Antimicrobial Resistance and Pathogenicity of Aliarcobacter butzleri Isolated from Poultry Meat.

Aliarcobacter butzleri (A. butzleri) is an emergent zoonotic food-related pathogen that can be transmitted through the consumption of poultry meat. Data regarding the pathogenicity and resistance of A. butzleri are still scarce, and the presence of virulent MDR strains of this zoonotic pathogen in poultry meat is an issue of particular concern to public health. This study aimed to characterize the pathogenicity and antimicrobial resistance profiles of A. butzleri strains isolated from poultry meat sold at retail markets in São Paulo, Brazil. The minimum inhibitory concentrations of 27 strains were determined using the broth microdilution method. The results showed that 77.7% of the isolates were resistant to clindamycin, 62.9% to florfenicol, 59.2% to nalidixic acid, 11.1% to azithromycin, 7.4% to ciprofloxacin and telithromycin, and 3.7% to erythromycin and tetracycline, although all were susceptible to gentamicin. Moreover, 55.5% of the virulent isolates were also multidrug-resistant (MDR). Three strains were selected for pathogenicity tests in vitro and in vivo. The tested strains expressed weak/moderate biofilm production and showed a diffuse adhesion pattern (3 h) in HeLa cells and toxicity in Vero cells (24 h). Experimental inoculation in 11-week-old chicks induced a transitory inflammatory enteritis. Intestinal hemorrhage and destruction of the intestinal crypts were observed in the rabbit ileal loop test. Considering the fact that Brazil is a major exporter of poultry meat, the data from this study point to the need of improvement of the diagnostic tools, as well as of the adoption of surveillance guidelines and more specific control strategies to ensure food safety, reducing the presence of pathogenic MDR strains in broilers.

Captive wild birds as reservoirs of enteropathogenic E. coli (EPEC) and Shiga-toxin producing E. coli (STEC)

ABSTRACT Psittacine birds have been identified as reservoirs of diarrheagenic Escherichia coli, a subset of pathogens associated with mortality of children in tropical countries. The role of other orders of birds as source of infection is unclear. The aim of this study was to perform the molecular diagnosis of infection with diarrheagenic E. coli in 10 different orders of captive wild birds in the state of São Paulo, Brazil. Fecal samples were analyzed from 516 birds belonging to 10 orders: Accipitriformes, Anseriformes, Columbiformes, Falconiformes, Galliformes, Passeriformes, Pelecaniformes, Piciformes, Psittaciformes and Strigiformes. After isolation, 401 E. coli strains were subjected to multiplex PCR system with amplification of genes eae and bfp (EPEC), stx1 and stx2 for STEC. The results of these tests revealed 23/401 (5.74%) positive strains for eae gene, 16/401 positive strains for the bfp gene (3.99%) and 3/401 positive for stx2 gene (0.75%) distributed among the orders of Psittaciformes, Strigiformes and Columbiformes. None of strains were positive for stx1 gene. These data reveal the infection by STEC, typical and atypical EPEC in captive birds. The frequency of these pathotypes is low and restricted to few orders, but the data suggest the potential public health risk that these birds represent as reservoirs of diarrheagenic E. coli.

Captive wild birds as reservoirs of enteropathogenic E. coli (EPEC) and Shiga-toxin producing E. coli (STEC).

Psittacine birds have been identified as reservoirs of diarrheagenic Escherichia coli, a subset of pathogens associated with mortality of children in tropical countries. The role of other orders of birds as source of infection is unclear. The aim of this study was to perform the molecular diagnosis of infection with diarrheagenic E. coli in 10 different orders of captive wild birds in the state of São Paulo, Brazil. Fecal samples were analyzed from 516 birds belonging to 10 orders: Accipitriformes, Anseriformes, Columbiformes, Falconiformes, Galliformes, Passeriformes, Pelecaniformes, Piciformes, Psittaciformes and Strigiformes. After isolation, 401 E. coli strains were subjected to multiplex PCR system with amplification of genes eae and bfp (EPEC), stx1 and stx2 for STEC. The results of these tests revealed 23/401 (5.74%) positive strains for eae gene, 16/401 positive strains for the bfp gene (3.99%) and 3/401 positive for stx2 gene (0.75%) distributed among the orders of Psittaciformes, Strigiformes and Columbiformes. None of strains were positive for stx1 gene. These data reveal the infection by STEC, typical and atypical EPEC in captive birds. The frequency of these pathotypes is low and restricted to few orders, but the data suggest the potential public health risk that these birds represent as reservoirs of diarrheagenic E. coli.

Pandemic extra-intestinal pathogenic Escherichia coli (ExPEC) clonal group O6-B2-ST73 as a cause of avian colibacillosis in Brazil.

Extra-intestinal pathogenic Escherichia coli (ExPEC) represent an emerging pathogen, with pandemic strains increasingly involved in cases of urinary tract infections (UTIs), bacteremia, and meningitis. In addition to affecting humans, the avian pathotype of ExPEC, avian pathogenic E. coli (APEC), causes severe economic losses to the poultry industry. Several studies have revealed overlapping characteristics between APEC and human ExPEC, leading to the hypothesis of a zoonotic potential of poultry strains. However, the description of certain important pandemic clones, such as Sequence Type 73 (ST73), has not been reported in food sources. We characterized 27 temporally matched APEC strains from diverse poultry farms in Brazil belonging to the O6 serogroup because this serogroup is frequently described as a causal factor in UTI and septicemia in humans in Brazil and worldwide. The isolates were genotypically characterized by identifying ExPEC virulence factors, phylogenetically tested by phylogrouping and multilocus sequence type (MLST) analysis, and compared to determine their similarity employing the pulsed field gel electrophoresis (PFGE) technique. The strains harbored a large number of virulence determinants that are commonly described in uropathogenic E. coli (UPEC) and sepsis associated E. coli (SEPEC) strains and, to a lesser extent in neonatal meningitis associated E. coli (NMEC), such as pap (85%), sfa (100%), usp (100%), cnf1 (22%), kpsMTII (66%), hlyA (52%), and ibeA (4%). These isolates also yielded a low prevalence of some genes that are frequently described in APEC, such as iss (37%), tsh, ompT, and hlyF (8% each), and cvi/cva (0%). All strains were classified as part of the B2 phylogroup and sequence type 73 (ST73), with a cluster of 25 strains showing a clonal profile by PFGE. These results further suggest the zoonotic potential of some APEC clonal lineages and their possible role in the epidemiology of human ExPEC, in addition to providing the first description of the O6-B2-ST73 clonal group in poultry.

Prevalence of avian pathogenic Escherichia coli (APEC) clone harboring sfa gene in Brazil.

Escherichia coli sfa+ strains isolated from poultry were serotyped and characterized by polymerase chain reaction (PCR) and amplified fragment length polymorphism (AFLP). Isolates collected from 12 Brazilian poultry farms mostly belonged to serogroup O6, followed by serogroups O2, O8, O21, O46, O78, O88, O106, O111, and O143. Virulence genes associated were: iuc 90%, fim 86% neuS 60%, hly 34%, tsh 28%, crl/csg 26%, iss 26%, pap 18%, and 14% cnf. Strains from the same farm presented more than one genotypic pattern belonging to different profiles in AFLP. AFLP showed a clonal relation between Escherichia coli sfa+ serogroup O6. The virulence genes found in these strains reveal some similarity with extraintestinal E. coli (ExPEC), thus alerting for potential zoonotic risk.

Ilhas de patogenicidade / Islas de patogenicidad / Pathogenicity islands

Ilhas de patogenicidade constituem segmentos de DNA inseridos no cromossomo bacteriano, que atribuem uma variedade de caracteristicas de virulência aos microorganismos que a possuem. Dentre as propriedades conferidas pelas PAIs destacam-se a capacidade de aderir e invadir o epitélio da célula hospedeira, produzir toxinas, captar ferro do meio ambiente e sintetizar o sitema de secreção tipo III, dispositivo molecular que permitem a translocação de moléculas efetoras para o interior da célula hospedeira. A capacidade de adquirir propriedades patogênicas em um único evento genético permite a evolução, bem como o surgimento de microorganismos patogênicos.

Las islas de patogenicidad (PAI) son segmentos de DNA insertados en el cromosoma bacteriano, las cuales atribuyen una variedad de características de virulencia a los microorganismos que las poseen. Entre las características atribuidas por las PAI se distinguen la capacidad de adhesión e invasión del epitelio de la célula hospedera, de producción de toxinas, de captación del hierro del medio ambiente y de sintetizar el sistema de secreción tipo III, el dispositivo molecular que permite la translocación de las moléculasde efectoras para el interior de las células hospederas. La capacidad de adquirir características patógenas en un único acontecimiento genético permite la evolución, así bien la emergencia de microorganismos patógenos.

Pathogenicity island are inserted DNA fragments in the bacterial chromosome that confer a variety of virulence traits to the microorganisms. Among this variety of virulence properties we can see: the ability of adhering and invading the host cells, toxins production, iron uptake and the synthesis of Type Three Secretion System, a molecular device that allows the effectors molecules translocation inside the host cells. The feature of acquiring virulence traits in one genetic event allows the formation of new pathogens, as well the pathogenic microorganism’s evolution.

Diversity of virulence profiles of Shiga toxin-producing Escherichia coli serotypes in food-producing animals in Brazil.

The prevalence, serotypes and virulence profiles of Shiga toxin-producing Escherichia coli (STEC) were investigated in 205 healthy beef and dairy cattle, and 106 goats reared in the southeastern region of Minas Gerais State, Brazil. The prevalence of STEC was 57.5% (61/106) in goats, 39.2%, (40/102) in beef cattle and 17.5% (18/103) in dairy cattle. Among the 514 STEC isolates, 40 different serotypes were found and some of them were identified in a specific host. STEC isolates harboring stx1 corresponded to 15.6% (28/180), 26.7% (16/60) and 24.1% (66/274) in beef cattle, dairy cattle and goats, respectively. stx2 was found in 30% (54/180), 53.3% (32/60) and 34.7% (95/274) of beef and dairy cattle, and goats. stx1 plus stx2 sequences were harbored by 54.4% (98/180), 20% (12/60) and 41.2% (113/274) of beef cattle, dairy cattle and goats, respectively. The eae sequence was found in 15% (9/60) and 0.6% (1/180) of STEC isolates from dairy and beef cattle, respectively, and the toxB gene was found only in one O157:H7 strain isolated from beef cattle. Strains with the genetic profiles stx2 ehxA iha saa and stx1 stx2 ehxA iha saa were the most prevalent among STEC isolates from cattle. Profiles stx1 stx2 ehxA iha, stx2, and stx1 iha accounted for 75.5% (207 /274) of the STEC isolates from goats. While STEC strains carrying either stx2 alone or associated with stx1 were found more frequently in cattle, those harboring sequences stx1c and stx2d alone or associated with stx1c predominated in goats. Our data show a diversity of STEC strains in food-producing animals, most of them carrying genes linked to severe forms of human diseases.

Expression of aggregative adherence to hela cells by Escherichia coli strains isolated from sick horses

The virulence attributes of 56 Escherichia coli strains isolated from sick horses (secretions of uterine cervices; gastrointestinal and lung fragments of necropsy; diarrheic feces, and tracheal washings) was examined by determining their adherence pattern to HeLa cells and searching for the presence of virulence genes of the various E. coli pathotypes. Two non-adherent strains presented astA, which encodes the enteroaggregative E. coli heat-stable toxin. Twenty-seven strains (48.2 percent) adhered to HeLa cells, 21 (77.8 percent) of which presented the aggregative adherence pattern (AA) that characterize the Enteroaggregative E. coli pathotype (EAEC). Nine of the strains presenting AA were isolated from secretions of uterine cervix, including one carrying virulence genes of the EAEC pathotype (aggR,aap,irp2, and pic). This is the first description of the AA phenotype amongst E. coli strains from sick horses. Such strains should be further evaluated regarding their potential role in the pathogenesis of diverse equine diseases and as reservoirs of human infections.

Características de virulência de 56 amostras de Escherichia coli isoladas de eqüinos doentes (secreção de colo uterino, fragmentos de necrópsia do trato gastrointestinal e de pulmões, fezes diarréicas e lavado traqueal) foram examinadas para determinar o padrão de aderência em células HeLa e pesquisar a presença de genes de virulência de vários patotipos de E. coli. Duas amostras não aderentes apresentaram astA, gene que codifica a toxina termo-estável de E. coli enteroagregativa. Das vinte e sete amostras (48,2 por cento) que aderiram a células HeLa, 21 (77,8 por cento) apresentaram o padrão de aderência agregativa (AA) que caracteriza o patotipo de E. coli Enteroagregativa (EAEC). Nove destas amostras que apresentaram AA foram isoladas de secreção de colo uterino, incluindo uma que apresentava genes de virulência de patotipos de EAEC (aggR,aap,irp2 e pic). Esta é a primeira descrição do fenótipo AA em amostras de cavalos doentes. Estas amostras deverão ser melhor avaliadas em relação a sua potencial função na patogênese de diferentes doenças eqüinas, bem como à possibilidade destes animais representarem um reservatório de infecções humanas causadas por esta bactéria.

Characterization of atypical enteropathogenic Escherichia coli strains that express typical localized adherence in HeLa cells in the absence of the bundle-forming pilus.

The characterization of nine atypical enteropathogenic Escherichia coli strains expressing localized adherence in HeLa cells in the absence of the bundle-forming pilus revealed a diversity of serotypes, plasmids, and virulence genes. Although the strains lacked known E. coli adhesin genes, the identification of new adhesins could contribute to the characterization of similar enteropathogenic E. coli isolates.

Some adhesins of avian pathogenic Escherichia coli (APEC) isolated from septicemic poultry in Brazil

Three hundred and fifty strains of E. coli isolated from septicemic poultry from seven states of Brazil were examined for presence of nine adhesion-encoding genes, hemagglutination and adherence to chicken tracheal cells (in vitro). Analysis of the strains by colony hybridization tests demonstrated that 93.7 percent of the isolates were fim +, 17 percent pap+ and 5.7 percent were sfa+. The mannose sensitive fimbriae occur with similar frequency in APEC isolated from all Brazilians states, while significant differences among pap and sfa genes distributions were observed. The results showed that 0.85 percent and 0.28 percent of APEC were positive for genes that encoded enteroaggregative adhesins and EPEC adherence factor, respectively. None of APEC was positive for DA, afa, Bfp and Eae probes. The adherence to chicken tracheal cells showed 96 percent positive strains, while hemagglutination assays showed 26.5 percent of the isolates were mannose sensitive and 21.7 percent were mannose resistant.

Trezentas e cinqüenta amostras de E. coli isoladas de aves com septicemia em sete estados do Brasil foram examinadas para a presença de nove genes codificadores de adesinas, hemaglutinação e aderência em células da traquéia (in vitro). A análise das amostras pela hibridização de colônias demonstrou que 93,7 por cento dos isolados eram fim +, 17 por cento pap+ e 5,7 por cento eram sfa+. As fímbrias manose sensíveis apresentaram uma distribuição uniforme em todos os estados do Brasil. No entanto, diferenças significativas na distribuição dos genes pap e sfa foram observadas. Os resultados mostraram que 0,85 por cento e 0,28 por cento das APEC foram positivas para os genes que codificam as adesinas enteroagregativas e o fator de aderência de EPEC, respectivamente. Nenhuma amostra foi positiva para as sondas DA, afa, Bfp e Eae. A aderência em células de traquéia de aves revelou 96 por cento de amostras positivas, enquanto os testes de hemaglutinação mostraram 26,5 por cento dos isolados mannose sensíveis e 21,7 por cento manose resistentes.

Genetic analysis of Escherichia coli strains carrying enteropathogenic Escherichia coli (EPEC) markers, isolated from children in Rio de Janeiro city, Brazil

No presente estudo, 47 amostras enteropatogênicas de Escherichia coli, previamente caracterizadas pelo sorotipo, fenótipo de aderência, habilidade de induzir a formação da lesão histopatológica e presença das seqüências genéticas eae, bfp e EAF, foram analisadas de acordo com o perfil de fragmentação do DNA cromossômico pela técnica de eletroforese em campo pulsado (PFGE), as variantes isoenzimáticas através da eletroforese de isoenzimas (MLEE) e a presença de seqüências específicas da região LEE (eae, espA, espB, tir) e respectivos alelos. A amplificação destas seqüências mostrou a presença de 18 padrões genéticos distintos. A tipagem do gene eae revelou que a maior parte das amostras apresentou intimina não-tipável (42%) seguida dos tipos alélicos b (35%), g e a (12% cada). A fragmentação do DNA cromossômico detectou um elevado polimorfismo genético entre as amostras estudadas e não foi observada uma correlação com os marcadores de virulência investigados. Por outro lado, a análise das variantes isoenzimáticas sugeriu uma distribuição clonal específica de variantes genéticas do locus eae, o que nos leva a indicar a sua utilização como um marcador promissor para definir as relações genéticas neste grupo de microrganismos.

Genetic analysis of Escherichia coli strains carrying enteropathogenic Escherichia coli (EPEC) markers, isolated from children in Rio de Janeiro city, Brazil

In the present study, 47 enteropathogenic Escherichia coli strains identified according to serotyping, presence of eae, bfp and EAF sequences, adherence phenotype and ability to induce attaching-effacing lesions were analyzed by pulsed-field gel electrophoresis (PFGE), multilocus enzyme electrophoresis (MLEE), and the presence of LEE genes (eae, espA, espB, tir) as well as the respective alleles. Amplification of LEE genes subtypes revealed 18 different pathotypes. Typing of the eae gene showed that most strains contained nontypable intimin (42%) followed by beta (35%), gamma and alpha genes (12% each). PFGE analysis revealed a variable degree of polymorphism among isolates and, in general, no clear correlation was observed among PFGE profiles and the virulence markers identified. Otherwise, grouping based on MLEE analysis showed a close association between eae allele and clonal cluster distribution leading us to indicate the eae profile as a promising marker to establish relatedness among such microorganisms.

No presente estudo, 47 amostras enteropatogênicas de Escherichia coli, previamente caracterizadas pelo sorotipo, fenótipo de aderência, habilidade de induzir a formação da lesão histopatológica e presença das seqüências genéticas eae, bfp e EAF, foram analisadas de acordo com o perfil de fragmentação do DNA cromossômico pela técnica de eletroforese em campo pulsado (PFGE), as variantes isoenzimáticas através da eletroforese de isoenzimas (MLEE) e a presença de seqüências específicas da região LEE (eae, espA, espB, tir) e respectivos alelos. A amplificação destas seqüências mostrou a presença de 18 padrões genéticos distintos. A tipagem do gene eae revelou que a maior parte das amostras apresentou intimina não-tipável (42%) seguida dos tipos alélicos beta (35%), gama e alfa (12% cada). A fragmentação do DNA cromossômico detectou um elevado polimorfismo genético entre as amostras estudadas e não foi observada uma correlação com os marcadores de virulência investigados. Por outro lado, a análise das variantes isoenzimáticas sugeriu uma distribuição clonal específica de variantes genéticas do locus eae, o que nos leva a indicar a sua utilização como um marcador promissor para definir as relações genéticas neste grupo de microrganismos.

Análise do potencial de virulência de amostras de Escherichia coli näo pertencentes a sorogrupos de EPEC, portadoras do gene eae e desprovidas das seqüências das sondas EAF e stx / Analysis of the virulence potential of Escherichia coli strains, that do not belong to EPEC serogroups, carrying eae genes and lacking EAF and stx sequences

A lesão "attaching and effacing" é um mecanismo de virulência importante de alguns enteropatógenos bacterianos humanos, como a Escherichia coli enteropatogênica (EPEC) e a E. coli produtora de toxina Shiga (STEC) (que inclui a E. coli enterohemorrágica - EHEC), Essa lesão é caracterizada pela aderência íntima da bactéria ao enterócito, destruição das microvilosidades e formação de estruturas semelhantes a pedestais, onde com freqüência se localizam as bactérias aderidas. O teste de FAS ("fluorescent actin staining") detecta a alta concentração de filamentos de actina polimerizada presentes no enterócito, logo abaixo do sítio de aderência bacteriana. A intimina é a proteína bacteriana, codificada pelo gene eae, responsável pela aderência íntima às células hospedeiras, na lesão A/E, o receptor da intimina, denominado Tir ("translocated intimin receptor"), é um produto bacteriano que é inserido na célula hospedeira durante a infecção. Estudos imunológicos e moleculares possibilitaram a descrição de variações na porção carboxi-terminal da molécula de intimina, Os genes envolvidos na formação da lesão A/E estão localizados em uma ilha de patogenicidade denominada "locus of enterocyte effacement" (LEE). Foram descritos pelo menos dois sítios diferentes de inserção da região LEE, que estão localizados adjacentes a selC e pheU, no cromossomo de E. col K-12. Além da lesão A/E, foram descritos outros fatores que colaboram com a patogenicidade de EPEC e STEC/EHEC. Na categoria das EPECS, esses fatores compreendem a presença do plasmídio EAF ("EPEC adherence factor"), que codifica o "bundle forming pilus" (BFP) e está associado à formação do padrão de aderência localizada (AL), em algumas linhagens celulares cultivadas "in vitro". Nas STEC/EHEC, compreendem a presença de seqüências stx, relacionadas à produçäo de toxinas Shiga. A descriçäo de que amostras de E. coli FAS+, desprovidas das seqüências das sondas DNA EAF e stx, haviam sido isoladas de casos de diarréia em algumas regiöes geográficas, colocou uma questäo sobre o papael dessas amostras na doença diarréica. Este estudo foi realizado para caracterizar o potencial de virulência de 59 amostras de E. coli que eram portadoras do gene eae e näo apresentavam similaridade com as sondas genéticas EAF e stx(E.coli eae + EAF-stx-), que haviam sido previamente isoladas e crianças com e sem diarréia em Säo Paulo. Foram pesquisados vários marcadores genotípicos e fenotípicos. Os ensaios...(au)