Escherichia coli vacuolating factor, involved in avian cellulitis, induces actin contraction and binds to cytoskeleton proteins in fibroblasts

Avian pathogenic Escherichia coli (APEC) isolated from avian cellulitis lesions produces a toxin, named Escherichia coli vacuolating factor (ECVF), that causes cell vacuolization and induces inflammatory response in broiler chicken. Methods We investigated the intracellular activities of ECVF in avian fibroblasts using fluorescence staining, electron microscopy, MTT and LDH measurements. As ECVF act specifically in avian cells, we performed blotting assay followed by mass spectrometry to better understand its initial intracellular protein recognition. Results ECVF induced actin contraction, mitochondrial damage and membrane permeability alterations. Ultrastructural analysis showed intracellular alterations, as nuclear lobulation and the presence of degraded structures inside the vacuoles. Moreover, ECVF induced cell death in fibroblasts. ECVF-biotin associates to at least two proteins only in avian cell lysates: alpha-actinin 4 and vinculin, both involved in cytoskeleton structure. Conclusion These findings demonstrated that ECVF plays an important role in avian cellulitis, markedly in initial steps of infection. Taken together, the results place this toxin as a target for drug and/or vaccine development, instead of the use of large amounts antibiotics.(AU)

Escherichia coli vacuolating factor, involved in avian cellulitis, induces actin contraction and binds to cytoskeleton proteins in fibroblasts

Avian pathogenic Escherichia coli (APEC) isolated from avian cellulitis lesions produces a toxin, named Escherichia coli vacuolating factor (ECVF), that causes cell vacuolization and induces inflammatory response in broiler chicken. Methods We investigated the intracellular activities of ECVF in avian fibroblasts using fluorescence staining, electron microscopy, MTT and LDH measurements. As ECVF act specifically in avian cells, we performed blotting assay followed by mass spectrometry to better understand its initial intracellular protein recognition. Results ECVF induced actin contraction, mitochondrial damage and membrane permeability alterations. Ultrastructural analysis showed intracellular alterations, as nuclear lobulation and the presence of degraded structures inside the vacuoles. Moreover, ECVF induced cell death in fibroblasts. ECVF-biotin associates to at least two proteins only in avian cell lysates: alpha-actinin 4 and vinculin, both involved in cytoskeleton structure. Conclusion These findings demonstrated that ECVF plays an important role in avian cellulitis, markedly in initial steps of infection. Taken together, the results place this toxin as a target for drug and/or vaccine development, instead of the use of large amounts antibiotics.(AU)

Occurrence of Escherichia coli in captive psittaciformes: antimicrobial susceptibility and virulence genes / Ocorrência de Escherichia coli em psitacídios cativos: suscetibilidade antimicrobiana e genes de virulência

Captive Psittaciformes may harbor Gram-negative bacteria in their digestive tract, mainly due to poor hygienic conditions and confinement. The present study was carried out with the objective of isolating and identifying Escherichia coli in samples collected from Psittaciformes cages in 50 commercial establishments in the metropolitan region of Goiania, with subsequent antimicrobial susceptibility testing and detection of virulence genes. A total of 141 samples of excreta and swab samples from feeders and water bowls were collected, totaling 423 samples. Escherichia coli was isolated from 9.7% (41/423) samples: 12% (17/141) in excreta, 8.5% (12/141) in feed, and 8.5% (12 /141) in waterers. To determine the susceptibility profile of E. coli isolates, resistance to ciprofloxacin 4.9% (2/41), gentamicin 17.0% (7/41), doxycycline 34.1% (14/41), florfenicol 34.1% (14/41), trimethoprim 39.0% (16/41), tetracycline 41.5% (17/41), enrofloxacin 43.9% (18/41), amoxicillin 48.8% (20/41), neomycin 61.0% (25/41), and sulfonamide 90.2% (37/41) was determined. In 20 isolates, resistance was determined at 4 or more antimicrobials, seven of excreta (7/17), five of feed (5/12), and eight of waterers (8/12). One of the isolates from the waterers showed resistance to all antimicrobials. The iss gene was detected in three isolates, the tsh gene in three, the papC gene in two, traT and eae genes were not detected. In this study, it can be concluded that Psittaciformes commercialized as pet are carry E. coli isolates resistant to most commonly used antimicrobials, mainly sulfonamides and neomycin, besides having virulence and serum resistance genes, which highlights the possibility of the to cause disease in humans.

Psittaciformes em cativeiro podem abrigar bactérias Gram-negativas em seu trato digestivo, principalmente devido a condições higiênicas inadequadas e ao confinamento. O presente estudo teve o objetivo de isolar e identificar Escherichia coli em amostras coletadas de gaiolas de Psittaciformes em 50 estabelecimentos comerciais da região metropolitana de Goiânia, com subsequentes testes de susceptibilidade antimicrobiana e detecção de genes de virulência. Foram coletadas 141 amostras de excrementos e suabes de alimentadores e bebedouros, totalizando 423 amostras. Escherichia coli foi isolada em 9,7% (41/423) amostras: 12% (17/141) em excrementos, 8,5% (12/141) em ração e 8,5% (12/141) em bebedouros. Os isolados de E. coli mostraram resistência à ciprofloxacina 4,9% (2/41), gentamicina 17,0% (7/41), doxiciclina 34,1% (14/41), florfenicol 34,1% (14/41), trimetoprim 39,0% (16/41), tetraciclina 41,5% (17/41), enrofloxacina 43,9% (18/41), amoxicilina 48,8% (20/41), neomicina 61,0% (25/41) e sulfonamida 90,2% (37 / 41) foi determinado. Multirresistência (resistência a quatro ou mais antimicrobianos) foi encontrada em 20 amostras, sete de excrementos (7/17), cinco de ração (5/12) e oito de bebedouros (8/12). Um dos isolados dos bebedouros apresentou resistência a todos os antimicrobianos. O gene iss foi detectado em três isolados, o gene tsh em três, o gene papC em dois, os genes traT e eae não foram detectados. Neste estudo, pode-se concluir que os Psittaciformes comercializados como animais de estimação são portadores de isolados de E. coli resistentes aos antimicrobianos mais utilizados, principalmente sulfonamidas e neomicina, além de possuir genes de virulência e resistência sérica, destacando a possibilidade de causar doenças em humanos.

Occurrence of Escherichia coli in captive psittaciformes: antimicrobial susceptibility and virulence genes / Ocorrência de Escherichia coli em psitacídios cativos: suscetibilidade antimicrobiana e genes de virulência

Captive Psittaciformes may harbor Gram-negative bacteria in their digestive tract, mainly due to poor hygienic conditions and confinement. The present study was carried out with the objective of isolating and identifying Escherichia coli in samples collected from Psittaciformes cages in 50 commercial establishments in the metropolitan region of Goiania, with subsequent antimicrobial susceptibility testing and detection of virulence genes. A total of 141 samples of excreta and swab samples from feeders and water bowls were collected, totaling 423 samples. Escherichia coli was isolated from 9.7% (41/423) samples: 12% (17/141) in excreta, 8.5% (12/141) in feed, and 8.5% (12 /141) in waterers. To determine the susceptibility profile of E. coli isolates, resistance to ciprofloxacin 4.9% (2/41), gentamicin 17.0% (7/41), doxycycline 34.1% (14/41), florfenicol 34.1% (14/41), trimethoprim 39.0% (16/41), tetracycline 41.5% (17/41), enrofloxacin 43.9% (18/41), amoxicillin 48.8% (20/41), neomycin 61.0% (25/41), and sulfonamide 90.2% (37/41) was determined. In 20 isolates, resistance was determined at 4 or more antimicrobials, seven of excreta (7/17), five of feed (5/12), and eight of waterers (8/12). One of the isolates from the waterers showed resistance to all antimicrobials. The iss gene was detected in three isolates, the tsh gene in three, the papC gene in two, traT and eae genes were not detected. In this study, it can be concluded that Psittaciformes commercialized as pet are carry E. coli isolates resistant to most commonly used antimicrobials, mainly sulfonamides and neomycin, besides having virulence and serum resistance genes, which highlights the possibility of the to cause disease in humans.(AU)

Psittaciformes em cativeiro podem abrigar bactérias Gram-negativas em seu trato digestivo, principalmente devido a condições higiênicas inadequadas e ao confinamento. O presente estudo teve o objetivo de isolar e identificar Escherichia coli em amostras coletadas de gaiolas de Psittaciformes em 50 estabelecimentos comerciais da região metropolitana de Goiânia, com subsequentes testes de susceptibilidade antimicrobiana e detecção de genes de virulência. Foram coletadas 141 amostras de excrementos e suabes de alimentadores e bebedouros, totalizando 423 amostras. Escherichia coli foi isolada em 9,7% (41/423) amostras: 12% (17/141) em excrementos, 8,5% (12/141) em ração e 8,5% (12/141) em bebedouros. Os isolados de E. coli mostraram resistência à ciprofloxacina 4,9% (2/41), gentamicina 17,0% (7/41), doxiciclina 34,1% (14/41), florfenicol 34,1% (14/41), trimetoprim 39,0% (16/41), tetraciclina 41,5% (17/41), enrofloxacina 43,9% (18/41), amoxicilina 48,8% (20/41), neomicina 61,0% (25/41) e sulfonamida 90,2% (37 / 41) foi determinado. Multirresistência (resistência a quatro ou mais antimicrobianos) foi encontrada em 20 amostras, sete de excrementos (7/17), cinco de ração (5/12) e oito de bebedouros (8/12). Um dos isolados dos bebedouros apresentou resistência a todos os antimicrobianos. O gene iss foi detectado em três isolados, o gene tsh em três, o gene papC em dois, os genes traT e eae não foram detectados. Neste estudo, pode-se concluir que os Psittaciformes comercializados como animais de estimação são portadores de isolados de E. coli resistentes aos antimicrobianos mais utilizados, principalmente sulfonamidas e neomicina, além de possuir genes de virulência e resistência sérica, destacando a possibilidade de causar doenças em humanos.(AU)

Highly Pathogenic and Multidrug Resistant Avian Pathogenic Escherichia Coli in Free-Range Chickens from Brazil

Avian pathogenic Escherichiacoli (APEC) virulence mechanism has been continuously studied and it is believed to be multifactorial and because of this, this work aimed to characterize potentially APEC strains isolated from free-range hens. Isolates were submitted to PCR for the detection of virulence genes, which were of high prevalence. In vivo inoculation of day-old chicks revealed that 49 of these strains were of high and intermediate pathogenicity. In addition, isolates were submitted to antimicrobials susceptibility test with the majority of the strains presenting multiresistance. Phylogenetic analysis showed a greater presence of potentially APEC isolates in-group B2. In addition, high heterogeneity was detected among the isolates byXbaI enzyme. Fifteen serogroups were identified, being the O8 the most frequent. These results strengthen the fact that a combination of diverse factors are associated with the pathogenicity APEC strains, as well as to highlight its importance to public health and that free-range hens can act as a reservoirs of potentially zoonoticbacteria.(AU)

Extended-Spectrum Beta-Lactamase Producing Strains of Escherichia Coli Isolated from Avian Cellulitis Lesions

Avian cellulitis causes significant losses to the poultry industry. Avian-pathogenic Escherichia coli (APEC) is the etiological agent of that disease. This microorganism has zoonotic potential and may act as reservoir of antimicrobial-resistance genes. In this context, the production of extended-spectrum B-lactamase (ESBL) is one of the main antimicrobial resistance mechanisms. The objective of this study was to determine the production of ESBL in an Escherichia coli (E. coli) strain isolated from avian cellulitis lesions. Twenty-two E. Coli isolates were harvested from cellulitis lesions in chicken carcasses in a commercial processing plant. Isolates were then submitted to virulence genotypic profile (iutA, hlyF, iss, ironN, ompT) analysis, antimicrobial susceptibility test, and detection of ESBL production. The results showed that 22.7% of the isolates presented five virulence genes, 9.1% four genes, 36.4% three genes, 13.6% two genes, and 18.2% one gene. The tested isolates showed resistance to ampicillin (90.9%), ceftiofur (54.5%), gentamicin (45.5%), tetracycline (72.1%), sulfamethoxazole/trimethoprim (54.5%), and enrofloxacin (54.5%). Furthermore, 77.3% of the isolates presented multidrug resistance (MDR) profile and 72.7% were positive for ESBL production. This study is the first description of ESBL-producing APEC isolated from avian cellulitis lesions, which suggests the need to establish efficient APEC control measures and programs to prevent flock productivity losses due to colibacillosis and public health risks.(AU)

Highly Pathogenic and Multidrug Resistant Avian Pathogenic Escherichia Coli in Free-Range Chickens from Brazil

Avian pathogenic Escherichiacoli (APEC) virulence mechanism has been continuously studied and it is believed to be multifactorial and because of this, this work aimed to characterize potentially APEC strains isolated from free-range hens. Isolates were submitted to PCR for the detection of virulence genes, which were of high prevalence. In vivo inoculation of day-old chicks revealed that 49 of these strains were of high and intermediate pathogenicity. In addition, isolates were submitted to antimicrobials susceptibility test with the majority of the strains presenting multiresistance. Phylogenetic analysis showed a greater presence of potentially APEC isolates in-group B2. In addition, high heterogeneity was detected among the isolates byXbaI enzyme. Fifteen serogroups were identified, being the O8 the most frequent. These results strengthen the fact that a combination of diverse factors are associated with the pathogenicity APEC strains, as well as to highlight its importance to public health and that free-range hens can act as a reservoirs of potentially zoonoticbacteria.

Extended-Spectrum Beta-Lactamase Producing Strains of Escherichia Coli Isolated from Avian Cellulitis Lesions

Avian cellulitis causes significant losses to the poultry industry. Avian-pathogenic Escherichia coli (APEC) is the etiological agent of that disease. This microorganism has zoonotic potential and may act as reservoir of antimicrobial-resistance genes. In this context, the production of extended-spectrum B-lactamase (ESBL) is one of the main antimicrobial resistance mechanisms. The objective of this study was to determine the production of ESBL in an Escherichia coli (E. coli) strain isolated from avian cellulitis lesions. Twenty-two E. Coli isolates were harvested from cellulitis lesions in chicken carcasses in a commercial processing plant. Isolates were then submitted to virulence genotypic profile (iutA, hlyF, iss, ironN, ompT) analysis, antimicrobial susceptibility test, and detection of ESBL production. The results showed that 22.7% of the isolates presented five virulence genes, 9.1% four genes, 36.4% three genes, 13.6% two genes, and 18.2% one gene. The tested isolates showed resistance to ampicillin (90.9%), ceftiofur (54.5%), gentamicin (45.5%), tetracycline (72.1%), sulfamethoxazole/trimethoprim (54.5%), and enrofloxacin (54.5%). Furthermore, 77.3% of the isolates presented multidrug resistance (MDR) profile and 72.7% were positive for ESBL production. This study is the first description of ESBL-producing APEC isolated from avian cellulitis lesions, which suggests the need to establish efficient APEC control measures and programs to prevent flock productivity losses due to colibacillosis and public health risks.

Detection of Enterobacteriaceae, antimicrobial susceptibility, and virulence genes of Escherichia coli in canaries (Serinus canaria) in northeastern Brazil / Detecção de enterobactérias, sensibilidade antimicrobiana e genes de virulência de Escherichia coli em canários belgas (Serinus canaria) da região Nordeste do Brasil

This study aimed to verify the presence of members from the Enterobacteriaceae family and determine antimicrobial susceptibility profiles of the isolates in canaries bred in northeastern Brazil; in addition, the presence of diarrheagenic Escherichia coli (DEC) and avian pathogenic Escherichia coli (APEC) was also verified in these birds. Samples were collected during an exhibition organized by the Brazilian Ornithological Federation in July 2015 in Fortaleza, Brazil. A total of 88 fecal samples were collected and submitted to pre-enrichment step using buffered peptone water, followed by enrichment with the following broths: brain-heart infusion, Rappaport-Vassiliadis, and Selenite-Cystine. Subsequently, aliquots were streaked on MacConkey, brilliant green and salmonella-shigella agar plates. Colonies were selected according to morphological characteristics and submitted to biochemical identification and antimicrobial susceptibility tests with disk-diffusion technique. E. coli strains were evaluated for the presence of eight DEC genes and five APEC genes through conventional polymerase chain reaction (PCR) screening. The most frequent species observed were Pantoea agglomerans (25%), Serratia liquefaciens (12.5%), and Enterobacter aerogenes (9.1%). A single rough strain of Salmonella enterica subsp. enterica was identified in one sample (1.1%). High resistance rates to amoxicillin (78.7%) and ampicillin (75.4%) were identified. Polymyxin B (9.8%), gentamycin (6.6%), and enrofloxacin (6.6%) were the most efficient antibiotics. The total number of multidrug-resistant strains (isolates resistant to more than three antimicrobial classes) was 23 (37.7%). Four E. coli strains were tested for the virulence genes, and two were positive for APEC virulence genes: one strain was positive for iutA and the other for hlyF...(AU)

O objetivo deste trabalho foi verificar a presença de enterobactérias e determinar o perfil de sensibilidade aos antimicrobianos dos isolados oriundos de canários belgas criados em cativeiro do Nordeste do Brasil, adicionalmente verificou-se a presença de Escherichia coli diarreiogênicas (DEC) e E. coli patogênica aviária (APEC) nesses animais. A colheita das amostras ocorreu durante uma exposição de canários belgas organizada pela Federação Ornitológica do Brasil (FOB), em julho de 2015, na cidade de Fortaleza, Ceará, Brasil. Um total de 88 amostras de fezes foram coletadas e submetidas a pré-enriquecimento utilizando água peptonada, caldo de enriquecimento Brain Heart Infusion, Rappaport-Vassiliadis e Selenito-Cistina. Fez-se triagem em placas de ágar MacConkey, Verde Brilhante e ágar Salmonella Shigella. As colônias foram selecionadas e submetidas à identificação bioquímica e susceptibilidade antimicrobiana. Estirpes de Escherichia coli foram avaliadas quanto a presença de 8 genes de virulência de DEC e cinco de APEC por reação em cadeia da polimerase convencional (PCR). As enterobactérias encontradas com maior frequência foram Pantoea agglomerans (25%), Serratia liquefaciens (12,5%) e Enterobacter aerogenes (9,1%). Uma única estirpe de Salmonella enterica subsp. enterica (rugosa) esteve presente em um dos isolados (1,1%). Altos percentuais de resistência foram encontrados para dois antibióticos: amoxicilina (78,7%) e ampicilina (75,4%). Polimixina B (9,8%), gentamicina (6,8%) e enrofloxacina (6,5%) foram os antibióticos com melhor eficiência. O total de estirpes multirresistentes (a mais de três classes de antimicrobianos) foi de 23 (37,7%). Das quatro estirpes de E. coli isoladas, duas foram positivas para os genes de APEC, sendo uma estipe para o gene iss e outra para os genes iutA e hlyF...(AU)

Detection of Enterobacteriaceae, antimicrobial susceptibility, and virulence genes of Escherichia coli in canaries (Serinus canaria) in northeastern Brazil / Detecção de enterobactérias, sensibilidade antimicrobiana e genes de virulência de Escherichia coli em canários belgas (Serinus canaria) da região Nordeste do Brasil

This study aimed to verify the presence of members from the Enterobacteriaceae family and determine antimicrobial susceptibility profiles of the isolates in canaries bred in northeastern Brazil; in addition, the presence of diarrheagenic Escherichia coli (DEC) and avian pathogenic Escherichia coli (APEC) was also verified in these birds. Samples were collected during an exhibition organized by the Brazilian Ornithological Federation in July 2015 in Fortaleza, Brazil. A total of 88 fecal samples were collected and submitted to pre-enrichment step using buffered peptone water, followed by enrichment with the following broths: brain-heart infusion, Rappaport-Vassiliadis, and Selenite-Cystine. Subsequently, aliquots were streaked on MacConkey, brilliant green and salmonella-shigella agar plates. Colonies were selected according to morphological characteristics and submitted to biochemical identification and antimicrobial susceptibility tests with disk-diffusion technique. E. coli strains were evaluated for the presence of eight DEC genes and five APEC genes through conventional polymerase chain reaction (PCR) screening. The most frequent species observed were Pantoea agglomerans (25%), Serratia liquefaciens (12.5%), and Enterobacter aerogenes (9.1%). A single rough strain of Salmonella enterica subsp. enterica was identified in one sample (1.1%). High resistance rates to amoxicillin (78.7%) and ampicillin (75.4%) were identified. Polymyxin B (9.8%), gentamycin (6.6%), and enrofloxacin (6.6%) were the most efficient antibiotics. The total number of multidrug-resistant strains (isolates resistant to more than three antimicrobial classes) was 23 (37.7%). Four E. coli strains were tested for the virulence genes, and two were positive for APEC virulence genes: one strain was positive for iutA and the other for hlyF. In conclusion, canaries in northeastern Brazil participating in exhibitions may present Salmonella spp., Escherichia coli and other enterobacteria in the intestinal microbiota with antimicrobial resistance. These results indicate that, although the E. coli strains recovered from canaries in this study have some virulence genes, they still do not fulfill all the requirements to be considered APEC.(AU)

O objetivo deste trabalho foi verificar a presença de enterobactérias e determinar o perfil de sensibilidade aos antimicrobianos dos isolados oriundos de canários belgas criados em cativeiro do Nordeste do Brasil, adicionalmente verificou-se a presença de Escherichia coli diarreiogênicas (DEC) e E. coli patogênica aviária (APEC) nesses animais. A colheita das amostras ocorreu durante uma exposição de canários belgas organizada pela Federação Ornitológica do Brasil (FOB), em julho de 2015, na cidade de Fortaleza, Ceará, Brasil. Um total de 88 amostras de fezes foram coletadas e submetidas a pré-enriquecimento utilizando água peptonada, caldo de enriquecimento Brain Heart Infusion, Rappaport-Vassiliadis e Selenito-Cistina. Fez-se triagem em placas de ágar MacConkey, Verde Brilhante e ágar Salmonella Shigella. As colônias foram selecionadas e submetidas à identificação bioquímica e susceptibilidade antimicrobiana. Estirpes de Escherichia coli foram avaliadas quanto a presença de 8 genes de virulência de DEC e cinco de APEC por reação em cadeia da polimerase convencional (PCR). As enterobactérias encontradas com maior frequência foram Pantoea agglomerans (25%), Serratia liquefaciens (12,5%) e Enterobacter aerogenes (9,1%). Uma única estirpe de Salmonella enterica subsp. enterica (rugosa) esteve presente em um dos isolados (1,1%). Altos percentuais de resistência foram encontrados para dois antibióticos: amoxicilina (78,7%) e ampicilina (75,4%). Polimixina B (9,8%), gentamicina (6,8%) e enrofloxacina (6,5%) foram os antibióticos com melhor eficiência. O total de estirpes multirresistentes (a mais de três classes de antimicrobianos) foi de 23 (37,7%). Das quatro estirpes de E. coli isoladas, duas foram positivas para os genes de APEC, sendo uma estipe para o gene iss e outra para os genes iutA e hlyF. Portanto, canários belgas criados em cativeiro no Brasil que participam de exposições podem apresentar Salmonella spp., Escherichia coli e outras enterobactérias em sua microbiota intestinal com resistência antimicrobiana. Estes resultados indicam que as estirpes de E. coli isoladas de canário belga no presente estudo apresentam alguns, mas não todos, genes de virulência para serem caracterizadas como E. coli patogênica para aves (APEC).(AU)

Colimetric index and virulence genes iss and iutA in Escherichia coli isolates in cellulitis of poultry carcasses under sanitary inspection / Índice colimétrico e genes de virulência iss e iutA em isolados de “Escherichia coli” em celulites de carcaças de frangos sob Inspeção Sanitária

Current study determines the population of total coliforms and Escherichia coli and identifies iss and iutA virulence genes in Escherichia coli strains isolated from cellulitis in poultry carcasses retrieved from a slaughterhouse. One hundred cellulitis lesions were collected between August 2013 and January 2014. The population of total coliforms and Escherichia coli was verified by Petrifilm rapid counting method (AOAC 998.8). Escherichia coli samples were analyzed for iss and iutA genes by Polymerase Chain Reaction (PCR) technique. Total coliforms were present in 96.0% (96/100) of the analyzed samples, with a population between 3.4 and 9.5 log CFU/g. Escherichia coli was present in 82.0% (82/100) of cellulitis samples and the population ranged between 1.0 and 9.0 log CFU/g. The iss gene was found in 89.0% of isolates and the iutA gene in 97.6%. High populations of total coliforms and Escherichiacoli in cellulitis samples indicate that hygienic-sanitary failures may have occurred in the production of broilers. When high prevalence of virulence genes under analysis, characteristic of Avian Pathogenic Escherichia coli (APEC) and possible zoonotic character of the pathotype are taken into account, it is important to highlight the need to adopt Good Manufacturing Practices, Standard Procedures of Operational Hygiene and Hazard Analysis and Critical Control Points in poultry slaughterhouses to ensure the safety of the final product.(AU)

Objetivou-se determinar a população de coliformes totais e de Escherichia coli e identificar os genes de virulência iss e iutA nas cepas de Escherichia coli isoladas de celulites em carcaças de frangos em um abatedouro. No periodo de agosto de 2013 a janeiro de 2014 foram coletadas 100 lesões de celulite, sendo verificada a população de coliformes totais e Escherichia coli, pelo método rápido de contagem Petrifilm (AOAC 998.8). As amostras de Escherichia coli foram analisadas para verificar a presença dos genes iss e iutA, utilizando a técnica de Reação em Cadeia da Polimerase (PCR). Houve a presença de coliformes totais em 96,0 % (96/100) das amostras analisadas, com a população entre 3,4 a 9,5 log UFC/g. Constatou-se também a presença de Escherichia coli em 82,0 % (82/100) das amostras de celulite, com população entre 1,0 e 9,0 log UFC/g. O gene iss foi encontrado em 89,0 % dos isolados e o gene iutA em 97,6 %. As populações elevadas de coliformes totais e de Escherichia coli nas amostras de celulite indicam que falhas higiênico-sanitárias podem ter ocorrido na produção dos frangos de corte. Considerando a prevalência elevada dos genes de virulência pesquisados, característicos de Escherichia coli Patogênica para Aves (APEC) e o possível caráter zoonótico deste patotipo, é importante ressaltar a necessidade da adoção das Boas Práticas de Fabricação, Procedimentos Padrão de Higiene Operacional e da Análise de Perigos e Pontos Críticos de Controle nos abatedouros, para garantir a inocuidade do produto final.(AU)

Colimetric index and virulence genes iss and iutA in Escherichia coli isolates in cellulitis of poultry carcasses under sanitary inspection / Índice colimétrico e genes de virulência iss e iutA em isolados de “Escherichia coli” em celulites de carcaças de frangos sob Inspeção Sanitária

Current study determines the population of total coliforms and Escherichia coli and identifies iss and iutA virulence genes in Escherichia coli strains isolated from cellulitis in poultry carcasses retrieved from a slaughterhouse. One hundred cellulitis lesions were collected between August 2013 and January 2014. The population of total coliforms and Escherichia coli was verified by Petrifilm rapid counting method (AOAC 998.8). Escherichia coli samples were analyzed for iss and iutA genes by Polymerase Chain Reaction (PCR) technique. Total coliforms were present in 96.0% (96/100) of the analyzed samples, with a population between 3.4 and 9.5 log CFU/g. Escherichia coli was present in 82.0% (82/100) of cellulitis samples and the population ranged between 1.0 and 9.0 log CFU/g. The iss gene was found in 89.0% of isolates and the iutA gene in 97.6%. High populations of total coliforms and Escherichiacoli in cellulitis samples indicate that hygienic-sanitary failures may have occurred in the production of broilers. When high prevalence of virulence genes under analysis, characteristic of Avian Pathogenic Escherichia coli (APEC) and possible zoonotic character of the pathotype are taken into account, it is important to highlight the need to adopt Good Manufacturing Practices, Standard Procedures of Operational Hygiene and Hazard Analysis and Critical Control Points in poultry slaughterhouses to ensure the safety of the final product.

Objetivou-se determinar a população de coliformes totais e de Escherichia coli e identificar os genes de virulência iss e iutA nas cepas de Escherichia coli isoladas de celulites em carcaças de frangos em um abatedouro. No periodo de agosto de 2013 a janeiro de 2014 foram coletadas 100 lesões de celulite, sendo verificada a população de coliformes totais e Escherichia coli, pelo método rápido de contagem Petrifilm (AOAC 998.8). As amostras de Escherichia coli foram analisadas para verificar a presença dos genes iss e iutA, utilizando a técnica de Reação em Cadeia da Polimerase (PCR). Houve a presença de coliformes totais em 96,0 % (96/100) das amostras analisadas, com a população entre 3,4 a 9,5 log UFC/g. Constatou-se também a presença de Escherichia coli em 82,0 % (82/100) das amostras de celulite, com população entre 1,0 e 9,0 log UFC/g. O gene iss foi encontrado em 89,0 % dos isolados e o gene iutA em 97,6 %. As populações elevadas de coliformes totais e de Escherichia coli nas amostras de celulite indicam que falhas higiênico-sanitárias podem ter ocorrido na produção dos frangos de corte. Considerando a prevalência elevada dos genes de virulência pesquisados, característicos de Escherichia coli Patogênica para Aves (APEC) e o possível caráter zoonótico deste patotipo, é importante ressaltar a necessidade da adoção das Boas Práticas de Fabricação, Procedimentos Padrão de Higiene Operacional e da Análise de Perigos e Pontos Críticos de Controle nos abatedouros, para garantir a inocuidade do produto final.

Characterization of avian pathogenic Escherichia coli isolated from free-range helmeted guineafowl

Abstract Avian pathogenic Escherichia coli (APEC) isolates from apparently healthy free range helmeted guineafowl were characterized. Most of them had a high frequency of virulence associated genes, multi drug resistance and high pathogenicity. We demonstrated that helmeted guineafowl have potential to transmit antibiotic resistant APEC to other species including humans.

Characterization of avian pathogenic Escherichia coli isolated from free-range helmeted guineafowl

Avian pathogenic Escherichia coli (APEC) isolates from apparently healthy free range helmeted guineafowl were characterized. Most of them had a high frequency of virulence associated genes, multi drug resistance and high pathogenicity. We demonstrated that helmeted guineafowl have potential to transmit antibiotic resistant APEC to other species including humans.(AU)

Atividade dos desinfetantes cloreto de benzalcônio e iodóforo sobre cepas de Escherichia coli patogênica aviária isoladas em frangos de corte / Activity of the disinfectants benzalkonium chloride and iodophor on “Escherichia coli” avianpathogenic strains isolated in broilers

Os objetivos deste estudo foram avaliar a eficácia dos desinfetantes cloreto de benzalcônio (QAC) e iodóforo (I) sobre 10 cepas APEC (Escherichia coli patogênica aviária), bem como verificar se a característica alta patogenicidade está associada a uma maior resistência a estes compostos. O método utilizado foi o de diluição através do teste qualitativo de suspensão. As variáveis estudadas foram: concentrações do QAC (300, 150, 75 e 50 ppm) e do I (100, 75, 50 e 25 ppm), tempos de contato (5, 10 e 20 minutos) e temperatura ±20°C. O QAC inativou todos os isolados nas concentrações de 300 e 150 ppm, em todos os tempos de contato, porém a 75 e 50 ppm no tempo de 5 minutos o desinfetante não foi eficaz para uma e quatro amostras, respectivamente. O I a 100 e 75 ppm inativou os isolados em todos os tempos avaliados, mas a 50 ppm um foi resistente e a 25 ppm oito foram resistentes em todos os tempos de exposição. A característica alta patogenicidade não pareceu promover resistência, quando comparado com a cepa padrão. Concluiu-se, nas condições do experimento, que os dois desinfetantes podem ser usados em procedimentos de higiene frente às cepas APEC, apenas levando-se em consideração a concentração de uso e o tempo de contato.(AU)

The objectives of this study were to evaluate the effectiveness of the disinfectant benzalkonium chloride (QAC) and iodophor (I) on 10 strains APEC (Escherichia coli pathogenic avian) and verify that the high feature pathogenicity provides protection factor against these compounds. The method used was the dilution by the qualitative suspension test. The variables studied were: the concentrations of QAC (300, 150, 75 and 50 ppm) and I (100, 75, 50 and 25 ppm), contact times (5, 10 and 20 minutes) and room temperature (20°C). The QAC inactivated all strains at concentrations of 300 and 150 ppm at all contact times, but at 75 and 50 ppm in time of 5 minutes was not effective disinfectant for one and four APEC strains, respectively. The I 100 and 75 ppm inactivated isolates in all time periods, but 50 ppm one was tough and 25ppm eight were resistant in all exposure times. The highly pathogenic feature does not appear to promote resistance when compared with the standard strain. It was concluded, under the experimental conditions, the two disinfectants can be used in front of the hygiene procedures APEC strains, only taking into account the use concentration and contact time.(AU)

Atividade dos desinfetantes cloreto de benzalcônio e iodóforo sobre cepas de Escherichia coli patogênica aviária isoladas em frangos de corte / Activity of the disinfectants benzalkonium chloride and iodophor on “Escherichia coli” avianpathogenic strains isolated in broilers

Os objetivos deste estudo foram avaliar a eficácia dos desinfetantes cloreto de benzalcônio (QAC) e iodóforo (I) sobre 10 cepas APEC (Escherichia coli patogênica aviária), bem como verificar se a característica alta patogenicidade está associada a uma maior resistência a estes compostos. O método utilizado foi o de diluição através do teste qualitativo de suspensão. As variáveis estudadas foram: concentrações do QAC (300, 150, 75 e 50 ppm) e do I (100, 75, 50 e 25 ppm), tempos de contato (5, 10 e 20 minutos) e temperatura ±20°C. O QAC inativou todos os isolados nas concentrações de 300 e 150 ppm, em todos os tempos de contato, porém a 75 e 50 ppm no tempo de 5 minutos o desinfetante não foi eficaz para uma e quatro amostras, respectivamente. O I a 100 e 75 ppm inativou os isolados em todos os tempos avaliados, mas a 50 ppm um foi resistente e a 25 ppm oito foram resistentes em todos os tempos de exposição. A característica alta patogenicidade não pareceu promover resistência, quando comparado com a cepa padrão. Concluiu-se, nas condições do experimento, que os dois desinfetantes podem ser usados em procedimentos de higiene frente às cepas APEC, apenas levando-se em consideração a concentração de uso e o tempo de contato.

The objectives of this study were to evaluate the effectiveness of the disinfectant benzalkonium chloride (QAC) and iodophor (I) on 10 strains APEC (Escherichia coli pathogenic avian) and verify that the high feature pathogenicity provides protection factor against these compounds. The method used was the dilution by the qualitative suspension test. The variables studied were: the concentrations of QAC (300, 150, 75 and 50 ppm) and I (100, 75, 50 and 25 ppm), contact times (5, 10 and 20 minutes) and room temperature (20°C). The QAC inactivated all strains at concentrations of 300 and 150 ppm at all contact times, but at 75 and 50 ppm in time of 5 minutes was not effective disinfectant for one and four APEC strains, respectively. The I 100 and 75 ppm inactivated isolates in all time periods, but 50 ppm one was tough and 25ppm eight were resistant in all exposure times. The highly pathogenic feature does not appear to promote resistance when compared with the standard strain. It was concluded, under the experimental conditions, the two disinfectants can be used in front of the hygiene procedures APEC strains, only taking into account the use concentration and contact time.

Classification of avian pathogenic Escherichia coli by a novel pathogenicity index based on an animal model

Background: Avian Pathogenic Escherichia coli is the main agent of colibacillosis, a systemic disease that causes considerable economic losses to the poultry industry. In vivo experiments are used to measure the ability of E. coli to be pathogenic. Generally, these experiments have proposed different criteria for results interpretation and did not take into account the death time. The aim of this study was to propose a new methodology for the classification of E. coli pathogenicity by the establishment of a pathogenicity index based in the lethality, death time and the ability of the strain to cause colibacillosis lesions in challenged animals. Materials, Methods & Results: A total of 293 isolates of E. coli were randomly selected to this study. The strains were isolated from cellulitis lesions, broiler bedding material or respiratory diseases and were previously confirmed through biochemical profile. The bacterial isolates were kept frozen at -20C. The strains were retrieved from stocks and cultured in brain-heart infusion broth overnight at 37C to obtain a final concentration of 109 UFC/mL. A total of 2940 one-dayold chicks from commercial breeding hens were randomly assigned to groups containing 10 animals and each group was subcutaneously inoculated in the abdominal region with 0.1 mL of the standard inoculum solution containing each of the strains. A control group [...](AU)

Classification of avian pathogenic Escherichia coli by a novel pathogenicity index based on an animal model

Background: Avian Pathogenic Escherichia coli is the main agent of colibacillosis, a systemic disease that causes considerable economic losses to the poultry industry. In vivo experiments are used to measure the ability of E. coli to be pathogenic. Generally, these experiments have proposed different criteria for results interpretation and did not take into account the death time. The aim of this study was to propose a new methodology for the classification of E. coli pathogenicity by the establishment of a pathogenicity index based in the lethality, death time and the ability of the strain to cause colibacillosis lesions in challenged animals. Materials, Methods & Results: A total of 293 isolates of E. coli were randomly selected to this study. The strains were isolated from cellulitis lesions, broiler bedding material or respiratory diseases and were previously confirmed through biochemical profile. The bacterial isolates were kept frozen at -20C. The strains were retrieved from stocks and cultured in brain-heart infusion broth overnight at 37C to obtain a final concentration of 109 UFC/mL. A total of 2940 one-dayold chicks from commercial breeding hens were randomly assigned to groups containing 10 animals and each group was subcutaneously inoculated in the abdominal region with 0.1 mL of the standard inoculum solution containing each of the strains. A control group [...]

TIPAGEM MOLECULAR, DETECÇÃO DE GENES DE VIRULÊNCIA E DETERMINAÇÃO DO POTENCIAL PATOGÊNICO DE ISOLADOS DE Escherichia coli DE PATOS (Cairina moschata), PERUS (Meleagris gallopavo), GALINHAS (Gallus gallus)

O objetivo geral deste trabalho é avaliar a semelhante filogenética e detectar genes de virulência de isolados de Escherichia coli de diferentes espécies de aves, bem como investigar seu potencial patogênico em ovos férteis de frangos de corte. No presente estudo foram realizados três experimentos voltados a análise, em diferentes aspectos, de E.coli isolados de variadas espécies de aves. No experimento 1, E.coli isoladas de patos e de perus foram inoculadas em pintos de frango de corte um dia para avaliar a patogenicidade das cepas. Foram testados, mediante PCR convencional, 50 isolados de E.coli originários de patos e 50 isolados de perus para detecção dos genes iss, iucD, papC e tsh, todos comumente encontrados em APEC. Dos 100 isolados de E.coli analisados, em 42% (42/100) detectou-se um ou mais genes pesquisados e dos isolados positivos apenas 14,2% (6/42) tinham os quatro genes estudados. Após a realização de PCR convencional, ocorreu a seleção das cepas positivas para os quatro genes comuns a APEC. Foram selecionados três isolados originários de patos e três de perus, purificados separadamente e elaborado seis inoculos na concentração de 10 UFC/mL e inoculados via saco aéreo em pintos de um dia para avaliação da patogenicidade desses seis isolados positivos para os quatro genes. Os pintos foram avaliados duas vezes ao dia durante dez dias em relação a taxa de mortalidade, sinais clínicos e achados macroscópicos. Os pintos desafiados apresentaram sinais respiratórios (espirros, descarga nasal e estertor) e lesões macroscópicas tais como aerossaculite, persistência do saco da gema, pericardite e exsudato nos seios nasais. Diante dos resultados, conclui-se que E.coli originária tanto de patos e quanto de perus são potencialmente patogênicas para pintos de framgos de corte desafiados com um dia de vida. No experimento 2, embriões de frangos de corte foram desafiados com isolados de E.coli positivos para os genes iss, tsh, papC e iucD oriundos de galinha caipira, frango de corte, pato e peru com objetivo de analisar o quadro clínico patológico e hematológico, além do desempenho de frangos de corte criados até os 42 dias de vida. Embriões com 18 dias de incubação foram desafiados, via câmara de ar, com 0,1 mL de quatro diferentes inóculos, de acordo com o tratamento, (T2 E.coli de galinha caipira, T3 E.coli de frango de corte, T4 E.coli de pato e T5 E.coli de peru) com concentração de 10 UFC/mL e com solução salina estéril (T1 grupo controle). Foram avaliados taxa de mortalidade embrionária e pós eclosão, sinais clínicos, alterações hematológicas, interferência do desempenho zootécnico e achados macroscópicos durante o período de incubação e criação até os 42 dias de vida. Aos 14, 28 e 42 dias, uma ave de cada uma das quatro repetições dos cinco tratamentos. Independente da espécie de origem, os isolados de E.coli não foram capazes de causar mortalidade significativa em embriões desafiados aos 18 dias de incubação. Ao nascer e até os 42 dias de vida, frangos de corte desenvolveram doença de caráter respiratório, piora no desempenho zootécnico e não houve alterações hematológica significativa. No experimento 3, 33 isolados de E.coli originários de patos, perus, galinhas e pombos foram avaliados mediante PFGE e teste de sensibilidade aos antimicrobianos com objetivo de caracterizar seus respectivos perfis filogenéticos e susceptibilidade aos antimicrobianos comumente utilizados na avicultura. E.coli originárias de patos, perus, galinhas e pombos agruparam-se em um grupo com 41,1% de similaridade. Entre os 33 isolados de E.coli observou-se resistência a diferentes antimicrobianos testados sulfametazole, sulfonamida, tetraciclina, cotrimoxazol e cloranfericol), principalmente a classe das tetraciclinas, seguida pelas fluoroquinolonas e sulfonamidas. Conclui-se que E.coli originárias de aves possuem perfis e comportamentos distintos.

The general objective of this work is to evaluate the phylogenetics and to detect virulence genes from Escherichia coli isolates from different bird species, as well as to investigate their pathogenic potential in fertile broiler eggs. In the present study three experiments were carried out to analyze, in different aspects, E. coli isolates from different bird species. In experiment 1, E. coli isolated from ducks and turkeys were inoculated into broiler chicks one day to evaluate the pathogenicity of the strains. Fifty isolates of E. coli originating from ducks and 50 isolates from turkeys were tested for the genes iss, iucD, papC and tsh, all commonly found in APEC. Of the 100 isolates of E.coli analyzed, 42% (42/100) detected one or more genes and only 14.2% (6/42) had the four genes studied. After PCR, the positive strains were selected for the four genes common to APEC. Three isolates from ducks and three turkeys were purified separately and six inoculants were inoculated at 107 CFU / mL and inoculated via air sac in one - day - old chicks to evaluate the pathogenicity of these six positive isolates for the four genes. The chicks were evaluated twice a day for ten days in relation to mortality rate, clinical signs and macroscopic findings. The challenged chicks presented respiratory signs (sneezing, nasal discharge and rales) and macroscopic lesions such as aerossaculitis, persistence of the yolk sac, pericarditis and exudate in the nasal sinuses. In view of the results, it is concluded that E.coli originating from both ducks and turkeys are potentially pathogenic for day-old challenged cuttlefish chicks. In the experiment 2, embryos of broiler chickens were challenged with E. coli isolates positive for the iss, tsh, papC and iucD genes from hen, broiler, duck and turkey to analyze the pathological and hematological conditions , besides the performance of broiler chickens kept up to 42 days of age. Embryos with 18 days of incubation were challenged, via air chamber, with 0.1 mL of four different inoculants, according to the treatment, (T2 - E.coli of hen chicken, T3 - E.coli of broiler, T4 - E.coli of duck and T5 - E. coli of turkey) with concentration of 107 CFU / mL and with sterile saline solution (T1 - control group). Embryonic and post - hatch mortality rates, clinical signs, hematological alterations, interference of zootechnical performance and macroscopic findings during the incubation period and rearing up to 42 days of life were evaluated. At 14, 28 and 42 days, one bird from each of the four replicates of the five treatments. Regardless of the species of origin, E.coli isolates were not able to cause significant mortality in embryos challenged at 18 days of incubation. At birth and up to 42 days of age, broiler chickens developed respiratory disease, worsened zootechnical performance and there were no significant haematological changes. In the experiment 3, 33 E. coli isolates from ducks, turkeys, chickens and pigeons were evaluated by PFGE and antimicrobial susceptibility test to characterize their respective phylogenetic profiles and susceptibility to antimicrobials commonly used in poultry farming. E. coli originated from ducks, turkeys, chickens and pigeons grouped into a group with 41.1% similarity. Resistance to different antimicrobials tested (ampicillin, amoxicillin, ceftiofur, doxycycline, enrofloxacin, neomycin, sulfametazole, sulfonamide, tetracycline, cotrimoxazole and chloranfericol) was observed among the 33 isolates of E. coli, mainly tetracyclines, followed by fluoroquinolones and sulfonamides. It is concluded that E.coli originating from birds have different profiles and behaviors.

In silico phylogenetic and virulence gene profile analyses of avian pathogenic Escherichia coli genome sequences / Análises in silico da filogenia e do perfil de genes associados à virulência, dos genomas de linhagens de Escherichia coli de origem aviária

Avian pathogenic Escherichia coli (APEC) infections are responsible for significant losses in the poultry industry worldwide. A zoonotic risk has been attributed to APEC strains because they present similarities to extraintestinal pathogenic E. coli (ExPEC) associated with illness in humans, mainly urinary tract infections and neonatal meningitis. Here, we present in silico analyses with pathogenic E. coli genome sequences, including recently available APEC genomes. The phylogenetic tree, based on multi-locus sequence typing (MLST) of seven housekeeping genes, revealed high diversity in the allelic composition. Nevertheless, despite this diversity, the phylogenetic tree was able to cluster the different pathotypes together. An in silico virulence gene profile was also determined for each of these strains, through the presence or absence of 83 well-known virulence genes/traits described in pathogenic E. coli strains. The MLST phylogeny and the virulence gene profiles demonstrated a certain genetic similarity between Brazilian APEC strains, APEC isolated in the United States, UPEC (uropathogenic E. coli) and diarrheagenic strains isolated from humans. This correlation corroborates and reinforces the zoonotic potential hypothesis proposed to APEC.(AU)

As infecções causadas por linhagens de Escherichia coli de origem aviária (APEC) são responsáveis por perdas significativas na indústria avícola em todo mundo. Risco zoonótico tem sido atribuído às linhagens APEC, devido às semelhanças existentes entre elas e linhagens de E. coli patogênicas extraintestinais (ExPEC) de origem humana, causadoras de infecções no trato urinário e meningite neonatal. Neste trabalho, apresentamos os resultados de análises in silico feitas a partir dos genomas de linhagens patogênicas de E. coli, incluindo genomas recentemente obtidos de linhagens APEC. Uma árvore filogenética foi obtida, com base na tipagem de sequência multilocus (MLST) de sete genes essenciais, revelando alta diversidade na composição de alelos, mas ainda assim possibilitando o agrupamento dos diferentes patótipos. Foi determinado também, para cada linhagem, o perfil gênico, por meio da presença ou ausência de 83 genes associados à virulência. A árvore filogenética e o perfil gênico demonstraram que existem semelhanças genéticas entre cepas APEC brasileiras, APEC isolada nos Estados Unidos, UPEC (uropathogenic E. coli) e linhagens produtoras de diarreia em humanos. Essa correlação corrobora e reforça a hipótese de que linhagens APEC apresentam potencial risco zoonótico.(AU)