Eduarda Silveira

Spread of multidrug-resistant Enterococcus to animals and humans: an underestimated role for the pig farm environment

OBJECTIVES 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. METHODS 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. RESULTS 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]. CONCLUSIONS 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.

Co-transfer of resistance to high concentrations of copper and first-line antibiotics among Enterococcus from different origins (humans, animals, the environment and foods) and clonal lineages

OBJECTIVES 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. METHODS 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. RESULTS 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). CONCLUSIONS 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.

Different genetic supports for the tet(S) gene in enterococci

ABSTRACT 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 CTn6000 variants of chromosomal location or composite platforms flanked by IS1216 located on plasmids (∼40 to 115 kb). The comparative analysis of all tet(S) genetic elements available in the GenBank databases suggests that CTn6000 might be the origin of a variety of tet(S)-carrying platforms that were mobilized to different plasmids.

Co-diversification of Enterococcus faecium Core Genomes and PBP5: Evidences of pbp5 Horizontal Transfer

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.

A tet(S/M) hybrid from CTn6000 and CTn916 recombination

We read with interest the article by Barile et al. (2012) which describes the first mosaic tet(S/M) gene encoding tetracycline resistance from foodborne strains of Streptococcus bovis. The mosaic tet genes described to date are hybrids of other ribosome protection type [tet(O), tet(32), tet(W)] which are particularly abundant among Firmicutes (Lactobacillus, Bifidobacterium, Streptococcus, Clostridium) isolated from animals (Stanton et al., 2005; Patterson et al., 2007; van Hoek et al., 2008; Palmieri et al., 2012). The origin and nomenclature of such genetically divergent mosaic tet genes remains controversial due to the lack of information about their genetic contexts and the identical size of the resulting proteins (Zheng et al., 2004, Stanton et al., 2005; Palmieri et al., 2012).

Relevance of tcrYAZB operon acquisition for Enterococcus survival at high copper concentrations under anaerobic conditions

UCIBIO/REQUIMTE, Departamento de Ciências Biológicas, Faculdade de Farmácia, Universidade do Porto, Porto, Portugal; Servicio de Microbiologia, Hospital Ramón y Cajal, Instituto Ramón y Cajal de Investigación Sanitaria (IRYCIS), Madrid, Spain; Centro de Investigación Biomédica en Red de Epidemiologı́a y Salud Pública (CIBER-ESP), Madrid, Spain; Unidad de Resistencia a Antibióticos y Virulencia Bacteriana asociada al Consejo Superior de Investigaciones Cientı́ficas (CSIC), Madrid, Spain; Faculdade de Ciências da Nutrição e Alimentação, Universidade do Porto, Porto, Portugal

Water supply and feed as sources of antimicrobial-resistant Enterococcus spp. in aquacultures of rainbow trout (Oncorhyncus mykiss), Portugal

The role of European fish farms in the spread of antimicrobial-resistance in the environment and food chain, as well as possible sources of their contamination by clinically relevant antimicrobial-resistance bacteria is scarcely known. This study aimed to assess the contribution of Portuguese rural trout farms on dispersion of Enterococcus with antimicrobial-resistance and putative virulence genes in the environment and food chain, as well as to identify farms contamination sources. We also assessed the presence of Enterococcus with low-levels of antimicrobial-resistance using epidemiological cut-offs (ECOFFs). Enterococcus spp. (n=391) from water/sediment recovered upstream, within and downstream trout tanks, feed, trout (2 aquacultures; no antibiotic use) and marketed trout (8 supermarkets) showed variable resistance to tetracycline, erythromycin, ciprofloxacin, chloramphenicol, quinupristin-dalfopristin, nitrofurantoin or aminoglycosides. Antimicrobial-resistance rates were similar among upstream, within and downstream trout tank samples (P>0.05), positioning water-supplying aquacultures as a source of multidrug-resistant (MDR) strains. Nevertheless, predominance of MDR E. faecium in feed, trout tanks and trout comparing to upstream samples, suggests feed as an additional aquaculture contamination source. The observation of E. faecium and E. faecalis susceptible to ampicillin and gentamicin by clinical breakpoints but with low-levels of resistance to those antimicrobials by ECOFFs breakpoints is of concern, as they might evolve throughout secondary genetic events to resistance levels with human clinical impact. Multiple MDR clones carrying copper tolerance (tcrB/cueO), putative virulence or other genes often associated with clinical strains (e.g. E. faecium with IS16/ptsD/sgrA) were observed, some in distinct samples (e.g. upstream and within trout tanks). They included major human and animal Enterococcus lineages, suggesting human and non-aquatic animal origins. The results highlight the need to define the maximum acceptance level of antimicrobial-resistance genes/bacteria to assess water quality and to monitor antimicrobial-resistance strains on feed, essential requirements to maintain a sustainable aquaculture production.