PLOS Medicine Evolution of extensively drug-resistant tuberculosis over four decades revealed by whole genome sequencing of Mycobacterium tuberculosis from KwaZulu-Natal, South Africa

Background: The largest global outbreak of extensively drug-resistant (XDR) tuberculosis (TB) was identified in Tugela Ferry, KwaZulu-Natal, South Africa in 2005. Understanding the antecedents and timing of emergence of drug resistance in this fatal outbreak of XDR will inform the control and prevention of drug-resistant TB. Methods and Findings: We performed whole genome sequencing and drug susceptibility testing on 337 clinical isolates of Mycobacterium tuberculosis (M. tb) collected in KwaZulu-Natal from 2008 to 2013, in addition to historical isolates, including one from the Tugela Ferry outbreak. We utilized an array of whole genome comparative techniques to assess the relatedness among strains, to establish the order of acquisition of drug resistance mutations including the timing of acquisitions leading to XDR-TB in the LAM4 spoligotype, and to calculate the number of independent evolutionary emergences of MDR and XDR. Our sequencing and analysis revealed a 50-member clone of XDR M. tb that was highly related to the Tugela Ferry XDR outbreak strain. We estimated that mutations conferring isoniazid and streptomycin resistance in this clone were acquired 50 years prior to the Tugela Ferry outbreak, with the subsequent emergence of multidrug resistance (MDR) and XDR occurring 30 and 40 years later, respectively. We observed frequent de novo evolution of MDR and XDR, with 56 and 9 independent evolutionary events, respectively. Isoniazid resistance evolved before rifampicin resistance 46 times, whereas rifampicin resistance evolved prior to isoniazid only twice. We identified additional putative compensatory mutations to rifampicin in this dataset. Conclusions: In the first whole genome-based analysis of the emergence of drug resistance among clinical isolates of M. tb, we show that the ancestral precursor of the LAM4 XDR outbreak strain in Tugela Ferry gained mutations to first line drugs at the beginning of the antibiotic era. Subsequent accumulation of step-wise resistance mutations, occurring over decades and prior to the explosion of HIV in this region, yielded MDR and XDR, permitting the emergence of compensatory mutations. Our results suggest that drug-resistant strains circulating today reflect not only vulnerabilities of current TB control efforts, but also those that date back fifty years. In drug-resistant TB, isoniazid resistance was overwhelmingly the initial resistance mutation to be acquired, which would not be detected by current rapid molecular diagnostics employed in South Africa that assess only rifampicin resistance. Suggested Reviewers: Stewart Cole Ecole Polytechnique Federale de Lausanne stewart.cole@epfl.ch A microbiologist and current director of the Global Health Institute at EPFL, Dr. Cole led the research group that first sequenced the genome of Mycobacterium tuberculosis Powered by Editorial Manager® and ProduXion Manager® from Aries Systems Corporation in its entirety. His familiarity with both microbiology and genomics as it relates to mycobacteria will be helpful in a providing a critical review of this manuscript. Valerie Mizrahi Institute of Infectious Diseases and Molecular Medicine valerie.mizrahi@uct.ac. za A premier South African molecular microbiologist and Director of the Institute of Infectious Disease and Molecular Medicine (IDM) in Cape Town, Dr. Mizrahi has extensive knowledge of tuberculosis, molecular epidemiology and the South African context. Sven Hoffner Karolinska Institutet sven.hoffner@ki.se A pre-eminent bacteriologist, who directs the WHO Supranational Reference Laboratory for drug-resistant TB has invaluable expertise in this field, and would likely serve as a savvy reviewer. Opposed Reviewers: Megan Murray Harvard School of Public Health