Staphylococcus aureus is a versatile pathobiont, exhibiting a broad host range, including humans, other mammals, and avian species. Host specificity determinants, virulence, and antimicrobial ...resistance genes are often shared by strains circulating at the animal-human interface. While transmission dynamics studies have shown strain exchange between humans and livestock, knowledge of the source, genetic diversification, and transmission drivers of S. aureus in wildlife lag behind.
In this work, we explore a wide array of S. aureus genomes from different sources in the Iberian Peninsula to understand population structure, gene content and niche adaptation at the human-livestock-wildlife nexus. Through Bayesian inference, we address the hypothesis that S. aureus strains in wildlife originate from humanized landscapes, either from contact with humans or through interactions with livestock. Phylogenetic reconstruction applied to whole genome sequence data was completed with a dataset of 450 isolates featuring multiple clones from the 1990–2022 period and a subset of CC398 strains representing the 2008–2022 period. Phylodynamic signatures of S. aureus from the Iberian Peninsula suggest widespread circulation of most clones among humans before jumping to other hosts. The number of transitions of CC398 strains within each host category (human, livestock, wildlife) was high (88.26 %), while the posterior probability of transitions from livestock to wildlife was remarkably high (0.99). Microbial genome-wide association analysis did not evidence genome rearrangements nor biomarkers suggesting S. aureus niche adaptation to wildlife, thus supporting recent spill overs. Altogether, our findings indicate that S. aureus isolates collected in the past years from wildlife most likely represent multiple introduction events from livestock. The clonal origin of CC398 and its potential to disseminate and evolve through different animal host species are highlighted, calling for management practices at the livestock-wildlife axis to improve biosecurity and thus restrict S. aureus transmission and niche expansion along gradients of human influence.
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•The origin of S. aureus strains in wildlife species was investigated by WGS.•Genome data suggest widespread circulation of clones among humans before host jumps.•Phylodynamics highlight high probability of transitions from livestock to wildlife.•S. aureus strains in wildlife likely represent recent spillover events from livestock.•Gradients of human influence potentially drive multi-host S. aureus transmission patterns.
One of the most important and exclusive characteristics of mycobacteria is their cell wall. Amongst its constituent components are two related families of glycosylated lipids, diphthioceranates and ...phthiocerol dimycocerosate (PDIM) and its variant phenolic glycolipids (PGL). PGL have been associated with cell wall impermeability, phagocytosis, defence against nitrosative and oxidative stress and, intriguingly, biofilm formation. In bacteria from the Mycobacterium tuberculosis complex (MTBC), the biosynthetic pathway of the phenolphthiocerol moiety of PGL depends upon the expression of several genes encoding type I polyketide synthases (PKS), namely ppsA-E and pks15/1 which constitute the PDIM + PGL locus, and that are highly conserved in PDIM/PGL-producing strains. Consensus has not been achieved regarding the genetic organization of pks15/1 locus and knowledge is lacking on its transcriptional signature. Here we explore publicly available datasets of transcriptome data (RNA-seq) from more than 100 MTBC experiments in 40 growth conditions to outline the transcriptional structure and signature of pks15/1, using a differential expression approach to infer the regulatory patterns involving these and related genes. We show that pks1 expression is highly correlated with fadD22, Rv2949c, lppX, fadD29 and, also, pks6 and pks12, with the first three putatively integrating into a polycistronic structure. We evidence dynamic transcriptional heterogeneity within the genes involved in phenolphtiocerol and phenolic glycolipid production, most exhibiting up-regulation upon acidic pH and antibiotic exposure and down-regulation under hypoxia, dormancy, and low/high iron concentration. We finally propose a model based on transcriptome data in which σD positively regulates pks1, pks15 and fadD22, while σB and σE factors exert negative regulation at an upper level.
Spreading of Mycobacterium bovis causing animal tuberculosis (TB) at livestock-wildlife-environment interfaces remains a significant problem. Recently, we provided evidence of widespread ...environmental contamination of an endemic animal TB setting with viable and dormant M. bovis cells able to recover metabolic activity, making indirect transmission via environmental contamination plausible. We now report the first whole genome sequences of M. bovis recovered from the environment. We establish epidemiological links at the environment-animal interface by phylogenomic comparison of these M. bovis genomes with those isolated from livestock and wild ungulates from the same area. Environmental and animal genomes are highly intertwined and distribute similarly into the same M. bovis lineages, supporting several instances of environmental contamination. This study provides compelling evidence of M. bovis excretion into the environment and viability maintenance, supporting the environment as a potential source of new infection. These insights have clear implications for policy formulation, advocating environmental surveillance and an ecosystem perspective in TB control programs.
We report the first whole genome sequences of M. bovis from the environment and establish epidemiological links at the environment-animal interface, demonstrating close phylogenomic relatedness of animal and environmental M. bovis. Definitive evidence of M. bovis excretion into the environment with viability maintenance is provided, supporting the environment as a potential source of new infection. Implications of this work include methodological innovations offering a tool to resolve indirect transmission chains and support customized biosecurity measures. Policy formulation aiming at the control of animal tuberculosis and cost mitigation should consider these findings, encouraging environmental surveillance in official eradication programmes.
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•M. bovis shed from TB-infected animals may remain viable in the environment.•Whole genome sequencing of environmental M. bovis is reported for the first time.•M. bovis lineages distribute similarly at the animal-environment interface.•Environmental and animal M. bovis genomes are phylogenetically highly intertwined.•The environment is a potential source of new infections in animal TB endemic areas.
Animal tuberculosis (TB), caused by Mycobacterium bovis, is maintained in Portugal in a multi-host system, with cattle, red deer and wild boar, playing a central role. However, the ecological ...processes driving transmission are not understood. The main aim of this study was thus to contribute to the reconstruction of the spatiotemporal history of animal TB and to refine knowledge on M. bovis population structure in order to inform novel intervention strategies. A collection of 948 M. bovis isolates obtained during long-term surveillance (2002-2016, 15 years) of cattle (n = 384), red deer (n = 303) and wild boar (n = 261), from the main TB hotspot areas, was characterized by spoligotyping and 8 to 12-loci MIRU-VNTR. Spoligotyping identified 64 profiles and MIRU-VNTR distinguished 2 to 36 subtypes within each spoligotype, enabling differentiation of mixed or clonal populations. Common genotypic profiles within and among livestock and wildlife in the same spatiotemporal context highlighted epidemiological links across hosts and regions, as for example the SB0119-M205 genotype shared by cattle in Beja district or SB0121-M34 shared by the three hosts in Castelo Branco and Beja districts. These genomic data, together with metadata, were integrated in a Bayesian inference framework, identifying five ancestral M. bovis populations. The phylogeographic segregation of M. bovis in specific areas of Portugal where the disease persists locally is postulated. Concurrently, robust statistics indicates an association of the most probable ancient population with cattle and Beja, providing a clue on the origin of animal TB epidemics. This relationship was further confirmed through a multinomial probability model that assessed the influence of host species on spatiotemporal clustering. Two significant clusters were identified, one that persisted between 2004 and 2010, in Beja district, with Barrancos county at the centre, overlapping the central TB core area of the Iberian Peninsula, and highlighting a significant higher risk associated to cattle. The second cluster was predominant in the 2012-2016 period, holding the county Rosmaninhal at the centre, in Castelo Branco district, for which wild boar contributed the most in relative risk. These results provide novel quantitative insights beyond empirical perceptions, that may inform adaptive TB control choices in different regions.
Staphylococcus aureus thrives at animal-human-environment interfaces. A large-scale work from our group indicated that antimicrobial resistance (AMR) in commensal S. aureus strains from wild ...ungulates is associated with agricultural land cover and livestock farming, raising the hypothesis that AMR genes in wildlife strains may originate from different hosts, namely via exchange of mobile genetic elements (MGE). In this work, we generate the largest available dataset of S. aureus draft genomes from wild ungulates in Portugal and explore their mobilome, which can determine important traits such as AMR, virulence, and host specificity, to understand MGE exchange. Core genome multi-locus sequence typing based on 98 newly generated draft genomes and 101 publicly available genomes from Portugal demonstrated that the genomic relatedness of S. aureus from wild ungulates assigned to livestock-associated sequence types (ST) is greater compared to wild ungulate isolates assigned to human-associated STs. Screening of host specificity determinants disclosed the unexpected presence in wildlife of the immune evasion cluster encoded in φSa3 prophage, described as a human-specific virulence determinant. Additionally, two plasmids, pAVX and pETB, previously associated with avian species and humans, respectively, and the Tn553 transposon were detected. Both pETB and Tn553 encode penicillin resistance through blaZ. Pangenome analysis of wild ungulate isolates shows a core genome fraction of 2133 genes, with isolates assigned to ST72 and ST3224 being distinguished from the remaining by MGEs, although there is no reported role of these in adaptation to wildlife. AMR related gene clusters found in the shell genome are directly linked to resistance against penicillin, macrolides, fosfomycin, and aminoglycosides, and they represent mobile ARGs. Altogether, our findings support epidemiological interactions of human and non-human hosts at interfaces, with MGE exchange, including AMR determinants, associated with putative indirect movements of S. aureus among human and wildlife hosts that might be bridged by livestock.
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•We generate a broad dataset of S. aureus draft genomes from wild ungulates.•CgMLST highlights genomic relatedness of S. aureus at the animal-human interface.•The human marker φSa3 prophage is present in ST72 isolates from wild ungulates.•Antimicrobial and heavy metals resistance is encoded in S. aureus shell genome.•MGEs signatures support epidemiological links at wildlife-livestock-human interfaces.
Genome sequencing has reinvigorated the infectious disease research field, 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 different lineages (European and African) to gain insights into the evolutionary forces that shape genetic diversification in M. bovis. Three distinct approaches were used to estimate signs of recombination. Globally, a small number of recombinant events was identified and confirmed by two independent methods with solid support. Still, recombination reveals a weaker effect on M. bovis diversity compared with mutation (overall r/m = 0.037). The differential 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 effort in future comparative genomics studies for which WGS of large datasets from different 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 identification 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.
Mycobacterium avium subsp. paratuberculosis (MAP) is the etiological agent of paratuberculosis, a chronic infection affecting ruminants and other species worldwide. Information on the ecological ...factors that increase infection risk at the livestock-wildlife-environment interface remains scarce. Thus, this work aimed at determining which factors modulate the exposure of a mammal community within a Mediterranean agro-forestry farmstead to MAP. Through field, molecular and ecological modeling approaches, MAP prevalence, distribution and spatial risk at the livestock-wildlife-environment was estimated in the study area by screening 436 samples (cattle, n = 150; wildlife, n = 206; soil, n = 80). Using molecular detection of IS900 as proxy, MAP was identified in ten wild mammal species. Being a central prey of mesocarnivores in Portugal, the high prevalence of MAP in the wild rabbit (19%) may be related with red fox's (22%). MAP was also detected in cattle managed in the farmstead (animal and herd prevalence, 54% and 100%) and in soil (44%), which may perpetuate intraspecies and interspecies transmission. Wildlife diversity showed a positive influence on MAP presence in wild mammals, while wildlife abundance showed a negative effect. Land use variables exerted distinct degrees of impact upon MAP detection in specific groups of mammals: mixed forest cover showed positive influence on carnivores, and shrubland showed positive effect on wild rabbits. The prevalence of MAP in cattle showed a negative influence on the detection of MAP in lagomorph, which may stem from wild rabbit lower density and avoidance of cattle areas. Based on explanatory variables, the spatial prediction of MAP occurrence in wildlife indicated two hotspots with increased exposure risk but future studies are needed to confirm this projection.
This work represents the most comprehensive molecular survey of MAP occurrence and determinants in Mediterranean agroecosystems leveraging the principles and tools of community ecology, debating potential biological and ecological effects underlying MAP transmission.
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•MAP was monitored at the wildlife-livestock interface (n = 436) in an agroforestry farmstead.•MAP DNA was detected in the scats of ten wild mammal species (n = 206).•MAP flow in this Mediterranean setting is endemic, with widespread soil contamination.•Wildlife diversity is a predictor of MAP occurrence in wild mammals.•Land cover distinctly affects MAP occurrence in specific groups of mammals.
Epidemiological surveillance of animal tuberculosis (TB) based on whole genome sequencing (WGS) of
has recently gained track due to its high resolution to identify infection sources, characterize the ...pathogen population structure, and facilitate contact tracing. However, the workflow from bacterial isolation to sequence data analysis has several technical challenges that may severely impact the power to understand the epidemiological scenario and inform outbreak response. While trying to use archived DNA from cultured samples obtained during routine official surveillance of animal TB in Portugal, we struggled against three major challenges: the low amount of
DNA obtained from routinely processed animal samples; the lack of purity of
DNA, i.e., high levels of contamination with DNA from other organisms; and the co-occurrence of more than one
strain per sample (within-host mixed infection). The loss of isolated genomes generates missed links in transmission chain reconstruction, hampering the biological and epidemiological interpretation of data as a whole. Upon identification of these challenges, we implemented an integrated solution framework based on whole genome amplification and a dedicated computational pipeline to minimize their effects and recover as many genomes as possible. With the approaches described herein, we were able to recover 62 out of 100 samples that would have otherwise been lost. Based on these results, we discuss adjustments that should be made in official and research laboratories to facilitate the sequential implementation of bacteriological culture, PCR, downstream genomics, and computational-based methods. All of this in a time frame supporting data-driven intervention.
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•Literature related to antimicrobial resistance of wild boar in Europe is reviewed.•Growing interest in antimicrobial resistance in wild boar is highlighted.•Wild boar are carriers of ...antimicrobial resistant bacteria.•Research trends and future directions are discussed.
Antimicrobial resistance (AMR) has been recognized as an emerging and growing problem worldwide. Knowledge concerning AMR bacteria circulating in wildlife is currently limited, although it could provide important insights into AMR emergence and persistence. Across Europe, wild boar (Sus scrofa) populations have dramatically increased their distribution and number over the last decades. In the context of AMR dynamics, wild boar is a perfect model species to unveil the emergence, spread and persistence of AMR at the human-livestock-wildlife interface. Here, we summarize the current knowledge on the importance of wild boar as a reservoir of antimicrobial resistant bacteria, and its possible use as sentinel species for surveillance. Analyses of available data showed a rising interest on this topic in the last years, highlighting the growing concern on wild boar potential role as AMR facilitator and it is foreseen that the importance of antimicrobial resistance research in wild boar will continue to increase in years to come. Available studieshave been focused on specific bacterial species, particularlyE. coli, Salmonellaspp. andEnterococcusspp., bioindicators of AMR, and have been mainly conducted in three countries: Spain, Portugal and Germany.Strikingly, AMR surveillance in wild boar is uneven and still poorly allocated as many wild boar high-density countries do not yet have publications on the topic.Overall, accumulated data showed thatwild boar are carriers of antimicrobial resistant bacteria, withvariation in the prevalence of bacterial species and thepercentage of resistance to different antibiotics. Thelack of harmonized sampling and testing protocols makes it difficult to compare AMR in wild boar.The need for the establishment of standardised protocols keen to provide quantitative comparable data is highlighted. We finally suggest the long-term monitoring of wild boar as a sentinel species for AMR surveillance in order to inform public policies on this topic.
In response to the rapid emergence and dissemination of antimicrobial resistant bacteria (ARB) and genes (ARGs), integrated surveillance systems are needed to address antimicrobial resistance (AMR) ...within the One Health Era. Wastewater analyses enable biomarker monitoring at the sewershed level, offering timely insights into pathogen circulation and ARB/ARGs trends originating from different compartments. During two consecutive epidemic waves of the COVID-19 pandemic in Portugal, taxonomic and functional composition of raw urban wastewater from two wastewater treatment plants (WWTPs) representing one million in equivalent population, located in the main urban areas of the country, were profiled by shotgun metagenomics. Hospital wastewater from two central hospitals located in the WWTPs catchment areas were also sequenced. The resistome and virulome were profiled using metagenomic assemblies without taxonomic constraint, and then specifically characterized for ESKAPE pathogens.
Urban and hospital wastewater exhibited specific microbiota signatures, Pseudomonadota dominated in the first and Bacteroidota in the latter. Correlation network analyses highlighted 85 (out of top 100) genera co-occurring across samples. The most frequent ARGs were classified in the multidrug, tetracyclines, and Macrolides, Lincosamides, Streptogramins (MLS) classes. Links established between AMR determinants and bacterial hosts evidenced that the diversity and abundance of ARGs is not restricted to ESKAPE, being also highly predominant among emergent enteropathogens, like Aeromonas and Aliarcobacter, or in the iron (II) oxidizer Acidovorax. The Aliarcobacter genus accumulated high abundance of sulphonamides and polymyxins ARGs, while Acinetobacter and Aeromonas hosted the highest abundance of ARGs against beta-lactams. Other bacteria (e.g. Clostridioides, Francisella, Vibrio cholerae) and genes (e.g. vanA-type vancomycin resistance) of public health interest were detected, with targeted monitoring efforts being needed to establish informative baseline data. Altogether, results highlight that wastewater monitoring is a valuable component of pathogen and AMR surveillance in healthy populations, providing a community-representative snapshot of public health trends beyond priority pathogens.
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•Urban and hospital wastewater exhibit specific microbiota signatures.•The diversity and abundance of ARGs is not restricted to ESKAPE pathogens.•The most abundant ARGs correspond to the most consumed antimicrobials in Europe.•The virulome and broad host-range plasmids were differentially enriched in WWTPs.•WBE is a powerful tool to track pathogen and AMR trends of public health interest.
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