title: Whole genome sequencing based characterization of extensively drug-resistant Mycobacterium tuberculosis isolates from Pakistan.
abstract: Improved molecular diagnostic methods for detection drug resistance in Mycobacterium tuberculosis (MTB) strains are required. Resistance to first- and second- line anti-tuberculous drugs has been associated with single nucleotide polymorphisms (SNPs) in particular genes. However, these SNPs can vary between MTB lineages therefore local data is required to describe different strain populations. We used whole genome sequencing (WGS) to characterize 37 extensively drug-resistant (XDR) MTB isolates from Pakistan and investigated 40 genes associated with drug resistance. Rifampicin resistance was attributable to SNPs in the rpoB hot-spot region. Isoniazid resistance was most commonly associated with the katG codon 315 (92%) mutation followed by inhA S94A (8%) however, one strain did not have SNPs in katG, inhA or oxyR-ahpC. All strains were pyrazimamide resistant but only 43% had pncA SNPs. Ethambutol resistant strains predominantly had embB codon 306 (62%) mutations, but additional SNPs at embB codons 406, 378 and 328 were also present. Fluoroquinolone resistance was associated with gyrA 91-94 codons in 81% of strains; four strains had only gyrB mutations, while others did not have SNPs in either gyrA or gyrB. Streptomycin resistant strains had mutations in ribosomal RNA genes; rpsL codon 43 (42%); rrs 500 region (16%), and gidB (34%) while six strains did not have mutations in any of these genes. Amikacin/kanamycin/capreomycin resistance was associated with SNPs in rrs at nt1401 (78%) and nt1484 (3%), except in seven (19%) strains. We estimate that if only the common hot-spot region targets of current commercial assays were used, the concordance between phenotypic and genotypic testing for these XDR strains would vary between rifampicin (100%), isoniazid (92%), flouroquinolones (81%), aminoglycoside (78%) and ethambutol (62%); while pncA sequencing would provide genotypic resistance in less than half the isolates. This work highlights the importance of expanded targets for drug resistance detection in MTB isolates.
authors.affiliation: Department of Pathology and Microbiology, Aga Khan University Hospital, Karachi, Pakistan; School of Nursing and Midwifery, The Aga Khan University Hospital, Karachi, Pakistan.
authors.forename: Asho
authors.initials: A
authors.lastname: Ali
authors.affiliation: Department of Pathology and Microbiology, Aga Khan University Hospital, Karachi, Pakistan.
authors.forename: Zahra
authors.initials: Z
authors.lastname: Hasan
authors.affiliation: Faculty of Infectious and Tropical Diseases, London School of Hygiene and Tropical Medicine, London, United Kingdom.
authors.forename: Ruth
authors.initials: R
authors.lastname: McNerney
authors.affiliation: Faculty of Infectious and Tropical Diseases, London School of Hygiene and Tropical Medicine, London, United Kingdom.
authors.forename: Kim
authors.initials: K
authors.lastname: Mallard
authors.affiliation: Pathogen Genomics Laboratory, King Abdullah University of Science and Technology (KAUST), Thuwal, Kingdom of Saudi Arabia; Sydney Emerging Infections and Biosecurity Institute and School of Public Health, Sydney Medical School, University of Sydney, Sydney, Australia.
authors.forename: Grant
authors.initials: G
authors.lastname: Hill-Cawthorne
authors.affiliation: Faculty of Infectious and Tropical Diseases, London School of Hygiene and Tropical Medicine, London, United Kingdom.
authors.forename: Francesc
authors.initials: F
authors.lastname: Coll
authors.affiliation: Pathogen Genomics Laboratory, King Abdullah University of Science and Technology (KAUST), Thuwal, Kingdom of Saudi Arabia.
authors.forename: Mridul
authors.initials: M
authors.lastname: Nair
authors.affiliation: Pathogen Genomics Laboratory, King Abdullah University of Science and Technology (KAUST), Thuwal, Kingdom of Saudi Arabia.
authors.forename: Arnab
authors.initials: A
authors.lastname: Pain
authors.affiliation: Faculty of Infectious and Tropical Diseases, London School of Hygiene and Tropical Medicine, London, United Kingdom.
authors.forename: Taane G
authors.initials: TG
authors.lastname: Clark
authors.affiliation: Department of Pathology and Microbiology, Aga Khan University Hospital, Karachi, Pakistan.
authors.forename: Rumina
authors.initials: R
authors.lastname: Hasan
mesh_terms: D000995:Antitubercular Agents / Q000494:pharmacology; D001426:Bacterial Proteins / Q000235:genetics; D002374:Catalase / Q000235:genetics; D024881:Drug Resistance, Bacterial / Q000235:genetics*; D054908:Extensively Drug-Resistant Tuberculosis / Q000382:microbiology*; D016680:Genome, Bacterial; D009169:Mycobacterium tuberculosis / Q000187:drug effects / Q000235:genetics* / Q000302:isolation & purification; D010088:Oxidoreductases / Q000235:genetics; D010430:Pentosyltransferases / Q000235:genetics; D020641:Polymorphism, Single Nucleotide
chemical_list: D000995:Antitubercular Agents; D001426:Bacterial Proteins; D010088:Oxidoreductases; D002374:Catalase; C480353:katG protein, Mycobacterium tuberculosis; C085257:InhA protein, Mycobacterium; C540269:EmbB protein, Mycobacterium tuberculosis; D010430:Pentosyltransferases