Over the last 10 to 15 years, increasing evidence suggests that persistence of Listeria monocytogenes in food processing plants for years or even decades is an important factor in the transmission of this foodborne pathogen and the root cause of a number of human listeriosis outbreaks. L. monocytogenes persistence in other food-associated environments (e.g., farms and retail establishments) may also contribute to food contamination and transmission of the pathogen to humans. Although L. monocytogenes persistence is typically identified through isolation of a specific molecular subtype from samples collected in a given environment over time, formal (statistical) criteria for identification of persistence are undefined. Environmental factors (e.g., facilities and equipment that are difficult to clean) have been identified as key contributors to persistence; however, the mechanisms are less well understood. Although some researchers have reported that persistent strains possess specific characteristics that may facilitate persistence (e.g., biofilm formation and better adaptation to stress conditions), other researchers have not found significant differences between persistent and nonpersistent strains in the phenotypic characteristics that might facilitate persistence. This review includes a discussion of our current knowledge concerning some key issues associated with the persistence of L. monocytogenes, with special focus on (i) persistence in food processing plants and other food-associated environments, (ii) persistence in the general environment, (iii) phenotypic and genetic characteristics of persistent strains, (iv) niches, and (v) public health and economic implications of persistence. Although the available data clearly indicate that L. monocytogenes persistence at various stages of the food chain contributes to contamination of finished products, continued efforts to quantitatively integrate data on L. monocytogenes persistence (e.g., meta-analysis or quantitative microbial risk assessment) will be needed to advance our understanding of persistence of this pathogen and its economic and public health impacts.

While the food-borne pathogen Listeria monocytogenes can persist in food associated environments, there are no whole-genome sequence (WGS) based methods to differentiate persistent from sporadic strains. Whole-genome sequencing of 188 isolates from a longitudinal study of L. monocytogenes in retail delis was used to (i) apply single-nucleotide polymorphism (SNP)-based phylogenetics for subtyping of L. monocytogenes, (ii) use SNP counts to differentiate persistent from repeatedly reintroduced strains, and (iii) identify genetic determinants of L. monocytogenes persistence. WGS analysis revealed three prophage regions that explained differences between three pairs of phylogenetically similar populations with pulsed-field gel electrophoresis types that differed by <3 bands. WGS-SNP-based phylogenetics found that putatively persistent L. monocytogenes represent SNP patterns (i) unique to a single retail deli, supporting persistence within the deli (11 clades), (ii) unique to a single state, supporting clonal spread within a state (7 clades), or (iii) spanning multiple states (5 clades). Isolates that formed one of 11 deli-specific clades differed by a median of 10 SNPs or fewer. Isolates from 12 putative persistence events had significantly fewer SNPs (median, 2 to 22 SNPs) than between isolates of the same subtype from other delis (median up to 77 SNPs), supporting persistence of the strain. In 13 events, nearly indistinguishable isolates (0 to 1 SNP) were found across multiple delis. No individual genes were enriched among persistent isolates compared to sporadic isolates. Our data show that WGS analysis improves food-borne pathogen subtyping and identification of persistent bacterial pathogens in food associated environments. Listeria monocytogenes is an opportunistic food-borne pathogen responsible for approximately 250 deaths per year in the United States (1). The annual costs of listeriosis, including morbidity, mortality, and lost wages, are estimated at $2.8 billion (2). A 2003 risk assessment attributed 90% of listeriosis cases in the United States to consumption of contaminated ready-to-eat (RTE) deli meats (3), and most (Ͼ60%) of U.S. listeriosis cases linked to RTE deli meats were estimated to be due contamination during retail handling and slicing (4, 5). Consequently, the retail deli environment is a focal point for listeriosis reduction efforts.L. monocytogenes can persist in food-associated environments for months to years (6-8). Persistent strains have been linked to outbreaks of food-borne disease. For example, L. monocytogenes that was responsible for an outbreak linked to 29 cases and 4 deaths persisted in the source plant for at least 12 years (9). Therefore, the food processing industry has implemented the seek-anddestroy process to identify and eliminate point sources of persistence through enhanced environmental monitoring, sanitation, and equipment and process design (10).One challenge for persistent pathogen control is to differentiate true persistence from repeated reintroduction of a give...

Sampling of agricultural and natural environments in two US states (Colorado and Florida) yielded 18 Listeria-like isolates that could not be assigned to previously described species using traditional methods. Using whole-genome sequencing and traditional phenotypic methods, we identified five novel species, each with a genome-wide average blast nucleotide identity (ANIb) of less than 85 % to currently described species. Phylogenetic analysis based on 16S rRNA gene sequences and amino acid sequences of 31 conserved loci showed the existence of four well-supported clades within the genus Listeria ; (i) a clade representing Listeria monocytogenes , L. marthii , L. innocua , L. welshimeri , L. seeligeri and L. ivanovii , which we refer to as Listeria sensu stricto, (ii) a clade consisting of Listeria fleischmannii and two newly described species, Listeria aquatica sp. nov. (type strain FSL S10-1188T = DSM 26686T = LMG 28120T = BEI NR-42633T) and Listeria floridensis sp. nov. (type strain FSL S10-1187T = DSM 26687T = LMG 28121T = BEI NR-42632T), (iii) a clade consisting of Listeria rocourtiae , L. weihenstephanensis and three novel species, Listeria cornellensis sp. nov. (type strain TTU A1-0210T = FSL F6-0969T = DSM 26689T = LMG 28123T = BEI NR-42630T), Listeria grandensis sp. nov. (type strain TTU A1-0212T = FSL F6-0971T = DSM 26688T = LMG 28122T = BEI NR-42631T) and Listeria riparia sp. nov. (type strain FSL S10-1204T = DSM 26685T = LMG 28119T = BEI NR- 42634T) and (iv) a clade containing Listeria grayi . Genomic and phenotypic data suggest that the novel species are non-pathogenic.

Maize, a staple food in many African countries including Kenya, is often contaminated by toxic and carcinogenic fungal secondary metabolites such as aflatoxins and fumonisins. This study evaluated the potential use of a low-cost, multi-spectral sorter in identification and removal of aflatoxin-and fumonisin-contaminated single kernels from a bulk of mature maize kernels. The machine was calibrated by building a mathematical model relating reflectance at nine distinct wavelengths (470-1,550 nm) to mycotoxin levels of single kernels collected from small-scale maize traders in open-air markets and from inoculated maize field trials in Eastern Kenya. Due to the expected skewed distribution of mycotoxin contamination, visual assessment of putative risk factors such as discoloration, moldiness, breakage, and fluorescence under ultraviolet light (365 nm), was used to enrich for mycotoxin-positive kernels used for calibration. Discriminant analysis calibration using both infrared and visible spectra achieved 77% sensitivity and 83% specificity to identify kernels with aflatoxin > 10 ng g-1 and fumonisin > 1,000 ng g-1 , respectively (measured by ELISA or UHPLC). In subsequent sorting of 46 market maize samples previously tested for mycotoxins, 0-25% of sample mass was rejected from samples that previously tested toxin-positive and 0-1% was rejected for previously toxin-negative samples. In most cases where mycotoxins were detected in sorted maize streams, accepted maize had lower mycotoxin levels than the rejected maize (21/25 accepted maize streams had lower aflatoxin than rejected streams, 25/27 accepted maize streams had lower fumonisin than rejected streams). Reduction was statistically significant (p<0.001), achieving an 83% mean reduction in each toxin. With further development, this technology could be used to sort maize at local hammer mills to reduce human mycotoxin exposure in Kenya, and elsewhere in the world, while at once reducing food loss, and improving food safety and nutritional status.

Postprocessing contamination in processing plants has historically been a significant source of Listeria monocytogenes in ready-to-eat delicatessen meats, and therefore a major cause of human listeriosis cases and outbreaks. Recent risk assessments suggest that a majority of human listeriosis cases linked to consumption of contaminated deli meats may be due to L. monocytogenes contamination that occurs at the retail level. To better understand the ecology and transmission of Listeria spp. in retail delicatessens, food and nonfood contact surfaces were tested for L. monocytogenes and other Listeria spp. in a longitudinal study conducted in 30 retail delis in three U.S. states. In phase I of the study, seven sponge samples were collected monthly for 3 months in 15 delis (5 delis per state) prior to start of daily operation; in phase II, 28 food contact and nonfood contact sites were sampled in each of 30 delis during daily operation for 6 months. Among the 314 samples collected during phase I, 6.8% were positive for L. monocytogenes. Among 4,503 samples collected during phase II, 9.5% were positive for L. monocytogenes; 9 of 30 delis showed low L. monocytogenes prevalence (<1%) for all surfaces. A total of 245 Listeria spp. isolates, including 184 Listeria innocua, 48 Listeria seeligeri, and 13 Listeria welshimeri were characterized. Pulsed-field gel electrophoresis (PFGE) was used to characterize 446 L. monocytogenes isolates. PFGE showed that for 12 of 30 delis, one or more PFGE types were isolated on at least three separate occasions, providing evidence for persistence of a given L. monocytogenes subtype in the delis. For some delis, PFGE patterns for isolates from nonfood contact surfaces were distinct from patterns for occasional food contact surface isolates, suggesting limited cross-contamination between these sites in some delis. This study provides longitudinal data on L. monocytogenes contamination patterns in retail delis, which should facilitate further development of control strategies in retail delis.

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The organic acids lactate and diacetate are commonly used in combination in ready-to-eat foods because they show synergistic ability to inhibit the growth of Listeria monocytogenes. Full-genome microarrays were used to investigate the synergistic transcriptomic responses of two L. monocytogenes strains, H7858 (serotype 4b) and F6854 (serotype 1/2a), to these two organic acids under conditions representing osmotic and cold stress encountered in foods. Strains were exposed to brain heart infusion (BHI) broth at 7°C with 4.65% water-phase (w.p.) NaCl at pH 6.1 with (i) 2% w.p. potassium lactate, (ii) 0.14% w.p. sodium diacetate, (iii) the combination of both at the same levels, or (iv) no organic acids as a control. RNA was extracted 8 h after exposure, during lag phase, to capture gene transcription changes during adaptation to the organic acid stress. Significant differential transcription of 1,041 genes in H7858 and 640 genes in F6854 was observed in at least one pair of the 4 different treatments. The effects of combined treatment with lactate and diacetate included (i) synergistic transcription differences for 474 and 209 genes in H7858 and F6854, respectively, (ii) differential transcription of genes encoding cation transporters and ABC transporters of metals, and (iii) altered metabolism, including induction of a nutrient-limiting stress response, reduction of menaquinone biosynthesis, and a shift from fermentative production of acetate and lactate to energetically less favorable, neutral acetoin. These data suggest that additional treatments that interfere with cellular energy generation processes could more efficiently inhibit the growth of L. monocytogenes.Listeria monocytogenes is a psychrotolerant food-borne pathogen that is of particular concern to the ready-to-eat-meat and -seafood industries because its ability to grow at temperatures as low as Ϫ0.4°C (70) reduces the ability of refrigeration to control the pathogen's growth in foods. Because L. monocytogenes generally contaminates foods at low levels (27) and has a high infectious dose (69), the ability of the organism to grow in foods is critical for its ability to cause disease (16). Foods do not support growth if they are stored frozen, have a pH of Յ4.4, have a water activity of Ͻ0.92, or incorporate some type of growth inhibiting measure (21), e.g., chemical preservatives (32). Ready-to-eat meat and seafood products have been identified as high-risk foods for listeriosis, as these foods support growth of L. monocytogenes (22), but it has also been predicted that reformulation of U.S. deli meats with effective growth inhibitors, e.g., the organic acids lactate and diacetate, would result in a 2-to 8-fold reduction of listeriosis due to consumption of these products (55). One particular advantage to using the combination of lactate and diacetate for L. monocytogenes control in meat and seafood products is that these inhibitors have been shown to have greater-than-additive, i.e., synergistic, growth-inhibiting effects (65), although the mechanisms...

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