Prevalence,characterization and antibiotic resistance of enterococci from traditional cheeses in Turkey

The aim of the present work is to provide information about Enterococcus strains isolated from traditional Turkish cheese samples in Ankara (Turkey), focusing on their prevalence, phenotypic and genotypic characteristics, and antibiotic resistance. A total of 213 probable enterococcal isolates isolated from 215 samples were identified by phenotypic and genotypic methods. As a result of 16S rDNA sequence analysis, 88 of the 213 enterococci strains were identified as Enterococcus faecium and 125 as Enterococcus faecalis. The E. faecalis strains (58.7%) were identified as the dominant species isolated from cheese samples in Turkey. The 213 Enterococcus strains were tested for susceptibility to 12 different antimicrobial agents. The resistance phenotype were as follow: nalidixic acid (100%), kanamycin (98.6%), rifampicin (78.4%), ampicillin (48.8%), ciprofloxacin (45.5%), erythromycin (18.8%), tetracycline (11.7%), penicillin G (5.6%), chloramphenicol (4.2%), gentamycin (3.8%) and streptomycin (1.4%). None of the strains was resistant to vancomycin. E. faecium strains showed more resistant phenotypes than E. faecalis strains as shown by the antibiotic resistance levels. It was also observed that the resistance of E. faecium and E. faecalis strains against the antibiotics was statistically significant (p ? 0.05). In total, 100% of E. faecium and 88.8% of E. faecalis strains were resistant to multiple drugs.     

Prevalence of enterococci in frozen dairy products and their pathogenicity

Enterococci were present in all the samples of frozen dairy products examined in the present study. The total bacterial counts as well as enterococcal counts were generally very high in Kulfi (a frozen product similar to ice cream) and Kulfi mix. Among 161 enterococcal isolates recovered, the predominant type was Streptococcus faecalis var. faecalis, the majority of isolates coming from Kulfi and Kulfi mix samples. Although 58 cultures produced deoxyribonuclease, only two of them exhibited thermonuclease (TNase) production. Hyaluronidase activity was shown by three strains. However, 31 strains were alpha haemolytic, while five showed beta haemolysis on rabbit blood agar. Out of these haemolytic strains tested, 21 produced lethal toxicity in mice and 20 strains showed culture virulence, postmortem studies revealed congestion of heart, liver, kidney and spleen. The two TNase positive strains of Streptococcus faecium produced fluid accumulation in ligated ileal loops of rabbits and distension in the gastrointestinal tracts of infant mice.     

Safety aspects of enterococci from the medical point of view

Enterococci occur in a remarkable array of environments. They can be found in soil, food, and water, and make up a significant portion of the normal gut flora of humans (10(5)-10(7)/g of stool) and animals. As other bacteria of the gut flora, enterococci can also cause infectious diseases. Most clinical isolates are Enterocococus faecalis, which account for 80-90% of clinical strains. Enterocococus faecium accounts for 5-10% of such isolates. Typical enterococcal infections occur in hospitalised patients with underlying conditions representing a wide spectrum of severity of illness and immune modulation. Enterococci today rank second to third in frequency among bacteria isolated from hospitalised patients. They are isolated from urinary tract infections, intra-abdominal and pelvic infections, bacteremias, wound and tissue infections, and endocarditis--often as part of a polymicrobial flora. Surprisingly, little is known about the factors that contribute to the ability of enterococci to cause infections. Many strains of E. faecalis produce a cytolysin (haemolysin) exhibiting tissue-damaging capacity. Further extracellular products often observed in clinical isolates are a proteinase (gelatinase), hyaluronidase, and extracellular superoxide. Furthermore, many of the clinical isolates possess the aggregation substance on the surface and an extracellular surface protein, both contributing to the adherence to eucaryotic cells. Some strains of E. faecalis, and many E. faecium strains are resistant to multiple antimicrobials. The ultimate role of all these factors in enterococcal pathogenicity remains to be determined. It was previously thought that enterococcal infections were endogenously acquired from the patient's own gut flora. A rather new concept that has emerged is that enterococcal disease is a two-stage process. There is an initial colonisation of the gastrointestinal tract by enterococcal strains possessing virulence traits and/or antibiotic resistance. Subsequently, this population spreads, often facilitated by antibiotic elimination of competitors. For a selected number of patients, there is subsequent tissue invasion from the gastrointestinal tract reservoir. From this concept, it can be deduced that enterococcal strains without virulence traits and antibiotic resistances exogenously transferred into the human gut via food products or probiotics will not represent any risk for immunocompetent individuals. In very severely immunocompromised patients, however, a risk for enterococcal disease by such strains cannot completely be excluded.     

Molecular diversity among natural populations of <Emphasis Type="Italic">Lactobacillus paracasei</Emphasis> and <Emphasis Type="Italic">Lactobacillus plantarum</Emphasis>/<Emphasis Type="Italic">paraplantarum</Emphasis> strains isolated from autochthonous dairy products

The diversity of 87 Lactobacillus paracasei and Lactobacillus plantarum/paraplantarum strains, previously identified from different autochthonous dairy products, was investigated by phenotypic and genotypic approaches. The increased resolution obtained using phenotypic and genotypic characterization allowed the level of strain heterogeneity detection to be widened. Phenotypic diversity was evaluated by studying biochemical characteristics of technological interest, including antimicrobial and proteinase activities, resistance to nisin, aggregation ability, production of exopolysaccharides, acetoin and diacetyl, citrate utilization, and antibiotic susceptibility. Genotypic diversity was generally evaluated by PCR amplification of repetitive bacterial DNA element fingerprinting using the (GTG)₅ primer [(GTG)₅-PCR]. Moreover, in cases where strains were not discriminated by (GTG)₅-PCR combined with phenotypic analysis, pulsed-field gel electrophoresis (PFGE) analysis was performed. The results indicate that L. plantarum/paraplantarum and L. paracasei natural isolates from artisanal dairy products are a gold mine in terms of diversity of strains and could be potentially interesting to dairy companies for the formulation of functional starter cultures in the production of innovative foods.     

Production of bacteriocins by <Emphasis Type="Italic">Enterococcus</Emphasis> spp. isolated from traditional,Iranian, raw milk cheeses,and detection of their encoding genes

Strong bacteriocins, or bacteriocins with a wide range of activity against pathogens and spoilage microorganisms, are actively sought for use as natural food preservatives. This work reports the inhibitory activity of 96 enterococcal isolates from two Iranian, raw milk cheeses against five indicator organisms (including Listeria innocua). Forty-eight isolates inhibited at least one indicator in spot agar assays. Of these, 20 isolates corresponding to 15 different strains were shown to produce bacteriocin-like substances in liquid cultures. PCR analysis revealed the genes coding for enterocins (enterococcal bacteriocins) A, B, P or X, or their combinations, in all but one of these 15 strains. In addition, the gene coding for enterocin 31 was detected in two strains. No amplification was obtained in one strain when using specific primers for all 13 bacteriocin genes sought. Three different enterocin genes were identified in most strains and four in one strain. Although the concomitant production of bacteriocins is still to be verified, producers of multiple enterocins could be of great technological potential as protective cultures in the cheese industry.     

Enterococci as probiotics and their implications in food safety

Enterococci belong to the lactic acid bacteria (LAB) and they are of importance in foods due to their involvement in food spoilage and fermentations, as well as their utilisation as probiotics in humans and slaughter animals. However, they are also important nosocomial pathogens that cause bacteraemia, endocarditis and other infections. Some strains are resistant to many antibiotics and possess virulence factors such as adhesins, invasins, pili and haemolysin. The role of enterococci in disease has raised questions on their safety for use in foods or as probiotics. Studies on the incidence of virulence traits among enterococcal strains isolated from food showed that some can harbour virulence traits, but it is also thought that virulence is not the result of the presence of specific virulence determinants alone, but is rather a more intricate process. Specific genetic lineages of hospital-adapted strains have emerged, such as E. faecium clonal complex (CC) 17 and E. faecalis CC2, CC9, CC28 and CC40, which are high risk enterococcal clonal complexes. These are characterised by the presence of antibiotic resistance determinants and/or virulence factors, often located on pathogenicity islands or plasmids. Mobile genetic elements thus are considered to play a major role in the establishment of problematic lineages. Although enterococci occur in high numbers in certain types of fermented cheeses and sausages, they are not deliberately added as starter cultures. Some E. faecium and E. faecalis strains are used as probiotics and are ingested in high numbers, generally in the form of pharmaceutical preparations. Such probiotics are administered to treat diarrhoea, antibiotic-associated diarrhoea or irritable bowel syndrome, to lower cholesterol levels or to improve host immunity. In animals, enterococcal probiotics are mainly used to treat or prevent diarrhoea, for immune stimulation or to improve growth. From a food microbiological point of view, the safety of the bacteria used as probiotics must be assured, and data on the major strains in use so far indicate that they are safe. The advantage of use of probiotics in slaughter animals, from a food microbiological point of view, lies in the reduction of zoonotic pathogens in the gastrointestinal tract of animals which prevents the transmission of these pathogens via food. The use of enterococcal probiotics should, in view of the development of problematic lineages and the potential for gene transfer in the gastrointestinal tract of both humans and animals, be carefully monitored, and the advantages of using these and new strains should be considered in a well contemplated risk/benefit analysis.     

Identification and characterization of Enterococcus spp. in Greek spontaneous sausage fermentation

A total of 108 enterococcal strains previously isolated from spontaneously fermented sausages were identified using phenotypic traits, pulsed-field gel electrophoresis (PFGE), and sequencing of the 16S rRNA gene. The proteolytic and lipolytic activities of these isolates and their ability to decarboxylate lysine, tyrosine, ornithine, and histidine and to produce antimicrobial compounds also were assessed. All strains were identified as Enterococcus faecium, and a lack of correlation between data derived from phenotypic and those derived from genotypic techniques was evident. Wide strain diversity was revealed by both phenotypic properties and PFGE strain typing results. Few strains were present in all batches, suggesting a possible persistence in the respective production plants. Neither proteolytic nor lipolytic activities were detected, and none of the strains decarboxylated lysine, tyrosine, ornithine, or histidine. A total of 42 E. faecium strains inhibited in vitro growth of Listeria monocytogenes, which suggests possible contribution of these strains to the safety of the end product and possible utilization of these strains as protective cultures.     

Phylogenetic analysis of Rhodococcus erythropolis based on the variation of ribosomal proteins as observed by matrix-assisted laser desorption ionization-mass spectrometry without using genome information

Rhodococcus erythropolis strains characterized as antibiotic producers can be classified into three groups according to their antibiotic spectrum and growth compatibility. Due to their high genotypic similarity, the taxonomic relationship of these strains has not been elucidated. In this study, ribosomal protein profiling using matrix-assisted laser desorption ionization-mass spectrometry (MALDI-MS) was employed to classify twenty-one strains of R. erythropolis (15 antibiotic producers and 6 non-antibiotic producers). In the first step in this method, a total of 30 intense peaks observed for purified ribosomal subunit proteins of the type strain (R. erythropolis JCM 3201^T) were selected as the reference peaks. The mass spectra observed for the cell lysates of each sample strain were then checked as to whether peaks were observed at the same masses of the reference peaks. The results of peak matching were processed by cluster analysis, generating a dendrogram. Four major clusters of the R. erythropolis strains corresponded to three antibiotic groups and the non-antibiotic group. Furthermore, the topology of the dendrogram was highly comparable with the phylogenetic tree based on DNA gyrase subunit B gene (gyrB) sequencing. These results indicate that our proposed ribosomal protein profiling method using MALDI-MS is a potentially reliable and sufficiently high-throughput technique for the taxonomic analysis of closely related bacterial strains without using DNA sequence information.     

Optimization and Partial Purification of Bacteriocins from Enterococcus spp. Indigenous to Pakistan

This study was conducted to optimize bacteriocin producing enterococcal strains isolated from indigenous fermented dairy products of Pakistan. Isolates IJ-06, IJ-21, and IJ-31 were identified as Enterococcus faecium and IJ-11 was identified as Enterococcus faecalis. All of these enterococcal isolates were catalase, gelatinase negative, and non-hemolytic on sterile sheep blood. Bacteriocin production trials confirmed E. faecium IJ-06, IJ- 21, and IJ- 31 as the potential producer of bacteriocin showing antimicrobial activity in cell-free supernatant (CFS). E. faecalis IJ-11 displayed activity after partial purification. Optimization of culture conditions for the production of bacteriocin by the selected strains showed that maximum production was achieved at 35–37°C with 1% inoculum after 16 h of incubation and at pH 7–8. The molecular mass of the partially purified enterocins falls in the range of 4.5–4.7 kDa. These enterocins were close to class IIa of bacteriocins and were highly active against Listeria monocytogenes, Bacillus subtilis, Bacillus cereus and other closely related strains.     

Identification and preliminary characterization of strains of enterococci and micrococci isolated from Arzúa raw cows'-milk cheese

152 enterococcal isolates and 88 micrococcal isolates obtained from secondary microflora of Arzúa raw cows'-milk cheese at various stages of ripening were characterized. The most frequent Enterococcus specie was E. faecalis. The most frequent Micrococcus specie was M. varians, followed by M. luteus and M. lylae; 27 micrococcal isolates could not be assigned to species. Although most strains of micrococci did not cause appreciable acidification of milk, the proportions of micrococcal isolates displaying proteolytic and lipolytic activities were higher than those for enterococcal isolates. The biochemical and enzymatic characteristics of isolates from both groups displayed considerable variability, even within the same species. In screening microorganisms of this type for suitability as components of cheese starter cultures, it is therefore necessary, as has been pointed out by previous authors, to work with single strains.     

Methods used for the isolation, enumeration, characterisation and identification of Enterococcus spp. 2. Pheno- and genotypic criteria

This paper reviews the methodology applied for the identification and characterisation of enterococci and covers phenotypic, genotypic and phylogenetic techniques. Although conventional phenotypic typing schemes are useful for rapid and simple identification of enterococcal species for routine applications, other methods like standardised sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE), multilocus enzyme electrophoresis (MLEE), antimicrobial susceptibility testing, serotyping, pyrolysis mass spectrometry (pyMS) and vibrational spectroscopic methods allow a more in-depth characterisation of enterococci. Many of the recently described enterococcal species exhibit deviations from hitherto so-called classical enterococci with regard to their phenotypical properties. Therefore, genotypic methods have to be used to clarify their possible assignment to the genus Enterococcus. In this review, special emphasis is given on recently developed polymerase chain reaction (PCR)-based typing methods such as random amplified polymorphic DNA (RAPD), amplified fragment length polymorphism (AFLP), specific and random amplification (SARA) and modifications of PCR-ribotyping as well as pulsed-field gel electrophoresis (PFGE) and partial sequence analysis. The use of PCR and probes for genus and species identification of enterococci is also considered like the application of sequence data of conserved DNA regions (e.g., ribosomal ribonucleic acid (rRNA) genes) in the case of species identification.     

Probiotic and Technological Properties of Enterococci Isolates from Infants and Cheese

Abstract

In the present study 16 enterococcal strains isolated from infant feces and/or Feta cheese were tested for their ability to metabolize in milk and resist specific conditions of the gastrointestinal tract, in order to finally select interesting strains for further studies, for their evaluation as dietary adjuncts. The strains were characterized by phenotypic criteria (API ID 32 STREP) and sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE). Whole-cell protein analysis by SDS-PAGE confirmed the taxonomic allocation of 14 strains suggested by phenotypic criteria as E. durans (4 strains), E. faecalis (3 strains), and E. faecium (7 strains). Two strains biochemically characterized as E. faecalis were assigned to other species by SDS-PAGE. All strains grew well in bile concentrations 0–1%. The strains were also able to withstand low pH (3.0) values for 4?h, eventhough for some strains a reduction by ～1–2 log₁₀ cfu/mL was observed. Most of the strains produced tyramine and all of them were susceptible to vancomycin. In addition, six out of seven E. faecium strains from either feces or cheese were β-haemolytic. The strains exhibited antagonistic activities towards enterococci, lactic acid bacteria, and Cl. sporogenes. A considerable phenotypic diversity was found among the test strains concerning their acidifying and proteolytic activities. Results of this study give useful information for the selection of appropriate strains with interesting probiotic and technological properties.     

Enterococci from Tolminc cheese: population structure, antibiotic susceptibility and incidence of virulence determinants

Microbiological analysis of ripened artisanal Tolminc cheese revealed the presence of an enterococcal population in numbers of up to 10(6) per g. All colonies, isolated from the citrate azide tween carbonate (CATC) enterococcal selective medium were Gram positive and coccal-shaped and were analysed with PhenePlate FS system. This system discriminated 10 PhP clusters among the 90 enterococcal isolates. From each cluster the most representative isolate for that particular type was selected for further study. The 10 representative enterococci were catalase negative and grew in the presence of NaCl (2%, 4% and 6.5%) and bile salts (0.06%). Genus specific primers confirmed all 10 enterococcal representatives as Enterococcus members, while species specific primers determined them further as strains of Enterococcus faecalis species. PCR for vanA and vanB genes detection, respectively, amplified no PCR products. The absence of van genes was confirmed with both disc and E-test, as isolates were susceptible to vancomycin according to the National Committee for Clinical Laboratory Standards (NCCLS). The results of disc tests with other antimicrobial agents (ampicillin, vancomycin, kanamycin, penicillin, erythromycin, neomycin, chloramphenicol, clindamycin, rifampin) did not differ much among the tested enterococci: they were all very resistant to clindamycin only. The incidence of enterococcus virulence determinants was as expected: all of the 10 E. faecalis strains tested possessed multiple determinants (between 7 and 11).     

The Cronobacter sp. in milk and dairy products: Detection and typing

Cronobacter is a group of important foodborne pathogens that have been implicated in meningitis, sepsis and necrotising enterocolitis, especially in neonates through consumption of contaminated powdered infant formula (PIF). The detection of PIF contamination is of great concern to the infant formula industry. In this article, phenotypic and genotypic methods for detecting and typing Cronobacter are discussed in relation to minimising and controlling the risk of Cronobacter sp. contamination. To reliably detect and type Cronobacter strains, the use of conventional microbiological methods in combination with molecular assays is recommended.     

Taxonomy, ecology and antibiotic resistance of enterococci from food and the gastro-intestinal tract

Apart from genotypic identification methods, there is a need for reliable conventional phenotypic identification schemes for simple and rapid determination of enterococcal species in food or in the gastro-intestinal tract (GIT). Only a limited number of enterococcal species is of importance for the ecology of the GIT or the food microflora, including E. faecalis, E. faecium, E. durans/hirae, E. gallinarum and E. casseliflavus. After genus identification the differentiation within these species can include, e.g. mannitol and arabinose fermentation and growth at 50 degrees C. Widely used commercial identification systems may fail to precisely identify rare species. Ecological aspects should also be taken into account. In the human GIT E. faecium is the most common species whereas in most animal species E. faecalis is at least present in the same amount. Especially in foods of animal origin (cheese, pork meat, beef, poultry meat) also E. faecalis is very frequent. This is of special interest as glycopeptide resistance is most often found in human clinical E. faecium strains as well as in E. faecium from the environment or animal samples and less frequent in E. faecalis strains. EU experts propose as safety criteria for probiotics in feed additives the exclusion of resistances or the lack of transferability. This proposal can also be applied to enterococci in foods. Specific resistances must be excluded, but transferability or acquisition of resistance (e.g. vancomycin) cannot be excluded per se. However, technologically used strains should differ from clinical strains concerning their resistance patterns and transfer rates.     

Evidence of mixed wild populations of Oenococcus oeni strains during wine spontaneous malolactic fermentations

Two hundred and four bacterial isolates from Rioja red wines undergoing spontaneous malolactic fermentation (MLF) were studied. Bacterial species was determined both by microbiological identification methods and by specific PCR analysis. Oenococcus oeni was shown to be the predominant species (98.5% of total isolates). Pulsed field gel electrophoresis (PFGE) of chromosomal DNA digested with SfiI was used to differentiate individual strains of O. oeni. A wide variety of restriction digest patterns were detected, which indicated a rich biodiversity of indigenous strains. Most fermentations (37 out of 41) showed from 2 to 6 clones growing in the same tank. Five O. oeni strains were the most frequently found, appearing in more than three of the 13 studied wineries, and most times in combination with other less frequently found strains. PFGE was shown to be a suitable method for strain differentiation, for monitoring individual strains and determining which strains actually survive and carry out MLF. A high genotypic heterogeneity of wild O. oeni strains was demonstrated and 90% of the studied wines showed mixed populations of O. oeni strains during MLF.     

Genotypic and technological characterization of enterococci isolated from artisanal Fiore Sardo cheese

A total of 118 enterococcal strains isolated from artisanal Fiore Sardo cheese were characterized technologically and genetically. The presence of potential virulence factors was also investigated. Strains were classified as Ec. faecium (84 strains), Ec. durans (24 strains) and Ec. faecalis (10 strains). RAPD-PCR analysis with two different primers (M13 and XD9) confirmed species identification and proved useful for the detection of interstrain variations, especially among Ec. faecium isolates. Most strains could hydrolyse casein and had weak acidifying activity in milk. None of the isolates produced lipolytic reactions. Gelatinase activity was observed in two strains of Ec. faecalis and one strain of Ec. durans. beta-Haemolysis on horses' blood was never detected in any of the strains, independently of species. Most strains produced tyramine from tyrosine but none decarboxylated lysine, histidine or ornithine. Overall, a wide spectrum of resistance was observed. Almost all strains were resistant to the aminoglycosides, gentamycin, kanamycin, streptomycin, neomycin, and to the semisynthetic penicillin, oxacillin, but resistance to vancomycin was not widespread among our strains: only one Ec. faecium and one Ec. durans strain were found to be vancomycin resistant. Our results show a certain diversity in technological traits of the enterococcal strains isolated from artisanal Fiore Sardo, together with a low incidence of some potentially pathogenic traits of health concern.     

Enterocin typing of enterococci isolated from dried infant foods

One hundred and fifty enterococcal isolates recovered from 16 market samples of infant foods and 35 from other sources were characterized and subjected to enterocin typing with 18 indicator strains. Among 150 enterococcal isolates, 114 (76%) were able to be typed by the indicator strains. Although 24 enterocin patterns were observed with these enterococci, the most prevalent types were X-9, 224, and 65-603. Occurrence of pattern X-9 either singly or in combination with many other types was most frequent. Many of the enterocin patterns in enterococcal isolates were recovered from samples of dairy water supply and hand washings of personnel working in a dairy plant that manufactured infant food; this suggests the possibility of these as sources of contamination. Enterocin typing of enterococci could prove useful in epidemiological studies.     

A survey on biotechnological potential and safety of the novel Enterococcus species of dairy origin, E. italicus

The biotechnological and safety properties of the novel enterococcal species of dairy origin, Enterococcus italicus, were investigated. The strains of the species showed technological characteristics related to their use as adjunct cultures in the production of artisanal cheeses. They were susceptible or poorly resistant to several clinical relevant antibiotics. Moreover, E. italicus strains were associated with low virulence profiles, as verified by screening for the presence of 33 different genes encoding antibiotic resistance and known virulence factors in the genus Enterococcus. From the data obtained, we deduce that the presence of E. italicus strains in cheeses results in a low health risk and that within the species new safe adjunct cultures for the dairy industry could be found.     

Antimicrobial resistance profiles of dairy and clinical isolates and type strains of enterococci

The susceptibility to 30 antimicrobial agents was determined by the disk diffusion method for a collection of 172 enterococcal strains, including 96 isolates from dairy sources, 50 isolates of human and veterinary origin, and 26 reference strains from 24 different enterococcal species. Results were analysed by hierarchic numerical methods to cluster strains and to group antimicrobials according to similarity profiles. Resistance to 17 of the 30 antimicrobials showed to be correlated, leading to four groups reflecting the mode of action: quinolones (ofloxacin, enrofloxacin, ciprofloxacin and norfloxacin); macrolides (erythromycin, spiramycin), phenicols (cloramphenicol) and tetracyclins (tetracycline, oxytetracyclin); aminoglycosides (gentamicin, kanamycin) and lincosamides (clindamycin); penicillins (amoxicillin, ampicillin, penicillin G, piperacillin) and carbapenems (imipenem). Overall, the genus Enterococcus behaved as resistant to lincomycin, colistin, polimixin B and, with a few exceptions in dairy isolates, to methicillin. In general, all isolates were susceptible to vancomycin, cloramphenicol and fusidic acid. Clusters containing only dairy isolates were susceptible to the majority of antimicrobials tested, as opposed to clusters constituted only by clinical enterococcal isolates. Among the clinical isolates, 62% were highly multiresistant. Low level gentamicin resistance was found to be associated with clinical enterococci. Among dairy isolates, those that clustered with clinical isolates were both resistant to gentamicin and identified as Enterococcus faecalis. Resistance to macrolides, quinolones, penicillins and imipenem was found to be associated also with clinical environments, mainly with multiresistant isolates, contrary to what is generally agreed as a characteristic of the genus. Veterinary clinical isolates were mainly grouped with the multiresistant clinical human isolates. The 26 reference enterococcal strains were distributed in clusters with different antibiotic resistance profiles and were mainly clustered with dairy isolates.