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.     

Genetic Markers Unique to Listeria monocytogenes Serotype 4b Differentiate Epidemic Clone II (Hot Dog Outbreak Strains) from Other Lineages

A small number of closely related strains of Listeria monocytogenes serotype 4b, designated epidemic clone I (ECI), have been implicated in numerous outbreaks of food-borne listeriosis described during the past two decades in Europe and North America. In 1998 to 1999, a multistate outbreak traced to contaminated hot dogs involved a different strain type of serotype 4b, with genetic fingerprints rarely encountered before. In spite of the profound economic and public health impact of this outbreak, the implicated bacteria (designated epidemic clone II [ECII]) have remained poorly characterized genetically, and nucleotide sequences specific for these strains have not been reported. Using genome sequence information, PCR, and Southern blots, we identified DNA fragments which appeared to be either absent or markedly divergent in the hot dog outbreak strains but conserved among other serotype 4b strains. PCR with primers derived from these fragments as well as Southern blots with the amplicons as probes readily differentiated ECII from other serotype 4b strains. The serotype 4b-specific region harboring these fragments was adjacent to inlA, which encodes a well-characterized virulence determinant. The findings suggest that ECII strains have undergone divergence in portions of a serotype-specific region that is conserved in other serotype 4b strains. Although the mechanisms that drive this divergence remain to be identified, DNA-based tools from this region can facilitate the detection and further characterization of strains belonging to this lineage.     

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.     

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.     

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. DNA sequences were deposited in the GenBank database under accession numbers DQ 812146 to DQ 812517, DQ 843664 to DQ 844598, and AY 512391 to AY 512502.     

Conservation of Genomic Localization and Sequence Content of Sau3AI-Like Restriction-Modification Gene Cassettes among Listeria monocytogenes Epidemic Clone I and Selected Strains of Serotype 1/2a

Listeria monocytogenes is a food-borne pathogen with a clonal population structure and apparently limited gene flow between strains of different lineages. Strains of epidemic clone I (ECI) have been responsible for numerous outbreaks and invariably have DNA that is resistant to digestion by Sau3AI, suggesting methylation of cytosine at GATC sites. A putative restriction-modification (RM) gene cassette has been identified in the genome of the ECI strain F2365 and all other tested ECI strains but is absent from other strains of the same serotype (4b). Homologous RM cassettes have not been reported among L. monocytogenes isolates of other serotypes. Furthermore, conclusive evidence for the involvement of this RM cassette in the Sau3AI resistance phenotype of ECI strains has been lacking. In this study, we describe a highly conserved RM cassette in certain strains of serotypes 1/2a and 4a that have Sau3AI-resistant DNA. In these strains the RM cassette was in the same genomic location as in the ECI reference strain F2365. The cassette included a gene encoding a putative recombinase, suggesting insertion via site-specific recombination. Deletion of the RM cassette in the ECI strain F2365 and the serotype 1/2a strain A7 rendered the DNA of both strains susceptible to Sau3AI digestion, providing conclusive evidence that the cassette includes a gene required for methylation of cytosine at GATC sites in both strains. The findings suggest that, in addition to its presence in ECI strains, this RM cassette and the accompanying genomic DNA methylation is also encountered among selected strains of other lineages.Listeria monocytogenes is a Gram-positive, facultative intracellular food-borne pathogen capable of causing severe disease (listeriosis) in animals and humans. Listeriosis most often affects pregnant women and their fetuses, neonates, the elderly, and immunocompromised individuals. The disease is predominantly transmitted via the consumption of contaminated foods and has a ca. 20% fatality rate (12, 27). Application of numerous genotyping methods has consistently shown that the organism has a clonal population structure with three major phylogenetic lineages: lineage I consists of strains of serotypes 1/2b, 3b, and 4b, while those of serotypes 1/2a, 1/2c, 3a, and 3c are clustered in lineage II; strains of serotypes 4a and 4c, along with certain serotype 4b strains, constitute lineage III (37, 38).Most epidemics of human listeriosis have involved a small number of closely related strains (epidemic clones), predominantly of serotype 4b (7, 35). The earliest identified clone, epidemic clone I (ECI), has been responsible for several major outbreaks in Europe and North America. In addition, strains of this clonal group are frequently encountered in sporadic illness (10, 28, 29). ECI strains have also been found to comprise a significant portion of the serotype 4b strains from foods and from the environments of food processing plants (10, 11, 40).Genomic DNA of ECI strains has been long known to resist digestion with Sau3AI, suggesting methylation of cytosine at GATC sites (41). Genome sequencing of the ECI strain F2365, implicated in the 1985 California outbreak of listeriosis, revealed a putative restriction-modification (RM) gene cassette with specificity for GATC sites (25). This RM cassette was harbored by all tested serotype 4b strains with Sau3AI-resistant DNA and was absent from those with DNA that could be digested with Sau3AI (40). These findings were in agreement with previous evidence that a fragment of the putative methyltransferase gene was specific to ECI and absent from other strains (14).In spite of extensive documentation for the presence of this putative RM cassette in ECI strains, and its apparent absence among other serotype 4b strains, limited information is available about the possible presence of the cassette among other lineages of L. monocytogenes. Furthermore, conclusive evidence for involvement of the cassette in the resistance of the DNA of ECI strains to Sau3AI digestion has been lacking. In this study, we investigated a panel of food-derived serotype 1/2a strains with Sau3AI-resistant DNA and characterized the genetic content and genomic localization of the RM cassette harbored by these strains. Furthermore, we employed deletion mutagenesis to assess the involvement of the RM cassette in Sau3AI resistance of the DNA of the ECI strain F2365, as well as of a serotype 1/2a strain harboring the cassette.     

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.     

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.     

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.     

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.     

Temperature-Dependent Phage Resistance of Listeria monocytogenes Epidemic Clone II

Listeria monocytogenes epidemic clone II (ECII) has been responsible for two multistate outbreaks in the United States in 1998-1999 and in 2002, in which contaminated ready-to-eat meat products (hot dogs and turkey deli meats, respectively) were implicated. However, ecological adaptations of ECII strains in the food-processing plant environment remain unidentified. In this study, we found that broad-host-range phages, including phages isolated from the processing plant environment, produced plaques on ECII strains grown at 37°C but not when the bacteria were grown at lower temperatures (30°C or below). ECII strains grown at lower temperatures were resistant to phage regardless of the temperature during infection and subsequent incubation. In contrast, the phage susceptibility of all other tested strains of serotype 4b (including epidemic clone I) and of strains of other serotypes and Listeria species was independent of the growth temperature of the bacteria. This temperature-dependent phage susceptibility of ECII bacteria was consistently observed with all surveyed ECII strains from outbreaks or from processing plants, regardless of the presence or absence of cadmium resistance plasmids. Phages adsorbed similarly on ECII bacteria grown at 25°C and at 37°C, suggesting that resistance of ECII strains grown at 25°C was not due to failure of the phage to adsorb. Even though the underlying mechanisms remain to be elucidated, temperature-dependent phage resistance may represent an important ecological adaptation of L. monocytogenes ECII in processed, cold-stored foods and in the processing plant environment, where relatively low temperatures prevail.Listeria monocytogenes is responsible for an estimated 2,500 cases of serious food-borne illness (listeriosis) and 500 deaths annually in the United States. It affects primarily pregnant women, newborns, the elderly, and adults with weakened immune systems. L. monocytogenes is frequently found in the environment and can grow at low temperatures, thus representing a serious hazard for cold-stored, ready-to-eat foods (18, 31).Two multistate outbreaks of listeriosis in the United States, in 1998-1999 and in 2002, respectively, were caused by contaminated ready-to-eat meats (hot dogs and turkey deli meats, respectively) contaminated by serotype 4b strains that represented a novel clonal group, designated epidemic clone II (ECII) (3, 4). ECII strains have distinct genotypes as determined by pulsed-field gel electrophoresis and various other subtyping tools, and harbor unique genetic markers (6, 8, 11, 19, 34). The genome sequencing of one of the isolates (L. monocytogenes H7858) from the 1998-1999 outbreak revealed the presence of a plasmid of ca. 80 kb (pLM80), which harbored genes mediating resistance to the heavy metal cadmium as well as genes conferring resistance to the quaternary ammonium disinfectant benzalkonium chloride (10, 29).Listeria phages (listeriaphage) have long been used for subtyping purposes (33), and extensive research has focused on the genomic characterization (2, 24, 26, 35), transducing potential (14), and biotechnological applications of selected phages (25). In addition, applications of listeriaphage as biocontrol agents in foods and the processing plant environment have been investigated (12, 15, 22). However, limited information exists on phages from processing plant environments and on the impact of environmental conditions on susceptibility of L. monocytogenes strains representing the major epidemic-associated clonal groups to such phages. We have found that strains harboring ECII-specific genetic markers can indeed be recovered from the environment of turkey-processing plants (9). Furthermore, environmental samples from such processing plants yielded phages with broad host range, which were able to infect L. monocytogenes strains of various serotypes, and different Listeria species (20). In this study, we describe the impact of growth temperature on susceptibility of L. monocytogenes ECII strains to phages, including phages isolated from turkey-processing plant environmental samples.     

Genetic markers unique to Listeria monocytogenes serotype 4b differentiate epidemic clone II (hot dog outbreak strains) from other lineages

A small number of closely related strains of Listeria monocytogenes serotype 4b, designated epidemic clone I (ECI), have been implicated in numerous outbreaks of food-borne listeriosis described during the past two decades in Europe and North America. In 1998 to 1999, a multistate outbreak traced to contaminated hot dogs involved a different strain type of serotype 4b, with genetic fingerprints rarely encountered before. In spite of the profound economic and public health impact of this outbreak, the implicated bacteria (designated epidemic clone II [ECII]) have remained poorly characterized genetically, and nucleotide sequences specific for these strains have not been reported. Using genome sequence information, PCR, and Southern blots, we identified DNA fragments which appeared to be either absent or markedly divergent in the hot dog outbreak strains but conserved among other serotype 4b strains. PCR with primers derived from these fragments as well as Southern blots with the amplicons as probes readily differentiated ECII from other serotype 4b strains. The serotype 4b-specific region harboring these fragments was adjacent to inlA, which encodes a well-characterized virulence determinant. The findings suggest that ECII strains have undergone divergence in portions of a serotype-specific region that is conserved in other serotype 4b strains. Although the mechanisms that drive this divergence remain to be identified, DNA-based tools from this region can facilitate the detection and further characterization of strains belonging to this lineage.     

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.     

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.     

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.     

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     

Multilocus Genotyping Assays for Single Nucleotide Polymorphism-Based Subtyping of Listeria monocytogenes Isolates

Listeria monocytogenes is responsible for serious invasive illness associated with consumption of contaminated food and places a significant burden on public health and the agricultural economy. We recently developed a multilocus genotyping (MLGT) assay for high-throughput subtype determination of L. monocytogenes lineage I isolates based on interrogation of single nucleotide polymorphisms (SNPs) via multiplexed primer extension reactions. Here we report the development and validation of two additional MLGT assays that address the need for comprehensive DNA sequence-based subtyping of L. monocytogenes. The first of these novel MLGT assays targeted variation segregating within lineage II, while the second assay combined probes for lineage III strains with probes for strains representing a recently characterized fourth evolutionary lineage (IV) of L. monocytogenes. These assays were based on nucleotide variation identified in >3.8 Mb of comparative DNA sequence and consisted of 115 total probes that differentiated 93% of the 100 haplotypes defined by the multilocus sequence data. MLGT reproducibly typed the 173 isolates used in SNP discovery, and the 10,448 genotypes derived from MLGT analysis of these isolates were consistent with DNA sequence data. Application of the MLGT assays to assess subtype prevalence among isolates from ready-to-eat foods and food-processing facilities indicated a low frequency (6.3%) of epidemic clone subtypes and a substantial population of isolates (>30%) harboring mutations in inlA associated with attenuated virulence in cell culture and animal models. These mutations were restricted to serogroup 1/2 isolates, which may explain the overrepresentation of serotype 4b isolates in human listeriosis cases.     

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.     

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.