Invading slugs (Arion vulgaris) can be vectors for Listeria monocytogenes.

AIMS: Listeriosis is a frequent silage-associated disease in ruminants. The slugs Arion vulgaris are invaders in gardens, vegetable crops and meadows for silage production. Field and laboratory studies were conducted to clarify whether slugs could host Listeria monocytogenes and thereby constitute a threat to animal feed safety. METHODS AND RESULTS: Selective culture of L. monocytogenes from 79 pooled slug samples (710 slugs) resulted in 43% positive, 16% with mean L. monocytogenes values of 405 CFU g(-1) slug tissues. Of 62 individual slugs cultured, 11% also tested positive from surface/mucus. Multilocus sequence typing analysis of 36 isolates from different slug pools identified 20 sequence types belonging to L. monocytogenes lineages I and II. Slugs fed ≅4·0 × 10(5)  CFUL. monocytogenes, excreted viable L. monocytogenes in faeces for up to 22 days. Excretion of L. monocytogenes decreased with time, although there were indications of a short enrichment period during the first 24 h. CONCLUSIONS: Arion vulgaris may act as a vector for L. monocytogenes. SIGNIFICANCE AND IMPACT OF THE STUDY: Highly slug-contaminated grass silage may pose a potential threat to animal feed safety.

Genetic diversity among Norwegian Vibrio parahaemolyticus.

AIMS: To examine the variability among environmental Vibrio parahaemolyticus (including trh+ isolates) from Norway, and to compare these to clinical isolates and isolates from imported foods. METHODS AND RESULTS: A total of 246 V. parahaemolyticus were successfully digested with NotI, and the fragments were separated by pulsed field gel electrophoresis (PFGE). The isolates could be divided into 72 clusters and 103 pulsotypes. Eleven clusters contained 4-31 environmental isolates, and the isolates within these clusters greatly varied with respect to origin. None of the trh+ and /or tdh+ isolates clustered with trh-/tdh- isolates. The trh+ environmental isolates included in the study belonged to two separate clusters. A subset of isolates was serotyped, and great serotype diversity was observed among the environmental V. parahaemolyticus. The clinical isolates included O3:K6 and O3:KUT, and these were identical or related to a pandemic reference strain by PFGE. CONCLUSIONS: Environmental V. parahaemolyticus (including trh+) were genetically diverse, but certain variants occurred throughout the coastal environment, and some were persistent over time. SIGNIFICANCE AND IMPACT OF THE STUDY: Although trh+ V. parahaemolyticus persisted in the Norwegian environment, no evidence indicated that indigenous isolates have caused disease.

The occurrence of Staphylococcus aureus on a farm with small-scale production of raw milk cheese.

In recent years, the small-scale production of raw milk products has increased in Norway, and there is some concern that such foods may pose a risk of staphylococcal food poisoning to consumers. The aim of the study was to evaluate potential sources of contamination of raw milk cheese with Staphylococcus aureus on a bovine dairy farm with small-scale production. Samples for bacteriological analyses (n = 144) were collected from the animals, the environment, processing equipments, from humans, and from cheeses at various stages of production. Staphylococcus aureus was isolated from 10 of 11 cows, the farmer, equipment, the environment, and the cheese. Seventy-five Staph. aureus isolates were genotyped by pulsed-field gel electrophoresis, tested for enterotoxin (SE) production by reversed passive latex agglutination, for SE genes by multiplex polymerase chain reaction, and for penicillin resistance by the cloverleaf method. Five different pulsotypes were identified and SE gene fragments were identified in 11 isolates, but no isolates produced SE or were penicillin resistant. Staphylococcus aureus was found throughout the farm, and appeared to be spread with the milk to the environment, equipment, and to products. One pulsotype dominated and was identified from most sample sites on the farm. Raw milk products are vulnerable to contamination with Staph. aureus. Strategies to reduce the occurrence of Staph. aureus in bulk milk are of particular importance on farms where milk is used for raw milk products.

The use of avoparcin as a growth promoter and the occurrence of vancomycin-resistant Enterococcus species in Norwegian poultry and swine production.

This study documents a strong and statistically significant association between the use of the glycopeptide avoparcin as a growth promoter in Norwegian poultry production and the occurrence of vancomycin-resistant Enterococcus species (VRE). Avoparcin was approved as a feed additive for broilers and turkeys in Norway in 1986 and was banned from June 1, 1995. In a survey conducted in Norway between June, 1995 and March, 1997, VRE were isolated from fecal samples from 106 out of 109 poultry houses previously exposed to avoparcin (97%) and from six out of 33 poultry houses never exposed to avoparcin (18%) (RR = 5.35). Samples from previously exposed poultry houses were collected in three time periods. The proportion of positive samples remained high (96-98%), in all three time periods indicating a persistence of vancomycin resistance among enterococci for more than a year and a half after the withdrawal of avoparcin. VRE were also isolated from six out of 10 poultry farmers living on farms previously exposed to avoparcin, and from none of 16 farmers living on farms never exposed to avoparcin. Moreover, VRE were isolated from 68 out of the 225 broiler carcasses investigated (30%). The resistance to vancomycin was a high-level type (MIC > or = 256 microg/ml) mediated by the vanA gene. For comparison, VRE could only be isolated from two out of 147 fecal samples from Norwegian flocks of swine (1%). Because avoparcin never has been used in Norwegian swine production, this observation strengthens the association between the use of avoparcin in animal husbandry and the occurrence of VRE.

Listeria monocytogenes in the smoked salmon industry.

Smoked salmon is sporadically contaminated with Listerial monocytogenes. Contamination levels are normally low and consumers are probably seldom exposed to risk concentrations. No clones of L. monocytogenes seem to be specific to smoked salmon, some clones found in smoked salmon having been isolated from several sources, including patients. Cold-smoking has been shown to eliminate L. monocytogenes in challenge tests at temperatures from 17.1 to 21.1 degrees C, while from 22.2 to 30 degrees C the bacteria survived. Under natural cold-smoking conditions (19 to 22 degrees C) the frequency and level of L. monocytogenes seems to decrease. Hot-smoking seems to eliminate the bacteria when smoke is applied during the whole heating process. The prevention of recontamination of both cold-smoked and hot-smoked salmon is therefore of great importance. L. monocytogenes multiply considerably in smoked salmon during storage. Growth is faster in challenge tests than in naturally-contaminated smoked salmon. The declared shelf-life under refrigeration should be shorter than that customarily stipulated by many producers. While the sources of L. monocytogenes in smoked salmon processing plants have still to be determined, raw salmon does not seem to be an important source. The main issue for producers is to prevent colonization of the processing environment and spread of the bacteria to products. This should be achieved by the systemic implementation of hygienic measures, including the HACCP approach.

Listeria monocytogenes in foods in Norway.

Three-hundred-and-eighty-two samples of different retail food items in Norway (imported soft cheese, raw chicken, minced meat, fermented sausages, vacuum-packed processed meat products, smoked salmon, peeled shrimps, raw minced fish) and 78 carcass samples (sheep, pig, cattle), were screened for Listeria monocytogenes. Of the 460 samples investigated, 78 were found to contain L. monocytogenes. Five of these contained greater than 10(3) cfu/g, four greater than 10(2) cfu/g, while the remainder were shown to contain L. monocytogenes only after enrichment. L. monocytogenes was isolated most frequently from raw chicken, sporadically from soft cheese, shrimps, processed meat products and smoked salmon, and not at all from carcasses and fermented sausages.

Characterization of plasmids from Listeria sp.

Out of 139 isolates of Listeria sp. (mainly L. monocytogenes) 107 (78%) contained extrachromosomal DNA. Plasmids from 51 of these isolates were investigated further and covered a range of 8 different-sized molecules as shown by agarose gel electrophoresis. Only one of the 107 isolates contained more than one plasmid. With the exception of one plasmid from an isolate of L. seeligeri, restriction analysis and hybridization experiments showed a high degree of homology between the different plasmids.

Contamination pattern of Listeria monocytogenes and other Listeria spp. in a salmon slaughterhouse and smoked salmon processing plant.

A smoked salmon processing plant including a smokehouse and a slaughterhouse was examined for the occurrence of Listeria monocytogenes and other Listeria spp. From a total of 475 samples the overall frequency of L. monocytogenes was 16%, while other Listeria spp. were found in 22% of the samples. L. monocytogenes was most often detected in samples from the smokehouse, where 29% of the environmental and 26% of the fish samples during processing contained the bacteria. 17% of the fish raw material to the smokehouse were contaminated, while 11% of the samples from vacuum-packed smoked salmon were positive for L. monocytogenes. The slaughterhouse was sporadically contaminated, but L. monocytogenes was not found in 50 samples of slaughtered fish. L. monocytogenes was found in the seawater outside the slaughterhouse. Multilocus enzyme electrophoresis divided the isolated L. monocytogenes strains into 11 electrophoretic types (ETs). One ET, ET-6, which is the most common ET in Norway, seemed to have colonized the smokehouse. Isolates from the seawater, from the slaughterhouse and from fish coming into the smokehouse, before filleting, were other ETs.

Growth of Listeria monocytogenes in vacuum-packed, smoked salmon, during storage at 4 degrees C.

Samples of smoked salmon of different hygienic quality were inoculated with low (6 cfu/g) and high (600 cfu/g) levels of a mixture of three strains of Listeria monocytogenes, after which they were vacuum-packed and stored at 4 degrees C for up to 5 weeks. L. monocytogenes grew well during storage in all the inoculated sample groups. Growth was, however, slightly faster in the fish with the better hygienic quality. The smoked salmon was still sensorically acceptable after 4 weeks. All three strains were found after 4 weeks in the fish with the better quality, while only two strains were recovered after the same time from the poorer quality salmon.

Differentiation of Listeria monocytogenes isolates by using plasmid profiling and multilocus enzyme electrophoresis.

Three hundred and seven Listeria monocytogenes isolates from various origins (clinical sources, raw chicken, seafoods, dairy and meat products and processing environments) were screened for plasmids. The overall frequency of L. monocytogenes isolates containing plasmids was 77%. The highest percentages of plasmid positive isolates were found from meat (89%), chicken (81%) and dairy products (64%), while clinical isolates had the lowest plasmid percentage (28%). Seven sizes of plasmids (21, 24, 27, 35, 40, 47 and 52 MDa) were distinguished. All sizes were represented in the meat isolates, clinical isolates contained only two of the plasmid sizes, while several different sizes of plasmids were found in the isolates from other origins. Plasmid profiling divided the isolates into ten plasmid pattern types. Multilocus enzyme electrophoresis of 75 isolates demonstrated 12 distinctive multilocus genotypes (ETs) which clustered into two groups: cluster A including serotype 1 and 4 isolates, and isolates not typable by Difco antisera serotype 1 and 4, and cluster B containing only serotype 1 isolates. No relationship between ETs and plasmid profiles could be demonstrated.

Occurrence of and a possible mechanism for resistance to a quaternary ammonium compound in Listeria monocytogenes.

In a study of 200 Listeria monocytogenes isolates, 10% were determined to be resistant to benzalkonium chloride (BC). Serial subcultivation of initially BC sensitive (BC(S)) and BC resistant (BC(R)) isolates in sublethal concentrations of BC resulted in enhanced and approximately equal resistance of all strains to the compound. Fifty per cent of the BC(R) isolates showed resistance to ethidium bromide (EB) as well. A proton motive force (pmf)-dependent efflux of EB was demonstrated in BC(R) isolates, and in originally sensitive strains adapted to grow in BC. This efflux was not found in BC(S) strains. The result indicate that BC can induce a broad resistance mechanism based on a pmf-driven efflux pump. There was no indication that this type of resistance was related to resistance to antibiotics.

Risk factors for contamination of smoked salmon with Listeria monocytogenes during processing.

Forty smoked salmon processing plants were examined for the occurrence of Listeria monocytogenes and other Listeria spp. in the smoked salmon and the drains. L. monocytogenes was detected in smoked salmon from 13 (33%) and in the drains samples from 25 (63%) of the plants. Other Listeria spp. were found in smoked salmon samples from 16 (40%) and in the drains of 30 (75%) of the plants. Multivariate analyses of data on hygiene, management, production facilities of the plants and bacteriological results showed that job rotation was the strongest expressed risk factor for isolation of L. monocytogenes from the smoked salmon (hazard ratio, HR = 11.0, p = 0.002). Well-maintained facilities (HR = 0.31, p = 0.064) and use of vats for salting of the fillets (HR = 0.33, p = 0.109), showed a preventive effect. L. monocytogenes in the drains was found to be a sensitive predictor for the presence of L. monocytogenes in the smoked salmon. In general, detection of other Listeria spp. in the smoked salmon or the drains could not be demonstrated to have any association with detection of L. monocytogenes.

A novel multiplex PCR for the identification of Vibrio parahaemolyticus, Vibrio cholerae and Vibrio vulnificus.

AIM: To establish a simple multiplex polymerase chain reaction (PCR) that will identify Vibrio parahaemolyticus, Vibrio cholerae and Vibrio vulnificus. METHODS AND RESULTS: A total of 429 Vibrio spp. from various origins were tested with the novel primers targeting toxR. The reverse primers were all designed to be species specific, while the forward primer was universal. The primers correctly identified all the V. parahaemolyticus, V. cholerae and V. vulnificus isolates tested. CONCLUSIONS: The toxR multiplex PCR works well when the initial colony morphology is known. If not, Vibrio alginolyticus might represent a diagnostic obstacle. SIGNIFICANCE AND IMPACT OF THE STUDY: The method provides a fast and reliable way of identifying the main Vibrio spp. involved in food-borne disease. The method could prove very useful for laboratories working with identification of these Vibrio spp.

Bacteriological quality of organically grown leaf lettuce in Norway.

AIM: To investigate bacteriological quality in organically grown leaf lettuce, including the presence of selected pathogenic bacteria, and to obtain information about organic lettuce production, including fertilizing regimes. METHODS AND RESULTS: Altogether 179 samples of Norwegian organically grown lettuce were collected from 12 producers. Escherichia coli was isolated from 16 of the lettuce samples, but in 12 of these contamination was sufficiently low (<100 CFU g(-1)) that they would be considered to be of acceptable bacteriological quality. Escherichia coli O157 and Salmonella were not detected in any of the samples. Listeria monocytogenes serogroups 1 and 4 were isolated from two samples. CONCLUSIONS: Organic lettuce produced in Norway was generally of acceptable bacteriological quality, but the results show that contamination of organic lettuce with E. coli and L. monocytogenes do occasionally occur. SIGNIFICANCE AND IMPACT OF THE STUDY: These results suggest that organically grown lettuce may be contaminated with E. coli and L. monocytogenes during cultivation.

Enterotoxigenic Staphylococcus aureus in bulk milk in Norway.

AIMS: To investigate the presence of enterotoxigenic Staphylococcus aureus in bulk milk and in a selection of raw milk products. METHODS AND RESULTS: Samples of bovine (n = 220) and caprine (n = 213) bulk milk, and raw milk products (n = 82) were analysed for S. aureus. Isolates were tested for staphylococcal enterotoxin (SE) production (SEA-SED) by reversed passive latex agglutination and for SE genes (sea-see, seg-sej) by multiplex PCR. Staphylococcus aureus was detected in 165 (75%) bovine and 205 (96.2%) caprine bulk milk samples and in 31 (37.8%) raw milk product samples. Enterotoxin production was observed in 22.1% and 57.3% of S. aureus isolates from bovine and caprine bulk milk, respectively, while SE genes were detected in 52.5% of the bovine and 55.8% of the caprine bulk milk isolates. SEC and sec were most commonly detected. A greater diversity of SE genes were observed in bovine vs caprine isolates. CONCLUSIONS: Staphylococcus aureus seems highly prevalent in Norwegian bulk milk and isolates frequently produce SEs and contain SE genes. Enterotoxigenic S. aureus were also found in raw milk products. SIGNIFICANCE AND IMPACT OF THE STUDY: Staphylococcus aureus in Norwegian bovine and caprine bulk milk may constitute a risk with respect to staphylococcal food poisoning from raw milk products.

Diversity of Staphylococcus aureus enterotoxin types within single samples of raw milk and raw milk products.

AIM: To find out if testing of up to 10 Staphylococcus aureus isolates from each sample from raw milk and raw milk products for staphylococcal enterotoxin (SE) might increase the chances of identifying potential sources of food intoxication. METHODS AND RESULTS: Altogether 386 S. aureus isolates were tested for the presence of SE by reversed passive latex agglutination (SET-RPLA), and SE genes (se) by a multiplex polymerase chain reaction (PCR). In 18 of 34 (53%) S. aureus positive samples a mixture of SE and/or se positive and negative isolates were identified. Multiplex PCR increased the number of potential SE producing strains, i.e. isolates that harboured se, with 51% among the product and 48% among the raw bovine milk isolates. Examination by pulsed-field gel electrophoresis mostly confirmed clonal similarity among isolates sharing SE/se profile, but did not further differentiate between them. CONCLUSIONS: Isolates of S. aureus collected from one sample may show great diversity in SE production and different plating media seem to suppress or favour different strains of S. aureus. SIGNIFICANCE AND IMPACT OF THE STUDY: Several isolates of S. aureus from each sample should be tested for enterotoxin production in cases with typical SE intoxication symptoms with methods that are able to reveal new SE/se.

Genetic variation among Staphylococcus aureus strains from Norwegian bulk milk.

Strains of Staphylococcus aureus obtained from bovine (n = 117) and caprine (n = 114) bulk milk were characterized and compared with S. aureus strains from raw-milk products (n = 27), bovine mastitis specimens (n = 9), and human blood cultures (n = 39). All isolates were typed by pulsed-field gel electrophoresis (PFGE). In addition, subsets of isolates were characterized using multilocus sequence typing (MLST), multiplex PCR (m-PCR) for genes encoding nine of the staphylococcal enterotoxins (SE), and the cloverleaf method for penicillin resistance. A variety of genotypes were observed, and greater genetic diversity was found among bovine than caprine bulk milk isolates. Certain genotypes, with a wide geographic distribution, were common to bovine and caprine bulk milk and may represent ruminant-specialized S. aureus. Isolates with genotypes indistinguishable from those of strains from ruminant mastitis were frequently found in bulk milk, and strains with genotypes indistinguishable from those from bulk milk were observed in raw-milk products. This indicates that S. aureus from infected udders may contaminate bulk milk and, subsequently, raw-milk products. Human blood culture isolates were diverse and differed from isolates from other sources. Genotyping by PFGE, MLST, and m-PCR for SE genes largely corresponded. In general, isolates with indistinguishable PFGE banding patterns had the same SE gene profile and isolates with identical SE gene profiles were placed together in PFGE clusters. Phylogenetic analyses agreed with the division of MLST sequence types into clonal complexes, and isolates within the same clonal complex had the same SE gene profile. Furthermore, isolates within PFGE clusters generally belonged to the same clonal complex.

Fluctuations in the occurrence of Escherichia coli O157:H7 on a Norwegian farm*.

AIM: To describe the distribution of Escherichia coli O157:H7 on a sporadically positive dairy farm and on possible contact farms over a one-year period. METHODS AND RESULTS: Environmental and faecal samples from all animals at the farm, and faecal samples from animals at contact farms were analysed for E. coli O157:H7 by immunomagnetic separation methods or VIDAS. Confirmed isolates were tested for cytotoxicity in the Vero cell assay and typed by PFGE. Escherichia coli O157:H7 (stx2 and eae) of the same PFGE type were isolated from cattle, sheep, hens and environmental samples at variable levels during summer and fall 2002, but were not detected in 2003. CONCLUSIONS: Escherichia coli O157:H7 had a widespread distribution on the farm investigated, but the original source of contamination could not be identified. The occurrence of this bacterium on the farm did not result in any detectable increase in gastrointestinal disease in the associated population. SIGNIFICANCE AND IMPACT OF THE STUDY: Despite a low endemic level of E. coli O157:H7 in the Norwegian cattle population, the growth and spread of this potentially important bacterium may occur.

Detection of Listeria monocytogenes in foods by immunomagnetic separation.

Immunomagnetic separation with immunomagnetic beads was used to isolate strains of Listeria monocytogenes both from pure cultures and from heterogeneous suspensions. The monoclonal antibodies used recognized all six strains of serotype 4 but only one of three strains of serotype 1. Coating procedure, incubation time, and number of immunomagnetic beads influenced the sensitivity of the isolation method. Less than 1 x 10(2) bacteria per ml in pure cultures and less than 2 x 10(2) bacteria per ml in enriched foods could be detected. The method represents a new approach to extraction and isolation of pathogenic bacteria directly from foods, after resuscitation, or from enrichment broths.

Molecular epidemiological survey of Listeria monocytogenes in broilers and poultry products.

AIMS: To investigate the prevalence of Listeria monocytogenes in poultry products, and to elucidate whether poultry products may be linked to listeriosis cases. A further goal was to identify contamination routes for L. monocytogenes to broiler carcasses. METHODS AND RESULTS: Poultry products (385 samples) were screened for L. monocytogenes. The recovered isolates and 19 patient isolates were characterized by multilocus enzyme electrophoresis and restriction enzyme analysis. The poultry isolates showed great genetic diversity, but no identical subclones were identified from poultry sources and patients. One slaughterhouse was examined in detail during a 16-month period. The contamination rates increased along the processing line, and one subclone was found during the whole period. Only low prevalence of the bacteria was revealed from broiler faeces. CONCLUSIONS: The prevalence of L. monocytogenes in poultry products was high, but no listeriosis cases was linked to poultry products. Broilers seem to be contaminated during the slaughter process, and specific strains may persist in the processing environment. Broiler faeces does not seem to be an important source of L. monocytogenes in poultry products. SIGNIFICANCE AND IMPACT OF THE STUDY: Preventive measures to avoid contamination of poultry products by L. monocytogenes must be taken in the processing plants.