Evolution of Extensively Drug-Resistant Tuberculosis over Four Decades: Whole Genome Sequencing and Dating Analysis of Mycobacterium tuberculosis Isolates from KwaZulu-Natal

Abstract

BackgroundThe 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.Methods and FindingsWe 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.ConclusionsIn 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 e...

Insights into the processes that drive the evolution of drug resistance in Mycobacterium tuberculosis

“…Indeed, some of these resistance mutations do not reduce bacterial fitness in the absence of treatment (Bergval, Schuitema, Klatser, & Anthony, 2009; Huitric et al., 2006; Mariam et al., 2004). It is worth noting that MDR and XDR strains associated with outbreaks often carried these mutations explaining the successful spread of these highly drug‐resistant strains in the community (Casali et al., 2014; Cohen et al., 2015; Niehaus, Mlisana, Gandhi, Mathema, & Brust, 2015; de Vos et al., 2013). All these observations suggest that the prevalent mutations in clinical isolates have been positively selected because of their low biological cost (Figure 1a) (Farhat et al., 2013; Osorio et al., 2013).…”

“…This hypothesis is supported by the lack of pnc A mutant clusters, thus reflecting a low transmission potential. However, clusters of pnc A mutants have been described in some specific MDR and XDR M. tuberculosis outbreaks in South Africa and in Argentina showing the successful transmission of these PZA‐resistant clones (Cohen et al., 2015; Eldholm et al., 2015; Müller, Chihota, et al., 2013). The development of experimental evolution studies will allow assessing the biological cost magnitude of pnc A mutations.…”

“…This could be the result of compensatory mechanisms to alleviate the fitness cost exerted by specific mutations (Brandis & Hughes, 2013; Brandis et al., 2012). It is worth noting that compensatory mutations are more commonly identified in the dominant MDR, pre‐XDR and XDR clones in high MDR TB burden countries, suggesting that high drug‐resistant mutants harbouring these mutations can be successfully transmitted in human populations (Casali et al., 2014; Cohen et al., 2015; Comas et al., 2012; Klopper et al., 2013; Li et al., 2016; de Vos et al., 2013). …”

“…Nevertheless, for almost all drug resistance‐associated genes, the predominance of some specific mutations, generally known to be associated with high level of resistance and low biological cost, has been described (see Table 2). As a result, combinations of at least two specific mutations, such as rpo B531, kat G315, rps L43, emb B306 and gyr A94, are favoured (Casali et al., 2014; Cohen et al., 2015; Nguyen, Nguyen, et al., 2017). Although the quality of the treatment undoubtedly plays a role in the emergence of particular drug resistance mutations, the strains with drug resistance‐associated mutations seem to have higher propensity to accumulate other drug resistance mutations in the same gene or in different genes (Bahrmand et al., 2009; Jagielski et al., 2014; Nguyen, Nguyen, et al., 2017; Shen et al., 2007).…”

“…Concerning the evolution of MDR strains, clinical and molecular studies suggest that isoniazid resistance, due to the kat G S315T mutation, has preceded the emergence of rpo B gene mutations leading to the acquisition of rifampicin resistance (Cohen et al., 2015; Gegia, Winters, Benedetti, van Soolingen, & Menzies, 2017; Manson et al., 2017; Salvatore et al., 2016). The combination of kat G315 and rpo B531 mutations with a rifampicin‐resistant fitness‐compensatory mutation (e.g., rpo C mutation) is favoured in clinical MDR isolates, suggesting that these genotypes lead to primary MDR infections.…”