Characterization of European Yersinia enterocolitica 1A strains using restriction fragment length polymorphism and multilocus sequence analysis.

UNLABELLED: Yersinia enterocolitica is currently divided into two subspecies: subsp. enterocolitica including highly pathogenic strains of biotype 1B and subsp. palearctica including nonpathogenic strains of biotype 1A and moderately pathogenic strains of biotypes 2-5. In this work, we characterized 162 Y. enterocolitica strains of biotype 1A and 50 strains of biotypes 2-4 isolated from human, animal and food samples by restriction fragment length polymorphism using the HindIII restriction enzyme. Phylogenetic relatedness of 20 representative Y. enterocolitica strains including 15 biotype 1A strains was further studied by the multilocus sequence analysis of four housekeeping genes (glnA, gyrB, recA and HSP60). In all the analyses, biotype 1A strains formed a separate genomic group, which differed from Y. enterocolitica subsp. enterocolitica and from the strains of biotypes 2-4 of Y. enterocolitica subsp. palearctica. Based on these results, biotype 1A strains considered nonpathogenic should not be included in subspecies palearctica containing pathogenic strains of biotypes 2-5. SIGNIFICANCE AND IMPACT OF THE STUDY: Yersinia enterocolitica strains are currently divided into six biotypes and two subspecies. Strains of biotype 1A, which are phenotypically and genotypically very heterogeneous, are classified as subspecies palearctica. In this study, European Y. enterocolitica 1A strains isolated from both human and nonhuman sources were characterized using restriction fragment length polymorphism and multilocus sequence analysis. The European biotype 1A strains formed a separate group, which differed from strains belonging to subspecies enterocolitica and palearctica. This may indicate that the current division between the two subspecies is not sufficient considering the strain diversity within Y. enterocolitica.

Isolation and characterization of Yersinia-specific bacteriophages from pig stools in Finland.

AIMS: Bacteriophages infect bacteria, and they are present everywhere in the world including the intestinal tracts of animals. Yersiniosis is a common foodborne infection caused by Yersinia enterocolitica and Yersinia pseudotuberculosis. As these bacteria are frequently isolated from pigs, we wanted to know whether Yersinia-specific bacteriophages are also present in the pig stools and, if so, whether there is a positive or negative association between the prevalence of the Yersinia phages and the pathogenic Yersinia in the stool samples. METHODS AND RESULTS: Altogether 793 pig stool samples collected between November 2010 and March 2012 from 14 Finnish pig farms were screened for the presence of bacteriophages able to infect Y. enterocolitica serotype O:3, O:5,27 or O:9 strains, or Y. pseudotuberculosis serotype O:1a, O:1b or O:3 strains. Yersinia phages were isolated from 90 samples from eight farms. Yersinia enterocolitica O:3 was infected by 59 phages, 28 phages infected serotypes O:3 and O:5,27, and eight phages infected serotypes O:3, O:5,27 and O:9, and Y. pseudotuberculosis O:1a by eight phages. Many phages originating from pigs in the same farm were identical based on their restriction enzyme digestion patterns; 20 clearly different phages were selected for further characterization. Host ranges of these phages were tested with 94 Yersinia strains. Six of the phages infected eight strains, 13 phages infected three strains, and one phage infected only one strain, indicating that the phages had a relatively narrow host range. CONCLUSIONS: There was a clear association between the presence of the host bacteria and specific phages in the stools. SIGNIFICANCE AND IMPACT OF THE STUDY: The isolated bacteriophages may have potential as biocontrol agents for yersiniosis in both humans and pigs in future, and as alternatives or in addition to antibiotics. To our knowledge, this is the first reported isolation of Yersinia-specific phages from pig stool samples.

Infant botulism with prolonged faecal excretion of botulinum neurotoxin and Clostridium botulinum for 7 months.

In Finland in April 2010, a 3-month old baby was diagnosed with type A infant botulism. He excreted botulinum neurotoxin and/or Clostridium botulinum in his faeces until November 2010. Five months of excretion was after clinical recovery and discharge from hospital. C. botulinum isolates recovered from the household dust in the patient's home were genetically identical to those found in the infant's stool samples. Long-term faecal excretion of C. botulinum may pose a possible health risk for the parents and others in close contact with the infant.

Multiple-locus variable-number tandem-repeat analysis in genotyping Yersinia enterocolitica strains from human and porcine origins.

Sporadic and epidemiologically linked Yersinia enterocolitica strains (n = 379) isolated from fecal samples from human patients, tonsil or fecal samples from pigs collected at slaughterhouses, and pork samples collected at meat stores were genotyped using multiple-locus variable-number tandem-repeat analysis (MLVA) with six loci, i.e., V2A, V4, V5, V6, V7, and V9. In total, 312 different MLVA types were found. Similar types were detected (i) in fecal samples collected from human patients over 2 to 3 consecutive years, (ii) in samples from humans and pigs, and (iii) in samples from pigs that originated from the same farms. Among porcine strains, we found farm-specific MLVA profiles. Variations in the numbers of tandem repeats from one to four for variable-number tandem-repeat (VNTR) loci V2A, V5, V6, and V7 were observed within a farm. MLVA was applicable for serotypes O:3, O:5,27, and O:9 and appeared to be a highly discriminating tool for distinguishing sporadic and outbreak-related strains. With long-term use, interpretation of the results became more challenging due to variations in more-discriminating loci, as was observed for strains originating from pig farms. Additionally, we encountered unexpectedly short V2A VNTR fragments and sequenced them. According to the sequencing results, updated guidelines for interpreting V2A VNTR results were prepared.

Factors related to the prevalence of pathogenic Yersinia enterocolitica on pig farms.

A survey of 788 pigs from 120 farms was conducted to determine the within-farm prevalence of pathogenic Yersinia enterocolitica and a questionnaire of management conditions was mailed to the farms afterwards. A univariate statistical analysis with carriage and shedding as outcomes was conducted with random-effects logistic regression with farm as a clustering factor. Variables with a P value <0·15 were included into the respective multivariate random-effects logistic regression model. The use of municipal water was discovered to be a protective factor against carriage and faecal shedding of the pathogen. Organic production and buying feed from a certain feed manufacturer were also protective against total carriage. Tonsillar carriage, a different feed manufacturer, fasting pigs before transport to the slaughterhouse, higher-level farm health classification, and snout contacts between pigs were risk factors for faecal shedding. We concluded that differences in management can explain different prevalences of Y. enterocolitica between farms.

Two cases of food-borne botulism in Finland caused by conserved olives, October 2011.

In October 2011 in Finland, two persons fell ill with symptoms compatible with botulism after having eaten conserved olives stuffed with almonds. One of these two died. Clostridium botulinum type B and its neurotoxin were detected in the implicated olives by PCR and mouse bioassay, respectively. The olives were traced back to an Italian manufacturer and withdrawn from the market. The public and other European countries were informed through media and Europe-wide notifications.

Evaluation of isolation methods for pathogenic Yersinia enterocolitica from pig intestinal content.

AIMS: The aim of this study was to evaluate the efficiency of four isolation methods for the detection of pathogenic Yersinia enterocolitica from pig intestinal content. METHODS AND RESULTS: The four methods comprised of 15 isolation steps using selective enrichments (irgasan-ticarcillin-potassium chlorate and modified Rappaport broth) and mildly selective enrichments at 4 or 25 degrees C. Salmonella-Shigella-desoxycholate-calcium chloride agar, cefsulodin-irgasan-novobiocin agar were used as plating media. The most sensitive method detected 78% (53/68) of the positive samples. Individual isolation steps using cold enrichment as the only enrichment or as a pre-enrichment step with further selective enrichment showed the highest sensitivities (55-66%). All isolation methods resulted in high numbers of suspected colonies not confirmed as pathogenic Y. enterocolitica. CONCLUSIONS: Cold enrichment should be used in the detection of pathogenic Y. enterocolitica from pig intestinal contents. In addition, more than one parallel isolation step is needed. SIGNIFICANCE AND IMPACT OF THE STUDY: The study shows that depending on the isolation method used for Y. enterocolitica, the detected prevalence of Y. enterocolitica in pig intestinal contents varies greatly. More selective and sensitive isolation methods need to be developed for pathogenic Y. enterocolitica.

Botulism and hot-smoked whitefish: a family cluster of type E botulism in France, September 2009.

A family cluster of three cases of type E botulism were identified in south-east France in September 2009. The suspected food source of infection was a vacuum packed hot-smoked whitefish of Canadian origin purchased by the family during a visit to Finland and consumed several weeks later in France on the day prior to symptom onset. No leftover fish was available to confirm this hypothesis. Vacuum packed hot-smoked whitefish has previously been associated with cases of type E botulism in multiple countries, including Finland, Germany, the United States and Israel.

Listeria monocytogenes is common in wild birds in Helsinki region and genotypes are frequently similar with those found along the food chain.

AIMS: To evaluate the prevalence and genetic diversity of Listeria monocytogenes in wild birds and to compare the genotypes with isolates previously collected from foods and food processing environments. METHODS AND RESULTS: Samples of wild birds' faeces (n = 212) were collected from a municipal landfill site and from urban areas in the Helsinki region and analysed by two-step enrichment and plating onto L. monocytogenes-selective agar. The overall prevalence of L. monocytogenes in bird faeces was 36% (95% CI 30-43%), and prevalence on the landfill site was significantly higher. All isolates were analysed with pulsed-field gel electrophoresis and compared with the L. monocytogenes profiles in an existing collection. Similar pulsotypes were found in birds and in isolates collected along the food chain. CONCLUSIONS: Birds commonly carry L. monocytogenes, and strains are frequently similar with those detected in foods and food processing environments. Thus, birds may disseminate L. monocytogenes in nature and may also contaminate foods when entering the food processing environments and outdoor market places. SIGNIFICANCE AND IMPACT OF THE STUDY: Populations of L. monocytogenes in wild birds and along the food processing chain overlap. Our findings add to the epidemiological data on this significant foodborne pathogen.

Acid and heat tolerance of persistent and nonpersistent Listeria monocytogenes food plant strains.

AIMS: Acid and heat tolerance of 17 persistent and 23 nonpersistent Listeria monocytogenes strains, recovered from three meat-processing plants, were investigated. METHODS AND RESULTS: The isolates were genotyped by pulsed-field gel electrophoresis and categorized into persistent strains according to the frequency of the strain and duration of the contamination. The persistent and nonpersistent strains were challenged to acidic conditions (pH 2.4 for 2 h, 1 mol l(-1) HCl were used to acidify the suspension) and to heat (55 degrees C for 40 min) to receive a reduction in cell count. Listeria monocytogenes strains showed large variation in acid tolerance (over 6 log units) and in heat tolerance (3 log units). The persistent strains showed higher tolerance to acidic conditions than the nonpersistent strains (Student's t-test, P = 0.02), but significant differences in heat tolerance between persistent and nonpersistent strains were not observed. CONCLUSIONS: The results indicate that acid tolerance may have an effect on the persistence of L. monocytogenes contamination. SIGNIFICANCE AND IMPACT OF THE STUDY: This study highlights the fact that there are great differences in acid and heat tolerances between L. monocytogenes strains, and the preventive measures should be designed to be effective against the most tolerant strains.

Prevalence of Pathogenic Yersinia enterocolitica in Finnish Slaughter Pigs.

The prevalence of human pathogenic Yersinia enterocolitica was determined in tonsil and intestinal content samples from 388 healthy fattening pigs at the four biggest Finnish slaughterhouses. These slaughterhouses process 73% of pigs in Finland. Tonsil samples were tested by PCR targeted for yadA, and intestinal samples were cultured. All pathogenic Y. enterocolitica isolates represented bioserotype 4/O:3. The prevalence of Y. enterocolitica in tonsil samples was 60% (95% confidence limit, 55.4 to 65.1%), and its prevalence in intestinal samples was 26% (95% confidence limit, 22.1 to 31.2%). The prevalence of Y. enterocolitica in tonsil and intestinal samples varied between the four slaughterhouses. The tonsil prevalence of Y. enterocolitica was higher in slaughterhouse B, and the prevalence in intestinal content was higher in slaughterhouse C. There were more positive results in both tonsil and intestinal samples in pigs coming from fattening farms than in pigs coming from farrowing-and-fattening farms. A seasonal variation was observed in the prevalence of Y. enterocolitica in intestinal samples, with the highest prevalence during July and August, but no seasonal variation was detected in tonsil samples.

Improvement in the lipid extraction of staphylococcal cells by lysostaphin treatment.

A new method for extracting lipids from Staphylococcus aureus is described in this paper. The extracts of cells treated with lysostaphin are compared with those from untreated cells. The recovery of lipids improved with the enzymatic treatment. Tentative identification of the components of the lipid extracts has been carried out.

Bayesian Estimation of the True Prevalence and of the Diagnostic Test Sensitivity and Specificity of Enteropathogenic Yersinia in Finnish Pig Serum Samples.

Bayesian analysis was used to estimate the pig's and herd's true prevalence of enteropathogenic Yersinia in serum samples collected from Finnish pig farms. The sensitivity and specificity of the diagnostic test were also estimated for the commercially available ELISA which is used for antibody detection against enteropathogenic Yersinia. The Bayesian analysis was performed in two steps; the first step estimated the prior true prevalence of enteropathogenic Yersinia with data obtained from a systematic review of the literature. In the second step, data of the apparent prevalence (cross-sectional study data), prior true prevalence (first step), and estimated sensitivity and specificity of the diagnostic methods were used for building the Bayesian model. The true prevalence of Yersinia in slaughter-age pigs was 67.5% (95% PI 63.2-70.9). The true prevalence of Yersinia in sows was 74.0% (95% PI 57.3-82.4). The estimates of sensitivity and specificity values of the ELISA were 79.5% and 96.9%.

Bayesian modelling to identify the risk factors for Yersinia enterocolitica contamination of pork carcasses and pluck sets in slaughterhouses.

The probability of contamination by pathogenic Yersinia enterocolitica of carcasses and pluck sets at slaughterhouse was determined by means of a Bayesian analysis. Prior information of the prevalence of Y. enterocolitica in faeces and the seroprevalence of Yersinia in serum of pigs collected at farms were obtained from previous studies and introduced in the models as beta prior informative distributions. Samples of intestinal content, tonsils, and swabs of carcasses and pluck set surfaces were collected at slaughterhouses. The posterior probabilities, odds ratio (OR) and their probability interval (PI) were calculated by means of a generalized linear model constructed in WinBugs. Occurrence of Y. enterocolitica in intestinal content (OR: 35.6, 95%PI 2.8-8285), tonsils (OR: 38.4, 95%PI 5.0-854), and pluck set (OR: 16.6, 95%PI 1.9-1111) was a risk for the contamination of pork carcasses, and an increased risk of contaminated pluck set was observed when Y. enterocolitica was isolated in intestinal content (OR: 40.6, 95%PI 2.1-10510) and tonsils (OR: 17.6, 95%PI 3.4-230.6). This increased risk indicated a potential cross-contamination at the slaughterhouse.

Identification of Clostridium botulinum with API 20 A, Rapid ID 32 A and RapID ANA II.

Three commercially available test systems for the identification of anaerobic bacteria were evaluated for the identification of 18 proteolytic group I and 69 non-proteolytic group II Clostridium botulinum, four Clostridium sporogenes and 18 non-toxigenic group II C. botulinum-like strains. All proteolytic C. botulinum strains were misidentified by the Rapid ID 32 A and RapID ANA II, while 14 strains and all C. sporogenes strains were identified as C. botulinum or C. sporogenes by the API 20 A. Reversely, all non-proteolytic C. botulinum strains were misidentified by the API 20 A while the Rapid ID 32 A recognized 67 and RapID ANA II 68 strains. All C. sporogenes strains were recognized by the RapID ANA II, while the Rapid ID 32 A recognized one strain. All non-proteolytic non-toxigenic strains were identified as C. botulinum group II by the Rapid ID 32 A, 17 strains by the RapID ANA II, and one strain by the API 20 A. The results show that these test systems do not provide a reliable method for identification of C. botulinum.

High prevalence of Yersinia enterocolitica 4:O3 on pig offal in southern Germany: a slaughtering technique problem.

Prevalence and contamination routes of pathogenic Yersinia enterocolitica were studied in Southern Germany. Tonsil and faeces samples of 50 fattening pigs, 140 offal samples and 120 minced meat samples were examined. Pig and offal samples were collected from a slaughterhouse approved by the European Union, and minced meat samples from two large meat factories. Yersinia enterocolitica was isolated using direct plating, overnight enrichment and selective enrichment in MRB and ITC broth. The isolates were bio- and serotyped, and pathogenicity was studied using two plasmid-encoded virulence markers: calcium dependence and Congo red absorption. The genotypes were studied with pulsed-field gel electrophoresis using NotI enzyme. Prevalence of pathogenic Y. enterocolitica 4:O3 was 60% and 10% in tonsils and faeces of fattening pigs, respectively. Besides tonsils, prevalence of pathogenic Y. enterocolitica 4:O3 was also high in other pluck set samples, including tongues, lungs, hearts, diaphragms and livers. However, the highest isolation rate was obtained from the tonsils. Kidneys, which were not attached to the pluck set and did not hang together with tonsils on the rack, had the lowest isolation rate. Yersinia enterocolitica 4:O3 was isolated from 12% of minced meat samples. A total of 25 NotI profiles were obtained from porcine samples. The most common genotype, NBI, found in tonsils was also the most common type recovered from offal and minced meat samples. The high contamination rate of tonsils, and the indistinguishable NotI profiles obtained from tonsils and offal indicate that the tonsils contaminate offal when they are removed and hung on the rack together. When the head, with the tonsils and tongue, is not removed prior to evisceration and is not handled and inspected separately, it is difficult to control the spread of Y. enterocolitica 4:O3 from tonsils to the carcass, and subsequently, to meat.

Different Yersinia enterocolitica 4:O3 genotypes found in pig tonsils in Southern Germany and Finland.

The distribution of different genotypes of Y. enterocolitica 4:O3 strains recovered from pig tonsils in Southern Germany and Finland in 1999-2000 was investigated. A total of 96 and 207 Y. enterococolitica 4:O3 isolates recovered from 47 and 66 tonsils of finishing pigs in Germany and Finland, respectively, were characterised with PFGE using NotI enzyme. In all, 39 different NotI profiles were obtained, only one of which, NB1, was found in both Germany and Finland. All strains were further characterised with ApaI and XhoI enzymes. When the 54 German and 74 Finnish strains were characterised with all three enzymes, 51 genotypes were obtained. The 23 genotypes found in German strains differed from the 28 found in Finnish strains. These results indicate that Y. enterocolitica 4:O3 genotypes have a differential geographical distribution and thus can be used in epidemiological studies.

Characterization of lactic acid bacteria isolated from vacuum-packed cooked ring sausages.

The bacterial populations of the surface layer and the centre of 15 spoiled vacuum-packed cooked ring sausages were characterized. About 95% of the total bacterial population in the surface layer and 55% at the centre were lactic acid bacteria. Another large bacterial group at the centre consisted of Bacillus spp. The lactic acid bacteria on the surface and at the centre were quite similar. Atypical streptobacteria, i.e. homofermentative psychrotrophic lactobacilli, were a major group of lactic acid bacteria in the surface layer of the spoiled sausages. Three main homofermentative groups could be observed on the basis of different carbohydrate patterns. Heterofermentative lactic acid bacteria belonged mainly to genus Leuconostoc. The proportion of leuconostocs in the spoiled sausages was also quite large. They could be divided into three main groups on the basis of different carbohydrate fermentation patterns. The lactic acid bacteria population of spoiled cooked ring sausages thus seemed to be heterogeneous. The strains isolated resembled strains observed by other workers in meat and meat products.

Minimum growth temperatures of Hafnia alvei and other Enterobacteriaceae isolated from refrigerated meat determined with a temperature gradient incubator.

Minimum growth temperatures of Hafnia alvei (n = 156) and other Enterobacteriaceae isolates (n = 162) from refrigerated meat samples (n = 88) and control strains of H. alvei (n = 81) from clinical and environmental samples were determined with a plate-type continuous temperature gradient incubator on nutrient agar. The dominant species, Hafnia alvei and Serratia liquefaciens had mean minimum growth temperatures of 2.6 (range, 0.2-3.7 degrees C) and 1.7 (range, 0.2-2.6 degrees C), respectively. Values for other species included: Enterobacter agglomerans, 1.3 (0.7-1.7 degrees C); Escherichia coli, 8.7 (8.4-8.9); Escherichia vulneris, 1.6 (0.8-2.6 degrees C); and Serratia fonticola, 2.0 (1.1-3.0 degrees C). The H. alvei reference strains did not differ markedly from the meat isolates, with the exception of the diarrhoeagenic eae A positive strains (10.6, 10.2-11.5 degrees C). The representatives of H. alvei hybridization groups (HG) 1 and 2 did not differ in their minimum growth temperatures. The observed heterogeneity of the minimum growth temperatures of many Enterobacteriaceae species may be explained by limitations of the systems used for identification of enterobacteria, inadequacy of the Enterobacteriaceae taxonomy or true growth temperature heterogeneity within the various species.

rRNA gene restriction patterns as a characterization tool for Lactobacillus sake strains producing ropy slime.

The rRNA gene restriction patterns (ribotypes) of 69 ropy slime producing Lactobacillus sake strains isolated mainly from vacuum-packaged meat products of ten meat plants were determined. Ribotypes were compared to the corresponding patterns of non-ropy L. sake strains, and also to other species of the genus Lactobacillus, Carnobacterium and Weissella associated with meat products. Ropy slime-producing L. sake strains were divided into four characteristic groups corresponding to the phenotypic carbohydrate grouping. No association between certain ribotypes and individual plants was detected. Ribotyping was unable to distinguish slime producing and non-ropy strains of L. sake group sharing the same carbohydrate pattern. Otherwise, ribotyping distinguished the ropy slime producing strains from the non-ropy L. sake reference strains and all L. sake strains from other species of the genus Lactobacillus, Carnobacterium and Weissella. These results suggest that ribotyping is a suitable method for detection and surveillance of the contamination of ropy slime-producing L. sake strains but the patterns alone cannot be used as markers of slime production capability.