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Contamination of sliced cooked meat products with a Lactobacillus sake starter strain was suspected to cause spoilage in the products before the end of the expected shelf life. Slicing and vacuum packaging of the cooked products was done in the room in which the fermented product was handled. Since L. sake strains are known to be a dominant part of spoilage microflora associated with vacuum-pack-aged meat products, a contamination study was performed. One hundred and eighteen strains were isolated from 6 spoiled vacuum packaged meat products and from the surfaces of the packaging room and adjac-ent refrigerators. DNA was isolated from these strains and cleaved using Eco RI and Hind III restriction endonucleases to obtain characteristic ribotypes. Corresponding ribotypes of the L. sake starter strain were compared to the 14 different patterns obtained from the strains growing in spoiled products and on surfaces by Eco RI digestions. The L. sake starter strain was shown to contaminate the packaging room and it was also isolated from one of the products. However, it was not a dominant strain in this product and it could not be linked to the other products. Our results indicated that handling the fermented product in the refrigerating and packaging rooms together with cooked products was not the major cause of spoilage in these products.

SUMMARYThe first reported bovine botulism outbreak in Finland is described. Nine out of 90 cattle on a dairy farm died after being fed non-acidified silage contaminated by animal carcasses. Type C botulinum neurotoxin gene was detected in one heifer by polymerase chain reaction (PCR) and the neurotoxin was detected by the mouse bioassay.Clostridium botulinumtype C was isolated from liver samples. The isolated strain was identified with amplified fragment length polymorphism (AFLP) analysis as group IIIC. botulinum. To our knowledge, this is the first time that a type C bovine botulism outbreak has been diagnosed by PCR and confirmed by subsequent isolation and AFLP identification of the disease strain. The importance of the acidification process in silage production to inhibitC. botulinumtoxin production in silage and thus to prevent further botulism outbreaks is emphasized. Nevertheless, preformed toxin in the carcass is not destroyed by acid.

Three Clostridium botulinum type E strains were sequenced for the botulinum neurotoxin (BoNT) gene cluster, and 11 type E strains, representing a wide biodiversity, were sequenced for the bont/E gene. The total length of the BoNT/E gene cluster was 12,908 bp, and a novel gene (partial) designated orfx3, together with the complete orfx2 gene, was identified in the three type E strains for the first time. Apart from orfx3, the structure and organization of the neurotoxin gene cluster of the three strains were identical to those of previously published ones. Only minor differences (

A total of 312 samples of sliced, vacuum packaged, cold-smoked pork from 15 meat processing plants in Latvia and Lithuania, obtained over a 15-month period from 2003 until 2004, were analyzed for the presence of Listeria monocytogenes at the end of their shelf-life. Overall, 120 samples (38%) tested positive for L. monocytogenes. Despite the long storing period, the levels of L. monocytogenes in cold-smoked pork products were low. Manufacturing processes were studied at seven meat processing plants. A new approach with a logistic multivariable regression model was applied to identify the main factors associated with L. monocytogenes contamination during the manufacturing of cold-smoked pork products. Brining by injection was a significant factor (odds ratio 10.66; P0.05) for contamination of product with L. monocytogenes. Moreover, long cold-smoking times (or = 12 h) had a significant predictive value (odds ratio 24.38; P0.014) for a sample to test positive for L. monocytogenes. Pulsed-field gel electrophoresis results indicated that various sources of L. monocytogenes contamination existed over periods of time in several meat processing plants. In two meat processing plants, persistent L. monocytogenes strains belonging to serotypes 1/2a and 1/2c were found.

http://www.elsevier.com/locate/issn/01681605 Microbiological and sensory changes in vacuum-packaged charcoal-broiled river lampreys from three lamprey processing plants were monitored as a function of time at 8°C. The lampreys were examined every 7 days up to 8 weeks for aerobic plate count (APC) and lactic acid bacteria (LAB). The highest mean APC and LAB were 6.01 log CFU/g and 4.86 log CFU/g, respectively. Only 6 out of 15 lots reached an APC value of 7.0 log CFU/g during storage. The sensory scores remained at the baseline levels after 8 weeks´ storage. Twenty-seven isolates were randomly picked from MRS agar and identified to species level using a 16S and 23S rDNA HindIII RFLP (ribotyping) database and sequencing of the 16S rRNA gene if no database match was obtained. Twelve of the 27 isolates were identified as Lactobacillus curvatus subsp. curvatus, and two Leuconostoc mesenteroides and one Weissella halotolerans strain were also detected. Twelve isolates were not identified by the LAB database. However, they possessed very high (99.9%) 16S gene sequence similarity with either Staphylococcus warneri or Staphylococcus pasteuri type strains. The LAB detected, with the exception of W. halotolerans, have commonly been associated with spoilage of fishery products, but in these vacuum-packaged lampreys they were not the dominant organisms within the developing spoilage population.

Amplified fragment length polymorphism (AFLP) analysis was applied to characterize 33 group I and 37 group II Clostridium botulinum strains. Four restriction enzyme and 30 primer combinations were screened to tailor the AFLP technique for optimal characterization of C. botulinum . The enzyme combination HindIII and HpyCH4IV, with primers having one selective nucleotide apiece (Hind-C and Hpy-A), was selected. AFLP clearly differentiated between C. botulinum groups I and II; group-specific clusters showed C. botulinum strains. In addition, group-specific fragments were detected in both groups. All strains studied were typeable by AFLP, and a total of 42 AFLP types were identified. Extensive diversity was observed among strains of C. botulinum type E, whereas group I had lower genetic biodiversity. These results indicate that AFLP is a fast, highly discriminating, and reproducible DNA fingerprinting method with excellent typeability, which, in addition to its suitability for typing at strain level, can be used for C. botulinum group identification.

http://www.elsevier.nl/locate/01681605 A total of 176 lactic acid bacteria (LAB) isolated from a typical Spanish blood sausage called “morcilla de Burgos” were identified by means of phenotypic characteristics and 16S rDNA RFLP (ribotyping). LAB were isolated from “morcilla” of different producers and in different storage periods, that includes unpackaged, vacuum and modified atmosphere packaged “morcilla” and vacuum packed and pasteurised “morcilla”. The knowledge of specific spoilage bacteria of ”morcilla de Burgos” will be useful to design new preservation methods to extent the shelf-life of this product. Identification made according to phenotypic and biochemical characteristics shows the majority of the isolates were heterofermentative LAB (93.2%) and eight different bacterial groups could be distinguished (A-G). W. viridescens was the main species detected (42%). In addition, Leuconostoc spp. (23.9%), W. confusa (11.4%) and Lactobacillus fructosus (5.7%) species were found. Few strains were phenotypically missidentified as Lb. sanfrancisco, Pediococcus spp., Lb. sakei/curvatus and Carnobacterium spp. and 11 strains remained unknown. Most of the leuconostocs were identified as Lc. mesenteroides and Lc. carnosum species. Ribotyping shows a quite good correlation with phenotypic methods, although it has been possible to identify 15 different clusters. W. viridescens and leuconostocs were also the predominant LAB. Strains identified as W. confusa by phenotypic characteristics were resolved in W. confusa and W. cibaria by ribotyping. Neither Carnobacterium piscicola nor Lb. sanfrancisco were identified by means of genotypic method. All Lb. fructosus strains and some more included in different phenotypic groups (17 strains in total) could not be associated with any reference strain (cluster VII). Although some discrepancies exists the combination of phenotypic and genotypic methods led to a better identification and characterization of the strains isolated from “morcilla de Burgos”.

A hydrophobic grid membrane filter-colony hybridization (HGMF-CH) method for the enumeration and isolation of cpe gene-carrying ( cpe -positive) Clostridium perfringens spores from feces was developed. A 425-bp DNA probe specific for the cpe gene was sensitive and specific when tested with bacterial DNA and pure cultures. The enumeration of cpe -positive C. perfringens by the HGMF-CH method proved to be as sensitive as nested PCR combined with the most-probable number technique when tested with fecal samples from healthy individuals. With the aid of the HGMF-CH method, positive hybridization signals were detected from two out of seven fecal samples obtained from healthy individuals. Furthermore, cpe -positive C. perfringens was successfully isolated from both of these samples. The detection of cpe -positive C. perfringens by the HGMF-CH method is dependent on the ratio of cpe- positive C. perfringens colonies to total C. perfringens colonies growing on the HGMF-tryptose-sulfite-cycloserine plate. cpe- positive C. perfringens could be isolated if the ratio of cpe- positive C. perfringens spores to total C. perfringens spores was 6 × 10 −5 or higher. The HGMF-CH method provides an aid in the investigation of fecal samples of patients suffering from food poisoning or other diseases caused by cpe -positive C. perfringens . The method also offers a new approach in the investigation of the epidemiology of cpe- positive C. perfringens strains.

Isolates 302, 334, 356, 377 and 379, detected in modified atmosphere packaged (MAP) broiler meat, together with LMG 12317T and LMG 13617, detected in dog tonsils, were analysed in a polyphasic taxonomy study including numerical analysis of ribopatterns and whole cell protein patterns, 16S rDNA sequence analysis, DNA-DNA hybridisation and determination of some phenotypic properties. The results indicated that these isolates represent a new species in the genus Enterococcus. The isolates showed classical phenotypic reactions for the genus Enterococcus with the exception of not possessing the Lancefield group D antigen. Isolates 334, LMG 12317T, and LMG 13617 showed the highest 16S rDNA sequence similarity (98.3-99.0%) with the Enterococcus pallens type strain. In the distance matrix tree based on 16S rDNA sequences, the three isolates were located in the Enterococcus avium –group with E. pallens as their closest phylogenic neighbour. Numerical analyses of whole cell protein patterns and HindIII/EcoRI ribotypes placed all seven isolates together in a single cluster separated from the E. avium group reference strains. The DNA-DNA hybridisation level between strains 334 and LMG 12317T was 93.5% confirming that they represent the same species. Low hybridisation levels (12-30%) were, on the other hand, obtained with the E. pallens and Enterococcus raffinosus type strains. The name Enterococcus hermanniensis sp. nov. is proposed, with strain LMG 12317T (=CCUG 48100 T) as the type strain. We would like to thank Ms. Henna Niinivirta for her excellent technical assistance. Financial support from the Academy of Finland (project 100479) is gratefully acknowledged. T.C. and P.V are indebted to the Fund for Scientific Research – Flanders (Belgium) for a position as postdoctoral fellow and research grants, respectively. T.C. also acknowledges the support from Belgian Federal Government (Federal Office for Scientific, Technical and Cultural Affairs).

Background. The vehicles and sources of Yersinia pseudotuberculosis infection are unknown. In Finland, clinical microbiology laboratories routinely report Y. pseudotuberculosis isolations and submit isolates for serotype analysis. In October 1998, the number of serotype O:3 infections increased markedly. Methods. Case patients with culture-confirmed Y. pseudotuberculosis O:3 infection were identified by use of laboratory-based surveillance. We conducted a population-based case-control study. Healthy community control subjects were matched by age, sex, and postal code. Isolates were subtyped by pulsed-field gel electrophoresis (PFGE). Results. Nationwide, 47 case patients were identified (age range, 277 years; median, 19 years). One patient with bacteremia died; 5 underwent appendectomies. We enrolled 38 case patients and 76 control subjects in the case-control study. Seventy-one percent of case patients and 42% of control subjects reported having eaten iceberg lettuce (matched odds ratio, 3.8; 95% confidence interval, 1.39.4); a dose-response relationship was found for increasing frequency of consumption. Of the 27 isolates obtained from case patients and tested in the analysis, all had indistinguishable PFGE patterns. Four lunch cafeterias that had served iceberg lettuce were associated with clusters of case patients. The lettuce was traced back to originating farms. Conclusions. Iceberg lettuce was implicated as the vehicle of a widespread foodborne Y. pseudotuberculosis outbreak. Ongoing laboratory-based surveillance and serotype analysis were essential in the rapid detection of infection. Cases of yersiniosis, which appear to be sporadic, may be part of unrecognized outbreaks caused by contaminated fresh produce.

Canine intestinal lactic acid bacterium (LAB) population in four fistulated dogs was cultured and enumerated using MRS agar. LAB levels ranging from 1.4x10(6) to 1.5x10(7) CFU ml(-1) were obtained in jejunal chyme. In the fecal samples 7.0x10(7) and 2.0x10(8) CFU g(-1) were detected. Thirty randomly selected isolates growing in the highest sample dilutions were identified to species level using numerical analysis of 16S and 23S rDNA restriction fragment length polymorphism patterns (ribotyping) and 16S rDNA sequence analysis. According to these results, Streptococcus alactolyticus was the dominant culturable LAB species in both feces and jejunal chyme. In addition, Lactobacillus murinus and Lactobacillus reuteri were detected.

During spring and autumn migrations, 468 fecal samples from 57 different species of migratory birds were collected in Sweden. In total, Yersinia spp. were isolated from 12.8% of collected samples. The most commonly found species was Yersinia enterocolitica , which was isolated from 5.6% of all collected samples, followed by Y. intermedia (3.8%), Y. frederiksenii (3.0%), Y. kristensenii (0.9%), Y. pseudotuberculosis (0.6%), and Y. rohdei (0.4%). The pathogenic, virF -positive Y. pseudotuberculosis strains were recovered from three thrushes. These strains belonged to the same bioserotype, 1/O:2, but had two different profiles as determined by pulsed-field gel electrophoresis with Not I and Spe I enzymes. In addition, 10 Y. enterocolitica strains, all from barnacle geese, belonged to bioserotype 3/O:3, which is associated with human disease. Two of the strains were pathogenic, carrying the virF gene on their plasmids. All pathogenic Y. pseudotuberculosis and Y. enterocolitica strains were recovered during the spring, and as the birds were caught during active migration they likely became infected at an earlier stage of the migration, thus potentially transporting these bacterial pathogens over long geographical distances.

http://www.elsevier.com/locate/01681605 A total of 296 lactic acid bacteria (LAB) isolated from spoiled, vacuum-packaged ‘gravad’ rainbow trout stored at 3ºC and 8ºC were characterised and identified using a molecular approach. The isolates were initially grouped according to their HindIII restriction endonuclease profiles and further identified to species level using a rRNA gene restriction pattern (ribotype) identification database. Lactobacillus sakei, Lactobacillus curvatus and Carnobacterium piscicola were the three main species detected. Only one isolate was identified as Carnobacterium divergens. Most of the carnobacteria were found in the samples stored at 3ºC. The relative proportion of L. sakei was higher in the samples stored at 8ºC.

Ten low-capacity slaughterhouses were examined for Listeria by collecting a total of 373 samples, of which 50, 250, and 73 were taken from carcasses, pluck sets, and the slaughterhouse environment, respectively. Six slaughterhouses and 9% of all samples were positive for Listeria monocytogenes. Of the samples taken from pluck sets, 9% were positive for L. monocytogenes, the highest prevalence occurring in tongue and tonsil samples, at 14% and 12%, respectively. Six of 50 (12%) carcasses were contaminated with L. monocytogenes. In the slaughterhouse environment, L. monocytogenes was detected in two, one, one, and one sample originating from the saws, drain, door, and table, respectively. Carcasses were contaminated with L. monocytogenes in those two slaughterhouses, where the mechanical saws, used for both brisket and back splitting, were also positive for L. monocytogenes. A total of 58 L. monocytogenes isolates were characterized by pulsed-field gel electrophoresis typing. The isolates were divided into 18 pulsotypes, 15 of which were detected in pluck sets. In two slaughterhouses, where the carcasses were contaminated with L. monocytogenes, the same pulsotypes were also recovered from splitting saws. In addition, identical pulsotypes were recovered from pluck sets. Our findings indicate that L. monocytogenes of tongue and tonsil origin may contaminate the slaughtering equipment that may in turn spread the pathogen to carcasses. Thus, it is of the utmost importance to follow good manufacturing practices and to have efficient cleaning and disinfection procedures to prevent equipment being contaminated with L. monocytogenes.

http://www.elsevier.nl/locate/01681605 Eighteen earlier well characterised Lactobacillus sake strains representing different slime production capabilities in vacuum-packaged meat products were analysed using repetitive element sequence-based PCR (rep-PCR). The single primers BOXA1R and RW3A and the primer pair REP1R-Dt & REP2R-Dt were evaluated for their applicability in L. sake genotyping. The five different patterns produced by RW3A were least revealing, with the discriminatory power equalling to ribotyping. BOXA1R and REP-primer pair both produced six different banding patterns and the combination of these results yielded seven different rep-types. Rep-PCR was concluded to have approximately the same discriminatory power as randomly amplified polymorphic DNA (RAPD) analysis, but it was inferior to pulsed-field gel electrophoresis (PFGE). How-ever, if the results of rep-PCR and RAPD were combined, the discrimination was comparable PFGE, with the exception that within Ribogroup I the non-slime-producing strains were not distinguished from weak slime producers. It was concluded that the combination of the two PCR-based typing techniques, rep-PCR and RAPD, would be a valuable tool in large scale contamination studies at the meat processing plants, since results can be obtained rapidly and fewer isolates need further analysis by PFGE.

Sites of Listeria monocytogenes contamination in a cold-smoked rainbow trout ( Oncorhynchus mykiss ) processing plant were detected by sampling the production line, environment, and fish at different production stages. Two lots were monitored. The frequency of raw fish samples containing L. monocytogenes was low. During processing, the frequency of fish contaminated with L. monocytogenes clearly rose after brining, and the most contaminated sites of the processing plant were the brining and postbrining areas. A total of 303 isolates from the raw fish, product, and the environment were characterized by pulsed-field gel electrophoresis (PFGE). PFGE yielded nine pulsotypes, which formed four clusters. The predominating L. monocytogenes pulsotypes of the final product were associated with brining and slicing, whereas contaminants of raw fish were not detected in the final product. Air-mediated contamination in the plant could not be proved. In accordance with these results, an L. monocytogenes eradication program was planned. The use of hot steam, hot air, and hot water seemed to be useful in eliminating L. monocytogenes . None of the control samples taken in the 5 months after the eradication program was implemented contained L. monocytogenes .

http://www.elsevier.nl/locate/01681605 The spoilage flora of vacuum-packaged, salted, cold-smoked rainbow trout fillets, with or without the addition of nitrate or nitrite, stored at 4°C and 8°C, was studied. Of 620 isolates, lactic acid bacteria were the major fraction (76%), predominating in all samples of spoiled product. However, the phenotypical tests used were insufficient to identify the lactic acid bacteria to the species level. Gram-positive, catalase-positive cocci, Gram-negative, oxidase-negative rods and Gram-negative, oxidase-positive rods were found in 6%, 16% and 2% of the samples, respectively. Of 39 Gram-positive, catalase-positive cocci, 29 were identified as staphylococci and 10 as micrococci. Eighty-five isolates were found to belong to the family Enterobacteriaceae, with 45 of those being Serratia plymuthica. Eleven isolates from the nitrate treated samples stored at 8°C were identified as Pseudomonas aeruginosa. The occurrence of P. aeruginosa and staphylococci in the nitrate-containing samples, stored at 8°C, may cause problems with respect to the safety of the product. The types of lactic acid and other bacteria in the spoilage flora were generally reduced by the addition of nitrate or nitrite to fillets.

Lactic acid bacteria are considered a major component of the microbial population found on various types of vacuum-packaged cooked sausages. Lactobacillus sake and Lactobacillus curvatus have been shown to be common species in these products. L. sake seems to form the predominant part of the spoilage population. Lactic acid bacterial growth on the surface of the sausages produces undesirable sensory attributes, such as sour aroma and taste. A specific spoilage phenomenon of commercial significance, characterized by long, stretchy, polysaccharide ropes between sausages or sausage slices, has also been found. L. sake strains play a major role in this spoilage phenomenon as well. Cooking of sausages during manufacturing destroys lactic acid bacteria on the surface of the sausages. Sausages are recontaminated with spoilage lactic acid bacteria mainly during the processing stages after cooking. During the chilling process, product contamination apparently results from exposure to airborne microorganisms. Workers and equipment are among the most likely sources of contamination during packaging and slicing. Spoilage strains originating from raw material may spread to other areas in the production facility (i.e., chill, slicing, and packaging rooms) via the air, workers and equipment. Decontamination methods can be used to prevent the growth of spoilage microorganisms and to extend the shelf life of vacuum-packaged sausages after packaging.

Hafnia alvei strains which possess the attachment-effacement gene (eaeA) may have clinical importance as new diarrhea-causing pathogens and should therefore be differentiated from other H. alvei strains. We characterized diarrheal H. alvei strains, which were positive in the PCR test for the eaeA gene, using biochemical tests not routinely used for identification of members of the family Enterobacteriaceae, and compared them with eaeA-negative strains isolated from different clinical and nonclinical sources to find characteristics useful for identification. Random amplified polymorphic DNA (RAPD)-PCR and partial sequencing of the 16S rRNA gene were utilized to study the genetic diversity of the isolates. The eaeA-positive strains were found to have many characteristic biochemical properties. Negative reactions in the 2-ketogluconate and histidine assimilation tests and a positive reaction in the 3-hydroxybenzoate assimilation test may be useful in routine diagnostics. Nearly identical RAPD-PCR profiles and identical 353-bp fragments of the 16S rRNA genes indicated little genetic diversity among the eaeA-positive strains. The low level of homology (92%) in the partial 16S rRNA genes of eaeA-positive and -negative H. alvei strains raises questions about the taxonomic positioning of eaeA-positive H. alvei.

Groups I (proteolytic) and II (nonproteolytic) C. botulinum are genetically and physiologically distinct groups of organisms, with both groups being involved with human botulism. Due to differences in spore heat resistance and growth characteristics, the two groups possess different types of human health risks through foods, drink, and the environment. The epidemiology of human botulism due to Groups I and II C. botulinum is poorly understood, largely due to insufficient characterization of disease isolates, and warrants thorough outbreak investigation with a particular attention to discrimination between the different physiological groups of C. botulinum. In this study, a PCR assay was developed to discriminate between Group I and Group II C. botulinum. The assay is based on the fldB associated with phenylalanine metabolism in proteolytic clostridia, and employs an internal amplification control targeted to conservative regions of 16S rrn in Groups I and II C. botulinum. The assay correctly identified all 36 Group I and 24 Group II C. botulinum strains, possessing a 100% exclusivity and inclusivity. The assay provides a substantial improvement in discriminating between the Groups I and II C. botulinum, which traditionally is based on a time-consuming and error-prone culture method. Differentiation between the physiological groups of C. botulinum is an essential step in investigation of human botulism outbreaks, and should be considered as a diagnostic corner-stone in order to improve our epidemiological understanding of human botulism.

The microbial numbers and the lactic acid bacteria population of the raw materials of cooked ring sausages were studied in order to determine the presence of the spoilage lactic acid bacteria of the sausages. The highest aerobic plate counts and lactic acid bacteria counts (up to the level of 108 and 105 CFU/g, respectively) were found in the pork skin emulsion and the meat trimmings. Lactic acid bacteria were also commonly found in the skim milk powder and the potato flour. Lactic acid bacteria isolates able to multiply at 8°C were recovered from all the raw materials studied except for the commercial spice mixture. These isolates could be divided into 11 different groups according to their morphological and biochemical characteristics. Isolates resembling the lactic acid bacteria strains responsible for the spoilage of vacuum-packed cooked ring sausages were found in the pork skin emulsion, the meat trimmings, and the potato flour. These raw materials can thus form a source for the spoilage bacteria of the sausages at a sausage processing plant.

Reprinted with permission from Journal of Food Protection. Copyright held by the International Association for Food Protection, Des Moines, U.S.A. Contamination routes of Listeria monocytogenes were examined in a chilled food processing plant that produced ready-to-eat and ready-to-reheat meals during an 8-year period by amplified fragment length polymorphism (AFLP) analysis. A total of 319 L. monocytogenes isolates were recovered from raw materials (n=18), the environment (n=77), equipment (n=193), and products (n=31), and 18 different AFLP types were identified, five of which were repeatedly found to be persistent types. The three compartments (I to III) of the plant showed markedly different contamination statuses. Compartment I, which produced cooked meals, was heavily contaminated with three persistent AFLP types. AFLP type A1 dominated, and it comprised 93% of the isolates of the compartment. Compartment II, which produced uncooked chilled food, was contaminated with four persistent and five nonpersistent AFLP types. The equipment of compartment III, which produced cooked ready-to-reheat meals, was free of contamination. In compartments that produced cooked meals, the cleaning routines, product types, and lack of compartmentalization seemed to predispose production lines to persistent contamination. The most contaminated lines harbored L. monocytogenes in coolers, conveyors, and packing machines. Good compartmentalization limited the flow of L. monocytogenes into the postheat -treatment area and prevented the undesired movement of equipment and personnel, thus protecting the production lines from contamination. In compartment II, grated cheese was shown to cause product contamination. Therefore, special attention should be paid to continuous quality control of raw ingredients when uncooked ready-to-eat foods are produced. In compartment II, reconstruction of the production line resulted in reduced prevalence rates of L. monocytogenes and elimination of two persistent AFLP types.

Reprinted with permission from Journal of Food Protection. Copyright held by the International Association for Food Protection, Des Moines, U.S.A. Persistent Listeria monocytogenes contamination of food industry equipment is a difficult problem to solve. Ultrasonic cleaning offers new possibilities for cleaning conveyors and other equipment that are not easy to clean. Ultrasonic cleaning was tested on three conveyor belt materials: polypropylene, acetal, and stainless steel (cold-rolled, AISI 304). Cleaning efficiency was tested at two temperatures (30 and 45 degrees C) and two cleaning times (30 and 60 s) with two cleaning detergents (KOH, and NaOH combined with KOH). Conveyor belt materials were soiled with milk-based soil and L. monocytogenes strains V1, V3, and B9, and then incubated for 72 h to attach bacteria to surfaces. Ultrasonic cleaning treatments reduced L. monocytogenes counts on stainless steel 4.61 to 5.90 log units; on acetal, 3.37 to 5.55 log units; and on polypropylene, 2.31 to 4.40 log units. The logarithmic reduction differences were statistically analyzed by analysis of variance using Statistical Package for the Social Sciences software. The logarithmic reduction was significantly greater in stainless steel than in plastic materials (P

SUMMARY Botulism is a potentially lethal paralytic disease caused by botulinum neurotoxin. Human pathogenic neurotoxins of types A, B, E, and F are produced by a diverse group of anaerobic spore-forming bacteria, including Clostridium botulinum groups I and II, Clostridium butyricum, and Clostridium baratii. The routine laboratory diagnostics of botulism is based on the detection of botulinum neurotoxin in the patient. Detection of toxin-producing clostridia in the patient and/or the vehicle confirms the diagnosis. The neurotoxin detection is based on the mouse lethality assay. Sensitive and rapid in vitro assays have been developed, but they have not yet been appropriately validated on clinical and food matrices. Culture methods for C. botulinum are poorly developed, and efficient isolation and identification tools are lacking. Molecular techniques targeted to the neurotoxin genes are ideal for the detection and identification of C. botulinum, but they do not detect biologically active neurotoxin and should not be used alone. Apart from rapid diagnosis, the laboratory diagnostics of botulism should aim at increasing our understanding of the epidemiology and prevention of the disease. Therefore, the toxin-producing organisms should be routinely isolated from the patient and the vehicle. The physiological group and genetic traits of the isolates should be determined.

http://www.elsevier.com/locate/issn/01681605 Lactic acid bacteria (LAB) isolated from marinated or non-marinated, modified atmosphere packaged (MAP) broiler leg products and air samples of a large-scale broiler meat processing plant were identified and analyzed for their phenotypic properties. Previously, these strains had been found to be coccal LAB. However, the use of a 16 and 23S rRNA gene RFLP database had not resulted in species identification because none of the typically meat-associated LAB type strains had clustered together with these strains in the numerical analysis of the RFLP patterns. To establish the taxonomic position of these isolates, 16S rRNA gene sequence analysis, numerical analysis of ribopatterns, and DNA–DNA hybridization experiments were done. The 16S rRNA gene sequences of three isolates possessed the highest similarities (over 99%) with the sequence of S. parauberis type strain. However, in the numerical analysis of HindIII ribopatterns, the type strain did not cluster together with these isolates. Reassociation values between S. parauberis type or reference strain and the strains studied varied from 82 to 97%, confirming that these strains belong to S. parauberis. Unexpectedly, most of the broiler meat-originating strains studied for their phenotypical properties did not utilize lactose at all and the same strains fermented also galactose very weakly, properties considered atypical for S. parauberis. This is, to our knowledge, the first report of lactose negative S. parauberis strains and also the first report associating S. parauberis with broiler slaughter and meat products.

Seven small-scale drinking water purification devices were evaluated for their capacity to eliminate botulinum neurotoxin (BoNT) type B from drinking water. Influent water inoculated with toxic Clostridium botulinum cultures and effluent purified water samples were tested for the presence of BoNT by using a standard mouse bioassay and two commercial rapid enzyme immunoassays (EIAs). The water purification devices based on filtration through ceramic or membrane filters with a pore size of 0.2 to 0.4 μm or irradiation from a low-pressure UV-lamp (254 nm) failed to remove BoNT from raw water (reduction of 10 units). A single device based on reverse osmosis was capable of removing the BoNT to a level below the detection limit of the mouse bioassay (reduction of >2.3 log 10 units). The rapid EIAs intended for the detection of BoNT from various types of samples failed to detect BoNT from aqueous samples containing an estimated concentration of BoNT of 396,000 ng/liter.

http://www.elsevier.com/locate/issn/01681605 In order to show which of the initial lactic acid bacteria (LAB) contaminants are also causing spoilage of a modified-atmosphere-packaged (MAP), marinated broiler leg product at 6ºC, LAB were enumerated and identified on the 2nd and 17th day following manufacture. A total of 8 fresh and 13 spoiled packages were studied for LAB levels. In addition, aerobic mesophilic bacteria and Enterobacteriaceae were determined. The average CFU/g values in the 8 fresh packages were 1.3 × 103, 9.8 × 103 and 2.6 × 102 on de Man Rogosa Sharpe agar (MRS), Plate Count Agar (PCA) and Violet Red Bile Glucose agar (VRBG), respectively. The commercial shelf life for the product had been set as 12 days and all packages analyzed on the 17th day were deemed unfit for human consumption by the sensory analysis. The corresponding CFU/g averages in the spoiled product were 1.4 × 109, 1.1 × 109 and 3.9 × 107 on MRS, PCA and VRBG agar, respectively. For characterization of LAB population, 104 colonies originating from the fresh and 144 colonies from the spoiled packages were randomly picked, cultured pure and identified to species level using a 16 and 23S rDNA HindIII RFLP (ribotyping) database. The results showed that enterococci (35.7% of the LAB population) were dominating in the fresh product whereas carnobacteria (59.7%) dominated among the spoilage LAB. Enterococcus faecalis, Carnobacterium piscicola and Carnobacterium divergens were the main species detected. In general, when the initial LAB population is compared to the spoilage LAB, a shift from homofermentative cocci towards carnobacteria, Lactobacillus sakei/curvatus and heterofermentative rods is seen in this marinated product.

The taxonomic position of two bovine strains, LMG 13603 and LMG 14595, assigned to the species Enterococcus raffinosus on the basis of biochemical features, was reinvestigated. Both reference strains and two other isolates, 6/1 (=LMG 22829) originating from a charcoal-broiled river lamprey and IE38.4 (=LMG 22830) from the air of a poultry slaughter by-product processing plant, occupied a clearly separate position, on the basis of sequence analysis of the housekeeping gene pheS (encoding the phenylalanyl-tRNA synthase α-subunit), relative to the type strain of E. raffinosus and all other enterococcal species with validly published names. 16S rRNA gene sequencing of strains LMG 13603, LMG 14595, 6/1 and IE38.4 confirmed their phylogenetic position in the Enterococcus avium species group, there being more than 99 % 16S rRNA gene sequence similarity to most members of the group, including E. raffinosus, and revealed Enterococcus pseudoavium as the closest phylogenetic relative (99·8–99·9 %). Further phenotypic and genotypic analyses using whole-cell-protein electrophoresis, (GTG)5-PCR fingerprinting, ribotyping and DNA–DNA hybridization experiments demonstrated that all four strains represent a novel enterococcal species, for which the name Enterococcus devriesei sp. nov. is proposed. The type strain is LMG 14595T (=CCM 7299T).

Clostridium botulinum type B was detected by multiplex PCR in the intestinal contents of a suddenly deceased 11-week-old infant and in vacuum cleaner dust from the patient's household. C. botulinum was also isolated from the deceased infant's intestinal contents and from the household dust. The genetic similarity of the two isolates was demonstrated by pulsed-field gel electrophoresis and randomly amplified polymorphic DNA analysis, thereby confirming that dust may act as a vehicle for infant botulism that results in sudden death.

The largest reported outbreak of type C botulism in fur production animals is described. Epidemiological investigation of 117 out of 157 (response rate, 74.5%) farms revealed that 44,130 animals died or were euthanized, while 8,033 animals with milder symptoms recovered. The overall death rate in all animals at risk was 21.7%. The death rates were significantly higher in blue and shadow foxes (24.2 and 27.8%, respectively) than in silver and blue silver foxes and minks (below 4%). All minks had been immunized against botulinum toxin type C. Deaths were associated with feed manufactured by a local processor, 83 of whose customer farms (70.9%) reported dead or sick animals. Five feedlots out of 19 delivered to the farms on the day preceding the onset of the outbreak (day 2) were associated with a death rate higher than 40%. These feedlots consisted of fresh feed processed on day 2 and feed processed 1 day earlier (day 1). In laboratory analysis, the day 2 feed contained botulinum toxin type C (>600 minimum lethal doses/g), while the day 1 feed did not contain toxin. Toxin was not detected in feed raw-material samples. Clostridium botulinum type C was detected by PCR in some feed components and in feed. However, as the feed temperature was continuously 8°C or below and the pH was continuously 5.6 or below according to the manufacturer, it seems unlikely that spore germination and toxin formation occurred during overnight storage. Hence, the events leading to toxin formation were not determined.

Thermal inactivation of nonproteolytic Clostridium botulinum type E spores was investigated in rainbow trout and whitefish media at 75 to 93°C. Lysozyme was applied in the recovery of spores, yielding biphasic thermal destruction curves. Approximately 0.1% of the spores were permeable to lysozyme, showing an increased measured heat resistance. Decimal reduction times for the heat-resistant spore fraction in rainbow trout medium were 255, 98, and 4.2 min at 75, 85, and 93°C, respectively, and those in whitefish medium were 55 and 7.1 min at 81 and 90°C, respectively. The z values were 10.4°C in trout medium and 10.1°C in whitefish medium. Commercial hot-smoking processes employed in five Finnish fish-smoking companies provided reduction in the numbers of spores of nonproteolytic C. botulinum of less than 10 3 . An inoculated-pack study revealed that a time-temperature combination of 42 min at 85°C (fish surface temperature) with >70% relative humidity (RH) prevented growth from 10 6 spores in vacuum-packaged hot-smoked rainbow trout fillets and whole whitefish stored for 5 weeks at 8°C. In Finland it is recommended that hot-smoked fish be stored at or below 3°C, further extending product safety. However, heating whitefish for 44 min at 85°C with 10% RH resulted in growth and toxicity in 5 weeks at 8°C. Moist heat thus enhanced spore thermal inactivation and is essential to an effective process. The sensory qualities of safely processed and more lightly processed whitefish were similar, while differences between the sensory qualities of safely processed and lightly processes rainbow trout were observed.

Listeria monocytogenes strains that were isolated from 314 human listeriosis cases in Finland during an 11-year period were analyzed by O:H serotyping and pulsed-field gel electrophoresis (PFGE). Serotyping divided the isolates into five serotypes, the most common being 1/2a (53%) and 4b (27%). During the study period, the number of cases caused by serotype 1/2a increased from 22% in 1990 to 67% in 2001, and those caused by serotype 4b decreased from 61 to 27%, respectively. PFGE with restriction enzyme Asc I divided the strains into 81 PFGE genotypes; among strains of serotypes 1/2a and 4b, 49 and 18 PFGE types were seen, respectively. PFGE type 1 (serotype 1/2a) was the most prevalent single type (37 strains). Together with six other, closely related PFGE types, PFGE type 1 formed a group of 71 strains, representing 23% of all 314 strains. Strains of PFGE type 1 have also been isolated from cold smoked fish, suggesting a source of human infections caused by this type. Moreover, PFGE type 24 (serotype 1/2c) was significantly associated with gender: 5% of 180 male subjects but none of 132 female subjects ( P = 0.012). An electronic database library was created from the PFGE profiles to make possible the prompt detection of new emerging profiles and the tracing of potential infection clusters in the future.

http://www.elsevier.com/locate/issn/01681605 Spoilage characterised by strong slime and gas formation affected some manufacture lots of 2 an acetic-acid Baltic herring (Culpea haerengus membras) preserve after few weeks’ storage 3 at 0-6°C. The product consisted of herring filets in acetic acid marinade containing sugar, 4 salt, allspice and carrot slices. Microbiological analyses of the spoiled product showed high 5 lactic acid bacterium (LAB) levels ranging from 4.5 ×108 to 2.4 × 109 CFU/g. Yeasts were 6 not detected in any of the herring samples. Since LAB contaminants seldom are associated 7 with fresh fish, LAB populations associated with marinade ingredients (carrots, allspice) 8 were also analysed. The highest LAB levels exceeding 107 CFU/g were detected in 9 equilibrium modified atmosphere packaged baby carrots whereas the levels detected in the 10 allspice samples did nor exceed 4.3 × 105. A total of 176 randomly selected LAB isolates 11 originating from herring, carrot and allspice samples were further identified to species level 12 using a 16 and 23S rRNA gene RFLP (ribotyping) database. Leuconostoc gelidum and 13 Leuconostoc gasicomitatum strains dominated both in the spoiled herring and carrot samples. 14 These species are heterofermentative producing CO2 from glucose and they also produce 15 dextran from sucrose. Inoculation of some commercial herring products with spoilage- 16 associated L. gelidum and L. gasicomitatum strains verified that these strains have the 17 capability of producing slime and gas in herring preserves although slime formation was not 18 as strong as in the original samples. Since L. gelidum and L. gasicomitatum strains were 19 commonly detected in carrots, carrot slices used for the fish marinade were considered to be 20 the probable source of these specific spoilage organisms. 21

http://www.elsevier.com/locate/issn/01681605 Lactic acid bacteria (LAB) in retail, modified-atmosphere-packaged (MAP), marinated broiler meat strips on sell-by day were mainly identified as Leuconostoc gasicomitatum. A total of 32 packages, 3 to 5 packages of 7 differently marinated broiler meat products, were studied at the end of the producer-defined shelf life (at 6ºC, 7 to 9 days depending on the manufacturer). Prior to the microbiological analyses, appearance and smell of the product was checked and pH measured. Bacteria were cultured on MRS and Tomato Juice Agar (TJA), Rogosa SL agar (SLA), Plate Count Agar (PCA) and Streptomycin Thallium Acetate Agar (STAA) for the enumeration of LAB, lactobacilli, total bacterial count and B. thermosphacta, respectively. The average CFU/g of the 32 packages was 2.3 × 108 on PCA. The highest bacterial average, 3.1 × 108, was recovered on TJA, the corresponding CFU/g averages on MRS and SLA being 2.3 × 108 and 1.3 × 108, respectively. Despite the high LAB numbers, radical spoilage changes such as unpleasant odor, slime production and formation of gas were not recognized. B. thermosphacta did not form a significant part of the bacterial population since none of the levels exceeded the spoilage threshold level of 105 CFU/g reported in previous studies. In order to characterize the dominating LAB population, as many as 85, 85 and 88 colonies from MRS, TJA and SLA, respectively, were randomly picked and cultured pure. LAB were identified to species level using a 16 and 23S rDNA HindIII RFLP (ribotyping) database. Fifty-six of the 170 isolates picked from the non-selective LAB media (MRS and TJA) were identified as Leuconostoc gasicomitatum, followed by Carnobacterium divergens (41 isolates), Lactobacillus sakei and Lactobacillus curvatus subsp. melibiosus (31 isolates) and Lactobacillus curvatus subsp. curvatus (20 isolates) species. SLA proved not to be completely selective for lactobacilli because the growth of Leuconostoc spp. was not inhibited, Carnobacterium spp. were the only species not detected on SLA.

Seventy-eight Enterococcus faecium strains from various sources were characterized by random amplified polymorphic DNA (RAPD)-PCR, amplified fragment length polymorphism (AFLP), and pulsed-field gel electrophoresis (PFGE) analysis of Sma I restriction patterns. Two main genomic groups (I and II) were obtained in both RAPD-PCR and AFLP analyses. DNA-DNA hybridization values between representative strains of both groups demonstrated a mean DNA-DNA reassociation level of 71%. PFGE analysis revealed high genetic strain diversity within the two genomic groups. Only group I contained strains originating from human clinical samples or strains that were vancomycin-resistant or beta-hemolytic. No differentiating phenotypic features between groups I and II were found using the rapid ID 32 STREP system. The two groups could be further subdivided into, respectively, four and three subclusters in both RAPD-PCR and AFLP analyses, and a high correlation was seen between the subclusters generated by these two methods. Subclusters of group I were to some extent correlated with origin, pathogenicity, and bacteriocinogeny of the strains. Host specificity of E. faecium strains was not confirmed.

This study was carried out in order to evaluate the contamination of the pig-slaughtering line with pathogenic Yersinia enterocolitica carrying the yadA gene. A total of 292 samples were collected from the slaughterhouse; 131 swab samples from pig carcasses, ears, livers, kidneys, and hearts; 89 swab samples from the environment; and 72 sedimentation samples from the air. All surface samples were studied with both the polymerase chain reaction (PCR) and culture methods. The contamination rate of edible pig offals was high with both methods. Using PCR, the detection rates of yadA-positive Y. enterocolitica for livers, kidneys, and hearts were 38, 86, and 63%, respectively, and using the culture method, the detection rates were 31, 69, and 50%, respectively. Pathogenic Y. enterocolitica was also detected from different environmental sites in the slaughterhouse. Using PCR, 13% of the surface samples from the environment were contaminated with yadA-positive Y. enterocolitica. PCR-positive samples were found on the brisket saw, the hook from which the pluck set (heart, lungs, esophagus, trachea, diaphragm, liver, kidneys, and tongue with tonsils) hang, the knife used for evisceration, the floors in the eviscerating area and the weighing area, the meat-cutting table, the aprons used by trimming workers, the computer used in the meat-inspection area, and the coffeemaker used by slaughterhouse workers. The respective detection rate (6%) was considerably lower when we used the culture method. Pathogenic Y. enterocolitica was isolated from the air in the bleeding area. Bioserotype 4/O:3 was the only pathogenic bioserotype isolated in this study. A total of 113 isolates of type 4/O:3 were characterized with pulsed-field gel electrophoresis using NotI and XbaI digests. By combining these profiles, nine different pulsotypes were obtained, the most common of which (1a) was found in 19 (61%) of 31 samples from different sites. This is the same type that has dominated in pig tonsils, which suggests that tonsils may be the source of Y. enterocolitica contamination in the slaughterhouse. The four pulsotypes (1a, 4g, 6g, and 19q) found on edible offals were the same as those found in tonsils, which supports our hypothesis that tonsils are the contamination source for the liver, heart, and kidneys.

In February 1999, an outbreak of listeriosis caused by Listeria monocytogenes serotype 3a occurred in Finland. All isolates were identical. The outbreak strain was first isolated in 1997 in dairy butter. This dairy began delivery to a tertiary care hospital (TCH) in June 1998. From June 1998 to April 1999, 25 case patients were identified (20 with sepsis, 4 with meningitis, and 1 with abscess; 6 patients died). Patients with the outbreak strain were more likely to have been admitted to the TCH than were patients with other strains of L. monocytogenes (60% vs. 8%; odds ratio, 17.3; 95% confidence interval, 2.8-136.8). Case patients admitted to the TCH had been hospitalized longer before cultures tested positive than had matched controls (median, 31 vs. 10 days; P = .008). An investigation found the outbreak strain in packaged butter served at the TCH and at the source dairy. Recall of the product ended the outbreak.

The genetic biodiversity of Clostridium botulinum type E strains was studied by pulsed-field gel electrophoresis (PFGE) with two macrorestriction enzymes ( Sma I- Xma I and Xho I) and by randomly amplified polymorphic DNA (RAPD) analysis with two primers (OPJ 6 and OPJ 13) to characterize 67 Finnish isolates from fresh fish and fishery products, 15 German isolates from farmed fish, and 10 isolates of North American or North Atlantic origin derived mainly from different types of seafood. The effects of fish species, processing, and geographical origin on the epidemiology of the isolates were evaluated. Cluster analysis based on macrorestriction profiles was performed to study the genetic relationships of the isolates. PFGE and RAPD analyses were combined and resulted in the identification of 62 different subtypes among the 92 type E isolates analyzed. High genetic biodiversity among the isolates was observed regardless of their source. Finnish and North American or North Atlantic isolates did not form distinctly discernible clusters, in contrast with the genetically homogeneous group of German isolates. On the other hand, indistinguishable or closely related genetic profiles among epidemiologically unrelated samples were detected. It was concluded that the high genetic variation was probably a result of a lack of strong selection factors that would influence the evolution of type E. The wide genetic biodiversity observed among type E isolates indicates the value of DNA-based typing methods as a tool in contamination studies in the food industry and in investigations of botulism outbreaks.

The minimum, optimum, and maximum growth temperatures of ropy slime-producing lactic acid bacteria able to spoil vacuum-packed cooked meat products were determined on MRS-agar with temperature-gradient incubator GradiplateR W10. The minimum growth temperatures of slime-producing lactobacilli and Leuconostoc mesenteroides strain D1 were below -1°C and 4°C, respectively. The low minimum growth temperature allows these bacteria to compete with other bacteria in meat processing plants and in meat products causing ropiness problems. The maximum growth temperatures varied between 36.6-39.8°C. The maximum growth temperature of lactobacilli seemed to be an unstable character. Single lactobacilli colonies were able to grow above the actual maximum growth temperature, which is determined as the edge of continuous growth of the bacteria. The significance of this phenomenon needs further study.

The distribution of Clostridium botulinum serotypes A, B, E, and F in Finnish trout farms was examined. A total of 333 samples were tested with a neurotoxin-specific PCR assay. C. botulinum type E was found in 68% of the farm sediment samples, in 15% of the fish intestinal samples, and in 5% of the fish skin samples. No other serotypes were found. The spore counts determined by the most-probable-number method were considerably higher for the sediments than for the fish intestines and skin; the average values were 2,020, 166, and 310 C. botulinum type E spores kg −1 , respectively. The contamination rates in traditional freshwater ponds and marine net cages were high, but in concrete ponds equipped with sediment suction devices the contamination rates were significantly lower. Pulsed-field gel electrophoresis (PFGE) typing of 42 isolates obtained in this survey and 12 North American reference strains generated 28 pulsotypes upon visual inspection, suggesting that there was extensive genetic diversity and that the discriminatory power of PFGE typing in C. botulinum type E was high. A numerical analysis of Sma I- Xma I macrorestriction profiles confirmed these findings, as it divided the 54 isolates into 15 clusters at a similarity level of 76%. For this material, this level of similarity corresponded to a three-band difference in the macrorestriction profiles, which indicated that there is no genotypic proof of a close epidemiological relationship among the clusters.

Pulsed-field gel electrophoresis (PFGE) was optimized for genomic analyses of Clostridium botulinum (nonproteolytic) group II. DNA degradation problems caused by extracellular DNases were overcome by fixation of cells with formaldehyde prior to isolation. A rapid (4-h) in situ DNA isolation method was also assessed and gave indistinguishable results. Genomic DNA from 21 strains of various geographical and temporal origins was digested with 15 rare-cutting restriction enzymes. Of these, Apa I, Mlu I, Nru I, Sma I, and Xho I gave the most revealing PFGE patterns, enabling strain differentiation. Twenty strains yielded PFGE patterns containing 13 pulsotypes. From summation of Mlu I, Sma I, and Xho I restriction fragments, the genome size of C. botulinum group II was estimated to be 3.6 to 4.1 Mb (mean ± standard deviation = 3,890 ± 170 kb). The results substantiate that after problems due to DNases are overcome, PFGE analysis will be a reproducible and highly discriminating epidemiological method for studying C. botulinum group II at the molecular level.

http://www.elsevier.nl/locate/01681605 Aseptically handled Frankfurters were treated with a commercial Lactobacillus alimentarius biopreservate and inoculated with different cell concentrations of four ropy slime-producing Lactobacillus sake strains. The packages were vacuum sealed and kept at 6°C for 28 days, after which the production of ropy slime was evaluated. The inoculation test was controlled by sealing the different control packages containing either aseptically manufactured sausages without any bacterial inoculation, packages containing biopreservate only or packages inoculated only with the four different ropy slime-producing strains. Authenticity of the biopreservate strain after the cold storage period was ascertained by performing EcoRI restriction endonuclease analysis of 30 randomly selected isolates originating from the biopreservate control packages. All patterns were identical to the pattern of the original L. alimentarius biopreservate strain. The biopreservate was found to be quite ineffective against the four ropy slime-producing L. sake strains. The strongest slime producers inoculated with approximately 1 CFU/cm2 could compete efficiently with the L. alimentarius having an onset concentration of 107 CFU/cm2 on sausage surfaces. This commercial biopreservate failed to occupy the vital niche of the four ropy slime-producing L. sake strains leading to spoilage in almost all packages.

Microbiological contamination of air and carcasses was studied in four slaughterhouses by using impactor samples taken at the back-splitting and weighing areas and by sampling carcasses with the swabbing method. Air flow was determined by an air-flow detector, and the movement of workers was observed. The air contamination level in the back-splitting areas (2.25 log CFU/100 liters of air) was generally higher than that in the weighing areas (2.03 log CFU/100 liters of air). Associations between the microbiological contamination of air and carcasses with the movements of workers were found. Layout of the slaughtering line was shown to be important in decreasing airborne contamination. Separation of the clean and unclean parts of the line as well as separation of the weighing area from the other clean parts of the line decreased the contamination level. It appears that airborne bacteria have an important role in carcass contamination.

Ribotyping was used to characterize 34 Clostridium perfringens strains isolated from 10 food poisoning cases and outbreaks over a 7-year period. Twelve different ribopatterns were generated by EcoRI digestion. In eight food poisoning cases and outbreaks, all of the ribotypes of each food and stool isolate were found to be identical. Two C. perfringens isolates showed unique patterns. Ribotyping was found to be a useful tool for determining the genetic relationship of C. perfringens isolates in the context of foodborne poisoning cases.

We found an epidemiological association of Hafnia alvei with diarrhea, because the organism was isolated from 12 of 77 (16%) adult Finnish tourists to Morocco who developed diarrhea and from 0 of 321 tourists without diarrhea (P < 0.001). From another group of 112 adult Finnish diarrheal patients, only 2 (2%) yielded H. alvei. In contrast to some Bangladeshi strains of H. alvei, the Finnish strains were negative for the attachment-effacement lesion by an in vitro fluorescent acting staining test and also did not show homology to the Escherichia coli attachment-effacement gene (eaeA) by PCR. These results suggest that a mechanism or mechanisms other than the attachment-effacement lesion may also be involved in the association of H. alvei with diarrhea.

The effect of NaNO(2) and NaCl on the growth of 24 lactic acid bacteria strains isolated from vacuum-packed cooked ring sausages were examined by analyzing different growth parameters with Bioscreen. NaNO(2) had a very limited effect on the growth of lactic acid bacteria at 50 and 100 mg/l but at 400 mg/l a more pronounced inhibitory effect was found. Bacterial growth was enhanced by 1–2% (w/v) of added NaCl, while NaCl concentrations above 3% (w/v) had a clear inhibitory effect. Leuconostoc isolates seemed to be more sensitive to sodium nitrite and sodium chloride than homofermentative lactobacilli strains. Among homofermentative lactobacilli, the strains resembling Lactobacillus curvatus were more sensitive to NaCl than those resembling Lactobacillus sake.

Three slide agglutination tests for identification of Staphylococcus aureus were compared. The agglutination tests used for evaluation were Staphaurex (Wellcome Diagnostics), Staphyslide-Test (BioMerieux), and ANI S. aureus TEST (Ani Biotech Oy). A total of 347 isolates were analyzed, including 288 strains of S. aureus, 49 of S. epidermis, 11 of S. intermedius, 12 strains of other staphylococci and 14 non-staphylococcal strains. One hundred of the S. aureus strains were isolates from cases of food poisoning, 129 from mastitis and 59 from other clinical cases. The sensitivities of the tests were also compared using diluted suspensions of S. aureus strains and with purified Protein A dilutions. The results showed that the sensitivities of the tests were 98.6 %, 97.9 % and 99.0 % for Staphaurex, Staphyslide-test and ANI S. aureus TEST, respectively. The specificities were 100 % for the Staphyslide test and 98.8 % for both the ANI S. aureus TEST and the Staphaurex test. The sensitivities measured with diluted S. aureus strain suspensions and Protein A solutions were equal with the Staphaurex and ANI S. aureus TEST.All the agglutination tests studied proved to be practical, easy to use and accurate for the rapid identification of S. aureus strains from culture isolates.

Pulsed-field gel electrophoresis (PFGE) was applied to the study of the similarity of 55 strains of proteolytic Clostridium botulinum ( C. botulinum group I) types A, AB, B, and F. Rare-cutting restriction enzymes ApaI, AscI, MluI, NruI, PmeI, RsrII, SacII, SmaI, and XhoI were tested for their suitability for the cleavage of DNA of five proteolytic C. botulinum strains. Of these enzymes, SacII, followed by SmaI and XhoI, produced the most convenient number of fragments for genetic typing and were selected for analysis of the 55 strains. The proteolytic C. botulinum species was found to be heterogeneous. In the majority of cases, PFGE enabled discrimination between individual strains of proteolytic C. botulinum types A and B. The different toxin types were discriminated at an 86% similarity level with both SacII and SmaI and at an 83% similarity level with XhoI. Despite the high heterogeneity, three clusters at a 95% similarity level consisting of more than three strains of different origin were noted. The strains of types A and B showed higher diversity than the type F organisms which formed a single cluster. According to this survey, PFGE is to be considered a useful tool for molecular epidemiological analysis of proteolytic C. botulinum types A and B. However, epidemiological conclusions based on PFGE data only should be made with discretion, since highly similar PFGE patterns were noticed, especially within the type B strains.

The effect of the pH on the boiling test was studied in 68 beef and 108 pork muscles. The pH had a significant effect on the sensory scores obtained from the boiling test. The effect was particularly pronounced in the odour of meat. In beef the odour scores remain steady for samples with a pH value under about 6.2 and start to increase rapidly in higher pH values. In pork the increase in scores appears to be linear. The odour observed in high pH meat was described by the judges as abnormal and ammonialike. In the case of meat with a high pH, the results of the boiling test at meat inspection should be interpreted with extreme caution.

The effect of cadmium (Cd) on the number of different faecal indicator bacteria in sewage, and on species composition of different indicator bacteria, was studied. Different amounts of Cd were added to aliquots of a sewage sample, and after 0, 4% and 24 h of Cd exposure at 20°C conforms, faecal coliforms, faecal streptococci and Clostridium perfringens were enumerated by the membrane filter method. The Cd-induced reduction in the number of coliforms and faecal coliforms during exposure was found to be greater than the decrease in the number of faecal streptococci. In the case of C. perfringens the Cd concentrations used produced no observable effect on the cell number. The addition of Cd changed the faecal coliforms and faecal streptococci density relationship. Escherichia coli seems to be more resistant to Cd than other coliforms and Streptococcus faecalis var. liquefaciens and Streptococcus durans more resistant to Cd than other faecal streptococci. No influence of Cd on gas production by faecal coliforms was observed. Faecal streptococci and C. perfringens seem to be better indicator bacteria than coliforms and faecal coliforms in evaluating the hygienic quality of Cd polluted sewage.