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

Cohen KA, Abeel T, Manson McGuire A, Desjardins CA, Munsamy V, Shea TP, Walker BJ, Bantubani N, Almeida DV, Alvarado L, Chapman SB, Mvelase NR, Duffy EY, Fitzgerald MG, Govender P, Gujja S, Hamilton S, Howarth C, Larimer JD, Maharaj K, Pearson MD, Priest ME, Zeng Q, Padayatchi N, Grosset J, Young SK, Wortman J, Mlisana KP, O'Donnell MR, Birren BW, Bishai WR, Pym AS, Earl AM - PLoS Med. (2015)

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

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.

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.

Methods and findings: 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.

No MeSH data available.


Related in: MedlinePlus

Molecular evolution and dating of drug resistance emergence within the Tugela Ferry XDR Clone.Midpoint rooted maximum-likelihood phylogeny of 107 M. tuberculosis isolates of the LAM4 spoligotype. The gray shaded box identifies the Tugela Ferry XDR Clone. KZN605, the historical XDR strain collected in Tugela Ferry during the outbreak, is a member of this clone. Two additional historical isolates, KZN1435 and KZN4207, are not members of the Tugela Ferry XDR Clone. Each evolutionary gain of a drug resistance mutation was assigned to its position on the phylogenetic tree by parsimony (colored circles). A–E traces the stepwise order of drug resistance acquisition in the Tugela Ferry XDR Clone and estimates the year when each mutation was gained. Gray bars indicate the 95% highest posterior density (HPD) intervals. (A) katG S315T (isoniazid); gidB 130 bp deletion (streptomycin); 1957 (95% HPD: 1937–1971); (B) inhA promoter -8 (isoniazid and ethionamide); 1964 (95% HPD: 1948–1976); (C) embB M306V (ethambutol); 1967 (95% HPD: 1950–1978); (D) rpoB L452P (rifampicin); pncA 1bp insertion (pyrazinamide); 1984 (95% HPD: 1974–1992); and (E) rpoB D435G (rifampicin); rrs 1400 (kanamycin); gyrA A90V (ofloxacin); 1995 (95% HPD: 1988–1999). The accumulation of individual drug-resistant mutations within a strain is denoted to the right of the phylogenetic tree. The dates of drug discovery are displayed at the bottom of the figure [53]. Four additional LAM4 strains on a distant branch were not included in this figure because of size constraints. Bootstrap values are provided for lettered nodes, and bootstrap values for all nodes are shown in S5 Fig.
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pmed.1001880.g003: Molecular evolution and dating of drug resistance emergence within the Tugela Ferry XDR Clone.Midpoint rooted maximum-likelihood phylogeny of 107 M. tuberculosis isolates of the LAM4 spoligotype. The gray shaded box identifies the Tugela Ferry XDR Clone. KZN605, the historical XDR strain collected in Tugela Ferry during the outbreak, is a member of this clone. Two additional historical isolates, KZN1435 and KZN4207, are not members of the Tugela Ferry XDR Clone. Each evolutionary gain of a drug resistance mutation was assigned to its position on the phylogenetic tree by parsimony (colored circles). A–E traces the stepwise order of drug resistance acquisition in the Tugela Ferry XDR Clone and estimates the year when each mutation was gained. Gray bars indicate the 95% highest posterior density (HPD) intervals. (A) katG S315T (isoniazid); gidB 130 bp deletion (streptomycin); 1957 (95% HPD: 1937–1971); (B) inhA promoter -8 (isoniazid and ethionamide); 1964 (95% HPD: 1948–1976); (C) embB M306V (ethambutol); 1967 (95% HPD: 1950–1978); (D) rpoB L452P (rifampicin); pncA 1bp insertion (pyrazinamide); 1984 (95% HPD: 1974–1992); and (E) rpoB D435G (rifampicin); rrs 1400 (kanamycin); gyrA A90V (ofloxacin); 1995 (95% HPD: 1988–1999). The accumulation of individual drug-resistant mutations within a strain is denoted to the right of the phylogenetic tree. The dates of drug discovery are displayed at the bottom of the figure [53]. Four additional LAM4 strains on a distant branch were not included in this figure because of size constraints. Bootstrap values are provided for lettered nodes, and bootstrap values for all nodes are shown in S5 Fig.

Mentions: The “historical” Tugela Ferry XDR strain, KZN605, was nested phylogenetically within a large clone of 50 LAM4 strains with predominantly phenotypic XDR (Fig 3). All of the strains within this clone (henceforth referred to as the Tugela Ferry XDR Clone) possessed the characteristic drug resistance mutations that were previously identified in XDR-TB strains circulating in Tugela Ferry during the outbreak [9,24], further indicating this clone’s continued prevalence within KwaZulu-Natal. Patients in whom the Tugela Ferry XDR Clone was isolated were from ten of the 11 districts within the province (Fig 2, panel B). In addition, the Tugela Ferry XDR Clone was not overrepresented among HIV-positive patients (p = 0.6750) (Table 2). This suggests that strains within this clone were neither geographically constrained nor restricted to immunodeficient hosts.


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

Cohen KA, Abeel T, Manson McGuire A, Desjardins CA, Munsamy V, Shea TP, Walker BJ, Bantubani N, Almeida DV, Alvarado L, Chapman SB, Mvelase NR, Duffy EY, Fitzgerald MG, Govender P, Gujja S, Hamilton S, Howarth C, Larimer JD, Maharaj K, Pearson MD, Priest ME, Zeng Q, Padayatchi N, Grosset J, Young SK, Wortman J, Mlisana KP, O'Donnell MR, Birren BW, Bishai WR, Pym AS, Earl AM - PLoS Med. (2015)

Molecular evolution and dating of drug resistance emergence within the Tugela Ferry XDR Clone.Midpoint rooted maximum-likelihood phylogeny of 107 M. tuberculosis isolates of the LAM4 spoligotype. The gray shaded box identifies the Tugela Ferry XDR Clone. KZN605, the historical XDR strain collected in Tugela Ferry during the outbreak, is a member of this clone. Two additional historical isolates, KZN1435 and KZN4207, are not members of the Tugela Ferry XDR Clone. Each evolutionary gain of a drug resistance mutation was assigned to its position on the phylogenetic tree by parsimony (colored circles). A–E traces the stepwise order of drug resistance acquisition in the Tugela Ferry XDR Clone and estimates the year when each mutation was gained. Gray bars indicate the 95% highest posterior density (HPD) intervals. (A) katG S315T (isoniazid); gidB 130 bp deletion (streptomycin); 1957 (95% HPD: 1937–1971); (B) inhA promoter -8 (isoniazid and ethionamide); 1964 (95% HPD: 1948–1976); (C) embB M306V (ethambutol); 1967 (95% HPD: 1950–1978); (D) rpoB L452P (rifampicin); pncA 1bp insertion (pyrazinamide); 1984 (95% HPD: 1974–1992); and (E) rpoB D435G (rifampicin); rrs 1400 (kanamycin); gyrA A90V (ofloxacin); 1995 (95% HPD: 1988–1999). The accumulation of individual drug-resistant mutations within a strain is denoted to the right of the phylogenetic tree. The dates of drug discovery are displayed at the bottom of the figure [53]. Four additional LAM4 strains on a distant branch were not included in this figure because of size constraints. Bootstrap values are provided for lettered nodes, and bootstrap values for all nodes are shown in S5 Fig.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4587932&req=5

pmed.1001880.g003: Molecular evolution and dating of drug resistance emergence within the Tugela Ferry XDR Clone.Midpoint rooted maximum-likelihood phylogeny of 107 M. tuberculosis isolates of the LAM4 spoligotype. The gray shaded box identifies the Tugela Ferry XDR Clone. KZN605, the historical XDR strain collected in Tugela Ferry during the outbreak, is a member of this clone. Two additional historical isolates, KZN1435 and KZN4207, are not members of the Tugela Ferry XDR Clone. Each evolutionary gain of a drug resistance mutation was assigned to its position on the phylogenetic tree by parsimony (colored circles). A–E traces the stepwise order of drug resistance acquisition in the Tugela Ferry XDR Clone and estimates the year when each mutation was gained. Gray bars indicate the 95% highest posterior density (HPD) intervals. (A) katG S315T (isoniazid); gidB 130 bp deletion (streptomycin); 1957 (95% HPD: 1937–1971); (B) inhA promoter -8 (isoniazid and ethionamide); 1964 (95% HPD: 1948–1976); (C) embB M306V (ethambutol); 1967 (95% HPD: 1950–1978); (D) rpoB L452P (rifampicin); pncA 1bp insertion (pyrazinamide); 1984 (95% HPD: 1974–1992); and (E) rpoB D435G (rifampicin); rrs 1400 (kanamycin); gyrA A90V (ofloxacin); 1995 (95% HPD: 1988–1999). The accumulation of individual drug-resistant mutations within a strain is denoted to the right of the phylogenetic tree. The dates of drug discovery are displayed at the bottom of the figure [53]. Four additional LAM4 strains on a distant branch were not included in this figure because of size constraints. Bootstrap values are provided for lettered nodes, and bootstrap values for all nodes are shown in S5 Fig.
Mentions: The “historical” Tugela Ferry XDR strain, KZN605, was nested phylogenetically within a large clone of 50 LAM4 strains with predominantly phenotypic XDR (Fig 3). All of the strains within this clone (henceforth referred to as the Tugela Ferry XDR Clone) possessed the characteristic drug resistance mutations that were previously identified in XDR-TB strains circulating in Tugela Ferry during the outbreak [9,24], further indicating this clone’s continued prevalence within KwaZulu-Natal. Patients in whom the Tugela Ferry XDR Clone was isolated were from ten of the 11 districts within the province (Fig 2, panel B). In addition, the Tugela Ferry XDR Clone was not overrepresented among HIV-positive patients (p = 0.6750) (Table 2). This suggests that strains within this clone were neither geographically constrained nor restricted to immunodeficient hosts.

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

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.

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.

Methods and findings: 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.

No MeSH data available.


Related in: MedlinePlus