The aim of this study was to discover the potential role of the pig farm environment in the spread of multidrug-resistant (MDR) Enterococcus strains, including high-risk clones, to animals and humans. Enterococcus isolates were recovered from a variety of samples (n = 82; swine, feed/medicines/antiseptics and pig farm facilities) from six Portuguese farms, most using antibiotics. Antimicrobial susceptibility/conjugation assays were performed by standard procedures, bacterial identification/screening of antibiotic resistance genes were performed by PCR and clonality was determined using PFGE/multilocus sequence typing. Enterococcus isolates resistant to antibiotics (n = 473) were recovered from samples of different origin (swine, feed/antiseptics, animal residues and pig farm facilities), but only the clinically relevant species Enterococcus faecium (n = 171) and Enterococcus faecalis (n = 78) were included for further comprehensive molecular analysis. Isolates resistant to vancomycin, ampicillin, tetracyclines, erythromycin and aminoglycosides were better recovered in Slanetz–Bartley medium with these antibiotics present than in media not supplemented with antibiotics (P < 0.05). E. faecium was more frequently resistant to ampicillin, ciprofloxacin or nitrofurantoin and E. faecalis to tetracyclines, chloramphenicol or aminoglycosides (P < 0.05). Glycopeptide and erythromycin resistance rates were similar in both species. The transfer of resistance to several antibiotics, including vancomycin and ampicillin, was demonstrated. Clones associated with human infections were detected in different samples from the same farm [E. faecium from sequence type (ST) 78 lineage and E. faecalis ST16; manure, waste lagoons, faeces and drinking water] and in geographically distant farms [E. faecium clonal complex (CC) 5; E. faecalis CC21 and ST16]. The pig farm environment has an underestimated potential role in the transmission of MDR Enterococcus to animals and, possibly, to humans. The continuous contact of swine with MDR Enterococcus by different routes (e.g. feed, dust, air and rooms) might decrease the impact of restrictive antibiotic use policies and reinforces the need for different and preliminary interventions at the husbandry management level.

We studied the occurrence of diverse copper (Cu) tolerance genes from Gram-positive bacteria and their co-transfer with antibiotic resistance genes among Enterococcus from diverse sources. Enterococcus (n = 922) of several species and from human, animal, environment and food samples were included. Antimicrobial and CuSO₄ susceptibility and conjugation assays were performed by standard procedures, bacterial screening of Cu and antibiotic resistance genes by PCR, and clonality by PFGE/multilocus sequence typing. tcrB and cueO genes occurred in 15% (n = 137/922) and 14% (n = 128/922) of isolates, respectively, with the highest occurrence in piggeries (P < 0.05). They were more frequent among Enterococcus faecium (tcrB: 23% versus 8% in Enterococcus faecalis and 12% in other species; cueO: 25% versus 5% and 9%, respectively; P < 0.05). A correlation between phenotypic and genotypic assays was observed for most E. faecium (CuSO₄ MIC₅₀ = 24 mM in tcrB/cueO⁺ versus CuSO₄ MIC₅₀ = 12 mM in tcrB/cueO⁻), but not for other species. Co-transfer of Cu tolerance (associated with tcrB, cueO or an unknown mechanism) with erythromycin, tetracycline, vancomycin, aminoglycosides or ampicillin resistance was demonstrated. A variety of PFGE types was detected among isolates carrying Cu tolerance mechanisms, some identified in sequence types (STs) often linked to human infections (E. faecium from ST18 and ST78 clonal lineages and E. faecalis clonal complex 2). Cu tolerance might contribute to the selection/maintenance of multidrug-resistant Enterococcus (including resistance to first-line antibiotics used to treat enterococcal infections) due to the use of Cu compounds (e.g. antiseptics/animal feed supplements). The distribution of the multicopper oxidase cueO and the co-transfer of ampicillin resistance along with Cu tolerance genes are described for the first time.

Ampicillin resistance has greatly contributed to the recent dramatic increase of a cluster of human adapted Enterococcus faecium lineages (ST17, ST18 and ST78) in hospital-based infections. Changes in the chromosomal pbp5 gene have been associated with different levels of ampicillin susceptibility, leading to protein variants (designated as PBP5 C-types to keep the nomenclature used in previous works) with diverse degrees of reduction in penicillin affinity. Our goal was to use a comparative genomics approach to evaluate the relationship between the diversity of PBP5 among E. faecium isolates of different phylogenomic groups as well as to assess the pbp5 transferability among isolates of disparate clonal lineages. The analyses of 78 selected E. faecium strains as well as published E. faecium genomes, suggested that the diversity of pbp5 mirrors the phylogenomic diversification of E. faecium. The presence of identical PBP5 C-types as well as similar pbp5 genetic environments in different E. faecium lineages and clones from quite different geographical and environmental origin was also documented and would indicate their horizontal gene transfer among E. faecium populations. This was supported by experimental assays showing transfer of large (≈180-280 kb) chromosomal genetic platforms containing pbp5 alleles, ponA (transglycosilase) and other metabolic and adaptive features, from E. faecium donor isolates to suitable E. faecium recipient strains. Mutation profile analysis of PBP5 from available genomes and strains from this study suggests that the spread of PBP5 C-types might have occurred even in the absence of a significant ampicillin resistance phenotype. In summary, genetic platforms containing pbp5 sequences were stably maintained in particular E. faecium lineages, but were also able to be transferred among E. faecium clones of different origins, emphasizing the growing risk of further spread of ampicillin resistance in this nosocomial pathogen.

The diversity of tet (S) genetic contexts of 13 enterococci from human, animal, and environmental samples from different geographical areas is reported. The tet (S) gene was linked to either CTn 6000 variants of chromosomal location or composite platforms flanked by IS 1216 located on plasmids (∼40 to 115 kb). The comparative analysis of all tet (S) genetic elements available in the GenBank databases suggests that CTn 6000 might be the origin of a variety of tet (S)-carrying platforms that were mobilized to different plasmids.

Antimicrobial resistance (AMR) poses a global threat to human, animal and environmental health. Among the multidisciplinary tasks aimed at collectively tackling the AMR crisis, surveillance, research and education stand as major priorities. Based on a crowdsourcing research strategy, the MicroMundo project, a partner of the Tiny Earth initiative in Spain and Portugal, has been developed and consolidated with success in the academic environment. The objectives are focused on promoting research and, especially, on bringing knowledge of One Health and microbiology concepts, as well as AMR awareness to the community. Following a service‐learning approach, MicroMundo integrates university and secondary/high school students in a citizen science‐based research project to collectively isolate microorganisms with the potential to produce new antibiotics from soil environments. Over the last 7 years, 32 MicroMundo hubs operating across 31 different Portuguese and Spanish universities have recruited thousands of teenagers in this quest. Here we review the outcome of this unprecedented effort from a scientific and an educational perspective.