RESUMO
AIM: The occurrence of virulence markers, serotypes and invasive ability were investigated in Shiga toxin-producing Escherichia coli (STEC) isolated from faecal samples of healthy dairy cattle at Rio de Janeiro State, Brazil. METHODS AND RESULTS: From 1562 stx-positive faecal samples, 105 STEC strains were isolated by immuno-magnetic separation (IMS) or plating onto MacConkey agar (MC) followed by colony hybridisation. Fifty (47·6%) strains belonged to nine serotypes (O8:H19, O22:H8, O22:H16, O74:H42, O113:H21, O141:H21, O157:H7, O171:H2 and ONT:H21). The prevalent serotypes were O157:H7 (12·4%), O113:H21 (6·7%) and O8:H19 (5·7%). Virulence genes were identified by polymerase chain reaction (PCR). E-hlyA (77·1%) was the more prevalent virulence marker, followed by espP (64·8%), saa (39%), eae (24·8%) and astA (21·9%). All O157:H7 strains carried the γ (gamma) variant of the locus of enterocyte effacement (LEE) genes and the stx2c gene, while the stx1/stx2 genotype prevailed among the eae-negative strains. None of the eae-positive STEC produced the localized adherence (LA) phenotype in HEp-2 or Caco-2 cells. However, intimate attachment (judged by the fluorescent actin staining test) was detected in some eae-positive strains, both in HEp-2 (23·1%) and in Caco-2 cells (11·5%). Most strains (87·5%) showed 'peripheral association' (PA) adherence phenotype to undifferentiated Caco-2 cells. Twenty-five (92·6%) of 27 strains invaded Caco-2 cells. The highest average value of invasion (9·6%) was observed among the eae-negative bovine strains from serotypes described in human disease. CONCLUSION: Healthy dairy cattle is a reservoir of STEC carrying virulence genes and properties associated with human disease. SIGNIFICANCE AND IMPACT OF THE STUDY: Although reports of human disease associated with STEC are scarce in Brazil, the colonization of the animal reservoir by potentially pathogenic strains offers a significant risk to our population.
Assuntos
Bovinos/microbiologia , Reservatórios de Doenças/microbiologia , Escherichia coli Shiga Toxigênica/isolamento & purificação , Animais , Brasil , Células CACO-2 , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Fezes/microbiologia , Humanos , Reação em Cadeia da Polimerase , Sorotipagem , Toxina Shiga/metabolismo , Escherichia coli Shiga Toxigênica/classificação , Escherichia coli Shiga Toxigênica/genética , Escherichia coli Shiga Toxigênica/fisiologia , Virulência/genética , Fatores de Virulência/genética , Fatores de Virulência/metabolismoRESUMO
The study was undertaken to determine the clonal relationship and the genetic diversity among Escherichia coli isolates by comparing a non-motile O157 variant with three O157:H7 EHEC isolates and one O55:H7 enteropathogenic E. coli (EPEC) strain. E. coli strains were characterized by sorbitol phenotype, multilocus enzyme electrophoresis, pulsed-field gel electrophoresis, random amplification polymorphic DNA, and the presence of specific virulence genes (stx, E-hly and LEE genes). Sorbitol fermentation was observed in O157:H- (strain 116I), O55:H7 and O157:H7 (strain GC148) serotypes. stx1 or stx2 and E-hly genes were only detected among O157:H7 isolates. LEE typing revealed specific allele distribution: eaegamma, tirgamma, espAgamma, espBgamma associated with EPEC O55:H7 and EHEC O157:H7 strains (B1/1 and EDL 933), eaealpha, tiralpha, espAalpha, espBalpha related to the 116I O157:H- strain and the GC148 strain presented non-typable LEE sequences. Multilocus enzyme profiles revealed two main clusters associated with specific LEE pathotypes. E. coli strains were discriminated by random amplification of polymorphic DNA-polymerase chain reaction and pulsed-field gel electrophoresis methodologies. The molecular approaches used in this study allowed the determination of the genetic relatedness among E. coli strains as well as the detection of lineage specific group markers.
Assuntos
Escherichia coli Enteropatogênica/classificação , Escherichia coli Enteropatogênica/genética , Escherichia coli O157/classificação , Escherichia coli O157/genética , Animais , Técnicas de Tipagem Bacteriana , Bovinos , Linhagem Celular , Impressões Digitais de DNA , DNA Bacteriano/genética , Eletroforese em Gel de Campo Pulsado , Escherichia coli Enteropatogênica/enzimologia , Infecções por Escherichia coli/microbiologia , Infecções por Escherichia coli/veterinária , Escherichia coli O157/enzimologia , Fermentação , Humanos , Técnica de Amplificação ao Acaso de DNA Polimórfico , Sorbitol/metabolismo , Fatores de Virulência/genéticaRESUMO
Antimicrobial resistance in Escherichia coli isolated from pet dogs can be considered a potential threat of infection for the human population. Our objective was to characterize the resistance pattern, extended spectrum beta-lactamase production and genetic relatedness of multiresistant E. coli strains isolated from dogs (n=134), their owners (n=134), and humans who claim to have no contact with dogs (n=44, control), searching for sharing of strains. The strains were assessed for their genetic relatedness by phylogenetic grouping and pulsed-field gel electrophoresis. Multiresistant E. coli strains were isolated from 42 (31.3%) fecal samples from pairs of dogs and owners, totaling 84 isolates, and from 19 (43.1%) control group subjects. The strains showed high levels of resistance to ampicillin, streptomycin, tetracycline, trimethoprim and sulfamethoxazole regardless of host species or group of origin. The blaTEM, blaCTX-M, and blaSHV genes were detected in similar proportions in all groups. All isolates positive for bla genes were ESBL producers. The phylogenetic group A was the most prevalent, irrespective of the host species. None of the strains belonging to the B2 group contained bla genes. Similar resistance patterns were found for strains from dogs, owners and controls; furthermore, identical PFGE profiles were detected in four (9.5%) isolate pairs from dogs and owners, denoting the sharing of strains. Pet dogs were shown to be a potential household source of multiresistant E. coli strains.
Assuntos
Farmacorresistência Bacteriana , Infecções por Escherichia coli/microbiologia , Infecções por Escherichia coli/veterinária , Escherichia coli/efeitos dos fármacos , Escherichia coli/enzimologia , Genótipo , beta-Lactamases/genética , Animais , Antibacterianos/farmacologia , Cães , Eletroforese em Gel de Campo Pulsado , Escherichia coli/genética , Fezes/microbiologia , Humanos , Tipagem Molecular , Animais de Estimação , FilogeniaRESUMO
Respiratory diseases are common in young horses but little is known about such infections in mule foals. This study aimed to characterize Escherichia coli and Klebsiella sp. isolated from tracheal wash (TW) and fecal samples (FS) of mule foals, with or without cytological evidence of respiratory disease. Strains were analyzed against 13 antimicrobials, for presence of Extended spectrum beta-lactamase (ESBL), and virulence genes. Phylogrouping and Randomic (RAPD)-PCR profiles were used to evaluate their genetic relatedness. E. coli strains from TW and FS showed greatest resistance to tetracycline, while Klebsiella strains were mainly resistant to ampicillin; multidrug resistance and ESBL production were also detected. The blaCTX gene prevailed among the E. coli isolates, while the blaSHV gene was more frequently found in K. pneumoniae. The fimH gene was detected in most of the isolates and multiple virulence factors were identified in three E. coli isolates. Most of the E. coli isolates belonged to the B1 phylogroup, but B2 strains displayed more virulence genes. The RAPD assay revealed genetic diversity among strains and was able to distinguish FS isolates from TW isolates. Knowledge of the bacteria associated with the respiratory tract of mule foals is important in the treatment of sick animals.(AU)
Doenças respiratórias são comuns em potros de equinos, porém pouco se sabe sobre tais infecções em potros de muar. Este estudo buscou caracterizar Escherichia coli e Klebsiella sp. isolados de lavados traqueais (TW) e amostras fecais (FS) de potros de muar com e sem evidências citológicas de doença respiratória. As amostras bacterianas foram testadas contra 13 antimicrobianos, para a presença de genes de resistência estendida às betalactamases (ESBL) e de virulência. Filogrupagem e perfis de PCR randômicos (RAPD) foram usados para avaliar sua relação genética. As amostras de E. coli de TW e FS mostraram maior resistência à tetraciclina, enquanto as amostras de Klebsiella foram mais resistentes à ampicilina; multirresistência e produção de ESBL também foram detectadas. O gene blaCTX foi mais frequente entre E. coli, enquanto o gene blaSHV foi mais encontrado entre K. pneumoniae. O gene fimH foi detectado na maioria dos isolados de E. coli, enquanto múltiplos genes de virulência foram identificados em três isolados de E. coli. A maioria dos isolados de E. coli pertenceu ao filogrupo B1, porém somente isolados do filogrupo B2 apresentaram mais genes de virulência. Os ensaios de RAPD demonstraram a diversidade genética entre as amostras e distinguiram amostras TW e FS. O conhecimento de bactérias associadas a infecções de trato respiratório de potros de muar é importante no tratamento de animais doentes.(AU)
Assuntos
Animais , Resistência Microbiana a Medicamentos , Equidae/microbiologia , Escherichia coli/genética , Escherichia coli/patogenicidade , Klebsiella/genética , Klebsiella/patogenicidade , Doenças Respiratórias/veterinária , VirulênciaRESUMO
Abstract Antimicrobial resistance in Escherichia coli isolated from pet dogs can be considered a potential threat of infection for the human population. Our objective was to characterize the resistance pattern, extended spectrum beta-lactamase production and genetic relatedness of multiresistant E. coli strains isolated from dogs (n = 134), their owners (n = 134), and humans who claim to have no contact with dogs (n = 44, control), searching for sharing of strains. The strains were assessed for their genetic relatedness by phylogenetic grouping and pulsed-field gel electrophoresis. Multiresistant E. coli strains were isolated from 42 (31.3%) fecal samples from pairs of dogs and owners, totaling 84 isolates, and from 19 (43.1%) control group subjects. The strains showed high levels of resistance to ampicillin, streptomycin, tetracycline, trimethoprim and sulfamethoxazole regardless of host species or group of origin. The blaTEM, blaCTX-M, and blaSHV genes were detected in similar proportions in all groups. All isolates positive for bla genes were ESBL producers. The phylogenetic group A was the most prevalent, irrespective of the host species. None of the strains belonging to the B2 group contained bla genes. Similar resistance patterns were found for strains from dogs, owners and controls; furthermore, identical PFGE profiles were detected in four (9.5%) isolate pairs from dogs and owners, denoting the sharing of strains. Pet dogs were shown to be a potential household source of multiresistant E. coli strains.
Assuntos
Animais , Cães , Humanos , Farmacorresistência Bacteriana , Infecções por Escherichia coli/microbiologia , Infecções por Escherichia coli/veterinária , Escherichia coli/efeitos dos fármacos , Escherichia coli/enzimologia , Genótipo , beta-Lactamases/genética , Antibacterianos/farmacologia , Eletroforese em Gel de Campo Pulsado , Escherichia coli/genética , Fezes/microbiologia , Tipagem Molecular , Animais de Estimação , FilogeniaRESUMO
This report aimed to study the interference in molecular testing for Ehrlichia canis and Anaplasma platys in blood of 155 dogs from the coastal region of Rio de Janeiro. Five Anaplasmataceae positive samples but negative for E. canis and A. platys, from microfilaremic animals, were chosen for sequencing. These sequences, when compared to Gen et Bank database, showed 88% to 100% similarity with Wolbachia spp. denoting an interference in the detection of DNA from other members of Anaplasmataceae, possibly due to a high concentration of Wolbachia spp. DNA.(AU)