Three studies were conducted to assess if the ability of Enterococcus spp. surviving the ensiling process and bovine gastrointestinal tract could impact risk of bovine mastitis. The first study determined ability of enterococci to survive 3 wk ensiling. Grass and corn crops were divided into 3 treatments: 2 commercial silage inoculants, 1 negative control. After wk 1, 2, and 3 of ensiling, dry matter and bacterial enumeration were performed. Addition of silage inoculant led to greater levels of enterococci in grass silage compared with negative control levels, but showed less difference in inoculated corn silage. The second study quantified enterococci shedding rates in lactating dairy cows. Using a 4 x 4 Latin Square design, lactating, ruminally fistulated Holsteins were inoculated with enterococcal isolates from silage inoculants, ensiled forages, or clinical mastitis cases. Over the 4-period study, each period consisted of rumen and fecal sampling (2 wk) followed by a wash period (10 d). There were no significant differences in rumen or fecal enterococci levels between the 4 treatments. Both rumen and fecal enterococci levels showed significant differences between baseline and treatment periods (period 3, 4). The third study analyzed similarity in enterococcal isolates of silage and bovine origin using pulsed-field gel electrophoresis patterns from SmaI restrictions. Dendogram analysis showed none of the isolates met or were greater than a 75% genetic similarity and produced a genetically diverse population. Results suggest Enterococcus spp. from silage inoculants are not likely to contribute to an increased risk of enterococcal bovine mastitis. / Master of Science

Multiresistenta bakterier så som meticillinresistenta Staphylococcus aureus (MRSA), bakterier som producerar extended-spectrum beta-lactamase (ESBL) och vancomycinresistenta enterokocker (VRE) är ett problem sedan årtionden tillbaka och som ökar för varje år. Idag på mikrobiologen, Unilabs Skövde, isoleras bakteriestammar från selektiva medier för just MRSA, ESBL och VRE på blodagar innan resistensbestämningarna utförs. Syftet med studien var därför att undersöka möjligheten att göra diskdiffusionstest direkt från de selektiva medierna och således kunna svara ut resultaten tidigare. Utvärdering av detta gjordes genom att undersöka om storleken på antibiotikazonerna för sammanlagt 64 isolat påverkades av att bakterierna som användes vuxit på ett selektivt agarmedium i förhållande till om de vuxit på blodagar. Resultatet visade vad som ansågs vara en normal variation på maximalt ±2 mm för alla parvisa zoner utom en på 3 mm. Av alla zoner som undersöktes för MRSA, ESBL och VRE var majoriteten identiska i antal millimeter, 62 %, 89 % och 98 % respektive. Baserat på det goda resultatet ansågs materialet vara tillräckligt stort för att göra bedömningen att metoden är utförbar. Med tanke på de positiva effekterna av att göra resistensbestämningar direkt från de selektiva agarmedierna görs rekommendationen till mikrobiologen, Unilabs Skövde, att övergå till denna metod. / Multiresistant bacteria such as methicillin-resistant Staphylococcus aureus (MRSA), extended-spectrum beta-lactamase (ESBL) producing bacteria and vancomycin-resistant enterococci (VRE) have been a problem for decades with an increasing rate. Today, at mikrobiologen, Unilabs Skövde, bacterial strains are isolated from selective media for MRSA, ESBL and VRE onto blood agar before the susceptibility testing. The aim of the study was to examine the possibility of disk diffusion susceptibility testing directly from the selective media and thus be able to reply the findings earlier. The zones of inhibition were examined for a total of 64 isolates after disk diffusion testing from both the selective and blood agar plates in order to evaluate if the zone sizes were affected. The results showed what was considered a normal variation of ±2 mm for all pairwise zones except for a difference in 3 mm. The majority of all zones tested for MRSA, ESBL and VRE had equally large zones, 62%, 89% and 98% respectively. Based on the good results, the material was considered enough to make the conclusion that the method is feasible. Considering the positive effects of making susceptibility testing directly from selective agar, a change to this method is recommended to mikrobiologen, Unilabs Skövde.

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / Enterococcus resistentes à vancomicina (VRE) são reconhecidos como importantes patógenos causadores de infecções nosocomiais, configurando um grave problema de saúde pública, principalmente pela escassez de opção terapêutica eficaz. O fenótipo de resistência VanA é o mais frequente, sendo definido pela resistência a altos níveis de vancomicina e teicoplanina. VanA é caracterizado por um conjunto gênico (vanRSHAXYZ) localizado no elemento genético móvel denominado transposon Tn1546. A diversidade de Tn1546 resulta de alterações estruturais promovidas por deleções ou integração de sequências de inserção (IS) que, exercem papel chave na evolução do elemento VanA, modificando os aspectos relacionados à sua transferência e expressão do fenótipo. O objetivo deste estudo foi caracterizar e avaliar o polimorfismo de elementos Tn1546 presentes em amostras de diferentes espécies de Enterococcus isoladas em instituições hospitalares do Estado do Rio de Janeiro no período de 2000 a 2012. Foram incluídas neste estudo 70 amostras VRE que foram caracterizadas quanto ao gênero, espécies e genótipo de resistência aos glicopeptídeos por métodos convencionais e PCR multiplex. A susceptibilidade a 17 antimicrobianos foi avaliada pelo método de difusão em ágar, e concentração inibitória mínima (CIM) para vancomicina e teicoplanina foi determinada por microdiluição em caldo. O tranposon foi obtido após lise das células bacterianas e amplificação por PCR longo, utilizando-se oligonucleotídeos específicos para a região repetida e invertida que flanqueia este elemento genético. A diversidade dos elementos Tn1546 foi avaliada por um conjunto de métodos moleculares que incluiu a análise do polimorfismo do tamanho de fragmentos de restrição (restriction fragment lenght polymorphism, RFLP), utilizando-se a endonuclease ClaI, amplificação de segmentos internos por PCR de sobreposição de oligonucleotídeos (overlapping PCR) e detecção de sequências de inserção (ISs). A caracterização em espécies considerada para as demais análises foi obtida pela metodologia de PCR de acordo com a seguinte distribuição: E. avium (N=6), E. faecalis (N=12), E. faecium (N=46), E. gallinarum (N=4) e E. raffinosus (N=2). Todas as amostras apresentaram o genótipo vanA. Nos testes de susceptibilidade aos antimicrobianos foi observado que todas as amostras foram multirresistentes, sendo resistente de 6 a 13 dentre os 17 antimicrobianos testados. A presença de elementos semelhantes ao arquétipo de Tn1546 foi observada em 61,5% das amostras; entretanto, 27 amostras apresentaram perfis variantes de Tn1546. Foram identificados nove perfis de RFLP, dentre 66 avaliadas, sendo o perfil I, prevalente e semelhante ao arquétipo de Tn1546. Não foi possível analisar quatro amostras por RFLP. Os produtos de amplificação de Tn1546 alterados, obtidos pela overlapping PCR e pelo rastreamento de IS, levaram à classificação de 15 tipos polimórficos, nomeados de A a O. A maioria dos Tn1546 polimórficos teve suas regiões de ORF1 e/ou ORF2 deletadas; e IS1542 juntamente com IS1216V foram as inserções mais frequentes, que em muitas situações compartilhavam a mesma região de inserção. IS19 foi detectada apenas na região vanS-vanH. Os dados apresentados neste estudo indicam que o polimorfismo de Tn1546 pode ser explorado no rastreamento de rotas de transmissão, acompanhamento da dispersão de elementos VanA e investigação da evolução de amostras VRE. / Vancomycin-resistant Enterococcus (VRE) is a leading cause of nosocomial infections, remaining as a public health concern in the last two decades. VanA phenotype is the most frequently encountered and it is responsible for high-level vancomycin and teicoplanin resistance. VanA is characterized by a gene cluster (vanRSHAXYZ) located on the mobile genetic element called Tn1546. The diversity of Tn1546 results from structural changes promoted by deletions or additions of insertion sequences (IS) that play a key role in the evolution of VanA element, changing its transferability and expression of phenotype. The aim of this study was to characterize and evaluate the polymorphism of Tn1546 genetic elements belong to VRE isolates obtained from patients attending in hospitals located in the Rio de Janeiro state, during the period from 2000 to 2012. Seventy VRE strains were included in this study. The strains were identified by conventional physiological testes and multiplex PCR, including the vancomycin resistance phenotype and genotype. Antimicrobial susceptibilities testing were carried out by disk diffusion method for 17 antimicrobials; and the minimal inhibitory concentrations (MIC) values to vancomycin and teicoplanin were evaluated by microdilution technique. Tn1546 were amplified by long-PCR using primers to inverted-repeat sequences flanking the transposon. Tn1546 diversity was evaluated by a set of molecular methods including restriction fragment length polymorphism (RFLP), using the endonuclease ClaI, overlapping PCR and detection of insertion sequence elements (IS). Among the 70 strains, 6 E. avium, 12 E. faecalis, 46 E. faecium, 4 E. gallinarum, and 2 E. raffinosus were characterized by multiplex PCR, as well as the vanA glycopeptide resistance determinant. All the strains were multirresistant, being resistant from 6-13 among the 17 antimicrobials tested. The presence of similar elements to the archetype of Tn1546 was observed in 61.5% of samples; however, 27 samples had variant profiles of Tn1546. Nine RFLP profiles were identified among 66 strains, and the profile I was the most frequent and showed to be similar to the archetype of Tn1546. It was not possible to analyze four strains by RFLP. The amplification products of Tn1546, obtained by overlapping PCR, and screening the IS elements led to the characterization of 15 different types, named A through O. Most Tn1546 had its polymorphisms based on deletion of the ORF1 and / or ORF2 regions; and IS1542 as the most frequent insertion element, which in many cases shared the same region of insertion with IS1216V. IS19 was detected only in vanS-vanH region. The data presented in this study indicate that the polymorphism of Tn1546 can be exploited in tracking transmission routes, monitoring the dispersion of VanA elements and investigation of the evolution of VRE strains.

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / Enterococcus resistentes à vancomicina (VRE) são reconhecidos como importantes patógenos causadores de infecções nosocomiais, configurando um grave problema de saúde pública, principalmente pela escassez de opção terapêutica eficaz. O fenótipo de resistência VanA é o mais frequente, sendo definido pela resistência a altos níveis de vancomicina e teicoplanina. VanA é caracterizado por um conjunto gênico (vanRSHAXYZ) localizado no elemento genético móvel denominado transposon Tn1546. A diversidade de Tn1546 resulta de alterações estruturais promovidas por deleções ou integração de sequências de inserção (IS) que, exercem papel chave na evolução do elemento VanA, modificando os aspectos relacionados à sua transferência e expressão do fenótipo. O objetivo deste estudo foi caracterizar e avaliar o polimorfismo de elementos Tn1546 presentes em amostras de diferentes espécies de Enterococcus isoladas em instituições hospitalares do Estado do Rio de Janeiro no período de 2000 a 2012. Foram incluídas neste estudo 70 amostras VRE que foram caracterizadas quanto ao gênero, espécies e genótipo de resistência aos glicopeptídeos por métodos convencionais e PCR multiplex. A susceptibilidade a 17 antimicrobianos foi avaliada pelo método de difusão em ágar, e concentração inibitória mínima (CIM) para vancomicina e teicoplanina foi determinada por microdiluição em caldo. O tranposon foi obtido após lise das células bacterianas e amplificação por PCR longo, utilizando-se oligonucleotídeos específicos para a região repetida e invertida que flanqueia este elemento genético. A diversidade dos elementos Tn1546 foi avaliada por um conjunto de métodos moleculares que incluiu a análise do polimorfismo do tamanho de fragmentos de restrição (restriction fragment lenght polymorphism, RFLP), utilizando-se a endonuclease ClaI, amplificação de segmentos internos por PCR de sobreposição de oligonucleotídeos (overlapping PCR) e detecção de sequências de inserção (ISs). A caracterização em espécies considerada para as demais análises foi obtida pela metodologia de PCR de acordo com a seguinte distribuição: E. avium (N=6), E. faecalis (N=12), E. faecium (N=46), E. gallinarum (N=4) e E. raffinosus (N=2). Todas as amostras apresentaram o genótipo vanA. Nos testes de susceptibilidade aos antimicrobianos foi observado que todas as amostras foram multirresistentes, sendo resistente de 6 a 13 dentre os 17 antimicrobianos testados. A presença de elementos semelhantes ao arquétipo de Tn1546 foi observada em 61,5% das amostras; entretanto, 27 amostras apresentaram perfis variantes de Tn1546. Foram identificados nove perfis de RFLP, dentre 66 avaliadas, sendo o perfil I, prevalente e semelhante ao arquétipo de Tn1546. Não foi possível analisar quatro amostras por RFLP. Os produtos de amplificação de Tn1546 alterados, obtidos pela overlapping PCR e pelo rastreamento de IS, levaram à classificação de 15 tipos polimórficos, nomeados de A a O. A maioria dos Tn1546 polimórficos teve suas regiões de ORF1 e/ou ORF2 deletadas; e IS1542 juntamente com IS1216V foram as inserções mais frequentes, que em muitas situações compartilhavam a mesma região de inserção. IS19 foi detectada apenas na região vanS-vanH. Os dados apresentados neste estudo indicam que o polimorfismo de Tn1546 pode ser explorado no rastreamento de rotas de transmissão, acompanhamento da dispersão de elementos VanA e investigação da evolução de amostras VRE. / Vancomycin-resistant Enterococcus (VRE) is a leading cause of nosocomial infections, remaining as a public health concern in the last two decades. VanA phenotype is the most frequently encountered and it is responsible for high-level vancomycin and teicoplanin resistance. VanA is characterized by a gene cluster (vanRSHAXYZ) located on the mobile genetic element called Tn1546. The diversity of Tn1546 results from structural changes promoted by deletions or additions of insertion sequences (IS) that play a key role in the evolution of VanA element, changing its transferability and expression of phenotype. The aim of this study was to characterize and evaluate the polymorphism of Tn1546 genetic elements belong to VRE isolates obtained from patients attending in hospitals located in the Rio de Janeiro state, during the period from 2000 to 2012. Seventy VRE strains were included in this study. The strains were identified by conventional physiological testes and multiplex PCR, including the vancomycin resistance phenotype and genotype. Antimicrobial susceptibilities testing were carried out by disk diffusion method for 17 antimicrobials; and the minimal inhibitory concentrations (MIC) values to vancomycin and teicoplanin were evaluated by microdilution technique. Tn1546 were amplified by long-PCR using primers to inverted-repeat sequences flanking the transposon. Tn1546 diversity was evaluated by a set of molecular methods including restriction fragment length polymorphism (RFLP), using the endonuclease ClaI, overlapping PCR and detection of insertion sequence elements (IS). Among the 70 strains, 6 E. avium, 12 E. faecalis, 46 E. faecium, 4 E. gallinarum, and 2 E. raffinosus were characterized by multiplex PCR, as well as the vanA glycopeptide resistance determinant. All the strains were multirresistant, being resistant from 6-13 among the 17 antimicrobials tested. The presence of similar elements to the archetype of Tn1546 was observed in 61.5% of samples; however, 27 samples had variant profiles of Tn1546. Nine RFLP profiles were identified among 66 strains, and the profile I was the most frequent and showed to be similar to the archetype of Tn1546. It was not possible to analyze four strains by RFLP. The amplification products of Tn1546, obtained by overlapping PCR, and screening the IS elements led to the characterization of 15 different types, named A through O. Most Tn1546 had its polymorphisms based on deletion of the ORF1 and / or ORF2 regions; and IS1542 as the most frequent insertion element, which in many cases shared the same region of insertion with IS1216V. IS19 was detected only in vanS-vanH region. The data presented in this study indicate that the polymorphism of Tn1546 can be exploited in tracking transmission routes, monitoring the dispersion of VanA elements and investigation of the evolution of VRE strains.

Master of Science / Department of Diagnostic Medicine/Pathobiology / Tiruvoor G. Nagaraja / Copper, as copper sulfate, is often supplemented at elevated concentrations in swine diets, particularly in piglets, to promote growth. Growth promotional effects of copper are believed to be similar to that of antibiotics in that gut microbial flora is altered to reduce loss of nutrients and suppress pathogens. Bacteria exposed to copper may acquire resistance, and in Enterococcus faecium and E. faecalis, resistance is conferred by a plasmid-borne transferable copper resistance (tcrB) gene. The plasmid also carries macrolide [erm(B)] and glycopeptide (vanA) antibiotics resistance genes. The objectives of the research were to 1) determine the prevalence of tcrB gene in fecal enterococci of piglets in relation to normal (16.5 ppm) and elevated level (125 ppm) of copper supplementation, 2) determine the relationship of tcrB gene and susceptibilities to copper, erythromycin, and vancomycin, and 3) determine the transferability of tcrB gene in enterococci by conjugation. Weaned piglets, housed in pens, fed normal (16.5 ppm; control) or elevated level of copper (125 ppm) were used. Fecal samples were collected weekly for isolation of enterococci. Isolates were speciated by multiplex PCR and sodA gene sequence analysis. The prevalence of tcrB-positive enterococcal isolates was higher (P < 0.05) in the copper supplemented group than the control group. The prevalence of tcrB was affected by sampling days (P < 0.05) with a significant treatment and sampling time interaction (P < 0.05). The tcrB positive isolates were either E. faecium or E. faecalis, and majority of isolates was E. faecium. The mean MIC of copper for tcrB-positive isolates (21.1 mM) was higher (P < 0.001) compared to tcrB-negative isolates (6.1 mM). All isolates were resistant to erythromycin, tetracyclines and susceptible to vancomycin. The transferability of the tcrB gene from tcrB-positive strains to tcrB-negative strains was demonstrated by conjugation. The potential link between tcrB and antibiotic resistance genes and the propensity of enterococci to transfer tcrB to other strains suggests the possibility that copper supplementation may exert selection pressure for antibiotic resistance. The positive association between copper supplementation and prevalence of tcrB gene has important implications for antimicrobial resistance and food safety, which warrants further investigation.

Doctor of Philosophy / Department of Diagnostic Medicine/Pathobiology / H. Morgan Scott / Copper, as copper sulfate, is often used as an alternative to in-feed antibiotics for growth promotion in both swine and cattle diets. Gut bacteria exposed to copper can acquire resistance, which among enterococci is conferred by a plasmid-borne transferable copper resistance gene (tcrB). The plasmid also carries tetracycline [tet(M)] and macrolide [erm(B)] resistance genes. Because of the genetic link between acquired copper (tcrB) and antibiotic resistance in Enterococcus spp., we hypothesized that copper supplementation may exert selection pressure for enterococci to become resistant to macrolides and tetracyclines, and possibly to other antibiotics. We conducted studies in cattle and swine to investigate the relationship between copper supplementation and the fecal prevalence of tcrB-positive enterococci, as well as its potential co-selection for macrolide and tetracycline resistance. The prevalence was higher in animals fed diets supplemented with elevated level of copper compared to normal level (P < 0.05). The tcrB-positive isolates belonged to either E. faecium or E. faecalis; the majority was E. faecium. All tcrB-positive isolates also contained both erm(B) and tet(M) genes; however, none of them harbored the vanA gene. Median minimum inhibitory concentrations (MIC) of copper for tcrB-positive and tcrB-negative enterococci were 22 mM and 4 mM, respectively (P < 0.0001). The overall prevalence of erm(B) and tet(M) genes among enterococcal isolates of cattle were 46.8 % and 57.5%, respectively; in contrast,100% of the swine isolates were positive for both erm(B) and tet(M) genes. The transferability of the tcrB gene was demonstrated by filter mating assay. Multi-locus variable number tandem repeat analysis revealed a genetically diverse population of enterococci. The finding of a strong association between the copper resistance gene and other antibiotic (tetracycline and tylosin) resistance determinants is significant because enterococci are potential pathogens and have the propensity to transfer resistance genes to other bacteria in the gut. The occurrence of vancomycin resistant enterococci in swine in the US is very rare. Strains of E. faecium positive for vanA, that confers resistance to vancomycin, were isolated and characterized from swine feces. The swine strains belonged to clonal complex 17, a well-adapted hospital clone throughout the world.

For production of double moulded chesses are used lactic acid bacteria, which can be present in a form of non-starter lactic acid bacteria or as starter or adjunct culture. Genera Lactobacillus spp. and Enterococcus spp. are prevalent microorganisms present in these cultures. Of course, these microorganisms are for us interesting because of their possibility of coagulation, proteolytic possibility, probiotic function and antibiotic resistance, but especially because of their decarboxylation abilities. Bacteria contain decarboxylation enzymes, which are able to decarboxylized free amino acid, which rising at proteolysis during process of manufacturing and cheese ripening. Biogenic amines are the result of proteolytic activity. Biogenic amines occur practically in all foodstuffs as a common product of metabolic processes. BA are mainly presented in fermented food (cheeses), where rice just microbial action. Typical representatives of biogenic amines, which occurs in double moulded cheeses (Sedlčanský Vltavín, Bresse bleu) and in blue cheeses (Bleu des Causses, Bleu d'Auvergne) are cadaverine, putrescine, tyramine a 2 fenylethylamine and in much smaller amount histamine, spermidine and spermine too. On assessment concentration of BA is used high pressure liquid chromatography with reverse phase (RP HPLC) with utilizing simple direct derivatization with dansyl chloride and detection by UV VIS detector.

Στην παρούσα μελέτη η βιοτυπία και η ανάλυση του DNA με ηλεκτρο-φόρηση σε παλλόμενο ηλεκτρικό πεδίο (PFGE) εφαρμόσθηκαν σε ένα σύνο-λο εντε-ρο-κόκ-κων που απομονώθηκαν από νερά αναψυχής και πόσιμα νερά, προκει-μένου να προσδιορισθεί πιθανή γενετική συγγένεια. Τα 200 στελέχη εντε-ρο--κόκκου απομονώθηκαν από 246 δείγματα νερών αναψυχής και 903 δείγματα πόσιμου νερού, από θάλασσα, νερό δικτύου, ποταμούς και πηγές της Ν.Δ. Ελλάδας. Η ταυτοποίηση σε επίπεδο είδους έγινε με το σύστημα API 20strep. Ο έλεγχος της ευαισθησίας στα αντιβιοτικά έγινε με προσδιορισμό της ελάχιστης ανασταλτικής πυκνότητας (MIC) με την μέθοδο ταινιών E-test. Η ηλεκτροφό-ρηση σε παλλόμενο ηλεκτρικό πεδίο εφαρμόσθηκε για τις γονοτυπικές δοκιμασίες. Ο χαρακτηρισμός σε επίπεδο είδους ταξινόμησε τους εντερόκοκκους σε 22 βιότυπους. Στην πλειοψηφία τους τα στελέχη ήσαν E. faecium (142 ή 71%), ακολουθούσε ο E. faecalis (40 ή 20%), o E. durans (9 ή 4,5%), o E. gallinarum (5 ή 2,5%) και o E. avium (4 ή 2%). Μεταξύ των στελεχών του E. faecium υπήρ-χε σχέση των βιοτύπων και της πηγής του δείγματος. Αυτό δεν παρατηρή-θη-κε στα στελέχη του E. faecalis ούτε στα υπόλοιπα είδη. Κανένα στέλεχος δεν βρέθηκε να παράγει β-λακταμάση. Δεν παρατηρήθηκε καμία σχέση μεταξύ της αντοχής στα αντιβιοτικά και της προέλευσης των στελεχών. Κανένα είδος εντεροκόκκου δεν παρουσίασε αντοχή στα γλυκοπεπτίδια. Κλινικά στελέχη E. faecium περιελήφθησαν στα γονοτυπικά πειράματα ως μάρτυρες για τους ήδη ταυτοποιημένους κλώνους που ενδημούν στο Πανεπιστημιακό Νοσοκομείο της Πάτρας. Η ανάλυση των τύπων PFGE μεταξύ των 104 στελεχών αποκάλυψε την παρουσία 18 κλώνων μεταξύ του E. faecium, 14 του E. faecalis, 2 του E. durans, 3 του E. gallinarum και ενός του E. avium. Αν και μεταξύ των εξετασθέ-ντων στελεχών παρατηρήθηκε γενετική ποικιλία, προσδιορίσθηκαν κοινοί κλώ-νοι μεταξύ διαφορετικών δειγμάτων νερών. Τα δενδρογράμματα αποκά-λυ-ψαν γενετική σχέση μεταξύ συγκεκριμέ-νων περιβαλλοντικών στελεχών του E. faecium και αυτών που ήσαν νοσοκομειακής προέλευσης. Για τη μελέτη της παρουσίας κλώνων μεταξύ εντεροκόκκων που απομονώνονται από το περιβάλλον πρέπει να εφαρμόζονται μέθοδοι ανάλυσης χρωμοσωμικού DNA. / In this study we have identified the enterococcal species isolated from different environmental sources and we have characterized their biotypes, antibiotic resistance patterns and PFGE types. Biotyping and DNA fingerprinting by pulsed-field gel electrophoresis was applied to a collection of enterococci recovered form recreational and drinking water, in order to identify possible genetic relationships. Clinical strains of hospital origin were compared to the environmental isolates. A total of 200 enterococci were isolated from 246 recreational water, and 900 drinking water. One hundred forty two isolates were characterized as Enterococcus faecium recovered from all sources, 40 E. faecalis, 9 E. durans, 5. E. gallinarum and E. avium. Biotypes, determined with API 20 strep, among E. faecium were correlated with certain environmental sources, while antibiotypes, determined with Etest, did not reveal any relationship with the sample origin. Even though genetic diversity was observed among the studied strains, common clonal types were also identified in different sources, suggesting a possible common origin of the enterococci. Cluster analysis revealed a genetic relationship between certain environmental E. faecium and clinical strains.