The open pan-genome architecture and virulence landscape of Mycobacterium bovis

Animal tuberculosis (TB) is an emergent disease caused by Mycobacterium bovis, one of the animal-adapted ecotypes of the Mycobacterium tuberculosis complex (MTC). In this work, whole-genome comparative analyses of 70 M. bovis were performed to gain insights into the pan-genome architecture. The comparison across M. bovis predicted genome composition enabled clustering into the core- and accessory-genome components, with 2736 CDS for the former, while the accessory moiety included 3897 CDS, of which 2656 are restricted to one/two genomes only. These analyses predicted an open pan-genome architecture, with an average of 32 CDS added by each genome and show the diversification of discrete M. bovis subpopulations supported by both core- and accessory-genome components. The functional annotation of the pan-genome classified each CDS into one or several COG (Clusters of Orthologous Groups) categories, revealing ‘transcription’ (total average CDSs, n=258), ‘lipid metabolism and transport’ (n=242), ‘energy production and conversion’ (n=214) and ‘unknown function’ (n=876) as the most represented. The closer analysis of polymorphisms in virulence-related genes in a restrict group of M. bovis from a multi-host system enabled the identification of clade-monomorphic non-synonymous SNPs, illustrating clade-specific virulence landscapes and correlating with disease severity. This first comparative pan-genome study of a diverse collection of M. bovis encompassing all clonal complexes indicates a high percentage of accessory genes and denotes an open, dynamic non-conservative pan-genome structure, with high evolutionary potential, defying the canons of MTC biology. Furthermore, it shows that M. bovis can shape its virulence repertoire, either by acquisition and loss of genes or by SNP-based diversification, likely towards host immune evasion, adaptation and persistence.info:eu-repo/semantics/publishedVersio

Genome-wide estimation of recombination, mutation and positive selection enlightens diversification drivers of Mycobacterium bovis

Genome sequencing has reinvigorated the infectious disease research feld, shedding light on disease epidemiology, pathogenesis, host–pathogen interactions and also evolutionary processes exerted upon pathogens. Mycobacterium tuberculosis complex (MTBC), enclosing M. bovis as one of its animal-adapted members causing tuberculosis (TB) in terrestrial mammals, is a paradigmatic model of bacterial evolution. As other MTBC members, M. bovis is postulated as a strictly clonal, slowly evolving pathogen, with apparently no signs of recombination or horizontal gene transfer. In this work, we applied comparative genomics to a whole genome sequence (WGS) dataset composed by 70 M. bovis from diferent lineages (European and African) to gain insights into the evolutionary forces that shape genetic diversifcation in M. bovis. Three distinct approaches were used to estimate signs of recombination. Globally, a small number of recombinant events was identifed and confrmed by two independent methods with solid support. Still, recombination reveals a weaker efect on M. bovis diversity compared with mutation (overall r/m= 0.037). The diferential r/m average values obtained across the clonal complexes of M. bovis in our dataset are consistent with the general notion that the extent of recombination may vary widely among lineages assigned to the same taxonomical species. Based on this work, recombination in M. bovis cannot be excluded and should thus be a topic of further efort in future comparative genomics studies for which WGS of large datasets from diferent epidemiological scenarios across the world is crucial. A smaller M. bovis dataset (n= 42) from a multi-host TB endemic scenario was then subjected to additional analyses, with the identifcation of more than 1,800 sites wherein at least one strain showed a single nucleotide polymorphism (SNP). The majority (87.1%) was located in coding regions, with the global ratio of non-synonymous upon synonymous alterations (dN/dS) exceeding 1.5, suggesting that positive selection is an important evolutionary force exerted upon M. bovis. A higher percentage of SNPs was detected in genes enriched into “lipid metabolism”, “cell wall and cell processes” and “intermediary metabolism and respiration” functional categories, revealing their underlying importance in M. bovis biology and evolution. A closer look on genes prone to horizontal gene transfer in the MTBC ancestor and included in the 3R (DNA repair, replication and recombination) system revealed a global average negative value for Taijima’s D neutrality test, suggesting that past selective sweeps and population expansion after a recent bottleneck remain as major evolutionary drivers of the obligatory pathogen M. bovis in its struggle with the host.info:eu-repo/semantics/publishedVersio

Worldwide Disseminated IncX4 Plasmid Carrying mcr-1 Arrives to Wild Mammal in Portugal

The mcr-1 gene spread is worldwide recognized as a public health threat at multidrug-resistant infections therapy level. Here, we report for the first time, to the best of our knowledge, the detection of the globally distributed IncX4 plasmid carrying mcr-1 (mcr-1/IncX4) in Escherichia coli isolated from a wild mammal in Portugal and Europe. This plasmid was found in a ST533 E. coli isolate with a multidrug-resistant profile, virulence potential, and possibly phylogenetically related to human isolates. Our work contributes to highlight the importance of antimicrobial resistance (AMR) surveillance in wildlife, an important compartment of the whole ecosystem often overlooked in the fight against AMR. IMPORTANCE Colistin resistance mediated by plasmids is recognized worldwide as an emergency problem connected with the whole ecosystem, since is well described in the interface of the human-animal-environment. The plasmid IncX4 is reported as one of the most prevalent plasmids harboring the gene mcr-1. On an European scale the plasmid IncX4 carrying mcr-1 has been described in humans, the environment, and animals, including wildlife, but only in wild birds. This study shows the first report of the plasmid IncX4 harboring mcr-1 in a wild mammal in Portugal and Europe, identified in a ST533 E. coli commensal that is, curiously, more related to isolates from humans than from livestock. Our findings show that the plasmid IncX4 harboring mcr-1 is well established in a colistin resistance drive embracing the whole ecosystem.info:eu-repo/semantics/publishedVersio

A walk on the wild side: Wild ungulates as potential reservoirs of multi-drug resistant bacteria and genes, including Escherichia coli harbouring CTX-M beta-lactamases

Extended-spectrum β-lactamases (ESBL)-producing Enterobacterales have been classified as critical priority pathogens by the World Health Organization (WHO). ESBL are universally distributed and, in 2006, were firstly reported on a wild animal. Understanding the relative contributions of wild animals to ESBL circulation in the environment is urgently needed. In this work, we have conducted a nationwide study in Portugal to investigate the occurrence of bacteria carrying clinically significant antimicrobial resistance genes (ARG), using widely distributed wild ungulates as model species. A total of 151 antimicrobial resistant-Enterobacterales isolates were detected from 181 wild ungulates: 50% (44/88) of isolates from wild boar (Sus scrofa), 40.3% (25/62) from red deer (Cervus elaphus), 41.4% (12/29) from fallow deer (Dama dama) and 100% (2/2) from mouflon (Ovis aries subsp. musimon). Selected isolates showed a diversified resistance profile, with particularly high values corresponding to ampicillin (71.5%) and tetracycline (63.6%). Enterobacterales strains carried blaTEM, tetA, tetB, sul2, sul1 or dfrA1 ARG genes. They also carried blaCTX-M-type genes, which are prevalent in human infections, namely CTX-M-14, CTX-M-15 and CTX-M-98. Strikingly, this is the first report of CTX-M-98 in wildlife. Almost 40% (n = 59) of Enterobacterales were multi-drug resistant. The diversity of plasmids carried by ESBL isolates was remarkable, including IncF, K and P. This study highlights the potential role of wild ungulates as environmental reservoirs of CTX-M ESBL-producing E. coli and in the spill-over of AMR bacteria and their determinants. Our findings suggest that wild ungulates are useful as strategic sentinel species of AMR in terrestrial environments, especially in response to potential sources of anthropogenic pollution, providing early warning of potential risks to human, animal and environmental health.info:eu-repo/semantics/publishedVersio

Emergence and Spread of Cephalosporinases in Wildlife: A Review

In the last decade, detection of antibiotic resistant bacteria from wildlife has received increasing interest, due to the potential risk posed by those bacteria to wild animals, livestock or humans at the interface with wildlife, and due to the ensuing contamination of the environment. According toWorld Health Organization, cephalosporins are critically important antibiotics to human health. However, acquired resistance to -lactams is widely distributed and is mainly mediated by extended-spectrum beta-lactamase and AmpC beta-lactamases, such as cephalosporinases. This work thus aimed to compile and analyse the information available on the emergence and dissemination of cephalosporinases in wildlife worldwide. Results suggest a serious scenario, with reporting of cephalosporinases in 46 countries from all continents (52% in Europe), across 188 host species, mainly birds and mammals, especially gulls and ungulates. The most widely reported cephalosporinases, CTX-M-1, CTX-M-14, CTX-M-15 and CMY-2, were also the most common in wild animals, in agreement with their ubiquity in human settings, including their association to high-risk clones of Escherichia coli (E. coli), such as the worldwide distributed CTX-M-15/ST131 E. coli. Altogether, our findings show that anthropogenic activities affect the whole ecosystem and that public policies pro-moting animal and environmental surveillance, as well as mitigation measures to avoid antimicrobial misuse and AMR spread, are urgently needed to be out in practise.info:eu-repo/semantics/publishedVersio

Emergence of colistin resistance genes (mcr-1) in Escherichia coli among widely distributed wild ungulates

The environment is considered a major reservoir of antimicrobial resistant microorganisms (AMR) and antimicrobial resistance genes (ARG). Colistin, a “last resort” antibiotic, is used for the treatment of severe infections caused by multidrug-resistant Gram-negative bacteria. The global dissemination of mobile colistin resistance genes (mcr) in natural and non-natural environments is a major setback in the fight against antimicrobial resistance. Hitherto, there is a limited number of studies screening this resistance determinant in bacteria from wildlife. In this study, we describe for the first time the detection of plasmid-mediated colistin resistance in Escherichia coli from wild ungulates in Portugal, which are also widely distributed across Europe. This information is critical to identify the importance of ungulates in the dissemination of resistant bacteria, and their corresponding genes, across the environment. Here, 151 resistant-Enterobacteriaceae isolated from 181 samples collected from different wild ungulate species throughout Portugal were screened for mcr genes. Four mcr-1-positive Escherichia coli were detected from four fallow deer individuals that were sampled in the same hunting ground. These four isolates harboured mcr-1-related IncP plasmids belonging to sequencing types ST155, ST533 and ST345 (n = 2), suggesting bacterial and/or plasmid circulation. All mcr-1-positive E. coli also showed other resistance phenotypes, including MDR, including the B1 commensal phylogenetic profile. All mcr-1-positive E. coli show additional resistance phenotypes, including MDR, including the B1 commensal phylogenetic profile. Our findings are upsetting, highlighting the global dissemination of colistin resistance genes in the whole ecosystem, which, under the One Health framework, emphasizes the urgent need for effective implementation of AMR surveillance and control in the human-animal-environment interfaces.info:eu-repo/semantics/publishedVersio

Mapping the scientific knowledge of antimicrobial resistance in food-producing animals

Antimicrobial resistance (AMR) can be highlighted as one of the most significant health concerns among the last decades, for which antimicrobial drug use in food-producing animals has contributed as one of the major drivers. Food-producing animals are one of the most important and rapidly expanding commercial agricultural sectors worldwide but there is currently limited knowledge on the temporal and geographical distribution of scientific research on antimicrobial resistance in food-producing animals. We provide a global overview of the spatial and temporal trends of scientific knowledge on AMR in food-producing animals. Peer-reviewed papers of AMR on food-producing animals were retrieved from the Web of Science, systemized and dissected. The final validated dataset contained 1341 occurrences observations covering the 1957–2018 period. There has been a shift of research efforts, both geographically and temporally, emphasizing regional differences in food animal production and changing practices in the food production industry. It becomes evident that many regions have been poorly surveyed, wherein intensified sampling and testing efforts should be most valuable. This systematization of knowledge will be crucial in helping to determine how to optimally allocate limited resources available for AMR monitor and control, aiding in the prediction where the threat of new resistant infections will be greatest. AMR research in food-producing animals in developing countries is markedly growing, reflecting changes in food animals production systems but also posing a particularly significant threat, not only due to intensive animal production, but also exacerbated by poor sanitation. We highlight that the use of antibiotics in food producing animals is pervasive, calling for urgent action. These findings raise the possibility to finetuning key priorities on AMR global issues.info:eu-repo/semantics/publishedVersio

Temporal and geographical research trends of antimicrobial resistance in wildlife - A bibliometric analysis

Antimicrobial resistance (AMR) is a complex and global problem. Despite the growing literature on AMR in the medical and veterinary settings, there is still a lack of knowledge on the wildlife compartment. The main aim of this study was to report the global trends in AMR research in wildlife, through a bibliometric study of articles found in the Web of Science database. Search terms were "ANTIMICROBIAL" OR "ANTIBIOTIC" AND "RESISTANT" OR "RESISTANCE" and "WILDLIFE" "MAMMAL" "BIRD" "REPTILE" "FERAL" "FREE RANGE". A total of 219 articles were obtained, published between 1979 and 2019. A rising interest in the last decades towards this topic becomes evident. During this period, the scientific literature was distributed among several scientific areas, however it became more multidisciplinary in the last years, focusing on the "One Health" paradigm. There was a geographical bias in the research outputs: most published documents were from the United States, followed by Spain, Portugal and the United Kingdom. The most productive institutions in terms of publication number were located in Portugal and Spain. An important level of international collaboration was identified. An analysis of the main keywords showed an overall dominance of "AMR", "E. coli", "genes", "prevalence", "bacteria", "Salmonella spp." and "wild birds". This is the first study providing a global overview of the spatial and temporal trends of research related to AMR in wildlife. Given the growth tendency over the last years, it is envisaged that scientific production will expand in the future. In addition to offering a broad view of the existing research trends, this study identifies research gaps both in terms of geographical incidence and in relation to unexplored subtopics. Unearthing scientific areas that should be explored in the future is key to designing new strategic research agendas in AMR research in wildlife and to inform funding programs.info:eu-repo/semantics/publishedVersio

Enterotoxin- and Antibiotic-Resistance-Encoding Genes Are Present in Both Coagulase-Positive and Coagulase-Negative Foodborne Staphylococcus Strains

Food poisoning by staphylococcal enterotoxins (SE) is a major cause of foodborne illness, often associated with coagulase-positive staphylococci (CPS). The increase in the number of methicillin-resistant Staphylococcus aureus (MRSA) strains is another major problem associated with CPS. However, reports of the association of SE and methicillin-resistant Staphylococcus with coagulase-negative staphylococci (CNS) are beginning to re-emerge. In this context, the aim of this study is to investigate the presence of staphylococcal enterotoxin genes and to characterize the phenotypic and genotypic antimicrobial resistance in 66 isolates of Staphylococcus spp. (47 CNS and 19 CPS) recovered from ready-to-eat (RTE) street food sold in Maputo, Mozambique. Seven virulence genes encoding SE (sea, seb, sec, sed and see) and two toxins (hlb and sak) were screened by multiplex PCR (MPCR). Antimicrobial resistance against 12 antibiotics was evaluated by the disk diffusion method. The presence of genes encoding resistance to penicillin, methicillin, vancomycin and erythromycin (blaZ, mecA, vancA, vancB, ermA, ermB and ermC) were also screened by PCR. At least one of the seven virulence genes assessed in this study was detected in 57.9% and 51% of CPS and CNS isolates, respectively. In CPS isolates, the most frequent gene was hlb (47.4%), followed by sec (15.8%) and sea, seb and sed genes with 5.3% each. In CNS isolates, the most frequent gene was sec (36.2%) followed by sak (17%), hlb (14.9%), sed (12.8%) and seb (6.4%). Five of the twelve CPS in which virulence genes were detected were also antibiotic-resistant. All the CNS isolates harboring virulence genes (n = 27, 57.4%) were antimicrobial-resistant. The prevalence of multidrug resistance was higher (59.6%) in CNS than in CPS (26.3%) isolates. Regarding the presence of antibiotic-resistance genes, blaZ (penicillin-resistant) was the most frequent in both CPS (42.1%) and CNS (87.2%), followed by the mecA (encoding methicillin resistance) and vancA genes (vancomycin-resistant), which represented 36.8% and 31.6% in CPS isolates and 46.8% in CNS isolates, respectively. The prevalence of vancomycin-resistant staphylococci has been increasing worldwide and, to our knowledge, this is the first study to report the occurrence of vancomycin-resistant staphylococci in Mozambique. These results emphasize the need to investigate CNS isolates in parallel with CPS, as both constitute public health hazards, given their potential to produce SE and spread antimicrobial resistance genes.info:eu-repo/semantics/publishedVersio

Whole Genome Sequencing Refines Knowledge on the Population Structure of Mycobacterium bovis from a Multi-Host Tuberculosis System

Classical molecular analyses of Mycobacterium bovis based on spoligotyping and Variable Number Tandem Repeat (MIRU-VNTR) brought the first insights into the epidemiology of animal tuberculosis (TB) in Portugal, showing high genotypic diversity of circulating strains that mostly cluster within the European 2 clonal complex. Previous surveillance provided valuable information on the prevalence and spatial occurrence of TB and highlighted prevalent genotypes in areas where livestock and wild ungulates are sympatric. However, links at the wildlife–livestock interfaces were established mainly via classical genotype associations. Here, we apply whole genome sequencing (WGS) to cattle, red deer and wild boar isolates to reconstruct the M. bovis population structure in a multi-host, multi-region disease system and to explore links at a fine genomic scale between M. bovis from wildlife hosts and cattle. Whole genome sequences of 44 representative M. bovis isolates, obtained between 2003 and 2015 from three TB hotspots, were compared through single nucleotide polymorphism (SNP) variant calling analyses. Consistent with previous results combining classical genotyping with Bayesian population admixture modelling, SNP-based phylogenies support the branching of this M. bovis population into five genetic clades, three with apparent geographic specificities, as well as the establishment of an SNP catalogue specific to each clade, which may be explored in the future as phylogenetic markers. The core genome alignment of SNPs was integrated within a spatiotemporal metadata framework to further structure this M. bovis population by host species and TB hotspots, providing a baseline for network analyses in different epidemiological and disease control contexts. WGS of M. bovis isolates from Portugal is reported for the first time in this pilot study, refining the spatiotemporal context of TB at the wildlife–livestock interface and providing further support to the key role of red deer and wild boar on disease maintenance. The SNP diversity observed within this dataset supports the natural circulation of M. bovis for a long time period, as well as multiple introduction events of the pathogen in this Iberian multi-host system.info:eu-repo/semantics/publishedVersio