title: Novel katG mutations causing isoniazid resistance in clinical M. tuberculosis isolates.
abstract: We report the discovery and confirmation of 23 novel mutations with previously undocumented role in isoniazid (INH) drug resistance, in catalase-peroxidase (katG) gene of Mycobacterium tuberculosis (Mtb) isolates. With these mutations, a synonymous mutation in fabG1 (g609a), and two canonical mutations, we were able to explain 98% of the phenotypic resistance observed in 366 clinical Mtb isolates collected from four high tuberculosis (TB)-burden countries: India, Moldova, Philippines, and South Africa. We conducted overlapping targeted and whole-genome sequencing for variant discovery in all clinical isolates with a variety of INH-resistant phenotypes. Our analysis showed that just two canonical mutations (katG 315AGC-ACC and inhA promoter-15C-T) identified 89.5% of resistance phenotypes in our collection. Inclusion of the 23 novel mutations reported here, and the previously documented point mutation in fabG1, increased the sensitivity of these mutations as markers of INH resistance to 98%. Only six (2%) of the 332 resistant isolates in our collection did not harbor one or more of these mutations. The third most prevalent substitution, at inhA promoter position -8, present in 39 resistant isolates, was of no diagnostic significance since it always co-occurred with katG 315. 79% of our isolates harboring novel mutations belong to genetic group 1 indicating a higher tendency for this group to go down an uncommon evolutionary path and evade molecular diagnostics. The results of this study contribute to our understanding of the mechanisms of INH resistance in Mtb isolates that lack the canonical mutations and could improve the sensitivity of next generation molecular diagnostics.
authors.affiliation: Biological & Medical Informatics Research Center, San Diego State University , San Diego, CA 92182, USA.
authors.forename: Jessica N
authors.initials: JN
authors.lastname: Torres
authors.affiliation: SA MRC Centre for TB Research, DST/NRF Centre of Excellence for Biomedical Tuberculosis Research, Division of Molecular Biology and Human Genetics, Stellenbosch University , Tygerberg, Cape Town 7505, South Africa.
authors.forename: Lynthia V
authors.initials: LV
authors.lastname: Paul
authors.affiliation: Department of Medicine, University of California , San Diego, La Jolla, CA 92093, USA.
authors.forename: Timothy C
authors.initials: TC
authors.lastname: Rodwell
authors.affiliation: SA MRC Centre for TB Research, DST/NRF Centre of Excellence for Biomedical Tuberculosis Research, Division of Molecular Biology and Human Genetics, Stellenbosch University , Tygerberg, Cape Town 7505, South Africa.
authors.forename: Thomas C
authors.initials: TC
authors.lastname: Victor
authors.affiliation: Biological & Medical Informatics Research Center, San Diego State University , San Diego, CA 92182, USA.
authors.forename: Anu M
authors.initials: AM
authors.lastname: Amallraja
authors.affiliation: Biological & Medical Informatics Research Center, San Diego State University , San Diego, CA 92182, USA.
authors.forename: Afif
authors.initials: A
authors.lastname: Elghraoui
authors.affiliation: Biological & Medical Informatics Research Center, San Diego State University , San Diego, CA 92182, USA.
authors.forename: Amy P
authors.initials: AP
authors.lastname: Goodmanson
authors.affiliation: Biological & Medical Informatics Research Center, San Diego State University , San Diego, CA 92182, USA.
authors.forename: Sarah M
authors.initials: SM
authors.lastname: Ramirez-Busby
authors.affiliation: Biological & Medical Informatics Research Center, San Diego State University , San Diego, CA 92182, USA.
authors.forename: Ashu
authors.initials: A
authors.lastname: Chawla
authors.affiliation: Biological & Medical Informatics Research Center, San Diego State University , San Diego, CA 92182, USA.
authors.forename: Victoria
authors.initials: V
authors.lastname: Zadorozhny
authors.affiliation: SA MRC Centre for TB Research, DST/NRF Centre of Excellence for Biomedical Tuberculosis Research, Division of Molecular Biology and Human Genetics, Stellenbosch University , Tygerberg, Cape Town 7505, South Africa.
authors.forename: Elizabeth M
authors.initials: EM
authors.lastname: Streicher
authors.affiliation: SA MRC Centre for TB Research, DST/NRF Centre of Excellence for Biomedical Tuberculosis Research, Division of Molecular Biology and Human Genetics, Stellenbosch University , Tygerberg, Cape Town 7505, South Africa.
authors.forename: Frederick A
authors.initials: FA
authors.lastname: Sirgel
authors.affiliation: Department of Biological Sciences, University of Arkansas , Fayetteville, AR, 72701, USA.
authors.forename: Donald
authors.initials: D
authors.lastname: Catanzaro
authors.affiliation: P. D. Hinduja National Hospital & Medical Research Centre, Department of Microbiology , Veer Savarkar Marg, Mahim, Mumbai 400 016, India.
authors.forename: Camilla
authors.initials: C
authors.lastname: Rodrigues
authors.affiliation: Tropical Disease Foundation , Makati 1230, Philippines.
authors.forename: Maria Tarcela
authors.initials: MT
authors.lastname: Gler
authors.affiliation: Microbiology and Morphology Laboratory, Institute of Phthisiopneumology , Chisinau, MD 2001, Moldova.
authors.forename: Valeru
authors.initials: V
authors.lastname: Crudu
authors.affiliation: Department of Medicine, University of California , San Diego, La Jolla, CA 92093, USA.
authors.forename: Antonino
authors.initials: A
authors.lastname: Catanzaro
authors.affiliation: Biological & Medical Informatics Research Center, San Diego State University , San Diego, CA 92182, USA.
authors.forename: Faramarz
authors.initials: F
authors.lastname: Valafar
mesh_terms: D000995:Antitubercular Agents / Q000494:pharmacology*; D001426:Bacterial Proteins / Q000235:genetics*; D002374:Catalase / Q000235:genetics*; D024881:Drug Resistance, Bacterial / Q000235:genetics*; D006801:Humans; D007538:Isoniazid / Q000494:pharmacology*; D008826:Microbial Sensitivity Tests; D009154:Mutation; D009169:Mycobacterium tuberculosis / Q000187:drug effects* / Q000235:genetics* / Q000302:isolation & purification; D010088:Oxidoreductases / Q000235:genetics; D011401:Promoter Regions, Genetic / Q000235:genetics; D014376:Tuberculosis / Q000382:microbiology
chemical_list: D000995:Antitubercular Agents; D001426:Bacterial Proteins; D010088:Oxidoreductases; D002374:Catalase; C480353:katG protein, Mycobacterium tuberculosis; C085257:InhA protein, Mycobacterium; D007538:Isoniazid
keywords: MDR-TB; Pyrosequencing; Sanger sequencing; isoniazid drug-resistance; katG; novel mutation; tuberculosis; whole-genome sequencing