title: Evolution of Extensively Drug-Resistant Tuberculosis over Four Decades: Whole Genome Sequencing and Dating Analysis of Mycobacterium tuberculosis Isolates from KwaZulu-Natal.
abstract: BACKGROUND
  The continued advance of antibiotic resistance threatens the treatment and control of many infectious diseases. This is exemplified by the largest global outbreak of extensively drug-resistant (XDR) tuberculosis (TB) identified in Tugela Ferry, KwaZulu-Natal, South Africa, in 2005 that continues today. It is unclear whether the emergence of XDR-TB in KwaZulu-Natal was due to recent inadequacies in TB control in conjunction with HIV or other factors. Understanding the origins of drug resistance in this fatal outbreak of XDR will inform the control and prevention of drug-resistant TB in other settings. In this study, we used whole genome sequencing and dating analysis to determine if XDR-TB had emerged recently or had ancient antecedents.
  
  
  RESULTS
  We performed whole genome sequencing and drug susceptibility testing on 337 clinical isolates of Mycobacterium tuberculosis collected in KwaZulu-Natal from 2008 to 2013, in addition to three historical isolates, collected from patients in the same province and including an isolate from the 2005 Tugela Ferry XDR outbreak, a multidrug-resistant (MDR) isolate from 1994, and a pansusceptible isolate from 1995. 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. tuberculosis 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 y prior to the Tugela Ferry outbreak (katG S315T [isoniazid]; gidB 130 bp deletion [streptomycin]; 1957 [95% highest posterior density (HPD): 1937-1971]), with the subsequent emergence of MDR and XDR occurring 20 y (rpoB L452P [rifampicin]; pncA 1 bp insertion [pyrazinamide]; 1984 [95% HPD: 1974-1992]) and 10 y (rpoB D435G [rifampicin]; rrs 1400 [kanamycin]; gyrA A90V [ofloxacin]; 1995 [95% HPD: 1988-1999]) prior to the outbreak, respectively. We observed frequent de novo evolution of MDR and XDR, with 56 and nine 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. One major limitation of this study is that the conclusions with respect to ordering and timing of acquisition of mutations may not represent universal patterns of drug resistance emergence in other areas of the globe.
  
  
  CONCLUSIONS
  In the first whole genome-based analysis of the emergence of drug resistance among clinical isolates of M. tuberculosis, 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 stepwise 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 50 y. 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.
authors.affiliation: Division of Pulmonary and Critical Care Medicine, Brigham and Women's Hospital, Harvard Medical School, Boston, Massachusetts, United States of America; KwaZulu-Natal Research Institute for TB and HIV (K-RITH), Durban, South Africa.
authors.forename: Keira A
authors.initials: KA
authors.lastname: Cohen
authors.affiliation: Broad Institute of MIT and Harvard, Cambridge, Massachusetts, United States of America; Delft Bioinformatics Lab, Delft University of Technology, Delft, The Netherlands.
authors.forename: Thomas
authors.initials: T
authors.lastname: Abeel
authors.affiliation: Broad Institute of MIT and Harvard, Cambridge, Massachusetts, United States of America.
authors.forename: Abigail
authors.initials: A
authors.lastname: Manson McGuire
authors.affiliation: Broad Institute of MIT and Harvard, Cambridge, Massachusetts, United States of America.
authors.forename: Christopher A
authors.initials: CA
authors.lastname: Desjardins
authors.affiliation: KwaZulu-Natal Research Institute for TB and HIV (K-RITH), Durban, South Africa.
authors.forename: Vanisha
authors.initials: V
authors.lastname: Munsamy
authors.affiliation: Broad Institute of MIT and Harvard, Cambridge, Massachusetts, United States of America.
authors.forename: Terrance P
authors.initials: TP
authors.lastname: Shea
authors.affiliation: Broad Institute of MIT and Harvard, Cambridge, Massachusetts, United States of America.
authors.forename: Bruce J
authors.initials: BJ
authors.lastname: Walker
authors.affiliation: Medical Research Council, Durban, South Africa.
authors.forename: Nonkqubela
authors.initials: N
authors.lastname: Bantubani
authors.affiliation: KwaZulu-Natal Research Institute for TB and HIV (K-RITH), Durban, South Africa; Center for Tuberculosis Research, Johns Hopkins School of Medicine, Baltimore, Maryland, United States of America.
authors.forename: Deepak V
authors.initials: DV
authors.lastname: Almeida
authors.affiliation: Broad Institute of MIT and Harvard, Cambridge, Massachusetts, United States of America.
authors.forename: Lucia
authors.initials: L
authors.lastname: Alvarado
authors.affiliation: Broad Institute of MIT and Harvard, Cambridge, Massachusetts, United States of America.
authors.forename: Sinéad B
authors.initials: SB
authors.lastname: Chapman
authors.affiliation: School of Laboratory Medicine and Medical Sciences, University of KwaZulu-Natal, Durban, South Africa; National Health Laboratory Service, Durban, South Africa.
authors.forename: Nomonde R
authors.initials: NR
authors.lastname: Mvelase
authors.affiliation: KwaZulu-Natal Research Institute for TB and HIV (K-RITH), Durban, South Africa.
authors.forename: Eamon Y
authors.initials: EY
authors.lastname: Duffy
authors.affiliation: Broad Institute of MIT and Harvard, Cambridge, Massachusetts, United States of America.
authors.forename: Michael G
authors.initials: MG
authors.lastname: Fitzgerald
authors.affiliation: KwaZulu-Natal Research Institute for TB and HIV (K-RITH), Durban, South Africa.
authors.forename: Pamla
authors.initials: P
authors.lastname: Govender
authors.affiliation: Broad Institute of MIT and Harvard, Cambridge, Massachusetts, United States of America.
authors.forename: Sharvari
authors.initials: S
authors.lastname: Gujja
authors.affiliation: Broad Institute of MIT and Harvard, Cambridge, Massachusetts, United States of America.
authors.forename: Susanna
authors.initials: S
authors.lastname: Hamilton
authors.affiliation: Broad Institute of MIT and Harvard, Cambridge, Massachusetts, United States of America.
authors.forename: Clinton
authors.initials: C
authors.lastname: Howarth
authors.affiliation: Broad Institute of MIT and Harvard, Cambridge, Massachusetts, United States of America.
authors.forename: Jeffrey D
authors.initials: JD
authors.lastname: Larimer
authors.affiliation: KwaZulu-Natal Research Institute for TB and HIV (K-RITH), Durban, South Africa.
authors.forename: Kashmeel
authors.initials: K
authors.lastname: Maharaj
authors.affiliation: Broad Institute of MIT and Harvard, Cambridge, Massachusetts, United States of America.
authors.forename: Matthew D
authors.initials: MD
authors.lastname: Pearson
authors.affiliation: Broad Institute of MIT and Harvard, Cambridge, Massachusetts, United States of America.
authors.forename: Margaret E
authors.initials: ME
authors.lastname: Priest
authors.affiliation: Broad Institute of MIT and Harvard, Cambridge, Massachusetts, United States of America.
authors.forename: Qiandong
authors.initials: Q
authors.lastname: Zeng
authors.affiliation: Centre for the AIDS Programme of Research in South Africa (CAPRISA), Durban, South Africa.
authors.forename: Nesri
authors.initials: N
authors.lastname: Padayatchi
authors.affiliation: KwaZulu-Natal Research Institute for TB and HIV (K-RITH), Durban, South Africa; Center for Tuberculosis Research, Johns Hopkins School of Medicine, Baltimore, Maryland, United States of America.
authors.forename: Jacques
authors.initials: J
authors.lastname: Grosset
authors.affiliation: Broad Institute of MIT and Harvard, Cambridge, Massachusetts, United States of America.
authors.forename: Sarah K
authors.initials: SK
authors.lastname: Young
authors.affiliation: Broad Institute of MIT and Harvard, Cambridge, Massachusetts, United States of America.
authors.forename: Jennifer
authors.initials: J
authors.lastname: Wortman
authors.affiliation: School of Laboratory Medicine and Medical Sciences, University of KwaZulu-Natal, Durban, South Africa; National Health Laboratory Service, Durban, South Africa.
authors.forename: Koleka P
authors.initials: KP
authors.lastname: Mlisana
authors.affiliation: Centre for the AIDS Programme of Research in South Africa (CAPRISA), Durban, South Africa; Division of Pulmonary, Allergy, and Critical Care Medicine, Columbia University College of Physicians and Surgeons, New York, United States of America; Department of Epidemiology, Columbia Mailman School of Public Health, New York, United States of America.
authors.forename: Max R
authors.initials: MR
authors.lastname: O'Donnell
authors.affiliation: Broad Institute of MIT and Harvard, Cambridge, Massachusetts, United States of America.
authors.forename: Bruce W
authors.initials: BW
authors.lastname: Birren
authors.affiliation: Center for Tuberculosis Research, Johns Hopkins School of Medicine, Baltimore, Maryland, United States of America.
authors.forename: William R
authors.initials: WR
authors.lastname: Bishai
authors.affiliation: KwaZulu-Natal Research Institute for TB and HIV (K-RITH), Durban, South Africa.
authors.forename: Alexander S
authors.initials: AS
authors.lastname: Pym
authors.affiliation: Broad Institute of MIT and Harvard, Cambridge, Massachusetts, United States of America.
authors.forename: Ashlee M
authors.initials: AM
authors.lastname: Earl
mesh_terms: D000328:Adult; D000995:Antitubercular Agents / Q000494:pharmacology*; D004196:Disease Outbreaks; D054908:Extensively Drug-Resistant Tuberculosis / Q000188:drug therapy / Q000453:epidemiology / Q000235:genetics*; D005260:Female; D016680:Genome, Bacterial; D006801:Humans; D008297:Male; D008826:Microbial Sensitivity Tests; D009154:Mutation; D009169:Mycobacterium tuberculosis / Q000187:drug effects / Q000235:genetics* / Q000302:isolation & purification; D017422:Sequence Analysis, DNA; D013019:South Africa / Q000453:epidemiology
chemical_list: D000995:Antitubercular Agents