Aims: To evaluate the public health significance of representative strains of two Aeromonas spp., mainly from freshwater fish, on the basis of production of virulence-associated factors and presence of the haemolytic genes aerA and hlyA. Methods and Results: Eleven strains of Aer. hydrophila, three strains of Aer. veronii biovar sobria (all from freshwater fish) and one strain of Aer. hydrophila from human diarrhoea were tested for potential virulence traits and for the presence of the haemolytic genes aerA and hlyA. Ten Aer. hydrophila isolates were aerA + hlyA + and two aerA + hlyA -. Aeromonas veronii biovar sobria isolates were aerA -hlyA -. Strains from the three genotypes showed enterotoxic activity in the suckling mouse assay. At 28°C, four Aer. hydrophila fish strains could be considered as potentially virulent (possessing at least two of these characteristics: haemolytic, cytotoxic and enterotoxic). One Aer. veronii biovar sobria strain and the clinical isolate were cytotoxic on Vero cells. When grown at 4°C, these six isolates fulfilled virulence criterion, but at 37°C, only one fish strain, an Aer. hydrophila, did. Conclusions: The potential health risk derived from the presence of Aer. hydrophila and Aer. veronii biovar sobria in ice-stored freshwater fish should not be underestimated. Significance and Impact of the Study: Expression of virulence factors is affected by temperature incubation and not always related to the presence of haemolytic genes.

Among 800 stool specimens from patients with diarrhea submitted by Primary Care Centers for routine analysis to the Hospital of León (NW Spain) Microbiology and Parasitology Service, 32 (4%) were tested positive for Aeromonas spp. Mixed infections with other enteric pathogens occurred in 12 patients. A. caviae was isolated from 23 clinical specimens. There were also patients infected with A. media, A. hydrophila, A. bestiarum, and A. veronii biovar veronii. All but three isolates carried one or more of the virulence genes. The incidence of the alt, hlyA, aerA, ast, and laf genes was 71.9, 28.1, 25.0, 18.8, and 9.4%, respectively. The alt(+)/ast(+) combination was detected in four isolates and the aerA(+)/hlyA(+) combination was detected in the two A. hydrophila isolates. None of the strains harbored the TTSS, stx1, or stx2 genes and nine bore plasmids. Thirty clinical isolates and a collection of 12 A. caviae and A. media strains obtained from León municipal drinking water over the study period were typed by pulsed-field gel electrophoresis (PFGE). PFGE patterns revealed genetic relatedness and persistence over time among water isolates and some clinical isolates. Interestingly, one A. caviae (aerA(-)/hlyA(-)/alt(+)/ast(-)/laf(+)) human isolate and two A. caviae (aerA(-)/hlyA(-)/alt(+)/ast(-)/laf(+)) drinking water isolates had indistinguishable PFGE patterns, suggesting waterborne infection.

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Even though worldwide production of rabbit meat is >1,000,000 tons, little information is available for rabbit meat microbiology. This study provides data on the prevalence of Salmonella, Escherichia coli O157:H7, Yersinia enterocolitica, Listeria spp., motile Aeromonas spp., and Staphylococcus aureus on rabbit meat. A total of 24 rabbit carcasses from two abattoirs and 27 rabbit meat packages from supermarket displays were examined. In addition to culturing methods, associated virulence genes were investigated by PCR in suspect isolates and samples. Neither Salmonella nor E. coli O157:H7 was detected. All samples were negative for virulence-associated invA, stx1, and stx2 genes. At one abattoir, two carcasses (3.9%) carried Y. enterocolitica yst-, and two were positive for the yst gene, although viable Y. enterocolitica cells were not recovered from these samples. Seven samples (13.7%) were contaminated with Listeria. Of them, three were positive for hly and iap genes (Listeria monocytogenes hly+ / iap+), two carried Listeria seeligeri, one carried Listeria ivanovii, and one carried Listeria innocua. For detectable motile Aeromonas spp. (average count, 1.77 +/- 0.62 log CFU/g), the contamination rate was 35.3%, although ca. 90% of the samples were positive for the aerA and/or hlyA genes. The majority of aeromonad isolates were Aeromonas hydrophila aerA+ / hlyA+. Aeromonas caviae, Aeromonas popoffii, Aeromonas schubertii, and the two biovars of Aeromonas veronii were also isolated. The prevalence of S. aureus contamination (average count, 1.37 +/- 0.79 log CFU/g) was 52.9%. Among 27 S. aureus isolates, two harbored genes for staphylococcal enterotoxin B (seb), and two harbored genes for staphylococcal enterotoxin C (sec). The remaining isolates were negative for sea, seb, sec, sed, and see.

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The antimicrobial activity of two packaging films (polypropylene-PP-and polyethylene terephthalate-PET-) coated with different concentrations (0, 4, 6 and 8%) of essential oil from Origanum vulgare (OR) and Ethyl Lauroyl Arginate HCl (LAE) was tested against two E. coli O157:H7 strains using in vitro systems and a raw milk sheep cheese model (Zamorano). The influence of the antimicrobials on the sensory attributes of cheese was also evaluated. For both strains, the MBC (minimum bactericidal concentration) values were identical to their respective MIC (minimum inhibitory concentration) values and lower for LAE (25 mg/l) than for OR (200-400 mg/l). PP and PET films coated with OR were tested by a vapour phase assay and the Japanish Standard method (JIS Z 2801:2000). Both films coated with LAE were tested by an agar diffusion method. Overall, in vitro tests were effective against both strains. The inhibitory activity depended on the active compound concentration, the target strain and the packaging material, PET being more effective than PP. For inoculated cheese slices, OR and LAE PP films did not effectively decrease E. coli O157:H7 counts after 7-days cold storage. PET films incorporating 6 and 8 % of OR and LAE significantly (p< 0.05) decreased the numbers of both strains and also did 4% for the reference and wild strain depending on the antimicrobial. LAE PP, OR PET and LAE PET did not significantly (p> 0.05) affect sensorial characteristics of Zamorano cheese. Packaging with PET films coated with ≥ 6% LAE concentrations might be useful in reducing E. coli O157:H7 numbers in sheep cheese.

A collection of 81 isolates of enteropathogenic Escherichia coli (EPEC) was obtained from samples of bulk tank sheep milk (62 isolates), ovine feces (4 isolates), sheep farm environment (water, 4 isolates; air, 1 isolate), and human stool samples (9 isolates). The strains were considered atypical EPEC organisms, carrying the eae gene without harboring the pEAF plasmid. Multilocus sequence typing (MLST) was carried out with seven housekeeping genes and 19 sequence types (ST) were detected, with none of them having been previously reported for atypical EPEC. The most frequent ST included 41 strains isolated from milk and human stool samples. Genetic typing by pulsed-field gel electrophoresis (PFGE) resulted in 57 patterns which grouped in 24 clusters. Comparison of strains isolated from the different samples showed phylogenetic relationships between milk and human isolates and also between milk and water isolates. The results obtained show a possible risk for humans due to the presence of atypical EPEC in ewes' milk and suggest a transmission route for this emerging pathogen through contaminated water.

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