Evaluation of virulence factors in clinical isolates of pathogenic E. coli in avian samples in Caloto, Colombia

Avian pathogenic E. coli (APEC), produces an extraintestinal infection in chickens, turkeys, and other types of birds, called colibacillosis, which is considered one of the main causes of economic losses due to morbidity, mortality, and discard of poultry carcasses. The objective of the present study was to characterize the genetic profile of the virulence factors of different isolates of avian E. coli in Caloto, Cauca, Colombia. Materials and methods: E. coli was isolated and identified by biochemical tests, from 47 clinical isolates. Subsequently, the DNA was extracted using Chelex. Three multiplex PCRs were designed to amplify 13 virulence factors (iroN, hlyF, iss, iutA, frz, vat, sitA, KpsM, sitD, fimH, pstB, sopB, and uvrY), using primers previously reported for each. At the end, the amplification products were verified on agarose gels. Each isolate was classified according to the number of virulence factors: group A (between 10 and 13), group B (between 5 and 9), and group C (4 or less). Discussion and Conclusions: we were able to identify the presence of a group of virulence factors in clinical isolates of APEC, which allows us to demonstrate that both the frequency and the profile of virulence factors in the isolated strains showed a different profile than the reported by other authors. The virulence genes pstB and fimH were detected in all our samples, and the iss gene was the one with the lowest frequency. Finally, according to the number of virulence factors, the group A was the most frequent.

La E. coli patógena aviar (APEC), produce una infección extraintestinal en pollos, pavos y otros tipos de aves, denominada colibacilosis, la cual es considerada una de las principales causas de pérdidas económicas por morbilidad, mortalidad y descarte de canales de aves. El objetivo del presente estudio fue caracterizar el perfil genético de los factores de virulencia de diferentes aislamientos de E. coli aviar en Caloto, Cauca, Colombia. Materiales y métodos: E. coli se aisló e identificó mediante pruebas bioquímicas, a partir de 47 aislamientos clínicos. Posteriormente, el ADN se extrajo utilizando Chelex. Se diseñaron tres PCR multiplex para amplificar 13 factores de virulencia (iroN, hlyF, iss, iutA, frz, vat, sitA, KpsM, sitD, fimH, pstB, sopB y uvrY), utilizando primers informados previamente para cada uno. Al final, los productos de amplificación fueron verificados en geles de agarosa. Cada aislamiento se clasificó según el número de factores de virulencia: grupo A (entre 10 y 13), grupo B (entre 5 y 9) y grupo C (4 o menos). Discusión y Conclusiones: pudimos identificar la presencia de un grupo de factores de virulencia en los aislados clínicos de APEC, lo que nos permite demostrar que tanto la frecuencia como el perfil de los factores de virulencia en las cepas aisladas presentaron un perfil diferente al reportado por otros autores. Los genes de virulencia pstB y fimH se detectaron en todas nuestras muestras, siendo el gen iss el de menor frecuencia. Finalmente, según el número de factores de virulencia, el grupo A fue el más frecuente.

Pathological, microbiological and immunohistochemical characterization of avian colibacillosis in broiler chickens of Mozambique

ABSTRACT: Avian colibacillosis is an acute and globally occurring infectious disease of domestic and wild birds caused by Escherichia coli, and it is associated with considerable economic losses mainly due to the morbidity and mortality associated. The present study aimed to describe the pathological, bacteriological and immunohistochemical aspects of avian colibacillosis in broiler chickens of Mozambique. Forty-nine broiler chicken presented anorexia, decreased weight gain, ataxia, diarrhea, dyspnea, and death in a clinical course of 3-5 days. The birds were raised in five farms (small, medium and large farms) with manual and automatic breeding system, with flocks ranging from 100 to 20,000 birds. At the necropsy, all birds had poor body condition, and the pericardium and the Glissons capsule of all avian exhibited different degrees of adherence often associated with severe fibrin deposition. The thoracic and abdominal air sacs were thickened and adhered to the costal wall. Mild, moderate or marked hepatomegaly associated with white pinpoint multifocal areas (100%, 49/49) and mild to moderate splenomegaly in 75.5% (37/49) with a mottled surface were observed. The lungs and kidney were enlarged and reddish. Histologically, a multiorgan fibrinoheterophilic polyserositis was observed in 75.5% of the cases (37/49), which were characterized by inflammatory infiltrates composed mainly of degenerative heterophils, macrophages and plasma cells, associated with fibrin deposits and intermixed by coccobacillary bacterial basophilic aggregates. These affected mainly the pericardium (28.6%, 14/49), the pleura (18.4%, 9/49), the Glissons capsule (10.2%, 5/49), the ventriculus (10.2, 5/33), and the proventriculus (8.2%, 4/49) serosa. Multifocal to coalescing areas of coagulative necrosis associated with similar inflammatory cells were observed mainly in the spleen (28.6%, 14/49), liver (24.5%, 12/49), and intestines (22.4%, 11/49). A similar infiltrate was also observed affecting the the lungs (16.3%, 8/49), the kidney (16.3%, 8/49) and the myocardium (14.3%, 7/49). Isolation and identification of E. coli was obtained in 12 cases through bacterial culture. Some organs (2 cases of each farms) were selected and submitted to immunohistochemistry anti-E. coli, and a positive stain was observed in all tested cases in liver (3/3), heart (4/4), spleen (1/1), lungs (4/4), intestines (4/4), bursa of Fabricius (1/1), ventriculus (1/1), and proventriculus (1/1) tissue sections. These results demonstrate that E. coli was the cause of mortality in these birds. Therefore, biosecurity and management measures should be employed to prevent and control the disease occurrence in Mozambiques poultry farming.

RESUMO: A colibacilose aviária é uma doença aguda de ocorrência mundial que acomete aves domésticas e silvestres, causada por Escherichia coli e resulta em perdas econômicas consideráveis devido à elevada morbidade e mortalidade das aves. O presente estudo teve o objetivo de descrever os aspectos patológicos, bacteriológicos e imuno-histoquímicos de colibacilose aviária em frangos de corte de Moçambique. Um total de 49 frangos de corte apresentaram anorexia, baixo ganho de peso, ataxia, diarreia, dispneia e morte em um curso clínico de 3 a 5 dias. As aves eram provenientes de 5 granjas (pequenas, média e grandes), com sistema de criação manual e automático, com rebanhos que variavam de 100 a 20.000 aves. À necropsia, todas as aves exibiam condição corporal ruim a caquética, além de pericárdio e cápsula de Glisson de todas aves (100%; n=49) com diferentes graus de aderência e deposição de fibrina de forma difusa acentuada. Os sacos aéreos torácicos e abdominais estavam espessados e aderidos à parede costal. Foi observado ainda hepatomegalia discreta, moderada a severa frequentemente associada com áreas multifocais puntiformes brancacentas (100%; 49/49), e esplenomegalia discreta a moderada, associado a áreas multifocais moteadas (75,5%; 37/49). Os pulmões e rins estavam aumentados e com coloração avermelhada. Histologicamente, observou-se majoritariamente serosite fibrinoheterofílica em 75,5% dos casos (37/49), caracterizadas por infiltrado inflamatório composto por heterófilos degenerados, macrófagos, linfócitos e plasmócitos, com deposição de fibrina entremeada por uma miríade de estruturas bacterianas cocobacilares. Esta lesão foi observada principalmente em pericárdio (28,6%; 14/49), pleura (18,4%; 9/49), cápsula de Glisson (10,2%; 5/49), ventrículo (10,2; 5/33) e em proventrículo (8,2%; 4/49). Áreas multifocais a coalescentes de necrose de coagulação associada a infiltrado inflamatório semelhante ao descrito foi observado principalmente no baço (28,6%; 14/49), fígado (24.5%; 12/49), e intestinos (8,2%; 4/49). Um infiltrado inflamatório semelhante também foi visualizado em pulmões (16,3%; 8/49), rins (16,3%; 8/49) e miocárdio (14,3%; 7/49), Colônias puras de E. coli foram identificadas e isoladas em 12 casos. Alguns órgãos (2 de cada granja) foram submetidos ao exame imuno-histoquímico anti-E. coli e marcação positiva foi visualizada em todos casos testados, como em fígado (3/3), coração (4/4), baço (1/1), pulmão (4/4), intestinos (4/4), bursa de Fabricius (1/1), rim (1/1), ventrículo (1/1) e proventrículo (1/1). Estes resultados demonstram que E. coli foi a causa de morte destas aves. Sendo assim, a adoção de boas medidas de biosseguridade e de manejo são indispensáveis para a prevenção e controle da ocorrência da doença nas granjas de frango de corte de Moçambique.

Virulence genes and resistance profile of Escherichia coli isolated in meat meal samples

Feed is the main route of transmission of pathogenic microorganisms and is responsible for a large part of the cost of poultry production, so the inclusion of alternative foods in diets for monogastrics has been a constant. Among alternative foods most used in modern poultry farming are animal meal, however, when contaminated they constitute a route of transmission of several pathogenic agents, including Escherichia coli. In addition, there is a zoonotic potential, as poultry products are intended for human consumption. The objective of this research was to detect virulence genes, as well as to evaluate the resistance profile of Escherichia coli isolates from meat meal samples. A total of 40 Escherichia coli isolates were analyzed and the virulence genes surveyed iss, ompT, hlyF, iutA, and fimA identified by Polymerase Chain Reaction (PCR). The antimicrobial agents tested were: amoxycillin (30 µg), ceftiofur (30 µg), ciprofloxacin (5 µg), doxycycline (30 µg), florfenicol (30 µg), fosfomycin (200 µg), gentamicin (10 µg), norfloxacin (10 µg) and oxacillin (1 µg). It was possible to observe the occurrence of the iss resistance gene in 100% of Escherichia coli isolates, followed by hlyF (85%), fimA (75%), ompT (17.5%) and iutA (5%). Regarding the simultaneous detection for the genes, a greater association between the genes iss, hlyF and fimA (60%) was verified. All isolates showed resistance to oxacillin (100%), followed by doxycycline (25%), amoxicillin (22.5%), norfloxacin (17.5%), ceftiofur (15%), florfenicol (12.5%), fosfomycin (12.5%), ciprofloxacin (10%) and gentamycin (2.5%). In this study, a variation of the multiple antimicrobial resistance index (IRMA) was observed between 0.22 and 0.77. The indiscriminate use of of antimicrobials as performance enhancers in production animals, may have contributed to the increase in antimicrobial resistance, with the occurrence of multiresistant Escherichia coli carrying virulence genes. Virulence genes present in Escherichia coli isolates are studied to understand the degree of influence they exert in the establishment of the disease, one of the most researched genes is the iss gene, involved in the processes that promote serum resistance. In this study, iss (100%) was present in all the isolates analyzed, although it is not the only mechanism used by these bacteria to reach internal organs and trigger an infection, this gene encodes an important mechanism associated with high levels of virulence. The second highest prevalence found was of the hlyF gene (85%), the high prevalence of hlyF suggests virulence potential, involved with the production of hemolysin and improvement of outer membrane vesicles associated with the release of toxins. The fimA gene (75%) was detected in a slightly lower percentage when compared to iss and hlyF. With the second lowest prevalence, the ompT gene (17.5%), is involved in a process that includes the proteolytic degradation of antimicrobial peptides and with the lowest prevalence the iutA gene (5%). Certain combinations of virulence genes make the strains easier to survive, adhere to, colonize and even the ability to develop septicemic conditions. Multiresistant E. coli strains, is a fact of concern for both animal and human health, since the presence of multiresistant strains, originating from poultry, can be transmitted from chicken carcasses. In this sense, the importance of sanitary control of the inputs used in animal feed is emphasized, as well as the prudent use of antimicrobials in animal production, with a view to producing a safe food, minimizing not only the economic losses, but also the risks to human health.(AU)

Pathological, microbiological and immunohistochemical characterization of avian colibacillosis in broiler chickens of Mozambique / Caracterização patológica, microbiológica e imuno-histoquímica de colibacilose em frangos de corte de Moçambique

Avian colibacillosis is an acute and globally occurring infectious disease of domestic and wild birds caused by Escherichia coli, and it is associated with considerable economic losses mainly due to the morbidity and mortality associated. The present study aimed to describe the pathological, bacteriological and immunohistochemical aspects of avian colibacillosis in broiler chickens of Mozambique. Forty-nine broiler chicken presented anorexia, decreased weight gain, ataxia, diarrhea, dyspnea, and death in a clinical course of 3-5 days. The birds were raised in five farms (small, medium and large farms) with manual and automatic breeding system, with flocks ranging from 100 to 20,000 birds. At the necropsy, all birds had poor body condition, and the pericardium and the Glisson's capsule of all avian exhibited different degrees of adherence often associated with severe fibrin deposition. The thoracic and abdominal air sacs were thickened and adhered to the costal wall. Mild, moderate or marked hepatomegaly associated with white pinpoint multifocal areas (100%, 49/49) and mild to moderate splenomegaly in 75.5% (37/49) with a mottled surface were observed. The lungs and kidney were enlarged and reddish. Histologically, a multiorgan fibrinoheterophilic polyserositis was observed in 75.5% of the cases (37/49), which were characterized by inflammatory infiltrates composed mainly of degenerative heterophils, macrophages and plasma cells, associated with fibrin deposits and intermixed by coccobacillary bacterial basophilic aggregates. These affected mainly the pericardium (28.6%, 14/49), the pleura (18.4%, 9/49), the Glisson's capsule (10.2%, 5/49), the ventriculus (10.2, 5/33), and the proventriculus (8.2%, 4/49) serosa. Multifocal to coalescing areas of coagulative necrosis associated with similar inflammatory cells were observed mainly in the spleen (28.6%, 14/49), liver (24.5%, 12/49), and intestines (22.4%, 11/49). A similar infiltrate was also observed affecting the the lungs (16.3%, 8/49), the kidney (16.3%, 8/49) and the myocardium (14.3%, 7/49). Isolation and identification of E. coli was obtained in 12 cases through bacterial culture. Some organs (2 cases of each farms) were selected and submitted to immunohistochemistry anti-E. coli, and a positive stain was observed in all tested cases in liver (3/3), heart (4/4), spleen (1/1), lungs (4/4), intestines (4/4), bursa of Fabricius (1/1), ventriculus (1/1), and proventriculus (1/1) tissue sections. These results demonstrate that E. coli was the cause of mortality in these birds. Therefore, biosecurity and management measures should be employed to prevent and control the disease occurrence in Mozambique's poultry farming.(AU)

A colibacilose aviária é uma doença aguda de ocorrência mundial que acomete aves domésticas e silvestres, causada por Escherichia coli e resulta em perdas econômicas consideráveis devido à elevada morbidade e mortalidade das aves. O presente estudo teve o objetivo de descrever os aspectos patológicos, bacteriológicos e imuno-histoquímicos de colibacilose aviária em frangos de corte de Moçambique. Um total de 49 frangos de corte apresentaram anorexia, baixo ganho de peso, ataxia, diarreia, dispneia e morte em um curso clínico de 3 a 5 dias. As aves eram provenientes de 5 granjas (pequenas, média e grandes), com sistema de criação manual e automático, com rebanhos que variavam de 100 a 20.000 aves. À necropsia, todas as aves exibiam condição corporal ruim a caquética, além de pericárdio e cápsula de Glisson de todas aves (100%; n=49) com diferentes graus de aderência e deposição de fibrina de forma difusa acentuada. Os sacos aéreos torácicos e abdominais estavam espessados e aderidos à parede costal. Foi observado ainda hepatomegalia discreta, moderada a severa frequentemente associada com áreas multifocais puntiformes brancacentas (100%; 49/49), e esplenomegalia discreta a moderada, associado a áreas multifocais moteadas (75,5%; 37/49). Os pulmões e rins estavam aumentados e com coloração avermelhada. Histologicamente, observou-se majoritariamente serosite fibrinoheterofílica em 75,5% dos casos (37/49), caracterizadas por infiltrado inflamatório composto por heterófilos degenerados, macrófagos, linfócitos e plasmócitos, com deposição de fibrina entremeada por uma miríade de estruturas bacterianas cocobacilares. Esta lesão foi observada principalmente em pericárdio (28,6%; 14/49), pleura (18,4%; 9/49), cápsula de Glisson (10,2%; 5/49), ventrículo (10,2; 5/33) e em proventrículo (8,2%; 4/49). Áreas multifocais a coalescentes de necrose de coagulação associada a infiltrado inflamatório semelhante ao descrito foi observado principalmente no baço (28,6%; 14/49), fígado (24.5%; 12/49), e intestinos (8,2%; 4/49). Um infiltrado inflamatório semelhante também foi visualizado em pulmões (16,3%; 8/49), rins (16,3%; 8/49) e miocárdio (14,3%; 7/49), Colônias puras de E. coli foram identificadas e isoladas em 12 casos. Alguns órgãos (2 de cada granja) foram submetidos ao exame imuno-histoquímico anti-E. coli e marcação positiva foi visualizada em todos casos testados, como em fígado (3/3), coração (4/4), baço (1/1), pulmão (4/4), intestinos (4/4), bursa de Fabricius (1/1), rim (1/1), ventrículo (1/1) e proventrículo (1/1). Estes resultados demonstram que E. coli foi a causa de morte destas aves. Sendo assim, a adoção de boas medidas de biosseguridade e de manejo são indispensáveis para a prevenção e controle da ocorrência da doença nas granjas de frango de corte de Moçambique.(AU)

Molecular Detection of the Virulence Genes in Escherichia coli Isolated from Healthy and Diarrheic Calves in Southern Iraq

Aim: To investigate the stx1, stx2, hly, cnf2, sfa/foc, afaI, papC, and afaC virulence-associated genes of pathogenic Escherichia coli, isolated from diarrheic and non-diarrheic calves. Materials and Methods: The genes to be investigated were first isolated from the stool samples obtained from 150 diarrheic and non-diarrheic calves, between the ages of one day and six months, during the period 2016 to 2017. Rectal swabs were aseptically and randomly collected from several herds, from different regions in the province of Basra. Results of Research: The polymerase chain reaction (PCR) results showed that pathogenic E. coli were detected in a total of 34 out of 41 (82.9%) distributed as 23 out of 26 (88.5%) and 11 out of 15 (73.3%) of the diarrheagenic and non-diarrheagenic calves, respectively. The results also showed that stx1, hlyA, and stx2genes showed a higher incidence of distribution in both diarrheagenic and non-diarrheagenic calves, in a percentage rate of 69.6%, 65.2%, 56.5% and 63.6%, 63.6%, 45.5%, respectively. Moreover, eight different virulence gene profiles were established in the present study. Most of the isolates analyzed had at least two or three gene arrangements and only four isolates were seen in a combination of four genes stx1, stx2, hlyA, and afaI. Conclusion: It is concluded that E. coli isolates from healthy and diarrheic calves that carried various virulence genes, of which the most frequent were stx1, stx2, and hlyA. A high percentage of these isolates are found in both diarrheic and non-diarrheic calves.

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.

Phylogenetic analysis, biofilm production, and antimicrobial resistance profile of Escherichia coli isolated from slaughtered pigs / Análise filogenética, produção de biofilme e perfil de resistência antimicrobiana de Escherichia coli isoladas de suínos abatidos

Enteric diseases of bacterial origin are frequent in the pig industry, of particular notoriety are the colibacillosis that mainly affect piglets and cause great damage to the swine industry worldwide. The aim of the study was to analyze phylogenetics, to detect biofilm production, and to determine antimicrobial resistance profile in 126 strains of Escherichia coli isolated from swabs obtained from fragments of the small intestines of 235 healthy pigs killed in slaughterhouses in Pernambuco (Brazil) using polymerase chain reaction (PCR), adherence to microplates test and disc diffusion technique. Of the analyzed samples, 88.10% (111/126) were classified in phylogenetic group B1; 4.76% (6/126) in group D; 3.97% (5/126) in group B2 and, 3.17% (4/126) in group A. Antimicrobial resistance rates observed were: lincomycin 100% (126/126), erythromycin 100% (126/126), chlortetracycline 94.44% (119/126), cephalothin 51.59% (65/126), ampicillin 38.89% (49/126), sulfamethoxazole + trimethoprim 37.3% (47/126), ciprofloxacin 19.84% (25/126), norfloxacin 14.29% (18/126), gentamicin 8.73% (11/126) and, chloramphenicol 5.55% (7/126). Multiple antibiotic resistance (MAR) ranged from 0.2 to 0.9. Of the strains tested 46.03% (58/126) produced biofilm, and 99.21% (125/126) of the strains exhibited multi-resistance. Further studies are required to elucidate the importance of each phylogenetic group in pigs and to prevent the propagation of multi-resistant E. coli strains.(AU)

Doenças entéricas de origem bacteriana são frequentes na indústria de suínos, destacando-se a colibacilose, que afeta principalmente leitões e causa grandes danos à indústria suína em todo o mundo. Cento e vinte e seis cepas de Escherichia coli foram isoladas de swabs obtidos de fragmentos de intestino delgado de 235 suínos saudáveis abatidos em matadouros de Pernambuco (Brasil). O objetivo do presente estudo foi analisar filogeneticamente essas cepas, bem como detectar a produção de biofilme e determinar o perfil de resistência antimicrobiana delas, utilizando-se a reação em cadeia da polimerase (PCR), o teste de adesão em microplacas e a técnica de disco-difusão. 88,10% (111/126) das amostras foram classificadas no grupo filogenético B1; 4,76% (6/126) no grupo D; 3,97% (5/126) no grupo B2; e 3,17% (4/126) no grupo A. As taxas de resistência antimicrobiana observadas foram: lincomicina 100% (126/126), eritromicina 100% (126/126), clortetraciclina 94,44% (119/126), cefalotina 51,59% (65/126), ampicilina 38,89% (49/126), sulfametoxazol + trimetoprima 37,3% (47/126), ciprofloxacina 19,84% (25/126), norfloxacina 14,29% (18/126), gentamicina 8,73% (11/126) e cloranfenicol 5,55% (7/126). O índice de resistência múltipla (IRMA) variou de 0,2 a 0,9. Entre as amostras, 46,03% (58/126) produziram biofilme e 99,21% (125/126) foram multirresistentes. São necessários mais estudos para elucidar a importância de cada grupo filogenético em suínos e evitar a propagação de estirpes de E. coli multirresistentes.(AU)

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.

Phylogenetic characterization of serum plus antibiotic-resistant extraintestinal Escherichia coli obtained from the liver of poultry carcasses in Pernambuco / Caracterização filogenética de Escherichia coli extraintestinal soro- e antibiótico-resistentes isoladas do fígado de carcaças de frango em Pernambuco

In this study, avian extraintestinal Escherichia coli obtained from the liver of poultry carcasses approved for human consumption in the State of Pernambuco-Brazil were tested for antibiotic plus serum-resistance. Liver samples (n=110) were obtained from one slaughterhouse and 88 bacterial isolates were identified as Escherichia coli. The antibiotic-resistance profiles of antibiotics used in human and/or veterinary practice were accessed by the disk-diffusion method. Phenotypes with high resistance to streptomycin (84.0%), tetracycline (44.7%), amikacin (29.8%), gentamicin (21.3%) and ciprofloxacin (21.3%) were identified. Resistance to antibiotics such as ceftazidime, amoxicillin-clavulanic acid and imipenem was also recorded. Twenty isolates with distinct antibiotic-resistance and susceptibility profiles were selected for serum resistance assays, phylogenetic characterization and detection of the iss gene. We have shown that multidrug resistant isolates were often simultaneously resistant to broiler and human sera. Phylogenetic characterization of serum- plus antibiotic-resistant isolates have shown three belonging to group D, eleven to group B1, one to group B2, and five to group A. We concluded that commensal E. coli strains isolated from the liver of healthy poultry carcasses can harbor and potentially share multidrug- plus virulence genes found in pathogenic pathotypes. This suspicion was not related to specific phylogenetic groups or presence of the iss gene.(AU)

Neste estudo, isolados de Escherichia coli extraintestinal aviária obtidos a partir do fígado de carcaças de aves aprovadas para consumo humano no Estado de Pernambuco-Brasil foram testados para resistência a antibióticos e soro. As amostras de fígado (n = 110) foram obtidas de um abatedouro, sendo 88 isolados bacterianos identificados como Escherichia coli. Os perfis de resistência a antibióticos de uso humano e/ou veterinário foram determinados pelo método de disco-difusão. Foram identificados fenótipos com alta resistência à estreptomicina (84,0%), tetraciclina (44,7%), amicacina (29,8%), gentamicina (21,3%) e ciprofloxacina (21,3%). A resistência a antibióticos utilizados na medicina humana e/ou veterinária, tais como a ceftazidima, amoxicilina-ácido clavulânico, estreptomicina e imipenem também foi registrada. Vinte amostras com perfis distintos de resistência/sensibilidade a antibióticos foram selecionadas para os ensaios de resistência ao soro, caracterização filogenética e detecção do gene iss. Foi demonstrado que isolados resistentes a múltiplas drogas foram também simultaneamente resistentes ao soro de frangos e ao soro humano. A caracterização filogenética desses isolados mostraram três pertencentes ao grupo D, onze ao grupo B1, um ao grupo B2 e cinco ao grupo A. Conclui-se que E. coli comensais isoladas do fígado de carcaças de aves saudáveis podem abrigar e potencialmente compartilhar genes de resistência a drogas e de virulência encontrados em patotipos patogênicos. Essa suspeita não foi relacionada com grupos filogenéticos específicos ou com a presença do gene iss.(AU)

Escherichia coli and Salmonella ser. Saintpaul natural co-infection in a free-living ruddy ground dove (Columbina talpacoti): a case report / Coinfecção por Escherichia coli e Salmonella ser. Saintpaul em Columbina talpacoti: relato de caso

This study reports a co-infection of Escherichia coli and Salmonella in a free-living ruddy ground dove (Columbina talpacoti) received at the Laboratory of Ornithological Studies of the State University of Ceará, Brazil. The bird presented diarrhea, leg paralysis and anorexia, and died shortly after. Necropsy was then performed and samples from lung, kidney, liver and intestine were collected for microbiological and histopathological analyses. Escherichia coli was isolated from cloacal swab, lung and kidney samples. Salmonella ser. Saintpaul was identified in liver and spleen samples. Escherichia coli isolates were tested for the presence of eight diagnostic genes for diarrheagenic pathotypes (STEC, ETEC, EPEC, EIEC, EAEC) with conventional polymerase chain reaction (PCR). EAEC was detected in the lung and kidney, and STEC in the intestine. In conclusion, Columbina talpacoti is susceptible to enteroaggregative Escherichia coli and Salmonella ser. Saintpaul infection, which may have public health implications.(AU)

Este estudo relata um caso de coinfecção por Escherichia coli e Salmonella ser. Saintpaul em uma rolinha-roxa (Columbina talpacoti) recebida pelo Laboratório de Estudos Ornitológicos da Universidade Estadual do Ceará, Brasil. A ave apresentava diarreia, paralisia nas pernas e anorexia, indo a óbito rapidamente. A necropsia foi realizada e amostras de pulmão, rim, fígado e intestino foram coletados para isolamento microbiológico e análise histopatológica. Escherichia coli foi identificada em amostras de suabe cloacal, pulmão e rim. Salmonella ser. Saintpaul foi identificada no fígado e baço. Isolados de E. coli foram testados para a presença de oito genes de diagnóstico para patotipos diarreiogênicos (STEC, ETEC, EPEC, EIEC, EAEC) através de reação em cadeia de polimerase (PCR) convencional. EAEC foi detectada no pulmão e rim, e STEC foi identificada no intestino. Em conclusão, Columbina talpacoti é suscetível a infecção por Escherichia coli enteroagregativa e Salmonella ser. Saintpaul, o que pode implicar em risco para a saúde pública.(AU)

Pathologic and microbiologic aspects of pet psittacine infected by Escherichia coli and Salmonella Typhimurium / Aspectos patológicos e microbiológicos de Psittaciformes de companhia infectados por Escherichia coli e Salmonella Typhimurium

The role of Escherichia coli in healthy microbiota of psittacine is controversial, and the presence of Salmonella sp. indicates possible disease. Therefore, this study aimed to identify the presence of E. coli and Salmonella spp. in a psittacine pet that died in Fortaleza, Brazil, correlating pathogenicity aspects of the isolates through the evaluation of lesions and antimicrobial susceptibility. Psittacine pets sent to the Laboratory of Ornithological Studies, State University of Ceará, that died in 2014 and 2015 were necropsied. Fragments of liver, kidneys, intestine, lung, heart, spleen and brain were collected for microbiological and histopathological analyses. Scores were attributed to lesions and isolated strains submitted to antimicrobial susceptibility test. From the seventy necropsied birds, nineteen were positive for E. coli and one for Salmonella Typhimurium. Congestive lesions and lymphoplasmocitic inflammatory infiltrate were observed varying from light to moderate and were the main findings. In the analyzed strains, multidrug resistance against different groups of antibiotics was observed. In conclusion, according to the results, E. coli strains and the Salmonella Typhimurium isolate produced significant lesions in the psittacine pets, and multidrug resistance may hinder treatments with antibiotics used in avian pet medicine.(AU)

A participação de Escherichia coli na microbiota saudável de Psicittaciformes e a de Salmonella spp. já indica possível doença. O objetivo deste estudo foi pesquisar a presença de E. coli e Salmonella spp. em psittaciformes de companhia na cidade de Fortaleza/Ceará, traçando os aspectos de patogenicidade destas cepas através das lesões e da sensibilidade antimicrobiana. Foram necropsiados os psittaciformes de companhia encaminhados ao Laboratório de Estudos Ornitológicos da Universidade Estadual do Ceará durante o período de 2014 a 2015. No momento da necropsia foram coletados fragmentos de fígado, rins, intestino, pulmão, coração, baço e encéfalo para posterior processamento microbiológico e histopatológico. As lesões foram graduadas e as cepas isoladas submetidas a antibiograma. Das setenta aves necropsiadas, dezenove foram positivas para E. coli e apenas uma para Salmonella Typhimurium. As lesões de congestão e infiltrado inflamatório linfoplasmocitário variaram de leve a moderado, e foram as principais lesões encontradas. Nas cepas analisadas foi constatada multiresistência a diferentes grupos de antibióticos testados. De acordo com os achados, pode-se concluir que os isolados de E. coli e Salmonella Typhimurium produziram lesões significativas em psittaciformes em Fortaleza, Brasil, e a multirresistência pode dificultar o tratamento com antibióticos usados na clínica de aves de companhia.(AU)

Virulence factors of Escherichia coli in relation to the importance of vaccination in pigs / Fatores de virulência da Escherichia coli associados à importância da vacinação nos suínos

ABSTRACT: Enterotoxigenic Escherichia coli (ETEC) is the major cause of diarrhea in newborn and weaned pigs. Bacteria adhesion to the host cell is considered a specific phenomenon among fimbrial and non-fimbrial adhesins with their respective receptors on enterocytes. Enteric disorders are related with the fimbriae F4 (K88), F5 (K99), F6 (987P), F41, and F18. In addition to ETEC, another category of E. coli , porcine pathogenic E. coli (PEPEC),can cause diarrhea in pigs; it produces the porcine attaching and effacing-associated (Paa) adhesin in, which is capable to cause a typical lesion known as an attaching and effacing (A/E) lesion. Immunization of sows with adhesin is important to stimulate the production of antibodies and their subsequent transfer to piglets through colostrum. The aim of this paper is to illustrate the main impacts of enteric diseases caused by E. coli in swine production and to highlight the importance of continuing research on this bacterium to improve disease prevention through vaccination.

RESUMO: Escherichia coli ( E. coli ) enterotoxigênica (ETEC) é considerada importante causa de diarreia em suínos neonatos e desmamados. A adesão da bactéria à célula do hospedeiro é considerada um fenômeno específico entre as adesinas fimbriais e não fimbriais com seus respectivos receptores nos enterócitos. Os distúrbios entéricos estão relacionados com as fímbrias F4 (K88), F5 (K99), F6 (987P), F41 e F18. Além da ETEC, outra categoria de E. coli pode causar diarreia nos suínos, denominada de PEPEC (porcine pathogenic E. coli ), a qual produz a adesina Paa (Porcine attaching adherence), capaz de provocar uma lesão típica denominada A/E (attaching and effacing). A imunização das matrizes com adesinas é importante para estimular a produção de anticorpos e a consequente transferência através do colostro aos leitões. O objetivo deste trabalho foi mostrar os principais impactos das doenças entéricas causadas por Escherichia coli na produção de suínos, e mostrar a importância de atualizar o estudo dessa bactéria para prevenir a doença através da vacinação.

Virulence factors, antimicrobial resistance, and plasmid content of Escherichia coli isolated in swine commercial farms / Fatores de virulência, resistência aos antimicrobianos, presença de plasmídeos em Escherichia coli isoladas de amostras clínicas e ambientais de suínos

Virulence factors and antimicrobial resistance patterns of Escherichia coli isolates were evaluated. A total of 80 E. coli isolates were evaluated, being 64 from clinical samples (intestinal content and fragments of organs from diarrheic piglets), seven from feces of clinically healthy piglets and sows, and nine environmental samples (five from facilities, two from feed, one from insect, and one from waste). Molecular characterization was performed by PCR detection of fimbriae and toxin genes and plasmid content determination. The isolates were also characterized according to their resistance or sensitivity to the following drugs: ampicillin, trimethoprim:sulfamethoxazole, tetracycline, amikacine, colistin, norfloxacin, florfenicol, enrofloxacin, cefalexin, trimethoprim, neomycin, chloramphenicol, and gentamicin. From 80 E. coli isolates, 53.8 percent were classified as enterotoxigenic E. coli (ETEC), 2.5 percent were shiga toxin-producing E. coli (STEC), and 43.8 percent showed a non specific pattern and were unclassified. One fecal isolate from non-diarrheic piglet was classified as ETEC by PCR. Clinical isolates showed resistance mainly for tetracycline and trimethoprim:sulfamethoxazole. Plasmidial DNA was observed in 70 isolates, being 78.5 percent of clinical isolates, 8.57 percent of non-diarrheic feces, and 12.8 percent of environment.

Os fatores de virulência e a resistência aos antimicrobianos foram avaliados em Escherichia coli. Um total de 80 isolados de E. coli, sendo 64 de amostras clínicas (conteúdo intestinal e fragmentos de órgãos de leitões diarreicos), sete das fezes de porcas e leitões saudáveis e nove de amostras ambientais (cinco de instalações, dois de alimentos, um de inseto e um de esterqueira). A caracterização molecular feita pela PCR objetivou detectar fimbrias e toxinas, bem como a determinação do conteúdo de plasmídeos. Os isolados foram caracterizados quanto à resistência ou sensibilidade às seguintes drogas: ampicilina, sulfazotrim, tetraciclina, amikacina, colistina, norfloxacina, florfenicol, enrofloxacina, cefalexina, trimetoprim, neomicina, cloranfenicol e gentamicina. Dos 80 isolados, 53,8 por cento foram classificados como E. coli enterotoxigênica (ETEC), 2,5 por cento como E. coli produtora de shiga toxina (STEC) e 43,8 por cento, por não apresentarem padrão específico, não foram classificadas. Pela PCR, um isolado de fezes de suíno sem diarreia foi classificado como ETEC. Os isolados das amostras clínicas foram principalmente resistentes à tetraciclina e à sulfazotrim. Em 70 isolados, observaram-se DNA plasmidial, destes 78,5 por cento foram obtidos de amostras clínicas, 8,57 por cento de leitões sadios e 12,8 por cento de amostras ambientais.

Occurrence of virulence-related sequences and phylogenetic analysis of commensal and pathogenic avian Escherichia coli strains (APEC) / Ocorrência de seqüências relacionadas com a virulência e análise filogenética de estirpes comensais e patogênicas de Escherichia coli aviário (APEC)

The presence of iron uptake (irp-2, fyuA, sitA, fepC, iucA), adhesion (iha, lpfA O157/O141, lpfA O157/O154, efa, toxB) and invasion (inv, ial-related DNA sequences and assignment to the four main Escherichia coli phylogenetic groups (A, B1, B2 e D) were determined in 30 commensal E. coli strains isolated from healthy chickens and in 49 APEC strains isolated from chickens presenting clinical signs of septicemia (n=24) swollen head syndrome (n=14) and omphalitis (n=11) by PCR. None of the strains presented DNA sequences related to the inv, ial, efa, and toxB genes. DNA sequences related to lpfA O157/O154, iucA, fepC, and irp-2 genes were significantly found among pathogenic strains, where iucA gene was associated with septicemia and swollen head syndrome and fepC and irp-2 genes were associated with swollen head syndrome strains. Phylogenetic typing showed that commensal and omphalitis strains belonged mainly to phylogenetic Group A and swollen head syndrome to phylogenetic Group D. Septicemic strains were assigned in phylogenetic Groups A and D. These data could suggest that clonal lineage of septicemic APEC strains have a multiple ancestor origin; one from a pathogenic bacteria ancestor and other from a non-pathogenic ancestor that evolved by the acquisition of virulence related sequences through horizontal gene transfer. Swollen head syndrome may constitute a pathogenic clonal group. By the other side, omphalitis strains probably constitute a non-pathogenic clonal group, and could cause omphalitis as an opportunistic infection. The sharing of virulence related sequences by human pathogenic E. coli and APEC strains could indicate that APEC strains could be a source of virulence genes to human strains and could represent a zoonotic risk.(AU)

A presença de seqüências de DNA associadas à capacidade de captação de ferro (irp-2, fyuA, sitA, fepC, iucA), adesão (iha, lpfA O157/O141, lpfA O157/O154, efa, toxB) e de invasão (inv, ial) e a classificação dentro dos quatro grupos filogenéticos principais de Escherichia coli (Grupos A, B1, B2 e D) foram determinadas, através de PCR, em 30 amostras comensais de E. coli isoladas de frangos e de 49 linhagens APEC (24 isoladas de frangos com septicemia, 14 isoladas de frangos com síndrome da cabeça inchada e 11 isoladas de embriões de galinhas com onfalite). Nenhuma das linhagens apresentou os genes inv, ial, efa, e toxB. Os genes lpfA O157/O154, iucA, fepC e irp-2 foram encontrados em freqüências significativas entre as amostras patogênicas. O gene iucA foi associado com amostras causadoras de septicemia e de síndrome da cabeça inchada. Os genes fepC e irp-2 foram associados a amostras causadoras de síndrome da cabeça inchada. A análise filogenética demonstrou que linhagens comensais e causadoras de onfalite pertenceram principalmente ao Grupo filogenético A, não patogênico. Amostras causadoras de síndrome da cabeça inchada pertenceram, em sua maioria, ao Grupo patogênico D. Linhagens causadoras de septicemia pertenceram aos Grupos A e D. Estes dados sugerem que linhagens APEC causadoras de septicemia provavelmente têm uma origem ancestral múltipla: uma derivada de uma linhagem patogênica e outra de uma linhagem não patogênica que possivelmente evoluiu através da aquisição horizontal de genes de virulência. Amostras causadoras de síndrome da cabeça inchada possivelmente constituem um grupo clonal patogênico. Por outro lado, amostras causadoras de onfalite possivelmente constituem um grupo clonal não patogênico, que, possivelmente causam onfalite devido a uma infecção oportunista. A presença de genes de virulência também encontrados em E. coli de origem humana pode indicar a possível ocorrência de zoonoses causadas por APEC.(AU)