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Two recent listeriosis outbreaks have occurred in the United States and Europe due to contaminated individually quick-frozen (IQF) vegetables. While one of the outbreaks was due to frozen vegetables considered ready-to-eat (RTE), the other was linked to frozen corn whose packaging contained cooking instructions and was considered not-ready-to-eat (NRTE). However, consumers may thaw certain frozen vegetables and consume without cooking. Since no data is available on the survivability of L. monocytogenes on IQF vegetables during frozen storage, this study examined the population of six different strains (comprising lineages 1/2a, 1/2b, and 4b) on IQF vegetables during long-term storage. Individual strains were inoculated onto an IQF vegetable mix at 4 log CFU/g and stored at -18 or -10°C for 360 days. Although fluctuations in populations of all strains were observed on the vegetables during storage, no significant differences based on strain, lineages, or temperature were observed. Overall, L. monocytogenes populations were only reduced by up to 0.47 and 0.59 log CFU/g after 360 days at -18 and -10°C, respectively. Results from this study suggest that L. monocytogenes is able to persist on IQF vegetables for extended time periods with no significant reduction in population. Future studies could evaluate the survival and growth of L. monocytogenes on IQF vegetables during thawing and storage., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Megan L. Fay, Bashayer A. Khouja reports financial support was provided by Oak Ridge Institute for Science and Education., (Published by Elsevier Inc.)

Raw (unpasteurized) milk is available for sale and direct human consumption within some states in the United States (US); it cannot be sold or distributed in interstate commerce. Raw milk may contain pathogenic microorganisms that, when consumed, may cause illness and sometimes may result in death. No comprehensive review for prevalence and levels of the major bacterial pathogens in raw milk in the US exists. The objective of the present research was to systematically review the scientific literature published from 2000 to 2019 to estimate the prevalence and levels of Listeria monocytogenes, Salmonella spp., Shiga toxin-producing Escherichia coli (STEC), and Campylobacter spp. in raw milk in the US. Peer-reviewed studies were retrieved systematically from PubMed®, Embase®, and Web of Science TM . The unique complete nonduplicate references were uploaded into the Health Assessment Work Collaborative (HAWC). Based on the selection criteria, twenty studies were included in the systematic review and meta-analysis. Comprehensive Meta-Analysis (CMA) was used for statistical analyses, specifically, random effects meta-analyses were used to synthesize raw bulk tank milk (BTM) and milk filters (MF) data. Data from studies using culture and non-culture-based detection methods were included. Forest plots generated in CMA (Biostat, Englewood, NJ) were used to visualize the results. The average prevalence (event rate) of L. monocytogenes, Salmonella spp., STEC, and Campylobacter spp. in raw BTM in the US was estimated at 4.3% (95% confidence intervals [CIs], 2.8-6.5%), 3.6% (95% CIs, 2.0-6.2%), 4.3% (95% CIs, 2.4-7.4%), and 6.0% (95% CIs, 3.2-10.9%), respectively. Estimated prevalence was generally larger in MF than in BTM. There was not enough data to perform a meta-analysis for the prevalence or levels of pathogens in raw milk from retail establishments or other milk categories., (Published by Elsevier Inc.)

Listeria monocytogenes is a foodborne pathogen and the causative agent of listeriosis, a disease associated with high fatality (20-30%) and hospitalization rates (>95%). ATP-Binding Cassette (ABC) transporters have been demonstrated to be involved in the general stress response. In previous studies, in-frame deletion mutants of the ABC transporter genes, LMOf2365_1875 and LMOf2365_1877, were constructed and analyzed; however, additional work is needed to investigate the virulence potential of these deletion mutants. In this study, two in vitro methods and one in vivo model were used to investigate the virulence potential of in-frame deletion mutants of ABC transporter genes. First, the invasion efficiency in host cells was measured using the HT-29 human cell line. Second, cell-to-cell spread activity was measured using a plaque forming assay. Lastly, virulence potential of the mutants was tested in the Galleria mellonella wax moth model. Our results demonstrated that the deletion mutant, ⊿LMOf2365_1875, displayed decreased invasion and cell-to-cell spread efficiency in comparison to the wild-type, LMOf2365, indicating that LMOf2365_1875 may be required for virulence. Furthermore, the reduced virulence of these mutants was confirmed using the Galleria mellonella wax moth model. In addition, the expression levels of 15 virulence and stress-related genes were analyzed by RT-PCR assays using stationary phase cells. Our results showed that virulence-related gene expression levels from the deletion mutants were elevated (15/15 genes from ⊿LMOf2365_1877 and 7/15 genes from ⊿LMOf2365_1875) compared to the wild type LMOf2365, suggesting that ABC transporters may negatively regulate virulence gene expression under specific conditions. The expression level of the stress-related gene, clpE, also was increased in both deletion mutants, indicating the involvement of ABC transporters in the stress response. Taken together, our findings suggest that ABC transporters may be used as potential targets to develop new therapeutic strategies to control L. monocytogenes., Competing Interests: The authors have declared that no competing interests exist.

Abstract: Listeria monocytogenes (Lm) is one of the leading causes of death because of foodborne illness, affecting the elderly, pregnant women, neonates, and people who are immunocompromised. Serologically, Lm can be classified into 13 serotypes, although only 4 are typically linked with food contamination and illness. Since 2000, a shift in serotypes involved in listeriosis outbreaks has been observed, suggesting that tracking of serotypes could help identify emerging trends. A PCR method developed in 2004 allowed detection of the four major serotypes as molecular serogroups, corresponding to broad phylogenetic groups. In this study, a novel quantitative PCR (qPCR) method was developed that uses two multiplex qPCRs, one to confirm the Listeria genus and Lm species and the second for Lm molecular serogrouping. This method was compared with the U.S. Food and Drug Administration Bacteriological Analytical Manual (BAM) method for Lm and the seroagglutination method, using a 208-strain panel. Comparison of the genus and species qPCR assay with the BAM methods found an equal or slightly higher accuracy for the qPCR method (>98%), compared with the BAM protocol (>96%), when evaluated against independent characterization data. Molecular serogrouping using the qPCR method (96.6%) was more accurate than the seroagglutination assay (75.6%). The qPCR method identified Lm 4bV strains, which could not be resolved using seroagglutination. The qPCR could not identify lineage III and IV serotype 4b strains but did correctly identify 16 of 18 lineage III and IV strains. The qPCR method performed genus identification for the Listeria species Lm, L. innocua, L. welshimeri, L. ivanovii, and L. seeligeri. In addition, the method performed species identification for Lm and classified Lm into six molecular serogroups: 2A, 2B, 2C, 4B, NT, and 4bV. This method provided a rapid and accurate confirmation of Lm and serogroup determinations; furthermore, it could help identify otherwise unlinked strains by enabling whole genome sequencing analysis based on broad phylogeny, independent of other information., (Published 2021 by the International Association for Food Protection. Not subject to U.S. Copyright.)

Various produce including cantaloupe, caramel-coated apples, and packaged salads, have been recognized in recent years as vehicles for listeriosis, a human foodborne disease caused by intracellular pathogen Listeria monocytogenes. Our knowledge regarding the role of these foods in L. monocytogenes virulence, however, is limited. Understanding their role in modulating L. monocytogenes virulence can be useful in risk assessments and for developing control measures. In this study, we employed the Galleria mellonella larvae model to evaluate virulence potential of fifteen clinical, environmental and food isolates of L. monocytogenes, related to three major outbreaks, after growth on different foods. The non-human pathogen Listeria innocua was also included in the panel. Strains were inoculated in parallel in 5ml of brain heart infusion (BHI) broth, and on the surfaces of cantaloupe and apple fragments (5g each) at about 105 colony forming units (CFU)/ml/fragment. One set of inoculated broth and food fragments was incubated at 10°C for 5 days while the second set was kept at 25°C for 3 days. L. monocytogenes cells were recovered from the fruits and BHI, washed twice, re-suspended in saline, and used to inoculate G. mellonella larvae at final concentrations of 106 and 105 CFU/larva. The larvae were incubated at 37°C and monitored for mortality (LT50-time taken to kill 50% of the larvae) and phenotypic changes over seven days. L. monocytogenes grown on cantaloupe and apple flesh surfaces resulted in higher virulence than when grown in BHI. L. monocytogenes infection at 106 CFU/larvae resulted in an average LT50 of ≤ 30, 36 and 47 hours on cantaloupe, apples and BHI, respectively. These results represent a 2.5-4-fold increased mortality compared with an LT50 ≥120 hours in larvae infected with the same doses of L. innocua grown in corresponding matrices. Similar trends were also recorded with doses of about 105 CFU /larvae., Competing Interests: The authors have declared that no competing interests exist.

Understanding a pathogen's response to food environments is imperative to develop effective control strategies as well as to elucidate the impact of foods on virulence potential. The purpose of this study was to assess transcriptional response of Listeria monocytogenes after growth in cantaloupe, as well as its impact on survival in synthetic gastric fluid (SGF). The transcriptional profiles of L. monocytogenes grown in cantaloupe or Brain Heart Infusion (BHI) under refrigeration were compared by a custom-designed microarray. A total of 286 and 175 genes were significantly up- and down-regulated, respectively, in L. monocytogenes grown in cantaloupe as compared to BHI (fold change ≥ 2.5 and adj. P < 0.05). The majority of upregulated genes belonged to functions related to amino acid and nucleotide metabolism, flagellar biosynthesis, and iron acquisition, while most downregulated genes belonged to carbohydrate metabolism. Notably, the branched chain amino acid (BCAA: leucine, isoleucine, valine) biosynthesis operon was shown to be highly upregulated as well as the purine and pyrimidine biosynthesis pathways. Transcript levels of several stress- and virulence-related genes were significantly altered, implying an impact of growth in cantaloupe on the virulence potential of L. monocytogenes. Enhanced survival of L. monocytogenes in SGF following growth in cantaloupe further demonstrated the impact of cantaloupe-associated growth on the pathogen's subsequent response to a host relevant stress., (Copyright © 2018. Published by Elsevier Ltd.)

The classical definition of a disease outbreak is the occurrence of cases of disease in excess of what would normally be expected in a community, geographical area or time period. The establishment of an outbreak then starts with the identification of an incidence of cases above the normally expected threshold during a given time period. Subsequently, the cases are examined using a variety of subtyping methods to identify potential linkages. As listeriosis disease has a long incubation period, relating a single source or multiple sources of contaminated food to clinical disease is challenging and time consuming. The vast majority of human listeriosis cases are caused by three serotypes, 1/2a, 1/2b, and 4b. Thus serotyping of isolates from suspected foods and clinical samples, although useful for eliminating some food sources, has a very limited discriminatory power. The advent of faster and more affordable sequencing technology, coupled with increased computational power, has permitted comparisons of whole Listeria genome sequences from isolates recovered from clinical, food, and environmental sources. These analyses made it possible to identify outbreaks and the source much more accurately and faster, thus leading to a reduction in number of illnesses as well as a reduction in economic losses. Initial DNA sequence information also facilitated the development of a simple molecular serotype protocol which allowed for the identification of major disease causing serotypes of L. monocytogenes, including a clade of 4b variant (4bV) strains of L. monocytogenes involved in at least 3 more recent listeriosis outbreaks in the US. Furthermore, data generated using whole genome sequence (WGS) analyses was successfully utilized to develop a pan-genomic DNA microarray as well as a single nucleotide polymorphism (SNP) based analysis. Herein, we present and compare, the two recently developed sub-typing technologies and discuss how these methods are not only important in outbreak investigations, but could also shed light on possible adaptations to different foods and environments., (Published by Elsevier Ltd.)

Listeria monocytogenes is a pathogen of significant concern in many ready to eat foods due to its ability to survive and multiply even under significant environmental stresses. Listeriosis in humans is a concern, especially to high-risk populations such as those who are immunocompromised or pregnant, due to the high rates of morbidity and mortality. Whole genome sequencing has become a routine part of assessing L. monocytogenes isolated from patients, and the frequency of different genetic subtypes associated with listeriosis is now being reported. The recent abundance of genome sequences for L. monocytogenes has provided a wealth of information regarding the variation in core and accessory genomic elements. Newly described accessory genomic regions have been linked to greater virulence capabilities as well as greater resistance to environmental stressors such as sanitizers commonly used in food processing facilities. This review will provide a summary of our current understanding of stress response and virulence phenotypes of L. monocytogenes, within the context of the genetic diversity of the pathogen.

Several animal models have been used to understand the molecular basis of the pathogenicity, infectious dose and strain to strain variation of Listeria monocytogenes. The greater wax worm Galleria mellonella, as an alternative model, provides some useful advantages not available with other models and has already been described as suitable for the virulence assessment of various pathogens including L. monocytogenes. The objectives of this study are: 1) confirming the usefulness of this model with a wide panel of Listeria spp. including non-pathogenic L. innocua, L. seeligeri, L. welshimeri and animal pathogen L. ivanovii; 2) assessment of virulence of several isogenic in-frame deletion mutants in virulence and stress related genes of L. monocytogenes and 3) virulence assessment of paired food and clinical isolates of L. monocytogenes from 14 major listeriosis outbreaks occurred worldwide between 1980 and 2015. Larvae injected with different concentrations of Listeria were incubated at 37°C and monitored over seven days for time needed to kill 50% of larvae (LT50) and to determine change of bacterial population in G. mellonella, 2 and 24 hours post-inoculation. Non-pathogenic members of Listeria and L. ivanovii showed significantly (P < 0.05) higher LT50 (lower virulence) than the wild type L. monocytogenes strains. Isogenic mutants of L. monocytogenes with the deletions in prfA, plcA, hly, actA and virR genes, also showed significantly (P < 0.05) higher LT50 than the wild type strain at the inoculum of 106CFU/larva. Food isolates had significantly (P < 0.05) lower virulence than the paired clinical isolates, at all three inoculum concentrations. L. monocytogenes strains related to non-invasive (gastroenteritis) outbreaks of listeriosis showed significantly (P < 0.05) lower virulence than isolates of the same serotype obtained from outbreaks with invasive symptoms. The difference, however, was dose and strain- dependent. No significant differences in virulence were observed among the serotype tested in this study.

Cut produce continues to constitute a significant portion of the fresh fruit and vegetables sold directly to consumers. As such, the safety of these items during storage, handling, and display remains a concern. Cut tomatoes, cut leafy greens, and cut melons, which have been studied in relation to their ability to support pathogen growth, have been specifically identified as needing temperature control for safety. Data are needed on the growth behavior of foodborne pathogens in other types of cut produce items that are commonly offered for retail purchase and are potentially held without temperature control. This study assessed the survival and growth of Listeria monocytogenes in cut produce items that are commonly offered for retail purchase, specifically broccoli, green and red bell peppers, yellow onions, canned green and black olives, fresh green olives, cantaloupe flesh and rind, avocado pulp, cucumbers, and button mushrooms. The survival of L. monocytogenes strains representing serotypes 1/2a, 1/2b, and 4b was determined on the cut produce items for each strain individually at 5, 10, and 25°C for up to 720 h. The modified Baranyi model was used to determine the growth kinetics (the maximum growth rates and maximum population increases) in the L. monocytogenes populations. The products that supported the most rapid growth of L. monocytogenes, considering the fastest growth and resulting population levels, were cantaloupe flesh and avocado pulp. When stored at 25°C, the maximum growth rates for these products were 0.093 to 0.138 log CFU/g/h and 0.130 to 0.193 log CFU/g/h, respectively, depending on the strain. Green olives and broccoli did not support growth at any temperature. These results can be used to inform discussions surrounding whether specific time and temperature storage conditions should be recommended for additional cut produce items.

Four listeriosis incidences/outbreaks, spanning 19 months, have been linked to Listeria monocytogenes serotype 4b variant (4bV) strains. Three of these incidents can be linked to a defined geographical region, while the fourth is likely to be linked. In this study, whole genome sequencing (WGS) of strains from these incidents was used for genomic comparisons using two approached. The first was JSpecies tetramer, which analyzed tetranucleotide frequency to assess relatedness. The second, the CFSAN SNP Pipeline, was used to perform WGS SNP analyses against three different reference genomes to evaluate relatedness by SNP distances. In each case, unrelated strains were included as controls. The analyses showed that strains from these incidents form a highly related clade with SNP differences of ≤101 within the clade and >9000 against other strains. Multi-Virulence-Locus Sequence Typing, a third standardized approach for evaluation relatedness, was used to assess the genetic drift in six conserved, known virulence loci and showed a different clustering pattern indicating possible differences in selection pressure experienced by these genes. These data suggest a high degree of relatedness among these 4bV strains linked to a defined geographic region and also highlight the possibility of alterations related to adaptation and virulence.

A precise and accurate method for enumeration of low level of Listeria monocytogenes in foods is critical to a variety of studies. In this study, paired comparison of most probable number (MPN) and direct plating enumeration of L. monocytogenes was conducted on a total of 1730 outbreak-associated ice cream samples that were naturally contaminated with low level of L. monocytogenes. MPN was performed on all 1730 samples. Direct plating was performed on all samples using the RAPID'L.mono (RLM) agar (1600 samples) and agar Listeria Ottaviani and Agosti (ALOA; 130 samples). Probabilistic analysis with Bayesian inference model was used to compare paired direct plating and MPN estimates of L. monocytogenes in ice cream samples because assumptions implicit in ordinary least squares (OLS) linear regression analyses were not met for such a comparison. The probabilistic analysis revealed good agreement between the MPN and direct plating estimates, and this agreement showed that the MPN schemes and direct plating schemes using ALOA or RLM evaluated in the present study were suitable for enumerating low levels of L. monocytogenes in these ice cream samples. The statistical analysis further revealed that OLS linear regression analyses of direct plating and MPN data did introduce bias that incorrectly characterized systematic differences between estimates from the two methods., (Published by Elsevier B.V.)

A most-probable-number (MPN) method was used to enumerate Listeria monocytogenes in 2,320 commercial ice cream scoops manufactured on a production line that was implicated in a 2015 listeriosis outbreak in the United States. The analyzed samples were collected from seven lots produced in November 2014, December 2014, January 2015, and March 2015. L. monocytogenes was detected in 99% (2,307 of 2,320) of the tested samples (lower limit of detection, 0.03 MPN/g), 92% of which were contaminated at <20 MPN/g. The levels of L. monocytogenes in these samples had a geometric mean per lot of 0.15 to 7.1 MPN/g. The prevalence and enumeration data from an unprecedented large number of naturally contaminated ice cream products linked to a listeriosis outbreak provided a unique data set for further understanding the risk associated with L. monocytogenes contamination for highly susceptible populations.

In an in-vitro assay, rabbit serum, but not human serum, killed Listeria monocytogenes, a foodborne pathogen. The aim of our study was to purify and partially characterize this killing factor. Listericidin was purified from rabbit serum by a single-step ion-exchange chromatography with DEAE-Sephadex A-50 and its antimicrobial activity was assessed by a microdilution method. Listericidin is a protein with a molecular weight of 9 kDa and an isoelectric point of 8.1. It kills L. monocytogenes at 4°C, 25°C, and 37°C, and its activity is resistant to heat (boiling) and acidic conditions (pH <2). Listericidin's activity is inhibited by sodium chloride and various growth media, is sensitive to proteolytic enzymes and is enhanced by calcium chloride, and is neutralized by monoclonal antibodies to human complement C3a. However, the listericidin reacts weakly with these antibodies in an ELISA. The first 33 N-terminal residues of listericidin (SVQLTEKRMDKVGQYTNKELRKXXEDGMRDNPM) have homology to various complement C3a components. Listericidin also kills other Listeria spp., Vibrio spp., Salmonella spp., Escherichia spp., Cronobacter spp., and Bacillus spp. The listericidin peptide purified in a single-step chromatography is pH and heat stable, and has a broad antimicrobial spectrum against major foodborne pathogens in addition to L. monocytogenes.

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.

Listeria monocytogenes, causative agent of human listeriosis, has been isolated from a wide variety of foods including deli meats, soft cheeses, cantaloupes, sprouts and canned mushrooms. Standard control measures for restricting microbial growth such as refrigeration and high salt are often inadequate as L. monocytogenes grows quite well in these environments. In an effort to better understand the genetic and physiological basis by which L. monocytogenes circumvents these controls, a transposon library of L. monocytogenes was screened for changes in their ability to grow in 7% NaCl and/ or at 5 °C. This work identified a transposon insertion upstream of an operon, here named lstABC, that led to a reduction in growth in 7% NaCl. In-frame deletion studies identified lstC which codes for a GNAT-acetyltransferase being responsible for the phenotype. Transcriptomic and RT-PCR analyses identified nine genes that were upregulated in the presence of high salt in the ΔlstC mutant. Further analysis of lstC and the genes affected by ΔlstC is needed to understand LstC's role in salt tolerance., (Published by Elsevier Ltd.)

Over 90% of the human listeriosis cases are caused by Listeria monocytogenes serotypes 1/2a, 1/2b and 4b strains. As an alternative to antigen-antibody based serotyping, a PCR-based method for serogrouping has been developed and validated. In this communication, we report an in-depth analysis of five 4b variant strains, four clinical isolates from Australia and one environmental isolate from USA. Although these five strains were serotype 4b by classical serotyping method, the serogrouping PCR profiles of these strains show the presence of a 1/2a-3a specific amplicon in addition to the standard 4b-4d-4e specific amplicons. These strains were further analyzed by pulsed field gel electrophoresis, binary gene typing, multi-locus variable-number-tandem-repeat analysis and a high density pan-genomic Listeria microarray. Using these sub-typing results, the clinical isolates were grouped into two distinct genomic groups- one of which could be part of an unidentified outbreak. The microarray results when compared with our database of other 4b outbreak isolates indicated that the serotype 4b variant strains represent very different genotypic profiles than the known reported 4b outbreak strains representing major epidemic clones. The acquisition of serotype 1/2a gene clusters by the 4b variant strains appears to be independent in origin, spanning large areas of geographical and temporal space and may indicate predisposition of some 4b strains towards accepting DNA from related organisms.

Listeria monocytogenes is a foodborne pathogen that is difficult to eliminate due to its ability to survive under different stress conditions such as low pH and high salt. To better control this pathogen in food, it is important to understand its survival mechanisms under these stress conditions. LMOf2365&#95;0442, 0443, and 0444 encode for phosphotransferase transport system (PTS) permease (fructose-specific IIABC components) that is responsible for sugar transport. LMOf2365&#95;0445 encodes for glycosyl hydrolase. These genes were induced by high pressure and inhibited under salt treatments; therefore, we hypothesized that genes encoding these PTS proteins may be involved in general stress responses. To study the function of these genes, deletion mutants of the PTS genes (LMOf2365&#95;0442, LMOf2365&#95;0443, and LMOf2365&#95;0444) and the downstream gene LMOf2365&#95;0445 were created in L. monocytogenes strain F2365. These deletion mutants were tested under different stress conditions. The growth of ∆LMOf2365&#95;0445 was increased under nisin (125 μg/mL) treatments compared to the wild-type (P < 0.01). The growth of ∆LMOf2365&#95;0442 in salt (brain-heart infusion medium with 5% NaCl) was significantly increased (P < 0.01), and ∆LMOf2365&#95;0442 showed increased growth under acidic conditions (pH 5.0) compared to the wild-type (P < 0.01). The results from phenotypic arrays demonstrated that some of these mutants showed slightly slower growth under different carbon sources and basic conditions. The results indicate that deletion mutants ∆LMOf2365&#95;0442 and ∆LMOf2365&#95;0445 were more resistant to multiple stress conditions compared to the wild-type, suggesting that they may contribute to the general stress response in L. monocytogenes. An understanding of the growth of these mutants under multiple stress conditions may assist in the development of intervention strategies to control L. monocytogenes in food., (© 2013 Institute of Food Technologists®)

As a vast majority of the human listeriosis cases are caused by serotypes 1/2a, 1/2b and 4b strains, it is imperative that strains from clinical as well as from food and environment are further characterised so that accurate and timely epidemiological determination of sources of the contamination can be established to minimise the disease burden. Recent developments in the field of genomics provide a great opportunity to use these tools towards the development of molecular sub-typing techniques with a greater degree of discrimination spanning the entire length of the genome. This brief review summarises a few of these DNA-based techniques with an emphasis on DNA microarray and other whole genome sequencing-based approaches and their usefulness in Listeria monocytogenes sub-typing and outbreak investigations.

Listeria monocytogenes can survive and grow in refrigerated temperatures and high-salt environments. In an effort to better understand the associated mechanisms, a library of ∼ 5,200 transposon mutants of LS411, a food isolate from the Jalisco cheese outbreak, were screened for their ability to grow in brain heart infusion (BHI) broth at 5°C or in the presence of 7% NaCl and two mutants with altered growth profiles were identified. The LS522 mutant has a transposon insertion between secA2 and iap and showed a significant reduction in growth in BHI broth at 5°C and in the presence of 7% NaCl. Reverse transcriptase quantitative PCR (RT-qPCR) revealed a substantial reduction in the expression of iap. Additionally, a hypothetical gene (met), containing a putative S-adenosylmethionine-dependent methyltransferase domain, downstream of iap had downregulated expression. In-frame deletion mutants of iap and met were created in LS411. The LS560 (LS411 Δiap) mutant showed reduced growth at 5°C and in the presence of 7% salt, confirming its role in cold and salt growth attenuation. Surprisingly, the LS655 (LS411 Δmet) mutant showed slightly increased growth during refrigeration, though no alteration was seen in salt growth relative to the wild-type strain. The LS527 mutant, containing an insertion 36 bp upstream of the gbu operon, showed reduced expression of the gbu transcript by RT-qPCR and also showed growth reduction at 5°C and in the presence of 7% salt. This attenuation was severely exacerbated when the mutant was grown under the combined stresses. Analysis of the gbu operon deletion mutant showed decreased growth in 7% salt and refrigeration, supporting the previously characterized role for this gene in cold and salt adaptation. These studies indicate the potential for an intricate relationship between environmental stress regulation and virulence in L. monocytogenes.

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.

Listeria monocytogenes, a foodborne bacterial pathogen, causes invasive and febrile gastroenteritis forms of listeriosis in humans. Both invasive and febrile gastroenteritis listeriosis is caused mostly by serotypes 1/2a, 1/2b and 4b strains. The outbreak strains of serotype 1/2a and 4b could be further classified into several epidemic clones but the genetic bases for the diverse pathophysiology have been unsuccessful. DNA microarray provides an important tool to scan the entire genome for genetic signatures that may distinguish the L. monocytogenes strains belonging to different outbreaks. We have designed a pan-genomic microarray chip (Listeria GeneChip) containing sequences from 24 L. monocytogenes strains. The chip was designed to identify the presence/absence of genomic sequences, analyze transcription profiles and identify SNPs. Analysis of the genomic profiles of 38 outbreak strains representing 1/2a, 1/2b and 4b serotypes, revealed that the strains formed distinct genetic clusters adhering to their serotypes and epidemic clone types. Although serologically 1/2a and 1/b strains share common antigenic markers microarray analysis revealed that 1/2a strains are further apart from the closely related 1/2b and 4b strains. Within any given serotype and epidemic clone type the febrile gastroenteritis and invasive strains can be further distinguished based on several genetic markers including large numbers of phage genome, and intergenic sequences. Our results showed that the microarray-based data can be an important tool in characterization of L. monocytogenes strains involved in both invasive and gastroenteritis outbreaks. The results for the first time showed that the serotypes and epidemic clones are based on extensive pan-genomic variability and the 1/2b and 4bstrains are more closely related to each other than the 1/2a strains. The data also supported the hypothesis that the strains causing these two diverse outbreaks are genotypically different and this finding might be important in understanding the pathophysiology of this organism.

To evaluate a simplified serotyping scheme, we used a combination of an antibody-based serogrouping assay that identified only type 1 and type 4 strains and a multiplex PCR-based serogrouping assay to analyze 362 L. monocytogenes isolates collected over more than 20 years. The multiplex PCR assay also incorporated a set of primers specific for L. monocytogenes hlyA gene to verify the species identification of these isolates. A subset (n = 120) of these isolates were also serotyped with the Denka Seiken serotyping scheme, which is often considered the "gold standard" for serotyping of L. monocytogenes. The results indicate that the multiplex PCR-based assay, in combination with an antibody-based serogrouping assay, correctly identified serotypes of 96% of the previously serotyped isolates. Compared with the Denka Seiken method, the combination method also performed better in identifying serotypes of 120 previously unserotyped L. monocytogenes isolates. Thus, the combination scheme appears to be a simple and rapid way to identify serotypes 1/2a, 1/2b, 1/2c, 3a, 3b, 3c, and 4b isolates, which are the predominant L. monocytogenes serotypes found in food, environmental, and clinical samples.

From 1986 to 2006, the incidence of listeriosis in the United States dropped from approximately seven to three cases per million population, a reduction that most likely reflects the joint efforts of industry, government, consumers, and academia. Herein, we describe the U.S. Food and Drug Administration (FDA) strategy over the past three decades to combat listeriosis. Specifically, we discuss early actions taken to address outbreaks during the 1980s, policy decisions regarding the presence of Listeria monocytogenes in FDA-regulated foods, FDA compliance programs with L. monocytogenes components, enforcement actions to remove L. monocytogenes-contaminated products from the market (i.e., recalls) or to prevent entry of such products into the market (i.e., import detentions and refusals), research milestones, outreach and education efforts, and selected special projects. Evolving demographic trends in the United States may pose a challenge to further reduction of the incidence of listeriosis.

The acid tolerance of a Listeria monocytogenes serotype 4b strain was studied by measuring its ability to survive at an acidic pH at 37 degrees C. The acid tolerance of L. monocytogenes was much lower than those of Escherichia coli O157:H7 and Shigella flexneri strains. This observation suggested a higher infective dose for L. monocytogenes than E. coli O157:H7 and Shigella. The susceptibility of L. monocytogenes to acidic pH was dependent upon growth medium pH and growth phase of the culture. Nisin and some other ionophores reduced the acid tolerance of both stationary-phase and log-phase cultures of L. monocytogenes. These studies indicated that nisin might be a useful candidate for controlling acid tolerance of L. monocytogenes.

Expression of listeriolysin O of Listeria monocytogenes as a function of different growth conditions was studied by performing a direct hemolysin assay, immunoblotting experiments, and an enzyme-linked immunosorbent assay. Expression of listeriolysin O was reduced at a lower growth temperatures (26 degrees C) and at higher glucose concentrations (> or = 0.3%) in the growth media. The effect of glucose appeared to be due to a change in the pH of the growth media.

A plasmid containing the cloned listeriolysin gene of Listeria monocytogenes was used as a probe to identify Listeria strains by DNA colony hybridization. The probe DNA was labeled with horseradish peroxidase in the presence of glutaraldehyde. After the hybridization and wash procedures, the hybrid molecules were detected by luminescence, which resulted from the oxidation of luminol by a horseradish peroxidase-hydrogen peroxide-coupled reaction. Of the 150 Listeria strains and 16 non-Listeria strains examined, the probe hybridized only with L. monocytogenes. The technique was also used to enumerate L. monocytogenes in artificially contaminated foods.

A clone containing 3.1 kb of Listeria DNA was selected from a gene library of Listeria monocytogenes Scott A strain. The Escherichia coli clone produced hemolysin on sheep blood agar and in sonicated extracts but very little in the culture supernatant. This 3.1-kb DNA fragment and a 650-bp HindIII fragment located within the listeriolysin gene were used as probes in a colony hybridization assay. Both probes were specific for L. monocytogenes and did not hybridize with any other Listeria strains at high stringency. Two synthetic probes, one from the 650-bp HindIII fragment and one from the carboxy-terminal region of the protein, were also specific for L. monocytogenes.

A 500-base-pair DNA fragment of a presumptive beta-hemolysin gene of Listeria monocytogenes has been used to identify this organism by a modified colony hybridization technique. We have cloned this DNA fragment into M13 bacteriophage vectors and sequenced it by a dideoxynucleotide sequencing technique. From this sequencing information, several oligodeoxyribonucleotides were synthesized and used as synthetic probes to identify L. monocytogenes. The probes were specific for L. monocytogenes and did not react with any other Listeria strains in a colony hybridization assay. In particular, one of these probes (AD07) was used to detect L. monocytogenes in artificially contaminated raw-milk and soft-cheese samples.

A fragment of about 500 base pairs of the beta-hemolysin gene from Listeria monocytogenes was used to screen different bacterial strains by DNA colony hybridization. The cells in the colonies were lysed by microwaves in the presence of sodium hydroxide. Of 52 different strains of Listeria species screened, only the DNA from beta-hemolytic (CAMP-positive) strains of L. monocytogenes hybridized with this probe.

A DNA probe was used to identify hemolytic Listeria monocytogenes in naturally contaminated dairy products: unpasteurized milk, ricotta cheese, and imported semisoft cheeses. Of 34 milk samples, 12 were suspected to contain hemolytic L. monocytogenes; 1 contained greater than 6000 viable organisms/g. The ricotta cheese, although temperature-abused, had a titer of 3.6 x 10(6) beta-hemolytic L. monocytogenes cells/g, whereas the semisoft cheeses reached a maximum of 5.6 x 10(6) cells/g. Pure cultures of L. monocytogenes isolated from both types of cheese were found positive by the CAMP test and the DNA probe.