Relatedness of Listeria monocytogenes Isolates Recovered from Selected Ready-To-Eat Foods and Listeriosis Patients in the United States

Pulsed-field gel electrophoresis and serotyping were performed for 544 isolates of Listeria monocytogenes, including 502 isolates recovered from contaminated samples from 31,705 retail ready-to-eat (RTE) food products and 42 isolates recovered from human cases of listeriosis. The isolates were from Maryland (294 isolates) and California (250 isolates) and were collected in 2000 and 2001. The isolates were placed into 16 AscI pulsogroups (level of relatedness within each group, ≥66%), 139 AscI pulsotypes (levels of relatedness, ≥25% to 100%), and eight serotypes (serotypes 1/2a, 1/2b, 1/2c, 3a, 3b, 4b, 4c, and 4d). The most frequently found pulsotypes belonged to either pulsogroup A (150 food isolates plus 4 clinical isolates) or pulsogroup B (104 food isolates plus 5 clinical isolates). The majority of the 502 food isolates were either serotype 1/2a (298 isolates) or serotype 1/2b (133 isolates), whereas the majority of the 42 clinical isolates were either serotype 1/2a (19 isolates) or serotype 4b (15 isolates). Additionally, 13 clinical isolates displayed pulsotypes also found in food isolates, whereas the remaining 29 clinical isolates displayed 24 unique pulsotypes. These data indicate that most (86%) of the L. monocytogenes subtypes found in the RTE foods sampled belonged to only two serotypes and that 90% of the isolates displayed 73 pulsotypes, with 107 isolates displaying pulsotype 1. These data should help define the distribution and relatedness of isolates found in RTE foods in comparison with isolates that cause listeriosis.     

Heavy Metal and Disinfectant Resistance of Listeria monocytogenes from Foods and Food Processing Plants

The persistence of Listeria monocytogenes in food processing plants and other ecosystems reflects its ability to adapt to numerous stresses. In this study, we investigated 138 isolates from foods and food processing plants for resistance to the quaternary ammonium disinfectant benzalkonium chloride (BC) and to heavy metals (cadmium and arsenic). We also determined the prevalence of distinct cadmium resistance determinants (cadA1, cadA2, and cadA3) among cadmium-resistant isolates. Most BC-resistant isolates were resistant to cadmium as well. Arsenic resistance was encountered primarily in serotype 4b and was an attribute of most isolates of the serotype 4b epidemic clonal group ECIa. Prevalence of the known cadmium resistance determinants was serotype associated: cadA1 was more common in isolates of serotypes 1/2a and 1/2b than 4b, while cadA2 was more common in those of serotype 4b. A subset (15/77 [19%]) of the cadmium-resistant isolates lacked the known cadmium resistance determinants. Most of these isolates were of serotype 4b and were also resistant to arsenic, suggesting novel determinants that may confer resistance to both cadmium and arsenic in these serotype 4b strains. The findings may reflect previously unrecognized components of the ecological history of different serotypes and clonal groups of L. monocytogenes, including exposures to heavy metals and disinfectants.     

Pulsed-field fingerprinting of listeriae: identification of genomic divisions for Listeria monocytogenes and their correlation with serovar.

Clamped homogeneous electric field (CHEF) electrophoresis was optimized for genomic analyses of Listeria monocytogenes. Various human, animal, food, and environmental isolates, as well as strains representing other Listeria species, were separately digested with rarely cutting endonucleases. Of 176 L. monocytogenes strains analyzed, the enzymes AscI and ApaI established 63 and 72 unique restriction endonuclease digestion profiles (REDP), respectively. The 22 non-L. monocytogenes strains exhibited 18 AscI and 19 ApaI unique REDP. Statistical analyses of REDP information using the Dice coincidence index and principal component analysis revealed two distinct genomic divisions of L. monocytogenes that also correlated with the flagellar (H) antigen type: division I contained serovar 1/2a, 1/2c, 3a, and 3c stains and division II contained serovar 1/2b, 3b, 4b, 4d, and 4e strains. Division I isolates digested with ApaI were further grouped into cluster IA (serovar 1/2c and 3c) and cluster IB (serovar 1/2a and 3a) strains. Likewise, division II isolates digested with ApaI were further grouped into cluster IIA (serovar 1/2b and 3b) and cluster IIB (serovar 4b, 4d, and 4e) strains. These data indicate that genotypic data generated by CHEF can be directly related to phenotypic data generated by serotyping for establishing the overall relatedness of isolates. Moreover, these data further substantiate that CHEF analysis is a reproducible and highly discriminating method for characterizing L. monocytogenes strains at the molecular level.     

Pulsed-field gel electrophoresis (PFGE) analysis of Listeria monocytogenes isolates from different sources and geographical origins and representative of the twelve serovars

Multiplex-PCR (MPCR) serogrouping and pulsed-field gel electrophoresis (PFGE) subtyping analysis are currently used by several public and private laboratories for the characterization of Listeria monocytogenes. In this study a set of 80 L. monocytogenes isolates belonging to the twelve serovars was used to investigate (i) the typeability of the rare serovars, (ii) the ability of PFGE analysis with ApaI and AscI to differentiate serovars within MPCR serogroups and (iii) the association of molecular types with the specific source or geographical origin of the isolates. With the exception of three isolates (rare serovars 4a and 4c) that were not amenable to restriction with ApaI, all the other analyzed isolates were subtyped by both enzymes. PFGE discriminated the 80 isolates into 62 combined ApaI and AscI PFGE patterns (pulsotypes), but could not differentiate serovars within MPCR serogroups, in which isolates from different serovars displaying the same pulsotype were found. Clustering analysis suggests that for some pulsotypes grouping according to Portuguese origin or source can be suggested. On the other hand, some L. monocytogenes clones are widely distributed. Two pulsotypes from Portuguese human isolates were identical to the ones displayed by human outbreak clones in the UK and in the USA and Switzerland, respectively, although they were not temporally matched. Computer-assisted data analysis of large and diverse PFGE type databases will improve the correct interpretation of subtyping data in epidemiological studies and in tracing routes and sources of contamination in the food industry.     

Genotyping by REA-PFGE of Listeria monocytogenes isolated from clinical, environmental and food samples

Listeria monocytogenes strains isolated from clinical food and environmental samples were genotyped by Restriction Enzyme Analysis with Pulsed Field Gel Electrophoresis (REA-PFGE) using ApaI and AscI enzymes according to PulseNet Europe procedure. Analysis of DNA fragments profiles obtained by AscI digestion demonstrated presence of 62 REA-PFGE profiles grouped in 2 lineages (FI, FII). Diversity of strains source among both lineages was observed. Statistical analysis showed, that strains isolated from clinical samples more frequently are included to lineage FI, then lineage FII. Non-clinical strains were more frequently included to lineage FII. Combined analysis of REA-PFGE profiles for ApaI and AscI enzymes showed 8 unique pulsotypes characteristic for two or more L. monocytogenes isolates. Moreover researched L. monocytogenes strains were analyzed by multiplex-PCR according Doumith et al methodology. PCR-group 4B was most frequent among strains isolated from clinical samples. Correlation between PCR-group and pulsotype was observed only in few cases.     

Heteroduplex mobility assay for the identification of Listeria sp and Listeria monocytogenes strains: application to characterisation of strains from sludge and food samples

One hundred and ten Listeria sp. isolates from sewage sludge were identified according to phenotypic and genotypic methods. The Listeria sp. strains isolated from five types of sludge from three sewage treatment plants in Angers (France) and the surrounding area included L. monocytogenes (55.5%), L. innocua (29.1%), L. seeligeri (13.6%) and L. welshimeri (1.8%). The majority of L. monocytogenes strains belonged to serotypes 4b, 1/2b and 1/2a. Moreover, a heteroduplex mobility assay based on the 16S rRNA sequences was tested for its ability to identify the six species of the genus Listeria. This study, performed on 283 Listeria sp. strains from human, food and sewage sludge samples, showed that all the species were distinguishable from one another. L. innocua and L. seeligeri showed respectively three and two distinct banding patterns. Within L. monocytogenes, four groups (I-IV) were defined. The majority of food and environmental isolates were clustered in group I and it is noteworthy that group IV clustered epidemiologic isolates and strains belonging to serotypes 4b, 1/2a and 1/2b.     

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.     

Combined sigB allelic typing and multiplex PCR provide improved discriminatory power and reliability for Listeria monocytogenes molecular serotyping

Conventional serotyping has traditionally been used to subtype Listeria monocytogenes, but has several limitations, including low discriminatory power and poor reproducibility. Molecular serotyping methods have been developed for L. monocytogenes, but generally show limited discriminatory power and high misclassification rates. We selected 157 Listeria isolates to evaluate a combination of a previously described multiplex PCR assay and sigB allelic typing as an alternative molecular serotyping and subtyping strategy for L. monocytogenes. While the multiplex PCR assay differentiated five L. monocytogenes subtypes (Simpson's Index of Discrimination [SID]=0.78), including classification of the most common disease-associated serotypes (1/2a, 1/2b, 1/2c, and lineage I 4b) into four distinct groups, it misclassified 3.8% of the isolates studied here. sigB allelic typing differentiated 29 subtypes (SID=0.87) and also allowed identification of lineage III L. monocytogenes, which could not be differentiated from the other Listeria spp. by the multiplex PCR assay. sigB allelic typing failed to differentiate serotype 1/2c and 1/2a isolates and one sigB allelic type included serotype 4b and 1/2b isolates. A molecular serotyping approach that combines multiplex PCR and sigB sequence data showed increased discriminatory power (SID=0.91) over either method alone as well as conventional serotyping (SID=0.87) and classifies the four major serotypes (i.e., 1/2a, 1/2b, 1/2c, and 4b) into unique subgroups with a lower misclassification rate as compared to the multiplex PCR assay. This combined approach also differentiates lineage I serotype 4b isolates from the genetically distinct serotype 4b isolates classified into lineage III.     

Prevalence and lineages of Listeria monocytogenes in Chinese food products

AIMS: This study was undertaken to investigate the prevalence and lineages of Listeria monocytogenes in different kinds of food products in local Chinese markets. METHODS AND RESULTS: A total of 2686 food samples and 645 water samples were collected and L. monocytogenes was isolated from 2.28% (76 of 3331) of all samples. The prevalence of L. monocytogenes (14 of 290, 4.83%) in raw meat products was significantly higher than that in other raw food products (P < 0.05). Among 844 ready-to-eat (RTE) food samples, 21 samples were positive for L. monocytogenes. RTE packaged food products from two supermarkets had a prevalence ranging from 0.00% to 25.00%. The prevalence of L. monocytogenes in meat products of freshly slaughtered hogs was 0.95% (four of 420), significantly lower than that in raw meat products in the retail markets (P < 0.05). Ten isolates were recovered from 645 water samples, which were collected after hands washing by shopkeepers or waiters. A total of 38 isolates were randomly selected for lineage classification based on the nucleotide variation of actA gene. Eighty percentage of isolates from RTE food products belonged to Lineage II while only 20% belonged to Lineage I. CONCLUSIONS: Food products in Chinese markets are contaminated with L. monocytogenes. Raw meat products have the highest contamination rates among all the raw food samples. RTE food products are more likely to be contaminated with Lineage II strains. SIGNIFICANCE AND IMPACT OF THE STUDY: The data presented here show the main contamination sources of L. monocytogenes in Chinese food products.     

Characterization of Listeria monocytogenes isolates from food animal clinical cases: PFGE pattern similarity to strains from human listeriosis cases

Twenty-one isolates of Listeria monocytogenes from food animal clinical cases that involved meningitis or meningoencephalitis, encephalitis, mastitis and abortion were characterized by serotyping and pulsed-field gel electrophoresis (PFGE) in order to improve our understanding of the genetic links between individual strains and strains recovered from human listeriosis cases. Results showed that five of the isolates were serotype 1/2a, six were 1/2b, nine were 4b, and one was untypeable. A caprine, two bovine and an ovine brain isolate shared identical PFGE patterns indicating that strains of L. monocytogenes are not host specific. Other isolates exhibited distinct patterns that were not shared, indicating a genetic diversity. Dendrogram analysis revealed that PFGE patterns of the isolates clustered primarily according to serotype. We compared the PFGE types obtained for these isolates with PFGE types for human clinical isolates present in the CDC national PulseNet database. Six (29%) of the twenty-one strains had patterns that were indistinguishable from pathogenic human isolates in the database. Our observations offer preliminary evidence that food animals could be significant reservoirs of L. monocytogenes that lead to human infections and support the inclusion of PFGE patterns of veterinary clinical isolates in the national PulseNet database for increased surveillance.     

Characteristics and frequency of detection of fecal Listeria monocytogenes shed by livestock, wildlife, and humans

Listeria monocytogenes is a facultative intracellular pathogen that can be carried asymptomatically in various animals and can be shed in feces. We investigated the prevalence and characteristics of L. monocytogenes isolated from livestock, wildlife, and human potential sources of contamination in 2 areas in Ontario, Canada. From February 2003 to November 2005, a total of 268 fecal samples were collected from different animals. Listeria monocytogenes was isolated using selective enrichment, isolation, and confirmation procedures, and 15 samples (6%) yielded to the isolation of 84 confirmed strains. Listeria monocytogenes was isolated from livestock (beef and dairy), wildlife (deer, moose, otter, and raccoon), and human (biosolids and septic) fecal sources. Thirty-two isolates were from serovar 1/2a, 34 from serovar 1/2b, 1 from serovar 3a, and 17 from serovar 4b. Listeria monocytogenes populations were resolved into 13 EcoRI ribotypes, and 18 ApaI and 18 AscI pulsotypes, with Simpson indexes of discrimination of 0.878 and 0.907, respectively. A majority (59%) of L. monocytogenes isolates exhibited potential virulence linked to the production of a functional internalin A, which was supported by higher entry into Caco-2 cells (9.3%) than isolates producing truncated and secreted internalin A (1.3% of entry). Listeria monocytogenes fecal isolates were on average resistant to 6.4 +/- 2.5 antibiotics out of 17 tested, and potentially virulent isolates exhibited an enhanced resistance to kanamycin, gentamicin, streptomycin, and rifampicin. Livestock, wildlife, and human L. monocytogenes fecal communities exhibited overlapping but distinct populations, and some genotypes and phenotypes were similar to those previously described for surface water isolates in the same area.     

A new perspective on Listeria monocytogenes evolution

Listeria monocytogenes is a model organism for cellular microbiology and host-pathogen interaction studies and an important food-borne pathogen widespread in the environment, thus representing an attractive model to study the evolution of virulence. The phylogenetic structure of L. monocytogenes was determined by sequencing internal portions of seven housekeeping genes (3,288 nucleotides) in 360 representative isolates. Fifty-eight of the 126 disclosed sequence types were grouped into seven well-demarcated clonal complexes (clones) that comprised almost 75% of clinical isolates. Each clone had a unique or dominant serotype (4b for clones 1, 2 and 4, 1/2b for clones 3 and 5, 1/2a for clone 7, and 1/2c for clone 9), with no association of clones with clinical forms of human listeriosis. Homologous recombination was extremely limited (r/m<1 for nucleotides), implying long-term genetic stability of multilocus genotypes over time. Bayesian analysis based on 438 SNPs recovered the three previously defined lineages, plus one unclassified isolate of mixed ancestry. The phylogenetic distribution of serotypes indicated that serotype 4b evolved once from 1/2b, the likely ancestral serotype of lineage I. Serotype 1/2c derived once from 1/2a, with reference strain EGDe (1/2a) likely representing an intermediate evolutionary state. In contrast to housekeeping genes, the virulence factor internalin (InlA) evolved by localized recombination resulting in a mosaic pattern, with convergent evolution indicative of natural selection towards a truncation of InlA protein. This work provides a reference evolutionary framework for future studies on L. monocytogenes epidemiology, ecology, and virulence.     

Competitive fitness of Listeria monocytogenes serotype 1/2a and 4b strains in mixed cultures with and without food in the U.S. Food and Drug Administration enrichment protocol

Thirteen different serotypes of the food-borne pathogen Listeria monocytogenes have been described. Serotype 4b strains are most often associated with illness, and serotype 1/2a strains are most often isolated from foods and processing plants. Different abilities to respond to stresses have been described for serotype 4b and 1/2a strains. One of the common enrichment protocols used to test foods for the presence L. monocytogenes is described in the U.S. Food and Drug Administration (FDA) Bacterial Analytical Manual (BAM). We compared three strains of L. monocytogenes serotype 4b and five strains of serotype 1/2a in direct competition with each other in two-strain mixed cultures by using the FDA BAM enrichment protocol, which includes both enrichment broth and selective agar, with and without added food to mimic the conditions that occur during attempts to isolate Listeria species from contaminated foods. Using a colony immunoblot procedure and analyzing over 112,000 colonies, we observed differences in strain fitness, but these differences were not attributable to serotype or genetic lineage.     

Atypical Listeria monocytogenes serotype 4b strains harboring a lineage II-specific gene cassette

Listeria monocytogenes is the etiological agent of listeriosis, a severe food-borne illness. The population of L. monocytogenes is divided into four lineages (I to IV), and serotype 4b in lineage I has been involved in numerous outbreaks. Several serotype 4b epidemic-associated clonal groups (ECI, -II, and -Ia) have been identified. In this study, we characterized a panel of strains of serotype 4b that produced atypical results with a serotype-specific multiplex PCR and possessed the lmo0734 to lmo0739 gene cassette that had been thought to be specific to lineage II. The cassette was harbored in a genomically syntenic locus in these isolates and in lineage II strains. Three distinct clonal groups (groups 1 to 3) were identified among these isolates based on single-nucleotide polymorphism-based multilocus genotyping (MLGT) and DNA hybridization data. Groups 1 and 2 had MLGT haplotypes previously encountered among clinical isolates and were composed of clinical isolates from multiple states in the United States. In contrast, group 3 consisted of clinical and environmental isolates solely from North Carolina and exhibited a novel haplotype. In addition, all group 3 isolates had DNA that was resistant to MboI, suggesting methylation of adenines at GATC sites. Sequence analysis of the lmo0734 to lmo0739 gene cassette from two strains (group 1 and group 3) revealed that the genes were highly conserved (>99% identity). The data suggest relatively recent horizontal gene transfer from lineage II L. monocytogenes into L. monocytogenes serotype 4b and subsequent dissemination among at least three distinct clonal groups of L. monocytogenes serotype 4b, one of which exhibits restrictions in regional distribution.     

Estimating size and diversity of nitrifying communities in deodorizing filters using PCR and immunofluorescence

Thirty isolates of Listeria monocytogenes and 18 of L. innocua obtained from different short-ripened cheeses manufactured in Asturias (northern Spain), were compared with each other and with reference strains using serotype, phage type and pulsed-field restriction endonuclease digestion profiles analysis of the total DNA. Restriction enzymes Apa I and Sma I defined five clusters in L. monocytogenes ( m1 to m5 ) and two main clusters in L. innocua ( i1 and i2 ). Cluster i2 was further arranged into three subclusters ( i2a , i2b and i2c ) based on the different Eco 52I ( Xma III) and Crf 42I ( Sac II) patterns of its isolates. Clusters of L. innocua were clearly different whereas those of L. monocytogenes were more closely related to each other. In this latter species, serotype 4b isolates ( m4 and m5 ) constituted a more homogeneous group than serogroup 1 isolates ( m1 , m2 and m3 ). Cluster m3 contained two strains of serotype 1/2a whereas m1 and m2 harboured strains of both serotypes, 1/2a and 1/2b. Therefore, the combined use of restriction patterns and serotype may be useful to differentiate L. monocytogenes strains showing identical restriction profiles but differing in serotype. The cheese source of Listeria strains proved that isolates from cluster m1 were repeatedly detected as a contaminant in the same type of cheese. Comparison of L. monocytogenes Apa I profiles showed a genetic proximity of m4 and m5 to the recognized pathogenic strains ATCC 13932 and NCTC 11994, responsible for meningitis cases in other countries. Finally, bacteriophage typing data indicated that m4 , the sole phage typable group, had a phage type resembling that of strains causing the Auckland (New Zealand) outbreak of listeriosis in 1969. These data suggest a wide distribution of closely related types which might cause, under several circumstances, sporadic cases of listeriosis.     

Distribution and characteristics of Listeria monocytogenes isolates from surface waters of the South Nation River watershed, Ontario, Canada

Listeria monocytogenes is a facultative intracellular pathogen thought to be widely distributed in the environment. We investigated the prevalence and characteristics of L. monocytogenes isolates from surface waters derived from catchments within the South Nation River watershed (Ontario, Canada). This watershed is dominated by urban and rural development, livestock and crop production, and wildlife habitats. From June to November 2005, a total of 314 surface water samples were collected biweekly from 22 discrete sampling sites characterized by various upstream land uses. Presumptive Listeria spp. were isolated using a selective enrichment and isolation procedure, and 75 L. monocytogenes isolates were identified based on colony morphology, hemolytic activity, and amplification of three pathogenicity genes: iap, inlA, and hlyA. Thirty-two of 314 (10%) surface water samples were positive for the presence of L. monocytogenes, but detection ranged between 0 and 27% depending on the sampling date. Isolates belonging to serovar group 1/2a, 3a (50%) and group 4b, 4d, 4e (32%) were dominant. L. monocytogenes populations were resolved into 13 EcoRI ribotypes and 21 ApaI and 21 AscI pulsotypes. These had Simpson indexes of discrimination of up to 0.885. Lineage I-related isolates were dominant (61%) during the summer, whereas lineage II isolates were dominant (77%) in the fall. Isolates were, on average, resistant to 6.1 +/- 2.1 antibiotics out of 17 tested. Half of the L. monocytogenes isolates exhibited potential virulence linked to the production of a functional internalin A, and some isolates were found to be moderately to highly virulent by in vitro Caco-2 plaque formation assay (up to 28% of entry). There was a statistically significant link between the occurrence of L. monocytogenes and proximity to an upstream dairy farm and degree of cropped land. Our data indicate that L. monocytogenes is widespread in the studied catchments, where it could represent a public health issue related to agricultural land use.     

Virulence genes and intimin types of Shiga-toxin-producing Escherichia coli isolated from cattle and beef products in Argentina.

A total of 153 Shiga-toxin-producing Escherichia coli (STEC) isolates from feces of cattle and beef products (hamburgers and ground beef) in Argentina were characterized in this study. PCR showed that 22 (14%) isolates carried stx1 genes, 113 (74%) possessed stx2 genes and 18 (12%) both stx1 and stx2. Intimin (eae), enterohemolysin (ehxA), and STEC autoagglutinating adhesin (saa) virulence genes were detected in 36 (24%), 70 (46%) and in 34 (22%) of the isolates, respectively. None of 34 saa-positive isolates carried the gene eae, and 31 were ehxA-positive. Fourteen (7 of serotype O26:H11 and 4 of serotype O5:H-) isolates had intimin b1, 16 isolates possessed intimin g1 (11 of serotype O145:H- and 5 of serotype O157:H7), 5 isolates had intimin type e1 (4 of serotypes O103:H- and O103:H2), and one isolate O111:H- showed intimin type q/g2. Although the 153 STEC isolates belonged to 63 different seropathotypes, only 12 accounted for 58% of isolates. Seropathotype ONT:H- stx2 (18 isolates) was the most common, followed by O171:H2 stx2 (12 isolates), etc. The majority (84%) of STEC isolates belonged to serotypes previously found in human STEC and 56% to serotypes associated with STEC isolated from patients with hemolytic uremic syndrome (HUS). Thus, this study confirms that cattle are a major reservoir of STEC pathogenic for humans. To our knowledge, this is the first study that described the presence of saa gene in STEC of serotypes O20:H19, O39:H49, O74:H28, O79:H19, O116:H21, O120:H19, O141:H7, O141:H8, O174:H21, and ONT:H21. The serotypes O120:H19 and O185:H7 were not previously reported in bovine STEC.     

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.     

Listeria monocytogenes serotypes in Italian meat products

Listeria monocytogenes was isolated and enumerated in Italian fresh ground beef, fresh pork meat and industrial sausages. All the samples contained less than 2000 L. monocytogenes /g of meat. The main serotype isolated was 1/2c (56.9%). Other serotypes isolated included 1/2a, 1/2b, 3c, 4b and 4c. A prevalence of less virulent serotypes over more virulent was thus noted. It seems that the low incidence of listeriosis from these products is related to the low concentration and virulence of L. monocytogenes present.     

An SNP-based PCR assay to differentiate between Listeria monocytogenes lineages derived from phylogenetic analysis of the sigB gene

The alternative sigma factor sigB gene is involved in the stress response regulation of Listeria monocytogenes, and contributes towards growth and survival in adverse conditions. This gene was examined to determine if it could be a useful indicator of lineage differentiation, similar to the established method based on ribotyping. The sigB sequence was resolved in four local L. monocytogenes strains and the phylogenetic relationship among these, and a further 21 sigB gene sequences from strains of different serotype and lineage including two Listeria innocua strains, obtained from the GenBank database were determined. The sigB nucleotide sequences of these 25 Listeria strains were then examined for single nucleotide polymorphic (SNP) sites that could differentiate between the three lineages. Based on nucleotide sequences L. monocytogenes lineage I/serotype 1/2b and 4b clustered together, lineage II/serotype 1/2a and 1/2c strains clustered together, lineage III/serotypes 4a and 4c strains clustered together and L. innocua strains clustered together as an outgroup. SNPs differentiating the three lineages were identified. Individual allele-specific PCR reactions based on these polymorphisms were successful in grouping known and a further 37 local L. monocytogenes isolates into the three lineages.