Environmental Listeria Research Articles

Examining Patterns of Persistent Listeria Contamination in Packinghouses Using Agent-Based Models

Persistent Listeria monocytogenes contamination may occur in a packinghouse if the pathogen successfully infiltrates the facility and reaches a harborage site, where it may be difficult to remove and may contaminate produce within the facility. There is a need for simulation-based decision support tools that can predict which equipment sites are more likely to undergo persistent contamination and simulate potential corrective actions to prevent this contamination. Thus, we adapted for longer term simulation two existing applications of an agent-based model of Listeria spp. hourly contamination dynamics in produce packinghouses. Next, we developed a novel approach to identify and analyze persistent and transient Listeria contamination patterns on simulated agents representing equipment sites and employees. Testing of corrective actions showed that methods that involved targeted, facility-specific, risk-based sanitation were the most effective in reducing both the likelihood and duration of persistent contamination. Generic approaches to controlling Listeria (e.g., more concentrated sanitizers) are unlikely to be successful and suggest that use of sanitation schedules produced through facility-specific root cause analysis and hygienic design are key in reducing persistence. Hourly Listeria contamination patterns also suggest that transient contamination may be mistaken for persistent contamination, depending on the frequency of environmental sampling. Likewise, as concentrations of Listeria on most contaminated agents were predicted to be very low, there is also a possibility to mistake persistence for transient contamination of sites, or even miss it outright, due to false-negative environmental Listeria monitoring results. These findings support that agent-based models may be valuable decision support tools, aiding in the identification of contamination patterns within packinghouses and assessing the viability of specific corrective actions.

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

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).

Genotyping of Listeria monocytogenes strains in Italian food-processing environments

Abstract Background In 2019, case fatality from Listeria monocytogenes (Lm) was higher than the previous years confirming Lm as a major public health problem. Food is the main source of Lm infection and cross-contamination from the food-processing environment (FPEs) is the most common route of contamination. The aim of this work was to characterize FPEs Lm strains collected from the National Reference Laboratory (NRL) for Lm in order to evaluate circulating clonal complexes (CCs) and their environmental adaptation. Methods NRL Lm database actually consists of 798 FPEs sequenced strains. In silico MLST was evaluated. Sequences related to prevalent CCs were further investigated using Pasteur's cgMLST scheme and detecting metal and detergent resistance genes and stress survival islet (SSI). Results MLST identified 25 CCs and 4 singleton. Nine isolates gave inexact match with existing alleles and were not classified. CC9 (17.4%) and CC121 (14.2%) were the prevalent clones. CC9 cgMLST analysis revealed a relatedness among strains isolated from the same establishment type (meat or dairy), even if sampled in different regions. Indeed, CC121 cgMLST clustering analysis was strictly associated to the point of sampling. Regarding stress adaptation genes, SSI1 and brcABC genes were detected in 100% and 2.9% of the CC9 strains, respectively. Meanwhile, SSI2 and transposon Tn6188 were detected, respectively, in 100% and 97.3% of CC121 strains. Conclusions Results confirmed, in Italian FPEs, prevalence of hypovirulent Lm strains, as previously observed in Italian food. Tested isolates highlighted adaptation to low pH and high salt concentration (SSI1 genes for CC9), to alkaline and oxidative stress (SSI2 genes for CC121) and benzalkonium chloride tolerance (Tn6188 in CC121) resulting in a best survival under stress conditions. Interestingly, CC9 clustering denoted allelic proximity among strains isolated in different Italian regions paying attention to clones spreading at national level. Key messages Listeria monocytogenes CC9 and CC121 prevalence in Italian establishment confirms environment is the main route of food contamination at production level. Prevalent food-processing environmental Italian Listeria monocytogenes strains denotes adaptation to environmental stress and clones spreading at national level.

Monitoring environmental microbiological safety in a frozen fruit and vegetable plant

Ensuring food safety is becoming progressively important in frozen foods due to increased demand and consumption worldwide. The Environmental Monitoring Program (EMP) shall support daily operational activities and monitor the lack of process hygiene & sanitation actions to achieve food safety. The aim of this study was implementation of EMP in the frozen fruit and vegetable factory to identify the place that allows for pathogen/indicator microorganism growth and create preventive actions to reduce or eliminate the risk of cross- contamination. A total of 400 swab samples, Zone 1 (n = 228), Zone 2 (n = 90), Zone 3 (n = 52), Zone 4 (n = 30) were examined for E. coli and Coliform, Total Plate Count (TPC), yeast and mold and environmental Listeria spp. The percentage of satisfying target value before vs after the implementation of EMP was 82% vs. 100% for E. coli, 61% vs. 86% for Coliforms, 48% vs. 77% for TPC and 63% vs. 80% for yeast & mold respectively for Zone 1. Environmental Listeria was not determined in any of the zones. Integration of EMP to food safety management systems is essential to prevent outbreaks, recalls and economic losses. In addition to depending on the developing technologies, EMP should be supported with new corrective actions.

Practical application of bacteriophage in food manufacturing facilities for the control of Listeria sp.

AbstractListeria monocytogenes is a foodborne pathogen with the ability to persist and form biofilm matrices in processing environments of food manufacturing facilities. Bacteriophages are bacterial viruses with host specific lethality. Published research on the application of phage to control Listeria sp. in manufacturing environments is limited. In this study, we have assessed the capacity of bacteriophage P100 (Listex™) to reduce incidence of Listeria sp. in the ready‐to‐eat (RTE) environment of refrigerated (4°C) and ambient (20°C) temperature facilities using two different application strategies. A moderate application applied as a single treatment every 24 hr over three days (2 × 107 PFU/ml) and an intensified application applied once every 6 hr over a 24 hr period (1 × 108 PFU/ml). Environmental nonfood contact surface (NFCS) samples were collected and analyzed for the presence of Listeria sp. before and after treatment. When the moderate treatment protocol was applied the incidence of positives decreased from 51.3 to 17.5% in the 4°C environment and from 67.5 to 23.1% in the 20°C production area. For the intensified phage treatment method, the initial positive rate in the 4°C environment ranged from 5 to 47.5%, with an overall 43% reduction in Listeria sp. In the 20°C facility, initial environmental Listeria sp. ranged from 15 to 50%, with an overall reduction of 32% after treatment with phage P100. Data indicate the application of Listeria specific phage P100 in RTE food production environments by either the moderate or intensified application method can reduce incidence and be considered an additional intervention strategy for controlling this pathogen on NFCS.

Evaluation of 3M™ loop-mediated isothermal amplification-based kit and 3M™ ready-to-use plating system for detection of Listeria in naturally contaminated leafy vegetables, chicken, and their related processing environments.

The effectiveness of two different rapid methods involving the 3M™ molecular detection assay Listeria and the 3M™ Petrifilm environmental Listeria Plate were evaluated for the rapid detection of Listeria from naturally contaminated vegetables and chicken-processing environments against the standard culture-based method. A total of 178 samples were examined for the presence of Listeria. A total of 47/178 (26.4%) by standard ISO culture-based method (EN ISO 11290-1), 42/178 (23.6%) by 3M™ MDA Listeria and 40/178 (22.5%) by 3M™ Petrifilm EL Plate showed positive results, respectively. The accuracy, sensitivity, specificity, positive predictive value, and negative predictive value for 3M™ MDA Listeria and 3M™ Petrifilm EL Plate were 97.2, 89.4, 99.3, 97.7, 96.4% and 96.1, 85.1, 100.0, 100.0, 94.9%, respectively. Based on the Cohen's Kappa value, there was a complete and robust concordance between 3M™ MDA Listeria (0.911) and 3M™ Petrifilm EL Plates (0.894) as compared to the standard culture-based method.

Evaluation of the CERTUS Environmental Listeria Species Detection Kit for the Detection of Listeria Species on Environmental Surfaces: AOAC Performance Tested MethodSM 101802.

CERTUS Environmental Listeria species Detection Kit (CERTUS EL Detection Kit) is a real-time, bio-contained assay designed to accurately detect Listeria species (L. grayi, L. innocua, L. ivanovii, L. marthii, L. monocytogenes, L. seeligeri, and L. welshimeri) from environmental surface matrixes using an antibody-coupled magnetic microparticle with a Surface Enhanced Raman Spectroscopy (SERS) nanoparticle technology test system paired with proprietary CERTUS EL Selective Growth Media and CERTUS Detection Unit. The method was evaluated for AOAC®Performance Tested MethodSM certification. Inclusivity and exclusivity, matrix studies, product consistency and stability were conducted to evaluate the CERTUS EL Detection Kit. In the matrix studies, stainless steel, ceramic tile, plastic (polystyrene) and sealed concrete environmental surfaces (4 × 4" test areas) were tested. No statistically significant differences were found by Probability of Detection analysis (POD) in any of the matrixes when results were compared to the U.S. Food and Drug Administration cultural microbiology reference method for Listeria. The CERTUS EL Detection Kit correctly identified all 50 target Listeria isolates and correctly excluded all 30 non-target strains that were analyzed. Probability of Detection analysis of CERTUS EL Detection Kit robustness, product consistency (lot-to-lot) and stability studies demonstrated no statistically significant differences, and no variation was observed between instruments. The data collected in these studies demonstrate that the CERTUS EL Detection Kit is a reliable method for the rapid and specific detection of Listeria from stainless steel, ceramic tile, plastic (polystyrene) and sealed concrete environmental surfaces.

Listeria cross contamination levels in raw ice cream mix can serve as a predictor of their potential presence as heat-injured cells

Listeriosis is a life-threatening infection caused by foods contaminated with Listeria monocytogenes. Some of the major ice cream recalls in recent years reaffirm the ability of this food-borne pathogen to survive in diverse dairy processing environments and cause cross contamination. Inspection reports revealed certain lapses in implementing adequate hygienic practices for Listeria persistence in the processing environment, leading to cross contamination of ice cream. The higher levels of cross contamination of raw ice cream mix might result in random heat-injured cells when exposed to minimum heat treatment (69°C for 30 min). These heat-injured cells could later recover under abusive storage and handling conditions and pose a health risk. Evidence about the presence of injured cells in ice cream mix may thus prove useful to establish the overall Listeria risk, which was the aim of this study. Challenge studies were conducted to evaluate the dose-dependent presence of heat-injured cells of Listeria. Ice cream mix formulations of 4 different types (36, 40, 42, and 45% total solids) were inoculated at 2.0, 3.0, and 4.0 log cfu/g levels of Listeria innocua (an established surrogate). The dose levels were selected based on a likely cross contamination on the raw side from environmental Listeria, especially due to their resident nature and growth in harborage sites. The samples were exposed to minimum heat treatment (69°C for 30 min) and the survivors, including heat-injured cells, were enumerated using standard protocols. A binary logistic regression model was fitted for evaluating the severity of risk. The influence of total solids, water activity, and pH variability were also studied on Listeria survival. The enrichment protocol, using buffered Listeria enrichment broth, followed by plating on modified oxford agar and Rapid L'mono medium, revealed the random presence of heat-injured cells in buffered Listeria enrichment broth, only at the highest dose level of 4+ logs. Any potential risk from heat-injured cells was thus limited only to the highest levels of cross contamination, irrespective of the type of the mix. Significantly, none of the pasteurized ice cream mix samples supported the recovery of any heat-injured cells of Listeria during 72 h holding at 7°C, even at the highest dose level of 4+ logs, under the conditions of experimentation. The level of cross contamination (dose) emerged as a predictor of the potential presence of heat-injured cells of Listeria exposed to minimum pasteurization treatment.

Reduction of Environmental Listeria Using Gaseous Ozone in a Cheese Processing Facility

A cheese processing facility seeking to reduce environmental Listeria colonization initiated a regime of ozonation across all production areas as an adjunct to its sanitation regimes. A total of 360 environmental samples from the facility were tested for Listeria over a 12-month period. A total of 15 areas before and 15 areas after ozonation were tested. Listeria isolations were significantly ( P < 0.001) reduced from 15.0% in the preozonation samples to 1.67% in the postozonation samples in all areas. No deleterious effects of ozonation were noted on the wall paneling, seals, synthetic floors, or cheese processing equipment. The ozonation regime was readily incorporated by sanitation staff into the existing good manufacturing practice program. The application of ozone may result in a significant reduction in the prevalence of Listeria in food processing facilities.

Complete Circular Genome Sequence and Temperature Independent Adaptation to Anaerobiosis of Listeria weihenstephanensis DSM 24698.

The aim of this study was to analyze the adaptation of the environmental Listeria weihenstephanensis DSM 24698 to anaerobiosis. The complete circular genome sequence of this species is reported and the adaptation of L. weihenstephanensis DSM 24698 to oxygen availability was investigated by global transcriptional analyses via RNAseq at 18 and 34°C. A list of operons was created based on the transcriptional data. Forty-two genes were upregulated anaerobically and 62 genes were downregulated anaerobically. The oxygen dependent gene expression of selected genes was further validated via qPCR. Many of the differentially regulated genes encode metabolic enzymes indicating broad metabolic adaptations with respect to oxygen availability. Genes showing the strongest oxygen-dependent adaption encoded nitrate (narGHJI) and nitrite (nirBD) reductases. Together with the observation that nitrate supported anaerobic growth, these data indicate that L. weihenstephanensis DSM 24698 performs anaerobic nitrate respiration. The wide overlap between the oxygen-dependent transcriptional regulation at 18 and 34°C suggest that temperature does not play a key role in the oxygen-dependent transcriptional regulation of L. weihenstephanensis DSM 24698.

Thermal inactivation and growth of Listeria monocytogenes during production and storage of caramel apples

During the fall of 2014, commercially produced pre-packaged caramel apples were linked to 35 cases of listeriosis in 12 states. In response, this study aimed to assess 1) the reduction of different outbreak and non-outbreak strains of Listeria monocytogenes during caramel dipping of apples, and 2) subsequent growth of the apple outbreak strains within caramel apples during storage at 22 and 4 °C. In aim 1, three unwaxed Jonathan apples were dip-inoculated with three different 4-strain L. monocytogenes cocktails (apple outbreak, unrelated outbreak or unrelated environmental) at ∼8 log CFU/apple, dried for 1 h, dipped for 5 s in caramel at 82, 88, 93 or 99 °C, cooled for 1 h at room temperature and assessed for survivors. In aim 2, Jonathan apples were spot-inoculated with the apple outbreak cocktail (∼3 log CFU/apple) at the stem juncture, dried for 1 h, pushed onto wooden sticks, and dipped in caramel at 82 °C. During storage at 4 and 22 °C for 28 and 14 days, respectively, four different apple sections (top, middle, bottom and core) were cut from three apples, homogenized and plated for Listeria. After dipping apples in caramel at 82 and 99 °C, the apple outbreak, unrelated outbreak and environmental Listeria strains decreased 2.0 ± 0.6 and 2.7 ± 0.1, 1.8 ± 0.3 and 2.6 + 0.1, and 1.7 ± 0.1 and 2.9 ± 0.2 logs, respectively, with the environmental cocktail significantly less heat resistant (P < 0.05) at 99 °C compared to the other two cocktails. After 14 days of storage at 22 °C, Listeria populations were significantly higher (P < 0.05) in the core (7.4 ± 0.6 log CFU/g) compared to the other three sections (4.9–5.4 log CFU/g). The same trend was seen for the core (7.7 ± 0.6 log CFU/g) and the other three sections (5.0–5.4 log CFU/g) after 28 days of storage at 4 °C. Since dipping in hot caramel cannot ensure pathogen elimination, producers of caramel apples should implement good agricultural practices, post-harvest preventive controls and refrigeration of the final product to minimize the risks from Listeria.

Genomic-based identification of host and environmental Listeria monocytogenes strains associated with an abortion outbreak in beef heifers

Listeria monocytogenes (LM) is a bacterial pathogen that causes late-term abortions in cattle, and definitive diagnosis and point-source determination may be challenging. In 2014, a diagnostic work-up of listerial abortions was initiated by a novel sample collection protocol. To determine the source(s) of the outbreak, LM strains isolated from cases and their environment were subjected to whole-genome sequencing and compared.

Seek and Destroy Process: Listeria monocytogenes Process Controls in the Ready-to-Eat Meat and Poultry Industry

The majority of human listeriosis cases appear to be caused by consumption of ready-to-eat (RTE) foods contaminated at the time of consumption with high levels of Listeria monocytogenes. Although strategies to prevent growth of L. monocytogenes in RTE products are critical for reducing the incidence of human listeriosis, control of postprocessing environmental contamination of RTE meat and poultry products is an essential component of a comprehensive L. monocytogenes intervention and control program. Complete elimination of postprocessing L. monocytogenes contamination is challenging because this pathogen is common in various environments outside processing plants and can persist in food processing environments for years. Persistent L. monocytogenes strains in processing plants have been identified as the most common postprocessing contaminants of RTE foods and the cause of multiple listeriosis outbreaks. Identification and elimination of L. monocytogenes strains persisting in processing plants is thus critical for (i) compliance with zero-tolerance regulations for L. monocytogenes in U.S. RTE meat and poultry products and (ii) reduction of the incidence of human listeriosis. The seek-and-destroy process is a systematic approach to finding sites of persistent strains (niches) in food processing plants, with the goal of either eradicating or mitigating effects of these strains. This process has been used effectively to address persistent L. monocytogenes contamination in food processing plants, as supported by peer-reviewed evidence detailed here. Thus, a regulatory environment that encourages aggressive environmental Listeria testing is required to facilitate continued use of this science-based strategy for controlling L. monocytogenes in RTE foods.

PDX-LIB, an Improvement on Selective Enrichment Media for Detection of Environmental Listeria spp.

PDX-LIB is a selective enrichment indicator broth developed for use as a diagnostic screening test for environmental Listeria spp. contamination. The test method was developed to provide a single step selective enrichment media incorporating a colorimetric indicator to identify presumptive Listeria-positive samples. The method permits high throughput screening of the sample population streamlining the number of steps to confirmatory testing. In the original AOAC Research Institute guided performance test method the PDX-LIB selection media was shown to be significantly more sensitive for detection of Listeria spp. on selected surfaces than the USDA method, which utilizes UVM media as its primary selection step. The present study has shown that the PDXLIB selection media permits substantially greater numbers of recoverable Listeria monocytogenes- positive samples in the AOAC surface study protocol than comparable samples enriched with UVM media. The data presented demonstrate that the PDX-LIB method is a more sensitive method than procedures utilizing UVM enrichment media as the primary step particularly with regard to detection of Listeria sp.

Occurrence and diversity of Listeria spp. in seafood processing plant environments

Listeria contamination in processing plant environments is a major issue for the seafood industry worldwide; faster and more reliable results are therefore desired for early detection and monitoring of environmental Listeria spp. This study aimed to gain a better understanding of the prevalence and diversity of Listeria spp., and to evaluate a rapid detection method, the 3M Molecular Detection Assay (MDA) Listeria, for its ability to detect Listeria spp. in environmental samples from seafood processing plants. Duplicate environmental sponge samples (n = 444) were collected from 152 different sites within three seafood processing plants, and analyzed for Listeria spp. by the MDA method (after 26 and 48 h of enrichment) and the U.S. Food and Drug Administration Bacteriological Analytical Manual method. Overall, detection of Listeria spp. by the two methods did not differ significantly (p > 0.05); 11 (4.9%) and 13 (5.9%) samples were positive for Listeria spp. by the MDA and FDA-BAM method, respectively. The sensitivity of the MDS was 87.0% (95% CI: 77.4–96.6%), specificity was 97.6% (95% CI: 95.5–99.7%), accuracy was 95.3%, and the positive predictive value was 89.4% (95% CI: 80.5–98.2%). Classification of 19 Listeria isolates by partial SigB sequencing analysis identified three allelic types. Twelve of these isolates were ATs 58 and 60 which were classified as Listeria monocytogenes lineage I and serotypes 1/2b, 3b, 4b, 4d, 4e, by multiplex-PCR serotyping. Six Listeria isolates were classified as Listeria innocua (AT31). Our data show that the 3M Molecular Detection Assay Listeria provides rapid and reliable results for detection and monitoring of Listeria spp., which are important for seafood processing plants. Effective Listeria monitoring programs will allow for improved development of Listeria control measures in order to minimize cross-contamination in finished products.

Antimicrobial susceptibility and antibiotic resistance gene transfer analysis of foodborne, clinical, and environmental Listeria spp. isolates including Listeria monocytogenes.

The aims of this study were to assess antibiotic resistance pheno-and genotypes in foodborne, clinical, and environmental Listeria isolates, as well as to elucidate the horizontal gene transfer potential of detected resistance genes. A small fraction of in total 524 Listeria spp. isolates (3.1%) displayed acquired antibiotic resistance mainly to tetracycline (n = 11), but also to clindamycin (n = 4) and trimethoprim (n = 3), which was genotypically confirmed. In two cases, a tetracycline resistance phenotype was observed together with a trimethoprim resistance phenotype, namely in a clinical L. monocytogenes strain and in a foodborne L. innocua isolate. Depending on the applied guidelines, a differing number of isolates (n = 2 or n = 20) showed values for ampicillin that are on the edge between intermediate susceptibility and resistance. Transferability of the antibiotic resistance genes from the Listeria donors, elucidated in vitro by filter matings, was demonstrated for genes located on transposons of the Tn916 family and for an unknown clindamycin resistance determinant. Transfer rates of up to 10−5 transconjugants per donor were obtained with a L. monocytogenes recipient and up to 10−7 with an Enterococcus faecalis recipient, respectively. Although the prevalence of acquired antibiotic resistance in Listeria isolates from this study was rather low, the transferability of these resistances enables further spread in the future. This endorses the importance of surveillance of L. monocytogenes and other Listeria spp. in terms of antibiotic susceptibility.

The effect of ozone and open air factor on surface-attached and biofilm environmental Listeria monocytogenes

The effects of gaseous ozone and open air factor (OAF) on environmental Listeria monocytogenes attached to three common food contact surfaces were investigated. Listeria monocytogenes on different food contact surfaces was treated with ozone and OAF. Microbiological counts, scanning electron microscopy (SEM) and atomic force microscopy (AFM) were performed. Ozone at 10 ppm gave <1-log reduction when L. monocytogenes was attached to stainless steel, while 45 ppm gave a log reduction of 3.41. OAF gave better log reductions than 10 ppm ozone, but lower log reductions than 45 ppm. Significant differences were found between surfaces. Biofilm organisms were significantly more resistant than those surface attached on stainless steel. SEM and AFM demonstrated different membrane and cell surface modifications following ozone or OAF treatment. The strain used demonstrated higher resistance to ozone than previous studies. This may be due to the fact that it was isolated from a food manufacturing premises that used oxidizing disinfectants. OAF was more effective at reducing the levels of the organism than an ozone concentration of 10 ppm. Pathogen management strategies must account for resistance of environmental strains when validating cleaning and disinfection. OAF has shown potential for surface decontamination compared with ozone. SEM and AFM are valuable tools for determining mechanisms of action of antimicrobial agents.

3M™ Petrifilm™ Environmental Listeria Plate

A validation study of the 3M Petrifilm Environmental Listeria (EL) Plate (3M Food Safety, St. Paul, MN) was conducted at Q Laboratories, Inc., Cincinnati, OH. The method was compared to the Health Canada MFHPB-30 reference method for the analysis of stainless steel environmental surfaces. Twenty replicates of the environmental surface were analyzed at a low and a high inoculum level. The low-level sampling area was inoculated with 0.2-2 CFU/5 cm2, and the high-level sampling area was inoculated with 2-5 CFU/5 cm2. Five control replicates were also analyzed at 0 CFU/5 cm2. There was no significant difference in the number of positives detected by the 3M Petrifilm EL Plate method and the Health Canada MFHPB-30 reference method for the environmental surface analyzed in this study.

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

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

Evaluation of environmental sampling methods and rapid detection assays for recovery and identification of Listeria spp. from meat processing facilities.

Studies that isolated Listeria spp. from the environment of two meat processing facilities were conducted. Samples were collected in the processing environment of the facilities with three different sampling methods (cotton swab, sterile sponge, and composite-ply tissues) to evaluate their ability to recover Listeria spp. A total of 240 samples for each sampling method were collected and tested. The cotton swab method of sampling was significantly (P < 0.01) less efficient in recovery of Listeria spp. than the sterile-sponge and composite-ply tissue methods, which were similar (P > 0.05) in their ability to recover Listeria spp. The specificity and sensitivity of four detection methods (conventional culture, Petrifilm Environmental Listeria Plates [ELP], lateral-flow immunoprecipitation [LFI], and automated PCR) were evaluated for identification of Listeria spp. Facilities were visited until a minimum of 100 positive and 100 negative samples per detection method were collected. The LFI and PCR methods were highly sensitive (95.5 and 99.1%, respectively) and specific (100%) relative to the culture method. The ELP method was significantly less efficient (P < 0.01) than LFI and PCR in detection of Listeria spp., with lower sensitivity (50.6%) and specificity (91.5%). Kappa values indicated excellent agreement of the LFI and PCR assays and moderate agreement of the ELP method to the culture method. Overall, ELP was easy to use but less efficient in detection of Listeria spp. from environmental samples, while the LFI and PCR methods were found to be excellent alternatives to culture, considering performance and time and labor inputs.