A contingency locus in prfA in a Listeria monocytogenes subgroup allows reactivation of the PrfA virulence regulator during infection in mice.

A nonhemolytic Listeria monocytogenes strain isolated from a fish processing plant was avirulent in a plaque-forming assay and in a subcutaneous mouse virulence assay. However, it showed 60% lethality (9/15 mice) when 109 CFU were intraperitoneally injected into mice. Hemolytic L. monocytogenes bacteria were recovered from liver and spleen of the deceased mice, and the pulsed-field gel electrophoresis patterns were indistinguishable for the nonhemolytic and the hemolytic isolates. Sequencing of prfA from the nonhemolytic strain revealed a duplication of 7 bp in the helix-turn-helix region, resulting in a truncated PrfA protein. We propose that the direct repeat of 7 bp causes a reversible inactivation of prfA and that slipped-strand mispairing regulates the phase variation in hemolytic activity and virulence. Nonhemolytic L. monocytogenes strains with identical duplications in prfA were isolated from several sources in France, as well as in Norway, suggesting that the reversible inactivation described in this study is not an isolated event.

The ability to enter into an avirulent viable but non-culturable (VBNC) form is widespread among Listeria monocytogenes isolates from salmon, patients and environment.

Media-based bacteriological testing will fail to detect non-culturable organisms and the risk of consuming viable but non-culturable (VBNC) Listeria monocytogenes is unknown. We have here studied whether L. monocytogenes obtained from seafoods, processing environment and clinical cases enter the VBNC state and assessed the virulence of the non-culturable forms of the bacteria. A number of 16 L. monocytogenes strains were starved in microcosm water at 4 degrees C until loss of culturability. Metabolic activity in the VBNC form was measured as ATP generation using a luciferase assay and membrane integrity was examined using the LIVE/DEAD BacLight assay. All tested L. monocytogenes strains entered the VBNC state after starvation in microcosm water. Ongoing mRNA synthesis of hly in VBNC L. monocytogenes cells re-incubated in culture medium indicated a potential virulence of these forms. Sodium pyruvate and replenishment of nutrient were used in attempts to resuscitate VBNC cells. However, VBNC L. monocytogenes were not resuscitated under these conditions. VBNC L. monocytogenes were tested for virulence in a cell plaque assay and by intraperitoneally inoculation in immunodeficient RAG1(-/-) mice. Inoculation of VBNC L. monocytogenes in immunodeficient mice did not cause morbidity, and plaque assay on HT-29 cells in culture indicated that the VBNC cells were avirulent. The results indicate that the risk of non-culturable L. monocytogenes in foods, when the VBNC state is induced by starvation, is negligible.

An outbreak of Escherichia coli O103:H25 - bacteriological investigations and genotyping of isolates from food.

During the spring of 2006, a national disease outbreak caused by Shiga toxin-producing Escherichia coli (STEC) O103:H25 was investigated in Norway. At the time of the outbreak the Norwegian School of Veterinary Science was the national reference laboratory for E. coli O157 in food, and the microbiological investigations to identify the food source were performed there. Food- and environmental samples (n=931) were collected by the Norwegian Food Safety Authorities following two different hypotheses i) that minced meat was the source of STEC, and ii) that fermented sausage was the source of STEC. Twenty seven food samples, all collected following the latter hypothesis contained eae-positive E. coli O103:H25, but none of these were stx-positive. By PFGE it was shown that isolates from one particular type of fermented sausage "morr sausage 1" were identical to the isolates from patients. Samples of sheep meat that were linked epidemiologically to meat used for sausage production also contained isolates identical or closely related to patient strains. The presented study underpins epidemiological indications that fermented sausage was the source of the outbreak, but points specifically to one particular brand of sausage as the source.

Occurrence of Vibrio parahaemolyticus, V. cholerae, and V. vulnificus in Norwegian Blue Mussels (Mytilus edulis).

Vibrio parahaemolyticus, V. cholerae, and V. vulnificus were isolated from 10.3%, 1.0%, and 0.1% of 885 blue mussel samples, respectively. Four of the samples contained trh(+) V. parahaemolyticus, while no tdh-positive isolates were detected. The V. cholerae isolates were non-O:1/non-O:139 serotypes and were ctxA negative.

Potential uptake of Escherichia coli O157:H7 from organic manure into crisphead lettuce.

To investigate the potential transfer of Escherichia coli O157:H7 from contaminated manure to fresh produce, lettuce seedlings were transplanted into soil fertilized with bovine manure which had been inoculated with approximately 10(4) CFU g(-1) E. coli O157:H7. The lettuce was grown for approximately 50 days in beds in climate-controlled rooms in a greenhouse. As the bacterium was not detected in the edible parts of the lettuce, the outer leaves of the lettuce, or the lettuce roots at harvest it was concluded that transmission of E. coli O157:H7 from contaminated soil to lettuce did not occur. The pathogen persisted in the soil for at least 8 weeks after fertilizing but was not detected after 12 weeks. Indigenous E. coli was detected only sporadically on the lettuce at harvest, and enterococci were not detected at all. The numbers of enterococci declined more rapidly than those of E. coli in the soil. Pseudomonas fluorescens, which inhibited growth of E. coli O157:H7 in vitro, was isolated from the rhizosphere.

Genetic variability among isolates of Listeria monocytogenes from food products, clinical samples and processing environments, estimated by RAPD typing.

RAPD analysis with four primers was used to examine the genetic relationship among 432 strains of Listeria monocytogenes isolated from clinical and veterinarian cases of listeriosis, dairy, vegetable, meat- and fish-based food items, environmental samples and samples collected from one transport terminal, one poultry-processing company and four Atlantic salmon-processing plants. The purpose of the study was to determine whether clinical isolates belonged to a specific genetic group, whether links could be made between food groups and clinical cases and whether specific genetic groups were associated with specific food products or processing units. There was great genetic variability among the isolates, which produced a total of 141 RAPD composites based on the RAPD analysis with four primers. The RAPD composites divided in two major clusters and clinical isolates were evenly distributed in both of them. None of the isolates from food products had the same RAPD composite as isolates from human patients, thus, no particular food commodity could be linked to clinical cases. Each food-processing environment was contaminated with more than one RAPD composite and the genetic variability found within each company was, in most cases, of approximately the same magnitude as the variability found when considering all the samples. In each plant, one or a few types persisted over time, indicating the presence of an established in-house flora. Our results indicate that most of the analysed cases of listeriosis were sporadic and, further, that these cases cannot be traced to a few specific food sources. We also found that no particular RAPD composite was better suited for survival in specific food types or food-processing environments, indicating that although differences may be found in virulence properties of individual strains, all L. monocytogenes must be treated as potentially harmful.