Resistant enterococci isolated from raw sheep’s milk and cheeses from South region of Brazil

Enterococci have been used as sentinel organisms for monitoring antimicrobial resistance in food, humans, and other animals. In this sense, the present study evaluated the antimicrobial susceptibility profile and the presence of genes associated with resistance to erythromycin (msrC and ermB) and tetracycline [tet(M) and/or tet(L)] in enterococci isolated from raw sheep’s milk and cheeses (colonial, feta-, and pecorino-type) from South region of Brazil. A total of 156 enterococci were isolated from milk (n=80) and cheese (n=76) samples, identified by MALDI-TOF. Enterococcus faecalis (50.6%; n=79) was the most frequent species isolated from both samples. According to in vitro susceptibility tests, enterococci strains were not susceptible to the most commonly antimicrobial agents used in human and veterinary medicine. The frequency of MDR strains in enterococci isolated from milk (53.7%) was higher than those from cheese (24.2%). The tet(M) gene was the most commonly detected among tetracycline not-susceptible strains. The present study provided the first evidence of antimicrobial not-susceptible enterococci in raw sheep’s milk and cheeses in South Brazil. Drug-resistant strains, particularly those that are MDR, constitute a One Health issue.Os enterococos têm sido usados como organismos sentinela para monitorar o padrão de suscetibilidade a antimicrobianos em alimentos, humanos e outros animais. Neste sentido, o presente estudo objetivou avaliar o perfil de susceptibilidade a antimicrobianos e os genes associados com a resistência a eritromicina (msrC and ermB) e à tetraciclina [tet(M) and/or tet(L)] em enterococos isolados de leite cru de ovelha e queijos (colonial, tipo-feta e tipo-pecorino) do Sul do Brasil. Um total de 156 enterococos foram isolados de leite (n=80) e queijo (n=76), identificados por MALDI-TOF. Enterococcus faecalis (50,6%; n=79) foi a espécie mais frequentemente isolada de ambas as amostras. De acordo com o teste de suscetibilidade in vitro, as cepas de enterococos não foram susceptíveis aos agentes antimicrobianos mais comumente utilizados na clínica humana e veterinária. A frequência de cepas de enterococos MDR isoladas do leite (53,7%) foi superior à do queijo (24,2%). O gene tet(M) foi o mais comumente detectado entre as cepas não susceptíveis à tetraciclina. O presente estudo fornece as primeiras evidências de enterococos não susceptíveis aos antimicrobianos em leite cru de ovelha e queijos no Sul do Brasil. Cepas resistentes a drogas, particularmente as que são MDR, representam uma preocupação de Saúde Única

Frequency of Clustered Regularly Interspaced Short Palindromic Repeats (CRISPRs) in non-clinical Enterococcus faecalis and Enterococcus faecium strains

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.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

Antimicrobial resistance and genetic relationships of enterococci from siblings and non-siblings Heliconius erato phyllis caterpillars

Background: Studies evaluating bacteria in insects can provide information about host–microorganism–environment interactions. The gut microbial community has a profound effect on different physiological functions of insects. Enterococcus spp. are part of the gut community in humans and other animals, as well as in insects. The presence and antimicrobial resistance profile of enterococci are well studied in different animals; however, data for Heliconius erato phyllis (Lepidoptera: Nymphalidae) do not yet exist. Therefore, the aims of this study were to evaluate the distribution of enterococcal species, their antimicrobial resistance profile and virulence genes, and the genetic relationships between enterococci isolated from fecal samples from sibling and non-sibling H. erato phyllis caterpillars collected from different sites in South Brazil. Methods: Three H. erato phyllis females were captured (two from a forest fragment and one from an urban area), and kept individually in open-air insectaries. Eggs were collected and caterpillars (siblings and non-siblings) were fed daily with Passiflora suberosa leaves. Fecal samples (n = 12) were collected from fifth-instar caterpillars, inoculated in selective medium, and 15 bacterial colonies were randomly selected from each sample. Enterococci were identified by PCR and MALDI-TOF, analyzed by disk diffusion antimicrobial susceptibility tests, and screened for resistance and virulence genes by PCR. The genetic relationships between the strains were determined using pulsed-field gel electrophoresis (PFGE). Results: A total of 178 enterococci strains were identified: E. casseliflavus (74.15%; n = 132), E. mundtii (21.34%; n = 38), E. faecalis (1.12%; n = 2) and Enterococcus sp. (3.37%; n = 6). High rates of resistance to rifampicin (56%) and erythromycin (31%) were observed; 120 (67.41%) of the isolates showed resistance to at least one antibiotic and six (3.37%) were multidrug-resistant. None of the erythromycin-resistant strains was positive for the erm(B) and msrC genes. The virulence genes esp, ace, and gelE were observed in 35%, 7%, and 1% of the strains, respectively. PFGE separated the enterococci into 22 patterns, four being composed of strains from sibling caterpillars. Conclusion: Enterococcus casseliflavus was the dominant species in fecal samples of fifth-instar caterpillars. Resistant enterococci strains may be related to environmental pollution or the resistome. The PFGE analysis showed genetic relationships between some strains, suggesting that the enterococci isolated from fecal samples of the sibling caterpillars might have come from common sources, e.g., via diet (herbivory) and/or vertical transmission (through the egg surface). Further studies will be conducted to better understand the role of Enterococcus in the microbial community of the gastrointestinal tract of these insects, and the mechanisms involved in acquisition and maintenance of enterococci

Antimicrobial resistance profiles in Enterococcus spp. isolates from fecal samples of wild and captive black capuchin monkeys (Sapajus nigritus) in South Brazil

The environment, human, and animals play an important role in the spread of antibioticresistant bacteria. Enterococci are members of the gastrointestinal tracts of humans and animals and represent important reservoirs of antibiotic resistance genes. Until today, few studies have examined antibiotic susceptibility in enterococci isolated from primates. Therefore, the present study investigated species distribution, antibiotic susceptibility, and resistance genes in enterococci isolated from wild and captive black capuchins monkeys (Sapajus nigritus) in Rio Grande do Sul, South Brazil. A total of 24 swabs/fecal samples were collected, including 19 from wild monkeys living in two forest fragments [São Sebastião do Caí (SSC) and Santa Cruz do Sul (SCS)], and five in captive [Parque Zoológico da Fundação Zoobotânica (ZOO)], between August 2016 and November 2017. Fifteen colonies were randomly selected from each sample. Enterococci were identified by MALDI-TOF, tested for susceptibility to 12 antibiotics; and screened for tet(S), tet(M), tet(L), msrC, and erm(B) genes by PCR. Two-hundred ninety-six enterococci were isolated (SSC n = 137; SCS n = 86; ZOO n = 73) and differences in Enterococcus species distribution were detected on three monkey groups, with low abundance in SCS (1 D = 0.2), followed by ZOO (1 D = 0.68), and SSC (1 D = 0.73). The enterococci frequently recovered include the following: Enterococcus faecalis (42.6%), E. hirae (29.1%), and E. faecium (15.9%). Antibioticnonsusceptible was observed in 202 (67.9%) strains. The rate of non-susceptibility to rifampicin, tetracycline, erythromycin, nitrofurantoin, chloramphenicol, and ampicillin was 46%, 26%, 22% and 19%, 13%, 0.3%, and 0.3%, respectively. All strains were susceptible to vancomycin, streptomycin, gentamycin, and linezolid. Forty-three (14.52%) isolates were identified as multidrug resistant (MDR), and the highest number of MDR enterococci were E. faecium recovered from wild monkeys living close to a hospital and water treatment plant. Elevated rates of antibiotic resistance genes msrC and tet(L) were isolates from ZOO. In conclusion, differences in the frequency of enterococci species, antibiotic-nonsusceptible and antibiotic resistance genes in all groups of monkeys were identified. These data suggest that anthropogenic activities could have an impact in the resistome of primate gut enterococci communities

Genetic diversity and virulence factors of Enterococcus spp. from fecal samples of sea turtles recovered on the north coast of Rio Grande do Sul, Brazil

Os Enterococcus spp. apresentam natureza ubiquitária e são encontrados no trato gastrointestinal de diversos animais. Entretanto, estudos desses microorganismos associados a tartarugas marinhas são escassos. Os objetivos deste estudo foram: isolar enterococos a partir de amostras fecais de tartarugas marinhas encontradas no Litoral Norte do Rio Grande do Sul; determinar a prevalência das espécies; avaliar seu perfil de suscetibilidade antimicrobiana; verificar a presença de genes relacionados à resistência e à virulência e; analisar sua diversidade genética. Um total de 158 enterococos foram identificados como E. faecalis (55,7%), E. faecium (23,4%), E. hirae (15,2%) e E. casseliflavus (5,7%). A maioria dos isolados foi suscetível aos antimicrobianos testados, no entanto, fenótipos de resistência foram encontrados para eritromicina (34,2%), rifampicina (32,9%) e tetraciclina (0,63%). O gene de resistência à eritromicina msrC foi encontrado em todos os E. faecium resistentes, já o gene erm(B), não foi detectado. Somente um isolado foi resistente à tetraciclina, e este não apresentou nenhum dos genes testados. Os genes de virulência, gelE e ace (98,86%), asa (68.18%) e cylA (40,90%) foram detectados somente em E. faecalis. A atividade de gelatinase e citolisina foi verificada em 87 e 19 isolados, respectivamente. A maioria dos enterococos não foi capaz de formar biofilme. A análise dos perfis gerados por PFGE revelou um grande número de clones. Em conclusão, diferentes espécies de enterococos compõem a microbiota do trato gastrointestinal das tartarugas marinhas e a presença de determinantes de resistência e virulência nestes animais pode estar relacionada a fatores antropogênicos ou, ainda, ter origem no resistoma ambiental.Enterococcus spp. shows an ubiquitous nature and are found in the gastrointestinal tract of several animals. However, studies of enterococus in sea turtles are scarce The aims of this study were: to isolate Enterococcus spp. from fecal samples of sea turtle found on the North coast of Rio Grande do Sul; to determine the prevalence of species; to evaluate their antimicrobial susceptibility profile; to check the presence of resistance and virulence related genes and; to analyse their genetic diversity. A total of 158 enterococci were identified as E. faecalis (55.7%) E. faecium (23.4%), E. hirae (15.2 %) and E. casseliflavus (5.7%). Most of the isolates were susceptible to the tested antimicrobials, however, resistance phenotypes were found for erythromycin (34.2%), rifampicin (32.9%) and tetracycline (0.63%). The erythromycin resistance gene msrC was detected in all E. faecium erythromycin resistant; moreover, the gene erm (B) was not detected. Only one sample was resistant to tetracycline, although it did not show any of the tetracycline resistant genes tested. Virulence genes gelE and ace (98.86%), asa (68.18%) and cylA (40.90%) were detected only in E. faecalis. The cytolysin and gelatinase activity was observed in 19 and 87 strains, respectively. Most enterococci were not able to form biofilm. The profile analysis generated by PFGE revealed a large number of clones. In conclusion, different species of enterococci were found in the microflora of sea turtle gastrointestinal tract and the presence of resistance and virulence determinants in these animals might be related to anthropogenic factors or even to an environmental resistome origin

Genetic diversity and virulence factors of Enterococcus spp. from fecal samples of sea turtles recovered on the north coast of Rio Grande do Sul, Brazil

Os Enterococcus spp. apresentam natureza ubiquitária e são encontrados no trato gastrointestinal de diversos animais. Entretanto, estudos desses microorganismos associados a tartarugas marinhas são escassos. Os objetivos deste estudo foram: isolar enterococos a partir de amostras fecais de tartarugas marinhas encontradas no Litoral Norte do Rio Grande do Sul; determinar a prevalência das espécies; avaliar seu perfil de suscetibilidade antimicrobiana; verificar a presença de genes relacionados à resistência e à virulência e; analisar sua diversidade genética. Um total de 158 enterococos foram identificados como E. faecalis (55,7%), E. faecium (23,4%), E. hirae (15,2%) e E. casseliflavus (5,7%). A maioria dos isolados foi suscetível aos antimicrobianos testados, no entanto, fenótipos de resistência foram encontrados para eritromicina (34,2%), rifampicina (32,9%) e tetraciclina (0,63%). O gene de resistência à eritromicina msrC foi encontrado em todos os E. faecium resistentes, já o gene erm(B), não foi detectado. Somente um isolado foi resistente à tetraciclina, e este não apresentou nenhum dos genes testados. Os genes de virulência, gelE e ace (98,86%), asa (68.18%) e cylA (40,90%) foram detectados somente em E. faecalis. A atividade de gelatinase e citolisina foi verificada em 87 e 19 isolados, respectivamente. A maioria dos enterococos não foi capaz de formar biofilme. A análise dos perfis gerados por PFGE revelou um grande número de clones. Em conclusão, diferentes espécies de enterococos compõem a microbiota do trato gastrointestinal das tartarugas marinhas e a presença de determinantes de resistência e virulência nestes animais pode estar relacionada a fatores antropogênicos ou, ainda, ter origem no resistoma ambiental.Enterococcus spp. shows an ubiquitous nature and are found in the gastrointestinal tract of several animals. However, studies of enterococus in sea turtles are scarce The aims of this study were: to isolate Enterococcus spp. from fecal samples of sea turtle found on the North coast of Rio Grande do Sul; to determine the prevalence of species; to evaluate their antimicrobial susceptibility profile; to check the presence of resistance and virulence related genes and; to analyse their genetic diversity. A total of 158 enterococci were identified as E. faecalis (55.7%) E. faecium (23.4%), E. hirae (15.2 %) and E. casseliflavus (5.7%). Most of the isolates were susceptible to the tested antimicrobials, however, resistance phenotypes were found for erythromycin (34.2%), rifampicin (32.9%) and tetracycline (0.63%). The erythromycin resistance gene msrC was detected in all E. faecium erythromycin resistant; moreover, the gene erm (B) was not detected. Only one sample was resistant to tetracycline, although it did not show any of the tetracycline resistant genes tested. Virulence genes gelE and ace (98.86%), asa (68.18%) and cylA (40.90%) were detected only in E. faecalis. The cytolysin and gelatinase activity was observed in 19 and 87 strains, respectively. Most enterococci were not able to form biofilm. The profile analysis generated by PFGE revealed a large number of clones. In conclusion, different species of enterococci were found in the microflora of sea turtle gastrointestinal tract and the presence of resistance and virulence determinants in these animals might be related to anthropogenic factors or even to an environmental resistome origin

Expressão gênica em meristemas florais de espécies de Eucalyptus

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Expressão gênica em meristemas florais de espécies de Eucalyptus

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Identificação genotípica, fatores de virulência e capacidade de formação de biofilme in vitro de enterococcus spp. isolados de leite bubalino no sul do Brasil

O consumo de leite de búfala e seus derivados têm aumentado significativamente em decorrência da procura por produtos mais nutritivos e com menor teor de colesterol. O queijo mussarela é o principal produto processado a partir do leite bubalino cru, entretanto, a presença de bactérias patogênicas podem trazer muitos prejuízos aos consumidores desses alimentos in natura. Neste sentido, o objetivo desse estudo foi identificar genotipicamente e caracterizar os fatores de virulência de enterococos isolados de amostras de leite cru de búfalas no sul do Brasil. Setenta e nove bactérias isoladas de leite bubalino foram submetidas a identificação genotípica para confirmação do gênero através da presença ou ausência do gene tuf e as espécies foram identificadas por PCR multiplex. Além disso, os enterococos foram avaliados quanto à capacidade de formação de biofilme e análise genotípica dos fatores de virulência agg, ace, gelE e produção da enzima gelatinase. Das setenta e nove amostras testadas, apenas 3 apresentaram resultados discordantes quando comparado as técnicas de identificação fenotípica e genotípica. Entre as espécies encontradas, E. faecalis foi a mais frequente entre os isolados, sendo também a que mais abrigou genes de virulência e a que mais teve representantes formadores de biofilme. Dessa forma, Enterococcus spp. são patógenos nosocomiais e a sua investigação, faz-se necessária para garantir segurança e qualidade dos alimentos que consumimos