Ashlee Earl and colleagues analyze whole-genome sequences from 5,310 Mycobacterium tuberculosis isolates from five continents. They find that resistance to isoniazid arises before rifampicin resistance across all of the lineages, geographical regions and time periods. Multidrug-resistant tuberculosis (MDR-TB), caused by drug-resistant strains of Mycobacterium tuberculosis, is an increasingly serious problem worldwide. Here we examined a data set of whole-genome sequences from 5,310 M. tuberculosis isolates from five continents. Despite the great diversity of these isolates with respect to geographical point of isolation, genetic background and drug resistance, the patterns for the emergence of drug resistance were conserved globally. We have identified harbinger mutations that often precede multidrug resistance. In particular, the katG mutation encoding p.Ser315Thr, which confers resistance to isoniazid, overwhelmingly arose before mutations that conferred rifampicin resistance across all of the lineages, geographical regions and time periods. Therefore, molecular diagnostics that include markers for rifampicin resistance alone will be insufficient to identify pre-MDR strains. Incorporating knowledge of polymorphisms that occur before the emergence of multidrug resistance, particularly katG p.Ser315Thr, into molecular diagnostics should enable targeted treatment of patients with pre-MDR-TB to prevent further development of MDR-TB.

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. 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. 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. Tuberculosis (TB)—a contagious bacterial disease that usually infects the lungs—is a global public health problem. Every year, about 9 million people develop active TB disease, and 1.5 million people die from the disease. Mycobacterium tuberculosis, the organism that causes TB, is spread in airborne droplets when people with TB cough. The symptoms of TB include cough, weight loss, and fever. Diagnostic tests for the disease include sputum smear microscopy (microscopic analysis of mucus coughed up from the lungs) and chest X-rays. TB can be cured by taking a regimen of multiple antibiotics daily for 6 mo. However, the emergence of multidrug-resistant tuberculosis (MDR-TB, TB with resistance to both isoniazid and rifampicin) and extensively drug-resistant tuberculosis (XDR-TB, MDR-TB with additional resistance to both quinolones and second-line injectable agents), together with the spread of HIV (which increases susceptibility to TB), is now threatening TB control efforts. MDR-TB is caused by M. tuberculosis strains that have acquired mutations (genetic changes) that make them resistant to isoniazid, rifampicin, and sometimes other anti-TB drugs; XDR-TB is caused by bacteria that are resistant to isoniazid, rifampicin, one or more fluoroquinolones (for example, ofloxacin), and at least one injectable second-line drug (for example, kanamycin). A better understanding of the origins of drug-resistant TB is essential for effective control of TB. Public health experts need to know whether the emergence of drug-resistant TB is caused by inadequacies in TB control or related to other factors such as the spread of HIV and whether new resistant strains of M. tuberculosis repeatedly emerge during XDR-TB outbreaks or whether the transmission of a single drug-resistant strain drives these outbreaks. Here, the researchers use whole genome sequencing and dating analysis to investigate the origin and evolution of an XDR-TB outbreak identified in 2005 in Tugela Ferry, KwaZulu-Natal, South Africa. The predominant strain of XDR M. tuberculosis isolated during this large XDR-TB outbreak belongs to a subfamily called LAM4. Since the outbreak began, XDR-TB has also been reported in hospitals across KwaZulu-Natal, and some of these outbreaks have been caused by bacterial strains not falling within the LAM4 spoligotype (“spoligotyping” characterizes M. tuberculosis strains based on the presence of unique DNA sequences in a specific region of the bacterial genome). The researchers tested the antibiotic susceptibility of 337 clinical isolates of M. tuberculosis collected in KwaZulu-Natal between 2008 and 2013 and of three historical isolates—two collected in the province in the mid-1990s and a third from the Tugela Ferry XDR outbreak. They sequenced the whole genome of these isolates and used comparative techniques to assess the isolates’ relatedness and to investigate the acquisition of drug resistance. This analysis revealed a 50-member clone of XDR bacteria among the isolates collected across KwaZulu-Natal that was highly related to the LAM4 strain (a clone is defined here as a set of strains in which each member differs by no more than ten single nucleotide polymorphisms [SNPs] from at least one other member; an SNP is a type of genetic variant). Mutations that conferred isoniazid resistance in this clone were acquired in about 1957; MDR and XDR strains emerged in about 1984 and 1995, respectively. The analysis also indicates that MDR and XDR evolved de novo 56 times and nine times, respectively, and that isoniazid resistance nearly always evolved before rifampicin resistance. These findings provide new information about the ordering and timing of the acquisition of drug-resistance mutations by M. tuberculosis in KwaZulu-Natal but do not necessarily represent the evolution of XDR-TB in other settings. Most notably, these findings indicate that the ancestral precursor of the Tugela Ferry XDR outbreak strain gained resistance to first-line antibiotics shortly after these antibiotics became available for clinical use. Subsequent stepwise accumulation of additional resistance mutations that occurred over decades led to the emergence of MDR and XDR strains. Importantly, the emergence of these strains occurred before the explosion of HIV in KwaZulu-Natal. Thus, these findings highlight the dire repercussions of the failure of historic attempts to control resistance to first-line anti-TB drugs and draw attention to the need for new anti-TB drugs to be used prudently to prevent early fixation of resistance and to protect the useful lifespan of these agents. Finally, the finding that isoniazid resistance is a key initiation event for progression to MDR and XDR suggests that TB control programs should test routinely for both isoniazid and rifampicin resistance to ensure early detection of drug-resistant TB. This list of resources contains links that can be accessed when viewing the PDF on a device or via the online version of the article at http://dx.doi.org/10.1371/journal.pmed.1001880.

Drug efflux is an important resistance mechanism in Mycobacterium tuberculosis . We found that verapamil, an efflux inhibitor, profoundly decreases the MIC of bedaquiline and clofazimine to M. tuberculosis by 8- to 16-fold. This exquisite susceptibility was noted among drug-susceptible and drug-resistant clinical isolates. Thus, efflux inhibition is an important sensitizer of bedaquiline and clofazimine, and efflux may emerge as a resistance mechanism to these drugs.

Alexander Pym, Ashlee Earl and colleagues use the whole-genome sequences from 498 strains of Mycobacterium tuberculosis to identify new genotypes conferring resistance to antitubercular drugs. They find that loss-of-function mutations in ald (Rv2780), encoding L-alanine dehydrogenase, are associated with unexplained drug resistance and demonstrate that these mutations confer resistance to D-cycloserine. A more complete understanding of the genetic basis of drug resistance in Mycobacterium tuberculosis is critical for prompt diagnosis and optimal treatment, particularly for toxic second-line drugs such as D-cycloserine. Here we used the whole-genome sequences from 498 strains of M. tuberculosis to identify new resistance-conferring genotypes. By combining association and correlated evolution tests with strategies for amplifying signal from rare variants, we found that loss-of-function mutations in ald (Rv2780), encoding L-alanine dehydrogenase, were associated with unexplained drug resistance. Convergent evolution of this loss of function was observed exclusively among multidrug-resistant strains. Drug susceptibility testing established that ald loss of function conferred resistance to D-cycloserine, and susceptibility to the drug was partially restored by complementation of ald. Clinical strains with mutations in ald and alr exhibited increased resistance to D-cycloserine when cultured in vitro. Incorporation of D-cycloserine resistance in novel molecular diagnostics could allow for targeted use of this toxic drug among patients with susceptible infections.

Tuberculosis (TB) is a global infectious threat that is intensified by an increasing incidence of highly drug-resistant disease. Whole-genome sequencing (WGS) studies of Mycobacterium tuberculosis, the causative agent of TB, have greatly increased our understanding of this pathogen. Since the first M. tuberculosis genome was published in 1998, WGS has provided a more complete account of the genomic features that cause resistance in populations of M. tuberculosis, has helped to fill gaps in our knowledge of how both classical and new antitubercular drugs work, and has identified specific mutations that allow M. tuberculosis to escape the effects of these drugs. WGS studies have also revealed how resistance evolves both within an individual patient and within patient populations, including the important roles of de novo acquisition of resistance and clonal spread. These findings have informed decisions about which drug-resistance mutations should be included on extended diagnostic panels. From its origins as a basic science technique, WGS of M. tuberculosis is becoming part of the modern clinical microbiology laboratory, promising rapid and improved detection of drug resistance, and detailed and real-time epidemiology of TB outbreaks. We review the successes and highlight the challenges that remain in applying WGS to improve the control of drug-resistant TB through monitoring its evolution and spread, and to inform more rapid and effective diagnostic and therapeutic strategies.