Evaluation of resistance genes and virulence factors in a food isolated Enterococcus durans with potential probiotic effect

AbstractEnterococci belong to the lactic acid bacteria (LAB) group, which are often considered to provide benefit to the host organism when consumed. However, these microorganisms have a potential as infective agents, being necessary to evaluate the presence of virulence factors and resistance to antibiotics to warrant the safe use of new strains as probiotic cultures. This study aimed to detect genes of potential virulence factors related with adhesion, aggregation, biofilm formation and resistance to vancomycin, in addition to evaluate the antibiotic susceptibility and adhesion capacity of Enterococcus durans LA18s, a strain previously isolated from Minas Frescal cheese. The PCR reactions with specific primers to detect genes of adhesion collagen protein (ace), aggregation substances (agg and asa), bopA (putative glycosyltransferase), bopB (beta-phosphoglucomutase), bopC (aldose 1-epimerase), and bopD (sugar-binding transcriptional regulator) were negative for E. durans LAB18s. In addition, the strain did not present the resistance genes vanA, vanC1 and vanC2/3, and exhibited sensibility to antibiotics commonly used in animal feed, such as erythromycin, tetracycline, vancomycin, gentamicin and penicillin. This strain also showed a strong capacity of biofilm formation and exhibited satisfactory auto-aggregative and hydrophobicity features. The results suggest that this strain can be safely used in animal feed

Colonization and antimicrobial resistance evaluation of Enterococcus sp. Isolated from cloacal swabs of broiler chickens

Bactérias comensais do intestino de frangos tornaram-se objeto frequente de estudos, pois a alta taxa de exportação desses produtos tem aumentado a preocupação com a qualidade e a sanidade de granjas no que se refere à nutrição animal, ganho de peso e doenças infecciosas. O objetivo do presente estudo foi verificar a influência de diferentes dietas para frangos de corte na colonização, assim como no fenótipo e genótipo de resistência antimicrobiana de isolados Enterococcus sp. Amostras de “swabs” cloacais de frangos de corte foram utilizadas para isolamento de Enterococcus sp. Os frangos foram submetidos a diferentes dietas contendo promotores de crescimento, probióticos, óleos essenciais e coccidiostáticos ionóforos, e divididos em grupos conforme o tratamento empregado. Foram obtidos 240 isolados de Enterococcus sp. confirmados para gênero através da técnica de Reação em Cadeia pela Polimerase (PCR), submetidos a testes bioquímicos e moleculares para identificação de espécie, determinados os perfis de susceptibilidade a diversos antimicrobianos e testados para a presença de genes de resistência tet(M), tet(L) e erm(B) por PCR. Observou-se uma alteração na composição ou no número de espécies de Enterococcus sp. de acordo com as dietas empregadas. Todas as dietas, independentes da presença ou não de coccidiostático ionóforo, apresentaram um aumento na freqüência de Enterococcus resistentes quando comparados com o grupo controle. No entanto, nos grupos que os frangos receberam coccidiostático ionóforo, a taxa de Enterococcus sp. isolados foi menor quando comparada aos isolados dos grupos que não receberam essa composição. Do total de isolados resistentes à tetraciclina, 94% e 30%, continham os genes tet(M) e tet(L), respectivamente. Dos isolados resistentes a eritromicina, 97,9% possuíam o gene de resistência erm(B). Não houve correlação das diferentes dietas para frangos de corte na colonização, fenótipo e genótipo de resistência antimicrobiana de isolados de Enterococcus sp.Commensal bacteria from the gut of chickens have been frequently object of studies because the high rate of exportation of these products the concern with the quality and health of poultry, in relation to animal nutrition, weight gain and infectious diseases has been increasing. The principal aim of this study was to verify the influence of different diets treated to broilers, in the colonization of Enterococcus strains, and as well the phenotype and genotype of resistance strains. Samples from cloacal swabs of broiler chickens were used for the isolation of strains of Enterococcus sp. The broilers were subjected to diets with growth promoter, probiotics, essential oils and ionophore coccidiostat and divided into groups according to treatment used. Two hundred forty isolates of Enterococcus sp. were confirmed the genus using the polymerase chain reaction (PCR), subjected to biochemical and molecular species identification, antibiotic susceptibility profile and verification of antibiotic resistance genes tet(M), tet(L) e erm(B) by PCR. Accordingly to the diets employed to the chickens a changing the composition or the number of Enterococcus sp. was observed. All diets, regardless to the presence or absence of ionophore coccidiostat showed an increase in the frequency of Enterococcus resistant when compared with the control group. However, the groups that received ionophore coccidiostat, the rate of resistant Enterococcus sp. was lower than the groups that did not administered this composition. The isolates that presented resistant to tetracycline, 94% and 30%, contained the genes tet(M) and tet(L), respectively. In the isolates resistant to erythromycin, 97.9% had the gene erm(B). There was no correlation of different diets for broiler chickens on colonization, phenotype and genotype resistance in the Enterococcus sp strains

Analysis of virulence factors in Enterococcus faecalis isolated from humans, foods and chickens

Este estudo objetivou investigar a distribuição e a relação entre os genes envolvidos com a virulência e formação de biofilme em 196 Enterococcus faecalis isolados de alimentos, clínicos e suabes cloacais de frangos de corte que receberam oocistos de Eimeria maxima e Eimeria acervulina e ração suplementada ou não com anticoccidiano. No primeiro experimento foi investigada a frequência dos genes agg, ace, tet(M), tet(L) e do operon bopABCD; e foi analisada a produção da enzima gelatinase em duas temperaturas de crescimento (36°C e 42°C). Assim como a capacidade de formar biofilme em meio de cultura suplementado com 10% de sangue, 10% de urina ou 0,75% de glicose por 70 Enterococcus faecalis isolados de frangos. Os Enterococcus faecalis isolados de frangos de corte que receberam oocistos de Eimeria maxima e Eimeria acervulina e ração suplementada ou não com anticoccidiano apresentaram capacidade de se adaptar a diferentes nichos biológicos, como por exemplo, sangue e urina. Foi observado um alto percentual dos genes dos fatores de virulência. No segundo experimento, foi avaliada a diversidade filogenética, com base no método de PCR, de 182 cepas isoladas de humanos, frangos de corte e alimentos. Estas cepas formaram quatro grupos filogenéticos (A, B, C e D) e quatro subgrupos (A1, A2, B1 e B2) com índice de similaridade entre 65 a 100%, demonstrando a adaptação de Enterococcus faecalis a diferentes ambientes. No terceiro experimento, foi determinada a capacidade de formação de biofilme de isolados clínicos e alimentares em diferentes meios de cultivo. Observou-se que o meio suplementado com 0,75% de glicose demonstrou ser o mais adequado para estabelecimento de forte aderência e, consequente formação do biofilme microbiano nos isolados de todas as origens. Em contrapartida, o meio suplementado com 10% de sangue foi o que registrou as maiores taxas de fraca formação de biofilme. Os estudos conduzidos demonstram características fenotípicas e genotípicas de Enterococcus faecalis que sugerem uma ampla adaptação ambiental entre os isolados pesquisados.This study aimed to investigate the distribution and the relationship between genes involved in virulence and biofilm formation in 196 Enterococcus faecalis isolates from food, clinical and cloacal swabs of broilers that received oocysts of Eimeria maxima and Eimeria acervulina and diet supplemented or not with anticoccidial. The first experiment investigated the frequency of agg, ace, tet(M), tet(L) genes and bopABCD operon; production of gelatinase enzyme was analyzed at two growth temperatures (36°C and 42°C). Thus the ability to form biofilm in culture medium supplemented with 10% of blood, 10% of urine and glucose 0.75% for 70 Enterococcus faecalis isolates of chicken. The Enterococcus faecalis isolates from broilers that received oocysts of Eimeria maxima and Eimeria acervulina and supplemented or not with anticoccidial showed the ability to adapt to different biological niches, such as blood and urine. A high percentage of genes of virulence factors were observed. In the second experiment, we evaluated the phylogenetic diversity based on PCR method of 182 strains isolated from humans, broilers and food. These strains formed four phylogenetic groups (A, B, C and D) and four subgroups (A1, A2, B1 and B2) with similarity index between 65 and 100%, demonstrating the adaptation of Enterococcus faecalis to different environments. In the third experiment, the ability of biofilm formation of clinical and food isolates in different culture media was determined. It was observed that the medium supplemented with 0.75% glucose was shown to be more suitable for the establishment of strong adhesion and subsequent formation of the biofilm isolates from all sources. In other hand, medium supplemented with 10% blood was reported that the highest rates of weak biofilm formation. Studies conducted show phenotypic and genotypic characteristics of Enterococcus faecalis that suggest a broad environmental adaptation among the isolates studied

Colonization and antimicrobial resistance evaluation of Enterococcus sp. Isolated from cloacal swabs of broiler chickens

Bactérias comensais do intestino de frangos tornaram-se objeto frequente de estudos, pois a alta taxa de exportação desses produtos tem aumentado a preocupação com a qualidade e a sanidade de granjas no que se refere à nutrição animal, ganho de peso e doenças infecciosas. O objetivo do presente estudo foi verificar a influência de diferentes dietas para frangos de corte na colonização, assim como no fenótipo e genótipo de resistência antimicrobiana de isolados Enterococcus sp. Amostras de “swabs” cloacais de frangos de corte foram utilizadas para isolamento de Enterococcus sp. Os frangos foram submetidos a diferentes dietas contendo promotores de crescimento, probióticos, óleos essenciais e coccidiostáticos ionóforos, e divididos em grupos conforme o tratamento empregado. Foram obtidos 240 isolados de Enterococcus sp. confirmados para gênero através da técnica de Reação em Cadeia pela Polimerase (PCR), submetidos a testes bioquímicos e moleculares para identificação de espécie, determinados os perfis de susceptibilidade a diversos antimicrobianos e testados para a presença de genes de resistência tet(M), tet(L) e erm(B) por PCR. Observou-se uma alteração na composição ou no número de espécies de Enterococcus sp. de acordo com as dietas empregadas. Todas as dietas, independentes da presença ou não de coccidiostático ionóforo, apresentaram um aumento na freqüência de Enterococcus resistentes quando comparados com o grupo controle. No entanto, nos grupos que os frangos receberam coccidiostático ionóforo, a taxa de Enterococcus sp. isolados foi menor quando comparada aos isolados dos grupos que não receberam essa composição. Do total de isolados resistentes à tetraciclina, 94% e 30%, continham os genes tet(M) e tet(L), respectivamente. Dos isolados resistentes a eritromicina, 97,9% possuíam o gene de resistência erm(B). Não houve correlação das diferentes dietas para frangos de corte na colonização, fenótipo e genótipo de resistência antimicrobiana de isolados de Enterococcus sp.Commensal bacteria from the gut of chickens have been frequently object of studies because the high rate of exportation of these products the concern with the quality and health of poultry, in relation to animal nutrition, weight gain and infectious diseases has been increasing. The principal aim of this study was to verify the influence of different diets treated to broilers, in the colonization of Enterococcus strains, and as well the phenotype and genotype of resistance strains. Samples from cloacal swabs of broiler chickens were used for the isolation of strains of Enterococcus sp. The broilers were subjected to diets with growth promoter, probiotics, essential oils and ionophore coccidiostat and divided into groups according to treatment used. Two hundred forty isolates of Enterococcus sp. were confirmed the genus using the polymerase chain reaction (PCR), subjected to biochemical and molecular species identification, antibiotic susceptibility profile and verification of antibiotic resistance genes tet(M), tet(L) e erm(B) by PCR. Accordingly to the diets employed to the chickens a changing the composition or the number of Enterococcus sp. was observed. All diets, regardless to the presence or absence of ionophore coccidiostat showed an increase in the frequency of Enterococcus resistant when compared with the control group. However, the groups that received ionophore coccidiostat, the rate of resistant Enterococcus sp. was lower than the groups that did not administered this composition. The isolates that presented resistant to tetracycline, 94% and 30%, contained the genes tet(M) and tet(L), respectively. In the isolates resistant to erythromycin, 97.9% had the gene erm(B). There was no correlation of different diets for broiler chickens on colonization, phenotype and genotype resistance in the Enterococcus sp strains

Species distribution and antimicrobial susceptibility of enterococci isolated from broilers infected experimentally with Eimeria spp and feed with diets containing different supplements

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

Virulence and biofilm formation by Enterococcus faecalis isolates from cloacal swabs of broilers infected with Eimeria spp

A dinâmica da microbiota no trato gastrointestinal (TG) de animais pode ser afetada por patógenos, tais como Eimeria spp. Os enterococos são bactérias saprófitas que colonizam o TG de mamíferos e aves. A influência sobre a microbiota intestinal está relacionada com a capacidade de adaptação das bactérias em se aderir às células hospedeiras e de colonizar as células das mucosas. O objetivo deste estudo foi analisar a frequência de genes de virulência ace, agg e operon do bopABCD em Enterococcus faecalis isolados de swabs cloacais de frangos de corte desafiados com Eimeria spp e alimentados com dietas padrões suplementadas ou não com anticoccidiano (monesina) e também avaliar a capacidade dessas cepas em formar biofilmes sob condições in vitro. Um total de 70 E. faecalis foram selecionadas e o gene agg foi mais freqüente em cepas isoladas de frangos de corte alimentados com anticoccidiano (92,3%) quando comparado ao grupo que não recebeu anticoccidiano (70,5%). Por outro lado, os genes ace e do operon bopABCD não demostraram nenhuma diferença significativa entre os dois grupos de frangos (P>0,005). Os E. faecalis isolados de frangos de corte alimentados com anticoccidiano demostraram uma maior frequência de fortes aderentes quando crescendo em meio suplementado com glicose (92,3-88,5%) e urina (77%), quando comparado com enterococos isolados de frangos que não receberam anticoccidiano. Observou-se que E. faecalis isolados de frangos tratados com anticoccidiano mostraram uma maior frequêencia dos genes dos fatores de virulência e de perfil de fortes formadores de biofilme, o que indica uma melhor adaptação dos isolados em ambiente intestinal saudável.The microbiota dynamics in the gastrointestinal tract (GT) of animals can be disrupted by pathogens, such as Eimeria spp. Enterococci are saprophytic bacteria that colonize the GT of mammals and birds. The influence on the intestinal microbiota is related to the adaptive capacity of bacteria to adhere to host cells and colonize the mucosal cells. The aim of this study was to analyze the frequency of virulence genes ace, agg and bopABCD operon in Enterococcus faecalis isolated from cloacal swabs of broilers challenged with Eimeria spp. and fed with a standard diet supplemented or not with anticoccidial (monensin), and, also to evaluate for the ability of these strains to form biofilms under in vitro conditions. A total of 70 E. faecalis were selected and the agg gene was more frequent in strains isolated from the broilers treated with anticoccidial (92.3%) when compared to the group that not received anticoccidial (70.5%). On the other hand, the ace and bopABCD operon genes showed no significant difference between the two groups of broilers (P>0.005). The E. faecalis isolated from the broilers treated with anticoccidial showed a higher frequency of strong biofilm formation when growing in medium supplemented with glucose (92.3- 88.5%) and urine (77%) when compared with enterococci isolated from broilers that not received anticoccidial. It was observed that E. faecalis isolated from broilers treated with anticoccidial showed a higher frequency of virulence factors genes and stronger biofilms formation, indicating better adaptation of the isolates in healthy intestinal environment

Comparison of virulence factors and genetic relationships of Enterococcus faecalis strains isolated from clinical, food and poultry samples

Enterococcus faecalis do not only inhabit the intestines of many animals, but also food and the environment. These microorganisms have intrinsic ability which enables them to persist in different environments. The aims of our study were (i) to carry out a comparative analysis of tetracycline resistance and virulence factor genes of Enterococcus faecalis isolates from food, poultry and clinical samples and (ii) to determine the genetic relationships of these factors among these isolates. A total of 182 E. faecalis were studied; 70, 52, and 60 strains were isolated from clinical samples, broiler cloacal swabs and food, respectively. Enterococcus faecalis isolates were submitted to research genes for virulence factors tet (L), tet (M), (bop ABCD, ace and agg) by PCR and grouped into clusters according to their genotype. The prevalence among all the genes studied could be considered high, ranging from 61.5 to 99.4% of the virulence factors of genes and 19.2 to 70.3% of the antimicrobial resistance genes, tet (L) and tet (M), respectively, where it was possible to obtain different genetic profiles. The enterococci isolated from food, humans and broiler cloacal swabs showed high genetic diversity, although some strains seemed to be closely related. The 182 isolates formed twelve different clusters independent of the origin of the samples or the diets used in the feeding of broilers, with the similarity index value ranging from 0.16 to 1.0, similarity coefficient, 0.70. In conclusion, enterococci isolated from food, humans and broiler cloacal swabs are genetically different. In addition, the analysis of virulence factors genes and tet genes by PCR proved to be an effective methodology for determining the microbial diversity of Enterococcus faecalis isolates of different environmental sources

Virulence and biofilm formation by Enterococcus faecalis isolates from cloacal swabs of broilers infected with Eimeria spp

A dinâmica da microbiota no trato gastrointestinal (TG) de animais pode ser afetada por patógenos, tais como Eimeria spp. Os enterococos são bactérias saprófitas que colonizam o TG de mamíferos e aves. A influência sobre a microbiota intestinal está relacionada com a capacidade de adaptação das bactérias em se aderir às células hospedeiras e de colonizar as células das mucosas. O objetivo deste estudo foi analisar a frequência de genes de virulência ace, agg e operon do bopABCD em Enterococcus faecalis isolados de swabs cloacais de frangos de corte desafiados com Eimeria spp e alimentados com dietas padrões suplementadas ou não com anticoccidiano (monesina) e também avaliar a capacidade dessas cepas em formar biofilmes sob condições in vitro. Um total de 70 E. faecalis foram selecionadas e o gene agg foi mais freqüente em cepas isoladas de frangos de corte alimentados com anticoccidiano (92,3%) quando comparado ao grupo que não recebeu anticoccidiano (70,5%). Por outro lado, os genes ace e do operon bopABCD não demostraram nenhuma diferença significativa entre os dois grupos de frangos (P>0,005). Os E. faecalis isolados de frangos de corte alimentados com anticoccidiano demostraram uma maior frequência de fortes aderentes quando crescendo em meio suplementado com glicose (92,3-88,5%) e urina (77%), quando comparado com enterococos isolados de frangos que não receberam anticoccidiano. Observou-se que E. faecalis isolados de frangos tratados com anticoccidiano mostraram uma maior frequêencia dos genes dos fatores de virulência e de perfil de fortes formadores de biofilme, o que indica uma melhor adaptação dos isolados em ambiente intestinal saudável.The microbiota dynamics in the gastrointestinal tract (GT) of animals can be disrupted by pathogens, such as Eimeria spp. Enterococci are saprophytic bacteria that colonize the GT of mammals and birds. The influence on the intestinal microbiota is related to the adaptive capacity of bacteria to adhere to host cells and colonize the mucosal cells. The aim of this study was to analyze the frequency of virulence genes ace, agg and bopABCD operon in Enterococcus faecalis isolated from cloacal swabs of broilers challenged with Eimeria spp. and fed with a standard diet supplemented or not with anticoccidial (monensin), and, also to evaluate for the ability of these strains to form biofilms under in vitro conditions. A total of 70 E. faecalis were selected and the agg gene was more frequent in strains isolated from the broilers treated with anticoccidial (92.3%) when compared to the group that not received anticoccidial (70.5%). On the other hand, the ace and bopABCD operon genes showed no significant difference between the two groups of broilers (P>0.005). The E. faecalis isolated from the broilers treated with anticoccidial showed a higher frequency of strong biofilm formation when growing in medium supplemented with glucose (92.3- 88.5%) and urine (77%) when compared with enterococci isolated from broilers that not received anticoccidial. It was observed that E. faecalis isolated from broilers treated with anticoccidial showed a higher frequency of virulence factors genes and stronger biofilms formation, indicating better adaptation of the isolates in healthy intestinal environment

Species distribution and antimicrobial susceptibility of enterococci isolated from broilers infected experimentally with Eimeria spp and feed with diets containing different supplements

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