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

In this search, a new pyrophosphate technique was proved. The technique was employed to single- nucleotide polymorphisms (SNPs), which diagnosis using a one-base extension reaction. Three Mycobacterium tuberculosis genes were chosen (Rpob, InhA, KatG) genes. Fifty-four specimens were used in this study fifty-three proved as drug-resistant specimens by The Iraqi Institute of Chest and Respiratory Diseases in Baghdad.; also one specimen was used as a negative control. The steps of this technique were by used a specific primer within each aliquot that has a short 3-OH end of the base of the target gene that was hybridized to the single-stranded DNA template. Then, the Taq polymerase enzyme and one of either α-thio-dATP, dTTP, dGTP, or dCTP were supplemented and incubated for 1 min. ATP is synthesis by convert Pyrophosphate freed by DNA polymerase using pyruvate phosphate dikinase (PPDK), and the amount of ATP estimates by the firefly luciferase reaction. This technique, which does not demand expensive equipment, can be applied to rapidly monitor a one-point mutation in the gene that causes drug-resistant in mycobacterium tuberculosis. The results showed a high variation in values of ATP formation through matching and mismatch bases added. So, this assay (which required only five minutes), enable to find the gene SNP causes resistant for the specific drug.

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Urgent Implementation in a Hospital Setting of a Strategy To Rule Out Secondary Cases Caused by Imported Extensively Drug-Resistant Mycobacterium tuberculosis Strains at Diagnosis

Journal of Clinical Microbiology ◽

10.1128/jcm.01718-16 ◽

2016 ◽

Vol 54 (12) ◽

pp. 2969-2974 ◽

Cited By ~ 10

Author(s):

Laura Pérez-Lago ◽

Miguel Martínez-Lirola ◽

Sergio García ◽

Marta Herranz ◽

Igor Mokrousov ◽

...

Keyword(s):

Mycobacterium Tuberculosis ◽

High Risk ◽

Primary Cultures ◽

Hospital Setting ◽

Drug Resistant ◽

Nucleotide Polymorphisms ◽

Content Type ◽

Extensively Drug Resistant ◽

Incident Cases ◽

Rule Out

Current migratory movements require new strategies for rapidly tracking the transmission of high-risk importedMycobacterium tuberculosisstrains. Whole-genome sequencing (WGS) enables us to identify single-nucleotide polymorphisms (SNPs) and therefore design PCRs to track specific relevant strains. However, fast implementation of these strategies in the hospital setting is difficult because professionals working in diagnostics, molecular epidemiology, and genomics are generally at separate institutions. In this study, we describe the urgent implementation of a system that integrates genomics and molecular tools in a genuine high-risk epidemiological alert involving 2 independent importations of extensively drug resistant (XDR) and pre-XDR BeijingM. tuberculosisstrains from Russia into Spain. Both cases involved commercial sex workers with long-standing tuberculosis (TB). The system was based on strain-specific PCRs tailored from WGS data that were transferred to the local node that was managing the epidemiological alert. The optimized tests were available for prospective implementation in the local node 33 working days after receiving the primary cultures of the XDR strains and were applied to all 42 new incident cases. An interpretable result was obtained in each case (directly from sputum for 27 stain-positive cases) and corresponded to the amplification profiles for strains other than the targeted pre-XDR and XDR strains, which made it possible to prospectively rule out transmission of these high-risk strains at diagnosis.

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Single nucleotide polymorphisms in Mycobacterium tuberculosis and the need for a curated database

Tuberculosis ◽

10.1016/j.tube.2012.11.002 ◽

2013 ◽

Vol 93 (1) ◽

pp. 30-39 ◽

Cited By ~ 32

Author(s):

David Stucki ◽

Sebastien Gagneux

Keyword(s):

Mycobacterium Tuberculosis ◽

Single Nucleotide Polymorphisms ◽

Nucleotide Polymorphisms ◽

Single Nucleotide

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Using Mycobacterium tuberculosis Single-Nucleotide Polymorphisms To Predict Fluoroquinolone Treatment Response

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.00076-19 ◽

2019 ◽

Vol 63 (7) ◽

Cited By ~ 3

Author(s):

Marva Seifert ◽

Edmund Capparelli ◽

Donald G. Catanzaro ◽

Timothy C. Rodwell

Keyword(s):

Mycobacterium Tuberculosis ◽

Single Nucleotide Polymorphisms ◽

Specific Resistance ◽

Therapeutic Targets ◽

World Health ◽

Population Pharmacokinetic ◽

Nucleotide Polymorphisms ◽

Single Nucleotide ◽

Content Type ◽

Level Increase

ABSTRACT Clinical phenotypic fluoroquinolone susceptibility testing of Mycobacterium tuberculosis is currently based on M. tuberculosis growth at a single critical concentration, which provides limited information for a nuanced clinical response. We propose using specific resistance-conferring M. tuberculosis mutations in gyrA together with population pharmacokinetic and pharmacodynamic modeling as a novel tool to better inform fluoroquinolone treatment decisions. We sequenced the gyrA resistance-determining region of 138 clinical M. tuberculosis isolates collected from India, Moldova, Philippines, and South Africa and then determined each strain’s MIC against ofloxacin, moxifloxacin, levofloxacin, and gatifloxacin. Strains with specific gyrA single-nucleotide polymorphisms (SNPs) were grouped into high or low drug-specific resistance categories based on their empirically measured MICs. Published population pharmacokinetic models were then used to explore the pharmacokinetics and pharmacodynamics of each fluoroquinolone relative to the empirical MIC distribution for each resistance category to make predictions about the likelihood of patients achieving defined therapeutic targets. In patients infected with M. tuberculosis isolates containing SNPs associated with a fluoroquinolone-specific low-level increase in MIC, models suggest increased fluoroquinolone dosing improved the probability of achieving therapeutic targets for gatifloxacin and moxifloxacin but not for levofloxacin and ofloxacin. In contrast, among patients with isolates harboring SNPs associated with a high-level increase in MIC, increased dosing of levofloxacin, moxifloxacin, gatifloxacin, or ofloxacin did not meaningfully improve the probability of therapeutic target attainment. We demonstrated that quantifiable fluoroquinolone drug resistance phenotypes could be predicted from rapidly detectable gyrA SNPs and used to support dosing decisions based on the likelihood of patients reaching therapeutic targets. Our findings provide further supporting evidence for the moxifloxacin clinical breakpoint recently established by the World Health Organization.

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Geographical Differences Associated with Single-Nucleotide Polymorphisms (SNPs) in Nine Gene Targets among Resistant Clinical Isolates of Mycobacterium tuberculosis

Journal of Clinical Microbiology ◽

10.1128/jcm.00857-13 ◽

2013 ◽

Vol 52 (5) ◽

pp. 1322-1329 ◽

Cited By ~ 16

Author(s):

M. Hoshide ◽

L. Qian ◽

C. Rodrigues ◽

R. Warren ◽

T. Victor ◽

...

Keyword(s):

Mycobacterium Tuberculosis ◽

Single Nucleotide Polymorphisms ◽

Clinical Isolates ◽

Nucleotide Polymorphisms ◽

Geographical Differences ◽

Single Nucleotide

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In-Depth Study of a Nosocomial Outbreak Caused by Extensively Drug-Resistant Pseudomonas aeruginosa Using Whole Genome Sequencing Coupled With a Polymerase Chain Reaction Targeting Strain-Specific Single Nucleotide Polymorphisms

American Journal of Epidemiology ◽

10.1093/aje/kwaa025 ◽

2020 ◽

Vol 189 (8) ◽

pp. 841-849

Author(s):

Fermín Acosta ◽

Ana Fernández-Cruz ◽

Sandra R Maus ◽

Pedro J Sola-Campoy ◽

Mercedes Marín ◽

...

Keyword(s):

Single Nucleotide Polymorphisms ◽

Whole Genome Sequencing ◽

Genome Sequencing ◽

Whole Genome ◽

Nucleotide Polymorphisms ◽

Outbreak Strain ◽

Single Nucleotide ◽

Specific Pcr ◽

Extensively Drug Resistant ◽

Polymerase Chain

Abstract In 2013–2014, an outbreak involving 14 patients infected by an extensively drug-resistant strain of Pseudomonas aeruginosa was detected in a hospital in Madrid, Spain. Our objective was to evaluate an alternative strategy for investigating the outbreak in depth by means of molecular and genomic approaches. Pulsed-field gel electrophoresis (PFGE) was applied as a first-line approach, followed by a more refined whole genome sequencing analysis. Single nucleotide polymorphisms identified by whole genome sequencing were used to design a specific polymerase chain reaction (PCR) for screening unsuspected cases infected by the outbreak strain. Whole genome sequencing alerted us to the existence of greater genetic diversity than was initially assumed, splitting the PFGE-associated outbreak isolates into 4 groups, 2 of which represented coincidental transmission unrelated to the outbreak. A multiplex allele-specific PCR targeting outbreak-specific single nucleotide polymorphisms was applied to 290 isolates, which allowed us to identify 25 additional cases related to the outbreak during 2011–2017. Whole genome sequencing coupled with an outbreak-strain-specific PCR enabled us to markedly redefine the initial picture of the outbreak by 1) ruling out initially suspected cases, 2) defining likely independent coincidental transmission events, 3) predating the starting point of the outbreak, 4) capturing new unsuspected cases, and 5) revealing that the outbreak was still active.

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Single Nucleotide Polymorphisms in Genes Associated with Isoniazid Resistance in Mycobacterium tuberculosis

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.47.4.1241-1250.2003 ◽

2003 ◽

Vol 47 (4) ◽

pp. 1241-1250 ◽

Cited By ~ 202

Author(s):

Srinivas V. Ramaswamy ◽

Robert Reich ◽

Shu-Jun Dou ◽

Linda Jasperse ◽

Xi Pan ◽

...

Keyword(s):

Mycobacterium Tuberculosis ◽

Single Nucleotide Polymorphisms ◽

Molecular Genetic ◽

Mycolic Acid ◽

Genetic Group ◽

Clinical Isolates ◽

Central Component ◽

Polymorphism Analysis ◽

Nucleotide Polymorphisms ◽

Single Nucleotide

ABSTRACT Isoniazid (INH) is a central component of drug regimens used worldwide to treat tuberculosis. Previous studies have identified resistance-associated mutations in katG, inhA, kasA, ndh, and the oxyR-ahpC intergenic region. DNA microarray-based experiments have shown that INH induces several genes in Mycobacterium tuberculosis that encode proteins physiologically relevant to the drug's mode of action. To gain further insight into the molecular genetic basis of INH resistance, 20 genes implicated in INH resistance were sequenced for INH resistance-associated mutations. Thirty-eight INH-monoresistant clinical isolates and 86 INH-susceptible isolates of M. tuberculosis were obtained from the Texas Department of Health and the Houston Tuberculosis Initiative. Epidemiologic independence was established for all isolates by IS6110 restriction fragment length polymorphism analysis. Susceptible isolates were matched with resistant isolates by molecular genetic group and IS6110 profiles. Spoligotyping was done with isolates with five or fewer IS6110 copies. A major genetic group was established on the basis of the polymorphisms in katG codon 463 and gyrA codon 95. MICs were determined by the E-test. Semiquantitative catalase assays were performed with isolates with mutations in the katG gene. When the 20 genes were sequenced, it was found that 17 (44.7%) INH-resistant isolates had a single-locus, resistance-associated mutation in the katG, mabA, or Rv1772 gene. Seventeen (44.7%) INH-resistant isolates had resistance-associated mutations in two or more genes, and 76% of all INH-resistant isolates had a mutation in the katG gene. Mutations were also identified in the fadE24, Rv1592c, Rv1772, Rv0340, and iniBAC genes, recently shown by DNA-based microarray experiments to be upregulated in response to INH. In general, the MICs were higher for isolates with mutations in katG and the isolates had reduced catalase activities. The results show that a variety of single nucleotide polymorphisms in multiple genes are found exclusively in INH-resistant clinical isolates. These genes either are involved in mycolic acid biosynthesis or are overexpressed as a response to the buildup or cellular toxicity of INH.

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Draft Genome Sequences of Two Extensively Drug-Resistant Strains ofMycobacterium tuberculosisBelonging to the Euro-American S Lineage

Genome Announcements ◽

10.1128/genomea.01771-15 ◽

2016 ◽

Vol 4 (2) ◽

Cited By ~ 2

Author(s):

Lesibana A. Malinga ◽

Thomas Abeel ◽

Christopher A. Desjardins ◽

Talent C. Dlamini ◽

Gail Cassell ◽

...

Keyword(s):

Draft Genome ◽

Drug Efflux ◽

Drug Resistant ◽

Nucleotide Polymorphisms ◽

Genome Sequences ◽

Single Nucleotide ◽

Resistant Tuberculosis ◽

Extensively Drug Resistant ◽

Resistant Strains ◽

Drug Resistant Strains

We report the whole-genome sequencing of two extensively drug-resistant tuberculosis strains belonging to the Euro-American S lineage. The RSA 114 strain showed single-nucleotide polymorphisms predicted to have drug efflux activity.

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