Persistence of animal and human glycopeptide-resistant enterococci on two Norwegian poultry farms formerly exposed to avoparcin is associated with a widespread plasmid-mediated vanA element within a polyclonal enterococcus faecium population

The evolutionary processes responsible for the long-term persistence of glycopeptide-resistant Enterococcus faecium (GREF) in nonselective environments were addressed by genetic analyses of E. faecium populations in animals and humans on two Norwegian poultry farms that were previously exposed to avoparcin. A total of 222 fecal GREF (n = 136) and glycopeptide-susceptible (n = 86) E. faecium (GSEF) isolates were obtained from farmers and poultry on three separate occasions in 1998 and 1999. Pulsed-field gel electrophoresis (PFGE) and plasmid DNA analyses discerned 22 GREF and 32 GSEF PFGE types within shifting polyclonal animal and human E. faecium populations and indicated the presence of transferable plasmid-mediated vanA resistance, respectively. Examples of dominant, persistent GREF PFGE types supported the notion that environmentally well-adapted GREF types may counteract the reversal of resistance. PFGE analyses, sequencing of the purK housekeeping gene, and partial typing of vanA-containing Tn1546 suggested a common animal and human reservoir of glycopeptide resistance. Inverse PCR amplification and sequence analyses targeting the right end of the Tn1546-plasmid junction fragment strongly indicated the presence of a common single Tn1546-plasmid-mediated element in 20 of 22 GREF PFGE types. This observation was further strengthened by vanY-vanZ hybridization analyses of plasmid DNAs as well as the finding of a physical linkage between Tn1546 and a putative postsegregation killing system for seven GREF PFGE types. In conclusion, our observations suggest that the molecular unit of persistence of glycopeptide resistance is a common mobile plasmid-mediated vanA-containing element within a polyclonal GREF population that changes over time. In addition, we propose that "plasmid addiction systems" may contribute to the persistence of GREF in nonselective environments.     

Molecular characterization of Enterococcus faecium isolated from hospitalized patients in Korea

AIMS: Multilocus sequence typing (MLST) was performed for vancomycin-resistant Enterococcus faecium (VREF) from diverse geographical areas in Korea to obtain insights into the genetic relationships with other molecular profiles. To understand the diversity of lineages, vancomycin-susceptible E. faecium (VSEF) were included. METHODS AND RESULTS: A total of 60 E. faecium isolates were analysed by MLST and esp profile. Molecular typing of Tn1546 of 30 VREF strains was evaluated by overlapping PCR of Tn1546 and DNA sequencing. Seven sequence types (ST) were found among 30 VSEF isolates, and four STs were found among 30 VREF isolates. The types most frequently encountered were ST 78 (26 isolates) and ST 203 (16 isolates). Of the 60 E. faecium isolates, 35 isolates were positive for the esp gene. On molecular typing of Tn1546, all VREF isolates were divided into four main types. Strains with the same ST showed divergence in Tn1546 types and strains with the same Tn1546 type represented different STs. CONCLUSIONS: An association between Tn1546 typing and MLST was not found. SIGNIFICANCE AND IMPACT OF THE STUDY: These results suggest that the horizontal spread of Tn1546 between strains plays a major role in the dissemination of vancomycin resistance in Korea.     

High-resolution melting genotyping of Enterococcus faecium based on multilocus sequence typing derived single nucleotide polymorphisms

We have developed a single nucleotide polymorphism (SNP) nucleated high-resolution melting (HRM) technique to genotype Enterococcus faecium. Eight SNPs were derived from the E. faecium multilocus sequence typing (MLST) database and amplified fragments containing these SNPs were interrogated by HRM. We tested the HRM genotyping scheme on 85 E. faecium bloodstream isolates and compared the results with MLST, pulsed-field gel electrophoresis (PFGE) and an allele specific real-time PCR (AS kinetic PCR) SNP typing method. In silico analysis based on predicted HRM curves according to the G+C content of each fragment for all 567 sequence types (STs) in the MLST database together with empiric data from the 85 isolates demonstrated that HRM analysis resolves E. faecium into 231 "melting types" (MelTs) and provides a Simpson's Index of Diversity (D) of 0.991 with respect to MLST. This is a significant improvement on the AS kinetic PCR SNP typing scheme that resolves 61 SNP types with D of 0.95. The MelTs were concordant with the known ST of the isolates. For the 85 isolates, there were 13 PFGE patterns, 17 STs, 14 MelTs and eight SNP types. There was excellent concordance between PFGE, MLST and MelTs with Adjusted Rand Indices of PFGE to MelT 0.936 and ST to MelT 0.973. In conclusion, this HRM based method appears rapid and reproducible. The results are concordant with MLST and the MLST based population structure.     

Antibiotic resistance and virulence traits in clinical and environmental Enterococcus faecalis and Enterococcus faecium isolates

This study compared virulence and antibiotic resistance traits in clinical and environmental Enterococcus faecalis and Enterococcus faecium isolates. E. faecalis isolates harboured a broader spectrum of virulence determinants compared to E. faecium isolates. The virulence traits Cyl-A, Cyl-B, Cyl-M, gel-E, esp and acm were tested and environmental isolates predominantly harboured gel-E (80% of E. faecalis and 31.9% of E. faecium) whereas esp was more prevalent in clinical isolates (67.8% of E. faecalis and 70.4% of E. faecium). E. faecalis and E. faecium isolated from water had different antibiotic resistance patterns compared to those isolated from clinical samples. Linezolid resistance was not observed in any isolates tested and vancomycin resistance was observed only in clinical isolates. Resistance to other antibiotics (tetracycline, gentamicin, ciprofloxacin and ampicillin) was detected in both clinical and water isolates. Clinical isolates were more resistant to all the antibiotics tested compared to water isolates. Multi-drug resistance was more prevalent in clinical isolates (71.2% of E. faecalis and 70.3% of E. faecium) compared to water isolates (only 5.7% E. faecium). tet L and tet M genes were predominantly identified in tetracycline-resistant isolates. All water and clinical isolates resistant to ciprofloxacin and ampicillin contained mutations in the gyrA, parC and pbp5 genes. A significant correlation was found between the presence of virulence determinants and antibiotic resistance in all the isolates tested in this study (p<0.05). The presence of antibiotic resistant enterococci, together with associated virulence traits, in surface recreational water could be a public health risk.     

Vancomycin-resistant Enterococcus faecium (VREF) from Norwegian poultry cluster with VREF from poultry from the United Kingdom and The Netherlands in an amplified fragment length polymorphism genogroup

The genetic relationship between 197 vancomycin-resistant Enterococcus faecium (VREF) isolates and 21 vancomycin-susceptible E. faecium isolates from Norwegian poultry was analyzed by amplified fragment length polymorphism (AFLP). The isolates were compared to 255 VREF isolates from various sources and countries. The Norwegian isolates constituted a relatively homogeneous population of E. faecium and clustered in a previously defined poultry AFLP genogroup.     

Antimicrobial susceptibility of<Emphasis Type="Italic">Enterococcus</Emphasis> species isolated from slovak bryndza cheese

Three hundred and ten enterococcal isolates (178 Enterococcus faecium, 68 E. durans, 49 E. faecalis, 8 E. italicus, 3 E. gallinarum, 3 E. casseliflavus, and 1 E. hirae) from Slovak Bryndza cheese were evaluated for susceptibility to nine antimicrobial agents (vancomycin, teicoplanin, ampicillin, streptomycin, gentamicin, erythromycin, rifampicin, nitrofurantoin, and ciprofloxacin). All enterococcal isolates from Bryndza cheese were susceptible to ampicillin, streptomycin, gentamicin, vancomycin, and teicoplanin as determined by the disk diffusion method. Vancomycin resistance genes vanA and vanB were not detected. Resistance rates of enterococcal isolates to rifampicin, erythromycin, ciprofloxacin, and nitrofurantoin were 24, 26, 2, and 1 %, respectively. Thirty-six % of E. faecium isolates and 22 % of the E. faecalis isolates were resistant to erythromycin. Resistance to rifampicin was similar in E. faecium (31 %) and E. faecalis (29 %). Both E. faecium and E. faecalis strains showed the same resistance to ciprofloxacin (2 %). E. durans isolates showed low levels of resistance to rifampicin, erythromycin, ciprofloxacin, and nitrofurantoin (1-4 %). Forty-eight (30 %) of the E. faecium isolates, two (3 %) of the E. durans isolates, and six (12 %) of the E. faecalis isolates exhibited multidrug resistance. The highest frequency of resistant enterococci was observed in Bryndza produced in winter season.     

Antimicrobial susceptibilities of enterococci isolated from faeces of broiler and layer chickens

Enterococci were isolated from faecal droppings of chickens in broiler and layer farms and the susceptibilities to nine therapeutic antimicrobial agents and six growth-promoting antibiotics were determined by the agar dilution method. Resistance to therapeutic antimicrobial agents such as ampicillin, clindamycin, erythromycin, streptomycin, tetracycline or tylosin was more frequent in enterococcal isolates from broiler farms than in those from layer farms. Resistance to ofloxacin was rare, occurring in only one (0.7%) of the Enterococcus faecium isolates from broiler farms. Resistance to growth-promoting antibiotics such as avilamycin, salinomycin and virginiamycin was common among isolates from broiler farms. Of the E. faecium isolates from broiler farms, 12.4% were resistant to avilamycin and 27.4% were resistant to virginiamycin. Resistance to salinomycin was detected in all enterococcal species, ranging from 12.4% of E. faecium isolates to 50% of E. hirae isolates.     

Galleria mellonella as a model for studying Enterococcus faecium host persistence

Enterococcus faecium is an opportunistic pathogen responsible for numerous outbreaks worldwide. The basis for the colonization capacities, host persistence and environmental stress response of the hospital-adapted clones emerging from E. faecium are poorly understood. In this study, we propose the use of Galleria mellonella as a simple nonmammalian model to assess E. faecium host persistence. Various strains (n = 10), including hospital-adapted, commensal or animal isolates and a SodA-deficient strain were used to assess the relevance of this model. Compared to Enterococcus faecalis, E. faecium strains do not appear very lethal in a Galleria killing assay. The ability of E. faecium strains to overcome host-immune responses and multiply within the host system was evaluated by monitoring bacterial loads following Galleria infection. Among the E. faecium strains, two hospital-adapted isolates displayed increased colonization ability. In contrast, inactivation of sodA, encoding a putative manganese-dependent superoxide dismutase, significantly reduced survival of E. faecium to Galleria defenses. Galleria appears to be a suitable and convenient surrogate model to study E. faecium survival to host defenses and the role of suspected virulence factors in the colonization process.     

Clinical isolates of Enterococcus faecium exhibit strain-specific collagen binding mediated by Acm,a new member of the MSCRAMM family

A collagen-binding adhesin of Enterococcus faecium, Acm, was identified. Acm shows 62% similarity to the Staphylococcus aureus collagen adhesin Cna over the entire protein and is more similar to Cna (60% and 75% similarity with Cna A and B domains respectively) than to the Enterococcus faecalis collagen-binding adhesin, Ace, which shares homology with Acm only in the A domain. Despite the detection of acm in 32 out of 32 E. faecium isolates, only 11 of these (all clinical isolates, including four vancomycin-resistant endocarditis isolates and seven other isolates) exhibited binding to collagen type I (CI). Although acm from three CI-binding vancomycin-resistant E. faecium clinical isolates showed 100% identity, analysis of acm genes and their promoter regions from six non-CI-binding strains identified deletions or mutations that introduced stop codons and/or IS elements within the gene or the promoter region in five out of six strains, suggesting that the presence of an intact functional acm gene is necessary for binding of E. faecium strains to CI. Recombinant Acm A domain showed specific and concentration-dependent binding to collagen, and this protein competed with E. faecium binding to immobilized CI. Consistent with the adherence phenotype and sequence data, probing with Acm-specific IgGs purified from anti-recombinant Acm A polyclonal rabbit serum confirmed the surface expression of Acm in three out of three collagen-binding clinical isolates of E. faecium tested, but in none of the strains with a non-functional pseudo acm gene. Introduction of a functional acm gene into two non-CI-binding natural acm mutant strains conferred a CI-binding phenotype, further confirming that native Acm is sufficient for the binding of E. faecium to CI. These results demonstrate that acm, which encodes a potential virulence factor, is functional only in certain infection-derived clinical isolates of E. faecium, and suggest that Acm is the primary adhesin responsible for the ability of E. faecium to bind collagen.     

Antibiotic resistance and virulence factors among clinical and food enterococci isolated in Slovakia

The resistance to antibiotics and the distribution of virulence factors in enterococci isolated from traditional Slovak sheep cheese bryndza was compared with strains from human infections. The occurrence of 4 enterococcal species was observed in 117 bryndza-cheese isolates. The majority of strains were identified as E. faecium (76 %) and E. faecalis (23 %). Several strains of E. durans and 1 strain of E. hirae were also present. More than 90 % of strains isolated from 109 clinical enterococci were E. faecalis, the rest belonged to E. faecium. The resistance to 6 antimicrobial substances (ampicillin, ciprofloxacin, higher concentration of gentamicin, nitrofurantoin, tetracycline and vancomycin) was tested in clinical and food enterococci. A higher level of resistance was found in clinical than in food strains and E. faecium had a higher resistance than E. faecalis; no resistance to vancomycin was detected. The occurrence of 3 virulence-associated genes, cylA (coding for hemolysin), gelE (coding for gelatinase) and esp (coding for surface protein) was monitored. Differences were found in the distribution of cylA gene between clinical and bryndza-cheese E. faecalis strains; in contrast to clinical strains (45 %), cylA gene was detected in 22 % of food isolates. The distribution of 2 other virulence factors, gelE and esp, was not significantly different in the two groups of E. faecalis strains. cylA and gelE genes were not detected in E. faecium but more than 70 % of clinical E. faecium were positive for esp, even thought none of the 79 E. faecium cheese isolates contained this gene.     

A Comparison of BOX-PCR and Pulsed-Field Gel Electrophoresis to Determine Genetic Relatedness of Enterococci from Different Environments

Genetic relatedness of enterococci from poultry litter to enterococci from nearby surface water and groundwater in the Lower Fraser Valley regions of British Columbia, Canada was determined. A new automated BOX-PCR and Pulsed-Field Gel Electrophoresis (PFGE) were used to subtype enterococcal isolates from broiler and layer litter and surface and groundwater. All surface water samples (n = 12) were positive for enterococci, as were 11% (3/28) of groundwater samples. Enterococcus faecium (n = 90) was isolated from all sources, while Enterococcus faecalis (n = 59) was isolated from all sources except layer litter. The majority of E. faecalis originated from broiler litter (28/59; 47.5%) while the majority of E. faecium were isolated from layer litter (29/90; 32.2%). E. faecalis grouped primarily by source using BOX-PCR. Isolates from water samples were dispersed more frequently among PFGE groups containing isolates from poultry litter. E. faecium strains were genetically diverse as overall clustering was independent of source by both molecular methods. Subgroups of E. faecium isolates based upon source (layer litter) were present in BOX-PCR groups. Three individual E. faecalis groups and two individual E. faecium groups were 100% similar using BOX-PCR; only one instance of 100% similarity among isolates using PFGE was observed. Although enterococci from litter and water sources were grouped together using BOX-PCR and PFGE, isolates originating from water could not be definitively identified as originating from poultry litter. Automation of BOX-PCR amplicon separation and visualization increased the reproducibility and standardization of subtyping using this procedure.     

A genetic element present on megaplasmids allows Enterococcus faecium to use raffinose as carbon source

Enterococcus faecium is a commensal of the gastrointestinal tract of humans and animals. Since the 1990s, it has also emerged as a nosocomial pathogen. Little is known about carbon metabolism of E. faecium even though the ability to utilize different sugars could be an important factor in adapting to different ecological niches. In this study we identify an E. faecium gene cluster that is responsible for the metabolism of the α-galactoside sugar raffinose. Phenotypic testing of seven E. faecium isolates of which the genomes were previously sequenced showed that one isolate (strain E980) could grow on raffinose. Genome analysis identified a gene cluster containing two genes encoding α-galactosidases (termed agaA and agaB) that was uniquely present in E980. The agaA and agaB genes were significantly more frequently found in strains that are phylogenetically related to E980 and were more prevalent in surveillance isolates from hospital and community sources than in isolates from clinical infections. Disruption of the α-galactosidase gene agaB, but not of agaA, disabled growth on raffinose in strain E980. In all strains agaA and agaB are carried on megaplasmids that are between 150 and 300 kb in size. Filter-mating experiments showed that the megaplasmid of E980 can be transferred to a plasmidless recipient which then gains the ability to grow on raffinose. The observation that raffinose utilization by E. faecium is a trait carried by megaplasmids indicates that these megaplasmids can have important roles in shaping the competitive fitness of E. faecium in the environment, for example by expanding the metabolic repertoire of this organism.     

Virulence factors and<Emphasis Type="Italic">in Vitro</Emphasis> adherence of<Emphasis Type="Italic">Enterococcus</Emphasis> strains to urinary catheters

The ability to adhere in vitro to urinary catheters and the presence of enterococcal virulence factors was determined in 30 Enterococcus urinary isolates (12 E. faecalis, 12 E. faecium, 3 E. casseliflavus, 3 E. gallinarum). Silicone, siliconized latex and polyvinyl chloride (PVC) were examined by sonication quantitative culture technique and scanning electron microscope. As compared to E. faecalis and E. faecium, E. casseliflavus and E. gallinarum displayed lower adhesion to all synthetic materials. All the tests performed showed higher adherence of all tested strains to siliconized latex and silicone than to PVC. Biofilmforming ability was observed in 5 E. faecalis but in none of the remaining strains. The gene coding enterococcal surface protein (Esp) was detected in 7 E. faecalis and 6 E. faecium strains. Gelatinase was found in 1 E. faecalis, 2 E. faecium and hemolysins were found in 6 E. faecalis and 1 E. faecium strains. All E. casseliflavus and E. gallinarum strainswere negative for these traits. Hydrophobic type of cell surface (measured by its affinity for n-hexadecane) was shown in a few isolates. Bacterial adherence was not significantly associated with the above pathogenic factors.     

Molecular confirmation of Enterococcus faecalis and E. faecium from clinical, faecal and environmental sources

AIMS: The genus Enterococcus includes opportunistic pathogens such as E. faecalis and E. faecium, and is also used to assess water quality. Speciation of enterococci in environmental studies can be particularly problematic, therefore protocols for unambiguous, DNA-based analysis could receive wide use in applications ranging from water quality monitoring to microbial source tracking. The goal of this work was to investigate the usefulness of PCR for speciation of putative, biochemically identified E. faecalis and E. faecium isolated from water, faeces and sewage. METHODS AND RESULTS: Putative enterococci (n = 139) were isolated on mEI agar from dog, human, gull and cow faeces, and from sewage, freshwaters and marine waters. A total of 128 isolates passed standard physiological tests for the genus, and were speciated by the API 20 Strep (APIStrep) biochemical test system. 42.2% were identified as E. faecalis, and all were confirmed by PCR. 19.5% were biochemically identified as E. faecium, but only seven were PCR-positive. CONCLUSIONS: The 16S rDNA of PCR-positive and PCR-negative E. faecium, including isolates that were inconclusively identified by APIStrep, was sequenced. All formed a monophyletic clade with E. faecium sequences in Genbank. SIGNIFICANCE AND IMPACT OF THE STUDY: Biochemical identification of E. faecalis agreed 100% with PCR assays, therefore a simple protocol of isolation on mEI followed by PCR should be useful for environmental studies. Discrepancies among biochemical identification, PCR confirmation and DNA sequencing were noted for E. faecium, indicating that routine isolation/identification of E. faecium from environmental samples is a much more difficult task.     

Identification of enterococci by ribotyping with horseradish-peroxidase-labelled 16S rDNA probes.

Enterococci are frequently associated with hospital-acquired infection. Identification of enterococci using conventional biochemical tests are often tedious to perform in a routine diagnostic laboratory and may give equivocal results. This study evaluates the usefulness of ribotyping by DNA hybridisation to identify 68 members of the bacterial genus Enterococcus characterised by a conventional test scheme. DNA probes (830 bp in size) were derived from the 16S rRNA gene of E. coli or E. faecalis by PCR, labelled with horseradish peroxidase and used in Southern blot hybridisations of enterococcal DNA digested with EcoRI. Unique ribotypes were obtained for 11 different species using 12 Enterococcus type strains. Ribotyping identified 44 E. faecalis isolates, 19 E. faecium isolates, two E. durans isolates and one E. avium isolate in concordance with results of the biochemistry tests. Two isolates that had ribotype patterns identical to the E. faecium type strain were unable to be definitively identified by biochemical tests. The results show that ribotyping is able to differentiate between E. faecium and E. faecalis and may be useful for identifying other enterococci in the hospital setting. In addition, ribotyping using DNA probes and enhanced chemiluminescence is a safe and more reproducible alternative to radiolabelling RNA in a clinical microbiology laboratory.     

Genotypic diversity, antimicrobial resistance, and virulence factors of human isolates and probiotic cultures constituting two intraspecific groups of Enterococcus faecium isolates

The intraspecific relationships among a collection of Enterococcus faecium isolates comprising probiotic cultures and human clinical isolates were investigated through the combined use of two high-resolution DNA-fingerprinting techniques. In addition, the incidences of antimicrobial resistance and virulence traits were investigated. A total of 128 E. faecium isolates from human clinical or nonclinical sources or used as probiotic cultures were subjected to fluorescent amplified fragment length polymorphism (FAFLP) fingerprinting and pulsed-field gel electrophoresis (PFGE) analysis of SmaI macrorestriction patterns. Susceptibilities to 16 antimicrobial agents were tested using broth microdilution, and the presence of the corresponding resistance genes was investigated using PCR. Multiplex PCR was used to detect the presence of the enterococcal virulence genes asa1, gelE, cylA, esp, and hyl. The results of the study showed that two intraspecific genomic groups (I and II) were obtained in FAFLP analysis. PFGE analysis demonstrated high variability within these two groups but also indicated that some probiotic cultures were indistinguishable and that a number of clinical isolates may be reisolations of commercial probiotic cultures. Compared to group II, which contained the majority of the probiotic isolates and fewer human clinical isolates, higher phenotypic and genotypic resistance frequencies were observed in group I. Two probiotic isolates were phenotypically resistant to erythromycin, one of which contained an erm(B) gene that was not transferable to enterococcal recipients. None of the probiotic E. faecium isolates demonstrated the presence of the tested virulence genes. The previously reported observation that E. faecium consists of two intraspecific genomic groups was further substantiated by FAFLP fingerprinting of 128 isolates. In combination with antimicrobial resistance and virulence testing, this grouping might represent an additional criterion in assessing the safety of new potential probiotic E. faecium isolates.     

Antibiotic resistance and genotypic characterization by PFGE of clinical and environmental isolates of enterococci

Fifty-four Enterococcus faecalis and 20 Enterococcus faecium isolates from clinical and non-human sources in Rome, Italy, were characterized by antibiotic resistance and pulsed field gel electrophoresis (PFGE). Resistance to vancomycin, teicoplanin, ampicillin, and ciprofloxacin was more frequent in E. faecium than in E. faecalis, whereas high-level resistance to aminoglycoside was found primarily in E. faecalis. Multi-resistance was found primarily among clinical isolates, but was also observed among environmental isolates. Common genotypes shared among clinical and environmental isolates were observed, however, the majority of isolates occurred as unique, source-specific clones. Several PFGE types were associated with shared features in their antibiotic resistance patterns; evidences of clonal spread between and within wards were also noted. This is the first report indicating clonal relatedness between human and environmental enterococci isolated in Italy.     

Detection of virulence factors in high-level gentamicin-resistant Enterococcus faecalis and Enterococcus faecium isolates from a Tunisian hospital

Phenotypic and genotypic determination of virulence factors were carried out in 46 high-level gentamicin-resistant (HLGR) clinical Enterococcus faecalis (n=34) and Enterococcus faecium (n=12) isolates recovered from different patients in La Rabta Hospital in Tunis, Tunisia, between 2000 and 2003 (all these isolates harboured the aac(6')-aph(2") gene). The genes encoding virulence factors (agg, gelE, ace, cylLLS, esp, cpd, and fsrB) were analysed by PCR and sequencing. The production of gelatinase and hemolysin, the adherence to caco-2 and hep-2 cells, and the capacity for biofilm formation were investigated in all 46 HLGR enterococci. The percentages of E. faecalis isolates harbouring virulence genes were as follows: gelE, cpd, and ace (100%); fsrB (62%); agg (56%); cylLLS (41.2%); and esp (26.5%). The only virulence gene detected among the 12 HLGR E. faecium isolates was esp (58%). Gelatinase activity was detected in 22 of the 34 E. faecalis isolates (65%, most of them with the gelE+-fsrB+ genotype); the remaining 12 isolates were gelatinase-negative (with the gelE+-fsrB- genotype and the deletion of a 23.9 kb fragment of the fsr locus). Overall, 64% of the cylLLS-containing E. faecalis isolates showed beta-hemolysis. A high proportion of our HLGR E. faecalis isolates, in contrast to E. faecium, showed moderate or strong biofilm formation or adherence to caco-2 and hep-2 cells.     

Persistence of vancomycin-resistant enterococci in New Zealand broilers after discontinuation of avoparcin use

Large amounts of tylosin, zinc-bacitracin, and avilamycin are currently used as prophylactics in New Zealand broiler production. Avoparcin was also used from 1977 to 2000. A total of 382 enterococci were isolated from 213 fecal samples (147 individual poultry farms) using enrichment broths plated on m-Enterococcus agar lacking antimicrobials. These isolates were then examined to determine the prevalence of antimicrobial resistance. Of the 382 isolates, 5.8% (22 isolates) were resistant to vancomycin, and 64.7% were resistant to erythromycin. The bacitracin MIC was > or =256 microg/ml for 98.7% of isolates, and the avilamycin MIC was > or =8 microg/ml for 14.9% of isolates. No resistance to ampicillin or gentamicin was detected. Of the 22 vancomycin-resistant enterococci (VRE) isolates, 18 (81.8%) were Enterococcus faecalis, 3 were Enterococcus faecium, and 1 was Enterococcus durans. However, when the 213 fecal enrichment broths were plated on m-Enterococcus agar containing vancomycin, 86 VRE were recovered; 66% of these isolates were E. faecium and the remainder were E. faecalis. Vancomycin-resistant E. faecium isolates were found to have heterogenous pulsed-field gel electrophoresis (PFGE) patterns of SmaI-digested DNA, whereas the PFGE patterns of vancomycin-resistant E. faecalis isolates were identical or closely related, suggesting that this VRE clone is widespread throughout New Zealand. These data demonstrate that vancomycin-resistant E. faecalis persists in the absence and presence of vancomycin-selective pressure, thus explaining the dominance of this VRE clone even in the absence of avoparcin.     

Antimicrobial resistance ofEnterococcus spp. isolates from raw beef and meat products

E. faecalis (67%) and E. faecium (13.7%) were most frequently isolated among enterococci that contaminate cooled and frozen processed meat, follow-up heat-treated meat products and unheated fermented dry salami. Most isolates of both species were resistant to cephalothin (95 and 83 %) and clindamycin (77 and 67%, respectively). Furthermore, E. faecalis and E. faecium isolates were resistant to erythromycin (44 and 72%), tetracycline (34.5 and 17.4%), and streptomycin (13.3 and 4.3%, respectively). Only a few of the isolates were resistant to ampicillin, ampicillin-sulbactam, chloramphenicol, and vancomycin while all isolates were susceptible to gentamicin, penicillin, and teicoplanin. During the production of heat-treated meat products, numbers of resistant isolates increased in spite of the decreasing enterococcal contamination of the samples. An opposite situation was found in the production of fermented dry salami.