Characterization of Vibrio Populations from Cultured European Seabass and the Surrounding Marine Environment with Emphasis on V. anguillarum.

Vibrio species are widely distributed and can be potentially pathogenic to aquatic organisms. In this study, we isolated Vibrio spp. from environmental samples (seawater, sediment, and fish swabs) collected over a three-year period from a fish farm in Mali Ston Bay in the Adriatic Sea, Croatia, and assess their distribution. A total of 48 seawater samples and 12 sediment samples, as well as gill and skin swabs from 110 farmed European seabass, were analysed for the presence of Vibrio. Vibrio strains were identified to the species level by MALDI TOF MS. The analysis revealed that V. alginolyticus was the predominant species in European seabass, followed by V. anguillarum. V. alginolyticus was isolated from the sediments, along with V. gigantis and V. pomeroyi, while V. chagasii, V. cyclitrophicus, V. fortis, V. gigantis, V. harveyi, V. pelagius, and V. pomeroyi were isolated from seawater. V. anguillarum was isolated only twice during two different spring seasons, once from a diseased sea bass and the second time from a healthy sea bass. We analysed these two isolates and found that they differ both genetically and in terms of resistance to antibiotics. Our results confirm the seasonality of vibriosis incidence and the presence of the pathogenic V. anguillarum, which increases the risk of vibriosis.

Influence of storage temperature on gene expression and virulence potential of Listeria monocytogenes strains grown in a salmon matrix.

Little is understood about the impact of environmental conditions on the virulence plasticity of Listeria monocytogenes strains grown in food. In this report, we monitored changes in the virulence properties of one high virulent (CCUG 3998) and one low virulent (442) L. monocytogenes strains grown on raw salmon (Salmo salar L.). The effect of temperature exposures (0 degrees C, 4 degrees C and 20 degrees C) on the expression levels of virulence genes (hlyA, actA, inlA and prfA), invasion into Caco-2 cells and in vivo mouse infection was analysed. Our results showed that L. monocytogenes virulence genes are differentially expressed when salmon is stored at different temperatures. Of the four virulence genes, the transcript levels for inlA were strongly affected, which correlated with the strain's virulence capacity as assessed by Caco-2 cells. In contrast to CCUG 3998, the virulence of strain 442 was altered with tested conditions. This strain maintains its low virulence status as far as salmon is stored at lower temperatures, but increases its virulence at higher temperatures. These results lead to the indication that exposure to abuse temperature conditions might influence the virulence potential of low pathogenic L. monocytogenes strains in salmon.

Multicenter validation of a multiplex PCR assay for differentiating the major Listeria monocytogenes serovars 1/2a, 1/2b, 1/2c, and 4b: toward an international standard.

The performance of a multiplex PCR assay that separates the four major serovars of the pathogenic Listeria monocytogenes into four distinct PCR groups was evaluated through a multicenter typing study. Identical panels of 90 Listeria isolates were distributed to five participating laboratories that were blind to the nature of the isolates. Isolates were characterized using the previously standardized protocol. Overall concordance was 96.6 to 100%, sufficient for the assay to be used as an alternative to serotyping and confidently applied in laboratories involved in L. monocytogenes typing.

An outbreak of staphylococcal food poisoning caused by enterotoxin H in mashed potato made with raw milk.

Mashed potato made with raw bovine milk was suspected to have been the source of a food poisoning outbreak. Almost 8 x 10(8)Staphylococcus aureus CFU g(-1) were detected in the mashed potato. S. aureus was also found in bulk milk from the farm that had supplied milk for the mashed potato. Isolates from mashed potato and bulk milk were positive for the gene encoding staphylococcal enterotoxin H (seh), and the corresponding protein toxin, SEH, was detected by ELISA in the mashed potato. Production of SEH by S. aureus isolates from mashed potato (n = 4) and bulk milk (n = 4) was also demonstrated by ELISA. Sequencing of seh from one mashed potato isolate and one bulk milk isolate confirmed that the gene was a variant seh, and that the genes in both isolates were identical. Macrorestriction of chromosomal DNA with Sma1 followed by pulsed-field gel electrophoresis of seh-positive S. aureus from mashed potato and bulk milk revealed indistinguishable banding patterns between isolates from both sources. It seems likely that raw bovine milk used in the preparation of mashed potato contained S. aureus that subsequently produced sufficient SEH in the mashed potato to cause food poisoning.

Quantification of Staphylococcus aureus in unpasteurised bovine and caprine milk by real-time PCR.

A sensitive and reproducible real-time PCR assay targeting the nuc gene of Staphylococcus aureus was applied for quantification of this microorganism in artificially and naturally contaminated raw milk samples. The S. aureus cell equivalents (SCEs) estimated by the real-time PCR method were two log scales higher than colony forming units (CFUs) estimated from a plate count method in artificially contaminated milk. The repeatability of the real-time PCR assay including the DNA isolation procedure was assessed by analysing the data derived from naturally contaminated samples. The relative standard deviation of the log-transformed data of four real-time PCR measurements including duplicate DNA isolations ranged between 11.3 and 1.0%. When analysing 80 bovine and 107 caprine naturally contaminated raw milk samples, the real-time PCR method yielded 19.3% more positive samples than the plate count method. With the exception of one sample, SCEs were always higher than CFUs. The difference between SCEs and CFUs was highly variable, and it was not possible to correlate real-time PCR-derived SCEs and CFUs. However, as each SCE detected by real-time PCR indicates a S. aureus cell, which is or has been present in the sample, this method offers the advantage of a retrospective analysis even of processed samples to aid food poisoning-related risk assessment.

Modified multiplex PCR method for detection of pyrogenic exotoxin genes in staphylococcal isolates.

A modified multiplex PCR method for detection of nine Staphylococcus aureus enterotoxin genes (sea, seb, sec, sed, see, seg, seh, sei, and sej) and one form of immunoreactive toxic shock syndrome toxin based on a previously published method (S. R. Monday and G. A. Bohach, J. Clin. Microbiol. 37:3411-3414, 1999) has been developed. The modified PCR protocol seems robust and gives reliable results.

Bacteriological analysis of fresh produce in Norway.

A total of 890 samples of fresh produce obtained from Norwegian markets were examined in order to assess the bacteriological quality of the products and their potential public health risk. The samples comprised lettuce, pre-cut salads, growing herbs, parsley and dill, mushrooms and strawberries. The samples were analysed for the presence of thermotolerant coliform bacteria (TCB), Escherichia coli O157, Salmonella spp., Listeria monocytogenes, Staphylococcus spp., and Yersinia enterocolitica. Neither Salmonella spp. nor E. coli O157 were isolated. For all product groups included, TCB were isolated from a small proportion of samples. Three samples harboured L. monocytogenes; one of the isolates belonging to serogroup 1 (champignons) and two of the isolates belonging to serogroup 4 (Chinese leaves and strawberries). Staphylococci were isolated from a relatively large proportion of the samples of strawberries and mushrooms. However, only four isolates were identified as S. aureus (non-toxinogenic). By the use of PCR, the presence of Y. enterocolitica was indicated in a few of the samples of lettuce, whilst no positive samples were found using a culturing method. The study shows that the occurrence of pathogenic bacteria and TCB in the products analysed was quite low. Nevertheless, the results indicate that the type of products analysed may contain pathogenic bacteria and thereby represent a risk to the consumers in regard to food-borne diseases.

Microbiological analysis of seed sprouts in Norway.

As part of larger survey of microbial contamination of fruits and vegetables in Norway, four different sprouted seed products were analysed for bacterial and parasitic contaminants (n = 300 for bacterial analyses and n = from 17 to 171 for parasite analyses, depending on parasite). Escherichia coli O157, Salmonella, Listeria monocytogenes, Cyclospora oocysts, Ascaris eggs and other helminth parasites were not detected in any of the sprout samples. Thermotolerant coliform bacteria (TCB) were isolated from approximately 25% of the sprout samples, with the highest percentage of TCB positive samples in alfalfa sprouts. Most TCB were Enterobacter spp. and Klebsiella. E. coli was isolated from 8 of 62 TCB positive mung bean sprout samples. Cryptosporidium oocysts were detected in 8% of the sprout samples and Giardia cysts were detected in 2% of the samples. All the Cryptosporidium positive samples, and most of the Giardia positive samples, were mung bean sprouts. Parasite concentrations in positive samples were low (between 1 and 3 oocysts/cysts per 50 g sprouts). Sprout irrigation water was also analysed for microbial contaminants. E. coli O157 and L. monocytogenes were not detected. TCB were isolated from approximately 40% of the water samples. Salmonella reading was isolated from three samples of spent irrigation water on 3 consecutive days. Cryptosporidium and Giardia were also isolated from spent irrigation water. Additionally, eight samples of unsprouted mung bean seed were analysed for Cryptosporidium oocysts and Giardia cysts. One or both of these parasites were detected in six of the unsprouted seed samples at concentrations of between 1 and 5 oocysts/cysts per 100 g unsprouted seed. Whilst our results support spent irrigation water as the most suitable matrix for testing for bacteria, unsprouted seed is considered the more useful matrix for analysing for parasite contaminants.