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, > or =66%), 139 AscI pulsotypes (levels of relatedness, > or =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.     

Molecular Typing by Pulsed-Field Gel Electrophoresis of Spanish Animal and Human Listeria monocytogenes Isolates

A total of 153 strains of Listeria monocytogenes isolated from different sources (72 from sheep, 12 from cattle, 18 from feedstuffs, and 51 from humans) in Spain from 1989 to 2000 were characterized by pulsed-field gel electrophoresis. The strains of L. monocytogenes displayed 55 pulsotypes. The 84 animal, 51 human, and 18 feedstuff strains displayed 31, 29, and 7 different pulsotypes, respectively, indicating a great genetic diversity among the Spanish L. monocytogenes isolates studied. L. monocytogenes isolates from clinical samples and feedstuffs consumed by the diseased animals were analyzed in 21 flocks. In most cases, clinical strains from different animals of the same flock had identical pulsotypes, confirming the existence of a listeriosis outbreak. L. monocytogenes strains with pulsotypes identical to those of clinical strains were isolated from silage, potatoes, and maize stalks. This is the first study wherein potatoes and maize stalks are epidemiologically linked with clinical listeriosis.     

Examination of Food Chain-Derived Listeria monocytogenes Strains of Different Serotypes Reveals Considerable Diversity in inlA Genotypes,Mutability, and Adaptation to Cold Temperatures

Listeria monocytogenes strains belonging to serotypes 1/2a and 4b are frequently linked to listeriosis. While inlA mutations leading to premature stop codons (PMSCs) and attenuated virulence are common in 1/2a, they are rare in serotype 4b. We observed PMSCs in 35% of L. monocytogenes isolates (n = 54) recovered from the British Columbia food supply, including serotypes 1/2a (30%), 1/2c (100%), and 3a (100%), and a 3-codon deletion (amino acid positions 738 to 740) seen in 57% of 4b isolates from fish-processing facilities. Caco-2 invasion assays showed that two isolates with the deletion were significantly more invasive than EGD-SmR (P < 0.0001) and were either as (FF19-1) or more (FE13-1) invasive than a clinical control strain (08-5578) (P = 0.006). To examine whether serotype 1/2a was more likely to acquire mutations than other serotypes, strains were plated on agar with rifampin, revealing 4b isolates to be significantly more mutable than 1/2a, 1/2c, and 3a serotypes (P = 0.0002). We also examined the ability of 33 strains to adapt to cold temperature following a downshift from 37°C to 4°C. Overall, three distinct cold-adapting groups (CAG) were observed: 46% were fast (<70 h), 39% were intermediate (70 to 200 h), and 15% were slow (>200 h) adaptors. Intermediate CAG strains (70%) more frequently possessed inlA PMSCs than did fast (20%) and slow (10%) CAGs; in contrast, 87% of fast adaptors lacked inlA PMSCs. In conclusion, we report food chain-derived 1/2a and 4b serotypes with a 3-codon deletion possessing invasive behavior and the novel association of inlA genotypes encoding a full-length InlA with fast cold-adaptation phenotypes.     

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.     

Identification and Characterization of Nucleotide Sequence Differences in Three Virulence-Associated Genes of Listeria monocytogenes Strains Representing Clinically Important Serotypes

Listeria monocytogenes is a Gram-positive, facultative intracellular bacterium that causes invasive, often fatal, disease in susceptible hosts. As a foodborne pathogen, the bacterium has emerged as a significant public health problem and has caused several epidemics in the United States and Europe. Three serotypes (1/2a, 1/2b, 4b) of L. monocytogenes are responsible for nearly 95% of all reported cases of human listeriosis. L. monocytogenes serotype 4b has caused all well-characterized foodborne epidemic outbreaks in North America and Europe between 1981 and 1993. However, most of the genetic studies to characterize virulence factors of L. monocytogenes have been done by using serotypes 1/2a and 1/2c. In this investigation, we examined three virulence-associated genes (hly encoding listeriolysin, plcA encoding phosphotidylinositol-specific phospholipase C, and inlA encoding internalin) of two serotype 4b and two serotype 1/2b strains. We chose these virulence-associated genes on the basis of published sequence differences among strains from Listeria subgroups containing serotypes 1/2a and 1/2c versus 4b, respectively. They correspond to sequence homologies that include very highly conserved (hlyA), highly conserved (plcA) and mostly conserved (inlA). We found by using nucleotide sequence analysis of the hly, plcA, and inlA genes, the two L. monocytogenes strains (including a strain associated with a foodborne disease outbreak in California in 1985) in this study, two serotype 1/2b strains from a study that we recently reported, and other similar published data for serotypes 1/2a, 1/2c, and 4b, had a high degree of sequence conservation at the gene and protein levels for all three genes. However, the sequences for the hly gene of L. monocytogenes strains of serotypes 1/2b and 4b were more closely related to each other and showed significant divergence from serotypes 1/2a and 1/2c. A unique nonsynonymous mutation was found in the hly gene of L. monocytogenes isolates that were associated with the 1985 California outbreak and were the epidemic phage type. When 158 L. monocytogenes isolates from the collection at the Centers for Disease Control and Prevention were screened, the mutation was found only in one other strain that had been isolated in California 3 years before the epidemic. Although the California epidemic clone was lactose negative, other L. monocytogenes serotype 4b isolates that were lactose negative did not possess the unique mutation observed in that epidemic clone. Received: 18 June 1997 / Accepted: 4 December 1997     

Genetic Diversity of Listeria monocytogenes Strains from a High-Prevalence Dairy Farm

Listeria monocytogenes is a significant food-borne human and veterinary pathogen. Contaminated silage commonly leads to disease in livestock, but the pervasive nature of the bacterium can make it difficult to identify the source of infection. An investigation of bovine listeriosis that occurred on a Pacific Northwest dairy farm (“farm A”) revealed that the clinical strain was closely related to fecal strains from asymptomatic cows, and that farm environment was heavily contaminated with a diversity of L. monocytogenes strains. In addition, the farm A clinical strain was closely related to clinical and environmental strains obtained 1 year prior from a second Northwest dairy farm (“farm B”). To investigate the source(s) of contamination on farm A, environmental samples were collected from farm A at two time points. Pulsed-field gel electrophoresis characterization of 538 isolates obtained from that farm identified 57 different AscI pulsovars. Fecal isolates obtained from individual cows were the most genetically diverse, with up to 94% of fecal samples containing more than one pulsovar. The maximum numbers of pulsovars and serotypes isolated from a fecal sample of one cow were 6 and 4, respectively. Serotype 1/2a was isolated most frequently at both time points. Microarray genotyping of bovine listeriosis, fecal, and silage strains from both farms identified four probes that differentiated listeriosis strains from environmental strains; however, no probe was common to both bovine listeriosis strains.     

Whole Genome Sequence Analysis Using JSpecies Tool Establishes Clonal Relationships between Listeria monocytogenes Strains from Epidemiologically Unrelated Listeriosis Outbreaks

In an effort to build a comprehensive genomic approach to food safety challenges, the FDA has implemented a whole genome sequencing effort, GenomeTrakr, which involves the sequencing and analysis of genomes of foodborne pathogens. As a part of this effort, we routinely sequence whole genomes of Listeria monocytogenes (Lm) isolates associated with human listeriosis outbreaks, as well as those isolated through other sources. To rapidly establish genetic relatedness of these genomes, we evaluated tetranucleotide frequency analysis via the JSpecies program to provide a cursory analysis of strain relatedness. The JSpecies tetranucleotide (tetra) analysis plots standardized (z-score) tetramer word frequencies of two strains against each other and uses linear regression analysis to determine similarity (r²). This tool was able to validate the close relationships between outbreak related strains from four different outbreaks. Included in this study was the analysis of Lm strains isolated during the recent caramel apple outbreak and stone fruit incident in 2014. We identified that many of the isolates from these two outbreaks shared a common 4b variant (4bV) serotype, also designated as IVb-v1, using a qPCR protocol developed in our laboratory. The 4bV serotype is characterized by the presence of a 6.3 Kb DNA segment normally found in serotype 1/2a, 3a, 1/2c and 3c strains but not in serotype 4b or 1/2b strains. We decided to compare these strains at a genomic level using the JSpecies Tetra tool. Specifically, we compared several 4bV and 4b isolates and identified a high level of similarity between the stone fruit and apple 4bV strains, but not the 4b strains co-identified in the caramel apple outbreak or other 4b or 4bV strains in our collection. This finding was further substantiated by a SNP-based analysis. Additionally, we were able to identify close relatedness between isolates from clinical cases from 1993–1994 and a single case from 2011 as well as links between two isolates from over 30 years ago. The identification of these potential links shows that JSpecies Tetra analysis can be a useful tool in rapidly assessing genetic relatedness of Lm isolates during outbreak investigations and for comparing historical isolates. Our analyses led to the identification of a highly related clonal group involved in two separate outbreaks, stone fruit and caramel apple, and suggests the possibility of a new genotype that may be better adapted for certain foods and/or environment.     

Population Structure of Listeria monocytogenes Serotype 4b Isolates from Sporadic Human Listeriosis Cases in the United States from 2003 to 2008

Listeria monocytogenes can cause severe food-borne disease (listeriosis). Numerous outbreaks have involved three serotype 4b epidemic clones (ECs): ECI, ECII, and ECIa. However, little is known about the population structure of L. monocytogenes serotype 4b from sporadic listeriosis in the United States, even though most cases of human listeriosis are in fact sporadic. Here we analyzed 136 serotype 4b isolates from sporadic cases in the United States, 2003 to 2008, utilizing multiple tools including multilocus genotyping, pulsed-field gel electrophoresis, and sequence analysis of the inlAB locus. ECI, ECII, and ECIa were frequently encountered (32, 17, and 7%, respectively). However, annually 30 to 68% of isolates were outside these ECs, and several novel clonal groups were identified. An estimated 33 and 17% of the isolates, mostly among the ECs, were resistant to cadmium and arsenic, respectively, but resistance to benzalkonium chloride was uncommon (3%) among the sporadic isolates. The frequency of clonal groups fluctuated within the 6-year study period, without consistent trends. However, on several occasions, temporal clusters of isolates with indistinguishable genotypes were detected, suggesting the possibility of hidden multistate outbreaks. Our analysis suggests a complex population structure of serotype 4b L. monocytogenes from sporadic disease, with important contributions by ECs and several novel clonal groups. Continuous monitoring will be needed to assess long-term trends in clonality patterns and population structure of L. monocytogenes from sporadic 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.     

Restriction Fragment Length Polymorphisms Detected with Novel DNA Probes Differentiate among Diverse Lineages of Serogroup 4 Listeria monocytogenes and Identify Four Distinct Lineages in Serotype 4b

Listeria monocytogenes of serotype 4b has been implicated in numerous outbreaks of food-borne listeriosis and in ca. 40% of sporadic cases. Strains of this serotype appear to be relatively homogeneous genetically, and molecular markers specific for distinct serotype 4b lineages have not been frequently identified. Here we show that DNA fragments derived from the putative mannitol permease locus of Listeria monocytogenes had an unexpectedly high potential to differentiate among different strains of serotype 4b when used as probes in Southern blotting of EcoRI-digested genomic DNA, yielding four distinct restriction fragment length polymorphism (RFLP) patterns. Strains of two epidemic-associated lineages, including the major epidemic clone implicated in several outbreaks in Europe and North America, had distinct RFLPs which differed from those of all other serotype 4b strains that we screened but which were encountered among strains of serotypes 1/2b and 3b. In addition, three serogroup 4 lineages were found to have unique RFLPs that were not encountered among any other L. monocytogenes strains. One was an unusual lineage of serotype 4b, and the other two were members of the serotype 4a and 4c group. The observed polymorphisms may reflect evolutionary relationships among lineages of L. monocytogenes and may facilitate detection and population genetic analysis of specific lineages.     

Genomic Characterization of Listeria monocytogenes Strains Involved in a Multistate Listeriosis Outbreak Associated with Cantaloupe in US

A multistate listeriosis outbreak associated with cantaloupe consumption was reported in the United States in September, 2011. The outbreak investigation recorded a total of 146 invasive illnesses, 30 deaths and one miscarriage. Subtyping of the outbreak associated clinical, food and environmental isolates revealed two serotypes (1/2a and 1/2b) and four pulsed-field gel electrophoresis two-enzyme pattern combinations I, II, III, and IV, including one rarely seen before this outbreak. A DNA-microarray, Listeria GeneChip®, developed by FDA from 24 Listeria monocytogenes genome sequences, was used to further characterize a representative sample of the outbreak isolates. The microarray data (in the form of present or absent calls of specific DNA sequences) separated the isolates into two distinct groups as per their serotypes. The gene content of the outbreak-associated isolates was distinct from that of the previously-reported outbreak strains belonging to the same serotypes. Although the 1/2b outbreak associated isolates are closely related to each other, the 1/2a isolates could be further divided into two distinct genomic groups, one represented by pattern combination I strains and the other represented by highly similar pattern combinations III and IV strains. Gene content analysis of these groups revealed unique genomic sequences associated with these two 1/2a genovars. This work underscores the utility of multiple approaches, such as serotyping, PFGE and DNA microarray analysis to characterize the composition of complex polyclonal listeriosis outbreaks.     

Ability of the Listeria monocytogenes Strain Scott A To Cause Systemic Infection in Mice Infected by the Intragastric Route

Listeriosis is an important food-borne disease that causes high rates of morbidity and mortality. For reasons that are not clear, most large outbreaks of human listeriosis involve Listeria monocytogenes serotype 4b. Relatively little is known about the pathogenesis of listeriosis following gastrointestinal exposure to food-borne disease isolates of L. monocytogenes. In the present study, we investigated the pathogenesis of systemic infection by the food-borne isolate Scott A in an intragastric (i.g.) mouse challenge model. We found that the severity of infection with L. monocytogenes Scott A was increased in mice made neutropenic by administration of monoclonal antibody RB6-8C5. This observation was similar to a previous report on a study with the laboratory strain L. monocytogenes EGD. Prior administration of sodium bicarbonate did not enhance the virulence of L. monocytogenes strain Scott A for i.g. inoculated mice. Following i.g. inoculation of mice, two serotype 4b strains of L. monocytogenes (Scott A and 101M) achieved a greater bacterial burden in the spleen and liver and elicited more severe histopathological damage to those organs than did a serotype 1/2a strain (EGD) and a serotype 1/2b stain (CM). Of the four strains tested, only strain CM exhibited poor survival in synthetic gastric fluid in vitro. The other three strains exhibited similar patterns of survival at pHs of greater than 5 and relatively rapid (<30 min) loss of viability at pHs of less than 5.0. Growth of L. monocytogenes Scott A at temperatures of 12.5 to 37°C did not affect its ability to cause systemic infection in i.g. inoculated mice. These observations suggest that the serotype 4b L. monocytogenes strains Scott A and 101M possess one or more virulence determinants that make them better able to cause systemic infection following inoculation via the g.i. tract than do the serotype 1/2 strains EGD and CM.     

Comparative analysis of CRISPR loci in different Listeria monocytogenes lineages

Listeria monocytogenes, an important food-borne pathogen, causes high mortality rate of listeriosis. Pan-genomic comparisons revealed the species genome of L. monocytogenes is highly stable but not completely clonal. The population structure of this species displays at least four evolutionary lineages (I–IV). Isolates of different lineages displayed distinct genetic, phenotypic and ecologic characteristics, which appear to affect their ability to be transmitted through foods and to cause human disease, as well as their ability to thrive in markedly phage-rich environments. CRISPR (clustered regularly interspaced short palindrome repeats), a recently described adaptive immunity system, not only confers defense against invading elements derived from bacteriophages or plasmids in many bacteria and archaeal, but also displays strains-level variations in almost any given endowed species. This work was aimed to investigate CRISPR diversity in L. monocytogenes strains of different lineages and estimated the potential practicability of the CRISPR-based approach to resolve this species’ biodiversity. Only a third of strains contained all three CRISPR loci (here defined as LMa, LMb and LMc) at same time. Combined the strain-level variations in presence/absence of each CRISPR locus and its relative size and spacer arrangements, a total of 29 CRISPR genotypes and 11 groups were defined within a collection of 128 strains covering all serotypes. The CRISPR-based approach showed powerful ability to subtype the more commonly food-borne isolates of serotype 1/2a (lineage II) and serotypes 1/2b (lineage I), but limited by the absence of typical CRISPR structure in many lineage I isolates. Strikingly, we found a long associated cas1 gene as well as two self-targeting LMb spacers accidently homologous with endogenous genes in a fraction of serotype 1/2a isolations, demonstrated that CRISPR I B system might involve in bacterial physiology besides antiviral immunity.     

Synergistic Effects of Sodium Chloride,Glucose, and Temperature on Biofilm Formation by Listeria monocytogenes Serotype 1/2a and 4b Strains

Biofilm formation by Listeria monocytogenes is generally associated with its persistence in the food-processing environment. Serotype 1/2a strains make up more than 50% of the total isolates recovered from food and the environment, while serotype 4b strains are most often associated with major outbreaks of human listeriosis. Using a microplate assay with crystal violet staining, we examined biofilm formation by 18 strains of each serotype in tryptic soy broth with varying concentrations of glucose (from 0.25% to 10.0%, wt/vol), sodium chloride (from 0.5% to 7.0%, wt/vol) and ethanol (from 1% to 5.0%, vol/vol), and at different temperatures (22.5°C, 30°C, and 37°C). A synergistic effect on biofilm formation was observed for glucose, sodium chloride, and temperature. The serotype 1/2a strains generally formed higher-density biofilms than the 4b strains under most conditions tested. Interestingly, most serotype 4b strains had a higher growth rate than the 1/2a strains, suggesting that the growth rate may not be directly related to the capacity for biofilm formation. Crystal violet was found to stain both bacterial cells and biofilm matrix material. The enhancement in biofilm formation by environmental factors was apparently due to the production of extracellular polymeric substances instead of the accumulation of viable biofilm cells.Listeria monocytogenes, a Gram-positive bacterium, is capable of causing severe food-borne infections in both humans and animals. The organism is ubiquitous in the environment and can grow in a wide variety of foods, including those stored at refrigeration temperatures. It is particularly difficult to eliminate this bacterium from ready-to-eat foods and food-processing equipment (19). The ability to form biofilms protects the bacterium from stresses in food-processing environments (13, 25). Among the 13 different serotypes described, serotypes 1/2a, 1/2b, and 4b are involved in the majority of human cases of listeriosis. Serotype 4b strains have accounted for most human outbreaks, whereas the majority of L. monocytogenes strains isolated from foods or food-processing plants belong to serotype 1/2a (19).Comparative studies to link the phenotypic attributes of L. monocytogenes strains to serotypes have obtained variable results. Buncic et al. (4) have shown that serotype 1/2a isolates were more resistant to antilisterial bacteriocins than serotype 4b strains at 4°C. They also found that 4b isolates exhibited greater resistance to heat treatments at 60°C and were easier to recover than 1/2a strains immediately following cold storage. Bruhn et al. (3) observed that 1/2a strains (lineage II) grew faster than 4b and 1/2b (lineage I) strains in commonly used enrichment broth media (University of Vermont media I and II). However, other studies have indicated that similar differences could not be linked to a serotype (14), and sequencing results have shown a syntenic relationship between strains of the two serotypes (27).Some L. monocytogenes strains have consistently been isolated from food-processing plants over many years (1, 28). Although several studies have been carried out to identify differences in cell adherence and biofilm formation among different serotypes, conflicting results were obtained. Lineage I isolates (including serotypes 4b, 1/2b, 3c, and 3b) were found to produce higher-density biofilms than lineage II isolates (including serotypes 1/2a, 1/2c, and 3a) (8, 28). However, this conclusion was not supported by other studies (1, 7, 18). For serotype 4b strains, the capacity to form biofilms was reduced when the nutrient level in a medium decreased, while serotype 1/2a strains were not similarly affected (11).It has been suggested that the formation of a biofilm is a stress response by bacterial cells (15, 16). Biofilm research under laboratory conditions may not reflect biofilm formation in the environment. To investigate the behavior of L. monocytogenes in biofilms, a simulated food-processing (SFP) system including several stresses was designed (30). The SFP system was used to study 1/2a and 4b strains in mixed-culture biofilms (31). Bacterial cells from a 1/2a cocktail predominated over 4b strains when exposed to the SFP system for 4 weeks, but no competitive inhibition was observed. Environmental factors, including temperature, sugar, salt, pH, and nutrients that are common in foods and food-processing environments, have been demonstrated to have impacts on L. monocytogenes adhesion and biofilm formation (25). The objectives of this study were to investigate and compare biofilm formation between L. monocytogenes serotype 1/2a strains and serotype 4b strains under a variety of environmental conditions, including different temperatures and varying concentrations of salt, sugar, and ethanol, and to examine the synergistic effects of these factors on biofilm formation by both serotypes.     

The characterization of Listeria spp. isolated from food products and the food‐processing environment

Aim: To enhance the information pertaining to the epidemiology of a collection of 378 Listeria spp. isolates obtained from several food‐processing plants in Ireland over a 3‐ year period (2004–2007). Methods and results: The collection was characterized by pulsed‐field gel electrophoresis (PFGE). The most prevalent pulse‐type was PFGE profile I (n = 14·5%) that consisted mainly of environmental Listeria spp. samples. Serotyping of 145 Listeria monocytogenes isolates was performed. The most common serovar was 1/2a and comprised 57·4% (n = 77) of the L. monocytogenes collection. The other serovars were as follows: 4b (14·1%, n = 19), 1/2b (9·7%, n = 13), 4c (4·4%, n = 6) and 1/2c (6·7%, n = 9), respectively. Eleven isolates were identified as non‐Listeria spp., the remaining ten L. monocytogenes isolates were nontypeable. The antimicrobial susceptibility testing revealed the antibiotic that isolates displayed the most resistance to was gentamicin (5%) followed by sulfamethoxazole‐trimethoprim (2%), tetracycline and ciprofloxacin (1·5%). Conclusions: The subtyping has indicated the diversity of the Listeria spp. The presence of serotype 1/2a, 1/2b and 4b in both raw and cooked ready‐to‐eat food products is a public health concern, as these serotypes are frequently associated with foodborne outbreaks and sporadic cases of human listeriosis. In addition, the emergence of antimicrobial‐resistant L. monocytogenes isolates could have serious therapeutic consequences. Significance and Impact of Study: The molecular subtyping and the further characterization of these isolates may be valuable particularly in the context of a suspected common source outbreak in the future.     

A Single-Nucleotide-Polymorphism-Based Multilocus Genotyping Assay for Subtyping Lineage I Isolates of Listeria monocytogenes

Listeria monocytogenes is a facultative intracellular pathogen responsible for food-borne disease with high mortality rates in humans and is the leading microbiological cause of food recalls. Lineage I isolates of L. monocytogenes are a particular public health concern because they are responsible for most sporadic cases of listeriosis and the vast majority of epidemic outbreaks. Rapid, reproducible, and sensitive methods for differentiating pathogens below the species level are required for effective pathogen control programs, and the CDC PulseNet Task Force has called for the development and validation of DNA sequence-based methods for subtyping food-borne pathogens. Therefore, we developed a multilocus genotyping (MLGT) assay for L. monocytogenes lineage I isolates based on nucleotide variation identified by sequencing 23,251 bp of DNA from 22 genes distributed across seven genomic regions in 65 L. monocytogenes isolates. This single-well assay of 60 allele-specific probes captured 100% of the haplotype information contained in approximately 1.5 Mb of comparative DNA sequence and was used to reproducibly type a total of 241 lineage I isolates. The MLGT assay provided high discriminatory power (Simpson's index value, 0.91), uniquely identified isolates from the eight listeriosis outbreaks examined, and differentiated serotypes 1/2b and 4b as well as epidemic clone I (ECI), ECIa, and ECII. In addition, the assay included probes for a previously characterized truncation mutation in inlA, providing for the identification of a specific virulence-attenuated subtype. These results demonstrate that MLGT represents a significant new tool for use in pathogen surveillance, outbreak detection, risk assessment, population analyses, and epidemiological investigations.     

Different assembly of acid and salt tolerance response in two dairy Listeria monocytogenes wild strains

A lack on the association between acid tolerance response (ATR) and osmotolerance response (OTR) among Listeria monocytogenes dairy isolates was found. In order to evaluate how wild L. monocytogenes isolates mount tolerance responses under a sub-lethal pH and a low sodium chloride concentration (pH 5.5 and 3.5 % [w/v] NaCl), a proteomic approach was used. The ATR and OTR of two L. monocytogenes cheese dairy isolates (strain T8, serotype 4b and A9, serotype 1/2b or 3b) were determined. The proteomes of the adapted and non-adapted cultures were evaluated by 2-DE. One strain displayed an ATR, but not an OTR and the other displayed an OTR, but not an ATR. The ATR positive strain showed the over-production of proteins related with protein synthesis, protein folding, attainment of reduction power, ribose production and cell wall. In contrast, in the OTR-positive-strain proteins related with glycolysis, general stress and detoxification were identified.     

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.     

Retail Survey of Brazilian Milk and Minas Frescal Cheese and a Contaminated Dairy Plant To Establish Prevalence, Relatedness, and Sources of Listeria monocytogenes Isolates

A study was designed to recover Listeria monocytogenes from pasteurized milk and Minas frescal cheese (MFC) sampled at retail establishments (REs) and to identify the contamination source(s) of these products in the corresponding dairy processing plant. Fifty milk samples (9 brands) and 55 MFC samples (10 brands) were tested from REs located in Juiz de Fora, Minas Gerais, Brazil. All milk samples and 45 samples from 9 of 10 MFC brands tested negative for L. monocytogenes; however, “brand F” of MFC obtained from REs 119 and 159 tested positive. Thus, the farm/plant that produced brand F MFC was sampled; all samples from the milking parlor tested negative for L. monocytogenes, whereas several sites within the processing plant and the MFC samples tested positive. All 344 isolates recovered from retail MFC, plant F MFC, and plant F environmental samples were serotype 1/2a and displayed the same AscI or ApaI fingerprints. Since these results established that the storage coolers served as the contamination source of the MFC, plant F was closed so that corrective renovations could be made. Following renovation, samples from sites that previously tested positive for the pathogen were collected from the processing environment and from MFC on multiple visits; all tested negative for L. monocytogenes. In addition, on subsequent visits to REs 159 and 119, all MFC samples tested negative for the pathogen. Studies are ongoing to quantify the prevalence, levels, and types of L. monocytogenes in MFC and associated processing plants to lessen the likelihood of listeriosis in Brazil.     

Heavy-Metal and Benzalkonium Chloride Resistance of Listeria monocytogenes Isolates from the Environment of Turkey-Processing Plants

The resistance of Listeria monocytogenes to cadmium and arsenic has been used extensively for strain subtyping. However, limited information is available on the prevalence of such resistance among isolates from the environment of food-processing plants. In addition, it is not known whether the resistance of such isolates to heavy metals may correlate with resistance to quaternary ammonium compounds extensively used as disinfectants in the food-processing industry. In this study, we characterized 192 L. monocytogenes isolates (123 putative strains) from the environment of turkey-processing plants in the United States for resistance to cadmium and arsenic and to the quaternary ammonium disinfectant benzalkonium chloride (BC). Resistance to cadmium was significantly more prevalent among strains of serotypes 1/2a (or 3a) and 1/2b (or 3b) (83% and 74%, respectively) than among strains of the serotype 4b complex (19%). Resistance to BC was encountered among 60% and 51% of the serotype 1/2a (or 3a) and 1/2b (or 3b) strains, respectively, and among 7% of the strains of the serotype 4b complex. All BC-resistant strains were also resistant to cadmium, although the reverse was not always the case. In contrast, no correlation was found between BC resistance and resistance to arsenic, which overall was low (6%). Our findings suggest that the processing environment of turkey-processing plants may constitute a reservoir for L. monocytogenes harboring resistance to cadmium and to BC and raise the possibility of common genetic elements or mechanisms mediating resistance to quaternary ammonium disinfectants and to cadmium in L. monocytogenes.