Whole genome sequencing of drug resistant Mycobacterium tuberculosis isolates from a high burden tuberculosis region of North West Pakistan

• Published in: Scientific reports 2019-10, Vol.9 (1), p.14996-9, Article 14996
• Main Authors: Jabbar, Abdul, Phelan, Jody E., de Sessions, Paola Florez, Khan, Taj Ali, Rahman, Hazir, Khan, Sadiq Noor, Cantillon, Daire M., Wildner, Leticia Muraro, Ali, Sajid, Campino, Susana, Waddell, Simon J., Clark, Taane G.
• Format: Article
• Language: English
• Subjects: 45; 45/23; 631/208/212/2304; 631/208/325; Adolescent; Adult; Antitubercular Agents - therapeutic use; Base Sequence; Decision making; Disease control; Disease transmission; DNA, Bacterial - genetics; Drug resistance; Drug Resistance, Multiple, Bacterial - genetics; Epidemiology; Ethambutol; Ethionamide; Female; Fluoroquinolones; Genomes; Humanities and Social Sciences; Humans; Isoniazid; Male; Microbial Sensitivity Tests; Middle Aged; multidisciplinary; Multidrug resistance; Mutation; Mycobacterium tuberculosis; Mycobacterium tuberculosis - genetics; Mycobacterium tuberculosis - isolation & purification; Pakistan - epidemiology; Phylogeny; Polymorphism, Single Nucleotide; Pyrazinamide; Rifampin; RpoB protein; Science; Science (multidisciplinary); Strains (organisms); Streptomycin; Tuberculosis; Tuberculosis, Multidrug-Resistant - drug therapy; Tuberculosis, Multidrug-Resistant - epidemiology; Tuberculosis, Multidrug-Resistant - microbiology; Tuberculosis, Multidrug-Resistant - transmission; Whole Genome Sequencing - methods; Young Adult
• ISSN: 2045-2322; 2045-2322
• DOI: 10.1038/s41598-019-51562-6

Tuberculosis (TB), caused by Mycobacterium tuberculosis bacteria, is a leading infectious cause of mortality worldwide, including in Pakistan. Drug resistant M. tuberculosis is an emerging threat for TB control, making it important to detect the underlying genetic mutations, and thereby inform treatment decision making and prevent transmission. Whole genome sequencing has emerged as the new diagnostic to reliably predict drug resistance within a clinically relevant time frame, and its deployment will have the greatest impact on TB control in highly endemic regions. To evaluate the mutations leading to drug resistance and to assess for evidence of the transmission of resistant strains, 81  M. tuberculosis samples from Khyber Pakhtunkhwa province (North West Pakistan) were subjected to whole genome sequencing and standard drug susceptibility testing for eleven anti-TB drugs. We found the majority of M. tuberculosis isolates were the CAS/Delhi strain-type (lineage 3; n = 57; 70.4%) and multi-drug resistant (MDR; n = 62; 76.5%). The most frequent resistance mutations were observed in the katG and rpoB genes, conferring resistance to isoniazid and rifampicin respectively. Mutations were also observed in genes conferring resistance to other first and second-line drugs, including in pncA (pyrazinamide), embB (ethambutol), gyrA (fluoroquinolones), rrs (aminoglycosides), rpsL, rrs and giB (streptomycin) loci. Whilst the majority of mutations have been reported in global datasets, we describe unreported putative resistance markers in katG , ethA (ethionamide), gyrA and gyrB (fluoroquinolones), and pncA . Analysis of the mutations revealed that acquisition of rifampicin resistance often preceded isoniazid in our isolates. We also observed a high proportion (17.6%) of pre-MDR isolates with fluoroquinolone resistance markers, potentially due to unregulated anti-TB drug use. Our isolates were compared to previously sequenced strains from Pakistan in a combined phylogenetic tree analysis. The presence of lineage 2 was only observed in our isolates. Using a cut-off of less than ten genome-wide mutation differences between isolates, a transmission analysis revealed 18  M. tuberculosis isolates clustering within eight networks, thereby providing evidence of drug-resistant TB transmission in the Khyber Pakhtunkhwa province. Overall, we have demonstrated that drug-resistant TB isolates are circulating and transmitted in North West Pakistan. Further, we have shown the usefulne