Host-mediated modification of Sau3AI restriction in Listeria monocytogenes: prevalence in epidemic-associated strains.

Most major food-related outbreaks of listeriosis have been traced to a cluster of genetically related strains of serovar 4b (epidemic clone). In spite of numerous searches, distinct bacteriologic or virulence-related features unique to these strains have eluded identification, although a restriction fragment length polymorphism (RFLP) characteristic of the epidemic clone has previously been described (W. Zheng and S. Kathariou, Appl. Environ. Microbiol. 61:4310-4314, 1995). We found that DNAs from 75 strains which were derived from three separate outbreaks and which had the epidemic clone-specific RFLP were also invariably resistant to digestion by Sau3AI and other restriction endonucleases sensitive to cytosine methylation at 5' GATC 3' sites. This modification of Sau3AI restriction was host mediated, as it did not persist when DNA was cloned and propagated in Escherichia coli, and was uncommon among other Listeria strains. Epidemic-associated strains with this modification were resistant to infection by phage propagated in a serotype 4b strain which was not known to be involved in an epidemic and which lacked the epidemic clone-specific RFLP. Screening for susceptibility to MboI digestion revealed that these epidemic strains lacked methylation of adenines at GATC sites. This type of modification was rare among Listeria strains and was found in only three (of eight screened) strains of serovar 1/2b, possibly representing one clonal lineage.     

A case of foodborne listeriosis in Sweden

A 70-year-old woman fell seriously ill overnight with meningitis and was admitted to hospital. Cerebrospinal fluid culture yielded Listeria monocytogenes . One of the first problems in solving a human case of listeriosis suspected to be foodborne is to find the foods likely to have been transmitting L. monocytogenes . Two enrichment procedures and a direct plating procedure were used for isolation of the bacteria from different food items collected from the patient's refrigerator, local retail store and producer. Samples of vacuum-packed products of sliced pork brawn, sliced cooked medwurst and berliner wurst of the same brand harboured L. monocytogenes . Serotyping and restriction enzyme analysis (REA) with pulsed-field gel electrophoresis (PFGE) were used to characterize and compare 41 isolates, including the human strain. At least three clones were present in the foods investigated, and one of these was identical to the human clone. This clone was present in samples of medwurst from the patient's refrigerator and the local retail store. This is, to our knowledge, the first proven foodborne case of listeriosis reported in Sweden.     

An updated review of Listeria monocytogenes in the pork meat industry and its products

Pork meat and processed pork products have been the sources of outbreaks of listeriosis in France and in other European countries during the last decade. The aim of this review is to understand how contamination, survival and growth of Listeria monocytogenes can occur in pork meat products. This study discusses the presence of L. monocytogenes in raw pork meat, in the processing environment and in finished products. The prevalence of L. monocytogenes generally increases from the farm to the manufacturing plants and this mainly due to cross-contamination. In many cases, this pathogen is present in raw pork meat at low or moderate levels, but foods involved in listeriosis outbreaks are those in which the organism has multiplied to reach levels significantly higher than 1000 CFU g(-1). In such cases, L. monocytogenes has been able to survive and/or to grow despite the hurdles encountered during the manufacturing and conservation processes. Accordingly, attention must be paid to the design of food-processing equipment and to the effectiveness of the cleaning and disinfecting procedures in factories. Finally, the production of safe pork meat products is based on the implementation of general preventive measures such as Good Hygiene Practices, Good Manufacturing and the Hazard Analysis Critical Control Point.     

Genetic Characteristics of Japanese Clinical Listeria monocytogenes Isolates

Listeria monocytogenes causes foodborne illnesses through consumption of ready-to-eat foods. Although 135-201annual listeriosis cases have been estimated in Japan, the details regarding the clinical isolates such as infection source, virulence level, and other genetic characteristics, are not known. In order to uncover the trends of listeriosis in Japan and use the knowledge for prevention measures to be taken, the genetic characteristics of the past human clinical isolates needs to be elucidated. For this purpose, multilocus tandem-repeat sequence analysis (MLTSA) and multi-virulence-locus sequence typing (MVLST) were used in this study. The clinical isolates showed a variety of genetically distant genotypes, indicating they were from sporadic cases. However, the MVLST profiles of 7 clinical isolates were identical to those of epidemic clone (EC) I isolates, which have caused several serious outbreaks in other countries, suggesting the possibility that they have strong virulence potential and originated from a single outbreak. Moreover, 6 Japanese food isolates shared their genotypes with ECI isolates, indicating that there may be risks for listeriosis outbreak in Japan. This is the first investigational study on genetic characteristics of Japanese listeriosis isolates. The listeriosis cases happened in the past are presumably sporadic, but it is still possible that some isolates with strong virulence potential have caused listeriosis outbreaks, and future listeriosis risks also exist.     

Epidemic clone I-specific genetic markers in strains of Listeria monocytogenes serotype 4b from foods

Listeria monocytogenes contamination of ready-to-eat foods has been implicated in numerous outbreaks of food-borne listeriosis. However, the health hazards posed by L. monocytogenes detected in foods may vary, and speculations exist that strains actually implicated in illness may constitute only a fraction of those that contaminate foods. In this study, examination of 34 serogroup 4 (putative or confirmed serotype 4b) isolates of L. monocytogenes obtained from various foods and food-processing environments, without known implication in illness, revealed that many of these strains had methylation of cytosines at GATC sites in the genome, rendering their DNA resistant to digestion by the restriction endonuclease Sau3AI. These strains also harbored a gene cassette with putative restriction-modification system genes as well as other, genomically unlinked genetic markers characteristic of the major epidemic-associated lineage of L. monocytogenes (epidemic clone I), implicated in numerous outbreaks in Europe and North America. This may reflect a relatively high fitness of strains with these genetic markers in foods and food-related environments relative to other serotype 4b strains and may partially account for the repeated involvement of such strains in human food-borne listeriosis.     

Characterization of Listeria monocytogenes recovered from 41 cases of sporadic listeriosis in Austria by serotyping and pulsed-field gel electrophoresis

41 clinical Listeria monocytogenes strains recovered from seven feto-maternal and 34 non-pregnancy associated cases of human listeriosis documented between 1997 and 2000 underwent serotyping and typing by pulsed-field gel electrophoresis (PFGE) applying the enzymes AscI, ApaI and SmaI. The pulsotypes of the clinical strains were compared to the pulsotypes of three L. monocytogenes strains isolated from healthy fecal carriers and nine reference strains isolated from seven outbreaks in Europe and the USA. The 41 clinical strains of Austrian provenance showed 37 pulsotypes. Five sets of two Austrian strains each were indistinguishable by PFGE typing. Epidemiological links were absent between these indistinguishable isolates. One unique pulsotype (AB) was found in three fecal isolates. Five pulsotypes (A, Q, R, AC and AD) were distinguished among the strains associated with outbreaks. Clusters consisting of two, five and six Austrian strains each were indistinguishable from the outbreak-associated pulsotypes A, Q and R, respectively, after PFGE analysis with AscI. Three strains of AscI pulsotype Q and five strains of AscI pulsotype R could be further differentiated by restriction with ApaI and SmaI. One strain each from sporadic cases shared a combined pulsotype with the outbreak strains of pulsotypes A and R, respectively. These PFGE data suggest that a similar genetic background can be found in strains which have been contributing to outbreaks world-wide and in isolates associated with sporadic listeriosis in Austria.     

Epidemic Clone I-Specific Genetic Markers in Strains of Listeria monocytogenes Serotype 4b from Foods

Listeria monocytogenes contamination of ready-to-eat foods has been implicated in numerous outbreaks of food-borne listeriosis. However, the health hazards posed by L. monocytogenes detected in foods may vary, and speculations exist that strains actually implicated in illness may constitute only a fraction of those that contaminate foods. In this study, examination of 34 serogroup 4 (putative or confirmed serotype 4b) isolates of L. monocytogenes obtained from various foods and food-processing environments, without known implication in illness, revealed that many of these strains had methylation of cytosines at GATC sites in the genome, rendering their DNA resistant to digestion by the restriction endonuclease Sau3AI. These strains also harbored a gene cassette with putative restriction-modification system genes as well as other, genomically unlinked genetic markers characteristic of the major epidemic-associated lineage of L. monocytogenes (epidemic clone I), implicated in numerous outbreaks in Europe and North America. This may reflect a relatively high fitness of strains with these genetic markers in foods and food-related environments relative to other serotype 4b strains and may partially account for the repeated involvement of such strains in human food-borne listeriosis.     

Series of incidents of Listeria monocytogenes non-invasive febrile gastroenteritis involving ready-to-eat meats

AIMS: A series of cases and outbreaks of febrile noninvasive gastrointestinal disease involving 31 identified cases was investigated in terms of the numbers and types of Listeria monocytogenes present in the suspect foods (ready-to-eat meats) and clinical samples from cases. METHODS AND RESULTS: Foods and faecal samples involved in the incidents were tested for the presence and number of L. monocytogenes. Isolates were typed by macrorestriction analysis using pulsed-field gel electrophoresis. The foods contained high levels of L. monocytogenes, in one case 1.8 x 10(7) g-1. Faecal samples contained L. monocytogenes for up to 15 d after the contaminated food was consumed. All isolates from the food and faecal samples were of serotype 1/2 and were indistinguishable from one another by macrorestriction typing. CONCLUSIONS: It is likely that the meats were contaminated either during their manufacture after they had been cooked or by underprocessing. The long shelf lives on these products would have allowed the contaminating L. monocytogenes to grow to the high numbers measured in this study, causing food poisoning as described. SIGNIFICANCE AND IMPACT OF THE STUDY: Outbreaks of febrile noninvasive listeriosis are relatively rare. This report adds ready-to-eat meats to the range of foods that have acted as vehicles for such outbreaks.     

Genetic diversity and molecular typing of Listeria monocytogenes in China

ABSTRACT: BACKGROUND: Listeria monocytogenes can cause invasive diseases in humans and farm animals and is frequently isolated from dairy products and poultry. Listeriosis is uncommon in China but L. monocytogenes has been isolated from foods and food processing environments in China. However little is known of genetic diversity of Chinese L. monocytogenes isolates and their relationships with global isolates. RESULTS: Two hundred and twelve isolates of L. monocytogenes from food sources from 12 provinces/cities in China were analysed by serotyping, Pulsed Field Gel Electrophoresis (PFGE) and Multi-locus Sequence Typing (MLST). The predominant serotypes are 1/2a, 1/2b and 1/2c accounting for 90.1% of the isolations. PFGE divided the isolates into 61 pulse types (PTs). Twenty nine PTs were represented by more than one isolates with PT GX6A16.0004 containing the most number of isolates. MLST differentiated the isolates into 36 STs, among which 15 were novel. The most common 3 STs were ST9 (29.1%), ST8 (10.7%) and ST87 (9.2%), accounting for 49.0% of the isolates. CONCLUSIONS: STs prevalent in other parts of the world are also prevalent in China including 7 STs (ST1-ST3, ST5, ST6, ST8, ST9) which caused maternal fetal infections or outbreaks, suggesting that these STs potentially can also cause severe human infections or outbreaks in China. Surveillance of these STs will provide important information for prevention of listeriosis. This study also enhances our understanding of genetic diversity of L. monocytogenes in China.     

Novel virulence gene and clustered regularly interspaced short palindromic repeat (CRISPR) multilocus sequence typing scheme for subtyping of the major serovars of Salmonella enterica subsp. enterica

Salmonella enterica subsp. enterica is the leading cause of bacterial food-borne disease in the United States. Molecular subtyping methods are powerful tools for tracking the farm-to-fork spread of food-borne pathogens during outbreaks. In order to develop a novel multilocus sequence typing (MLST) scheme for subtyping the major serovars of S. enterica subsp. enterica, the virulence genes sseL and fimH and clustered regularly interspaced short palindromic repeat (CRISPR) loci were sequenced from 171 clinical isolates from nine Salmonella serovars, Salmonella serovars Typhimurium, Enteritidis, Newport, Heidelberg, Javiana, I 4,[5],12:i:-, Montevideo, Muenchen, and Saintpaul. The MLST scheme using only virulence genes was congruent with serotyping and identified epidemic clones but could not differentiate outbreaks. The addition of CRISPR sequences dramatically improved discriminatory power by differentiating individual outbreak strains/clones. Of particular note, the present MLST scheme provided better discrimination of Salmonella serovar Enteritidis strains than pulsed-field gel electrophoresis (PFGE). This method showed high epidemiologic concordance for all serovars screened except for Salmonella serovar Muenchen. In conclusion, the novel MLST scheme described in the present study accurately differentiated outbreak strains/clones of the major serovars of Salmonella, and therefore, it shows promise for subtyping this important food-borne pathogen during investigations of outbreaks.     

Outbreak of Uncommon O4 Non-Agglutinating Salmonella Typhimurium Linked to Minced Pork,Saxony-Anhalt,Germany, January to April 2013

Introduction

In January 2013, the National Reference Centre for Salmonella (NRC) detected a salmonellosis cluster in Saxony-Anhalt, Germany, caused by uncommon O4 non-agglutinating, monophasic Salmonella (S.) Typhimurium DT193. Circulating predominant monophasic S. Typhimurium DT193 clones typically display resistance phenotype ASSuT. We investigated common exposures to control the outbreak, and conducted microbiological investigations to assess the strains’ phenotype.

Methods

We conducted a case-control study defining cases as persons living or working in Saxony-Anhalt diagnosed with the O4 non-agglutinating strain between January and March 2013. We selected two controls contemporarily reported with norovirus infection, frequency-matched on residence and age group, per case. We interviewed regarding food consumption, especially pork and its place of purchase. We calculated odds ratios (ORs) with 95% confidence intervals (95% CI) using logistic regression. The NRC investigated human and food isolates by PCR, SDS-PAGE, MLST, PFGE, MLVA and susceptibility testing.

Results

Altogether, 68 O4 non-agglutinating human isolates were confirmed between January and April 2013. Of those, 61 were assigned to the outbreak (median age 57 years, 44% female); 83% cases ≥ 60 years were hospitalized. Eating raw minced pork from butcheries within 3 days was associated with disease (31 cases, 28 controls; OR adjusted for sex: 3.6; 95% CI: 1.0-13). Phage type DT193 and MLST ST34 were assigned, and isolates’ lipopolysaccharide (LPS) matched control strains. Isolates linked to Saxony-Anhalt exhibited PFGE type 5. ASSuT- and ACSSuT phenotype proportions were 34 and 39% respectively; 54% were resistant to chloramphenicol. Three pork isolates matched the outbreak strain.

Discussion

Raw minced pork was the most likely infection vehicle in this first reported outbreak caused by O4 non-agglutinating, mostly chloramphenicol-resistant S. Typhimurium DT193. High hospitalization proportions demand awareness on the risk of consumption of raw pork among elderly. LPS analysis indicated O4 expression; therefore, testing with antisera from different lots is recommendable in unexpected agglutination reactions.     

Real-time PCR assay to differentiate Listeriolysin S-positive and -negative strains of Listeria monocytogenes

Due to the severity of the food-borne infection listeriosis, strict legislation governs the detectable and permissible limits at which Listeria monocytogenes is permitted in foods. These requirements, coupled with the ubiquitous nature of L. monocytogenes strains and the potential for epidemic outbreaks, mean that the pathogen can devastate affected sectors of the food industry. Although almost all L. monocytogenes strains have the potential to cause listeriosis, those implicated in the vast majority of epidemics belong to a subset of strains belonging to evolutionary lineage I. It has been established that a significant proportion of these strains, including those implicated in the majority of outbreaks, produce an additional hemolysin, designated listeriolysin S (LLS), which may be responsible for the enhanced virulence of these strains. In order to ultimately establish this definitively, it is important to first be able to rapidly discriminate between LLS-positive and -negative strains. Here, after essential genes within the LLS-encoding cluster, Listeria pathogenicity island 3, were identified by deletion mutagenesis, a real-time PCR assay which targets one such gene, llsX, was developed as a means of identifying LLS-positive L. monocytogenes. The specificity of the assay was validated against a panel of 40 L. monocytogenes strains (20 of which were LLS positive) and 25 strains representative of other Listeria species. Furthermore, 1 CFU of an LLS-positive strain per 25 g/ml of spiked foods was detected in less than 30 h when the assay was coupled with culture enrichment. The detection limit of this assay was 10 genome equivalents.     

Significant shift in median guinea pig infectious dose shown by an outbreak-associated Listeria monocytogenes epidemic clone strain and a strain carrying a premature stop codon mutation in inlA

Listeria monocytogenes contains (i) epidemic clone (EC) strains, which have been linked to the majority of listeriosis outbreaks worldwide and are overrepresented among sporadic cases in the United States, and (ii) strains commonly isolated from ready-to-eat foods that carry a mutation leading to a premature stop codon (PMSC) in inlA, which encodes the key virulence factor internalin A (InlA). Internalin A binds certain isoforms of the cellular receptor E-cadherin to facilitate crossing the intestinal barrier during the initial stages of an L. monocytogenes infection. Juvenile guinea pigs, which express the human isoform of E-cadherin that binds InlA, were intragastrically challenged with a range of doses of (i) an EC strain associated with a listeriosis outbreak or (ii) a strain carrying a PMSC mutation in inlA. Recovery of L. monocytogenes from tissues (i.e., liver, spleen, mesenteric lymph nodes, and ileum) was used to develop strain-specific dose-response curves on the basis of individual and combined organ data. Modeling of individual and combined organ data revealed an approximate 1.2 to 1.3 log(10) increase in the median infectious dose for the strain carrying a PMSC in inlA relative to that for the EC strain. Inclusion of the strain parameter significantly improved the goodness of fit for individual and combined organ models, indicating a significant shift in median infectious dose for guinea pigs challenged with an inlA PMSC strain compared to that for guinea pigs challenged with an EC strain. Results from this work provide evidence that the L. monocytogenes dose-response relationship is strain specific and will provide critical data for enhancement of current risk assessments and development of future risk assessments.     

Risk Factors for Sporadic Non-Pregnancy Associated Listeriosis in Germany—Immunocompromised Patients and Frequently Consumed Ready-To-Eat Products

Non-pregnancy associated (N-PA) listeriosis, caused by Listeria monocytogenes, is a rare but severe disease, and is predominantly food-borne. Most cases appear sporadic and their infection vehicle remains unknown. Incidence has increased since 2008 in Germany. We aimed to identify underlying conditions and foods associated with sporadic N-PA listeriosis in Germany. We performed a nationwide case-control study from March 2012-December 2013. Cases were sporadic N-PA listeriosis patients notified to public health. Control subjects were age (40–65 years, 66–75 years, ≥76 years) frequency-matched persons from a nationwide random telephone sample. A structured questionnaire collected information on underlying diseases, therapies and >60 food items. We conducted multivariable logistic regression analysis, adjusting for host factors identified by causal diagram theory, and calculated population attributable fractions. We enrolled 109 cases and 1982 controls. Cases’ median age was 69 years, 55% were male, 44% received immunosuppressive therapy within 3 months prior to illness onset; a further 28% had at least one immunocompromising disease. In multivariable analysis, immunosuppressive therapy (OR 8.8, 95%CI 4.9–15.6), immunocompromising disease (OR 2.7; 95%CI 1.4–5.2), gastric acid suppression (OR 3.0; 95%CI 1.4–6.3), the consumption of cold cooked sausages (OR 2.6; 95%CI 1.6–4.4), the preferred consumption of packaged cheese (OR 2.1; 95%CI 1.3–3.5) and pre-sliced cheese (OR 2.2; 95%CI 1.3–3.7) were significantly associated with N-PA listeriosis. These foods accounted for 59% of all cases. Typical high risk foods, e.g. cold seafood, certain types of cheeses, tended to be negatively associated with disease. In conclusion, immunosuppressive therapy and frequently consumed ready-to-eat foods are the main risk factors for sporadic N-PA listeriosis in Germany. To reduce their risk, immunocompromised persons should consume the identified foods well before the ‘use-by’ date. The microbiological criteria for Listeria monocytogenes in ready-to-eat foods may insufficiently protect persons who are markedly immunocompromised.     

First outbreak of food poisoning caused by Salmonella enterica subspecies enterica serovar Berta in Italy

Aims:  To provide an epidemiologic interpretation of a suspected outbreak of food poisoning caused by Salmonella enterica subspecies enterica serovar Berta strains isolated from humans and from the leftovers of the implicated foods (cream, dairy‐based desserts and eggs). Methods and Results:  We have correlated the similarity between the strains through genotyping with Pulsed Field Gel Electrophoresis (PFGE), studying antimicrobial sensitivity patterns and epidemiological investigation. The clonal origin of the outbreak was confirmed by all laboratory tests. PFGE analysis of the restriction profiles obtained with XbaI and SpeI revealed a certainly correlation from the strains isolated from the various sources, while the antimicrobial sensitivity pattern was the same in all cases, with all strains sensitive to all antibiotics tested. Conclusions:  Poor hygiene conditions in the facility concerned, lack of hygiene in food handling, high summer temperatures and positive cultures from asymptomatic staff could all be implicated in the infection, with food being the means through which it spread. Significance and Impact of the Study:  This study describes the first outbreak of food poisoning caused by Salmonella enterica subspecies enterica serovar Berta (Salmonella Berta) reported in Italy. It confirms the importance of correlating epidemiological investigations with genotyping and phenotyping to understand the dynamics of infection.     

Bias in the Listeria monocytogenes enrichment procedure: lineage 2 strains outcompete lineage 1 strains in University of Vermont selective enrichments

Listeria monocytogenes can be isolated from a range of food products and may cause food-borne outbreaks or sporadic cases of listeriosis. L. monocytogenes is divided into three genetic lineages and 13 serotypes. Strains of three serotypes (1/2a, 1/2b, and 4b) are associated with most human cases of listeriosis. Of these, strains of serotypes 1/2b and 4b belong to lineage 1, whereas strains of serotype 1/2a and many other strains isolated from foods belong to lineage 2. L. monocytogenes is isolated from foods by selective enrichment procedures and from patients by nonselective methods. The aim of the present study was to investigate if the selective enrichment procedure results in a true representation of the subtypes of L. monocytogenes present in a sample. Eight L. monocytogenes strains (four lineage 1 strains and four lineage 2 strains) and one Listeria innocua strain grew with identical growth rates in the nonselective medium brain heart infusion (BHI), but differed in their growth rate in the selective medium University of Vermont medium I (UVM I). When coinoculated in UVM I, some strains completely outgrew other strains. This outcome was dependent on the lineage of L. monocytogenes rather than the individual growth rate of the strains. When inoculated at identical cell densities in UVM I, L. innocua outcompeted L. monocytogenes lineage 1 strains but not lineage 2 strains. In addition, lineage 2 L. monocytogenes strains outcompeted lineage 1 L. monocytogenes strains in all combinations tested, indicating a bias in strains selected by the enrichment procedures. Bias also occurred when coinoculating two lineage 2 or lineage 1 strains; however, it did not appear to correlate with origin (clinical versus food). Identical coinoculation experiments in BHI suggested that the selective compounds in UVM I and II influenced this bias. The results of the present study demonstrate that the selective procedures used for isolation of L. monocytogenes may not allow a true representation of the types present in foods. Our results could have a significant impact on epidemiological studies, as lineage 1 strains, which are often isolated from clinical cases of listeriosis, may be suppressed during enrichment by other L. monocytogenes lineages present in a food sample.     

Neutral Genomic Microevolution of a Recently Emerged Pathogen,Salmonella enterica Serovar Agona

Salmonella enterica serovar Agona has caused multiple food-borne outbreaks of gastroenteritis since it was first isolated in 1952. We analyzed the genomes of 73 isolates from global sources, comparing five distinct outbreaks with sporadic infections as well as food contamination and the environment. Agona consists of three lineages with minimal mutational diversity: only 846 single nucleotide polymorphisms (SNPs) have accumulated in the non-repetitive, core genome since Agona evolved in 1932 and subsequently underwent a major population expansion in the 1960s. Homologous recombination with other serovars of S. enterica imported 42 recombinational tracts (360 kb) in 5/143 nodes within the genealogy, which resulted in 3,164 additional SNPs. In contrast to this paucity of genetic diversity, Agona is highly diverse according to pulsed-field gel electrophoresis (PFGE), which is used to assign isolates to outbreaks. PFGE diversity reflects a highly dynamic accessory genome associated with the gain or loss (indels) of 51 bacteriophages, 10 plasmids, and 6 integrative conjugational elements (ICE/IMEs), but did not correlate uniquely with outbreaks. Unlike the core genome, indels occurred repeatedly in independent nodes (homoplasies), resulting in inaccurate PFGE genealogies. The accessory genome contained only few cargo genes relevant to infection, other than antibiotic resistance. Thus, most of the genetic diversity within this recently emerged pathogen reflects changes in the accessory genome, or is due to recombination, but these changes seemed to reflect neutral processes rather than Darwinian selection. Each outbreak was caused by an independent clade, without universal, outbreak-associated genomic features, and none of the variable genes in the pan-genome seemed to be associated with an ability to cause outbreaks.     

Identification and characterization of pathogenic Yersinia enterocolitica isolates by PCR and pulsed-field gel electrophoresis

Approximately 550 to 600 yersiniosis patients are reported annually in Sweden. Although pigs are thought to be the main reservoir of food-borne pathogenic Yersinia enterocolitica, the role of pork meat as a vehicle for transmission to humans is still unclear. Pork meat collected from refrigerators and local shops frequented by yersiniosis patients (n=48) were examined for the presence of pathogenic Yersinia spp. A combined culture and PCR method was used for detection, and a multiplex PCR was developed and evaluated as a tool for efficient identification of pathogenic food and patient isolates. The results obtained with the multiplex PCR were compared to phenotypic test results and confirmed by pulsed-field gel electrophoresis (PFGE). In all, 118 pork products (91 raw and 27 ready-to-eat) were collected. Pathogenic Yersinia spp. were detected by PCR in 10% (9 of 91) of the raw pork samples (loin of pork, fillet of pork, pork chop, ham, and minced meat) but in none of the ready-to-eat products. Isolates of Y. enterocolitica bioserotype 4/O:3 were recovered from six of the PCR-positive raw pork samples; all harbored the virulence plasmid. All isolates were recovered from food collected in shops and, thus, none were from the patients' home. When subjected to PFGE, the six isolates displayed four different NotI profiles. The same four NotI profiles were also present among isolates recovered from the yersiniosis patients. The application of a multiplex PCR was shown to be an efficient tool for identification of pathogenic Y. enterocolitica isolates in naturally contaminated raw pork.     

Bias in the Listeria monocytogenes Enrichment Procedure: Lineage 2 Strains Outcompete Lineage 1 Strains in University of Vermont Selective Enrichments

Listeria monocytogenes can be isolated from a range of food products and may cause food-borne outbreaks or sporadic cases of listeriosis. L. monocytogenes is divided into three genetic lineages and 13 serotypes. Strains of three serotypes (1/2a, 1/2b, and 4b) are associated with most human cases of listeriosis. Of these, strains of serotypes 1/2b and 4b belong to lineage 1, whereas strains of serotype 1/2a and many other strains isolated from foods belong to lineage 2. L. monocytogenes is isolated from foods by selective enrichment procedures and from patients by nonselective methods. The aim of the present study was to investigate if the selective enrichment procedure results in a true representation of the subtypes of L. monocytogenes present in a sample. Eight L. monocytogenes strains (four lineage 1 strains and four lineage 2 strains) and one Listeria innocua strain grew with identical growth rates in the nonselective medium brain heart infusion (BHI), but differed in their growth rate in the selective medium University of Vermont medium I (UVM I). When coinoculated in UVM I, some strains completely outgrew other strains. This outcome was dependent on the lineage of L. monocytogenes rather than the individual growth rate of the strains. When inoculated at identical cell densities in UVM I, L. innocua outcompeted L. monocytogenes lineage 1 strains but not lineage 2 strains. In addition, lineage 2 L. monocytogenes strains outcompeted lineage 1 L. monocytogenes strains in all combinations tested, indicating a bias in strains selected by the enrichment procedures. Bias also occurred when coinoculating two lineage 2 or lineage 1 strains; however, it did not appear to correlate with origin (clinical versus food). Identical coinoculation experiments in BHI suggested that the selective compounds in UVM I and II influenced this bias. The results of the present study demonstrate that the selective procedures used for isolation of L. monocytogenes may not allow a true representation of the types present in foods. Our results could have a significant impact on epidemiological studies, as lineage 1 strains, which are often isolated from clinical cases of listeriosis, may be suppressed during enrichment by other L. monocytogenes lineages present in a food sample.