Tuberculosis in Swiss captive Asian elephants: microevolution of Mycobacterium tuberculosis characterized by multilocus variable-number tandem-repeat analysis and whole-genome sequencingle-number tandem-repeat analysis and whole-genome sequencing

Zoonotic tuberculosis is a risk for human health, especially when animals are in close contact with humans. Mycobacterium tuberculosis was cultured from several organs, including lung tissue and gastric mucosa, of three captive elephants euthanized in a Swiss zoo. The elephants presented weight loss, weakness and exercise intolerance. Molecular characterization of the M. tuberculosis isolates by spoligotyping revealed an identical profile, suggesting a single source of infection. Multilocus variable-number of tandem-repeat analysis (MLVA) elucidated two divergent populations of bacteria and mixed infection in one elephant, suggesting either different transmission chains or prolonged infection over time. A total of eight M. tuberculosis isolates were subjected to whole-genome sequence (WGS) analysis, confirming a single source of infection and indicating the route of transmission between the three animals. Our findings also show that the methods currently used for epidemiological investigations of M. tuberculosis infections should be carefully applied on isolates from elephants. Moreover the importance of multiple sampling and analysis of within-host mycobacterial clonal populations for investigations of transmission is demonstrated

The genetic background modulates the evolution of fluoroquinolone-resistance in Mycobacterium tuberculosis

Fluoroquinolones (FQ) form the backbone in experimental treatment regimens against drug-susceptible tuberculosis. However, little is known on whether the genetic variation present in natural populations of Mycobacterium tuberculosis (Mtb) affects the evolution of FQ-resistance (FQ-R). To investigate this question, we used nine genetically distinct drug-susceptible clinical isolates of Mtb and measured their frequency of resistance to the FQ ofloxacin (OFX) in vitro. We found that the Mtb genetic background led to differences in the frequency of OFX-resistance (OFX-R) that spanned two orders of magnitude and substantially modulated the observed mutational profiles for OFX-R. Further, in vitro assays showed that the genetic background also influenced the minimum inhibitory concentration and the fitness effect conferred by a given OFX-R mutation. To test the clinical relevance of our in vitro work, we surveyed the mutational profile for FQ-R in publicly available genomic sequences from clinical Mtb isolates, and found substantial Mtb lineage-dependent variability. Comparison of the clinical and the in vitro mutational profiles for FQ-R showed that 51% and 39% of the variability in the clinical frequency of FQ-R gyrA mutation events in Lineage 2 and Lineage 4 strains, respectively, can be attributed to how Mtb evolves FQ-R in vitro. As the Mtb genetic background strongly influenced the evolution of FQ-R in vitro, we conclude that the genetic background of Mtb also impacts the evolution of FQ-R in the clinic

An African origin for Mycobacterium bovis

Mycobacterium bovis; and; Mycobacterium caprae; are two of the most important agents of tuberculosis in livestock and the most important causes of zoonotic tuberculosis in humans. However, little is known about the global population structure, phylogeography and evolutionary history of these pathogens.; We compiled a global collection of 3364 whole-genome sequences from; M.bovis; and; M.caprae; originating from 35 countries and inferred their phylogenetic relationships, geographic origins and age.; Our results resolved the phylogenetic relationship among the four previously defined clonal complexes of; M.bovis; , and another eight newly described here. Our phylogeographic analysis showed that; M.bovis; likely originated in East Africa. While some groups remained restricted to East and West Africa, others have subsequently dispersed to different parts of the world.; Our results allow a better understanding of the global population structure of; M.bovis; and its evolutionary history. This knowledge can be used to define better molecular markers for epidemiological investigations of; M.bovis; in settings where whole-genome sequencing cannot easily be implemented.; During the last few years, analyses of large globally representative collections of whole-genome sequences (WGS) from the human-adapted Mycobacterium tuberculosis complex (MTBC) lineages have enhanced our understanding of the global population structure, phylogeography and evolutionary history of these pathogens. In contrast, little corresponding data exists for M. bovis, the most important agent of tuberculosis in livestock. Using whole-genome sequences of globally distributed M. bovis isolates, we inferred the genetic relationships among different M. bovis genotypes distributed around the world. The most likely origin of M. bovis is East Africa according to our inferences. While some M. bovis groups remained restricted to East and West Africa, others have subsequently dispersed to different parts of the world driven by cattle movements

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

Whole genome sequencing and spatial analysis identifies recent tuberculosis transmission hotspots in Ghana

Whole genome sequencing (WGS) is progressively being used to investigate the transmission dynamics of; Mycobacterium tuberculosis; complex (MTBC). We used WGS analysis to resolve traditional genotype clusters and explored the spatial distribution of confirmed recent transmission clusters. Bacterial genomes from a total of 452 MTBC isolates belonging to large traditional clusters from a population-based study spanning July 2012 and December 2015 were obtained through short read next-generation sequencing using the illumina HiSeq2500 platform. We performed clustering and spatial analysis using specified R packages and ArcGIS. Of the 452 traditional genotype clustered genomes, 314 (69.5%) were confirmed clusters with a median cluster size of 7.5 genomes and an interquartile range of 4-12. Recent tuberculosis (TB) transmission was estimated as 24.7%. We confirmed the wide spread of a Cameroon sub-lineage clone with a cluster size of 78 genomes predominantly from the Ablekuma sub-district of Accra metropolis. More importantly, we identified a recent transmission cluster associated with isoniazid resistance belonging to the Ghana sub-lineage of lineage 4. WGS was useful in detecting unsuspected outbreaks; hence, we recommend its use not only as a research tool but as a surveillance tool to aid in providing the necessary guided steps to track, monitor, and control TB

Red squirrels in the British Isles are infected with leprosy bacilli

Leprosy, caused by infection with Mycobacterium leprae or the recently discovered Mycobacterium lepromatosis, was once endemic in humans in the British Isles. Red squirrels in Great Britain (Sciurus vulgaris) have increasingly been observed with leprosy-like lesions on the head and limbs. Using genomics, histopathology, and serology, we found M. lepromatosis in squirrels from England, Ireland, and Scotland, and M. leprae in squirrels from Brownsea Island, England. Infection was detected in overtly diseased and seemingly healthy animals. Phylogenetic comparisons of British and Irish M. lepromatosis with two Mexican strains from humans show that they diverged from a common ancestor around 27,000 years ago, whereas the M. leprae strain is closest to one that circulated in Medieval England. Red squirrels are thus a reservoir for leprosy in the British Isles

Revised Interpretation of the Hain Lifescience GenoType MTBC To Differentiate Mycobacterium canettii and Members of the Mycobacterium tuberculosis Complex.

Using 894 phylogenetically diverse genomes of the Mycobacterium tuberculosis complex (MTBC), we simulated in silico the ability of the Hain Lifescience GenoType MTBC assay to differentiate the causative agents of tuberculosis. Here, we propose a revised interpretation of this assay to reflect its strengths (e.g., it can distinguish some strains of Mycobacterium canettii and variants of Mycobacterium bovis that are not intrinsically resistant to pyrazinamide) and limitations (e.g., Mycobacterium orygis cannot be differentiated from Mycobacterium africanum)

Phylogenomics of Mycobacterium africanum reveals a new lineage and a complex evolutionary history.

Human tuberculosis (TB) is caused by members of the Mycobacterium tuberculosis complex (MTBC). The MTBC comprises several human-adapted lineages known as M. tuberculosis sensu stricto, as well as two lineages (L5 and L6) traditionally referred to as Mycobacterium africanum. Strains of L5 and L6 are largely limited to West Africa for reasons unknown, and little is known of their genomic diversity, phylogeography and evolution. Here, we analysed the genomes of 350 L5 and 320 L6 strains, isolated from patients from 21 African countries, plus 5 related genomes that had not been classified into any of the known MTBC lineages. Our population genomic and phylogeographical analyses showed that the unclassified genomes belonged to a new group that we propose to name MTBC lineage 9 (L9). While the most likely ancestral distribution of L9 was predicted to be East Africa, the most likely ancestral distribution for both L5 and L6 was the Eastern part of West Africa. Moreover, we found important differences between L5 and L6 strains with respect to their phylogeographical substructure and genetic diversity. Finally, we could not confirm the previous association of drug-resistance markers with lineage and sublineages. Instead, our results indicate that the association of drug resistance with lineage is most likely driven by sample bias or geography. In conclusion, our study sheds new light onto the genomic diversity and evolutionary history of M. africanum, and highlights the need to consider the particularities of each MTBC lineage for understanding the ecology and epidemiology of TB in Africa and globally

Phylogenomics of Mycobacterium africanum reveals a new lineage and a complex evolutionary history

Coscolla et al.Human tuberculosis (TB) is caused by members of the Mycobacterium tuberculosis complex (MTBC). The MTBC comprises several human-adapted lineages known as M. tuberculosis sensu stricto, as well as two lineages (L5 and L6) traditionally referred to as Mycobacterium africanum . Strains of L5 and L6 are largely limited to West Africa for reasons unknown, and little is known of their genomic diversity, phylogeography and evolution. Here, we analysed the genomes of 350 L5 and 320 L6 strains, isolated from patients from 21 African countries, plus 5 related genomes that had not been classified into any of the known MTBC lineages. Our population genomic and phylogeographical analyses showed that the unclassified genomes belonged to a new group that we propose to name MTBC lineage 9 (L9). While the most likely ancestral distribution of L9 was predicted to be East Africa, the most likely ancestral distribution for both L5 and L6 was the Eastern part of West Africa. Moreover, we found important differences between L5 and L6 strains with respect to their phylogeographical substructure and genetic diversity. Finally, we could not confirm the previous association of drug-resistance markers with lineage and sublineages. Instead, our results indicate that the association of drug resistance with lineage is most likely driven by sample bias or geography. In conclusion, our study sheds new light onto the genomic diversity and evolutionary history of M. africanum , and highlights the need to consider the particularities of each MTBC lineage for understanding the ecology and epidemiology of TB in Africa and globally.M.C. is supported by the Ramón y Cajal programme from the Ministerio de Ciencia, Innovación y Universidades. This work was supported by the European Society of Clinical Microbiology and Infectious Diseases (ESCMID) (research award to M.C.), Ministerio de Ciencia, Innovación y Universidades (grant number RTI2018-094399-A-I00 to M.C.) and Consellería de Educació de la Generalitat Valenciana (grant number SEJI/2019/011 to M.C.), the Swiss National Science Foundation (grants 310030_188888, IZRJZ3_164171, IZLSZ3_170834 and CRSII5_177163 to S. G.), the European Research Council (883582-ECOEVODRTB to S. G.) and Wellcome (grant number 097134/Z/11/Z to D. Y.-M).Peer reviewe

Whole-genome sequencing for drug resistance profile prediction in Mycobacterium tuberculosis

Whole-genome sequencing allows rapid detection of drug-resistant; Mycobacterium tuberculosis; isolates. However, the availability of high-quality data linking quantitative phenotypic drug susceptibility testing (DST) and genomic data have thus far been limited. We determined drug resistance profiles of 176 genetically diverse clinical; M. tuberculosis; isolates from the Democratic Republic of the Congo, Ivory Coast, Peru, Thailand, and Switzerland by quantitative phenotypic DST for 11 antituberculous drugs using the BD Bactec MGIT 960 system and 7H10 agar dilution to generate a cross-validated phenotypic DST readout. We compared DST results with predicted drug resistance profiles inferred by whole-genome sequencing. Classification of strains by the two phenotypic DST methods into resistotype/wild-type populations was concordant in 73 to 99% of cases, depending on the drug. Our data suggest that the established critical concentration (5 mg/liter) for ethambutol resistance (MGIT 960 system) is too high and misclassifies strains as susceptible, unlike 7H10 agar dilution. Increased minimal inhibitory concentrations were explained by mutations identified by whole-genome sequencing. Using whole-genome sequences, we were able to predict quantitative drug resistance levels for the majority of drug resistance mutations. Predicting quantitative levels of drug resistance by whole-genome sequencing was partially limited due to incompletely understood drug resistance mechanisms. The overall sensitivity and specificity of whole-genome-based DST were 86.8% and 94.5%, respectively. Despite some limitations, whole-genome sequencing has the potential to infer resistance profiles without the need for time-consuming phenotypic methods