistan

Frequency of extensively drug-resistant tuberculosis in Pakistan increased from 1.5% in 2006 to 4.5% in 2009 (p<0.01). To understand the epidemiology, we genotyped selected strains by using spoligotyping, mycobacterial interspersed repetitive units–variable number of tandem repeats, and IS6110 restriction fragment length polymorphism analysis

Genotyping and drug resistance patterns of M. tuberculosis strains in Pakistan

<p>Abstract</p> <p>Background</p> <p>The incidence of tuberculosis in Pakistan is 181/100,000 population. However, information about transmission and geographical prevalence of <it>Mycobacterium tuberculosis </it>strains and their evolutionary genetics as well as drug resistance remains limited. Our objective was to determine the clonal composition, evolutionary genetics and drug resistance of <it>M. tuberculosis </it>isolates from different regions of the country.</p> <p>Methods</p> <p><it>M. tuberculosis </it>strains isolated (2003–2005) from specimens submitted to the laboratory through collection units nationwide were included. Drug susceptibility was performed and strains were spoligotyped.</p> <p>Results</p> <p>Of 926 <it>M. tuberculosis </it>strains studied, 721(78%) were grouped into 59 "shared types", while 205 (22%) were identified as "Orphan" spoligotypes. Amongst the predominant genotypes 61% were Central Asian strains (CAS ; including CAS1, CAS sub-families and Orphan Pak clusters), 4% East African-Indian (EAI), 3% Beijing, 2% poorly defined TB strains (T), 2% Haarlem and LAM (0.2). Also TbD1 analysis (<it>M. tuberculosis </it>specific deletion 1) confirmed that CAS1 was of "modern" origin while EAI isolates belonged to "ancestral" strain types.</p> <p>Prevalence of CAS1 clade was significantly higher in Punjab (P < 0.01, Pearsons Chi-square test) as compared with Sindh, North West Frontier Province and Balochistan provinces. Forty six percent of isolates were sensitive to five first line antibiotics tested, 45% were Rifampicin resistant, 50% isoniazid resistant. MDR was significantly associated with Beijing strains (P = 0.01, Pearsons Chi-square test) and EAI (P = 0.001, Pearsons Chi-square test), but not with CAS family.</p> <p>Conclusion</p> <p>Our results show variation of prevalent <it>M. tuberculosis </it>strain with greater association of CAS1 with the Punjab province. The fact that the prevalent CAS genotype was not associated with drug resistance is encouraging. It further suggests a more effective treatment and control programme should be successful in reducing the tuberculosis burden in Pakistan.</p