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)

Co-relationship between Escherichia coli in broiler cellulitis and liver lesions / Correlação entre a presença de Escherichia coli em lesões de celulite e fígados de frango

Pathogenic strains of Escherichia coli may invade the subcutaneous tissue of poultry and cause cellulitis, whilst the pathogen may also cause lesions in internal organs such as the liver. Current paper co-relates Escherichia coli and virulence genes characteristic of Avian Pathogenic Escherichia coli (APEC) in broilers´ cellulitis and liver lesions. One hundred carcasses were retrieved from the production chain in an avian abattoir in the state of Bahia, Brazil, between August 2013 and January 2014, due to detection of cellulitis lesions. Cellulitis and liver samples were retrieved aseptically to quantify E. coli by Petrifilm count fast method (3M Company) (AOAC 998.8). Virulent genes iss and iutA were removed from E. coli isolates by Polymerase Chain Reaction (PCR). Escherichia coli was isolated from 82.0% of broilers removed from the production chain and the bacterium was concomitantly detected in cellulitis and liver lesions in 40.0% of broilers. E. coli counts ranged between 1.00 and 4.73 log CFU/g in liver lesions and between 2.00 and 9.00 log UFC/g in cellulitis lesions. Virulent genes iutA and iss were detected in 97.56% and 89.02% of E. coli isolates, respectively. Genotype analysis demonstrated the concomitant amplification of genes iutA and iss in 60.0% (n=40) of samples of cellulitis and liver lesions in which the simultaneous isolation of E. coli occurred. There was a positive and significant co-relationship (r=0.22; p 0.05) between the variables occurrence of E. coli isolated from liver samples and the occurrence of E. coli isolated from cellulitis lesions. There were also positive and significant co-relationships between populations of E. coli from liver isolates and cellulitis lesions (r=0.46; p 0.05) when E. coli isolated in the liver and in cellulitis lesions was detected. Since results showed a relationship between E. coli in cellulitis and liver lesions and possible systemic infection, the occurrence of cellulitis lesions as a criterion for total discarding of carcass may be suggested.(AU)

Cepas patogênicas de Escherichia coli podem invadir o tecido subcutâneo das aves e provocar celulite aviária e este patógeno pode provocar lesões nos órgãos internos, como o fígado. Desta forma, objetivou-se correlacionar a presença de Escherichia coli e os genes de virulência característicos de Escherichia coli Patogênica para Aves (APEC) nas lesões de celulite e nos fígados dos frangos. Entre agosto de 2013 a janeiro de 2014, foram retiradas 100 carcaças da linha de produção por apresentarem lesões de celulite em um matadouro avícola da Bahia (Brasil). Foram coletadas amostras de celulite e fígados de frango assepticamente para quantificação de E. coli pelo método rápido de contagem Petrifilm (3M Company) (AOAC 998.8). Em seguida foi realizada a pesquisa dos genes de virulência iss e iutA nos isolados de E. coli utilizando a Reação em Cadeia da Polimerase (PCR). Escherichia coli foi isolada em 82,00% das aves retiradas da linha de produção e a bactéria foi detectada concomitantemente nas lesões de celulite e fígado em 40,00% das aves. As contagens de E. coli variaram de 1,00 a 4,73 log UFC/g nos fígados e de 2,00 a 9,00 log UFC/g nas lesões de celulite. Os genes de virulência iutA e iss foram encontrados em 97,56% e 89,02% dos isolados de E. coli, respectivamente. A análise genotípica revelou a amplificação concomitante dos genes iutA e iss em 60,00% (n=40) das amostras de lesões de celulite e fígado nas quais houve o isolamento simultâneo de E. coli. Foi observada correlação positiva e significativa (r=0,22; p 0,05) entre as variáveis ocorrência de E. coli isolada das amostras dos fígados e ocorrência E. coli isolada das lesões de celulite e, nos casos em que foi detectada a ocorrência de E. coli isolada em fígado e lesões de celulite, correlações positivas e significativas também foram evidenciadas entre as populações de E. coli dos isolados dos fígados e das lesões de celulite, (r=0,46; p 0,05). Assim ficou evidenciada a relação entre E. coli presente nas lesões de celulite e no fígado e uma possível infecção sistêmica, desta forma, sugere-se que a presença de lesões de celulite seja utilizada como critério para o descarte total da carcaça.(AU)

Co-relationship between Escherichia coli in broiler cellulitis and liver lesions

Abstract Pathogenic strains of Escherichia coli may invade the subcutaneous tissue of poultry and cause cellulitis, whilst the pathogen may also cause lesions in internal organs such as the liver. Current paper co-relates Escherichia coli and virulence genes characteristic of Avian Pathogenic Escherichia coli (APEC) in broilers´ cellulitis and liver lesions. One hundred carcasses were retrieved from the production chain in an avian abattoir in the state of Bahia, Brazil, between August 2013 and January 2014, due to detection of cellulitis lesions. Cellulitis and liver samples were retrieved aseptically to quantify E. coli by Petrifilm count fast method (3M Company) (AOAC 998.8). Virulent genes iss and iutA were removed from E. coli isolates by Polymerase Chain Reaction (PCR). Escherichia coli was isolated from 82.0% of broilers removed from the production chain and the bacterium was concomitantly detected in cellulitis and liver lesions in 40.0% of broilers. E. coli counts ranged between 1.00 and 4.73 log CFU/g in liver lesions and between 2.00 and 9.00 log UFC/g in cellulitis lesions. Virulent genes iutA and iss were detected in 97.56% and 89.02% of E. coli isolates, respectively. Genotype analysis demonstrated the concomitant amplification of genes iutA and iss in 60.0% (n=40) of samples of cellulitis and liver lesions in which the simultaneous isolation of E. coli occurred. There was a positive and significant co-relationship (r=0.22; p 0.05) between the variables occurrence of E. coli isolated from liver samples and the occurrence of E. coli isolated from cellulitis lesions. There were also positive and significant co-relationships between populations of E. coli from liver isolates and cellulitis lesions (r=0.46; p 0.05) when E. coli isolated in the liver and in cellulitis lesions was detected. Since results showed a relationship between E. coli in cellulitis and liver lesions and possible systemic infection, the occurrence of cellulitis lesions as a criterion for total discarding of carcass may be suggested.

Resumo Cepas patogênicas de Escherichia coli podem invadir o tecido subcutâneo das aves e provocar celulite aviária e este patógeno pode provocar lesões nos órgãos internos, como o fígado. Desta forma, objetivou-se correlacionar a presença de Escherichia coli e os genes de virulência característicos de Escherichia coli Patogênica para Aves (APEC) nas lesões de celulite e nos fígados dos frangos. Entre agosto de 2013 a janeiro de 2014, foram retiradas 100 carcaças da linha de produção por apresentarem lesões de celulite em um matadouro avícola da Bahia (Brasil). Foram coletadas amostras de celulite e fígados de frango assepticamente para quantificação de E. coli pelo método rápido de contagem Petrifilm (3M Company) (AOAC 998.8). Em seguida foi realizada a pesquisa dos genes de virulência iss e iutA nos isolados de E. coli utilizando a Reação em Cadeia da Polimerase (PCR). Escherichia coli foi isolada em 82,00% das aves retiradas da linha de produção e a bactéria foi detectada concomitantemente nas lesões de celulite e fígado em 40,00% das aves. As contagens de E. coli variaram de 1,00 a 4,73 log UFC/g nos fígados e de 2,00 a 9,00 log UFC/g nas lesões de celulite. Os genes de virulência iutA e iss foram encontrados em 97,56% e 89,02% dos isolados de E. coli, respectivamente. A análise genotípica revelou a amplificação concomitante dos genes iutA e iss em 60,00% (n=40) das amostras de lesões de celulite e fígado nas quais houve o isolamento simultâneo de E. coli. Foi observada correlação positiva e significativa (r=0,22; p 0,05) entre as variáveis ocorrência de E. coli isolada das amostras dos fígados e ocorrência E. coli isolada das lesões de celulite e, nos casos em que foi detectada a ocorrência de E. coli isolada em fígado e lesões de celulite, correlações positivas e significativas também foram evidenciadas entre as populações de E. coli dos isolados dos fígados e das lesões de celulite, (r=0,46; p 0,05). Assim ficou evidenciada a relação entre E. coli presente nas lesões de celulite e no fígado e uma possível infecção sistêmica, desta forma, sugere-se que a presença de lesões de celulite seja utilizada como critério para o descarte total da carcaça.

Antibiotic resistance and virulence factors among Escherichia coli isolates from avian organic fertilizer / Resistência a antibióticos e fatores de virulência entre isolados de Escherichia coli provenientes de fertilizante orgânico aviário

Brazilian poultry industry generates large amounts of organic waste, such as chicken litter, which is often used in agriculture. Among the bacteria present in organic fertilizer are members of the Enterobacteriaceae family, such as Escherichia coli. The aim of this study was to analyze the profile of virulence factors and antimicrobial resistance of E. coli isolates from avian organic fertilizer. A total of 47 E. coli strains were tested through Polymerase chain reaction to detect virulence genes (hlyF, iss, ompT, iutA and iroN). Fourteen antimicrobials were used to test antimicrobial susceptibility in the strains. Genes characteristic of Avian Pathogenic E. coli (APEC) were reported among the strains, with the hlyF, iss and ompT genes being the most prevalent. The isolates showed high resistance (˃50%) to tetracycline, gentamicin, cefotaxime, nitrofurantoin, trimethoprim-sulfamethoxazole and ampicillin. Multidrug resistance was reported in a great number of strains (>70%). The results showed the presence of APEC cells with virulence genes and antimicrobial resistance after 15 days of the windrowing process in poultry houses, it means this process should be improved to eliminate these cells.(AU)

A indústria avícola brasileira gera grandes quantidades de resíduos orgânicos, como a cama de frango, utilizada frequentemente na agricultura. Entre as bactérias presentes neste fertilizante orgânico estão os membros da família Enterobacteriaceae, entre eles a Escherichia coli. O objetivo deste estudo foi analisar o perfil de fatores de virulência e resistência antimicrobiana de isolados de E. coli provenientes do fertilizante orgânico aviário. Um total de 47 cepas de E. coli foram testadas por meio da reação em cadeia da polimerase para detectar genes de virulência (hlyF, iss, ompT, iutA e iroN). Quatorze antimicrobianos foram utilizados para testar a susceptibilidade antimicrobiana nos isolados. Genes característicos de E. coli Patogênica Aviária (APEC) foram encontrados entre os isolados, sendo os genes hlyF, iss e ompT os mais prevalentes. Os isolados apresentaram alta resistência (˃50%) à tetraciclina, gentamicina, cefotaxima, nitrofurantoína, trimetoprima-sulfametoxazole e ampicilina. Múltipla resistência a drogas antimicrobianas foi encontrada em grande número de isolados (>70%). Os resultados obtidos mostraram a presença de células APEC portando genes de virulência e resistência a antimicrobianos após 15 dias de processo de empilhamento nas granjas, indicando que o processo necessita de um aperfeiçoamento para eliminar estas células.(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)

Antimicrobial resistance, diarrheagenic and avian pathogenic virulence genes in Escherichia coli from poultry feed and the ingredients / Resistência antimicrobiana, genes de virulência diarreiogênicos e patogênicos para aves em Escherichia coli isoladas de ingredientes e ração de frangos

Diarrheagenic (DEC) and avian pathogenic Escherichia coli (APEC) are associated with intestinal and extra-intestinal infections (ExPEC), respectively. We aimed to analyze the antimicrobial susceptibility, gene encoding virulence factors associated to DEC and APEC, and phylogenetic classification in E. coli isolated from 320 samples of feed and ingredients. Antimicrobial susceptibility was performed using the disk diffusion method and Multiple Antibiotic Resistance (MAR) Index and Multi-Drug Resistance (MDR) were calculated. Phylogenetic classification was performed on samples harboring DEC and/or APEC virulence-associated genes. A total of 110 E. coli strains were isolated in 15% (49/320) of the evaluated inputs (n=13 vegetable meal; n=33 animal meal, n=3 feed). In general, the isolates showed the highest rates of antimicrobial resistance to sulfonamide and cefazolin and 18% (20/110) were multi-drug resistant. MAR index of feed samples was the highest (0.467). Six and five strains had APEC and DEC virulence-associated genes, respectively, and belonging to phylogenetic groups A and B1. These findings point to the need for strict microbiological control during the production process of these foods.(AU)

Escherichia coli diarreiogênicas (DEC) e patogênicas para aves (APEC) são associadas a infecções intestinais e extraintestinais (ExPEC), respectivamente. O objetivo do presente trabalho foi avaliar a sensibilidade antimicrobiana, a presença de genes que codificam os fatores de virulência relacionados à DEC e APEC, e a classificação filogenética em E. coli isoladas de 320 amostras de ração para frangos e ingredientes. A sensibilidade antimicrobiana foi determinada pelo método disco-difusão e calculou-se o índice de resistência múltipla aos antimicrobianos (IRMA) e a resistência a múltiplas drogas (MDR). Nas amostras que possuíam genes de virulência relacionados à DEC e/ou APEC, foi realizada a classificação filogenética. Foram isoladas 110 amostras de E. coli em 15% (49/320) dos insumos avaliados (n=13 farelos vegetais; n=33 farinhas de origem animal; n=3 rações). De forma geral, os isolados apresentaram as maiores frequências de resistência antimicrobiana à sulfonamida e à cefazolina e 18% (20/110) foram resistentes a múltiplas drogas. O IRMA das rações foi o mais alto (0,467). Os genes que codificam fatores de virulência associados à APEC e DEC foram detectados em seis e cinco isolados, respectivamente, pertencentes aos grupos filogenéticos A e B1. Os resultados demonstram a necessidade de rigoroso controle microbiológico durante o processo de produção desses alimentos.(AU)

Antimicrobial resistance, diarrheagenic and avian pathogenic virulence genes in Escherichia coli from poultry feed and the ingredients / Resistência antimicrobiana, genes de virulência diarreiogênicos e patogênicos para aves em Escherichia coli isoladas de ingredientes e ração de frangos

Diarrheagenic (DEC) and avian pathogenic Escherichia coli (APEC) are associated with intestinal and extra-intestinal infections (ExPEC), respectively. We aimed to analyze the antimicrobial susceptibility, gene encoding virulence factors associated to DEC and APEC, and phylogenetic classification in E. coli isolated from 320 samples of feed and ingredients. Antimicrobial susceptibility was performed using the disk diffusion method and Multiple Antibiotic Resistance (MAR) Index and Multi-Drug Resistance (MDR) were calculated. Phylogenetic classification was performed on samples harboring DEC and/or APEC virulence-associated genes. A total of 110 E. coli strains were isolated in 15% (49/320) of the evaluated inputs (n=13 vegetable meal; n=33 animal meal, n=3 feed). In general, the isolates showed the highest rates of antimicrobial resistance to sulfonamide and cefazolin and 18% (20/110) were multi-drug resistant. MAR index of feed samples was the highest (0.467). Six and five strains had APEC and DEC virulence-associated genes, respectively, and belonging to phylogenetic groups A and B1. These findings point to the need for strict microbiological control during the production process of these foods.(AU)

Escherichia coli diarreiogênicas (DEC) e patogênicas para aves (APEC) são associadas a infecções intestinais e extraintestinais (ExPEC), respectivamente. O objetivo do presente trabalho foi avaliar a sensibilidade antimicrobiana, a presença de genes que codificam os fatores de virulência relacionados à DEC e APEC, e a classificação filogenética em E. coli isoladas de 320 amostras de ração para frangos e ingredientes. A sensibilidade antimicrobiana foi determinada pelo método disco-difusão e calculou-se o índice de resistência múltipla aos antimicrobianos (IRMA) e a resistência a múltiplas drogas (MDR). Nas amostras que possuíam genes de virulência relacionados à DEC e/ou APEC, foi realizada a classificação filogenética. Foram isoladas 110 amostras de E. coli em 15% (49/320) dos insumos avaliados (n=13 farelos vegetais; n=33 farinhas de origem animal; n=3 rações). De forma geral, os isolados apresentaram as maiores frequências de resistência antimicrobiana à sulfonamida e à cefazolina e 18% (20/110) foram resistentes a múltiplas drogas. O IRMA das rações foi o mais alto (0,467). Os genes que codificam fatores de virulência associados à APEC e DEC foram detectados em seis e cinco isolados, respectivamente, pertencentes aos grupos filogenéticos A e B1. Os resultados demonstram a necessidade de rigoroso controle microbiológico durante o processo de produção desses alimentos.(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)

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)

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.

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)

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)

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.

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.

Survey on pathogenic Escherichia coli and Salmonella spp. in captive cockatiels (Nymphicus hollandicus)

Abstract We surveyed healthy captive cockatiels (Nymphicus hollandicus) for Escherichia coli and Salmonella spp. Cloacal swabs were collected from 94 cockatiels kept in commercial breeders, private residencies and pet shops in the cities of São Paulo/SP and Niterói/RJ (Brazil). Three strains of E. coli from each individual were tested for the presence of ExPEC-, APEC- and DEC-related genes. We evaluated the blaTEM, blaSHV, blaOXA, blaCMY, blaCTX-M, tetA, tetB, aadA, aphA, strAB, sul1, sul2, sul3, qnrA, qnrD, qnrB, qnrS, oqxAB, aac (6)-Ib-cr, qepA resistance genes and markers for plasmid incompatibility groups. Salmonella spp. was not detected. E. coli was isolated in 10% of the animals (9/94). Four APEC genes (ironN, ompT, iss and hlyF) were detected in two strains (2/277%), and iss (1/274%) in one isolate. The highest resistance rates were observed with amoxicillin (22/2782%), ampicillin (21/2779%), streptomycin (18/2767%), tetracycline (11/2741%). Multiresistance was verified in 59% (16/27) of the isolates. We detected strAB, bla TEM, tetA, tetB, aadA, aphaA, sul1, sul2, sul3 resistance genes and plasmid Inc groups in 20 (74%) of the strains. E. coli isolated from these cockatiels are of epidemiological importance, since these pets could transmit pathogenic and multiresistant microorganisms to humans and other animals.

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)

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.