Haemolytic Escherichia coli isolated from dogs with diarrhea have characteristics of both uropathogenic and necrotoxigenic strains

Twenty-four haemolytic Escherichia coli strains were isolated from dogs with diarrhea. The strains were serotyped and analysed by polymerase chain reaction (PCR) for genes encoding virulence factors associated with E. coli that cause diarrhea in animals. Adhesion antigen production was deduced from haemagglutination experiments. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) of heat extracts was also used as an indication for the production of adhesive structures. The majority of the strains was shown to produce this type of virulence factor. Adhesion and invasion tests of the strains and Caco-2 cells showed that all strains adhered and that two were invasive. The two invasive strains were positive in the intimin PCR and one of them also contained genes encoding CS31A. The PCR for heat stable toxin (ST) was positive in only four strains, as was the presence of F17 fimbrial genes. Surprisingly, 19 strains had intact P fimbrial operons, coding for an adhesin involved in urinary tract infection (UTI). The cytotoxic necrotising factor 1 (CNF1) gene, also mainly found in UTI was likewise detected in these 19 strains. Cytolethal distending toxin (Cdt) genes were found in five strains. The high number of strains positive for CNF1 and P fimbriae prompted us to test the strains in a multiplex PCR used to test E. coli isolated from UTI in various species for 30 virulence associated genes. The data showed that the majority of the diarrhea isolates have virulence factor profiles highly similar to UTI E. coli isolates from dogs. This raises the question whether these isolates are real intestinal pathogens or "innocent bystanders". However, since CNF1 producing necrotoxic E. coli (NTEC) strains isolated from humans, pigs and calves with diarrhea appear to be highly related to our strains, it might be that in dogs this type of isolate is capable of causing not only UTI, but also diarrhea. If this is the case and this type of isolate is "bifunctional", domestic animals likely constitute a reservoir of NTEC strains which can be also pathogenic for humans.     

Prevalence and identity of cdt-related sequences in necrotoxigenic Escherichia coli

The cytolethal distending toxins (CDT) are responsible for the mitosis block at G2/M and the cycle arrest of cells in culture. Escherichia coli isolated from humans and animals with intestinal and extra-intestinal diseases can be positive for the production of a CDT-like cytopathic effect or for the presence of cdt-related genes. The purpose of this study was to compare the prevalence and the identity of cdt-related sequences in necrotoxigenic E. coli (NTEC). A collection of 98 bovine type 2 NTEC (NTEC2) and 45 bovine, 20 canine, 3 feline, 65 human and 129 porcine type 1 NTEC (NTEC1) isolates was studied by colony hybridisation and PCR assays specific for the cdtB genes encoding the B sub-unit of the CDT-I, CDT-II, CDT-III and CDT-IV toxins produced by E. coli. cdtB-III sequences were frequent amongst bovine NTEC2, since 83% of these isolates were positive by colony hybridisation and/or PCR, whereas cdtB-related sequences were rare amongst NTEC1, since only 2 bovine (4%), 3 canine (15%), 10 human (15%) and 13 porcine (10%) of these isolates were positive. The 28 probe-positive NTEC1 harboured cdtB-IV sequences (13 isolates), cdtB-I sequences (10 isolates), or still unidentified cdt-related sequences (5 isolates). After comparison with previously published and unpublished results of phenotypic assay on cell cultures, existence of other cdt-related sequences is suggested amongst NTEC1. The differences between NTEC1 and NTEC2 in their CDT profiles may have implication for the pathogenesis of those two classes of pathogenic E. coli.     

Prevalence and characteristics of necrotoxigenic Escherichia coli (NTEC) strains isolated from diarrhoeic dairy calves.

Fecal samples from 246, 1-90-days old diarrhoeic dairy calves in 72 herds were screened for the presence of cytotoxic necrotizing factors (CNF)-producing Escherichia coli (NTEC). NTEC were detected by tissue culture assays and PCR in 39 (15.8%) of the diarrheic calves, and the majority of these animals (34 of 39, ca. 87.2%) were infected by NTEC producing CNF2. Calves were grouped according to their age (1-7 days, 8-14 days, 15-21 days, 22-30 days and 31-90 days) and analyses of prevalence were done by the Mantel-Haenzsel chi2-test for trend. A significant age-associated increase in the prevalence of NTEC producing CNF2 (p<0.0001) was found. Eighty-one (8.4%) of the 958 E. coli isolates from the 246 diarrheic calves were positive for CNF in the tissue culture assays. These strains were analyzed by PCR and this technique showed that three (3.7%) strains were CNF1-positive and 75 (92.6%) were CNF2-positive. Moreover, three of the strains positive in the tissue culture assays were negative by PCR. These strains were subsequently assayed in several biological tests (rabbit skin test, mouse intraperitoneal test and mouse footpad test) which showed that they were really NTEC, probably producing CNF2, but with some different properties to classical strains producing CNF2. NTEC strains producing CNF2 belonged to different serogroups (O2, O7, O9, O14, O15, O41, O43, O45, O55, O76, O86, O88, O109, O115, O123, O128, O153 and O159) than strains producing CNF1 (O11 and O32) or PCR-negative strains (O111). Moreover, a strong association between CNF2 and F17 fimbriae was found (78.6% of CNF2-positive strains were F17-positive, whereas only 22.9% of CNF2-negative strains were F17-positive).     

Characterization of Necrotoxigenic Escherichia coli (NTEC) Strains Isolated from Healthy Calves in Poland

Faecal samples from 132 healthy, 4–8‐week‐old calves from four different farms were examined for necrotoxigenic Escherichia coli (NTEC) producing the cytotoxic necrotizing factors type 1 (CNF1) and type 2 (CNF2). CNF2 genes were detected by polymerase chain reaction in 24 (6.1%) of the 396 E. coli strains tested; these strains were found in 18 (13.6%) calves used in the study. None of the 396 E. coli isolates examined possessed the gene encoding CNF1. Overall, 28.8% of E. coli examined expressed the F17 fimbrial antigen. A strong association between CNF2 toxin and F17 fimbriae was found (62.5% of CNF2‐positive strains were F17‐positive). Moreover, six out of 24 NTEC strains had the Stx1 or the Stx2 shiga toxin genes, and three additional isolates possessed the eae genetic marker of the intimin protein.     

Presence of pap-, sfa-, and afa-related sequences in necrotoxigenic Escherichia coli isolates from cattle: evidence for new variants of the AFA family.

Necrotoxigenic Escherichia coli (NTEC) are associated with intestinal and extraintestinal diseases in animals and human beings and produce Cytotoxic Necrotizing Factor 1 (CNF1) or 2 (CNF2). Fourty-three NTEC1, 42 NTEC2, and 32 CNF-negative isolates from cattle were tested by colony DNA hybridization, by plasmid DNA hybridization and by PCR assays for the presence of DNA sequences homologous to the operons coding for fimbrial (PAP/PRS, SFA/FIC, and F17) and afimbrial (AFA/Dr) adhesins of extraintestinal E. coli. Most NTEC1 isolates hybridized with the PAP probes and either the SFA probe (37%) or the AFA probes (49%). Most NTEC2 isolates, in contrast, hybridized with the F17 probe (45%), the AFA probes (19%), or the F17 and AFA probes (22%). A probe-positive plasmid was identified in each of the 19 NTEC2 isolates studied. They all hybridized with the CNF2 toxin probe (Vir plasmids) and most of them with the F17 (6 plasmids) or AFA (7 plasmids) probes. PCR amplification was obtained with 6 of the 11 NTEC isolates tested for the papGII/prsG genes; with all 5 NTEC isolates tested for the sfa and related operons; but with none of the 18 NTEC isolates tested for the afa and related operons. pap-, sfa-, and afa-related sequences are thus present in NTEC isolates from cattle in addition to f17-related operons and may code for adhesins corresponding to specific colonization factors. f17- and afa-related sequences can be located on the Vir plasmids along with the cnf2 gene. Existence of new variants of the AFA/Dr family is evident from the negative results of this family-specific PCR assay.     

Characteristics of necrotoxigenic Escherichia coli isolated from septicemic and diarrheic calves between 1958 and 1970

A total of 434 Escherichia coli isolated from septicemic calves between 1958 and 1965 and 430 E. coli isolated from diarrheic calves between 1967 and 1970 were studied by colony hybridisation and PCR assays for the presence of the cnf1- and the cnf2-like genes. They were also studied for the presence of genes coding for putative virulence factors associated with the CNF toxins including F17-, Pap- and Sfa-fimbrial adhesins and the recently described CDT-III toxin and AfaVIII-afimbrial adhesin. Thirty (7%) of the 434 septicemic strains were positive for CNF by colony hybridisation. Twenty-six were confirmed as necrotoxigenic E. coli type 2 (NTEC2) and four as NTEC1 by PCR. Thirty-five (8%) of the 430 diarrheic strains were positive for CNF by colony hybridisation. Five of them were studied by PCR and confirmed as NTEC1. The 26 septicemic NTEC2 strains and 20 of the 35 diarrheic NTEC including three of the five NTEC1 were positive for CDT-III. All adhesins studied were present in NTEC as well as in non-NTEC. NTEC1 were mainly Pap-, Sfa- and/or Afa8-positive, whereas NTEC2 were mainly F17- and/or Afa8-positive. This study shows that necrotoxigenic E. coli with their associated adhesins and toxins were present in calves as early as 1958, but their prevalence seems to have increased since that time.     

Characterization of enterotoxigenic E. coli (ETEC), Shiga-toxin producing E. coli (STEC) and necrotoxigenic E. coli (NTEC) isolated from diarrhoeic Mediterranean water buffalo calves (Bubalus bubalis)

Two hundred and twenty Escherichia coli isolates from 314 Mediterranean water buffalo calves less than 4 weeks old affected by severe diarrhoea with a lethal outcome were characterized for the presence of the virulence factors LT, ST, Stx1, Stx2, haemolysins, intimin, CNF1, CNF2, CDT-I, CDT-II, CDT-III, CDT-IV, and F17-related fimbriae (F17a, F17b, F17c, F17d). The prevalence of ETEC, STEC and NTEC were 1.8%, 6.8% and 20.9%, respectively. The ETEC isolates were all LT-positive and ST-negative. The STEC isolates were all Stx and intimin-positive, with Stx1 (80%) more frequent than Stx2 (27%). The NTEC isolates were all CNF and Hly-positive, with CNF2 (83%) more frequent than CNF1 (22%). Susceptibility assays to 11 antimicrobials displayed high rates of resistance (>30%) to antimicrobials tested. These data show that the most prevalent strains in diarrhoeic water buffalo calves were NTEC, mostly CNF2 and HlyA-positive, with strong associations CNF2/CDT-III and CNF2/F17c.     

Comparison of necrotoxigenic Escherichia coli isolates from farm animals and from humans

Necrotoxigenic Escherichia coli (NTEC) isolated from animals and humans can belong to the same serogroups/types and produce or carry the genes coding for fimbrial and afimbrial adhesins of the same family, P, S, F17, and/or AFA, raising the question of a potential zoonotic source of human infection. The main purpose of this study was to compare 239 NTEC1 strains (45 from cattle, 65 from humans and 129 from piglets) and 98 NTEC2 strains from cattle, using a uniform and standardized typing scheme. The O serogroups and the biotypes recognized amongst NTEC1 and NTEC2 strains were quite varied, although some were more frequently observed (serogroups O2, O4, O6, O8, O18, O78, and O83 and biotypes 1, 2, 5, 6, and 9). Hybridization, results with gene probes for the P family (PAP probe), S family (SFA probe), AFA family (AFA probe), F17 family (F17 probe) of fimbrial and afimbrial adhesins, could differentiate most NTEC1 strains, which are PAP-, SFA- and/or AFA-positive, from NTEC2 strains, which are mainly F17- and/or AFA-positive, but were of no help in differentiating between NTEC1 strains from cattle, humans, and piglets. All but seven (98%) NTEC1 and NTEC2 strains were serum resistant, 199 (59%) produced an aerobactin, and colicin (I, V, or unidentified) was produced by 22-34% of them. On the other hand, more than 90% of the NTEC1 strains were haemolytic on sheep blood agar compared with only 40% of the NTEC2 strains. Production of a classical haemolysin, active on sheep erythrocytes, and hybridization with the PAP probe were associated in a majority of NTEC1 strains (63-81%), but very rarely in NTEC2 strains (3%). Production of enterohaemolysin and hybridization with the PAP probe were much less frequently associated in NTEC strains (1-9%). It was thus possible neither to completely differentiate NTEC1 strains from cattle, humans, and pigs, nor to define a signature for the NTEC strains. Necrotoxigenic E. coli must still be identified on the basis of the production of the Cytotoxic Necrotizing Factors 1 or 2 (or of their encoding genes) and complete differentiation of NTEC1 strains from cattle, humans, and piglets, use additionnal methods.     

The afa-related gene cluster in necrotoxigenic and other Escherichia coli from animals belongs to the afa-8 variant

Six hundred and nine necrotoxigenic Escherichia coli type 1 and 2 (NTEC1 and NTEC2) and non-NTEC isolated in Western and Southern Europe, North Africa and Canada from diseased calves, pigs, humans, poultry, and 55 isolated from asymptomatic calves were studied for the identification of afa-related sequences to the recently described afa-7 and afa-8 gene cluster variants from two bovine Escherichia coli (Lalioui et al., 1999). Colony hybridization and PCR assays for the afaD-7, afaE-7, afaD-8 and afaE-8 identified the afa-related sequences to the afa-8 gene cluster in most (67/79; 85%) of the E. coli positive with the Afa-f family probe and in 14 additional strains negative with the Afa-f probe. No E. coli was positive for the afa-7 gene cluster. The existence of afa-8 positive strains was thus confirmed among bovine E. coli and for the first time among porcine, poultry and human E. coli. Sequencing of the afaE-8 amplicon of nine strains from the different host species showed a high degree of conservation (>95% at the DNA level; >92% at the amino-acid level). The afa-8 gene cluster was more frequent in E. coli from diseased calves (18%) than from piglets (12%), humans (6%) and poultry (5%). Bovine NTEC2 (26%) were more frequently positive than NTEC 1 (20%) and non-NTEC (11%). E. coli isolated from asymptomatic calves were rarely positive: one NTEC2 (3%) and no non-NTEC. The afa-8 gene cluster was located on the Vir plasmid in 11/23 NTEC2, but no plasmid localization was detected in NTEC1 or non-NTEC.     

Escherichia coli as a pathogen in dogs and cats.

Certain strains of Escherichia coli behave as pathogens in dogs and cats causing gastro-intestinal and extra-intestinal diseases. Among the five known groups of diarrhoeagenic E. coli, namely enteropathogenic E. coli (EPEC), enterotoxigenic E. coli (ETEC), enteroinvasive E. coli (EIEC), shiga-toxin producing E. coli (STEC) and enteroaggregative E. coli (EAggEC), only EPEC and ETEC were clearly associated with enteric disease in young dogs. ETEC isolates from diarrhoeic dogs were found to be positive for the heat-stable enterotoxins STa and STb but negative for heat-labile enterotoxin (LT). Canine ETEC were found to be different from those of other animals and humans by their serotypes, production of alpha-haemolysin and adhesive factors and by the production of uncharacterized types of enterotoxins by some ETEC. Canine EPEC could be distinguished from EPEC of humans or other animals by their serotypes and by the eae-protein intimin which mediates intimate adherence of EPEC to intestinal mucosa cells. STEC were occasionally isolated from faeces of healthy and diarrhoeic dogs but their role in canine diarrhoea is not yet well known. EIEC and EAggEC were not reported to occur in dogs or cats. Very little is known on diarrhoegenic E. coli in cats and further epidemiological investigations on this subject are needed. Besides its role in gastro-intestinal infections, E. coli can cause infections of the urogenital tract and systemic disease in dogs and cats. Extra-intestinal pathogenic E. coli strains from dogs and cats belong to a limited number of serotypes and clonal groups and are frequently found as a part of the normal gut flora of these animals. Many of these E. coli strains carry P-fimbriae and produce alpha-haemolysin and a necrotizing cytotoxin (CNF1). Some of the frequently isolated types of extra-intestinal pathogenic E. coli from dogs, cats and humans were found to be highly genetically related but showed differences in their P-fimbrial adhesins which determine host specificity. Transmission of extra-intestinal and enteral pathogenic E. coli between dogs and humans was reported. Further research is needed, however, to determine the role of dogs and cats as transmission vectors of pathogenic E. coli strains to other animals and humans.     

Cloning and Sequencing of cnf1 from Escherichia coli Incriminated in Mink and Bovine Colibacillosis

Colibacillosis is responsible for significant losses to the mink and cattle industries. Previous work in our laboratory and by others has suggested that possession of cnf1, the gene encoding cytotoxic necrotizing factor (CNF1), may contribute to the virulence of isolates of E. coli from mink and cattle. The cnf1 gene from E. coli isolated from a mink with colisepticaemia and a bovid with scours was amplified and cloned as a 3.5 kb fragment, and the fragment was sequenced. The cnf1 sequences from the mink and bovine isolates of E. coli were compared to each other and to cnf1 sequences of E. coli from urinary tract and diarrhoea-associated infections of humans. The difference was only 7 nucleotides between the cnf1 sequences of the mink and bovine isolates of E. coli, which translated into 7 differences in amino acids. The cnf1 sequence of the mink isolate of E. coli had 15 nucleotide differences from the cnf1 sequences of the human isolate of E. coli (GenBank X70670), which translated into 11 differences in amino acids between these proteins. The cnf1 sequence of the bovine isolate of E. coli had 14 nucleotide differences from the cnf1 sequence of the human isolate of E. coli (GenBank X70670), which translated into 10 differences in amino acids between these proteins. The highly conserved sequences of the amino acids of CNF1 proteins make them a promising target for detection and control of the CNF1-producing E. coli involved in disease among various host species.     

Characteristics of alpha-hemolytic strains of Escherichia coli isolated from dogs with gastroenteritis

Twenty-four hemolysin producing (Hly+) strains of Escherichia coli isolated from dogs with gastroenteritis were investigated for their virulence markers and their phenotypic properties. The strains were distributed over eleven known E. coli O-serogroups and most of them were heterogeneous for their phenotypes. All strains were found to produce alpha-hemolysin which was detected by Southern hybridization and colony immunoblotting using a specific gene probe and a monoclonal antibody. Eight strains were carrying plasmids encoding alpha-hemolysin sequences (hly-plasmids) and 16 strains carried chromosomal hly-determinants. Twelve of the strains showed enterotoxic activities which were tested for in different assays. Among these, three O42:H37 and two O70:H-strains carrying hly-plasmids were found to harbour other plasmids encoding the heat-stable enterotoxin STA1. The other seven strains showing enterotoxicity in the ileal loop or the suckling mouse assay were negative for STA1, STA2, or LT. None of the 24 strains were positive for invasiveness or for production of Vero (Shiga-like) toxins. The production of alpha-hemolysin was closely associated with the production of cytotoxic necrotizing factor (CNF), which was detected in 17 of 24 strains. Of these, 16 elaborated CNF1 and one strain produced an unknown CNF type. Surprisingly, all strains carrying ST-plasmids and six of eight strains carrying hly-plasmids were negative for CNF. Thus, in canine E. coli strains CNF production seems to be closely associated with production of chromosomally encoded alpha-hemolysin whereas hly-plasmids are more often associated with ST-producing, CNF negative isolates.     

Virulence genes in bla(CTX-M) Escherichia coli isolates from chickens and humans

The aim of this study was to determine the presence of virulence genes in isolates of CTX-M Escherichia coli from diseased chickens, from healthy chickens and from urinary tract infections in people. Three CTX-M E. coli strains from three different instances of disease in poultry (two of which were E. coli related) were tested for bla(CTX-M) sequence type and replicon type. Additionally, they were tested for the presence of 56 virulence genes (encoding fimbriae, adhesins, toxins, microcins and iron acquisition genes) using a micro-array. Results were compared to the virulence genes present in isolates from 26 healthy chickens and from 10 people with urinary tract infections. All genes found in isolates from diseased birds, including the astA (heat stable toxin) and tsh (temperature sensitive haemagglutinin) genes which have previously been associated with colibacillosis in chickens, were also present in isolates from healthy birds. However, 6/10 of the virulence genes found were exclusive to isolates from humans. Genes exclusive to chicken isolates included ireA (sidephore receptor), lpfA (long polar fimbriae), mchF (microcin transporter protein) and tsh whilst genes exclusive to human isolates included ctdB (cytolethal distending toxin), nfaE (non-fimbrial adhesion), senB (plasmid encoded enterotoxin) and toxB (toxin B). The results support previous findings that CTX-M E. coli strains in chickens are generally different from those causing disease in humans, but genes such as astA and tsh in isolates from diseased birds with colisepticaemia were also present in isolates from healthy birds.     

Necrotoxigenic Escherichia coli type-2 invade and cause diarrhoea during experimental infection in colostrum-restricted newborn calves

There exists experimental evidence that necrotoxigenic Escherichia coli (NTEC) strains producing the cytotoxic necrotising factor 1 cause intestinal and extra-intestinal disease in piglets. On the other hand, no experimental model has been developed with NTEC strains producing the cytotoxic necrotising factor 2. In all, 14 colostrum-restricted calves were orally challenged with two strains isolated from the faeces of a diarrheic calf (B20a) or from the heart blood of a septicaemic calf (1404). All calves had diarrhoea which lasted until euthanasia in eight of them. In those calves, diarrhoea was correlated with the faecal excretion of the challenge strains. At necropsy, vascular congestion of the intestinal mucosa, hypertrophy of the mesenteric lymph nodes (MLN) and some congestion of the lungs were observed. Bacteriology confirmed the colonisation of the intestine by the challenge strains which were also recovered from the heart blood, the lungs and/or the liver. Histological sections confirmed enterocolitis, lymphadenitis and limited bronchopneumonia. In the intestinal tissue sections, bacteria testing positive in an in situ DNA hybridisation assay with a CNF2 probe were observed. Those results were confirmed by immunohistochemistry with a polyclonal anti-O78 and a monoclonal anti-F17b antisera. Three of the five control calves receiving either saline or a CNF(-), F17a strain (25KH09) had no clinical signs or lesions. The other two presented a profuse liquid diarrhoea but those calves were positive for the presence of K99(+) E. coli. In this model, both NTEC2 strains were thus, able to colonise the intestine, to cause long-lasting diarrhoea and to invade the blood stream with localisation in various internal organs in colostrum-restricted conventional newborn calves.     

Identification of the F17 fimbrial subunit- and adhesin-encoding (f17A and f17G) gene variants in necrotoxigenic Escherichia coli from cattle, pigs and humans

Putative colonization factors of the F17 family of fimbrial adhesins have been identified in necrotoxigenic Escherichia coli Type 1 and Type 2 (NTEC1 and NTEC2) from calves, pigs, and humans. The f17A and f17G gene variants, coding respectively for the major subunit and for the adhesin of the F17 fimbriae, were typed in 70 E. coli carrying f17-related sequences (15 NTEC1, 51 NTEC2, and four non-NTEC) by colony hybridisation with gene probes derived from the different f17A gene variants (a, b, c, and d) and by PCRs specific for each f17A and f17G (I and II) gene variants. Typing of f17A genes was not possible by colony hybridisation, as most 70 E. coli were positive with more than one gene probe. On the other hand, the PCRs allowed the typing of the f17A gene in 37 E. coli and of the f17G gene in all 70 E. coli. The f17Ab gene variant was detected in 13 NTEC2; the f17Ac, in all 15 NTEC1, six NTEC2 and two non-NTEC; and the f17Ad, in one non-NTEC. Seven additional NTEC2 were positive with the PCRs for two variants: f17Ab and f17Ac in three of them; f17Ac and f17Ad in four of them. Either these seven NTEC2 harbour two variants or the variant present can be detected by two PCRs. The remaining 25 NTEC2 and one non-NTEC tested negative with the PCRs for the four f17A gene variants, suggesting the existence of other variant(s). In contrast, all 70 E. coli were positive with the PCR for the f17GII gene variant and none with the PCR for the f17GI gene variant. The f17-related sequences were present on the CNF2/Vir plasmids in 27 out of the 46 NTEC2 from which plasmid DNA could be extracted: all but one of those positive for the f17Ab gene variant and various proportions of those positive for other variants. In contrast, no plasmid carried f17-related sequences in NTEC1 and non-NTEC.     

Prevalence and genetic profiles of Shiga toxin-producing Escherichia coli strains isolated from buffaloes, cattle, and goats in central Vietnam

We investigated the prevalence of Shiga toxin-producing Escherichia coli (STEC) in 568 healthy domestic animals (buffaloes, cattle, and goats) from 98 farms in the central region of Vietnam. The aims of this study were to determine if the prevalence of STEC in South East Asia is similar to that in other parts of the world, to characterize the virulence gene profiles from the recovered STEC and to determine if the recovered STEC belong to serotypes commonly associated with human disease. STEC and intimin-positive strains were recovered from 27% of buffaloes, 23% of cattle, and 38.5% of goats. Seventy percent of buffalo farms, 60% of cattle farms and 100% goat farms were positive for STEC. Of 170 STEC strains, 99 carried both stx1 and stx2 genes, 36 carried the stx2 gene, and 35 carried the stx1 gene. The eae gene was found in six caprine isolates, but not in buffalo or bovine isolates. Among 173 E. coli strains (170 STEC and 3 intimin-positive), 110 carried the ehxA gene, 106 possessed the saa gene. Further characterization of stx subtypes demonstrated that among 134 stx1-containing isolates, 107 belonged to the stx1c subtype and 27 were the stx1 subtype. Of the 132 stx2-containing isolates, 36 were stx2, 34 were stx2c, 43 were stx2d subtype, 3 belonged to stx2g, and 16 strains were stx2d(act). The stx2c variant was dominant in strains isolated from buffalo while the stx2d variant occurred more frequently in caprine isolates. Only 9 (5%) STEC strains contained genes encoding for serotypes O26, O91, O121, O145, and O157 LPS, which are more frequently associated with human infections. The results of this study provide data for understanding of epidemiology of STEC among domestic animals in Vietnam and indicate that buffaloes are also an important reservoir of STEC.     

AFA and F17 adhesins produced by pathogenic Escherichia coli strains in domestic animals.

AFA and F17 are afimbrial and fimbrial adhesins, respectively, produced by pathogenic Escherichia coli strains in domestic animals. F17-related fimbriae are mainly detected on bovine and ovine E. coli associated with diarrhoea or septicaemia. The F17-G adhesin subunits recognize N-acetyl-D-glucosamine (GlcNAc) receptors present on bovine intestinal cells. Some F17 subtypes also bind to GlcNAc receptors present on human uroepithelial and intestinal Caco-2 cells or to the laminin contained in the basement of mammalian membranes. F17 is often associated with other virulence factors (aerobactin, serum resistance, CNF2 toxin, K99, CS31A or AFA adhesins) on pathogenic E. coli. A cluster of only four genes is required to synthesize functional F17-related fimbrial structures. The hypothesis of multifunctional F17 fimbrial subunits is supported by the fact that: i) the N-terminal part of the adhesin subunit participates in receptor recognition, whereas the C-terminal part is required for biogenesis of the fimbrial filament; and ii) the interaction between structural and adhesin subunits seems to be crucial for the initiation of monomer polymerization. Recently, determinants related to the afa gene clusters from human pathogenic E. coli associated with intestinal and extra-intestinal infections were identified in strains isolated from calves and piglets with diarrhoea and septicaemia. Two afa-related gene clusters, designated afa-7 and afa-8, that encode afimbrial adhesins were cloned and characterized from bovine pathogenic E. coli. These animal afa gene clusters were plasmid and chromosome borne and were expressed by strains that produced other virulence factors such as CNF toxins, F17, PAP and CS31A adhesins. A high frequency of afa-8 and a low prevalence of afa-7 among bovine E. coli isolates were suggested by preliminary epidemiological studies. As with the human afa gene clusters, the animal ones encode an adhesive structure composed of two proteins: AfaE which mediates adhesion to epithelial cells and AfaD which is an invasin.     

Comparison of Clostridioides difficile strains from animals and humans: First results after introduction of C. difficile molecular typing and characterization at the Istituto Zooprofilattico Sperimentale of Piemonte,Liguria e Valle d’Aosta,Italy

PCR ribotypes (RTs027 and 078) are known causes of Clostridioides difficile infection (CDI) in humans. Molecular typing and characterization of 39 C. difficile strains isolated from samples from humas and animals in 2016–2018 indicated an overlap of RTs between community-acquired patients (CA-CDI) and domestic animals from the same geographical area; 14 RTs were identified: 12 RTs were positive for toxins A/B; RT078, RT080 and RT126 were also positive for binary toxin (CDT). Most of the RTs from the animals (RTs020, 078, 106, 126) were also detected in the samples from humans. Strains grouped into three clusters: cluster I included prevalently human strains, mainly RT 018; clusters II and III included strains from humans and animals, mainly RT078 and RT020. The CA-CDI strains suggested animals as a reservoir of C. difficile isolated together with other microorganisms from animals, highlighting the association of enteric pathogens as a cause of infection and death.     

Occurrence of virulence genes among Campylobacter jejuni and Campylobacter coli isolates from domestic animals and children

The presence of the flaA, cadF, cdtB and iam genes of Campylobacter spp. was determined with the PCR method. The materials to investigate were 56 C. jejuni and 23 C. coli strains isolated from clinical samples (children and domestic animals). It was found that all of the Campylobacter spp. isolates from children with diarrhoea and domestic animals had cadF gene, responsible for adherence. The flaA gene was present in all Campylobacter spp. isolates derived from children and cats. Occurrence of flaA gene was confirmed in 100% of C. jejuni strains obtained from dogs. The high prevalence of the cdtB gene associated with toxin production was observed in this study (100%-Campylobacter spp. isolates obtained from dogs and cats, 97.9%-Campylobacter spp. isolates from children). The isolates showed a wide variation for the presence of iam gene. The lowest prevalence (23.5%) was detected in Campylobacter spp. obtained from dogs. The highest rates of iam detection (91.6%) were revealed in C. coli isolates from children.     

Genotypic and phenotypic characterisation of Escherichia coli strains associated with porcine pyelonephritis

Urinary tract infection (UTI) is a severe problem in humans as well as in many domestic animals like pigs. The most frequent infectious agent in UTI is uropathogenic Escherichia coli. Such strains have been extensively characterised with respect to virulence and fitness factors as well as clonal type when it comes to human isolates. However, relatively little has been done to characterise the corresponding porcine strains. On this background we have analysed 20 porcine pyelonephritis E. coli strains isolated from infected pig kidneys. The strains were quite distinct from that of human uropathogenic strains with regards to adhesion profile and haemolysin production. Also, the clonal profiles differed from that of human infections since our strains all belonged to the E. coli clonal groups A and B1.