Sorbitol-fermenting, β-glucuronidase-positive, Shiga toxin-negative Escherichia coli O157:H7 in free-ranging red deer in South-Central Spain.

We investigated the prevalence of Escherichia coli O157:H7 in free-ranging red deer in south-central Spain, to assess their potential as reservoir hosts of sorbitol-fermenting (SF) E. coli O157:H7 strains, which are emerging causes of hemolytic uremic syndrome in Europe. Fecal samples from 264 hunter-harvested Iberian red deer (Cervus elaphus) were collected in 25 different game estates and examined for E. coli O157:H7 by culture and PCR. E. coli O157:H7 was detected and isolated in 4 of the 25 game estates sampled (16%) and the isolates obtained (four in total) were further phenogenotypically characterized. One of them was biochemically typical of E. coli O157:H7, that is, neither fermented sorbitol nor exhibited β-glucuronidase (GUD) activity, and carried genes encoding Shiga toxins (Stx) 1 and 2, the intimin subtype γ1, the enterohemorrhagic E. coli (EHEC)-hemolysin, and the ter gene cluster. The rest of the isolates (three of four) fermented sorbitol, exhibited GUD activity after 18-24 h incubation, and carried genes encoding the intimin subtype γ1 and the EHEC-hemolysin, although no Stx-encoding genes were detected. All these atypical isolates carried the sfp gene cluster, lacked the ter gene cluster, and were unable to grow on cefixime tellurite sorbitol MacConkey agar, which are typical features of SF E. coli O157:H7 strains isolated from patients. In total, SF, GUD-positive, Stx-negative E. coli O157:H7 strains were isolated in 3 of the 25 game estates sampled (12%), with an overall sample-level prevalence of 1.1% (3/264). Our findings indicate that free-ranging red deer may be one of the possible reservoir hosts of Stx-negative derivatives of SF E. coli O157:H7.

Díaz S ，Vidal D ，Herrera-León S ，Sánchez S ... -  《-》

Presence of Shiga toxin-producing E. coli O157:H7 in a survey of wild artiodactyls.

García-Sánchez A ，Sánchez S ，Rubio R ，Pereira G ，Alonso JM ，Hermoso de Mendoza J ，Rey J ... -  《VETERINARY MICROBIOLOGY》

Escherichia coli O157:H7 and non-O157 Shiga toxin-producing E. coli in healthy cattle, sheep and swine herds in Northern Spain.

Three-hundred and forty-five herds (17 swine, 122 dairy sheep, 124 beef and 82 dairy cattle) were investigated for prevalence of Shiga toxin-producing Escherichia coli (STEC). Rectal faecal samples were selectively enriched and then examined by immunodetection techniques (Immunomagnetic Separation with anti-E. coli O157 Dynabeads, ImmunoMagnetic cell Separation (IMS) and automated enzyme-linked fluorescent immunoassay using VIDAS) and polymerase chain reaction (PCR) (rfbE and fliC genes) to assess the prevalence of E. coli O157:H7. Prevalence of non-O157 STEC was estimated by PCR screening for stx genes of 10 lactose-positive colonies grown on MacConkey agar after enrichment. PCR was used on all STEC isolates to detect stx(1), stx(2), eaeA and E-hlyA genes. Both immunodetection methods showed a moderate-good level of agreement (kappa = 0.649) but IMS showed 87.5% complementary sensitivity. Prevalence of positive herds for E. coli O157:H7 was estimated at 8.7% for sheep and 3.8% for cattle, whereas all the porcine herds tested negative. Non-O157 STEC were also absent from swine, but were isolated more frequently from ovine (50.8%) than bovine herds (35.9%). Within-herd prevalences of excretion of E. coli O157:H7 established by individual testing of 279 sheep (six herds) and 30 beef cattle (one herd) were 7.3% and 6.7% respectively. PCR analysis of 49 E. coli O157:H7 and 209 non-O157 isolates showed a different distribution of virulence genes. All E. coli O157:H7 were stx(2) gene-positive, eaeA was detected in 95.9%, and the toxigenic profile stx(2)/eaeA/E-hlyA was present in 75.5% of the isolates. Among the non-O157 STEC, prevalence of eaeA was significantly lower (5.3%) and E-hlyA was present in 50.2% of the isolates but only sporadically associated with eaeA. stx(2) was predominant in non-O157 isolates from cattle, whereas in sheep the combination stx(1)/stx(2) was more prevalent. This study demonstrated the wide distribution of STEC in ruminant herds, which represent an important reservoir for strains that pose a potential risk for human infections.

Oporto B ，Esteban JI ，Aduriz G ，Juste RA ，Hurtado A ... -  《Zoonoses and Public Health》

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.

Sánchez S ，Martínez R ，Rey J ，García A ，Blanco J ，Blanco M ，Blanco JE ，Mora A ，Herrera-León S ，Echeita A ，Alonso JM ... -  《-》

Phylogeny, clinical associations, and diagnostic utility of the pilin subunit gene (sfpA) of sorbitol-fermenting, enterohemorrhagic Escherichia coli O157:H-.

The plasmid-borne sfpA gene encodes the pilin subunit in sorbitol-fermenting (SF) enterohemorrhagic Escherichia coli (EHEC) O157:H-. We investigated the distribution of sfpA among 600 E. coli isolates comprising the complete E. coli standard reference (ECOR) and diarrheagenic E. coli (DEC) strain collections and clinical isolates associated with enteric disease. sfpA was detected in DEC3F SF EHEC O157:H- strain 493/89, each of 107 SF EHEC O157:H- clinical isolates, and 14 Shiga toxin-negative SF E. coli O157:H- strains which contained eae, which encodes gamma-intimin, and fliC, which encodes the H7 antigen. sfpA was absent from all other strains, including the ECOR strain collection, all non-SF EHEC O157:H7 strains, and all E. coli O55:H7 strains (E. coli O55:H7 is the postulated ancestor of Shiga toxin-producing E. coli [STEC] O157). These results suggest that there was a single acquisition of the sfpA gene in the nonmotile SF E. coli O157 branch, presumably after the eae-encoding pathogenicity island (the locus of enterocyte effacement) was acquired and motility was lost. We then applied the sfpA PCR in combination with rfbO157, stx, and eae PCRs to screen 636 stool samples from patients with diarrhea or hemolytic-uremic syndrome for SF STEC O157:H-. In 27 cases, the simultaneous presence of the sfpA, eae, and rfbO157 amplicons indicated the presence of SF E. coli O157:H- strains, and the result was subsequently confirmed by isolation. All but two of these strains possessed stx2. None of the other stool samples was positive by the sfpA PCR; 59 of these stool samples contained EHEC O157:H7. The sfpA gene can be recommended as a target for screening for SF E. coli O157:H-.

Friedrich AW ，Nierhoff KV ，Bielaszewska M ，Mellmann A ，Karch H ... -  《-》