Characterization of Listeria monocytogenes isolated from wildlife in central New York.

BACKGROUND: Listeria monocytogenes (Lm) present in farming soil and food-processing facilities threatens food safety, but little is known about the carriage of Lm by wildlife. OBJECTIVES: We estimated the prevalence of faecal Lm shedding among wildlife admitted to a veterinary medical teaching hospital in central New York and characterized a subset of the Lm isolates. METHODS: Wildlife samples were collected between May 2018 and December 2019. We characterized the Lm isolates by assessing the growth at three temperatures approximating the body temperatures of reptiles (25°C), mammals (37°C), and birds (42°C) and identifying genotypic characteristics related to transmission and virulence. RESULTS: The apparent prevalence of faecal Lm shedding was 5.6% [18/324; 95% confidence interval (CI), 3.3%-8.6%]. Among 13 isolates that represented two lineages and 11 clonal complexes, three and five isolates were grouped into the same SNP clusters with human clinical isolates and environmental isolates, respectively. However, specific SNP difference data showed that Lm from wildlife was generally not closely related (>22 SNP differences) to Lm from human clinical sources and the food-processing environment. While the stress response locus SSI-2 was absent, SSI-1 was found in four isolates. Virulence genes prfA, plcA, hly, mpl, actA, plcB, inlA, inlB, inlC, inlE, inlH, inlJ, and inlK were present, without any premature stop codons, in all isolates. Virulence loci Listeria pathogenicity island 3 (LIPI-3) and LIPI-4, which have been linked to hypervirulence, and inlG were found in four, three, and seven isolates, respectively. CONCLUSIONS: Wildlife represents a potential reservoir for genetically diverse and putatively hypervirulent Lm strains. No statistically significant association between growth parameters and hosts was observed. However, compared to lineage I isolates, lineage II isolates showed significantly (p < 0.05) faster growth at 25°C and significantly slower growth at 42°C, suggesting that wildlife Lm isolates that belong to lineages I and II differ in their ability to grow at 25°C and 42°C.

Assessment of Reference Method Selective Broth and Plating Media with 19 Listeria Species Highlights the Importance of Including Diverse Species in Listeria Method Evaluations.

ABSTRACT: Reference methods developed for detection of Listeria monocytogenes are commonly used for detection of Listeria at the genus level. Improved method performance data are needed because this genus has expanded from 6 to 26 species and now includes several Listeria sensu lato species, which can have phenotypes distinct from those of Listeria sensu stricto. We evaluated growth of 19 Listeria species, including 12 recently described Listeria sensu lato species, using the media specified by (i) the U.S. Food and Drug Administration (FDA) Bacteriological Analytical Manual, (ii) the U.S. Department of Agriculture (USDA) Microbiology Laboratory Guidebook, and (iii) the International Organization for Standardization (ISO). The FDA broth enrichment procedure allowed all species to grow to detectable levels (≥4 log CFU/mL), yielded the highest mean growth (7.58 log CFU/mL), and was the only procedure with which no Listeria sensu lato species yielded significantly higher growth than did a comparison Listeria sensu stricto species. With the USDA and ISO broth enrichment procedures, several Listeria sensu lato species yielded significantly higher growth than did either Listeria seeligeri or Listeria ivanovii, suggesting that these two Listeria sensu stricto species could be outgrown by Listeria sensu lato species. On selective and differential agar media, L. seeligeri, L. ivanovii, and Listeria grayi produced colonies with atypical morphology and/or growth of these species was inhibited (which may lead to incorrect classification of a sample as negative), whereas several newly described Listeria sensu lato species grew to high levels and produced colonies with typical morphology. Overall, our study results indicate that the ability to detect various Listeria species can be impacted by the specific broth and selective and differential agar used. Our data can help guide selection of appropriate media and detection methods for environmental Listeria monitoring programs and methods that are most likely to detect the targeted Listeria groups (e.g., Listeria sensu stricto, which appear to be the most appropriate index organisms for the pathogen L. monocytogenes).

Landscape, Water Quality, and Weather Factors Associated With an Increased Likelihood of Foodborne Pathogen Contamination of New York Streams Used to Source Water for Produce Production.

There is a need for science-based tools to (i) help manage microbial produce safety hazards associated with preharvest surface water use, and (ii) facilitate comanagement of agroecosystems for competing stakeholder aims. To develop these tools an improved understanding of foodborne pathogen ecology in freshwater systems is needed. The purpose of this study was to identify (i) sources of potential food safety hazards, and (ii) combinations of factors associated with an increased likelihood of pathogen contamination of agricultural water Sixty-eight streams were sampled between April and October 2018 (196 samples). At each sampling event separate 10-L grab samples (GS) were collected and tested for Listeria, Salmonella, and the stx and eaeA genes. A 1-L GS was also collected and used for Escherichia coli enumeration and detection of four host-associated fecal source-tracking markers (FST). Regression analysis was used to identify individual factors that were significantly associated with pathogen detection. We found that eaeA-stx codetection [Odds Ratio (OR) = 4.2; 95% Confidence Interval (CI) = 1.3, 13.4] and Salmonella isolation (OR = 1.8; CI = 0.9, 3.5) were strongly associated with detection of ruminant and human FST markers, respectively, while Listeria spp. (excluding Listeria monocytogenes) was negatively associated with log10 E. coli levels (OR = 0.50; CI = 0.26, 0.96). L. monocytogenes isolation was not associated with the detection of any fecal indicators. This observation supports the current understanding that, unlike enteric pathogens, Listeria is not fecally-associated and instead originates from other environmental sources. Separately, conditional inference trees were used to identify scenarios associated with an elevated or reduced risk of pathogen contamination. Interestingly, while the likelihood of isolating L. monocytogenes appears to be driven by complex interactions between environmental factors, the likelihood of Salmonella isolation and eaeA-stx codetection were driven by physicochemical water quality (e.g., dissolved oxygen) and temperature, respectively. Overall, these models identify environmental conditions associated with an enhanced risk of pathogen presence in agricultural water (e.g., rain events were associated with L. monocytogenes isolation from samples collected downstream of dairy farms; P = 0.002). The information presented here will enable growers to comanage their operations to mitigate the produce safety risks associated with preharvest surface water use.

Extended Enrichment Procedures Can Be Used To Define False-Negative Probabilities for Cultural Gold Standard Methods for Salmonella Detection, Facilitating Comparisons between Gold Standard and Alternative Methods.

ABSTRACT: Evaluation of alternative detection methods for foodborne pathogens typically involves comparisons against a "gold standard" culture method, which may produce false-negative (FN) results, particularly under worst-case scenarios such as low contamination levels, difficult-to-detect strains, and challenging food matrices (e.g., matrices with a water activity of <0.6). We used extended enrichment times (up to 72 h for both primary and secondary enrichments) to evaluate a gold standard method for Salmonella detection (the U.S. Food and Drug Administration Bacteriological Analytical Manual [BAM] method) in two low-water-activity foods (dry pet food and chocolate) inoculated at low contamination levels (most probable number ca. 1/25 g) with five Salmonella strains. Strains were selected to include those with a poor ability to grow in enrichment media. Among the 100 pet food and 100 chocolate samples tested, 53 and 50, respectively, were positive with the standard BAM method, and 57 and 59, respectively, were positive with the extended BAM method. Thus, the FN probabilities for the standard BAM method were 7% for pet food and 15% for chocolate. An alternative enzyme immunoassay method for detection of Salmonella in chocolate produced FN probabilities of 6 and 20% when compared against the standard and extended BAM methods, respectively. Detection of Salmonella Mississippi was significantly reduced with the alternative method (P = 0.023) compared with the extended BAM method. We calculated a composite reference standard to further define FN probabilities based on variable results from multiple assays (the standard BAM, extended BAM, and alternative methods). Based on this standard, the enzyme immunoassay for Salmonella detection in chocolate had a 28% FN probability and the standard and extended BAM methods had 23 and 9% FN probabilities, respectively. These results provide a framework for how inclusion of extended enrichment times can facilitate evaluation of alternative detection methods.

Complex Interactions Between Weather, and Microbial and Physicochemical Water Quality Impact the Likelihood of Detecting Foodborne Pathogens in Agricultural Water.

Agricultural water is an important source of foodborne pathogens on produce farms. Managing water-associated risks does not lend itself to one-size-fits-all approaches due to the heterogeneous nature of freshwater environments. To improve our ability to develop location-specific risk management practices, a study was conducted in two produce-growing regions to (i) characterize the relationship between Escherichia coli levels and pathogen presence in agricultural water, and (ii) identify environmental factors associated with pathogen detection. Three AZ and six NY waterways were sampled longitudinally using 10-L grab samples (GS) and 24-h Moore swabs (MS). Regression showed that the likelihood of Salmonella detection (Odds Ratio [OR] = 2.18), and eaeA-stx codetection (OR = 6.49) was significantly greater for MS compared to GS, while the likelihood of detecting L. monocytogenes was not. Regression also showed that eaeA-stx codetection in AZ (OR = 50.2) and NY (OR = 18.4), and Salmonella detection in AZ (OR = 4.4) were significantly associated with E. coli levels, while Salmonella detection in NY was not. Random forest analysis indicated that interactions between environmental factors (e.g., rainfall, temperature, turbidity) (i) were associated with likelihood of pathogen detection and (ii) mediated the relationship between E. coli levels and likelihood of pathogen detection. Our findings suggest that (i) environmental heterogeneity, including interactions between factors, affects microbial water quality, and (ii) E. coli levels alone may not be a suitable indicator of food safety risks. Instead, targeted methods that utilize environmental and microbial data (e.g., models that use turbidity and E. coli levels to predict when there is a high or low risk of surface water being contaminated by pathogens) are needed to assess and mitigate the food safety risks associated with preharvest water use. By identifying environmental factors associated with an increased likelihood of detecting pathogens in agricultural water, this study provides information that (i) can be used to assess when pathogen contamination of agricultural water is likely to occur, and (ii) facilitate development of targeted interventions for individual water sources, providing an alternative to existing one-size-fits-all approaches.

Pre-Harvest Survival and Post-Harvest Chlorine Tolerance of Enterohemorrhagic Escherichia coli on Lettuce.

In the field, foodborne pathogens such as enterohemorrhagic Escherichia coli (EHEC) are capable of surviving on produce over time, yet little is known about how these pathogens adapt to this environment. To assess the impact of pre-harvest environmental conditions on EHEC survival, we quantified survival on romaine lettuce under two relative humidity (75% and 45%) and seasonal conditions (March and June). Greenhouse-grown lettuce was spray-inoculated with EHEC and placed in a growth chamber, mimicking conditions typical for June and March in Salinas Valley, California. Bacteria were enumerated on days 0, 1, 3, and 5 post-inoculation. Overall, we found that the effect of relative humidity on EHEC survival depended on the seasonal conditions. Under June seasonal conditions, higher relative humidity led to lower survival, and lower relative humidity led to greater survival, five days post-inoculation. Under March seasonal conditions, the impact of relative humidity on EHEC survival was minimal over the five days. The bacteria were also tested for their ability to survive a chlorine decontamination wash. Inoculated lettuce was incubated under the June 75% relative humidity conditions and then washed with a 50 ppm sodium hypochlorite solution (40 ppm free chlorine). When incubated under June seasonal conditions for three to five days, EHEC strains showed increased tolerance to chlorine (adj. p < 0.05) compared to chlorine tolerance upon inoculation onto lettuce. This indicated that longer incubation on lettuce led to greater EHEC survival upon exposure to chlorine. Subsequent transcriptome analysis identified the upregulation of osmotic and oxidative stress response genes by EHEC after three and five days of incubation on pre-harvest lettuce. Assessing the physiological changes in EHEC that occur during association with pre-harvest lettuce is important for understanding how changing tolerance to post-harvest control measures may occur.

Enhanced Sanitation Standard Operating Procedures Have Limited Impact on Listeria monocytogenes Prevalence in Retail Delis.

In a recent longitudinal surveillance study in 30 U.S. retail delicatessens, 9.7% of environmental surfaces were positive for Listeria monocytogenes, and we found substantial evidence of persistence. In this study, we aimed to reduce the prevalence and persistence of L. monocytogenes in the retail deli environment by developing and implementing practical and feasible intervention strategies (i.e., sanitation standard operating procedures; SSOPs). These SSOPs were standardized across the 30 delis enrolled in this study. SSOP implementation was verified by systems inherent to each retailer. Each deli also was equipped with ATP monitoring systems to verify effective sanitation. We evaluated intervention strategy efficacy by testing 28 food and nonfood contact surfaces for L. monocytogenes for 6 months in all 30 retail delis. The efficacy of the intervention on the delis compared with preintervention prevalence level was not statistically significant; we found that L. monocytogenes could persist despite implementation of enhanced SSOPs. Systematic and accurate use of ATP monitoring systems varied widely among delis. The findings indicate that intervention strategies in the form of enhanced daily SSOPs were not sufficient to eliminate L. monocytogenes from highly prevalent and persistently contaminated delis and that more aggressive strategies (e.g., deep cleaning or capital investment in redesign or equipment) may be necessary to fully mitigate persistent contamination.

Escherichia coli transfer from simulated wildlife feces to lettuce during foliar irrigation: A field study in the Northeastern United States.

Wildlife intrusion has been associated with pathogen contamination of produce. However, few studies have examined pathogen transfer from wildlife feces to pre-harvest produce. This study was performed to calculate transfer coefficients for Escherichia coli from simulated wildlife feces to field-grown lettuce during irrigation. Rabbit feces inoculated with a 3-strain cocktail of non-pathogenic E. coli were placed in a lettuce field 2.5-72 h before irrigation. Following irrigation, the E. coli concentration on the lettuce was determined. After exclusion of an outlier with high E. coli levels (Most Probable Number = 5.94*108), the average percent of E. coli in the feces that transferred to intact lettuce heads was 0.0267% (Standard Error [SE] = 0.0172). Log-linear regression showed that significantly more E. coli transferred to outer leaves compared to inner leaves (Effect = 1.3; 95% Confidence Interval = 0.4, 2.1). Additionally, the percent of E. coli that transferred from the feces to the lettuce decreased significantly with time after fecal placement, and as the distance between the lettuce and the feces, and the lettuce and the sprinklers increased. These findings provide key data that may be used in future quantitative risk assessments to identify potential intervention strategies for reducing food safety risks associated with fresh produce.

Survival of Escherichia coli on Lettuce under Field Conditions Encountered in the Northeastern United States.

Although wildlife intrusion and untreated manure have been associated with microbial contamination of produce, relatively few studies have examined the survival of Escherichia coli on produce under field conditions following contamination (e.g., via splash from wildlife feces). This experimental study was performed to estimate the die-off rate of E. coli on preharvest lettuce following contamination with a fecal slurry. During August 2015, field-grown lettuce was inoculated via pipette with a fecal slurry that was spiked with a three-strain cocktail of rifampin-resistant nonpathogenic E. coli. Ten lettuce heads were harvested at each of 13 time points following inoculation (0, 2.5, 5, and 24 h after inoculation and every 24 h thereafter until day 10). The most probable number (MPN) of E. coli on each lettuce head was determined, and die-off rates were estimated. The relationship between sample time and the log MPN of E. coli per head was modeled using a segmented linear model. This model had a breakpoint at 106 h (95% confidence interval = 69, 142 h) after inoculation, with a daily decrease of 0.70 and 0.19 log MPN for 0 to 106 h and 106 to 240 h following inoculation, respectively. These findings are consistent with die-off rates obtained in similar studies that assessed E. coli survival on produce following irrigation. Overall, these findings provide die-off rates for E. coli on lettuce that can be used in future quantitative risk assessments.

Evaluation of Rapid Molecular Detection Assays for Salmonella in Challenging Food Matrices at Low Inoculation Levels and Using Difficult-to-Detect Strains.

Assays for detection of foodborne pathogens are generally initially evaluated for performance in validation studies carried out according to guidelines provided by validation schemes (e.g., AOAC International or the International Organization for Standardization). End users often perform additional validation studies to evaluate the performance of assays in specific matrices (e.g., specific foods or raw material streams of interest) and with specific pathogen strains. However, these types of end-user validations are typically not well defined. This study was conducted to evaluate a secondary end user validation of four AOAC-validated commercial rapid detection assays (an isothermal nucleic acid amplification, an immunoassay, and two PCR-based assays) for their ability to detect Salmonella in two challenging matrices (dry pet food and dark chocolate). Inclusivity was evaluated with 68 diverse Salmonella strains at low population levels representing the limit of detection (LOD) for each assay. One assay detected all strains at the LOD, two assays detected multiple strains only at 10 times the LOD, and the fourth assay failed to detect two strains (Salmonella bongori and S. enterica subsp. houtenae) even at 1,000 times the LOD; this assay was not further evaluated. The three remaining assays were subsequently evaluated for their ability to detect five selected Salmonella strains in food samples contaminated at fractional levels. Unpaired comparisons revealed no significant difference between the results for each given assay and the results obtained with the reference assay. However, analysis of paired culture-confirmed results revealed assay false-negative rates of 4 to 26% for dry pet food and 12 to 16% for dark chocolate. Overall, our data indicate that rapid assays may have high false-negative rates when performance is evaluated under challenging conditions, including low-moisture matrices, strains that are difficult to detect, injured cells, and low inoculum levels.

Aerobic plate counts and ATP levels correlate with Listeria monocytogenes detection in retail delis.

Listeria monocytogenes is a foodborne pathogen that causes an estimated 1,591 cases of illness and 255 deaths annually in the United States, the majority of which are attributed to ready-to-eat deli meats processed in retail delis. Because retail delis distribute product directly to consumers, rapid methods to validate cleaning and sanitation are needed to improve retail food safety. This study investigated the relationships among ATP levels, standard aerobic plate count (APC), and L. monocytogenes presence in fully operational delis. Fifteen full-service delis were concurrently sampled for ATP, APC, and L. monocytogenes during preoperational hours once monthly for 3 months. Fifteen additional delis were recruited for 6 months of operational sampling (n = 30). A 1-log increase in APC was equivalent to a 3.3-fold increase in the odds of detecting L. monocytogenes (P < 0.001) and a 1.9-log increase in L monocytogenes population (P = 0.03). An ATP level increase of 1 log relative light unit correlated to a 0.22-log increase in APC (P < 0.001). A preoperational ATP level mean increase by 1 log relative light unit increased the odds of detecting L. monocytogenes concurrently fourfold. A 0.5-log increase in mean ATP level during preoperational sampling corresponded to a 2% increase in the predicted L. monocytogenes prevalence during operation (P < 0.01). Additionally, 10 statistically representative sites were identified and recommended for use in sanitation monitoring programs. Our data support the use of ATP as a rapid method to validate effective cleaning and sanitation to reduce L. monocytogenes in retail delis.

Listeria monocytogenes and Listeria spp. contamination patterns in retail delicatessen establishments in three U.S. states.

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.

Listeria floridensis sp. nov., Listeria aquatica sp. nov., Listeria cornellensis sp. nov., Listeria riparia sp. nov. and Listeria grandensis sp. nov., from agricultural and natural environments.

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 S10-1188(T) = DSM 26686(T) = LMG 28120(T) = BEI NR-42633(T)) and Listeria floridensis sp. nov. (type strain FSL S10-1187(T) = DSM 26687(T) = LMG 28121(T) = BEI NR-42632(T)), (iii) a clade consisting of Listeria rocourtiae, L. weihenstephanensis and three novel species, Listeria cornellensis sp. nov. (type strain TTU A1-0210(T) = FSL F6-0969(T) = DSM 26689(T) = LMG 28123(T) = BEI NR-42630(T)), Listeria grandensis sp. nov. (type strain TTU A1-0212(T) = FSL F6-0971(T) = DSM 26688(T) = LMG 28122(T) = BEI NR-42631(T)) and Listeria riparia sp. nov. (type strain FSL S10-1204(T) = DSM 26685(T) = LMG 28119(T) = BEI NR- 42634(T)) and (iv) a clade containing Listeria grayi. Genomic and phenotypic data suggest that the novel species are non-pathogenic.

Implementation of statistical tools to support identification and management of persistent Listeria monocytogenes contamination in smoked fish processing plants.

Listeria monocytogenes persistence in food processing plants is a key source of postprocessing contamination of ready-to-eat foods. Thus, identification and elimination of sites where L. monocytogenes persists (niches) is critical. Two smoked fish processing plants were used as models to develop and implement environmental sampling plans (i) to identify persistent L. monocytogenes subtypes (EcoRI ribotypes) using two statistical approaches and (ii) to identify and eliminate likely L. monocytogenes niches. The first statistic, a binomial test based on ribotype frequencies, was used to evaluate L. monocytogenes ribotype recurrences relative to reference distributions extracted from a public database; the second statistic, a binomial test based on previous positives, was used to measure ribotype occurrences as a risk factor for subsequent isolation of the same ribotype. Both statistics revealed persistent ribotypes in both plants based on data from the initial 4 months of sampling. The statistic based on ribotype frequencies revealed persistence of particular ribotypes at specific sampling sites. Two adaptive sampling strategies guided plant interventions during the study: sampling multiple times before and during processing and vector swabbing (i.e., sampling of additional sites in different directions [vectors] relative to a given site). Among sites sampled for 12 months, a Poisson model regression revealed borderline significant monthly decreases in L. monocytogenes isolates at both plants (P = 0.026 and 0.076). Our data indicate elimination of an L. monocytogenes niche on a food contact surface; niches on nonfood contact surfaces were not eliminated. Although our data illustrate the challenge of identifying and eliminating L. monocytogenes niches, particularly at nonfood contact sites in small and medium plants, the methods for identification of persistence we describe here should broadly facilitate science-based identification of microbial persistence.

Persistent Listeria monocytogenes subtypes isolated from a smoked fish processing facility included both phage susceptible and resistant isolates.

Contamination of Ready-To-Eat foods with Listeria monocytogenes can typically be traced back to post-processing contamination from environmental sources; contamination is often linked to subtypes that persist in food associated environments. Although phage-based biocontrol strategies have been proposed for controlling this pathogen, information on the efficacy of phage treatment against diverse L. monocytogenes subtypes from food associated environments is still limited. We identified subtypes that were repeatedly found ("persistent") in a smoked fish processing facility by using EcoRI ribotyping data for isolates obtained in 1998-2009. PFGE analysis of 141 isolates (9 ribotypes) supported persistence for up to 11 years. Characterization of selected isolates, representing persistent subtypes, against a panel of 28 listeriaphages showed a wide range of likelihood of phage susceptibility, ranging from 4.6% (for 7 ribotype DUP-1043A isolates) to 95.4% (for 7 ribotype DUP-1044A isolates). In challenge studies with 10(5) and 10(6) CFU/ml L. monocytogenes, using phage cocktails and a commercial phage product at different phage-host ratios, one isolate (ribotype DUP-1043A) was not affected by any treatment. A reduction in L. monocytogenes counts of up to 4 log units was observed, after 8 h of treatment, in isolates of two ribotypes, but subsequent re-growth occurred. Survivor isolates obtained after 24 h of treatment showed decreased susceptibility to individual phages included in the phage cocktail, suggesting rapid emergence of resistant subtypes.

Prevalence, distribution, and diversity of Listeria monocytogenes in retail environments, focusing on small establishments and establishments with a history of failed inspections.

Despite growing concerns about cross-contamination of ready-to-eat foods with Listeria monocytogenes, our knowledge about the ecology and transmission of L. monocytogenes in retail establishments has remained limited. We conducted a cross-sectional study to characterize the prevalence, distribution, and subtype diversity of L. monocytogenes in 120 New York State retail deli establishments that were hypothesized to present an increased risk for environmental L. monocytogenes contamination (i.e., small establishments and establishments with a history of failed New York State Agriculture and Markets inspections). Analysis of these data along with previously reported data for 121 predominantly larger retail establishments in New York State identified establishment size, geographic location, and inspection history as significant predictors of L. monocytogenes presence and prevalence. The odds of an establishment being L. monocytogenes positive were approximately twice as high for large establishments, establishments located in New York City, or establishments with poor inspection history (as compared with establishments without these attributes), even though correlation between location and inspection history complicated interpretation of results. Within an establishment, L. monocytogenes was significantly more prevalent on nonfood contact surfaces than on food contact surfaces; prevalence was particularly high for floors and in floor drains, sinks, the dairy case, and milk crates. L. monocytogenes subtype diversity differed between sites, with lineage I isolates significantly associated with nonfood contact surfaces and lineage II isolates significantly associated with food contact surfaces. Isolates belonging to the same ribotype were often found dispersed across multiple sites within an operation.

Molecular ecology of Listeria monocytogenes and other Listeria species in small and very small ready-to-eat meat processing plants.

A longitudinal study was conducted to track Listeria contamination patterns in ready-to-eat meats from six small or very small meat processing plants located in three states over 1 year. A total of 688 environmental sponge samples were collected from nonfood contact surfaces during bimonthly visits to each plant. Overall, L. monocytogenes was isolated from 42 (6.1%) environmental samples, and its prevalence ranged from 1.7 to 10.8% across different plants. Listeria spp., other than L. monocytogenes, were isolated from 9.5% of samples overall, with the prevalence ranging from 1.5 to 18.3% across different plants. The prevalence of L. monocytogenes correlated well with that of other Listeria spp. for some but not all plants. One L. monocytogenes isolate representing each positive sample was characterized by molecular serotyping, EcoRI ribotyping, and pulsed-field gel electrophoresis typing. Seven sample sites tested positive for L. monocytogenes on more than one occasion, and the same ribotype was detected more than once at five of these sites. Partial sigB sequencing was used to speciate other Listeria spp. isolates and assign an allelic type to each isolate. Other Listeria spp. were isolated more than once from 14 sample sites, and the same sigB allelic type was recovered at least twice from seven of these sites. One plant was colonized by an atypical hemolytic L. innocua strain. Our findings indicate that small and very small meat processing plants that produce ready-to-eat meat products are characterized by a varied prevalence of Listeria, inconsistent correlation between contamination by L. monocytogenes and other Listeria spp., and a unique Listeria molecular ecology.

Molecular and phenotypic characterization of Listeria monocytogenes from U.S. Department of Agriculture Food Safety and Inspection Service surveillance of ready-to-eat foods and processing facilities.

A panel of 501 Listeria monocytogenes isolates obtained from the U.S. Department of Agriculture Food Safety and Inspection Service monitoring programs for ready-to-eat (RTE) foods were subtyped by multilocus genotyping (MLGT) and by sequencing the virulence gene inlA, which codes for internalin. MLGT analyses confirmed that clonal lineages associated with previous epidemic outbreaks were rare (7.6%) contaminants of RTE meat and poultry products and their production environments. Conversely, sequence analyses revealed mutations leading to 11 different premature stop codons (PMSCs) in inlA, including three novel PMSC mutations, and revealed that the frequency of these virulence-attenuating mutations among RTE isolates (48.5%) was substantially higher than previously appreciated. Significant differences (P < 0.001) in the frequency of inlA PMSCs were observed between lineages and between major serogroups, which could partially explain differences in association of these subtypes with human listeriosis. Interrogation of single-nucleotide polymorphisms responsible for PMSCs in inlA improved strain resolution among isolates with the 10 most common pulsed-field gel electrophoresis (PFGE) patterns, 8 of which included isolates with a PMSC in inlA. The presence or absence of PMSCs in inlA accounted for significant differences (P < 0.05) in Caco-2 invasion efficiencies among isolates with identical PFGE patterns, and the proportion of PulseNet entries from clinical sources was significantly higher (P < 0.001) for PFGE patterns exclusively from isolates with full-length inlA. These results indicated that integration of PFGE and DNA sequence-based subtyping provides an improved framework for prediction of relative risk associated with L. monocytogenes strains from RTE foods.

Listeria marthii sp. nov., isolated from the natural environment, Finger Lakes National Forest.

Four isolates (FSL S4-120(T), FSL S4-696, FSL S4-710, and FSL S4-965) of Gram-positive, motile, facultatively anaerobic, non-spore-forming bacilli that were phenotypically similar to species of the genus Listeria were isolated from soil, standing water and flowing water samples obtained from the natural environment in the Finger Lakes National Forest, New York, USA. The four isolates were closely related to one another and were determined to be the same species by whole genome DNA-DNA hybridization studies (>82 % relatedness at 55 degrees C and >76 % relatedness at 70 degrees C with 0.0-0.5 % divergence). 16S rRNA gene sequence analysis confirmed their close phylogenetic relatedness to Listeria monocytogenes and Listeria innocua and more distant relatedness to Listeria welshimeri, L. seeligeri, L. ivanovii and L. grayi. Phylogenetic analysis of partial sequences for sigB, gap, and prs showed that these isolates form a well-supported sistergroup to L. monocytogenes. The four isolates were sufficiently different from L. monocytogenes and L. innocua by DNA-DNA hybridization to warrant their designation as a new species of the genus Listeria. The four isolates yielded positive reactions in the AccuProbe test that is purported to be specific for L. monocytogenes, did not ferment L-rhamnose, were non-haemolytic on blood agar media, and did not contain a homologue of the L. monocytogenes virulence gene island. On the basis of their phenotypic characteristics and their genotypic distinctiveness from L. monocytogenes and L. innocua, the four isolates should be classified as a new species within the genus Listeria, for which the name Listeria marthii sp. nov. is proposed. The type strain of L. marthii is FSL S4-120(T) (=ATCC BAA-1595(T) =BEIR NR 9579(T) =CCUG 56148(T)). L. marthii has not been associated with human or animal disease at this time.