Integrative Assessment of Reduced Listeria monocytogenes Susceptibility to Benzalkonium Chloride in Produce Processing Environments.

For decades, quaternary ammonium compounds (QAC)-based sanitizers have been broadly used in food processing environments to control foodborne pathogens such as Listeria monocytogenes. Still, there is a lack of consensus on the likelihood and implication of reduced Listeria susceptibility to benzalkonium chloride (BC) that may emerge due to sublethal exposure to the sanitizers in food processing environments. With a focus on fresh produce processing, we attempted to fill multiple data and evidence gaps surrounding the debate. We determined a strong correlation between tolerance phenotypes and known genetic determinants of BC tolerance with an extensive set of fresh produce isolates. We assessed BC selection on L. monocytogenes through a large-scale and source-structured genomic survey of 25,083 publicly available L. monocytogenes genomes from diverse sources in the United States. With the consideration of processing environment constraints, we monitored the temporal onset and duration of adaptive BC tolerance in both tolerant and sensitive isolates. Finally, we examined residual BC concentrations throughout a fresh produce processing facility at different time points during daily operation. While genomic evidence supports elevated BC selection and the recommendation for sanitizer rotation in the general context of food processing environments, it also suggests a marked variation in the occurrence and potential impact of the selection among different commodities and sectors. For the processing of fresh fruits and vegetables, we conclude that properly sanitized and cleaned facilities are less affected by BC selection and unlikely to provide conditions that are conducive for the emergence of adaptive BC tolerance in L. monocytogenes. IMPORTANCE Our study demonstrates an integrative approach to improve food safety assessment and control strategies in food processing environments through the collective leveraging of genomic surveys, laboratory assays, and processing facility sampling. In the example of assessing reduced Listeria susceptibility to a widely used sanitizer, this approach yielded multifaceted evidence that incorporates population genetic signals, experimental findings, and real-world constraints to help address a lasting debate of policy and practical importance.

Enterobacteriaceae, coliform, yeast, and mold contamination patterns in peanuts compared to production, storage, use practices, and knowledge of food safety among growers in Senegal.

Peanuts and peanut products are significant revenue sources for smallholder farmers in the Senegalese peanut basin. However, microbial contamination during production and storage can greatly affect market access for producers. Peanut products have emerged as possible sources of foodborne illness, encouraging discussions on international standards for peanuts. In this study, we interviewed 198 households throughout the Senegalese peanut basin to assess current production practices, storage methods, and producers' prior knowledge of microbial contamination using a 162-question survey. A member of each household orally completed the survey with a trained enumerator and the results were compared to microbiological results obtained from peanut samples collected at the time of the interview using linear regression and an analysis of variance model. Samples were collected from stored peanuts at each household; peanuts were shelled and total Enterobacteriaceae, coliform, and yeast and mold populations were enumerated. Of the 198 samples analyzed, 13.0% and 13.6% were greater than the upper detection limits for Enterobacteriaceae and coliforms, respectively. A total of 21.2% of samples were above the detection limit for yeast and mold populations. Only 22.7% and 18.7% of producers were aware of pathogenic bacteria or aflatoxins, respectively; there were no significant differences in observed microbial populations between household who took preventative measures against microbial contamination and those who did not. Additionally, four households reported washing their kitchen utensils before using them to eat and 60.1% reported always washing their hands before eating. Enumerators were asked to report peanut storage container type and if the containers were stored off the ground at the time of collection. While the interaction between storage container type and if the container was stored off the ground was significant for Enterobacteriaceae and coliforms, it was not significant for yeast and mold. Additionally, when storage container type and if peanuts were stored off the ground were included in the regression model, these methods were predictive of contamination levels for Enterobacteriaceae and coliforms. To our knowledge, this is the first study to analyze the relationship among Enterobacteriaceae, coliforms, and yeast and mold contamination and producer knowledge of Senegalese peanuts. These results provide preliminary data to inform future studies to determine pathogen prevalence and impactful preventative measures to minimize microbial contamination of peanuts produced in Senegal.

Lytic Capacity Survey of Commercial Listeria Phage Against Listeria spp. with Varied Genotypic and Phenotypic Characteristics.

Listeria monocytogenes is regularly isolated from food processing environments and is endemic in some facilities. Bacteriophage have potential as biocontrol strategies for L. monocytogenes. In this study, the lytic capacity of a commercial Listeria phage cocktail was evaluated against a library of 475 Listeria spp. isolates (426 L. monocytogenes and 49 other Listeria spp.) with varied genotypic and phenotypic characteristics. The lytic capacity of the Listeria phages was measured by spot assays where lysis was scored on a scale of 0-3 (0 = no lysis; 1 = slight lysis; 2 = moderate lysis; 3 = confluent lysis). Only 5% of all tested Listeria spp. isolates, including L. monocytogenes, were either moderately or highly susceptible (score 2 or 3) to lysis by Listeria phage when scores were averaged across temperature and phage concentration; 155 of 5700 treatment (multiplicity of infection [MOI] and temperature) and characteristic (genotype, sanitizer tolerance, and attachment capacity) combinations resulted in confluent lysis (score = 3). Odds ratios for susceptibility to lysis were calculated using multinomial logistic regression. The odds of susceptibility to lysis by phage decreased (p < 0.05) if the L. monocytogenes isolate was previously found to persist or if the phage-bacteria culture was incubated at 30°C; neither isolate persistence or temperature was significant (p ≥ 0.05) when all factors were considered. In addition, lytic efficacy varied (p < 0.05) among pulse field gel electrophoresis (PFGE) pulsotypes and may be affected by host MOI (p < 0.05). There was no effect (p > 0.05) of attachment capacity or sanitizer tolerance on phage susceptibility. This study underscores the complexity of using Listeria phage as a biocontrol for Listeria spp. in food processing facilities and highlights that phage susceptibility is most greatly impacted by genotype. Further studies are needed to evaluate these findings within a processing environment.

Ground beef microbiome changes with antimicrobial decontamination interventions and product storage.

Ground beef makes up more than half of the beef consumed in the U.S. market. Although numerous studies have been conducted on microbial safety and shelf life of ground beef limited work has been done using a culture-independent approach. While past studies have allowed for the evaluation of a few organisms of interest, there is limited work on the microbial community associated with fresh ground beef. In order to have a more complete picture of the microbial ecology of the product, a culture-independent approach utilizing 16S rRNA gene amplicon sequencing was used. The objectives of this study were to characterize the fresh ground beef microbiome and the effect that antimicrobial interventions and antioxidants, applied to beef trim before grinding, and product storage have on community composition using 16S rRNA gene amplicon sequencing. Beef trimmings were treated with antimicrobials and an antioxidant. Samples were ground, loafed, and overwrapped before being packaged in modified-atmosphere packaging. Samples were in dark storage for 21 days followed by five days in retail display. Periodically during storage, samples were collected for microbiological analysis and DNA isolation. Due to low microbial biomass, only 52 of 210 samples were included in the final analysis. These samples represented two antimicrobial treatments (peroxyacetic acid, and a sulfuric acid and sodium sulfate blend) and a control, from day-15 of dark storage and day-5 of retail display. As sample age increased, so did the number of raw reads (P < 0.001) and aerobic plate counts (P < 0.001), which were correlated (r = 0.94, P = 0.017). Across all samples, lactic acid bacteria were most abundant followed by Enterobacteriaceae; several rare taxa were also identified (namely Geobacillus, Thermus, and Sporosarcina). Antimicrobial treatment altered the bacterial alpha (P < 0.001) and beta (P = 0.001) diversity, while storage day altered alpha (P = 0.001) diversity. Enterobacteriaceae relative abundance differed (P < 0.05) among treatments and was highest in control samples. In addition to confirming previously described dominant microbial differences in culture-dependent results, these data identified genera not typically associated with ground beef and allowed for study of shifts in the entire microbiome and not just a subset of indicator organisms.

Genetics and microbiology of meat.

Detection, reduction, and public health monitoring of foodborne pathogens has advanced in precision and efficiency as technology has progressed. Here, we look back on the evolution of food safety management and public health, and attempt to provide a view of how the technology and tools have changed, and how emerging technologies and tools may impact how we manage food safety and public health in the future. With the revolution of gene editing techniques (e.g. CRISPR-Cas9, etc.), Next Generation Sequencing (NGS) and "omics"-based technologies, along with the bioinformatics tools that go with them, we now have a very new array of tools that can impact foodborne disease management. In addition to overall improvement in food safety, these tools have helped understand antibiotic resistance and virulence factors in meat to a higher degree than ever before. These technological advancements will allow food safety to move beyond strain-level characterization and control of pathogens to pinpointing genes of public health concern.