Clonal study of avian Escherichia coli strains by fliC conserved-DNA-sequence regions analysis

The clonal relationship among avian Escherichia coli strains and their genetic proximity with human pathogenic E. coli, Salmonela enterica, Yersinia enterocolitica and Proteus mirabilis, was determined by the DNA sequencing of the conserved 5' and 3'regions fliC gene (flagellin encoded gene). Among 30 commensal avian E. coli strains and 49 pathogenic avian E. coli strains (APEC), 24 commensal and 39 APEC strains harbored fliC gene with fragments size varying from 670bp to 1,900bp. The comparative analysis of these regions allowed the construction of a dendrogram of similarity possessing two main clusters: one compounded mainly by APEC strains and by H-antigens from human E. coli, and another one compounded by commensal avian E. coli strains, S. enterica, and by other H-antigens from human E. coli. Overall, this work demonstrated that fliC conserved regions may be associated with pathogenic clones of APEC strains, and also shows a great similarity among APEC and H-antigens of E. coli strains isolated from humans. These data, can add evidence that APEC strains can exhibit a zoonotic risk.

Occurrence of virulence-related sequences and phylogenetic analysis of commensal and pathogenic avian Escherichia coli strains (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.

Analysis of Escherichia coli isolated from bovine mastitic milk

Mastitis has been recognized for some time as the most costly disease in dairy herds. From February to November 2004, 670 samples of bovine mastitic milk from which 231 Escherichia coli strains were isolated, were collected from two Brazilian states. The strains were screened for the presence of Shiga toxin-producing (stx 1 and stx 2) and intimin (eae) genes. Twenty (8.6%) strains were detected by PCR to harbor the Shiga toxin genes (8 the stx 1 gene, 12 the stx 2 gene and none both of them). Two (0.8%) of the Escherichia coli strains studied were eae positive non Shiga toxin-producing. The strains were also examined for resistance to 12 antimicrobial agents. The predominantly observed resistance was to tetracycline (92.2%), streptomycin (90.4%), nalidixic acid (88.3%), amikacin (86.5%) and cephalothin (84.8%). Multidrug resistance was found among 152 isolates (65.8%).

Virulence factors of avian pathogenic Escherichia coli (APEC)

Avian pathogenic Escherichia coli (APEC) strains cause a great diversity of diseases in birds and are responsible for great economic losses in the avian industry. To date, several studies have been carried out to better understand the APEC pathogenesis for a possible development of tools which could prevent the economics losses caused by these strains. This review discusses the virulence factors described do date to be expressed by these strains and the advances made to understand and identify virulence determinants present in APEC.

Perfil de sensibilidade antimicrobiana e detec����o do gene ISS pela rea����o em cadeia da polimerase na tipifica����o de Escherichia coli patog��nica em codornas de corte sob inspe����o sanit��ria

A patogenicidade das cepas de Escherichia coli est�� relacionada �� express��o de fatores de virul��ncia encontrados em elementos gen��ticos denominados plasm��dios. O patotipo APEC, respons��vel por diferentes tipos de doen��as em aves, pode apresentar o gene iss que aumenta a resist��ncia das cepas de E. coli aos efeitos l��ticos do soro, al��m da resist��ncia a diversos antimicrobianos. Este estudo foi conduzido para detectar E. coli em traqu��ias de codornas destinadas ao abate e avaliar, pela presen��a do gene iss e o perfil de susceptibilidade antimicrobiana, o potencial patog��nico para aves e humanos dos isolados obtidos. Foram coletadas 180 traqu��ias de codornas para detec����o de E. coli, determina����o do perfil de resist��ncia a agentes antimicrobianos e posterior detec����o, por rea����o em cadeia da polimerase (PCR), do gene iss. Das traqu��ias analisadas, 8,9 % (16/180) foram positivas para E. coli, sendo obtidos 20 isolados deste agente. A maioria dos isolados foi resistente �� Tetraciclina (16/20), seguida pela Ceftazidima (13/20) e ��cido Nalid��xico (12/20), sendo apenas um resistente �� Amoxicilina. A detec����o do gene iss ocorreu em 55% (11/20) dos isolados. A presen��a do gene iss e a resist��ncia a m��ltiplos antimicrobianos dos isolados obtidos neste estudo pode indicar um poss��vel potencial patog��nico das cepas de E. coli tanto para codornas quanto para outros tipos de aves e animais e mesmo para o ser humano que fique em contato com as mesmas.

Serogroups and virulence genes of Escherichia coli isolated from psittacine birds

Escherichia coli isolates from 24 sick psittacine birds were serogrouped and investigated for the presence of genes encoding the following virulence factors: attaching and effacing (eae), enteropathogenic E. coli EAF plasmid (EAF), pili associated with pyelonephritis (pap), S fimbriae (sfa), afimbrial adhesin (afa), capsule K1 (neu), curli (crl, csgA), temperature-sensitive hemagglutinin (tsh), enteroaggregative heat-stable enterotoxin-1 (astA), heat-stable enterotoxin -1 heat labile (LT) and heat stable (STa and STb) enterotoxins, Shiga-like toxins (stx1 and stx2), cytotoxic necrotizing factor 1 (cnf1), haemolysin (hly), aerobactin production (iuc) and serum resistance (iss). The results showed that the isolates belonged to 12 serogroups: O7; O15; O21; O23; O54; O64; O76; O84; O88; O128; O152 and O166. The virulence genes found were: crl in all isolates, pap in 10 isolates, iss in seven isolates, csgA in five isolates, iuc and tsh in three isolates and eae in two isolates. The combination of virulence genes revealed 11 different genotypic patterns. All strains were negative for genes encoding for EAF, EAEC, K1, sfa, afa, hly, cnf, LT, STa, STb, stx1 and stx2. Our findings showed that some E. coli isolated from psittacine birds present the same virulence factors as avian pathogenic E. coli (APEC), uropathogenic E. coli (UPEC) and Enteropathogenic E. coli (EPEC) pathotypes.

Phylogenetic and pathotype analysis of Escherichia coli swine isolates from Southern Brazil

The current study evaluated the presence of virulence factors by a multiplex PCR technique and then phylogenetically classified the studied strains into groups A, B1, B2 and D, according to Clermont et al. (2000), in 152 intestinal and extraintestinal swine isolates of Escherichia coli. Seventy seven isolates tested were positive for virulence factors. Phylogenetic characterization placed 21 samples into group A, 65 into B1, 19 into B2 and 47 into D. Fourteen urine samples were classified as uropathogenic E. coli (UPEC), nine were both UPEC and enterotoxigenic E. coli (ETEC) and four were ETEC only. The most common phylogenetic classifications were B1 and D groups. Of the analyzed fecal samples, 25 were classified as ETEC. Phylogenetically, the group of higher occurrence was B1, followed by B2, A and D. For the small intestine samples, 20 were classified as ETEC. Phylogenetic analysis found groups B1 and A to be the most commons in these samples. Six isolated tissue samples were classified as ETEC and most of them were designated as group D by phylogenetic classification. The phylogenetic analysis could be employed in veterinary laboratories in the E. coli isolates screening, including the possibility of vaccine strain selection and epidemiological searches.

Resist��ncia antimicrobiana e perfil plasmidial de Escherichia coli isolada de frangos de corte e poedeiras comerciais no Estado de Pernambuco

Embora existam linhagens de Escherichia coli n��o patog��nicas para aves, muitas outras possuem a capacidade de causar s��rios danos �� sa��de das mesmas, sendo capazes de ocasionar diferentes tipos de processos infecciosos. As linhagens patog��nicas s��o denominadas Avian Pathogenic Escherichia coli (APEC), possuindo genes relacionados ao processo de patog��nese em epissomos (plasm��dios) ou no cromossomo. A presen��a de plasm��dios, contendo genes de resist��ncia a antibi��ticos em linhagens avi��rias, patog��nicas ou n��o, indicam a possibilidade de transfer��ncia g��nica lateral entre diferentes tipos de linhagens facilitando tamb��m a transfer��ncia de genes de patogenicidade ou virul��ncia. Objetivou-se com este estudo avaliar o perfil de sensibilidade a antibi��ticos (13) de diferentes amostras (35) de E. coli isoladas de aves comerciais do Estado de Pernambuco apresentando, ou n��o, sinais cl��nicos de processos infecciosos e correlacionar esta resist��ncia com a presen��a de plasm��dios. Os testes utilizados demonstraram que 94,28% dos isolados foram resistentes a tr��s ou mais antibi��ticos, com a lincomicina apresentando o maior percentual de resist��ncia (100%). Na Concentra����o Inibit��ria M��nima (CIM) observou-se multirresist��ncia a v��rios antimicrobianos. A presen��a de plasm��dios foi detecada em 80,0% (28/35) dos isolados, com 16 isolados apresentando plasm��dios com peso molecular aproximado de 88 MDa. Tamb��m foi verificada a presen��a de linhagens apresentando plasm��dios de v��rios tamanhos. Concluiu-se que isolados de E. coli resistentes a antimicrobianos utilizados na avicultura est��o presentes no Estado de Pernambuco, tanto em frangos de corte quanto em poedeiras comerciais. A presen��a de plasm��dios detectados na maioria dos isolados pode estar associada �� resist��ncia aos antimicrobianos e sugere a presen��a de poss��veis genes relacionados �� patogenicidade. Monitorar a resist��ncia a antibi��ticos em bact��rias isoladas de animais torna-se um fator determinante para elei����o e ��xito do tratamento, bem como a possibilidade de elimina����o daquelas que possuem plasm��dios para se evitar a transfer��ncia de genes relacionados �� patogenicidade.

Molecular detection of enteropathogenic Escherichia coli in asymptomatic captive psittacines

Psittaciformes are one of the most endangered groups of birds, and several Brazilian species are classified between vulnerable and critically endangered. It is thus necessary to identify agents that cause infections in captive wild animals and to assess the risks posed thereof and to design interventions to minimize the possibility of disease outbreaks, leading to the conservation of endangered species. The purpose of this study was to identify enteropathogenic Escherichia coli (EPEC) cloacal isolates from asymptomatic psittacines in captivity and evaluate the distribution of the EPEC pathotype. Cloacal swabs were obtained from 46 asymptomatic birds, and resulting isolates were tested by polymerase chain reaction (PCR) for the presence of the attaching and effacing gene (eae) and bundle-forming pilus structural gene (bfpA) of EPEC. Samples from several species were tested, and three samples were found to be positive for the eae and bfpA genes and characterized as typical EPEC. This is the first report of this pathotype in asymptomatic psittacines. Although certain E. coli strains are more pathogenic than others, various factors should be considered when determining the potential of E. coli isolates to cause disease in captive psittacines. Birds that are positive for the EPEC (typical) strain could be zoonotic sources of infection, and may have acquired these strains through contact with humans or domestic animals. These findings may also be valuable for the long-term management of endangered species ex situ as one EPEC sample was isolated from a Red-tailed Amazon (Amazona brasiliensis).

Research of Salmonella spp. and evaluation of pathogenicity, cytotoxicity of Escherichia coli isolates proceeding from sparrows (Passer domesticus)

The aim of this study was to research the occurrence of Salmonella spp. and Escherichia coli in feces samples of sparrows, as well as to identify the pathogenicity, cytotoxicity and sensitivity profile of the isolates to antimicrobial use. Two hundred and twenty eight sparrows were captured in eight farms. The in vitro pathogenicity test was performed by the isolates culture on congo red-magnesium oxalate Agar, whilst the in vivo pathogenicity test was performed in one day-old chicks. In order to study the cytotoxic effects of indicators, samples were inoculated into Vero cells. The results obtained for Escherichia coli isolation confirmed the presence of this microorganism in 30 (13.2%) of the evaluated samples. Out of those isolates, 10 (33.3%) presented the capacity of absorbing ongo red. As for in vivo pathogenicity a 68.0% of mortality rate of the evaluated samples was observed. Out of 20 isolates tested for cytotoxin production, none of them presented cytotoxic effect in the Vero cells. The Salmonella spp was isolated only in one sample (0.04%), and it was identified as Salmonella enterica subspecies houtenae. Results obtained through this research indicate the need for new studies to identify other virulence factors of E. coli samples and to delineate the phylogenetic profile of the isolates in order to establish a relation with colibacillosis outbreaks in chickens and broilers in the studied region, as well as to analyze the critical points in the aviculture productive chain to identify the source of Salmonella enterica subspecies houtenae.

Suscetibilidade a desinfetantes e perfil de resist��ncia a antimicrobianos em isolados de Escherichia coli

A colibacilose, causada por Escherichia coli, �� a enfermidade ent��rica de maior impacto na produ����o de su��nos, podendo levar �� morte do animal. Esta bact��ria possui grande capacidade de desenvolver resist��ncia a m��ltiplos antimicrobianos e a desinfetantes. Desta forma, estudos que abordem mecanismos de resist��ncia e perfil de amostras de campo tornam-se necess��rios. E. coli �� amplamente utilizada como modelo de estudos que exploram a resist��ncia intr��nseca e extr��nseca a multidrogas. Neste trabalho, buscou-se verificar o perfil de sensibilidade de 62 isolados de E. coli de su��nos frente a tr��s desinfetantes e a 13 antimicrobianos. Ainda, em 31 destes isolados foi pesquisada a presen��a de mecanismo de efluxo. Dos tr��s desinfetantes avaliados, o cloreto de alquil dimetil benzil am��nio+poliexietilenonilfenileter foi o que se mostrou mais eficaz (100%), seguido do glutaralde��do+cloreto de alquil dimetil benzil am��nio (95,2%) e do cloreto de alquil dimetil benzil am��nio (88,8%). Dentre os antimicrobianos testados, observou-se maior resist��ncia para a tetraciclina (62,2%) e maior sensibilidade para o florfenicol (88,6%). A alta sensibilidade dos isolados frente aos desinfetantes pode estar relacionada �� aus��ncia de mecanismo de efluxo. O ��ndice de resist��ncia m��ltipla m��dio aos antimicrobianos foi de 0,52, o que demonstra um perfil multirresistente dos isolados, conduzindo para a necessidade do uso racional destas drogas em suinocultura.

Detec����o de fatores de virul��ncia de Escherichia coli e an��lise de Salmonella spp. em psitac��deos

A flora ent��rica dos psitac��deos �� composta principalmente por bact��rias Gram positivas. Bact��rias Gram negativas, como Escherichia coli e Salmonella spp., apresentam elevado potencial patog��nico, sendo consideradas indicativo de problemas de manejo, que poder��o culminar em manifesta����o de doen��as em decorr��ncia de fatores estressantes, dietas deficientes e superlota����o, combinados com alta carga bacteriana no ambiente. O objetivo deste trabalho foi avaliar a presen��a de Salmonella spp., Escherichia coli e os fatores de virul��ncia dos genes iss e iutA dos isolados de E. coli. Analisou-se um total de 44 amostras provenientes de psitac��deos criados em cativeiro, sendo estas 15 fragmentos de ��rg��os de aves submetidas a exame de necropsia e tamb��m 29 amostras de swabs de cloaca e ingl��vio de papagaios-char��o (Amazona pretrei) criados em cativeiro. Nenhuma amostra foi positiva para Salmonella spp. Nas amostras de E. coli detectou-se ambos os fatores de virul��ncia pesquisados.

Utiliza����o da vacina Escherichia coli J5 na imuniza����o de vacas leiteiras contra mastites causadas por E. coli

Avaliou-se a utiliza����o da vacina Escherichia coli J5, na imuniza����o de vacas leiteiras, para preven����o e controle da mastite causada por E. coli atrav��s da an��lise da preval��ncia das infec����es intramam��rias (IMM) no p��s-parto, ocorr��ncia e intensidade dos casos cl��nicos de mastite nos primeiros 100 dias de lacta����o, influ��ncia na contagem de c��lulas som��ticas (CCS) e produ����o de leite. O grupo experimental foi composto de 187 animais, divididos em 2 grupos, vacas vacinadas e n��o vacinadas. As imuniza����es ocorreram 60 dias antes do parto, 30 dias antes do parto e na primeira semana p��s-parto. No dia da secagem e sete dias ap��s o parto foram coletadas amostras para diagn��stico microbiol��gico dos pat��genos causadores de mastite. A ocorr��ncia de casos cl��nicos foi verificada pelo teste da caneca durante as ordenhas sendo registrados os dados relacionados �� intensidade. Amostras foram coletadas mensalmente, a partir do d��cimo dia de lacta����o, para avalia����o da CCS. A produ����o de leite foi registrada mensalmente nos primeiros 100 dias de lacta����o. Verificou-se no grupo vacinado, redu����o na preval��ncia de E. coli no p��s-parto, na ocorr��ncia de casos cl��nicos por E.coli nos primeiros 100 dias de lacta����o bem como na intensidade destes casos cl��nicos. N��o foram observadas diferen��as estat��sticas significativas na CCS, entretanto vacas vacinadas apresentaram maior produ����o de leite, comparadas ��s vacas n��o vacinadas. A vacina����o com E. coli J5 foi eficaz em reduzir a preval��ncia de infec����es intramam��rias (IMM) ao parto, ocorr��ncia e intensidade dos casos cl��nicos e aumento na produ����o de leite nos primeiros 100 dias de lacta����o.

Identification of new flagellin-encoding fliC genes in Escherichia coli isolated from domestic animals using RFLP-PCR and sequencing methods

Identification of Escherichia coli requires knowledge regarding the prevalent serotypes and virulence factors profiles allows the classification in pathogenic/non-pathogenic. However, some of these bacteria do not express flagellar antigen invitro. In this case the PCR-restriction fragment length polymorphism (RFLP-PCR) and sequencing of the fliC may be suitable for the identification of antigens by replacing the traditional serology. We studied 17 samples of E. coli isolated from animals and presenting antigen H nontypeable (HNT). The H antigens were characterized by PCR-RFLP and sequencing of fliC gene. Three new flagellin genes were identified, for which specific antisera were obtained. The PCR-RFLP was shown to be faster than the serotyping H antigen in E. coli, provided information on some characteristics of these antigens and indicated the presence of new genes fliC.

Some virulence genes of Escherichia coli isolated from cloacal swabs of healthy Alagoas Curassows (Pauxi mitu) in Brazil

Birds of the Cracidae family (curassows, guans, and chachalacas) are endemic of the Neotropics and 50 species are currently classified. Brazil has 22 species, seven of which are considered threatened. The Alagoas Curassow (Pauxi mitu) species is considered extinct in the wild; but about 120 birds are alive in captivity. Conservation of this species depends entirely on correct management. Health reports of both wildlife and captive curassows are rare. In this study the presence of Escherichia coli was evaluated in 23 healthy Alagoas Curassows from two private breeding centres. E. coli was isolated from cloacal swabs, and the presence of genes encoding cytotoxic necrotising factor 1 (cnf1), alpha-haemolysin (hly), aerobactin (iuc), serum resistance (iss) and the following adhesions: S fimbriae (sfa), pili associated with pyelonephritis (pap) and temperature-sensitive haemagglutinin (tsh) were investigated. E. coli was isolated from 78.3% (18/23) of the birds, and the percentage of curassows colonized by E. coli was similar between the two facilities. From the 22 E. coli isolates, 15 (68.2%) were positive for at least one virulence factor by PCR, and the most frequently found gene was iss (50%). No curassows had clinical signs of disease. Nevertheless, the presence of some E. coli strains may be a concern to the wildlife in captivity. Additional health surveillance studies are essential to guarantee successful conservation programmes for threatened cracids in Brazil.