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Official food control is intended to ensure food safety in the food business. In Finland, inspections of food service are performed using a 4-point risk-based grading system. This study compared routine inspection results of outbreak and nonoutbreak establishments in restaurants and institutional catering to investigate whether certain inspection results were associated with the occurrence of foodborne outbreaks. Also a more specific sample of outbreak establishments was defined using strength of evidence registered for each outbreak. Grade distributions of specific inspected items were compared separately. No significant differences were seen in restaurants but in institutional catering significantly poorer inspection results (p 0.05) were detected in items concerning the order and cleanliness of facilities, surfaces and equipment in outbreak establishments. Effective correction of noncompliances in cleanliness of the food handling environment and equipment and constant maintenance of a favourable situation is essential in ensuring a high level of consumer safety in food service.

Clostridium botulinum is a significant food safety concern due to its ability to produce highly potent neurotoxin and resistant endospores. Vegetarian sausages have become a popular source of plant protein and alternative for meat products. While vegetarian sausages have not been linked to botulism, numerous outbreaks due to preserved vegetables suggest a frequent occurrence of C. botulinum spores in the raw material. The product formulation of vegetarian sausages involves limited NaCl and preservatives, and shelf-lives may be several months. The safety of vegetarian sausages thus relies mainly on heat treatment and chilled storage. The main food safety concern is C. botulinum Group II that can grow and produce toxin at refrigeration temperatures. Here we show a high overall prevalence (32%) of C. botulinum in 74 samples of vegetarian sausages from seven producers. Both Groups I and II strains and genes for neurotoxin types A, B, E and F were detected in the products. The highest cell counts (1200 spores/kg) were observed for C. botulinum Group II in products with remaining shelf-lives of 6 months at the time of purchase. We conclude that vacuum-packaged vegetarian sausage products frequently contain C. botulinum spores and may possess a high risk of C. botulinum growth and toxin production. Chilled storage below 3°C and thorough reheating before consumption are warranted.

Maximum growth temperature and growth limits in Luria-Bertani broth at different pH values and NaCl and ethanol concentrations were determined for 49 Yersinia pseudotuberculosis strains representing serotypes O:1, O:2, O:3, O:4, and O:5. In addition, the ability of the strains to grow at 0°C and the growth parameters at 1°C were determined. The maximum growth temperatures measured by Gradiplate temperature incubator varied between 42.2 and 43.7°C. All strains were able to grow at 0°C in Luria-Bertani broth within 17 days of incubation. At 1°C, differences were observed among strains in the maximum growth rates and area under the curve values based on optical density data, which suggests that some Y. pseudotuberculosis strains adapt faster to colder conditions. The mean maximum growth rates and area under the curve values at 1°C, as well as the mean maximum growth temperatures, were statistically significantly higher among serotype O:1 strains compared with O:3 strains and among biotype 1 compared with biotype 2 strains. All strains grew at pH 4.5, whereas none of the strains were able to grow at pH 4.2. The highest pH at which growth was observed varied between 9.0 and 9.3. For 14 strains the maximum NaCl concentration at which growth was observed was 4.8%, whereas 35 of the strains were able to grow at 5.0% NaCl. None of the strains showed growth at 5.2% NaCl. All strains were able to grow at 4.5% ethanol concentration (v/v), whereas 5.0% ethanol concentration was completely inhibitory to all strains. The observed limited physiological diversity among various Y. pseudotuberculosis strains may stem from the genetic homogeneity of the species.

Pigs are considered the main reservoir of Yersinia enterocolitica, and hence, understanding the ecology of this foodborne pathogen at the farm level is crucial. We calculated Bayesian estimates for the ability of a commercial enzyme-linked immunosorbent assay (ELISA) diagnostic test kit to detect antibodies against pathogenic Yersinia in pigs. The sensitivity and specificity of the test were 75.4% and 98.1%, respectively. We also studied the dynamics of Y. enterocolitica infection in 3 farrow-to-finish pig farms by following the same 30 pens of pigs through their lifetime from farrowing unit to slaughterhouse. Each farm was sampled 4 times, and 864 fecal and 730 serum samples were collected altogether. Pathogenic Y. enterocolitica 4/O:3 was isolated from 31.6% of the fecal samples by culturing, and Yersinia antibodies were detected in 38.2% of the serum samples with the commercial ELISA test. The pathogen was not isolated from farrowing units or all-in/all-out weaning units. However, in the weaning and fattening units using continuous management systems, the pathogen was isolated from every pen at some point of the study. After the pigs were transported into slaughterhouse, 150 tonsils were collected and 96.7% were positive by culturing. Among the strains isolated from feces and tonsils, 56 different genotypes of pathogenic Y. enterocolitica 4/O:3 were found by multilocus variable-number tandem-repeat analysis (MLVA). Finally, we collected tonsils of 266 sows from 115 farrowing farms, and Y. enterocolitica 4/O:3 was detected in 6.0% of the samples by the culture method, whereas 77.1% of the tonsils were serologically positive; the estimate for true seroprevalence was 95.8%. In conclusion, sows may not be the main source of Y. enterocolitica for piglets, although sows may still play a role in maintaining Y. enterocolitica in pig farms. Instead, pigs appear to get this foodborne pathogen mainly during the fattening period, especially if continuous management is applied.

Psychrotrophic foodborne pathogens, such as enteropathogenic Yersinia, which are able to survive and multiply at low temperatures, require cold shock proteins (Csps). The Csp superfamily consists of a diverse group of homologous proteins, which have been found throughout the eubacteria. They are related to cold shock tolerance and other cellular processes. Csps are mainly named following the convention of those in Escherichia coli. However, the nomenclature of certain Csps reflects neither their sequences nor functions, which can be confusing. Here, we performed phylogenetic analyses on Csp sequences in psychrotrophic enteropathogenic Yersinia and E. coli. We found that representative Csps in enteropathogenic Yersinia and E. coli can be clustered into six phylogenetic groups. When we extended the analysis to cover Enterobacteriales, the same major groups formed. Moreover, we investigated the evolutionary and structural relationships and the origin time of Csp superfamily members in eubacteria using nucleotide-level comparisons. Csps in eubacteria were classified into five clades and 12 subclades. The most recent common ancestor of Csp genes was estimated to have existed 3585 million years ago, indicating that Csps have been important since the beginning of evolution and have enabled bacterial growth in unfavorable conditions.

Pigs are considered the main reservoir of

Psychrotrophic foodborne pathogens, such as enteropathogenic Yersinia, which are able to survive and multiply at low temperatures, require cold shock proteins (Csps). The Csp superfamily consists of a diverse group of homologous proteins, which have been found throughout the eubacteria. They are related to cold shock tolerance and other cellular processes. Csps are mainly named following the convention of those in Escherichia coli. However, the nomenclature of certain Csps reflects neither their sequences nor functions, which can be confusing. Here, we performed phylogenetic analyses on Csp sequences in psychrotrophic enteropathogenic Yersinia and E. coli. We found that representative Csps in enteropathogenic Yersinia and E. coli can be clustered into six phylogenetic groups. When we extended the analysis to cover Enterobacteriales, the same major groups formed. Moreover, we investigated the evolutionary and structural relationships and the origin time of Csp superfamily members in eubacteria using nucleotide-level comparisons. Csps in eubacteria were classified into five clades and 12 subclades. The most recent common ancestor of Csp genes was estimated to have existed 3585 million years ago, indicating that Csps have been important since the beginning of evolution and have enabled bacterial growth in unfavorable conditions.

Yersinia pseudotuberculosis is an important foodborne pathogen threatening modern food safety due to its ability to survive and grow at low temperatures. DEAD-box RNA helicase CsdA has been shown to play an important role in the low-temperature growth of psychrotrophic Y. pseudotuberculosis. A total of five DEAD-box RNA helicase genes (rhlB, csdA, rhlE, dbpA, srmB) have been identified in Y. pseudotuberculosis IP32953. However, their role in various stress conditions used in food production is unclear. We studied the involvement of the DEAD-box RNA helicase-encoding genes in the cold tolerance of Y. pseudotuberculosis IP32953 using quantitative real-time reverse transcription (RT-qPCR) and mutational analysis. Quantitative RT-PCR revealed that mRNA transcriptional levels of csdA, rhlE, dbpA and srmB were significantly higher after cold shock at 3 degrees C compared to non-shocked culture at 28 degrees C, suggesting the involvement of these four genes in cold shock response at the transcriptional level. The deletion of csdA ceased growth, while the deletion of dbpA or srmB significantly impaired growth at 3 degrees C, suggesting the requirement of these three genes in Y. pseudotuberculosis at low temperatures. Growth of each DEAD-box RNA helicase mutant was also investigated under pH, osmotic, ethanol and oxidative stress conditions. The five helicase-encoding genes did not play major roles in the growth of Y. pseudotuberculosis IP32953 under pH, osmotic, ethanol or oxidative stress.

The prevalence, counts and genetic diversity of Listeria monocytogenes in ready-to-eat (RTE) vacuum and modified atmosphere packaged meat and fish products was studied in Estonia. Within two consecutive years 370 RTE food samples were collected at retail level from which 11% were found to be positive for L. monocytogenes. Contamination was higher among RTE fish products (17%) than in RTE meat products (6%). Generally, the counts of L. monocytogenes in positive products remained under ten colony forming units (CFU) per gram of product. Only 1.6% of the RTE meat and fish products contained L. monocytogenes in range of 10–100 CFU/g and 0.3% more than 100 CFU/g at the end of shelf-life. The food category containing highest L. monocytogenes prevalence was RTE lightly salted fish products with the prevalence of 32%. Only one (0.3%) RTE food sample exceeded the 100 CFU/g food safety criterion set out in the EU Regulation 2073/2005. Pulsed-field gel electrophoresis (PFGE) characterization of the isolates showed an overall similarity higher than 70%, and nine clusters based on 100% similarity were revealed. PFGE genotyping revealed that the few predominant pulsotypes were associated with particular food plants.

Bacteria have evolved a number of mechanisms for coping with stress and adapting to changing environmental conditions. Many bacteria produce small cold shock proteins (Csp) as a response to rapid temperature downshift (cold shock). During cold shock, the cell membrane fluidity and enzyme activity decrease, and the efficiency of transcription and translation is reduced due to stabilization of nucleic acid secondary structures. Moreover, protein folding is inefficient and ribosome function is hampered. Csps are thought to counteract these harmful effects by serving as nucleic acid chaperons that may prevent the formation of secondary structures in mRNA at low temperature and thus facilitate the initiation of translation. However, some Csps are non-cold inducible and they are reported to be involved in various cellular processes to promote normal growth and stress adaptation responses. Csps have been shown to contribute to osmotic, oxidative, starvation, pH and ethanol stress tolerance as well as to host cell invasion. Therefore, Csps seem to have a wider role in stress tolerance of bacteria than previously assumed. Yersinia enterocolitica and Yersinia pseudotuberculosis are enteropathogens that can spread through foodstuffs and cause an enteric infection called yersiniosis. Enteropathogenic Yersinia are psychrotrophs that are able to grow at temperatures close to 0oC and thus they set great challenges for the modern food industry. To be able to efficiently control psychrotrophic Yersinia during food production and storage, it is essential to understand the functions and roles of Csps in stress response of enteropathogenic Yersinia.

A total of 253 multiple-locus variable-number tandem-repeat analysis (MLVA) types among 634 isolates were discovered while studying the genetic diversity of porcine Yersinia enterocolitica 4/O:3 isolates from eight different European countries. Six variable-number tandem-repeat (VNTR) loci V2A, V4, V5, V6, V7, and V9 were used to study the isolates from 82 farms in Belgium (n = 93, 7 farms), England (n = 41, 8 farms), Estonia (n = 106, 12 farms), Finland (n = 70, 13 farms), Italy (n = 111, 20 farms), Latvia (n = 66, 3 farms), Russia (n = 60, 10 farms), and Spain (n = 87, 9 farms). Cluster analysis revealed mainly country-specific clusters, and only one MLVA type consisting of two isolates was found from two countries: Russia and Italy. Also, farm-specific clusters were discovered, but same MLVA types could also be found from different farms. Analysis of multiple isolates originating either from the same tonsils (n = 4) or from the same farm, but 6 months apart, revealed both identical and different MLVA types. MLVA showed a very good discriminatory ability with a Simpson's discriminatory index (DI) of 0.989. DIs for VNTR loci V2A, V4, V5, V6, V7, and V9 were 0.916, 0.791, 0.901, 0.877, 0.912, and 0.785, respectively, when studying all isolates together, but variation was evident between isolates originating from different countries. Locus V4 in the Spanish isolates and locus V9 in the Latvian isolates did not differentiate (DI 0.000), and locus V9 in the English isolates showed very low discriminatory power (DI 0.049). The porcine Y. enterocolitica 4/O:3 isolates were diverse, but the variation in DI demonstrates that the well discriminating loci V2A, V5, V6, and V7 should be included in MLVA protocol when maximal discriminatory power is needed.

The effect of three different concentrations of sodium nitrite (0, 75, and 120 mg/kg) on growth and toxigenesis of group II (nonproteolytic) Clostridium botulinum type B was studied in Finnish wiener-type sausage, bologna-type sausage, and cooked ham. A low level of inoculum (2.0 log CFU/g) was used for wiener-type sausage and bologna-type sausage, and both low (2.0 log CFU/g) and high (4.0 log CFU/g) levels were used for cooked ham. The products were formulated and processed under simulated commercial conditions and stored at 8°C for 5 weeks. C. botulinum counts were determined in five replicate samples of each nitrite concentration at 1, 3, and 5 weeks after thermal processing. All samples were positive for C. botulinum type B. The highest C. botulinum counts were detected in nitrite-free products. Toxigenesis was observed in nitrite-free products during storage, but products containing either 75 or 120 mg/kg nitrite remained nontoxic during the 5-week study period, suggesting that spores surviving the heat treatment were unable to germinate and develop into a toxic culture in the presence of nitrite. The results suggest that the safety of processed meat products with respect to group II C. botulinum type B can be maintained even with a reduced concentration (75 mg/kg) of sodium nitrite.

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.

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.

Botulinum neurotoxin (BoNT) is produced by the anaerobic bacterium Clostridium botulinum during its vegetative growth. C. botulinum strains are commonly found in the environment, animals and in food raw materials. The bacterium forms dormant endospores, which do not produce toxins. However, the spores are highly resistant to environmental stress and thus maintain the toxic potential in environmental and agricultural systems and the food chains that occasionally provide conditions favourable for spore germination and outgrowth into a toxic culture. Besides causing botulism to man and animals, BoNTs are widely exploited by medical industries in treatment of spastic diseases. Thoroughly validated, sensitive and specific assays for neurotoxin detection are thus needed not only for laboratory diagnostics of botulism outbreaks but also for potency testing of the pharmaceutical neurotoxin products.

DNA-pohjaiset genotyypitysmenetelmät ovat nykyään tärkeitä työkaluja bakteerien tunnistamisessa ja sukulaisuussuhteiden selvittämisessä. Eri genotyypitysmenetelmien soveltuvuudessa eri bakteerilajeille sekä erilaisiin tutkimustarkoituksiin on kuitenkin eroja. Tässä väitöstutkimuksessa selvitettiin amplified fragment length polymorphism (AFLP) –menetelmän soveltuvuutta elintarvikevälitteisten tautia-aiheuttavien bakteerien Listeria monocytogeneksen, Clostridium botulinumin ja Clostridium perfringensin geneettiseen tyypittämiseen. Väitöstutkimuksessa kehitettiin AFLP-menetelmästä hyvin erotteleva sovellutus L. monocytogenes, C. botulinum ja C. perfringens kantojen karakterisointiin. Menetelmän todettiin olevan hyvin toistettava, nopea sekä helppokäyttöinen. Tutkimuksessa todettiin myös, että AFLP-menetelmää voidaan käyttää apuna eri listeria- ja klostridi-lajien tunnistamisessa. Menetelmästä on hyötyä etenkin klostridi-lajien diagnostiikassa, sillä nykyisin käytössä olevat diagnostiset menetelmät ovat osin puutteellisia. Kehitetyn AFLP sovellutuksen avulla luodut AFLP-tyypitystulokset voidaan kerätä tietokantoihin, joita voidaan hyödyntää mm. epidemiologisissa tutkimuksissa, elintarviketeollisuuden kontaminaatioreittien selvittämisessä, ruokamyrkytysepidemia selvityksissä sekä listeria- ja klostridi-lajien tunnistamisessa. Väitöstyössä tutkittiin AFLP-menetelmän avulla L. monocytogeneksen kontaminaatioreittejä eineksiä valmistavassa elintarvikelaitoksessa kahdeksan vuoden aikana. Siivousrutiinien, valmistettavan tuotteen ja osastoinnin todettiin vaikuttavan tuotantoympäristön kontaminaatiotilanteeseen kuumennettuja eineksiä valmistavilla osastoilla. Osastolla, jolla valmistettiin eineksiä, joiden valmistusprosessiin ei kuulu kuumennuskäsittelyä, raaka-aineiden todettiin saastuttavan valmiita tuotteita. Siten raaka-aineiden laatuun tulisi kiinnittää erityistä huomiota kun valmistetaan eineksiä, joita ei kuumennuskäsitellä. Lisäksi tutkimuksessa osoitettiin, että tuotantolinjan rakenteelliset muutokset voivat edesauttaa elintarvikelaitosta pääsemään eroon listeriakontaminaatiosta. Tutkimuksessa selvitettiin myös elintarvikkeiden tuotantoympäristössä pitkäkestoisen (persistoivan) kontaminaation aiheuttavien sekä satunnaisesti (sporadisesti) esiintyvien L. monocytogenes –kantojen välisiä eroja AFLP- ja pulssikenttäelektroforeesi-menetelmillä. Vaikka persistoivien kantojen todettiin eroavan sporadisista kannoista, ei persistoivilla kannoilla todettu olevan yhteistä geneettistä alkuperää. In epidemiological studies, techniques that effectively discriminate between individual bacterial strains are essential. Recent developments in molecular techniques necessitate an ongoing need to tailor new genotyping methods for optimal characterization of different bacterial species and to evaluate their performance and suitability for research purposes. In this thesis amplified fragment length polymorphism (AFLP) analysis was tailored for optimal characterization of Listeria monocytogenes and Clostridium botulinum. The suitability of the developed AFLP protocol to type L. monocytogenes, C. botulinum and Clostridium perfringens at strain level was evaluated. AFLP proved to be a highly reproducible, easy-to-use, relatively fast and highly discriminative approach. When AFLP was applied to L. monocytogenes strains, its discriminatory power was shown to equal that of PFGE, which is considered the current gold standard for molecular fingerprinting of L. monocytogenes. These features make AFLP analysis a useful alternative to other genotyping methods in, for example, outbreak investigations and contamination route studies. Since phenotypic identification of Clostridium isolates is laborious, the suitability of AFLP for genomic species identification was assessed. The AFLP technique was applied to 129 strains representing 24 different Clostridium species. AFLP differentiated all species tested, except for Clostridium ramosum and Clostridium limosum. AFLP also differentiated between six different Listeria species. If AFLP profiles of well-defined strains are collected in identification libraries, the database can be a valuable additional tool for identification of Clostridium and Listeria species. Due to high throughput of samples, AFLP proved to be especially suitable for screening large numbers of isolates. AFLP was also used to trace contamination routes of L. monocytogenes in a chilled food processing plant producing ready-to-eat and ready-to-reheat meals during an 8-year period. Cleaning routines, product type and degree of compartmentalization seemed to have an influence on the contamination status in compartments that produced cooked meals. In addition, raw materials were shown to cause contamination of uncooked meals. Thus, special attention should be paid to quality control of raw ingredients when uncooked ready-to-eat meals are produced. This work also demonstrated that structural adjustments of a production line may facilitate the eradication of L. monocytogenes from the food processing environment. AFLP and PFGE analysis of sporadic L. monocytogenes strains and strains that cause persistent plant contamination revealed that persistent strains differ from sporadic strains. However, no specific evolutionary lineage of persistent strains was observed.

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.

Factors influencing Clostridium botulinum contamination in the honey production environment were evaluated in a 3-year survey. A number of 1,168 samples from 100 apiaries and related facilities were analysed for the presence of C. botulinum types A, B, E and F, using multiplex polymerase chain reaction targeted to botA, botB, botE and botF genes. Production methods and environmental factors were registered using a questionnaire and by personal observation. Clostridium botulinum was shown to be a common finding throughout the whole honey production chain, and the type most frequently detected was group I type B. In a pulsed-field gel electrophoresis (PFGE) analysis of 202 group I type B isolates, only six different PFGE profiles were observed, of which two clearly distinct profiles predominated. This may indicate the existence of at least two different genetic lineages. The high prevalence of C. botulinum in soil and in samples associated with beeswax suggests the accumulation of soil-derived botulinal spores in wax. Additionally, according to Spearman's rank order correlation and multivariate analysis, production hygiene-dependent factors have a significant influence on the contamination, and thus the number and frequency of C. botulinum spores in honey could possibly be diminished by increasing hygienic level in honey production.

Summary Nine restriction enzyme combinations and 108 different primer combinations were initially tested for suitability for amplified fragment length polymorphism (AFLP) analysis of Listeria monocytogenes; the combination of Hin dIII and Hpy CH4IV showed consistently strong signals on gels, amplified an adequate number of DNA fragments and detected polymorphism among closely related strains based on Asc I macrorestriction profiles. AFLP also distinguished between L. monocytogenes, L. innocua, L. ivanovii, L. seeligeri, L. welshimeri and L. grayi species. All Listeria species showed species-specific clusters, with less than 33% similarity between different species. A total of 34 L. monocytogenes strains were characterised by using both AFLP and pulsed-field gel electrophoresis (PFGE). The results of AFLP analysis of L. monocytogenes strains were in concordance with those obtained by PFGE. Both methods identified 29 different genotypes of L. monocytogenes and had a high discrimination index (>0.999). By combining the results of AFLP and PFGE, subtype discrimination was further improved. Numerical analysis of both AFLP and PFGE profiles yielded three genomic groups of L. monocytogenes strains. AFLP was found to be faster and less labour-intensive than PFGE. We conclude that the AFLP protocol is a highly discriminatory, reproducible and valuable tool in characterisation of Listeria strains and may also be suitable for Listeria species identification.

This study was set up to evaluate the genetic similarity or dissimilarity of persistent and sporadic Listeria monocytogenes strains existing in eleven food processing facilities, including fish, dairy, meat and poultry processing plants. In each plant persistent and sporadic strains were selected on the basis of PFGE typing results. A total of 17 strains representing persistent strains and 38 sporadic strains originating from eleven food processing plants were included in the study. PFGE macrorestriction patterns of persistent and sporadic strains from different processing plants were compared and the strains were further studied by amplified fragment length polymorphism (AFLP), being a characterisation method giving more whole genome based information. The 17 persistent and 38 sporadic strains showed 14 and 35 pulsotypes, 14 and 28 AFLP types, respectively. The combination of PFGE and AFLP typing results yielded a total of 48 genotypes. Thirteen of 15 genotypes presented by persistent strains were only associated with persistent strains and similarly 94% (33/35) of genotypes showed by sporadic strains were recovered among sporadic strains only. Our results showed that L. monocytogenes strains causing persistent contamination differ from sporadic strains. In AFLP analysis persistent strains did not, however, form any specific clusters and neither was there any difference between the known two genomic groups. These results indicate that even though persistent strains differ from sporadic strains there seems not to be any specific evolutional lineage of persistent strains.

An amplified fragment length polymorphism (AFLP) method was applied to 129 strains representing 24 different Clostridium species, with special emphasis on pathogenic clostridia of medical or veterinary interest, to assess the potential of AFLP for identification of clostridia. In addition, the ability of the same AFLP protocol to type clostridia at the strain level was assessed by focusing on Clostridium perfringens strains. All strains were typeable by AFLP, so the method seemed to overcome the problem of extracellular DNase production. AFLP differentiated all Clostridium species tested, except for Clostridium ramosum and Clostridium limosum , which clustered together with a 45% similarity level. Other Clostridium species were divided into species-specific clusters or occupied separate positions. Wide genetic diversity was observed among Clostridium botulinum strains, which were divided into seven species-specific clusters. The same AFLP protocol was also suitable for typing C. perfringens at the strain level. A total of 29 different AFLP types were identified for 37 strains of C. perfringens ; strains initially originating from the same isolate showed identical fingerprinting patterns and were distinguished from unrelated strains. AFLP proved to be a highly reproducible, easy-to-perform, and relatively fast method which enables high throughput of samples and can serve in the generation of identification libraries. These results indicate that the AFLP method provides a promising tool for the identification and characterization of Clostridium species.