Claudio Zweifel

Serotypes, intimin variants and other virulence factors of eae positive Escherichia coli strains isolated from healthy cattle in Switzerland. Identification of a new intimin variant gene (eae-η2)

BackgroundEnteropathogenic Escherichia coli (EPEC) and Shigatoxin-producing Escherichia coli (STEC) share the ability to introduce attaching-and-effacing (A/E) lesions on intestinal cells. The genetic determinants for the production of A/E lesions are located on the locus of enterocyte effacement (LEE), a pathogenicity island that also contains the genes encoding intimin (eae). This study reports information on the occurrence of eae positive E. coli carried by healthy cattle at the point of slaughter, and on serotypes, intimin variants, and further virulence factors of isolated EPEC and STEC strains.ResultsOf 51 eae positive bovine E. coli strains, 59% were classified as EPEC and 41% as STEC. EPEC strains belonged to 18 O:H serotypes, six strains to typical EPEC serogroups. EPEC strains harbored a variety of intimin variants with eae-β1 being most frequently found. Moreover, nine EPEC strains harbored ast A (EAST1), seven bfpA (bundlin), and only one strain was positive for the EAF plasmid. We have identified a new intimin gene (η2) in three bovine bfpA and astA-positive EPEC strains of serotype ONT:H45. STEC strains belonged to seven O:H serotypes with one serotype (O103:H2) accounting for 48% of the strains. The majority of bovine STEC strains (90%) belonged to five serotypes previously reported in association with hemolytic uremic syndrom (HUS), including one O157:H7 STEC strain. STEC strains harbored four intimin variants with eae-ε1 and eae-γ1 being most frequently found. Moreover, the majority of STEC strains carried only stx 1 genes (13 strains), and was positive for ehxA (18 strains) encoding for Enterohemolysin. Four STEC strains showed a virulence pattern characteristic of highly virulent human strains (stx 2 and eae positive).ConclusionOur data confirm that ruminants are an important source of serologically and genetically diverse intimin-harboring E. coli strains. Moreover, cattle have not only to be considered as important asymptomatic carriers of O157 STEC but can also be a reservoir of EPEC and eae positive non-O157 STEC, which are described in association with human diseases.

Prevalence and characteristics of methicillin-resistant coagulase-negative staphylococci from livestock, chicken carcasses, bulk tank milk, minced meat, and contact persons

BackgroundMethicillin-resistant coagulase-negative staphylococci (MR-CNS) are of increasing importance to animal and public health. In veterinary medicine and along the meat and milk production line, only limited data were so far available on MR-CNS characteristics. The aim of the present study was to evaluate the prevalence of MR-CNS, to identify the detected staphylococci to species level, and to assess the antibiotic resistance profiles of isolated MR-CNS strains.ResultsAfter two-step enrichment and growth on chromogenic agar, MR-CNS were detected in 48.2% of samples from livestock and chicken carcasses, 46.4% of samples from bulk tank milk and minced meat, and 49.3% of human samples. Using matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS), 414 selected MR-CNS strains belonged to seven different species (S. sciuri, 32.6%; S. fleurettii, 25.1%; S. haemolyticus, 17.4%; S. epidermidis, 14.5%, S. lentus, 9.2%; S. warneri, 0.7%; S. cohnii, 0.5%). S. sciuri and S. fleurettii thereby predominated in livestock, BTM and minced meat samples, whereas S. epidermidis and S. haemolyticus predominated in human samples. In addition to beta-lactam resistance, 33-49% of all 414 strains were resistant to certain non-beta-lactam antibiotics (ciproflaxacin, clindamycin, erythromycin, tetracycline).ConclusionsA high prevalence of MR-CNS was found in livestock production. This is of concern in view of potential spread of mecA to S. aureus (MRSA). Multiresistant CNS strains might become an emerging problem for veterinary medicine. For species identification of MR-CNS isolated from different origins, MALDI-TOF MS proved to be a fast and reliable tool and is suitable for screening of large sample amounts.

ESBL-producing uropathogenic Escherichia coli isolated from dogs and cats in Switzerland

Extended-spectrum β-lactamase (ESBL)-producing Escherichia (E.) coli have emerged in human and veterinary medicine. The aim of this study was to investigate the presence of ESBL-producers among uropathogenic E. coli isolated from dogs and cats and to characterize detected ESBL-producing isolates by antibiotic susceptibility testing, identification of ESBL genes, multi-locus sequence typing (MLST), detection of putative virulence genes, and analysis of E. coli phylogroups. Among the 107 E. coli isolates derived from urinary samples (59 from dogs, 40 from cats), eight isolates from four different animals (two dogs, two cats) were found to be ESBL-producers. These isolates were of ST533/CTX-M-15/TEM/phylogroup B1 (four strains from one dog), ST410/CTX-M-15/TEM/phylogroup A (three strains, one from a dog and two from a cat), and ST648/CTX-M-15/phylogroup D (one strain from a cat). In terms of putative virulence factors, all isolates harbored lpfA, sat, and tsh, whereas iss was only detected in strains of ST533. Thus, ESBL-producers were detected among uropathogenic E. coli from Swiss companion animals and the eight CTX-M-15-producing isolates belonged to three sequence types (ST410, ST533, ST648) and three E. coli phylogroups (A, B1, D). For the first time, E. coli of ST533 carrying bla(CTX-M-15) were thereby detected in a dog.

Microbiological contamination of pig carcasses at different stages of slaughter in two European Union-approved abattoirs.

At sequential steps of slaughter (scalding, dehairing, singeing, polishing, trimming, washing, and chilling), 200 pig carcasses from two abattoirs were examined for total viable bacteria count (TVC) and the presence of Enterobacteriaceae and coagulase-positive Staphylococcus (CPS) by the wet-dry double-swab technique at the neck, belly, back, and ham. Before scalding, mean TVCs ranged from 5.0 to 6.0 log CFU cm(-2), and Enterobacteriaceae and CPS were detected on all carcasses. At abattoir A, mean TVCs and the percentage of Enterobacteriaceae-positive carcasses were reduced (P < 0.05) after scalding (1.9 log CFU cm(-2) and 12%, respectively), singeing (1.9 log CFU cm(-2) and 66%, respectively), and blast chilling (2.3 log CFU cm(-2) and 17%, respectively) and increased (P < 0.05) after dehairing (3.4 log CFU cm(-2) and 100%, respectively) and polishing (2.9 log CFU cm(-2)). The proportion of CPS-positive samples decreased to < or = 10% after scalding and remained at this level. At abattoir B, mean TVCs and the percentages of Enterobacteriaceae- and CPS-positive carcasses were reduced (P < 0.05) after scalding (2.4 log CFU cm(-2) and 29 and 20%, respectively), polishing (3.7 log CFU cm(-2)), and chilling (2.6 log CFU cm(-2) and 55 and 77%, respectively) and increased (P < 0.05) after the combined dehairing-singeing (4.7 log CFU cm(-2) and 97 and 100%, respectively). Among sites, the neck tended to yield higher levels of contamination from trimming to chilling at both abattoirs (P < 0.05). Consequently, scalding, singeing, and chilling may be integrated in a hazard analysis critical control point (HACCP) system for pig slaughter. As indicated by the higher levels of contamination on carcasses after dehairing-singeing and the following stages at abattoir B, each abattoir should develop its own baseline data and should customize HACCP systems to match process- and site-specific circumstances.

Microbiological contamination of cattle and pig carcasses at five abattoirs determined by swab sampling in accordance with EU Decision 2001/471/EC

A total of 800 cattle carcasses (abattoir A: n=200; B: n=150; C: n=150; D: n=150, E: n=150) and 650 pig carcasses (abattoir A: n=200; B: n=150; C: n=150; D: n=150) were examined at five Swiss abattoirs with an annual slaughtering capacity >10 million kg. Weekly, 10 cattle and 10 pig carcasses were sampled at four sites by the wet-dry double swab technique. From each carcass the samples were pooled and examined for total viable counts (TVC) and Enterobacteriaceae. At the abattoirs, mean log TVCs from cattle carcasses ranged from 2.1 to 3.1 cm(-2) and those from pig carcasses from 2.2 to 3.7 cm(-2). Daily TVC mean log values showed significant differences between abattoirs (P<0.05), whereas no significant differences were detected between animal species. On average, Enterobacteriaceae were detected (i) in low counts, (ii) on 31.0% of cattle and on 20.2% of pig carcasses, and (iii) more frequently and in higher counts on cattle than on pig carcasses (P<0.05). Data from this study indicate that the wet-dry double swab technique is suitable to determine microbiological contamination of cattle and pig carcasses in accordance with EU Decision 2001/471/EC. For samples obtained by the non-destructive technique from cattle and pig carcasses, the following microbiological performance criteria are proposed: Daily mean log values are acceptable, marginal, and unacceptable for TVC when they are <3.00, 3.00-4.00, >4.00 cm(-2), and for Enterobacteriaceae when they are <1.00, 1.00-2.00, and >2.00 cm(-2). However, such values have to be seen merely as baselines. It is important to implement a monitoring system based on abattoir-specific data and criteria as permitted by quality control chart methods.

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.

Occurrence and genotypes of Campylobacter in broiler flocks, other farm animals, and the environment during several rearing periods on selected poultry farms.

On 15 Swiss poultry farms, broiler flocks, other farm animals, and the environment were examined during consecutive rearing periods to investigate the occurrence and genetic diversity of Campylobacter. Of the 5154 collected samples, 311 (6%) from 14 farms were Campylobacter positive by culture. Amongst the positive samples, 228 tested positive for Campylobacter jejuni and 92 for Campylobacter coli. Positive samples originated from broilers, the broiler houses, cattle, pigs, bantams, laying hens, a horse, and a mouse. Feed, litter, flies, and the supply air to the broiler house tested negative. By flagellin gene typing (fla-RFLP) and pulsed-field gel electrophoresis (PFGE), 917 Campylobacter isolates were genotyped. Additionally, amplified fragment length polymorphism (AFLP) analysis was performed on 15 assorted strains. On eight farms, matching genotypes were isolated from broiler flocks and other farm animals: Certain genotypes from cattle (farms H, K, L, and M), pigs (farms D and P), or laying hens (farm L) were subsequently found in the broiler flocks, whereas other genotypes initially present in the broiler flocks turned up in cattle (farms A, D, and O). These results emphasize the importance of other farm animals on poultry farms for broiler flock colonization. Indications of persistent contamination of the broiler house were evident on four farms (C, D, I, and L) where matching genotypes were detected in consecutive broiler flocks, but not concurrently in other samples. By fla-RFLP, PFGE, and confirmed by AFLP, some genotypes proofed to be identical across different farms.

Microbiological monitoring of sheep carcass contamination in three Swiss abattoirs

At three Swiss abattoirs, 580 sheep carcasses were examined at 10 sites by the wet-dry double-swab technique. The aim of this study was to obtain data on microbiological contamination at the abattoirs and to develop a procedure for monitoring slaughter hygiene. Median aerobic plate counts (APCs) (log CFU/cm2) ranged from 2.5 to 3.8, with the brisket and neck sites showing the most extensive contamination. Enterobacteriaceae were detected on 68.1% of the carcasses and in 15.2% of the samples. The proportion of positive results ranged from 2.6% (for the hind leg and the flank at abattoir C) to 42.2% (for the perineal area at abattoir A). The percentage of samples testing positive for stx genes by polymerase chain reaction was 36.6%. A significant relationship between APC and the detection of Shiga toxin-producing Escherichia coli (STEC) was found for abattoirs A and B (depending on sampling site), whereas a significant relationship between Enterobacteriaceae and STEC detection was confirmed only for abattoir A (P < 0.05). In 57.1% of the 56 isolated non-O157 strains, stx2 genes were detected, and most of them were stx2d positive. Additional virulence factors were detected in 50% of the STEC strains, with 8.9% of these strains being eae positive, 50% being EHEC-hlyA positive, and 3.6% being astA positive. For the determination of carcass contamination, the monthly examination of 10 sheep carcasses for APC and Enterobacteriaceae counts in the neck, brisket, and perineal areas is recommended. This procedure is a valuable tool for the verification of slaughter hygiene according to hazard analysis critical control point principles.

Characteristics of shiga toxin-producing Escherichia coli isolated from Swiss raw milk cheese within a 3-year monitoring program.

Food is an important vehicle for transmission of Shiga toxin-producing Escherichia coli (STEC). To assess the potential public health impact of STEC in Swiss raw milk cheese produced from cows, goats, and ewes milk, 1,422 samples from semihard or hard cheese and 80 samples from soft cheese were examined for STEC, and isolated strains were further characterized. By PCR, STEC was detected after enrichment in 5.7% of the 1,502 raw milk cheese samples collected at the producer level. STEC-positive samples comprised 76 semihard, 8 soft, and 1 hard cheese. By colony hybridization, 29 STEC strains were isolated from 24 semihard and 5 soft cheeses. Thirteen of the 24 strains typeable with O antisera belonged to the serogroups O2, O22, and O91. More than half (58.6%) of the 29 strains belonged to O:H serotypes previously isolated from humans, and STEC O22:H8, O91:H10, O91:H21, and O174:H21 have also been identified as agents of hemolytic uremic syndrome. Typing of Shiga toxin genes showed that stx(1) was only found in 2 strains, whereas 27 strains carried genes encoding for the Stx(2) group, mainly stx(2) and stx(2vh-a/b). Production of Stx(2) and Stx(2vh-a/b) subtypes might be an indicator for a severe outcome in patients. Nine strains harbored hlyA (enterohemorrhagic E. coli hemolysin), whereas none tested positive for eae (intimin). Consequently, semihard and hard raw milk cheese may be a potential source of STEC, and a notable proportion of the isolated non-O157 STEC strains belonged to serotypes or harbored Shiga toxin gene variants associated with human infections.

Characterization of attaching and effacing Escherichia coli (AEEC) isolated from pigs and sheep

BackgroundAttaching and effacing Escherichia coli (AEEC) are characterized by their ability to cause attaching-and-effacing (A/E) lesions in the gut mucosa of human and animal hosts leading to diarrhoea. The genetic determinants for the production of A/E lesions are located on the locus of enterocyte effacement (LEE), a pathogenicity island that also contains the genes encoding intimin (eae). This study reports data on the occurrence of eae positive E. coli carried by healthy pigs and sheep at the point of slaughter, and on serotypes, intimin variants, and further virulence factors of isolated AEEC strains.ResultsFaecal samples from 198 finished pigs and 279 sheep were examined at slaughter. The proportion of eae positive samples was 89% for pigs and 55% for sheep. By colony dot-blot hybridization, AEEC were isolated from 50 and 53 randomly selected porcine and ovine samples and further characterized. Strains of the serotypes O2:H40, O3:H8 and O26:H11 were found in both pigs and sheep. In pigs O2:H40, O2:H49, O108:H9, O145:H28 and in sheep O2:H40, O26:H11, O70:H40, O146:H21 were the most prevalent serotypes among typable strains. Eleven different intimin types were detected, whereas γ2/θ was the most frequent, followed by β1, ε and γ1. All but two ovine strains tested negative for the genes encoding Shiga toxins. All strains tested negative for the bfp A gene and the EAF plasmid. EAST1 (ast A) was present in 18 of the isolated strains.ConclusionOur data show that pigs and sheep are a source of serologically and genetically diverse intimin-harbouring E. coli strains. Most of the strains show characteristics of atypical enteropathogenic E. coli. Nevertheless, there are stx-negative AEEC strains belonging to serotypes and intimin types that are associated with classical enterohaemorrhagic E. coli strains (O26:H11, β1; O145:H28, γ1).