Genevieve Sullivan

Listeria monocytogenes is of particular concern to the produce industry due to its frequent presence in natural environments as well as its ability to survive in packinghouses and fresh-cut processing facilities over time. The use of whole-genome sequencing, which provides high discriminatory power for the characterization of Listeria isolates, along with detailed source data (isolation date and sample location) shows that the presence of Listeria in produce operations appears to be due to random and continued reintroduction as well as to the persistence of highly related strains in both packinghouses and fresh-cut facilities.

The complex environment of a produce packinghouse can facilitate the spread of pathogens such asListeria monocytogenesin unexpected ways. This can lead to finished product contamination and potential foodborne disease cases. There is a need for simulation-based decision support tools that can test different corrective actions and are able to account for a facility’s interior cross-contamination dynamics. Thus, we developed agent-based models ofListeriacontamination dynamics for two produce packinghouse facilities; agents in the models represented equipment surfaces and employees, and models were parameterized using observations, values from published literature and expert opinion. Once validated with historical data fromListeriaenvironmental sampling, each model’s baseline conditions were investigated and used to determine the effectiveness of corrective actions in reducing prevalence of agents contaminated withListeriaand concentration ofListeriaon contaminated agents. Evaluated corrective actions included reducing incomingListeria, modifying cleaning and sanitation strategies, and reducing transmission pathways, and combinations thereof. Analysis ofListeriacontamination predictions revealed differences between the facilities despite their functional similarities, highlighting that one-size-fits-all approaches may not always be the most effective means for selection of corrective actions in fresh produce packinghouses. Corrective actions targetingListeriaintroduced in the facility on raw materials, implementing risk-based cleaning and sanitation, and modifying equipment connectivity were shown to be most effective in reducingListeriacontamination prevalence. Overall, our results suggest that a well-designed cleaning and sanitation schedule, coupled with good manufacturing practices can be effective in controlling contamination, even if incomingListeriaspp. on raw materials cannot be reduced. The presence of water within specific areas was also shown to influence corrective action performance. Our findings support that agent-based models can serve as effective decision support tools in identifyingListeria-specific vulnerabilities within individual packinghouses and hence may help reduce risks of food contamination and potential human exposure.

Listeria monocytogenes (LM) contamination of produce can often be traced back to the environment of packinghouses and fresh-cut facilities. As there is limited information on the detection, prevalence, and distribution of this pathogen in produce operations, environmental “routine sampling” plans for LM and other Listeria spp. were developed and implemented in three packinghouses and five fresh-cut facilities in the US. For “routine sampling”, a total of 2,014 sponge samples were collected over 6 - 8 separate samplings per operation, performed over one year; vector and pre-production samples (n=156) were also collected as needed to follow up on positive findings. In addition, a single “validation sampling” visit by an outside expert was used to evaluate the routine sampling. Among the 2,014 routine sponge samples collected, 35 and 30 were positive for LM and Listeria species other than LM (“LS”), respectively. LM prevalence varied from 0.8% to 5.8% for packinghouses and <0.4% to 1.6% for fresh-cut facilities. Among the 394 validation sponge samples, 23 and 13 were positive for LM and LS, respectively. Validation sampling found statistically significantly higher LM prevalence compared to routine sampling for 3/8 operations. For all samples collected, up to eight isolates per sample were characterized by sequencing of sigB, which allowed for classification into sigB allelic types. Among the 97 samples with more than one Listeria isolate characterized, 28 had more than one sigB allelic type present, including 18 sponges that were positive for LM and another Listeria spp and 13 sponges that were positive for more than one LM subtype. This indicates that collection of multiple isolates is necessary to capture Listeria diversity present in produce operations. Additionally, 17/77 sponges that were positive for LM were positive at only one enrichment time (i.e., 24 or 48h), indicating that LM testing after two different enrichment times provides enhanced sensitivity.

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 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. Worst-case conditions provide for stringent evaluation of detection methods. Extended enrichment can assess FN probabilities of culture methods. BAM methods can have up to a 15% FN probability.

Postprocessing cross-contamination of cheese can lead to both food safety issues and significant losses due to spoilage. Pulsed light (PL) treatment, consisting of short, high-energy, broad-spectrum light pulses, has been proven effective in reducing the microbial load on cheese surface. As PL treatment effectiveness is limited by light-cheese interactions, the possibility to improve its effectiveness by combining it with the antimicrobial nisin was explored. The effect of natamycin, which is added to cheeses as an antifungal agent, on PL effectiveness was also investigated. Pseudomonas fluorescens, Escherichia coli ATCC 25922, and Listeria innocua were used as challenge microorganisms. Bacterial cultures in stationary growth phase were diluted to initial inoculum levels of 5 or 7 log cfu per cheese slice. Slices of sharp white Cheddar cheese and white American singles were cut in rectangles of 2.5 × 5 cm. For cheese slices receiving antimicrobial treatment before PL, slices were dipped in natamycin or nisin, spot inoculated with 100 μL of bacterial suspension, and then treated with PL. Cheese slices receiving PL treatment before antimicrobials were spot inoculated, treated with PL, and then treated with antimicrobials. The PL fluence levels from 1.02 to 12.29 J/cm2 were used. Survivors were enumerated by standard plate counting or the most probable number technique, as appropriate. All treatments were performed in triplicate, and the data were analyzed using a general linear model. Treatment with nisin or natamycin before PL decreased the effectiveness of PL for all bacteria tested. For instance, PL reduced P. fluorescens on Cheddar cheese by 2.19 ± 0.27 log after 6.14 J/cm2, whereas combination treatments at the same PL fluence yielded barely 1 log reduction. Inactivation of L. innocua on Cheddar was only 0.78 ± 0.01 log when using PL after nisin, compared with a 1.30 ± 0.76 log reduction by nisin alone. This was attributed to the absorption of UV light by the 2 antimicrobials, which diminished the UV fluence received by the bacteria. Increased inactivation was obtained when antimicrobials were applied after PL. On process cheese, a maximum reduction of 3.73 ± 0.96 log of L. innocua was obtained at 9.22 J/cm2 for PL followed by nisin, compared with 3.01 ± 0.48 by PL alone. This study demonstrates that antimicrobials may increase the antimicrobial effectiveness of PL on cheese surface, but the order of treatments is critical.

The objective of this study was to examine the effect of pulsed light (PL) treatment on the color, oxidative stability, and onset of molding of Cheddar cheese. Slices of sharp white Cheddar cheese of 2.5 × 5 cm were treated on one side with PL doses from 1.02 to 12.29 J/cm2, sealed in polyethylene bags, and stored at 6°C for up to 1 mo. Peroxide value, color parameters, and the onset of molding were evaluated. No significant changes in color or peroxide value were observed for PL-treated samples compared with the untreated controls. Pulsed light was able to significantly delay surface molding during refrigerated storage, with a PL dose of 9.22 J/cm2 delaying the onset of molding by 7 d. The effect of PL on the taste, appearance, and acceptability of Cheddar cheese slices treated with a PL dose of 9.22 J/cm2 on each side was assessed. In triangle tests, 60 untrained panelists were unable to detect significant differences between the control and PL-treated samples, although PL had a significant effect on overall liking, flavor, and appearance. These findings suggest that although PL can be effective for surface decontamination of cheese, it may have some detrimental effects on sensory properties.

Cheese products are susceptible to postprocessing cross-contamination by bacterial surface contamination during slicing, handling, or packaging, which can lead to food safety issues and significant losses due to spoilage. This study examined the effectiveness of pulsed-light (PL) treatment on the inactivation of the spoilage microorganism Pseudomonas fluorescens, the nonenterohemorrhagic Escherichia coli ATCC 25922 (nonpathogenic surrogate of Escherichia coli O157:H7), and Listeria innocua (nonpathogenic surrogate of Listeria monocytogenes) on cheese surface. The effects of inoculum level and cheese surface topography and the presence of clear polyethylene packaging were evaluated in a full factorial experimental design. The challenge microorganisms were grown to early stationary phase and subsequently diluted to reach initial inoculum levels of either 5 or 7 log cfu/slice. White Cheddar and process cheeses were cut into 2.5×5 cm slices, which were spot-inoculated with 100 µL of bacterial suspension. Inoculated cheese samples were exposed to PL doses of 1.02 to 12.29 J/cm(2). Recovered survivors were enumerated by standard plate counting or the most probable number technique, as appropriate. The PL treatments were performed in triplicate and data were analyzed using a general linear model. Listeria innocua was the least sensitive to PL treatment, with a maximum inactivation level of 3.37±0.2 log, followed by P. fluorescens, with a maximum inactivation of 3.74±0.8 log. Escherichia coli was the most sensitive to PL, with a maximum reduction of 5.41±0.1 log. All PL inactivation curves were nonlinear, and inactivation reached a plateau after 3 pulses (3.07 J/cm(2)). The PL treatments through UV-transparent packaging and without packaging consistently resulted in similar inactivation levels. This study demonstrates that PL has strong potential for decontamination of the cheese surface.