AIMS:To investigate phenotypic and genotypic aspects of sorbitol-negative or slow-fermenting Escherichia coli, suspected to belong to O157 serogroup, isolated in Italy. METHODS AND RESULTS:Milk samples originating from goats and cows were screened for the presence of E. coli O157 with cultural methods. Sorbitol-negative or slow-fermenting strains were subjected to phenotypic characterization, antibiotic resistance profiles, PCR reactions for detection of toxins (stx(1) and stx(2)) and intimin (eae(GEN) and eae(O157)) genes and clustering by pulsed field gel electrophoresis (PFGE). Only one strain revealed to be O157. Susceptibility to 11 antibiotics highlighted the high resistance to tetracycline (50%), sulfonamide and streptomycin (33%). The stx(2) gene was detected in two strains; only the strain identified as O157 exhibited an amplicon for both eae genes. PFGE identified seven distinct XbaI macrorestriction patterns at a similarity level of 41%. CONCLUSIONS:The use of sorbitol fermentation as cultural method is not sufficient for STEC discrimination while PCR assay proved to be a valuable method. SIGNIFICANCE AND IMPACT OF THE STUDY:The study reports presence of Shiga toxin-producing E. coli in raw milk, signalling a potential risk for humans.

Due to the heat resistance ofBacillus cereus, its potential pathogenic character, the capability to grow in milk and reported diseases upon consumption of dairy products, the organism should be considered as hazardous in pasteurized milk. Human exposure experiments based on (1) enquiring about the storage conditions (time and temperature) of pasteurized milk in households in The Netherlands and (2) performing of storage tests at 6, 8, 10 and 12°C, respectively, demonstrated the following: pasteurized milk is consumed within 2–12 days after pasteurization and is stored at temperatures varying from <5–13°C. Visually spoiled milk is not consumed anymore. Based on the distribution of both the storage time and temperature of milk in private households, approximately 7 and 4% of the total portions of milk consumed in The Netherlands could contain >105and >106B. cereusml−1, respectively. Taking into account the high portion of milk consumed (approximately 109–1010portions/year) and the absence of epidemiological evidence that milk is causing foodborne diseases due toB. cereus, the dose–response relationship of this organism may need to be considered. Results of the storage tests, based on the worst case situation, provided useful information on the effect of the risk factors, storage temperature and time, to human exposure. However, such information can be obtained more easily by applying predictive models. Models also enable us to find out the initial number of spores which can be considered as a third risk factor.

Twelve strains of Bacillus cereus isolated from different food products and foodborne disease outbreaks, and able to grow at temperatures < 7 degrees C, were characterised. Generation times at 7 degrees C varied from 9.4 h up to 75 h. Lag phase of the vegetative cells at 7 degrees C was strongly influenced by the previous temperature history of the cells. Preincubation at 37 degrees C increased the duration of the lag phase drastically. The heat resistance at 90 degrees C (D-90 degrees C-values in min) for spores produced at 30 degrees C varied from 2.2 to 9.2 min for 11 strains. One strain, however, showed a D-90 degrees C-value of > 100 min. Germination of spores in milk was delayed compared to those grown in brain heart infusion broth (BHI). All strains showed production of the diarrheal type enterotoxin in BHI. Addition of 50 IU of nisin to skim milk resulted in a decrease of numbers for 9 of the 12 strains tested. At a nisin concentration of 250 IU, a decrease in bacterial numbers was observed for all strains tested.

Detection of the coccoid form of Campylobacter jejuni with the use of the polymerase chain reaction (PCR) was examined. Coccoid cells of this pathogen, formed at different temperatures, showed different detection characteristics in the PCR. For spirals and cocci formed at 4 degrees C and 12 degrees C, the detection limit was about 2x10(3) cells/PCR. However, for detection of coccoid cells formed at 25 degrees C and 37 degrees C, at least 2X10(4) cells per PCR were needed. PCR was also performed on homogenates in peptone saline solution and enrichment broths of chicken meat and chicken liver that were artificially contaminated with cocci formed at 4 degrees C. PCR-products of these samples could not be demonstrated clearly.

The effect of lithium chloride on the growth ofListeria was studied in enrichment broths to determine optimum levels for this inhibitor.Listeria monocytogenes was able to grow in concentrations up to 2%, but above this level inhibition was noted.Enterococcus faecalis, a major competitive organism, was inhibited by low levels of LiCl. Competitive flora able to grow in the presence of LiCl are normally inhibited by nalidixic acid except for staphylococci. Substrates such as skimmed milk powder do not affect the inhibition of competitors by LiCl significantly. Trypaflavine was shown not to inhibit significantly theListeria strain studied in the presence of LiCl. The combination of LiCl, trypaflavine and nalidixic acid should be studied further in enrichment procedures forL. monocytogenes.

The occurrence of Listeria spp. was investigated in 17 food factories (representing six different product groups), 35 Dutch households and in a sawmill. The aim of this study was to assess their distribution in these environments, possible measures for their control and the likelihood of human exposure to these organisms from sources other than foods or faeces. In food factories, listerias were found in drains, floors, standing water, residues and food-contact surfaces in descending order of frequency. In two dry culinary food units, no samples were found to be contaminated. These results indicate that dry conditions and the restriction of food residues contribute to the control of these organisms. From kitchens in 35 households chosen at random, seven (20%) were found to be contaminated with listerias. They were isolated from refrigerators (one swab), dish cloths (six from seven samples) and from two dustbins. It seems that dish cloths could be an important source of these organisms in the home. In the environment of the sawmill, L. ivanovii was isolated more frequently than any other species. This was interpreted in terms of the availability of xylose, a wood breakdown product, in such environments. Although listeriosis outbreaks have been associated with foods, most cases are of unknown origin. This study indicates that listerias are common in many environments and that epidemiological studies should concentrate on these as well as foods. In view of the large number of sources of listerias in the natural environment, the domestic environment and in raw foods prepared in the home, human beings are exposed to these organisms on a regular basis. Scrupulous personal and domestic hygiene to diminish cross-contamination could be an important factor in the prevention of human listeriosis.