Resumo O objetivo do estudo apresentado é caracterizar o perfil de resistência e virulência de enterococos isolados de viveiros escavados e tanques de alvenaria (6 amostras) de uma pisicultura no Sul do Brasil. As amostras foram cultivadas em meio seletivo, 10 colônias foram selecionadas aleatoriamente de cada amostra, que foram identificadas por MALDI-TOF e testadas contra 13 antimicrobianos. A presença de genes de resistência (tetL, tetM, tetS, ermB e msrC) e virulência (ace, esp, agg, cylA e gelE) foi determinada por PCR. Foram identificados 79 enterococos, sendo Entecococcus faecalis (44,3%) e E. casseliflavus (36,7%) as espécies mais frequentes isoladas. Sessenta e cinco cepas (82,3%) eram resistentes a pelo menos um dos antimicrobianos testados, enquanto 27 (34,2%) eram multirresistentes. As porcentagens gerais de isolados resistentes a antimicrobianos foram: 58,2% para rifampicina, 40,5% para fluoroquinolonas, 36,7% para eritromicina e 30,4% para tetraciclina. Os genes tetL e tetM foram encontrados em 57,7% das cepas resistentes à tetraciclina; e msrC em 31,01% das cepas resistentes à eritromicina. Os fatores de virulência mais comumente detectados foram ace e gelE. Embora limitados a uma única fazenda, esses dados indicam que a aquicultura pode ser uma fonte de enterococos resistentes e virulentos. Este estudo é o primeiro passo para melhorar nosso entendimento da distribuição, resistência e perfil de virulência em enterococos isolados de ambientes de piscicultura no sul do Brasil.

Abstract The aim of present study is to characterize the resistance and virulence profile of enterococci isolated from aquaculture excavated ponds and masonry tanks (6 samples) in southern Brazil. Samples were cultured in selective medium, 10 colonies were randomly selected from each sample, which were identified by MALDI-TOF and tested against 13 antimicrobials. The presence of resistance (tetL, tetM, tetS, ermB and msrC) and virulence (ace, esp, agg, cylA and gelE) genes were determined by PCR. A total of 79 enterococci were identified, and Entecococcus faecalis (44.3%) and E. casseliflavus (36.7%) were the most prevalent species isolated. Sixty-five strains (82.3%) were resistant to at least one of the antimicrobials tested, whereas 27 (34.2%) strains were multiresistant. The overall percentages of antimicrobial resistant isolates were: 58.2% to rifampicin, 40.5% to fluoroquinolones, 36.7% to erythromycin and 30.4% to tetracycline. The tetL and tetM genes were found in 57.7% of the tetracycline-resistant strains; and msrC in 31.01% of erythromycin-resistant strains. The most frequently detected virulence factors were ace and gelE genes. Although limited to a single farm, these data suggest that aquaculture may be a reservoir of resistant and virulent enterococci. This study is the first step towards enhancing our understandingof distribution, resistance and virulence profile in enterococci isolated from fish farming environments in the south Brazil.

Resumo A fidelidade dos genomas é defendida por mecanismos conhecidos como sistemas de repetições palindrômicas curtas agrupadas e regularmente interespaçadas (CRISPRs). Três tipos de sistemas CRISPR II (CRISPR1-cas, CRISPR2 e CRISPR3-cas) têm sido identificados em cepas de enterococos isolados de amostras clínicas e ambientais. O objetivo deste estudo foi observar a distribuição dos CRISPR1-cas, CRISPR2 e CRISPR3-cas em cepas não-clínicas de Enterococcus faecalis e Enterococcus faecium isoladas de amostras alimentícias e fecais, incluindo animais marinhos selvagens. A presenca dos CRISPRs foi determinada por PCR em 120 cepas de enterococos, sendo 67 E. faecalis e 53 E. faecium. É o primeiro relato da presença do sistema CRISPRs nas estirpes E. faecalis e E. faecium isoladas de amostras fecais de animais marinhos selvagens. Os resultados mostraram que em cepas não-clínicas, os CRISPRs foram mais frequentemente detectados em E. faecalis do que em E. faecium. E as frequências de CRISPR1-cas e CRISPR2 foram maiores (60%) em cepas de E. faecalis isoladas de fezes de animais, quando comparadas à amostras de alimentos. Ambas as cepas apresentaram baixas freqüências de CRISPR3-cas (8,95% e 1,88%). Em conclusão, as diferenças nos habitats das espécies de enterococos podem estar relacionadas com os resultados observados na distribuição dos sistemas CRISPRs.

Abstract The fidelity of the genomes is defended by mechanism known as Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) systems. Three Type II CRISPR systems (CRISPR1- cas, CRISPR2 and CRISPR3-cas) have been identified in enterococci isolates from clinical and environmental samples. The aim of this study was to observe the distribution of CRISPR1-cas, CRISPR2 and CRISPR3-cas in non-clinical strains of Enterococcus faecalis and Enterococcus faecium isolates from food and fecal samples, including wild marine animals. The presence of CRISPRs was evaluated by PCR in 120 enterococci strains, 67 E. faecalis and 53 E. faecium. It is the first report of the presence of the CRISPRs system in E. faecalis and E. faecium strains isolated from wild marine animal fecal samples. The results showed that in non-clinical strains, the CRISPRs were more frequently detected in E. faecalis than in E. faecium. And the frequencies of CRISPR1-cas and CRISPR2 were higher (60%) in E. faecalis strains isolated from animal feces, compared to food samples. Both strains showed low frequencies of CRISPR3-cas (8.95% and 1.88%). In conclusion, the differences in the habitats of enterococcal species may be related with the results observe in distribution of CRISPRs systems.

Resumo Os poliomavírus humanos JC e BK (JCPyV e BKPyV) são virus ubíquos, espécie-específicos, pertencentes à família Polyomaviridae. Estes vírus são conhecidos por serem excretados pela urina humana, sendo considerados potenciais indicadores de contaminação por águas residuais urbanas. Buscando acessar a distribuição de JCPyV e BKPyV em amostras de águas coletadas de uma estação de tratamento de esgoto e de um arroio canalizado de Porto Alegre, Brasil, duas técnicas de nested-PCR foram otimizadas e aplicadas às amostras coletadas. Os amplificados obtidos foram submetidos ao sequenciamento e suas sequências analisadas com base em sequências de poliomavírus humanos previamente depositadas no GenBank. Doze de 30 amostras de água (40%) foram positivas para JCPyV, enquanto 6 amostras (20%) foram positivas para BKPyV. Os resultados do sequenciamento confirmaram a presença dos subtipos 1 e 3 de JCPyV, enquanto apenas os BKPyV Ia e Ib foram encontrados. Este estudo demonstra pela primeira vez a presença de poliomavírus humanos em águas superficiais e em amostras coletadas em uma estação de tratamento de esgoto na região sul do Brasil.

Abstract The human polyomaviruses JC and BK (JCPyV and BKPyV) are ubiquitous, species-specific viruses that belong to the family Polyomaviridae. These viruses are known to be excreted in human urine, and they are potential indicators of human wastewater contamination. In order to assess the distribution of both JCPyV and BKPyV in urban water samples collected from a sewage treatment plant (STP) and from a canalized water stream of Porto Alegre, Brazil, two nested-PCR assays were optimized and applied to the samples collected. The amplicons obtained were submitted to sequencing, and the sequences were analyzed with sequences of human polyomaviruses previously deposited in GenBank. Twelve out of 30 water samples (40%) were JCPyV positive, whereas six samples (20%) were BKPyV positive. The sequencing results confirmed the presence of JCPyV subtypes 1 and 3, whereas only BKPyV Ia and Ib were found. This study shows for the first time the presence of human polyomaviruses in surface water and in samples collected in a sewage treatment plant in southern Brazil.

The present report aimed to perform a molecular epidemiological survey by investigating the presence of virulence factors in E. faecalis isolated from different human clinical (n = 57) and food samples (n = 55) in Porto Alegre, Brazil, collected from 2006 to 2009. In addition, the ability to form biofilm in vitro on polystyrene and the β-haemolytic and gelatinase activities were determined. Clinical strains presented a higher prevalence of aggregation substance (agg), enterococcal surface protein (esp) and cytolysin (cylA) genes when compared with food isolates. The esp gene was found only in clinical strains. On the other hand, the gelatinase (gelE) and adherence factor (ace) genes had similar prevalence among the strains, showing the widespread occurrence of these virulence factors among food and clinical E. faecalis strains in South Brazil. More than three virulence factor genes were detected in 77.2% and 18.2% of clinical and food strains, respectively. Gelatinase and β-haemolysin activities were not associated with the presence of gelE and cylA genes. The ability to produce biofilm was detected in 100% of clinical and 94.6% of food isolates, and clinical strains were more able to form biofilm than the food isolates (Student's t-test, p < 0.01). Results from the statistical analysis showed significant associations between strong biofilm formation and ace (p = 0.015) and gelE (p = 0.007) genes in clinical strains. In conclusion, our data indicate that E. faecalis strains isolated from clinical and food samples possess distinctive patterns of virulence factors, with a larger number of genes that encode virulence factors detected in clinical strains.

The ability of antibiotic resistant E. faecalis and E. faecium isolated from food to form biofilm at different temperatures in the absence or presence of 0.75% glucose was evaluated. A synergistic effect on biofilm at 10 °C, 28 °C, 37 °C and 45 °C and glucose was observed for E. faecalis and E. faecium.

Resistant bacteria in animal can be spread to environment and to humans. Poultry feed and infections caused by Eimeria spp. are important factors in determining the intestinal microbial communities. The aim of this study was to verify the prevalence of species and antimicrobial susceptibility of Enterococcus isolated from broilers fed with different supplements and infected experimentally with Eimeria spp. Broilers were divided in eight groups, fed with diets supplemented with a combination of antimicrobial, ionophore-coccidiostatics, probiotic, essential oil. At 14 days old all birds, except the control, received a solution containing oocysts of Eimeria spp. Samples of cloacal swabs from broilers were collected. A total of 240 Enterococcus sp. strains were isolated, confirmed genus by PCR, classified as species, tested for antimicrobial susceptibility and screened by PCR for the presence of tet(L), tet(M) and erm(B) genes. The overall distribution of species isolated from fecal samples was E. faecalis (40%), followed by E. casseliflavus/E. gallinarum (10.8%), E. mundtii (10.8%), E. faecium (10.8%), E. columbae (5.8%) and E. gallinarum (4.2%). Changes in the composition or frequency of Enterococcus species were observed in all dietary supplementation. Antimicrobial susceptibility tests showed resistance phenotypes a range of antibiotics, especially used in humans such as, streptomycin, penicillin, rifampicin and vancomycin. There was no correlation between different supplementation for broilers and antimicrobial resistance and the presence of tet(M), tet(L) and erm(B) genes. Dietary supplementation had effect on the Enterococcus sp. colonization, but did not have significant effect on the phenotype and genotype of antimicrobial resistance in enterococci.