Foodborne pathogen persistence in the food processing environment
Brandt, Alex L.
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Emerging evidence suggests that persistence of foodborne pathogens within food processing facilities represents a key factor in their introduction into the food chain and that pathogen persistence has contributed to several multi-state foodborne illness outbreaks and expansive recalls. While Listeria monocytogenes ecology has been extensively documented in various food processing facilities, until recently, no standardized definitions of persistence had been developed and applied to these ecological data, and few studies had assessed the impact of control measures on Listeria persistence in the meat processing environment. Also, very little information has been generated on the ecology of Salmonella enterica and Escherichia coli O157:H7 in food processing facilities, and thus much remains to be understood about the extent to which they persist in these premises. Additionally, little is known about population compositions for other bacteria that are present in sites of recurrent Listeria contamination, some of which may directly influence Listeria’s growth at the site. The first phase of this study investigated foodborne pathogen persistence in meat processing facilities using an integrated strategy of longitudinal sampling, molecular subtyping, and two statistical tests for persistence. Four meat processing facilities were enrolled, and environmental sites and food products were sampled and tested for Listeria, S. enterica, and E. coli O157:H7 for a six-month period. All isolates were subtyped using molecular and serological methods, and subtyping data were statistically analyzed. Across all facilities, six-month prevalences ranged from 1.21% to 16.97% for L. monocytogenes, 6.07% to 28.18% for non-pathogenic Listeria, 0.00% to 6.97% for S. enterica, and 0.00% to 1.21% for E. coli O157:H7. Drain and floor sites had the highest prevalence for Listeria and S. enterica. A binomial test for subtype frequency identified persistent strains of L. monocytogenes and non-pathogenic Listeria species in each facility, and persistent strains of S. enterica in three facilities. Another binomial test, based on previous subtype isolation as a risk factor for current subtype isolation, identified a smaller number of persistent Listeria strains, and identified no persistent S. enterica strains. Nine and seven sampling sites that were persistently colonized with specific strains of L. monocytogenes and non-pathogenic Listeria, respectively, were identified via the binomial frequency test. The second phase of the study assessed impacts of control measure implementation on Listeria persistence. In-plant trainings were delivered to personnel in all four facilities, and significant increases in knowledge were observed in three facilities and in the entire group of participants. Three facilities were enrolled in a follow-up intervention and sampling program for Listeria. Each facility implemented physical changes and interventions suggested during in-plant trainings, and also implemented changes autonomously. Significant short and long-term behavior changes regarding use of facility-specific footwear and footwear sanitation were observed for one facility, while a significant change in hygienic item use (e.g. hairnets and gloves) was seen for another. Persistent Listeria strains were still isolated in each facility, but all three facilities had decreased mid-shift prevalences of Listeria between the initial and follow-up periods among a subset of sites that were i) positive for Listeria at least once during the initial period and ii) were followed for the entirety of the study; the decrease was significant for two facilities. Processing room sites of two facilities saw significantly decreased Listeria prevalences after boot baths were installed. Directional sampling among a select group of sites in the three facilities demonstrated that contamination patterns at adjacent areas may not match those observed at the original site. Also, sampling in a newly-constructed area of one facility demonstrated that Listeria strains that persisted in older areas of the facility began to colonize the newly-constructed areas. The last portion of the study focused on the population composition of culturable bacteria at sites of recurrent Listeria contamination. Nine sites from three facilities were sampled, and up to ten colonies with different morphologies on Brain Heart Infusion Agar were selected from each sample. DNA sequencing was peformed on a portion of the 16S rRNA gene of each isolate. Bacterial genera were assigned to each isolate by comparison of sequence data with that of the Ribosomal Database Project. Pseudomonas predominated the isolates collected from each facility, while Staphylococcus, Psychrobacter, Aeromonas and Enterobacteriaceae were also among the most predominant. Stenotrophomonas and Kocuria, both of which have been shown to increase L. monocytogenes counts and cell densities in co-culture, were isolated at certain sites. Lactococcus, Shewanella, Carnobacterium, and Serratia, which all have detrimental effects on L. monocytogenes, were also isolated. Overall the results of the study helped to fill several knowledge gaps regarding pathogen persistence in meat processing facilities especially with regard to the prevalence and extent of persistence of E. coli O157:H7 and S. enterica, the application of statistical measures to achieve standardized definitions of persistence, and the utility of trainings and control measure implementation to reduce prevalence of pathogens. This information has applicability for all meat processors and will likely be of use in their pathogen control programs, but it is especially useful for those that are small and very small, have limited knowledge of pathogen persistence, and have few resources to combat it. Furthermore, this work also highlights several topics that remain to be addressed with regard to pathogen persistence, including persistence of S. enterica and E. coli O157:H7 in other food processing environments (especially low moisture foods), the study of interactions between bacterial populations at sites of recurrent Listeria contamination, and characterization of persistent-state Listeria gene expression profiles in sites of persistence.