Prevalence of Shiga toxin-producing Escherichia coli, Salmonella spp. and Campylobacter spp. in large game animals intended for consumption: relationship with management practices and livestock influence.

Although wild ruminants have been identified as reservoirs of Shiga-toxin producing Escherichia coli (STEC), little information is available concerning the role of Salmonella spp. and Campylobacter spp. in large game species. We evaluated the presence of these pathogens in faeces (N=574) and carcasses (N=585) sampled from red deer (N=295), wild boar (N=333) and other ungulates (fallow deer, mouflon) (N=9). Animal sampling was done in situ from 33 hunting estates during two hunting seasons. Salmonella spp. and Campylobacter spp. strains associated with human campylobacteriosis were infrequently detected indicating that both pathogens had a limited zoonotic risk in our study area. The overall STEC prevalence in animals was 21% (134/637), being significantly higher in faeces from red deer (90 out of 264). A total of 58 isolates were serotyped. Serotypes O146:H- and O27:H30 were the most frequent in red deer and the majority of isolates from red deer and wild boar were from serotypes previously found in STEC strains associated with human infection, including the serotype O157:H7. The STEC prevalence in red deer faeces was significantly higher with the presence of livestock (p<0, 01) where high densities of red deer (p<0.001) were present. To the best of our knowledge, this is the first study reporting the occurrence of Salmonella spp. and STEC in carcasses of large game animals. Furthermore, this study confirmed by pulsed-field gel electrophoresis (PFGE) that cross contamination of STEC during carcass dressing occurred, implying the likelihood of these pathogens entering into the food chain.

Pheno-genotypic characterisation of Escherichia coli O157:H7 isolates from domestic and wild ruminants.

Shiga toxin-producing Escherichia coli (STEC) O157:H7 represents a major public health concern worldwide, with ruminants recognised as their main natural reservoir. The aim of this work was to determine the phenotypic features and genetic relationships of 46 E. coli O157:H7 isolates obtained from sheep, cattle and deer faeces and from unpasteurised goat milk in Spain over a period of 11 years. Characterisation was performed by polymerase chain reaction (PCR), phage typing and pulsed-field gel electrophoresis (PFGE). An atypical E. coli O157:H7 strain (sorbitol-fermenting and beta-glucuronidase positive) originating from deer faeces was detected. Genes encoding Shiga toxins were detected in 69.6% of isolates, all of them carrying only the stx(2) gene. The isolates were from nine different phage types, although 67.4% were restricted to only three: PT14, PT34 and PT54. PT54 was the most prevalent phage type and contained isolates from cattle, sheep and deer. Majority of the isolates were from phage types previously found in strains associated with human infection. XbaI-PFGE identified 33 different types and 11 groups of closely related types (more than 85% similarity), one of which included 21 (45.7%) isolates originating from different animal species, including deer. These results indicate common origin or inter-species spread of genetically similar E. coli O157:H7 isolates and contribute to earlier investigations identifying deer as a natural source of E. coli O157:H7. The study also highlights the emergence of phenotypic variants of E. coli O157:H7, which may not be identified by routine culture methods or by biochemical tests used to characterise serotype O157:H7.

Detection and characterisation of O157:H7 and non-O157 Shiga toxin-producing Escherichia coli in wild boars.

The aim of this work was to determine the prevalence and characteristics of Escherichia coli O157:H7 and non-O157 Shiga toxin-producing E. coli (STEC) in free-ranging wild boars killed during the hunting season in southwest Spain. Faecal samples from 212 wild boars (Sus scrofa) were collected and examined for STEC. Characterisation of isolates was performed by PCR, serotyping, phage typing, and pulsed-field gel electrophoresis (PFGE). E. coli O157:H7 and non-O157 STEC were isolated from 7 (3.3%) and 11 (5.2%) animals, respectively, and the resulting 19 isolates were characterised. The PCR procedure indicated that 4 isolates carried the stx(1) gene, 12 carried the stx(2) gene, and 1 contained both of these genes. The ehxA, eae, and saa genes were detected in 13, 8, and 1 of the isolates, respectively. The eae-positive isolates comprised the types eae-gamma 1 and eae-zeta. The isolates belonged to 11 O:H serotypes, including 4 new serotypes not previously reported within STEC strains, and the majority of them were from serotypes previously associated with human infection. E. coli O157:H7 isolates belonged to phage types associated with severe human illness: PT14, PT34, and PT54. Indistinguishable PFGE types were found in E. coli O157:H7 isolates recovered from a wild boar and from a human patient with diarrhoea living in the same geographic area.

Detection and characterisation of Shiga toxin-producing Escherichia coli other than Escherichia coli O157:H7 in wild ruminants.

Shiga toxin-producing Escherichia coli (STEC) are an important group of emerging pathogens, with ruminants recognised as their main natural reservoir. The aim of this work was to establish the prevalence of non-O157 STEC in free-ranging wild ruminants in the Extremadura region of Spain and to characterise them phenogenotypically. Faecal samples were collected from 243 wild ruminants, including Cervus elaphus, Capreolus capreolus, Dama dama and Ovis musimon and were examined for STEC using both phenotypic (Vero cells) and genotypic (PCR and PFGE) methods. Shiga toxin-producing Escherichia coli were isolated from 58 (23.9%) of the samples and a total of 65 isolates were characterised. A PCR method indicated that 11 (16.9%) strains carried the stx(1) gene, 44 (67.7%) carried the stx(2) gene and 10 (15.4%) carried both these genes. The ehxA gene was detected in 37 (57%) of the isolates but none contained either the eae or saa genes. The isolates were from a total of 12 'O' serogroups, although 80% were restricted to the O2, O8, O128, O146, O166 and O174 serogroups. The most commonly isolated STEC bacteria, which were from the O146 serogroup, exhibited a high degree of polymorphism as indicated by PFGE. Shiga toxin-producing Escherichia coli isolates of serogroups O20, O25, O166, O171, O174 and O176 had not previously been found in wild ruminants. This is the first study to confirm that wild ruminants in Spain are a reservoir of STEC and are thus a potential source of human infection.

A longitudinal study of verotoxin-producing Escherichia coli in two dairy goat herds.

A longitudinal study was conducted on two dairy farms to investigate the pattern of shedding of verotoxin-producing Escherichia coli (VTEC) in goats. Faecal samples were taken from 20 goat kids once weekly during the first 4 weeks of life and then once every month for the next 5 months of life, and from 18 replacement animals and 15 adults once every month for 12 months. The proportion of samples containing VTEC was higher for replacement animals and adults (85.7% and 78.7%, respectively) than for goat kids (25.4%). About 90% of the VTEC colonies isolated from healthy goats belonged to five serogroups (O33, O76, O126, O146 and O166) but the most frequent serogroups of these isolates, except one, were different in the two herds studied. E. coli O157:H7 was found in three goat kids on only one occasion. None of the VTEC isolates, except the three E. coli O157:H7 isolates, was eae-positive. The patterns of shedding of VTEC in goat kids were variable, but, in contrast, most of the replacement animals and adults were persistent VTEC shedders. Our results show that isolates of VTEC O33, O76, O126, O146 and O166 are adapted for colonising the intestine of goats but that, in contrast, infection with VTEC O157:H7 in goats seems to be transient.

Serotypes, virulence genes, and intimin types of Shiga toxin-producing Escherichia coli (STEC) and enteropathogenic E. coli (EPEC) isolated from calves in São Paulo, Brazil.

Shiga toxin-producing Escherichia coli (STEC), is the most important recently emerged group of foodborne pathogens. Ruminants, especially cattle, have been implicated as a principal reservoir of STEC, undercooked ground beef and raw milk being the major vehicles of foodborne outbreaks. Enteropathogenic E. coli (EPEC) strains are defined as eae-harboring diarrheagenic E. coli that possess the ability to form A/E lesions on intestinal cells and that do not possess Shiga toxin genes. In order to determine the occurrence, serotypes and virulence markers of STEC and EPEC strains, 546 fecal samples from 264 diarrheic calves and 282 healthy calves in beef farms in São Paulo, Brazil, were screened by PCR. STEC and EPEC were isolated in 10% and 2.7% of the 546 animals, respectively. Although IMS test was used, the STEC serotype O157:H7 was not detected. The most frequent serotypes among STEC strains were O7:H10, O22:H16, O111:H(-), O119:H(-) and O174:H21, whereas O26:H11, O123:H11 and O177:H11 were the most prevalent among EPEC strains. In this study, serotypes not previously reported were found among STEC strains: O7:H7, O7:H10, O48:H7, O111:H19, O123:H2, O132:H51, O173:H(-), and O175:H49. The eae gene was detected in 25% of the STEC and 100% of EPEC strains. The intimin type theta/gamma2 was the most frequent among STEC, whereas the intimin beta1 was the most frequent intimin type among EPEC strains. To our knowledge, this is the first report of the occurrence of the new intimin muB in one strain of animal origin. This new intimin was detected in one atypical EPEC strain of serotype O123:H? isolated from diarrheic cattle. The enterohemolysin (ehxA) was detected in 51% of the STEC and 80% of the EPEC strains, whereas STEC autoagglutinating adhesin (saa) virulence gene was detected only in those STEC strains negative for eae gene. All 15 bovine EPEC strains isolated in this study were negative for both eaf and bfp genes. Our data shows that in Brazil cattle are not only a reservoir of STEC and atypical EPEC, but also a potential source of infection in humans, since the important STEC serotypes previously described and associated with severe diseases in humans, such as O111:H(-), O113:H21, O118:H16, and O174:H21 were isolated.

Serotypes, virulence genes, intimin types and PFGE profiles of Escherichia coli isolated from piglets with diarrhoea in Slovakia.

Two hundred and fifty Escherichia coli isolates from diarrhoeic and healthy piglets were serotyped and tested for the presence of virulence genes for fimbriae, intimin, heat-labile (LT) and heat-stable (STa and STb) enterotoxins, Stx toxins, and enteroaggregative heat-stable 1 (EAST1) enterotoxin by polymerase chain reaction (PCR). Although 220 isolates from diarrhoeic piglets belonged to 43 O serogroups and 77 O:H serotypes, 60% were of one of the 10 serogroups O2, O8, O15, O54, O84, O101, O141, O147, O149 and O157, and 60% belonged to only 10 serotypes (O8:H-, O54:H-, O84:H7, O101:H-, O141:H-, O141:H4, O147:H-, O149:H10, O163:H-, and ONT:H-). PCR showed that 79% of 220 isolates carried genes for at least one of the virulence factors tested. The gene encoding for EAST1 was the most prevalent (65%) followed by those encoding for STb (49%), LT (42%), STa (13%), and Stx2e (4%). Eighty-three (38%) of the 220 E. coli isolates carried the gene for F4 (K88), whereas genes for F18, F5 (K99), F41, F6 (P987), F17, and intimin (eae) were detected in 9%, 3%, 3%, 3%, 1%, and 3%, respectively. Seropathotype O149:H10:F4:LT/STb/EAST1 (70 isolates) was the most common, representing 32% of isolates. Pulsed-field gel electrophoresis (PFGE) analysis with XbaI of 15 O149:H10 representative isolates from diarrhoeic piglets distinguished 14 types. The 15 isolates exhibited a wide variability of distinct restriction patterns though all belonged to the same serotype (O149:H10), and all but one showed identical virulence determinants (F4, LT, STb, and EAST1). Among 30 isolates from healthy piglets only two virulence genes were detected: EAST1 (26%) and eae (17%). In total, 12 isolates were positives for the eae gene: five isolates had intimin beta1, four possessed intimin theta and three showed intimin type xiB. This is believed to be the first study describing the presence of intimin type xiB in E. coli of porcine origin.

Escherichia coli O157 and non-O157 Shiga toxin-producing Escherichia coli in fecal samples of finished pigs at slaughter in Switzerland.

Fecal samples from 630 slaughtered finisher pigs were examined by PCR to assess the shedding of Escherichia coli O157 (rfbE) and Shiga toxin-producing E. coli (STEC, stx). The proportion of positive samples was 7.5% for rfbE and 22% for stx. By colony hybridization, 31 E. coli O157 and 45 STEC strains were isolated, and these strains were further characterized by phenotypic and genotypic traits. Among E. coli O157 strains, 30 were sorbitol positive, 30 had an H type other than H7, and none harbored stx genes. Intimin (eae), enterohemolysin (ehxA), EAST1 (astA), and porcine A/E-associated protein (paa) were present in 10, 3, 26, and 6% of strains. Among them, one eae-gamma1-positive O157:H7 strain testing positive for ehxA and astA and two eae-alpha1-positive O157:H45 strains were classified as enteropathogenic E. coli (EPEC). The O157:H45 EPEC harbored the EAF plasmid and the bfpA gene, factors characteristic for typical EPEC. The isolated STEC strains (43 sorbitol positive) belonged to 11 O:H serotypes, including three previously reported in human STEC causing hemolytic uremic syndrome (O9:H-, O26:H-, and O103:H2). All but one strain harbored stx2e. The eae and ehxA genes, which are strongly correlated with human disease, were present in only one O103:H2 strain positive for stx1 and paa, whereas the astA gene was found more frequently (14 strains). High prevalence of STEC was found among finisher pigs, but according to the virulence factors the majority of these strains seem to be of low virulence.

Prevalence, serotypes and virulence genes of Shiga toxin-producing Escherichia coli isolated from ovine and caprine milk and other dairy products in Spain.

The aim of this study was to determinate the prevalence, serotypes and virulence genes of Shiga toxin-producing Escherichia coli (STEC) strains isolated from different dairy products (DP) in Spain with the purpose of determining whether DP represent a potential source of STEC pathogenic for humans. A total of 502 DP were examined from 64 different ovine and caprine flocks and 6 dairy plants in Extremadura (Western Spain). Samples were collected monthly between March 2003 and June 2004 and included 360 unpasteurised milk obtained from the bulk tank, 103 fresh cheese curds and 39 cheeses. Samples obtained were examined for STEC using genotypic (PCR) methods. STEC strains were detected from 39 (10.8%) bulk tank, 4 (3.9%) fresh cheese curds and 2 (5%) cheese, whereas O157:H7 serotype were isolated from one (0.3%) bulk tank. A total of 9 STEC strains (O27:H18, O45:H38, O76:H19, O91:H28, O157:H7, ONT:H7, ONT:H9 and ONT:H21) were identified in this study. One of them, the serotype O27:H18, has not been reported previously as STEC. PCR showed that 3 strains carried stx1 genes, 5 possessed stx2 genes and 1 both stx1 and stx2. Whereas all STEC caprine isolates showed ehxA genes, only O157:H7 serotype showed eae virulence genes. The strain O157:H7 isolated possessed intimin type gamma1 and belonged to phage type 31. This study confirms that dairy product is an important reservoir of STEC pathogenic for humans.

Serotypes, virulence genes and intimin types of verotoxin-producing Escherichia coli and enteropathogenic E. coli isolated from healthy dairy goats in Spain.

Faecal samples from 222 healthy dairy goats on 12 farms in Spain, as well as bulk tank milk samples of these farms, were screened for the presence of verotoxin-producing Escherichia coli (VTEC) and enteropathogenic E. coli (EPEC). VTEC and EPEC were isolated in 47.7 and 7.7% of the animals, respectively. VTEC were isolated more frequently from adults and replacement animals than from goat kids. In contrast, EPEC were detected more frequently from goat kids than from replacement animals and adults. VTEC or EPEC strains were not detected in the bulk tank milk samples. Although a selective enrichment protocol was used, the serotype O157:H7 was not detected. The most frequent serotypes among the 106 VTEC strains isolated from goats were O5:H-, O76:H19, O126:H8, O146:H21, ONT:H- and ONT:H21. None VTEC strain was eae-positive. The absence of the eae gene in the VTEC strains could indicate that these strains are less virulent for humans that the classical eae-positive enterohaemorrhagic E. coli types. However, 16% of VTEC strains isolated from healthy goats belonged to serotypes associated with haemolytic uraemic syndrome in humans. The ehxA gene was detected in 84.9 and 52.9% of the VTEC and EPEC from goats, respectively. The beta1, theta/gamma2 and zeta were the most frequent intimin types among the 17 EPEC strains studied and the most prevalent serotypes of these strains were O156:H25 and O177:H11. Our data show that in Spain healthy goats are an important reservoir of VTEC and EPEC, and a potential source of infection for humans.

Characterization of Shiga toxin-producing Escherichia coli isolated from aquatic environments.

This study reports the phenotypic and genotypic characterization of 144 Shiga toxin-producing Escherichia coli (STEC) strains isolated from urban sewage and animal wastewaters using a Shiga toxin 2 gene variant (stx(2))-specific DNA colony hybridization method. All the strains were classified as E. coli and belonged to 34 different serotypes, some of which had not been previously reported to carry the stx(2) genes (O8:H31, O89:H19, O166:H21 and O181:H20). Five stx(2) subtypes (stx(2), stx(2c), stx(2d), stx(2e) and stx(2g)) were detected. The stx(2), stx(2c), stx(2d) and stx(2e) subtypes were present in urban sewage and stx(2e) was the only stx(2) subtype found in pig wastewater samples. The stx(2c) and stx(2g) were more associated with cattle wastewater. One strain was positive for the intimin gene (eae) and five strains of serotypes were positive for the adhesin encoded by the saa gene. A total of 41 different seropathotypes were found. On the basis of occurrence of virulence genes, most non-O157 STEC strains are assumed to be low-virulence serotypes.

Phenotypic and genotypic characteristics of non-O157 Shiga toxin-producing Escherichia coli (STEC) from Swiss cattle.

A total of 42 Shiga toxin-producing (STEC) strains from slaughtered healthy cattle in Switzerland were characterized by phenotypic and genotypic traits. The 42 sorbitol-positive, non-O157 STEC strains belonged to 26 O:H serotypes (including eight new serotypes) with four serotypes (O103:H2, O113:H4, O116:H-, ONT:H-) accounting for 38.1% of strains. Out of 16 serotypes previously found in human STEC (71% of strains), nine serotypes (38% of strains) were serotypes that have been associated with hemolytic-uremic syndrome (HUS). Polymerase chain reaction (PCR) analysis showed that 18 (43%) strains carried the stx1 gene, 20 strains (48%) had the stx2 gene, and four (9%) strains had both stx1 and stx2 genes. Of strains encoding for stx2 variants, 63% were positive for stx2 subtype. Enterohemolysin (ehxA), intimin (eae), STEC autoagglutinating adhesin (saa) were detected in 17%, 21%, and 19% of the strains, respectively. Amongst the seven intimin-positive strains, one possessed intimin type beta1 (O5:H-), one intimin gamma1 (O145:H), one intimin gamma2/theta, (O111:H21), and four intimin epsilon (O103:H2). The strains belonged to 29 serovirotypes (association between serotypes and virulence factors). O103:H2 stx1eae-epsilon ehxA, O116:H- stx2, and ONT:H- stx2c were the most common accounting for 29% of the strains. Only one strain (2.4%) of serovirotype O145:H- stx1stx2eae-gamma1ehxA showed a pattern of highly virulent human strains. This is the first study providing characterization data of bovine non-O157 STEC in Switzerland, and underlining the importance of the determination of virulence factors (including intimin types) in addition to serotypes to assess the potential pathogenicity of these strains for humans.

Serotypes and virulence genes of ovine non-O157 Shiga toxin-producing Escherichia coli in Switzerland.

Sixty ovine STEC strains were examined with the aim (i) to serotype the strains, (ii) to characterize virulence factors, and (iii) to discuss possible associations between these factors and to assess the potential pathogenicity of these strains for humans. The 60 sorbitol-positive, non-O157 STEC strains belonged to 19 O:H serotypes, whereas 68% were of five serotypes (O87:H16, O91:H-, O103:H2, O128:H2, O176:H4). 52% belonged to serotypes reported in association with HUS. Five serotypes were not previously reported in sheep strains. Of the 47 strains encoding for stx1 variants, 57% were stx1c- and of the 45 encoding for stx2 variants, 80% were stx2d-positive. Eighty-two percent of the strains showed further putative virulence factors: 13% were eae-, 60% ehxA- and 67% saa-positive. The associations between harboring (i) eae and stx1, stx2, ehxA or no saa and (ii) saa and stx1c or stx2d were significant (P<0.05). The strains belonged to 27 seropathotypes (association between serotypes and virulence factors), but 57% belonged to only six and O91:H-stx1 stx2d saa and O128:H2 stx1c stx2d ehxA saa were the most common. Seven of the eight intimin-positive strains harbored eae. Four strains of serotype O103:H2 and O121:H10 harboring stx2, eae and ehxA showed virulence factors typical for strains associated with severe human disease. However, according to the virulence factors, the majority of the ovine non-O157 STEC strains are assumed low-virulence variants. Nevertheless, as long as the contribution and interaction of these factors in milder disease remains unclear P, a certain risk for humans cannot be excluded.

Fecal shedding of Escherichia coli O157, Salmonella, and Campylobacter in Swiss cattle at slaughter.

Fecal samples from 2,930 slaughtered healthy cattle were examined with the following goals: (i) to monitor the shedding of Escherichia coli O157, Salmonella, and Campylobacter in cattle; and (ii) to further characterize the isolated strains. The percentage of the 2,930 samples that tested positive for E. coli O157 by PCR was 1.6%. Thirty-eight strains from different animals that agglutinated with Wellcolex E. coli O157 were isolated. Of the six sorbitol-negative strains, five tested positive for stx2 genes (two times for stx2c and three times for stx2), and one strain tested positive for stx1 and stx2c genes. All sorbitol-negative strains belonged to the serotypes O157:H7- and O157:H7 and harbored the eae type gamma 1 and ehxA genes. The 32 sorbitol-positive strains tested negative for stx genes and belonged to the serotypes O157:H2, O157:H7, O157:H8, O157:H12, O157:H19, O157:H25, O157:H27, O157:H38, O157:H43, O157:H45, and O157:H-. All O157:H45 strains harbored the eae subtype alpha 1 and therefore seem to be atypical enteropathogenic E. coli strains. Whereas none of 1,000 examined samples was positive for Salmonella, 95 of 935 (10.2%) samples were positive for Campylobacter, and all strains were identified as C. jejuni. Sixteen Campylobacter strains were resistant to tetracycline, five were resistant to nalidixic acid/ciprofloxacin, four were resistant to streptomycin, and one was resistant to nalidixic acid/ciprofloxacin and streptomycin. Fecal shedding of zoonotic pathogens in slaughter animals is strongly correlated with the hazard of carcass contamination. Therefore, the maintenance of slaughter hygiene is of crucial importance.

Serotypes, virulence genes, and intimin types of Shiga toxin (verotoxin)-producing Escherichia coli isolates from cattle in Spain and identification of a new intimin variant gene (eae-xi).

A total of 514 Shiga toxin-producing Escherichia coli (STEC) isolates from diarrheic and healthy cattle in Spain were characterized in this study. PCR showed that 101 (20%) isolates carried stx(1) genes, 278 (54%) possessed stx(2) genes, and 135 (26%) possessed both stx(1) and stx(2). Enterohemolysin (ehxA) and intimin (eae) virulence genes were detected in 326 (63%) and in 151 (29%) of the isolates, respectively. STEC isolates belonged to 66 O serogroups and 113 O:H serotypes (including 23 new serotypes). However, 67% were of one of these 15 serogroups (O2, O4, O8, O20, O22, O26, O77, O91, O105, O113, O116, O157, O171, O174, and OX177) and 52% of the isolates belonged to only 10 serotypes (O4:H4, O20:H19, O22:H8, O26:H11, O77:H41, O105:H18, O113:H21, O157:H7, O171:H2, and ONT:H19). Although the 514 STEC isolates belonged to 164 different seropathotypes (associations between serotypes and virulence genes), only 12 accounted for 43% of isolates. Seropathotype O157:H7 stx(2) eae-gamma1 ehxA (46 isolates) was the most common, followed by O157:H7 stx(1) stx(2) eae-gamma1 ehxA (34 isolates), O113:H21 stx(2) (25 isolates), O22:H8 stx(1) stx(2) ehxA (15 isolates), O26:H11 stx(1) eae-beta1 ehxA (14 isolates), and O77:H41 stx(2) ehxA (14 isolates). Forty-one (22 of serotype O26:H11) isolates had intimin beta1, 82 O157:H7 isolates possessed intimin gamma1, three O111:H- isolates had intimin type gamma2, one O49:H- strain showed intimin type delta, 13 (six of serotype O103:H2) isolates had intimin type epsilon and eight (four of serotype O156:H-) isolates had intimin zeta. We have identified a new variant of the eae intimin gene designated xi (xi) in two isolates of serotype O80:H-. The majority (85%) of bovine STEC isolates belonged to serotypes previously found for human STEC organisms and 54% to serotypes associated with STEC organisms isolated from patients with hemolytic uremic syndrome. Thus, this study confirms that cattle are a major reservoir of STEC strains pathogenic for humans.

Serotypes, virulence genes, and intimin types of Shiga toxin (verotoxin)-producing Escherichia coli isolates from human patients: prevalence in Lugo, Spain, from 1992 through 1999.

We have analyzed the prevalence of Shiga toxin-producing Escherichia coli (STEC) in stool specimens of patients with diarrhea or other gastrointestinal alterations from the Xeral-Calde Hospital of Lugo City (Spain). STEC strains were detected in 126 (2.5%) of 5,054 cases investigated, with a progressive increase in the incidence from 0% in 1992 to 4.4% in 1999. STEC O157:H7 was isolated in 24 cases (0.5%), whereas non-O157 STEC strains were isolated from 87 patients (1.7%). STEC strains were (after Salmonella and Campylobacter strains) the third most frequently recovered enteropathogenic bacteria. A total of 126 human STEC isolates were characterized in this study. PCR showed that 43 (34%) isolates carried stx(1) genes, 45 (36%) possessed stx(2) genes and 38 (30%) carried both stx(1) and stx(2). A total of 88 (70%) isolates carried an ehxA enterohemolysin gene, and 70 (56%) isolates possessed an eae intimin gene (27 isolates with type gamma1, 20 with type beta1, 8 with type zeta, 5 with type gamma2, and 3 with type epsilon). STEC isolates belonged to 41 O serogroups and 66 O:H serotypes, including 21 serotypes associated with hemolytic uremic syndrome and 30 new serotypes not previously reported among human STEC strains in other studies. Although the 126 STEC isolates belonged to 81 different seropathotypes (associations between serotypes and virulence genes), only four accounted for 31% of isolates. Seropathotype O157:H7 stx(1) stx(2) eae-gamma1 ehxA was the most common (13 isolates) followed by O157:H7 stx(2) eae-gamma1 ehxA (11 isolates), O26:H11 stx(1) eae-beta1 ehxA (11 isolates), and O111:H- stx(1) stx(2) eae-gamma2 ehxA (4 isolates). Our results suggest that STEC strains are a significant cause of human infections in Spain and confirm that in continental Europe, infections caused by STEC non-O157 strains are more common than those caused by O157:H7 isolates. The high prevalence of STEC strains (both O157:H7 and non-O157 strains) in human patients, and their association with serious complications, strongly supports the utilization of protocols for detection of all serotypes of STEC in Spanish clinical microbiology laboratories.

Prevalence and characterization of Vero cytotoxin-producing Escherichia coli isolated from diarrhoeic and healthy sheep and goats.

Faecal samples from 146 diarrhoeic lambs and goat kids, and from 511 healthy sheep and goats were screened for the presence of Vero cytotoxin-producing Escherichia coli (VTEC). In healthy sheep and goats, VTEC were isolated in 24.4 and 16.2% of the animals, respectively. Moreover, VTEC were detected in 3.1 and 5.9% of the diarrhoeic lambs and goat kids, respectively. These data suggest that VTEC seems not to be associated with diarrhoea in lambs and goat kids. Only four VTEC strains were eae-positive. The absence of the eae gene in most of these VTEC strains could indicate that these strains are less virulent for humans that the classical eae-positive enterohaemorrhagic E. coli types. However, almost half (42.9%) and 12.2% of VTEC strains isolated from healthy sheep and goats, respectively, belonged to serotypes associated with severe diseases in humans.

Serotypes, virulence genes, and intimin types of Shiga toxin (verotoxin)-producing Escherichia coli isolates from healthy sheep in Spain.

Fecal swabs obtained from 1,300 healthy lambs in 93 flocks in Spain in 1997 were examined for Shiga toxin-producing Escherichia coli (STEC). STEC O157:H7 strains were isolated from 5 (0.4%) animals in 4 flocks, and non-O157 STEC strains were isolated from 462 (36%) lambs in 63 flocks. A total of 384 ovine STEC strains were characterized in this study. PCR showed that 213 (55%) strains carried the stx(1) gene, 10 (3%) possessed the stx(2) gene, and 161 (42%) carried both the stx(1) and the stx(2) genes. Enterohemolysin (ehxA) and intimin (eae) virulence genes were detected in 106 (28%) and 23 (6%) of the STEC strains, respectively. The STEC strains belonged to 35 O serogroups and 64 O:H serotypes (including 18 new serotypes). However, 72% were of 1 of the following 12 serotypes: O5:H-, O6:H10, O91:H-, O117:H-, O128:H-, O128:H2, O136:H20, O146:H8, O146:H21, O156:H-, O166:H28, and ONT:H21 (where NT is nontypeable). Although the 384 STEC strains belonged to 95 different seropathotypes (associations between serotypes and virulence genes), 49% of strains belonged to only 11. O91:H- stx(1) stx(2) (54 strains) was the most common seropathotype, followed by O128:H- stx(1) stx(2) (33 strains) and O6:H10 stx(1) (25 strains). Three strains of serotypes O26:H11, O156:H11, and OX177:H11 had intimin type beta1; 5 strains of serotype O157:H7 possessed intimin type gamma1; and 15 strains of serotypes O49:H-, O52:H12, O156:H- (12 strains), and O156:H25 had the new intimin, intimin type zeta. The majority (82%) of ovine STEC strains belonged to serotypes previously found to be associated with human STEC strains, and 51% belonged to serotypes associated with STEC strains isolated from patients with hemolytic-uremic syndrome. Thus, this study confirms that healthy sheep are a major reservoir of STEC strains pathogenic for humans.

Microbiological quality of raw goat's and ewe's bulk-tank milk in Switzerland.

A total of 407 samples of bulk-tank milk (344 of goat's milk and 63 of ewe's milk) collected from 403 different farms throughout Switzerland, was examined. The number of farms investigated in this study represents 8% of the country's dairy-goat and 15% of its dairy-sheep farms. Standard plate counts and Enterobacteriaceae counts were performed on each sample. Furthermore, the prevalence of Staphylococcus aureus, Campylobacter spp., Shiga toxin-producing Escherichia coli, Salmonella spp., and Mycobacterium avium ssp. paratuberculosis was studied. The median standard plate count for bulk-tank milk from small ruminants was 4.70 log cfu/ml (4.69 log cfu/ml for goat's milk and 4.78 log cfu/ml for ewe's milk), with a minimum of 2.00 log cfu/ml and a maximum of 8.64 log cfu/ml. Enterobacteriaceae were detected in 212 (61.6%) goat's milk and 45 (71.4%) ewe's milk samples, whereas S. aureus was detected in 109 (31.7%) samples of goat's milk and 21 (33.3%) samples of ewe's milk. Campylobacter spp. and Salmonella spp. were not isolated from any of the samples. However, 16.3% of the goat's milk and 12.7% of the ewe's milk samples were polymerase chain reaction (PCR)-positive for Shiga toxin-producing E. coli. Seventy-nine (23.0%) goat's tank-milk and 15 (23.8%) ewe's tank-milk samples were PCR-positive for insertion sequence 900, providing presumptive evidence for the presence of M. avium ssp. paratuberculosis. These results form the basis for determining the microbiological quality standards for goat's and ewe's milk. Moreover, the data presented form part of the risk assessment program for raw milk from small ruminants in Switzerland.

Serobiotypes and virulence genes of Escherichia coli strains isolated from diarrheic and healthy rabbits in Brazil.

A total of 178 Escherichia coli isolates from diarrheic and healthy rabbits in the São Paulo State (Brazil) were serobiotyped and investigated by PCR for the presence of virulence genes. Among the 90 (50.6%) isolates which possessed the eae gene, 74 were from diarrheic animals and all but one encoded intimin beta. Sixty five (72.2%) of the eae+ isolates had insertion of the locus of enterocyte effacement locus in the pheU locus, 11 (12.2%) in the selC and 14 (15.6%) did not insert in either of these loci. All isolates were negative for genes of the E. coli enterotoxins, Stx1, Stx2, CNF1, CNF2 and EHEC hemolysin. The O132:H2 serotype was dominant, being present in 63 isolates (70%) of the 90 eae+ isolates, and 57 of the 63 isolates of this serotype belonged to biotype 30. PCR detected the gene for AF/R2 fimbriae in 75 (83.3%) of the 90 eae+ isolates. Adherence to HeLa cells was best detected following 6h incubation and a positive fluorescence actin staining (FAS) test was given by 52 isolates. These data show that isolates of E. coli associated with diarrhea in rabbits in Brazil possess the genotype and phenotype typically associated with rabbit enteropathogenic E. coli (EPEC). We conclude that EPEC that possess the eae gene are a common cause of diarrhea in Brazilian rabbit farms and that the pathogenic eae+ AF/R2+ isolates of O132:H2:B30 serobiotype are especially predominant.