J.A. Santos

Numbers and types of microorganisms in vacuum-packed cold-smoked freshwater fish at the retail level.

Fifty-four packages (each one belonging to a different lot) of vacuum-packed cold-smoked salmon (30) and trout (24) produced by six Spanish smokehouses were obtained at retail level after 3 weeks storage at 2+/-1 degrees C. Sensorial, chemical, physicochemical and microbiological characteristics were examined. Overall, pH, a(w), salt content in water phase, aerobic plate counts at 30 and 25 degrees C. levels of Enterobacteriaceae, lactic acid bacteria (LAB), fungi and presumptive aeromonads and staphylococci are in agreement with available data on lightly preserved fish products. Psychrotrophic clostridia ranged between 1.71 and 2.21 log CFU/g. Levels of ethanol were highly variable and not significantly related (p > 0.05) to sensory scores or to microbial numbers. Salmonella, Escherichia coli and Listeria monocytogenes were not detected in any sample. Listeriae other than L. monocytogenes were isolated from three packages. Levels of Staphylococcus aureus lower than 4 log CFU/g were also found in three packages. Among 377 bacteria randomly isolated from aerobic 25 degrees C plate counts, LAB predominated, with Carnobacterium (C. piscicola) and Lactobacillus (eight species) being the genera most frequently found. The second and third major groups were Enterobacteriaceae and Micrococcaceae, respectively. Proteus vulgaris, P. mirabilis and Serratia liquefaciens were dominant among Enterobacteriaceae and coagulase-negative staphylococci among Micrococcaceae. Minor microbial groups such as aerobic gram-negative bacilli (Acinetobacter; Moraxella and Pseudomonas), Brochothrix, Aeromonas, Bacillus and Vibrio constituted less than 17% of the total flora.

Virulence markers in Aeromonas hydrophila and Aeromonas veronii biovar sobria isolates from freshwater fish and from a diarrhoea case

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.

Occurrence of foodborne pathogenic bacteria in retail prepackaged portions of marine fish in Spain

Aims:  To survey the presence of indigenous and nonindigenous foodborne bacterial pathogens in displayed prepacked portions of fresh marine fish.

PCR detection of potentially pathogenic aeromonads in raw and cold‐smoked freshwater fish

Aims: Development of a PCR assay for detection of aeromonads carrying the hlyA and/or aerA genes in fish. 
Methods and Results: The protocol involves an overnight selective enrichment step in tryptic soy broth yeast extract containing 10 µg ml‐1 of ampicillin followed by extraction of DNA and PCR amplification of two haemolysin genes that contribute to the virulence of Aer. hydrophila. This procedure can detect initial populations of 1–10 cfu g−1 within 24 h in artificially contaminated samples. In naturally contaminated fish, both genes were detected in 13 out of 14 fresh fish lots (aeromonads levels between < 1 and 5·42 log cfu g−1) and in 4 out of 16 lots of vacuum‐packed cold‐smoked fish (aeromonads levels between < 1 and 3·37 log cfu g−1). Before enrichment, dominant species were Aer. hydrophila HG1 (aerA+hlyA+), Aer. bestiarum HG2 (aerA+hlyA+) and Aer. caviae HG4 (aerA–hlyA–). After enrichment, Aer. hydrophila HG1 (aerA+hlyA+) was dominant. 
Conclusions: Fresh fish and even smoked fish carry hlyA+ and/or aerA+ aeromonads that can be detected by PCR within 24 h. 
Significance and Impact of the Study: The PCR assay described offers considerable potential as a rapid method with specificity, sensitivity and simplicity.

Foodborne pathogenic bacteria in prepackaged fresh retail portions of farmed rainbow trout and salmon stored at 3 °C

Twelve lots of fresh unskinned fillets of rainbow trout (Oncorhynchus mykiss) and 10 lots of fresh sliced salmon (Salmo salar) prepacked in trays wrapped with an oxygen-permeable film were obtained immediately after packing from two supermarkets having in-plant facilities for packaging wet fish. During storage at 3 °C, Listeria innocua was detected in eight lots of trout fillets after 4 days storage. L. monocytogenes was recovered from a single lot also contaminated with L. innocua. Initial numbers of aeromonads were significantly (p<0.05) lower in trout fillets (3.35±0.62 log cfu g−1) than in salmon slices (4.20±0.89 log cfu g−1). In both fish products, these bacteria significantly (p<0.05) increased up until spoilage. Most Aeromonas spp. isolates from trout fillets were assigned to A. veronii biovar sobria HG8 (hybridisation group 8), A. caviae HG4, A. eucrenophila HG6, A. hydrophila HG1 and A. veronii biovar veronii HG10. Strains of HG12 (A. schubertii), HG4 and HG8 formed the majority of aeromonads recovered from salmon slices.

Identification and epidemiological relationships of Aeromonas isolates from patients with diarrhea, drinking water and foods.

A collection of Aeromonas isolates obtained over a three-year period in the same geographic area (León, NW of Spain) was characterized by (GTG)₅-PCR fingerprinting, amplified fragment length polymorphism (AFLP) analysis and gyrB gene sequence analysis. The isolates originated from human diarrheal stools (29 isolates), potable water (13 isolates), rabbit meat (13 isolates) and marine fish (5 isolates). The distribution of Aeromonas species varied with the strain source. Aeromonas caviae HG4 and Aeromonas media HG5 were predominant in clinical and water isolates, respectively, whereas motile Aeromonas salmonicida HG3 strains were most frequently found in fish and meat. Molecular typing revealed several genotypic relationships among specific isolate subsets: (i) two clones of A. media HG5 persisted in drinking water over the study period, (ii) different patients harbored identical or closely related clones during several months, and (iii) clonal relatedness was observed in two sets of water and human isolates. The first of these sets comprised nine water isolates and two human A. media HG5 isolates, whereas the other one included a water isolate and a human isolate of A. caviae HG4. The latter finding suggests that Aeromonas transmission in the studied region followed a waterborne route. Interestingly, the three human isolates closely related to water isolates were recovered in a period of four days in June 2006 from non-related patients without underlying medical conditions that tested negative for other enteric pathogens. The data imply the transmission through contaminated water of strains of the A. caviae group that can produce disease in humans.

Molecular and phenotypic characterization of nonmotile Gram- negative bacteria associated with spoilage of freshwater fish

Aims:  Characterization of nonmotile bacteria associated with freshwater fish spoilage and that phenotypically resembled Psychrobacter spp.

Characterization of coagulase-positive staphylococci isolated from tank and silo ewe milk

The prevalence of coagulase-positive staphylococci (CPS) was studied among 390 samples of ewe milk. Fifty-seven (14.85%) samples of tank milk and all samples (6) of silo milk gave a positive result on Baird-Parker agar base supplemented with rabbit plasma fibrinogen, whereas amplification of the coagulase (coa) gene was successful in 6 (1.56%) samples of tank milk and in all silo samples. Phenotypic and genetic analysis of 153 isolates identified 151 (98.69%) as Staphylococcus aureus. Amplification of the coa gene was positive for 149 isolates (97.39%). The staphylococcal enterotoxin (SE) C gene was detected in 116 strains (75.82%), whereas more than one SE gene was carried in 5 strains (3.26%). None of the isolates harbored the genes for SEE or for methicillin resistance. A high prevalence of CPS carrying enterotoxin genes should be of concern because ewe milk is mainly processed into raw milk cheeses. The detection of the coa gene from milk samples could help to assess the microbiological safety of raw milk intended for direct use in the dairy industry.

Some technological properties of Penicillium roqueforti strains isolated from a home-made blue cheese

Nine strains of Penicillium roqueforti isolated from a traditional Spanish blue cheese (Valdeón cheese) along with two commercial strains were investigated for their ability to grow at different concentrations of salt and at different temperatures as well as for their proteolytic and lipolytic activities. Low concentrations of salt (1‐3%) were stimulating for all the strains, with 1% salt being the concentration with the highest stimulating effect in nearly all. The rate of growth at 10°C was 2‐3 times lower than at 25°C, the optimum temperature for the species. None of the strains, including the commercial cultures, showed proteolytic activity on casein agar, while all of them were lipolytic on tributyrin agar.

Relationship among specific bacterial counts and total bacterial and somatic cell counts and factors influencing their variation in ovine bulk tank milk

To analyze the relationship among the counts of different organisms and total bacterial count (BTTBC) and somatic cell count (BTSCC) as determined in dairy laboratories in ovine bulk tank milk, 751 bulk tank milk samples from 205 dairy sheep flocks belonging to Consortium for Ovine Promotion (CPO) were collected between January and December 2011. Four samplings were carried out in each flock, once per season, throughout 1 yr. Variables analyzed were bulk tank counts of thermoduric, psychrotrophic, coliform, and gram-positive catalase-negative cocci (GPCNC) bacterial groups. Thermoduric, psychrotrophic, and coliform species were significantly related to BTTBC, whereas GPCNC were correlated with both BTTBC and BTSCC variables. Highest counts were for psychrotroph and coliform groups, and a moderate to high correlation (r=0.51) was found between both variables, indicating that poor cleaning practices in the flocks tend to select for less-resistant organisms, such as gram-negative rods. In addition, BTTBC correlated with BTSCC (r=0.42). Some variation factors for specific bacterial counts, such as breed, season, milking type, dry therapy, and milk yield, were also analyzed. Flock information was collected from flock books, annual audits, and the CPO traceability system. Psychrotrophs and coliforms had elevated counts in winter, whereas GPCNC were higher in summer and in hand-milked flocks. Dry therapy contributed to the reduction in psychrotrophic bacteria; therefore, some strains of mammary pathogens could also be psychrotrophic bacteria. Results of this study would be helpful for troubleshooting milk quality problems and developing premium payment systems in dairy sheep.