Multilevel population genetic analysis of vanA and vanB Enterococcus faecium causing nosocomial outbreaks in 27 countries (1986-2012).

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.

Clinical response to pandemic H1N1 influenza virus from a fatal and mild case in ferrets.

BACKGROUND: The majority of pandemic 2009 H1N1 (A(H1N1)pdm09) influenza virus (IV) caused mild symptoms in most infected patients, however, a greater rate of severe disease was observed in healthy young adults and children without co-morbid conditions. The purpose of this work was to study in ferrets the dynamics of infection of two contemporary strains of human A(H1N1)pdm09 IV, one isolated from a patient showing mild disease and the other one from a fatal case. METHODS: Viral strains isolated from a patient showing mild disease-M (A/CastillaLaMancha/RR5661/2009) or from a fatal case-F (A/CastillaLaMancha/RR5911/2009), both without known comorbid conditions, were inoculated in two groups of ferrets and clinical and pathological conditions were analysed. RESULTS: Mild to severe clinical symptoms were observed in animals from both groups. A clinical score distribution was applied in which ferrets with mild clinical signs were distributed on a non-severe group (NS) and ferrets with severe clinical signs on a severe group (S), regardless of the virus used in the infection. Animals on S showed a significant decrease in body weight compared to animals on NS at 4 to 7 days post-infection (dpi). Clinical progress correlated with histopathological findings. Concentrations of haptoglobin (Hp) and serum amyloid A (SAA) increased on both groups after 2 dpi. Clinically severe infected ferrets showed a stronger antibody response and higher viral titres after infection (p = 0.001). CONCLUSIONS: The severity in the progress of infection was independent from the virus used for infection suggesting that the host immune response was determinant in the outcome of the infection. The diversity observed in ferrets mimicked the variability found in the human population.

Audouin's gull, a potential vehicle of an extended spectrum ß-lactamase producing Salmonella Agona.

The genome of a multidrug-resistant Salmonella Agona isolated from Larus audouinii (Audouin's gull) in Spain was examined. The isolate showed high levels of resistance to different antimicrobials, including third generation cephalosporins and fluoroquinolones, which is a public health concern as those being used to treat severe salmonellosis in humans. Whole genome sequencing revealed the strain being multilocus sequence type ST13, and eight resistance genes (aadA2, aadB, blaCTX-M-9,blaDHA-1, qnrA1, tetA, sul1 and dfrA16) belonging to seven antimicrobial classes were confirmed, as well as the presence of two plasmids. Migratory Audouin's gulls have the ability to cover long distances during annual movements. Therefore, they have the potential to disseminate multidrug-resistant Salmonella and resistance genes in the environment and over great geographic distances, contributing to the global dissemination of resistance genes.

Heterogeneous pathological outcomes after experimental pH1N1 influenza infection in ferrets correlate with viral replication and host immune responses in the lung.

The swine-origin pandemic (p) H1N1 influenza A virus causes mild upper-respiratory tract disease in most human patients. However, some patients developed severe lower-respiratory tract infections with fatal consequences, and the cause of these infections remain unknown. Recently, it has been suggested that different populations have different degrees of susceptibility to pH1N1 strains due to host genetic variations that are associated with inappropriate immune responses against viral genetic characteristics. Here, we tested whether the pathologic patterns of influenza strains that produce different disease outcomes in humans could be reproduced in a ferret model. Our results revealed that the severities of infection did not correspond to particular viral isolate and were not associated with the clinical phenotypes of the corresponding patients. Severe pathological outcomes were associated with higher viral replication, especially in alveolar areas, and with an exacerbated innate cellular immune response that was characterised by substantial phagocytic and cytotoxic cell migration into the lungs. Moreover, detrimental innate cellular responses were linked to the up-regulation of several proinflammatory cytokines and chemokines and the down-regulation of IFNα in the lungs. Additionally, severe lung lesions were associated with greater up-regulations of pro-apoptotic markers and higher levels of apoptotic neutrophils and macrophages. In conclusion, this study confirmed that the clinicopathological outcomes of pH1N1 infection in ferrets were not only due to viral replication abilities but also depended on the hosts' capacities to mount efficient immune responses to control viral infection of the lung.

Antimicrobial resistance of zoonotic and commensal bacteria in Europe: the missing link between consumption and resistance in veterinary medicine.

The emergence of resistance in food animals has been associated to the consumption of antimicrobials in veterinary medicine. Consequently, monitoring programs have been designed to monitor the occurrence of antimicrobial resistant bacteria. This study analyses the amount of antimicrobial agents used in nine European countries from 2005 to 2011, and compares by univariate analysis the correlations between consumptions of each of the following antimicrobial classes; tetracycline, penicillins, cephalosporins, quinolones and macrolides. An overview of resistance in zoonotic and commensal bacteria in Europe focusing on Salmonella, Escherichia coli, Campylobacter sp. and Enterococcus sp., during the same period of time based on monitoring programs is also assessed. With the exception of cephalosporins, linear regressions showed strong positive associations between the consumption of the four different antimicrobial classes. Substantial differences between countries were observed in the amount of antimicrobials used to produce 1 kg of meat. Moreover, large variations in proportions of resistant bacteria were reported by the different countries, suggesting differences in veterinary practice. Despite the withdrawn of a specific antimicrobial from "on farm" use, persistence over the years of bacteria resistant to this particular antimicrobial agent, was still observed. There were also differences in trends of resistance associated to specific animal species. In order to correlate the use of antimicrobial agents to the presence of resistance, surveillance of antimicrobial consumption by animal species should be established. Subsequently, intervention strategies could be designed to minimize the occurrence of resistance.

Dissemination of clonal Salmonella enterica serovar Typhimurium isolates causing salmonellosis in Mauritius.

Salmonella enterica serotype Typhimurium is one of the leading causes of salmonellosis in Mauritius, where it has also been associated with outbreaks of foodborne illness. However, little is known about its molecular epidemiology in the country. This study was therefore undertaken to investigate the clonality and source of Salmonella Typhimurium in Mauritius by studying human, food, and poultry isolates by pulsed-field gel electrophoresis (PFGE) and antibiotic minimum inhibitory concentration determination. Forty-nine isolates collected between 2008 and 2011 were analyzed, including 25 stool isolates from foodborne illness outbreaks and sporadic gastroenteritis cases, four blood isolates, one postmortem colon isolate, 14 food isolates, and five poultry isolates. All isolates were pansusceptible to the 16 antibiotics tested, except for two isolates that were resistant to sulfamethoxazole and trimethoprim. Overall characterization of the isolates by PFGE digested with XbaI and BlnI resulted in eight different patterns. The largest of the clusters in the composite dataset consisted of 20 isolates, including two raw chicken isolates, four poultry isolates, and nine human stool isolates from two outbreaks. A second cluster consisted of 18 isolates, of which 12 originated from human blood and stool samples from both sporadic and outbreak cases. Six food isolates were also found in this cluster, including isolates from raw and grilled chicken, marlin mousse, and cooked pork. One poultry isolate had a closely related PFGE pattern. The results indicate that one clone of Salmonella Typhimurium found in poultry has been causing outbreaks of foodborne illness in Mauritius and another clone that has caused many cases of gastrointestinal illness and bacteremia in humans could also be linked to poultry. Thus, poultry appears to be a major reservoir for Salmonella Typhimurium in Mauritius. Initiating on-farm control strategies and measures against future dissemination may substantially reduce the number of cases of salmonellosis in the country.

Expansion of a plasmid classification system for Gram-positive bacteria and determination of the diversity of plasmids in Staphylococcus aureus strains of human, animal, and food origins.

An expansion of a previously described plasmid classification was performed and used to reveal the plasmid content of a collection of 92 Staphylococcus aureus strains of different origins. rep genes of other genera were detected in Staphylococcus. S1 pulsed-field gel electrophoresis (PFGE) hybridizations were performed with 18 representative S. aureus strains, and a high number of plasmids of different sizes and organizations were detected.

Antimicrobial resistance in Salmonella spp. recovered from patients admitted to six different hospitals in Tehran, Iran from 2007 to 2008.

The objective of this study was to assess the genotypic diversity associated with antimicrobial susceptibility of Salmonella serovars isolated from patients arriving with diarrhoea to six hospitals of Tehran, Iran. During 2007-2008, a cross-sectional convenience study was performed. Stool samples were screened for the presence of Salmonella, serotyped, tested for antimicrobial susceptibility using disk diffusion and examined for the presence of relevant resistance genes and integrons by PCR. A total of 1,120 patients were screened for the presence of Salmonella. Out of 71 Salmonella isolates recovered, the following serovars were identified: 17 Typhi, 14 Paratyphi C, 13 Enteritidis, 11 Paratyphi B, 10 Paratyphi A and six Infantis. Most resistance was observed towards sulfamethoxazole (30%), tetracyclines (25%), nalidixic acid (22%), spectinomycin (17%), trimethoprim (15%), ampicillin (14%) and kanamycin (14%). The tetracycline resistance genes tet(A), tet(B), and tet(G) were found in 28%, 14% and 6% of the tetracycline resistant isolates, respectively. The genes aadA, aadB, strA, strB and aphA1-Iab were present in 83%, 55%, 34%, 1% and 17% of the aminoglycoside resistant isolates, respectively. Additionally, bla (PSE) and bla (TEM) ß-lactamase genes were detected in 63% and 18% of the ampicillin-resistant isolates. The 23 sulphonamide resistant isolates harboured sul1 and intI1 genes, typical to class 1 integrons. Nine of these isolates also yielded amplicons for intI2 (class 2 integrons). The presence of multi-drug resistant Salmonella may compromise the successful treatment of enteric infection diseases. The enforcement of strict prescription practices will help to minimise the emergence of resistance.

Antimicrobial-resistant Shigella infections from Iran: an overlooked problem?

OBJECTIVES: In this study, we wanted to assess the level of antimicrobial resistance, the presence of genes encoding resistance to cephalosporins and plasmid-mediated quinolone resistance (PMQR), and genetic relatedness among Shigella isolates obtained from Iranian patients. METHODS: A total of 44 Shigella isolates were collected from Iranian patients admitted to Milad Hospital, Tehran, Iran, during 2008-10. Of these, 37 were serotyped and characterized by MIC determination. A subset of eight suspected extended-spectrum ß-lactamase (ESBL) producers (six Shigella sonnei phase II and two Shigella flexneri type 1b) were examined for the presence of genes encoding cephalosporin resistance. The presence of PMQR was assessed in one S. flexneri isolate exhibiting low-level resistance to ciprofloxacin and susceptibility to nalidixic acid. PFGE was performed on 25 S. sonnei phase II isolates. RESULTS: Of the isolates, 25 (68%) were S. sonnei phase II, with 5 (14%) S. flexneri, 5 (14%) Shigella dysenteriae type 2, and 2 (5%) Shigella boydii type 2. Resistance to at least threeclasses of antimicrobials was detected in all species. The presence of bla(CTX-M-15) and the AmpC ß-lactamase producer bla(CMY-2) was confirmed in five and one S. sonnei phase II isolates, respectively. One of the two S. flexneri type 1b that contained bla(CTX-M-15) also harboured a qnrS1 gene. PFGE identified sevenPFGE profiles; the main cluster included 15 of the strains, suggesting low genetic diversity between isolates or the presence of an endemic clone in Iran. CONCLUSIONS: This is the first known description of ESBL-producing and AmpC ß-lactamase-producing Shigella and of PMQR Shigella in Iran. The emergence of CTX-15, CMY-2 and qnrS1 genes may compromise the treatment of shigellosis. Strategies to minimize the spread of ESBL-producing and AmpC-ß-lactamase-producing Shigella should be implemented.

Establishing streptomycin epidemiological cut-off values for Salmonella and Escherichia coli.

This study was conducted to elucidate the accuracy of the current streptomycin epidemiological cut-off value (ECOFF) for Escherichia coli and Salmonella spp. A total of 236 Salmonella enterica and 208 E. coli isolates exhibiting MICs between 4 and 32 mg/L were selected from 12 countries. Isolates were investigated by polymerase chain reaction for aadA, strA, and strB streptomycin resistance genes. Out of 236 Salmonella isolates, 32 (13.5%) yielded amplicons for aadA (n = 23), strA (n = 9), and strB (n = 11). None of the 60 Salmonella isolates exhibiting MIC 4 mg/L harbored resistance genes. Of the Salmonella isolates exhibiting MICs 8 mg/L, 16 mg/L, and 32 mg/L, 1.6%, 15%, and 39%, respectively, tested positive for one or more genes. For most monitoring programs, the streptomycin ECOFF for Salmonella is wild type (WT) ≤32 or ≤16 mg/L. A cut-off value of WT ≤32 mg/L would have misclassified 13.5% of the strains as belonging to the WT population, since this proportion of strains harbored resistance genes and exhibited MICs ≤32 mg/L. Out of 208 E. coli strains, 80 (38.5%) tested positive for aadA (n = 69), strA (n = 18), and strB (n = 31). Of the E. coli isolates exhibiting MICs of 4 mg/L, 8 mg/L, 16 mg/L, and 32 mg/L, 3.6%, 17.6%, 53%, and 82.3%, respectively, harbored any of the three genes. Based on the European Committee on Antimicrobial Susceptibility Testing guidelines (ECOFF ≤16 mg/L), 25% of the E. coli strains presenting MIC ≤16 mg/L would have been incorrectly categorized as belonging to the WT population. The authors recommend an ECOFF value of WT ≤16 mg/L for Salmonella and WT ≤8 mg/L for E. coli.

Presence of glycopeptide-encoding plasmids in enterococcal isolates from food and humans in Denmark.

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.

Epidemiological investigation of Salmonella enterica serovar Kedougou in Thailand.

OBJECTIVE: Salmonella enterica serovar Kedougou is among the top 10 serovars reported in northern Thailand. The objective of this study was to identify risk factors associated with Salmonella Kedougou infection in Thailand and to compare the molecular types and antimicrobial resistance with Salmonella Kedougou isolates of human origin from United States and of animal origin from the United Kingdom. METHODS: Data from 13,976 Salmonella infections of which 253 were Salmonella Kedougou collected in Thailand between 2002 and 2008 were analyzed by logistic regression. Antimicrobial susceptibility testing and pulsed-field gel electrophoresis (PFGE) were performed on selected Salmonella Kedougou strains causing infections in Thailand (n = 66), and compared to isolates from the United States (n = 5) and the United Kingdom (n = 20). RESULTS: Logistic analysis revealed season (hot/dry; p = 0.023), region (northern Thailand; p < 0.001), and specimen (stool; p < 0.001) as significant risk factors associated with Salmonella Kedougou infection compared to other nontyphoid Salmonella. Of the Salmonella Kedougou isolates of human origin, 84% exhibited resistance to at least three antimicrobial classes. Three strains recovered from human stool in Thailand were resistant to third-generation cephalosporins: two harbored bla(CTX-M-63) and one bla(CMY-2). PFGE revealed 45 unique clusters. Isolates obtained from humans in Thailand and the United States presented identical PFGE profiles suggesting a travel association, whereas the majority of the animal isolates from United Kingdom clustered separately. CONCLUSIONS: This study reveals Salmonella Kedougou as a major cause of human infections in northern Thailand especially during the hot period and suggests a global spread probably due to travel. The clonal types causing infections in humans differed from those observed in animals in United Kingdom, which suggests the absence of an epidemiological link and could suggest differences in virulence. The high frequency of antimicrobial resistance, including emergence of resistance to fluoroquinolones and third-generation cephalosporins, might pose problems for treatment of infections.

Host range of enterococcal vanA plasmids among Gram-positive intestinal bacteria.

OBJECTIVES: The most prevalent type of acquired glycopeptide resistance is encoded by the vanA transposon Tn1546 located mainly on transferable plasmids in Enterococcus faecium. The limited occurrence in other species could be due to the lack of inter-species transferability and/or stability of Tn1546-containing plasmids in other species. We investigated the in vitro transferability of 14 pre-characterized vanA-containing plasmids hosted by E. faecium (n = 9), Enterococcus faecalis (n = 4) and Enterococcus raffinosus (n = 1) into several enterococcal, lactobacterial, lactococcal and bifidobacterial recipients. METHODS: A filter-mating protocol was harmonized using procedures of seven partner laboratories. Donor strains were mated with three E. faecium recipients, three E. faecalis recipients, a Lactobacillus acidophilus recipient, a Lactococcus lactis recipient and two Bifidobacterium recipients. Transfer rates were calculated per donor and recipient. Transconjugants were confirmed by determining their phenotypic and genotypic properties. Stability of plasmids in the new host was assessed in long-term growth experiments. RESULTS: In total, 282 enterococcal matings and 73 inter-genus matings were performed and evaluated. In summary, intra-species transfer was far more frequent than inter-species transfer, if that was detectable at all. All recipients of the same species behaved similarly. Inter-genus transfer was shown for broad host range control plasmids (pIP501/pAMß1) only. Acquired resistance plasmids remained stable in the new host. CONCLUSIONS: Intra-species transfer of enterococcal vanA plasmids was far more frequent than transfer across species or genus barriers and may thus explain the preferred prevalence of vanA-containing plasmids among E. faecium. A reservoir of vanA plasmids in non-enterococcal intestinal colonizers does not seem to be reasonable.

Porcine-origin gentamicin-resistant Enterococcus faecalis in humans, Denmark.

During 2001-2002, high-level gentamicin-resistant (HLGR) Enterococcus faecalis isolates were detected in 2 patients in Denmark who had infective endocarditis and in pigs and pork. Our results demonstrate that these isolates belong to the same clonal group, which suggests that pigs are a source of HLGR E. faecalis infection in humans.

Presence of pRI1: a small cryptic mobilizable plasmid isolated from Enterococcus faecium of human and animal origin.

This study focused on the molecular characterization of a small cryptic, mobilizable plasmid (6038 bp) sequenced from an E. faecium 9631160-1 of poultry origin. Sequence analysis of pRI1 revealed seven open reading frames. pRI1 contained an IS 100% identical to ISEfa4. This insertion element disrupted a putative mobilization gene (mobA) which presented 99% similarity to the one described in plasmid pJS42 (NC_010291). pRI1 harbored a cluster of four coding sequences which exhibited a homology to those described in contig 658 (from nucleotide 8940 to nt 10515) of E. faecium DO. In addition, a rep almost identical to the repA from the pEFNP1 and pKQ10 plasmids from E. faecium was also identified. Presence of the pRI1 replication initiation gene (rep) was analyzed in a panel of 159 E. faecium isolates of human and animal origin from different European countries, of which 60 tested positive for the presence of pRI1-rep. Conjugation experiments verified transfer of the pRI1 together with conjugative plasmids harboring resistance to vancomycin and streptogramin. The presence of pRI1 in enterococcal isolates geographically separated and from different origin demonstrates the ability of enterococci to acquire and transfer mobile genetic elements, emphasizing the need for further studies to reveal the meaning and role that these cryptic plasmids play in nature.

Relevance of hot spots in the evolution and transmission of Tn1546 in glycopeptide-resistant Enterococcus faecium (GREF) from broiler origin.

OBJECTIVES: Glycopeptide-resistant enterococci are still present within the broiler sector, despite the EU ban of avoparcin more than a decade ago. In the present study, we have developed a rapid method for screening the flanking regions at the integration point of Tn1546 in glycopeptide-resistant Enterococcus faecium isolated from broiler farms. METHODS: Total DNA was digested, ligated and amplified using primers from inside Tn1546. The resulting amplicons were purified and sequenced. Two new primers were designed based on obtained sequences. RESULTS: Two main insertion points have been repeatedly found in isolates from the UK (n = 150). The first insertion point revealed that 25 isolates harboured Tn1546 positioned in a sequence with 96% homology to a streptomycin adenyltransferase gene (AY604739) from a Staphylococcus intermedius plasmid. At this insertion point, a direct repeat (GTCCT) was duplicated as previously described, indicating transposition at the target site. Furthermore, this 'hot spot' was also detected in isolates from Norway (2/8) and Denmark (17/20). The second insertion point detected in 45 isolates from the UK revealed integration into an Inc18-like plasmid, most likely by a process of target site recombination. CONCLUSIONS: The presence of a common insertion point for isolates from different geographical areas could suggest the insertion of Tn1546 by transposition in a plasmid-specific site, followed by genetic rearrangement both inside the transposon and in the flanking regions.

A longitudinal study to assess the persistence of vancomycin-resistant Enterococcus faecium (VREF) on an intensive broiler farm in the United Kingdom.

Seven years after the ban of avoparcin, VREF could still be isolated within sectors of the UK broiler industry. The aim of this study was to assess whether there is a carryover of VREF between consecutive flocks of birds, to conduct a preliminary investigation of possible routes of entry of VREF into broiler houses and to follow the dynamics of VREF shed by growing birds. A series of nine visits were made to two of six houses on a conventional broiler farm. A total of 343 vanA VREF were recovered from environmental (95/843) and faecal (248/416) samples. Significant differences were observed in the carryover of VREF between pre- and postcohort postcleaning and disinfection visits (RR 0.57, P=0.006). Ninety-nine percent of the VREF isolates were resistant to more than five antimicrobials, with 42 isolates (n=49) positive for erm(B) and 32 (n=40) for vat(E). Pulsed field gel electrophoresis (PFGE) typing identified 50 PFGE types within 15 different PFGE clusters of 90% similarity, demonstrating a high level of genetic diversity within VREF populations from epidemiologically related broiler flocks and broiler houses. Further characterization of Tn1546 from different clones showed a low diversity of Tn-types, suggesting horizontal transfer of resistance determinants between different genetic clones. Thus, this study does not only show the persistence of VREF but also of multi-drug resistant lineages of VREF.

Transposon characterization of vancomycin-resistant Enterococcus faecium (VREF) and dissemination of resistance associated with transferable plasmids.

OBJECTIVES: VanA glycopeptide resistance has persisted on broiler farms in the UK despite the absence of the antimicrobial selective pressure, avoparcin. This study aimed to investigate the contribution of horizontal gene transfer of Tn1546 versus clonal spread in the dissemination of the resistance. METHODS AND RESULTS: One hundred and one vancomycin-resistant Enterococcus faecium isolated from 19 unrelated farms have been investigated. Tn1546 characterization by long PCR and ClaI-digestions of amplicons showed a very low diversity of Tn types (n=4) in comparison to the high genotypic diversity demonstrated by PFGE (n=62). Conjugation experiments were carried out to assess the transfer of vancomycin resistance. Co-transfer of vanA together with erm(B) positioned on the same conjugative plasmid containing a replicon similar to pRE25 was demonstrated and also the presence of different plasmid replicons, associated with antimicrobial resistance on several unrelated farms. CONCLUSIONS: Horizontal transfer of vancomycin resistance may play a more important role in the persistence of antimicrobial resistance than clonal spread. The presence of different plasmid replicons, associated with antimicrobial resistance on several unrelated farms, illustrates the ability of these enterococci to acquire and disseminate mobile genetic elements within integrated livestock systems.