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Objectives: Vancomycin-resistant Enterococcus faecium (VREfm) have been increasingly reported since the 1980s. Despite the high number of published studies about VRE epidemiology, the dynamics and evolvability of these microorganisms are still not fully understood. A multilevel population genetic analysis of VREfm outbreak strains since 1986, representing the first comprehensive characterization of plasmid content in E. faecium, was performed to provide a detailed view of potential transmissible units., Methods: From a comprehensive MeSH search, we identified VREfm strains causing hospital outbreaks (1986-2012). In total, 53 VanA and 18 VanB isolates (27 countries, 5 continents) were analysed and 82 vancomycin-susceptible E. faecium (VSEfm) were included for comparison. Clonal relatedness was established by PFGE and MLST (goeBURST/Bayesian Analysis of Population Structure, BAPS). Characterization of van transposons (PCR mapping, RFLP, sequencing), plasmids (transfer, ClaI-RFLP, PCR typing of relaxases, replication-initiation proteins and toxin-antitoxin systems, hybridization, sequencing), bacteriocins and virulence determinants (PCR, hybridization, sequencing) was performed., Results: VREfm were mainly associated with major human lineages ST17, ST18 and ST78. VREfm and VSEfm harboured plasmids of different families [RCR, small theta plasmids, RepA_N (pRUM/pLG1) and Inc18] able to yield mosaic elements. Tn1546-vanA was mainly located on pRUM/Axe-Txe (USA) and Inc18-pIP186 (Europe) plasmids. The VanB2 type (Tn5382/Tn1549) was predominant among VanB strains (chromosome and plasmids)., Conclusions: Both strains and plasmids contributed to the spread and persistence of vancomycin resistance among E. faecium. Horizontal gene transfer events among genetic elements from different clonal lineages (same or different species) result in chimeras with different stability and host range, complicating the surveillance of epidemic plasmids., (© The Author 2016. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.)

Enterococci and especially glycopeptides-resistant enterococci (GRE) are a growing concern due to their ability to cause infections in hospitals. Transmission of antimicrobial resistance between reservoirs such as animals, meat, and humans are in most cases linked to transmission of mobile genetic elements (MGE) such as plasmids and transposons. Presence of MGE was tested in all GRE isolated from food in Denmark in 2005-2007 including the first vanA mediated Enterococcus faecalis isolated from food. The ability of these plasmids to transfer and persist among enterococci was investigated using newly developed techniques for classification of plasmids. Replicons associated with sex pheromone-inducible plasmids were detected in all GR E. faecalis, whereas GR Enterococcus faecium contained plasmids known to be widely distributed among enterococci. vanA resistance is common in E. faecium isolates from meat and animals in Europe and is rarely found in E. faecalis. This article describes the first characterization of MGE from vanA mediated E. faecalis, thus linking this resistance genotype to pheromone responding plasmids.

Objectives: A plasmid-encoded multidrug efflux pump, OqxAB, identified in Escherichia coli of porcine origin, was tested for substrate specificity against selected antibiotics, detergents and disinfectants. The ability of horizontal transfer to food-borne pathogens of the Enterobacteriaceae family was also investigated., Methods: The MICs of selected substrates were determined with a broth dilution assay using two isogenic E. coli strains, except for the presence of the oqxAB operon. A derivative of the plasmid encoding OqxAB (pOLA52) was constructed and horizontal transfer to Salmonella Typhimurium, Klebsiella pneumoniae, Kluyvera sp. and Enterobacter aerogenes was investigated. The effect of the presence of the OqxAB pump on susceptibility for selected compounds was investigated using broth dilution assays., Results: The OqxAB pump conferred antimicrobial resistance or reduced susceptibility towards a variety of substrates in E. coli. These included animal growth promoters, antimicrobials, disinfectants and detergents. pOLA52 could readily be transferred to enterobacterial pathogens. Transconjugants showed reduced susceptibility towards chloramphenicol, ciprofloxacin and olaquindox., Conclusions: The plasmid-encoded OqxAB pump has a wide substrate specificity and can be transferred between Enterobacteriaceae conferring reduced susceptibility to a multitude of substrates. These results could indicate some dependence on the outer membrane proteins present in the different species.

Unlabelled: A method for direct detection of antibiotic resistance genes in soil samples has been developed. The tetracycline resistance gene, tet(M), was used as a model. The method was validated on Danish farmland soil that had repeatedly been treated with pig manure slurry containing resistant bacteria. The tet(M) gene was directly detected in 10-80% of the samples from the various farmland soils and could be detected in all samples tested after selective enrichment. To validate the obtained results, the method was applied to garden soil samples where lower prevalence of resistance was found., Result: A detection limit of 10(2)-10(3) copies of the tet(M) gene per gram of soil (in a Bacillus cereus group bacterium) was achieved. tet(M) gene was detected in soil samples with the highest prevalence on farmland treated with pig manure slurry.

In this study, internal size variations in the VanA gene cluster Tn1546, encoding resistance to glycopeptides, is described. Studies of previously uncharacterized size variations of an internal region, encoding the vanX and vanY genes of Tn1546, revealed that these variations were due to the presence of the IS sequence, IS1216V. This IS sequence has previously been found integrated in Tn1546. Integration of the IS1216V element created both deletions and a duplication in a non-essential region of Tn1546. In several isolates, the entire vanY gene was deleted, proving that this gene is non-essential for vancomycin resistance.

Differences were examined among 24 distinct elements mediating VanA-type glycopeptide resistance in enterococci isolated from hospital patients and non-human sources in the UK. The methods used included long-PCR restriction fragment length polymorphism (L-PCR RFLP) analysis and DNA hybridization. All elements had conserved vanRSHAX genes, but variation occurred upstream of vanR and downstream of vanX. Twenty-one VanA elements had significant alterations upstream of vanR in the transposition genes orf1 and orf2: either parts of these genes were absent or they were disrupted by IS1216V or IS3-like insertion sequences. Among VanA elements with alterations downstream of vanX, seven lacked vanY, one lacked both vanY and vanZ, and ten had copies of insertion sequence IS1216V between vanX and vanY. All VanA elements of group D (from geographically and temporally diverse enterococci) were characterized by the presence of an IS1216V/IS3-like/orf1 complex and a point mutation in vanX, both of which were absent from the other 23 groups of VanA elements. This finding is consistent with the dissemination of a stable resistance element. We conclude that L-PCR RFLP analysis, combined with DNA hybridization, merits further development for studying the evolution and epidemiology of VanA resistance elements in enterococci.

The satA gene encoding streptogramin A resistance was detected in virginiamycin-resistant Enterococcus faecium isolates from pigs and broilers. The satA gene was present in 22 of 89 (25%) virginiamycin-resistant E. faecium isolates. It was shown that the satA gene and other gene(s) encoding streptogramin resistance could be transferred between isogenic E. faecium strains at frequencies ranging from 2.3 x 10(-4) to 2.2 x 10(-3) transconjugants per donor.