A landscape of genomic alterations at the root of a near-untreatable tuberculosis epidemic

Abstract Background Atypical Beijing genotype Mycobacterium tuberculosis strains are widespread in South Africa and have acquired resistance to up to 13 drugs on multiple occasions. It is puzzling that these strains have retained fitness and transmissibility despite the potential fitness cost associated with drug resistance mutations. Methods We conducted Illumina sequencing of 211 Beijing genotype M. tuberculosis isolates to facilitate the detection of genomic features that may promote acquisition of drug resistance and restore fitness in highly resistant atypical Beijing forms. Phylogenetic and comparative genomic analysis was done to determine changes that are unique to the resistant strains that also transmit well. Minimum inhibitory concentration (MIC) determination for streptomycin and bedaquiline was done for a limited number of isolates to demonstrate a difference in MIC between isolates with and without certain variants. Results Phylogenetic analysis confirmed that two clades of atypical Beijing strains have independently developed resistance to virtually all the potent drugs included in standard (pre-bedaquiline) drug-resistant TB treatment regimens. We show that undetected drug resistance in a progenitor strain was likely instrumental in this resistance acquisition. In this cohort, ethionamide (ethA A381P) resistance would be missed in first-line drug-susceptible isolates, and streptomycin (gidB L79S) resistance may be missed due to an MIC close to the critical concentration. Subsequent inadequate treatment historically led to amplification of resistance and facilitated spread of the strains. Bedaquiline resistance was found in a small number of isolates, despite lack of exposure to the drug. The highly resistant clades also carry inhA promoter mutations, which arose after ethA and katG mutations. In these isolates, inhA promoter mutations do not alter drug resistance, suggesting a possible alternative role. Conclusion The presence of the ethA mutation in otherwise susceptible isolates from ethionamide-naïve patients demonstrates that known exposure is not an adequate indicator of drug susceptibility. Similarly, it is demonstrated that bedaquiline resistance can occur without exposure to the drug. Inappropriate treatment regimens, due to missed resistance, leads to amplification of resistance, and transmission. We put these results into the context of current WHO treatment regimens, underscoring the risks of treatment without knowledge of the full drug resistance profile

Evaluation of Nanopore sequencing for Mycobacterium tuberculosis drug susceptibility testing and outbreak investigation: a genomic analysis

Mycobacterium tuberculosis whole-genome sequencing (WGS) has been widely used for genotypic drug susceptibility testing (DST) and outbreak investigation. For both applications, Illumina technology is used by most public health laboratories; however, Nanopore technology developed by Oxford Nanopore Technologies has not been thoroughly evaluated. The aim of this study was to determine whether Nanopore sequencing data can provide equivalent information to Illumina for transmission clustering and genotypic DST for M tuberculosis

Progenitor strain introduction of Mycobacterium bovis at the wildlife-livestock interface can lead to clonal expansion of the disease in a single ecosystem

Mycobacterium bovis infects multiple wildlife species and domesticated cattle across South Africa, and negatively impacts on livestock trade and movement of wildlife for conservation purposes. M. bovis infection was first reported in the Kruger National Park (KNP) in South Africa during the 1990s, and has since spread to infect numerous animal host species throughout the park and across South Africa. Whole genome sequencing data of 17 M. bovis isolates were analyzed to investigate the genomic diversity among M. bovis isolates causing disease in different animal host species from various locations in South Africa. M. bovis strains analyzed in this study are geographic rather than host species-specific. The clonal expansion of M. bovis in the KNP highlights the effect of an introduction of a transmissible infectious disease leading to a rising epidemic in wildlife, and emphasizes the importance of disease control and movement restriction of species that serve as disease reservoirs. In conclusion, the point source introduction of a single M. bovis strain type in the KNP ecosystem lead to an M. bovis outbreak in this area that affects various host species and poses an infection risk in neighboring rural communities where HIV prevalence is high.Supplementary data: Supplemental Methods; Supplemental Data 1, Table S1; Supplemental Data 2, Table S2The South African Medical Research Council, the National Research Foundation, and faculty baseline funding from King Abdullah University of Science and Technology (KAUST, Thuwal, Saudi Arabia) awarded to A Pain. MA Miller is funded by the South African Research Chairs Initiative of the Department of Science and Technology and National Research Foundation (NRF) of South Africa, award number UID 86949.http://www.elsevier.com/locate/meegid2018-07-30hj2017Veterinary Tropical Disease

Characterizing epidemiological and genotypic features of Mycobacterium bovis infection in wild dogs (Lycaon pictus)

Mycobacterium bovis (M. bovis) infects a wide range of wildlife species and has recently been discovered in the endangered African wild dog (Lycaon pictus). This study aimed to characterize the epidemiology of tuberculosis (TB) in wild dogs in endemic areas of South Africa. We describe 12 TB cases in wild dogs from Kruger National Park (KNP), Hluhluwe–iMfolozi Park (HiP) and a private facility in Hoedspruit from 2015 to 2017. Spoligotyping was used to identify the disease-causing M. bovis strain in these cases, and whole-genome sequencing was performed on 5 M. bovis isolates (KNP = 2 and HiP = 3) to investigate genomic diversity as well as the relationship to other isolates found in these geographical areas. Three distinct strain types were responsible for the M. bovis infections in this species. The SB0121 strain was observed in wild dogs from KNP, whereas SB0130 was responsible for infection in wild dogs from HiP. A novel strain, SB2681, was also identified in the HiP wild dogs. Whole-genome sequence analysis suggests that different infection sources exist among these wild dogs and that inter-species transmission most likely occurred between wildlife predators and prey located within shared geographical areas. This study highlights the importance of regular disease surveillance to identify and characterize potential threats for successful control of infection and protection of endangered species.The South African Medical Research Council and the National Research Foundation.http://wileyonlinelibrary.com/journal/tbedhj2022Paraclinical Science

Ancient and recent differences in the intrinsic susceptibility of Mycobacterium tuberculosis complex to pretomanid

OBJECTIVES: To develop a robust phenotypic antimicrobial susceptibility testing (AST) method with a correctly set breakpoint for pretomanid (Pa), the most recently approved anti-tuberculosis drug. METHODS: The Becton Dickinson Mycobacterial Growth Indicator Tube™ (MGIT) system was used at six laboratories to determine the MICs of a phylogenetically diverse collection of 356 Mycobacterium tuberculosis complex (MTBC) strains to establish the epidemiological cut-off value for pretomanid. MICs were correlated with WGS data to study the genetic basis of differences in the susceptibility to pretomanid. RESULTS: We observed ancient differences in the susceptibility to pretomanid among various members of MTBC. Most notably, lineage 1 of M. tuberculosis, which is estimated to account for 28% of tuberculosis cases globally, was less susceptible than lineages 2, 3, 4 and 7 of M. tuberculosis, resulting in a 99th percentile of 2 mg/L for lineage 1 compared with 0.5 mg/L for the remaining M. tuberculosis lineages. Moreover, we observed that higher MICs (≥8 mg/L), which probably confer resistance, had recently evolved independently in six different M. tuberculosis strains. Unlike the aforementioned ancient differences in susceptibility, these recent differences were likely caused by mutations in the known pretomanid resistance genes. CONCLUSIONS: In light of these findings, the provisional critical concentration of 1 mg/L for MGIT set by EMA must be re-evaluated. More broadly, these findings underline the importance of considering the global diversity of MTBC during clinical development of drugs and when defining breakpoints for AST

A New Phylogenetic Framework for the Animal-Adapted Mycobacterium tuberculosis Complex

Tuberculosis (TB) affects humans and other animals and is caused by bacteria from the Mycobacterium tuberculosis complex (MTBC). Previous studies have shown that there are at least nine members of the MTBC infecting animals other than humans; these have also been referred to as ecotypes. However, the ecology and the evolution of these animal-adapted MTBC ecotypes are poorly understood. Here we screened 12,886 publicly available MTBC genomes and newly sequenced 17 animal-adapted MTBC strains, gathering a total of 529 genomes of animal-adapted MTBC strains. Phylogenomic and comparative analyses confirm that the animal-adapted MTBC members are paraphyletic with some members more closely related to the human-adapted Mycobacterium africanum Lineage 6 than to other animal-adapted strains. Furthermore, we identified four main animal-adapted MTBC clades that might correspond to four main host shifts; two of these clades are hypothesized to reflect independent cattle domestication events. Contrary to what would be expected from an obligate pathogen, MTBC nucleotide diversity was not positively correlated with host phylogenetic distances, suggesting that host tropism in the animal-adapted MTBC seems to be driven by contact rates and demographic aspects of the host population rather by than host relatedness. By combining phylogenomics with ecological data, we propose an evolutionary scenario in which the ancestor of Lineage 6 and all animal-adapted MTBC ecotypes was a generalist pathogen that subsequently adapted to different host species. This study provides a new phylogenetic framework to better understand the evolution of the different ecotypes of the MTBC and guide future work aimed at elucidating the molecular mechanisms underlying host range

Fatal Tuberculosis in a Free-Ranging African Elephant and One Health Implications of Human Pathogens in Wildlife

Tuberculosis (TB) in humans is a global public health concern and the discovery of animal cases of Mycobacterium tuberculosis (Mtb) infection and disease, especially in multi-host settings, also has significant implications for public health, veterinary disease control, and conservation endeavors. This paper describes a fatal case of Mtb disease in a free-ranging African elephant (Loxodonta africana) in a high human TB burden region. Necropsy revealed extensive granulomatous pneumonia, from which Mtb was isolated and identified as a member of LAM3/F11 lineage; a common lineage found in humans in South Africa. These findings are contextualized within a framework of emerging Mtb disease in wildlife globally and highlights the importance of the One Health paradigm in addressing this anthroponotic threat to wildlife and the zoonotic implications

Ancient and recent differences in the intrinsic susceptibility of Mycobacterium tuberculosis complex to pretomanid.

OBJECTIVES: To develop a robust phenotypic antimicrobial susceptibility testing (AST) method with a correctly set breakpoint for pretomanid (Pa), the most recently approved anti-tuberculosis drug. METHODS: The Becton Dickinson Mycobacterial Growth Indicator Tube™ (MGIT) system was used at six laboratories to determine the MICs of a phylogenetically diverse collection of 356 Mycobacterium tuberculosis complex (MTBC) strains to establish the epidemiological cut-off value for pretomanid. MICs were correlated with WGS data to study the genetic basis of differences in the susceptibility to pretomanid. RESULTS: We observed ancient differences in the susceptibility to pretomanid among various members of MTBC. Most notably, lineage 1 of M. tuberculosis, which is estimated to account for 28% of tuberculosis cases globally, was less susceptible than lineages 2, 3, 4 and 7 of M. tuberculosis, resulting in a 99th percentile of 2 mg/L for lineage 1 compared with 0.5 mg/L for the remaining M. tuberculosis lineages. Moreover, we observed that higher MICs (≥8 mg/L), which probably confer resistance, had recently evolved independently in six different M. tuberculosis strains. Unlike the aforementioned ancient differences in susceptibility, these recent differences were likely caused by mutations in the known pretomanid resistance genes. CONCLUSIONS: In light of these findings, the provisional critical concentration of 1 mg/L for MGIT set by EMA must be re-evaluated. More broadly, these findings underline the importance of considering the global diversity of MTBC during clinical development of drugs and when defining breakpoints for AST

A phylogenomic- and proteomic investigation into the evolution and biological characteristics of the members of the group 2 Latin-American Mediterranean (LAM) genotype of Mycobacterium tuberculosis

Thesis (PhD)--Stellenbosch University, 2014.ENGLISH ABSTRACT: Mycobacterium tuberculosis (M. tuberculosis) is the causative agent of tuberculosis (TB), a disease that affects millions of people world-wide. The species M. tuberculosis consists of a large number of different strains that can be grouped into at least 40 different known strain families. Many of the strains present with different pathogenic characteristics and host adaptations. The F11 LAM strains and Beijing strains currently have a nearly equal representation in the population of Cape Town, making up a total of 45% of all strains in this setting. The Latin-American Mediterranean (LAM) family of M. tuberculosis is proved to be the cause of a large percentage of TB cases worldwide and it is the predominant strain in high-prevalence regions such as the Western Cape and KwaZulu-Natal regions of South Africa, Zambia, Zimbabwe, and South America. This project aimed to investigate the evolution and biological characteristics of the members of the principle genetic group (PGG) 2 Latin-American Mediterranean (LAM) genotype of M. tuberculosis using a combination of whole genomic and proteomic analyses, coupled to mycobacterial molecular epidemiological techniques. The evolution of M. tuberculosis strain families from the Western Cape Province of South Africa proved to be consistent with previous evolutionary scenarios for M. tuberculosis isolated from other parts of the world. This genome-wide SNP-based phylogeny for the evolution of M. tuberculosis offers novel insight into the unique global representation of the M. tuberculosis isolates from the Western Cape, South Africa. The evolutionary scenario presented confirms six LAM sub-lineages, namely IS6110 RFLP families F9, F11, F13, F14, F15, and F26. A subset of sub-lineage defining SNPs was determined for each of the six LAM sub-lineages. The genomic changes in the LAM genotype strains observed through the SNP analysis presented here mostly occur in the genes involved in the cell wall, cell processes, intermediary metabolism and respiration. The same phenomenon was observed when the non-redundant SNPs of the non-LAM isolates were functionally annotated. The functional classification of the regulated proteins in the representative of the LAM RDRio lineage of M. tuberculosis suggests that proteins involved in the lipid metabolism, intermediary metabolism and respiration may be the key to the pathogenic effectiveness of the RDRio LAM lineage. A combination of the LAM SNP analysis and the LAM RDRio/non-RDRio comparison showed that the overall genomic- and proteomic features involved in the cell wall and cell processes of the LAM genotype differ to a large extent from what is seen in the reference strain, M. tuberculosis H37Rv. This genome wide phylogenetic study is the first of its kind in a South African context, and not only presents a robust phylogeny of the M. tuberculosis strain families, and specifically the LAM lineage, but also gives the first ever insight into the protein differences which distinguishes RDRio and non-RDRio M. tuberculosis strains from each other.AFRIKAANSE OPSOMMING: Mycobacterium tuberculosis (M. tuberculosis) is die mikrobiese agent wat tuberkulose (TB), 'n siekte wat miljoene mense wêreldwyd affekteer, veroorsaak. Die spesie M. tuberculosis bestaan uit 'n groot aantal verskillende stamme wat in ten minste 40 verskillende bekende stam-families gegroepeer word. Baie van die stamme toon verskillende patogeniese eienskappe en gasheer aanpassings. Die F11 LAM stam en Beijing stam het tans 'n byna gelyke verteenwoordiging in die bevolking van Kaapstad, wat 'n totaal opmaak van 45% van stamme wat in hierdie gebied gevind word. Die Latyns-Amerikaanse Meditereense (LAM) familie van M. tuberculosis is bewys om die oorsaak van 'n groot persentasie van TB-gevalle wêreldwyd te wees, en dit is die oorheersende stam in hoë voorkoms streke soos die Wes-Kaap en KwaZulu-Natal streke van Suid-Afrika, Zambië, Zimbabwe en Suid-Amerika. Hierdie projek het ten doel gehad om die evolusie en biologiese eienskappe van die lede van die basiese genetiese groep (BGG) 2 Latyns-Amerikaanse Meditereense (LAM) genotipe van M. tuberculosis te ondersoek deur gebruik te maak van 'n kombinasie van heel genoom en proteoom analise, gekoppel aan mikobakteriële molekulêre epidemiologiese tegnieke. Die evolusie van M. tuberculosis stam families van die Wes-Kaap Provinsie van Suid-Afrika blyk om in ooreenstemming te wees met vorige evolusionêre scenario's vir M. tuberculosis wat in ander dele van die wêreld geïsoleer is. Die genoom-wye enkelnukleotied polimorfisme-gebaseerde filogenetiese hipotese vir die evolusie van M. tuberculosis bied nuwe insig in die unieke wêreldwye verteenwoordiging van die M. tuberculosis isolate van die Wes-Kaap, Suid-Afrika. Die evolusionêre scenario wat hier aangetoon word bevestig ses LAM sub-lyne, naamlik IS6110 RFLP families F9, F11, F13, F14, F15, en F26. 'n Versameling sub-lyn definiërende enkelnukleotied polimorfismes was bepaal vir elk van die ses LAM sub-afstammelinge. Die genomiese veranderinge wat waargeneem is in die LAM-genotipe isolate deur die enkelnukleotied polimorfisme analise wat hier aangebied word, is meestal in die gene wat betrokke is in die selwand, selprosesse, intermediêre metabolisme en respirasie. Dieselfde verskynsel is waargeneem wanneer die nie-oorbodige enkelnukleotied polimorfismes van die nie-LAM isolate funksioneel geannoteer is. Die funksionele klassifikasie van die gereguleerde proteïene in die verteenwoordiger van die LAM RDRio-lyn van M. tuberculosis dui daarop dat die proteïene wat betrokke is in die lipiedmetabolisme, intermediêre metabolisme en respirasie die sleutel tot die patogene doeltreffendheid van die RDRio-LAM-lyn kan wees. 'n Kombinasie van die LAM enkelnukleotied polimorfisme analise en die LAM-RDRio/nie-RDRio vergelyking het getoon dat die totale genomiese- en proteomiese kenmerke wat verwant is aan selwand en selprosesse van die LAM genotipe tot ʼn groot mate verskil van wat gesien word in die verwysing stam, M. tuberculosis H37Rv. Hierdie genoom-wye filogenetiese studie is die eerste van sy soort in 'n Suid-Afrikaanse konteks, en bied nie net ‗n robuuste filogenie van die M. tuberculosis stam families, en spesifiek die LAM genotipe van M. tuberculosis nie, maar gee ook die eerste keer ooit insig in die proteïen verskille wat RDRio en nie-RDRio M. tuberculosis stamme van mekaar onderskei