Multiple drug-susceptibility screening in Mycobacterium bovis: new nucleotide polymorphisms in the embB gene among ethambutol susceptible strains

Objectives: Pyrazinamide-resistant Mycobacterium bovis isolates of animal origin were assessed for drug susceptibility to five antituberculosis drugs by the agar based Middlebrook 7H11 method as gold standard as well as by a simplified, dichotomous resazurin microtitre assay (d-REMA). Methods: A total of 53 M. bovis isolates were typed and tested against isoniazid, rifampin, streptomycin, ethambutol, kanamycin and the control drug pyrazinamide. On the basis of the results obtained, pncA and embB genes were PCR-amplified and DNA-sequenced for all isolates. Results: All M. bovis isolates, classified into 21 spoligotype/MIRU-VNTR profiles, were resistant to pyrazinamide by both methods, as expected. The pncA gene sequencing confirmed the presence of the resistance-conferring H57D mutation. All strains were found to be susceptible to the other five drugs by the agar based gold standard method. The d-REMA was in agreement with these results for all five drugs, with the exception of 12 isolates, which showed ambiguous and therefore inconclusive results in ethambutol testing. Mutations in the embB gene were observed in all 53 isolates: four new single-nucleotide polymorphisms were identified. No association was found between embB genetic profiles and ethambutol resistance results by the gold standard. Conclusion: All M. bovis isolates were sensitive to the most common antituberculosis drugs used for treatment. There was a good agreement between the d-REMA assay and the agar based reference method. Among ethambutol susceptible isolates, four new embB mutations were found

Mycobacterium microti at the environment and wildlife interface

10openInternationalItalian coauthor/editorAn unexpected high presence of Mycobacterium microti in wild boar in Northern Italy (Garda Lake) has been reported since 2003, but the factors contributing to the maintenance of this pathogen are still unclear. In this study, we investigated the presence of M. microti in wild rodents and in water and soil samples collected at wild boar aggregation areas, such as watering holes, with the aim of clarifying their role in M. microti transmission. In total, 8 out of 120 captured animals tested positive for the Mycobacterium tuberculosis complex (MTBC) as assessed by real-time PCR, and six samples were confirmed to be M. microti. A strain with a genetic profile similar to those previously isolated in wild boars in the same area was isolated from one sample. Of the 20 water and 19 mud samples, 3 and 1, respectively, tested positive for the presence of MTBC, and spacer oligotype SB0118 (vole type) was detected in one sample. Our study suggests that wild rodents, in particular Apodemus sylvaticus, Microtus sp. and Apodemus flavicollis, play roles in the maintenance of M. microti infections in wild boar through ingestion or by contact with either infected excreta or a contaminated environment, such as at animal aggregation sitesopenTagliapietra, V.; Boniotti, M.B.; Mangeli, A.; Karaman, I.; Alborali, G.; Chiari, M.; D’Incau, M.; Zanoni, M.; Rizzoli, A.; Pacciarini, M.L.Tagliapietra, V.; Boniotti, M.B.; Mangeli, A.; Karaman, I.; Alborali, G.; Chiari, M.; D’Incau, M.; Zanoni, M.; Rizzoli, A.; Pacciarini, M.L

A new nomenclature for the livestock-associated Mycobacterium tuberculosis complex based on phylogenomics

Background: The bacteria that compose the Mycobacterium tuberculosis complex (MTBC) cause tuberculosis (TB) in humans and in different animals, including livestock. Much progress has been made in understanding the population structure of the human-adapted members of the MTBC by combining phylogenetics with genomics. Accompanying the discovery of new genetic diversity, a body of operational nomenclature has evolved to assist comparative and molecular epidemiological studies of human TB. By contrast, for the livestock-associated MTBC members, Mycobacterium bovis, M. caprae and M. orygis, there has been a lack of comprehensive nomenclature to accommodate new genetic diversity uncovered by emerging phylogenomic studies. We propose to fill this gap by putting forward a new nomenclature covering the main phylogenetic groups within M. bovis, M. caprae and M. orygis. Methods: We gathered a total of 8,747 whole-genome sequences (WGS) from public sources and 39 newly sequenced strains, and selected a subset of 839 WGS, representative of the worldwide diversity of M. bovis, M. caprae and M. orygis. We used phylogenetics and genetic diversity patterns inferred from WGS to define groups. Results: We propose to divide M. bovis, M. caprae and M. orygis, in three main phylogenetic lineages, which we named La1, La2 and La3, respectively. Within La1, we identified several monophyletic groups, which we propose to classify into eight sublineages (La1.1-La1.8). These differed in geographic distribution, with some being geographically restricted and others globally widespread, suggesting different expansion abilities. To ease molecular characterization of these MTBC groups by the community, we provide phylogenetically informed, single nucleotide polymorphisms that can be used as barcodes for genotyping. These makers were implemented in a new test suit in KvarQ, a platform-independent, open-source tool. Conclusions: Our results contribute to an improved classification of the genetic diversity within the livestock-associated MTBC, which will benefit future molecular epidemiological and evolutionary studies

Geo-epidemiology of animal tuberculosis and Mycobacterium bovis genotypes in livestock in a small, high-incidence area in Sicily, Italy

IntroductionThe persistence of animal tuberculosis (TB) in livestock is a major concern in Sicily, Italy. The objective of this study was to elucidate the transmission dynamics of M. bovis infection in a highly circumscribed, and at the same time geographically diverse, high-risk area of the island through an in-depth geo-epidemiological investigation of TB in cattle and black pigs raised in small-scale extensive farms across the district of Caronia.MethodsWe used genotype analysis coupled with geographic information system (GIS) technology and phylogenetic inference to characterize the spatial distribution of TB and M. bovis genotypes in livestock and the genetic relationships between M. bovis isolates. A total of 589 M. bovis isolates collected from slaughtered cattle (n = 527) and Sicilian black pigs (n = 62) over a 5-year period (2014–2018) were included in the study.ResultsTB was widespread throughout the district and was most frequent in the north-central area of the district, especially along one of the district’s streams. We identified a total of 62 M. bovis genotypes. Identical genetic profiles were isolated from both neighboring and non-neighburing herds. The 10 most frequent genotypes, accounting for 82% of M. bovis isolates, showed geographic specificities in that they tended to cluster in specific spatial niches. The landscape structure of these niches—i.e. steep slopes, rocky ridges, meadows and streams—is likely to have had a significant influence on the distribution of TB among livestock in Caronia. Higher concentrations of TB were observed along streams and in open meadows, while rocky ridges and slopes appeared to have hampered the spread of TB.DiscussionThe geographical distribution of TB cases among livestock in Caronia is consistent with several epidemiological scenarios (e.g., high density of infected herds along the streams or in hilly plateau where livestock share pastures). Landscape structure is likely to play an important role in the transmission and persistence of M. bovis infection across the district. Additional potential risk factors, such as livestock trading and extensive breeding methods, are also discussed. Our results will contribute to the improvement of surveillance, control and eradication activities of TB in Sicily by the implementation of ad hoc TB control measures, especially in farms located along streams, sharing common pastures or with mixed animal species

Genotype diversity and distribution of Mycobacterium bovis from livestock in a small, high-risk area in northeastern Sicily, Italy.

Bovine tuberculosis (bTB) caused by Mycobacterium bovis is an important re-emerging disease affecting livestock, wildlife and humans. Epidemiological studies are crucial to identifying the source of bTB infection, and its transmission dynamics and host preference, and thus to the implementation of effective strategies to contain it. In this study, we typed M. bovis isolates from livestock, and investigated their genetic diversity and distribution. A total of 204 M. bovis isolates were collected from cattle (n = 164) and Sicilian black pigs (n = 40) reared in a limited area of the province of Messina, northeastern Sicily, an area that had previously been identified as having the highest incidence of bTB in livestock on the island. All M. bovis isolates were typed by both spoligotyping and 12-loci MIRU-VNTR analysis. Results from both methods were then combined in order to improve the discriminatory power of M. bovis typing. We identified 73 combined genetic profiles. Thirty-five point six percent of the profiles were common to at least two animals, whereas 64.4% of profiles occurred in only one animal. A number of genetic profiles were predominant in either cattle or black pigs. We identified common genetic patterns in M. bovis isolates originating not only from neighboring districts, but also from non-neighboring districts. Our findings suggest that bTB is widespread in our setting, and is caused by a large number of genetically diverse M. bovis strains. The ecology and farming practices characteristic of the area may explain the substantial M. bovis heterogeneity observed, and could represent obstacles to bTB eradication

Molecular Typing of Mycobacterium bovis Strains Isolated in Italy from 2000 to 2006 and Evaluation of Variable-Number Tandem Repeats for Geographically Optimized Genotyping▿

Spoligotyping and exact tandem repeat (ETR) analysis of Mycobacterium bovis and M. caprae isolated strains has been routinely carried out in Italy since 2000 to obtain a database of genetic profiles and support traditional epidemiological investigations. In this study, we characterized 1,503 M. bovis and 57 M. caprae isolates obtained from 2000 to 2006 in 747 cattle herds mainly located in northern Italy. We identified 81 spoligotypes and 113 ETR profiles, while the combination of spoligotyping/ETR analysis differentiated 228 genotypes, with genotypic diversity indices of 0.70 (spoligotyping), 0.94 (ETR-A to -E typing), and 0.97 (spoligotyping/ETR-A to -E typing), respectively. Despite the high degree of resolution obtained, the spoligotyping/ETR methods were not discriminative enough in the case of genotypes characterized by the combination of SB0120, the predominant spoligotype in Italy, with the most common ETR profiles. To obtain a more informative subset of typing loci, 24 mycobacterial interspersed repetitive unit-variable-number tandem repeat (MIRU-VNTR) markers were evaluated by analyzing a panel of 100 epidemiologically unrelated SB0120 isolates. The panel was differentiated into 89 profiles with an overall genotypic diversity of 0.987 that could be also achieved by using a minimal group of 13 loci: ETR-A, -B, and -E; MIRU 26 and 40; and VNTR 2163a, 2163b, 3155, 1612, 4052, 1895, 3232, and 3336. The allelic diversity index and the stability of single loci was evaluated to provide the most discriminative genotyping method for locally prevalent strains