Knowledge attainment, learning approaches, and self-perceived study burnout among European veterinary students.

Introduction: This study investigates the relationship between approaches to learning, self-perceived study burnout, and the level of knowledge among veterinary students. Veterinary educational programs are under regular development and would benefit greatly from detailed feedback on students' knowledge, proficiency, influencing factors, and coping mechanisms. Methods: The VetRepos consortium developed and calibrated an item repository testing knowledge across the entire veterinary curriculum. Two hundred forty-eight students from seven European veterinary institutions took the VetRepos test, comprising a subset of the repository. They also responded to a questionnaire assessing deep and unreflective learning approaches and self-perceived study burnout, represented by exhaustion and cynicism. Structural equation modeling analyzed the relationship between these latent traits and the VetRepos test score. Results: The model failed the exact-fit test but was retained based on global fit indices, inter-item residual correlations, and standardized residual covariances. Root Mean Square Error of Approximation with robust standard errors and scaled test statistic was 0.049 (95% confidence interval 0.033-0.071), scaled and robust Comparative Fit Index 0.95 (0.90-0.98), and scaled Standardized Root Mean Square Residual 0.056 (0.049-0.071). Measurement invariance across study years was not violated (ΔCFI = 0.00, χ2 = 3.78, Δdf = 4, p = 0.44), but it could not be confirmed between genders or universities. The VetRepos test score regressed on the study year [standardized regression coefficient = 0.68 (0.62-0.73)], showed a negative regression on the unreflective learning approach [-0.25 (-0.47 to -0.03)], and a positive regression on the deep approach [0.16 (0.03-0.28)]. No direct association with perceived burnout was observed; however, a significant, medium-sized association was found between the unreflective approach and self-perceived study burnout. No significant differences in learning approaches or perceived burnout were found between study years. Discussion: The most important source of variance in VetRepos test scores, unrelated to the study year, was the learning approach. The association between the VetRepos test score and self-perceived burnout was indirect. Future research should complement this cross-sectional approach with longitudinal and person-oriented studies, further investigating the relationship between study burnout and learning approaches.

Campylobacter species and genotype distribution in Finnish beef liver - Retail liver juice ideal for isolation and quantification.

Campylobacteriosis, primarily caused by Campylobacter jejuni and C. coli, is the main bacterial zoonosis worldwide. While poultry is recognized as the main reservoir, bovines are considered another important reservoir for Campylobacter spp. found in human infections. In contrast to chicken, retail beef is seldom contaminated by Campylobacter species. However, beef liver is recognized to be frequently contaminated and has been linked to human infections via epidemiological investigations. Our aims were to evaluate the prevalence of Campylobacter spp. inside and on the surface of beef liver pieces at retail in Finland and to analyse the population in more detail using whole genome sequencing (WGS) to assess the public health relevance. A total of 90 retail beef livers were studied using both enrichment of the external peptone-saline rinse of the liver piece and direct culture from the inside after surface sterilization. Furthermore, 46 of the livers were also studied using direct culture of retail beef liver juice collected from the bottom of the consumer package to estimate the concentration of Campylobacter species. Overall, 44 (49 %) of the samples were positive for Campylobacter species, C. jejuni, C. fetus and C. lari being identified in 42 %, 8.9 % and 1.1 % of the samples, respectively. Direct culture of retail liver juice was a sensitive and convenient method for Campylobacter spp. detection, resulting in 48 % prevalence and a mean concentration of 49 cfu/ml (maximum 335 cfu/ml). Two samples (2.2 %), containing large hepatic ducts, were positive for C. jejuni internally, representing multilocus sequence typing (MLST) sequence type ST-19 and ST-21. WGS, core genome phylogeny and core genome MLST revealed that in most cases only one clearly distinct clone of clinically relevant C. jejuni or C. fetus was isolated from a single lot of samples. However, in some cases several distinct clones were identified simultaneously even from a single liver piece. In epidemiological investigations, it is thus highly advisable to genotype multiple isolates to capture the whole diversity of Campylobacter spp. from suspected food sources. Good kitchen hygiene, avoidance of cross-contamination and thorough cooking are important for limiting the transmission of campylobacteriosis.

Results of routine inspections in restaurants and institutional catering establishments associated with foodborne outbreaks in Finland.

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.

Prudent Antimicrobial Use Is Essential to Prevent the Emergence of Antimicrobial Resistance in Yersinia enterocolitica 4/O:3 Strains in Pigs.

Yersinia enterocolitica is a psychrotrophic zoonotic foodborne pathogen. Pigs are considered the main reservoir of Y. enterocolitica 4/O:3, which is the most commonly isolated bioserotype in many European countries. Consuming pork contaminated with Y. enterocolitica can be a health threat, and antimicrobial-resistant strains may complicate the treatment of the most severe forms of yersiniosis. We analyzed the antimicrobial resistance of 1,016 pathogenic porcine Y. enterocolitica 4/O:3 strains originating from Belgium, Estonia, Finland, Germany, Italy, Latvia, Russia, Spain, and the United Kingdom. Based on available reports, we also compared antimicrobial sales for food production animals in these countries, excluding Russia. Antimicrobial resistance profiles were determined using a broth microdilution method with VetMIC plates for 13 antimicrobial agents: ampicillin, cefotaxime, ceftiofur (CTF), chloramphenicol (CHL), ciprofloxacin, florfenicol, gentamicin, kanamycin, nalidixic acid (NAL), streptomycin (STR), sulfamethoxazole (SME), tetracycline (TET), and trimethoprim (TMP). The antimicrobial resistance of Y. enterocolitica 4/O:3 strains varied widely between the countries. Strains resistant to antimicrobial agents other than ampicillin were rare in Estonia, Finland, Latvia, and Russia, with prevalence of 0.7, 0.4, 0, and 8.3%, respectively. The highest prevalence of antimicrobial resistance was found in Spanish and Italian strains, with 98 and 61% of the strains being resistant to at least two antimicrobial agents, respectively. Resistance to at least four antimicrobial agents was found in 34% of Spanish, 19% of Italian, and 7.1% of English strains. Antimicrobial resistance was more common in countries where the total sales of antimicrobials for food production animals are high and orally administered medications are common. Our results indicate that antimicrobials should be used responsibly to treat infections, and parenteral medications should be preferred to orally administered mass medications.

Comparative Genome Analysis and Spore Heat Resistance Assay Reveal a New Component to Population Structure and Genome Epidemiology Within Clostridium perfringens Enterotoxin-Carrying Isolates.

Clostridium perfringens causes a variety of human and animal enteric diseases including food poisoning, antibiotic-associated diarrhea, and necrotic enteritis. Yet, the reservoirs of enteropathogenic enterotoxin-producing strains remain unknown. We conducted a genomic comparison of 290 strains and a heat resistance phenotyping of 30 C. perfringens strains to elucidate the population structure and ecology of this pathogen. C. perfringens genomes shared a conserved genetic backbone with more than half of the genes of an average genome conserved in >95% of strains. The cpe-carrying isolates were found to share genetic context: the cpe-carrying plasmids had different distribution patterns within the genetic lineages and the estimated pan genome of cpe-carrying isolates had a larger core genome and a smaller accessory genome compared to that of 290 strains. We characterize cpe-negative strains related to chromosomal cpe-carrying strains elucidating the origin of these strains and disclose two distinct groups of chromosomal cpe-carrying strains with different virulence characteristics, spore heat resistance properties, and, presumably, ecological niche. Finally, an antibiotic-associated diarrhea isolate carrying two copies of the enterotoxin cpe gene and the associated genetic lineage with the potential for the emergence of similar strains are outlined. With C. perfringens as an example, implications of input genome quality for pan genome analysis are discussed. Our study furthers the understanding of genome epidemiology and population structure of enteropathogenic C. perfringens and brings new insight into this important pathogen and its reservoirs.

High prevalence of Clostridium botulinum in vegetarian sausages.

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.

Shopping Detail Information and Home Freezer Sampling Confirmed the Role of Commercial, Modified-Atmosphere Packaged Meatballs as a Vehicle for Listeriosis in Finland.

In November 2016, an elderly patient was diagnosed with Listeria monocytogenes bacteremia in Finland. Grocery store loyalty card records and microbiological investigation of foods found in the home fridge and freezer of the patient revealed commercial, modified-atmosphere packaged meatballs as the source of the infection. Investigation of the meatball production plant revealed that the floor drain samples were contaminated with the same L. monocytogenes strain as those isolated from the patient and meatballs. Ready-to-eat meatballs were likely contaminated after heat treatment from the production environment before packaging. Long-term cold storage, modified-atmosphere conditions, and the absence of competing bacteria presumably enhanced the growth of L. monocytogenes. We recommend that collection of shopping details and home fridge and freezer sampling should be part of surveillance of all cases of L. monocytogenes infections to complement information obtained from in-depth interviews.

Insights into the Phylogeny and Evolution of Cold Shock Proteins: From Enteropathogenic Yersinia and Escherichia coli to Eubacteria.

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.

Role of DEAD-box RNA helicase genes in the growth of Yersinia pseudotuberculosis IP32953 under cold, pH, osmotic, ethanol and oxidative stresses.

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°C compared to non-shocked culture at 28°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°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.

Prevalence and Dynamics of Pathogenic Yersinia enterocolitica 4/O:3 Among Finnish Piglets, Fattening Pigs, and Sows.

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.

Changes in Transcriptome of Yersinia pseudotuberculosis IP32953 Grown at 3 and 28°C Detected by RNA Sequencing Shed Light on Cold Adaptation.

Yersinia pseudotuberculosis is a bacterium that not only survives, but also thrives, proliferates, and remains infective at cold-storage temperatures, making it an adept foodborne pathogen. We analyzed the differences in gene expression between Y. pseudotuberculosis IP32953 grown at 3 and 28°C to investigate which genes were significantly more expressed at low temperature at different phases of growth. We isolated and sequenced the RNA from six distinct corresponding growth points at both temperatures to also outline the expression patterns of the differentially expressed genes. Genes involved in motility, chemotaxis, phosphotransferase systems (PTS), and ATP-binding cassette (ABC) transporters of different nutrients such as fructose and mannose showed higher levels of transcripts at 3°C. At the beginning of growth, especially genes involved in securing nutrients, glycolysis, transcription, and translation were upregulated at 3°C. To thrive as well as it does at low temperature, Y. pseudotuberculosis seems to require certain cold shock proteins, especially those encoded by yptb3585, yptb3586, yptb2414, yptb2950, and yptb1423, and transcription factors, like Rho, IF-1, and RbfA, to maintain its protein synthesis. We also found that genes encoding RNA-helicases CsdA (yptb0468), RhlE (yptb1214), and DbpA (yptb1652), which unwind frozen secondary structures of nucleic acids with cold shock proteins, were significantly more expressed at 3°C, indicating that these RNA-helicases are important or even necessary during cold. Genes involved in excreting poisonous spermidine and acquiring compatible solute glycine betaine, by either uptake or biosynthesis, showed higher levels of transcripts at low temperatures. This is the first finding of a strong connection between the aforementioned genes and the cold adaptation of Y. pseudotuberculosis. Understanding the mechanisms behind the cold adaptation of Y. pseudotuberculosis is crucial for controlling its growth during cold storage of food, and will also shed light on microbial cold adaptation in general.

Growth of Yersinia pseudotuberculosis Strains at Different Temperatures, pH Values, and NaCl and Ethanol Concentrations.

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.

Cold Shock Proteins: A Minireview with Special Emphasis on Csp-family of Enteropathogenic Yersinia.

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 0°C 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.

Large Diversity of Porcine Yersinia enterocolitica 4/O:3 in Eight European Countries Assessed by Multiple-Locus Variable-Number Tandem-Repeat Analysis.

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.

Inhibition of toxigenesis of group II (nonproteolytic) Clostridium botulinum type B in meat products by using a reduced level of nitrite.

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.

An 8-year surveillance of the diversity and persistence of Listeria monocytogenes in a chilled food processing plant analyzed by amplified fragment length polymorphism.

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.

Identification of Clostridium species and DNA fingerprinting of Clostridium perfringens by amplified fragment length polymorphism analysis.

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.

Contamination routes of Clostridium botulinum in the honey production environment.

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.

Efficient DNA fingerprinting of Clostridium botulinum types A, B, E, and F by amplified fragment length polymorphism analysis.

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 <10% similarity between proteolytic and nonproteolytic 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.

Characterisation of persistent and sporadic Listeria monocytogenes strains by pulsed-field gel electrophoresis (PFGE) and amplified fragment length polymorphism (AFLP).

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.