Location: Meat Safety and Quality
Project Number: 3040-42000-021-008-T
Project Type: Trust Fund Cooperative Agreement
Start Date: Mar 1, 2022
End Date: Feb 28, 2023
Objective:
Available evidence suggests that biofilms may play an important role in product contamination such as HEPs at processing plants. Current sanitization studies generally focus on single species pathogen biofilms, but not take into consideration the fact that pathogens are harbored in natural mixed biofilms, which are commonly seen at commercial plants. Further, sanitizer effectiveness can be significantly different due to the interactions between the environmental microorganisms and the pathogens. Our previous study found that certain drain biofilm communities preferentially protected E. coli O157:H7 or Salmonella. We further observed that certain sanitizers, even though not highly effective in deactivating biofilms, reduced overall Salmonella prevalence at certain plants likely due to the environmental microbial community change induced by such treatment that was unfavorable for Salmonella recruitment and survival (ex: bromine in beef spray chill). Therefore the hypothesis of this project is that the different natural biofilm communities at processing plants can either enhance or inhibit Salmonella survival against sanitization and subsequently affect Salmonella prevalence rate.
Specific objectives:
1. Evaluate efficacy of commercial sanitizers against Salmonella harbored within environmental mixed biofilms.
2. Measure biofilm forming ability and community structure of environmental biofilms before and after sanitization.
3. Compare environmental microbial communities and Salmonella survival in mixed biofilms before and after sanitization to determine the impact of different sanitizers on controlling Salmonella.
Approach:
Sample collection: We will collect floor drain samples from four different processing plants (two each: beef and pork) before and after sanitization as representation of the environmental microorganisms. The drains selected will span from hot box to coolers and fabrication.
General microbiology & biofilm assays: Each sample will be examined for general bacterial counts (APC, mesophiles, psychrophiles, Enterobacteriaceae, and coliforms) using 3M PetriFilm. Salmonella presence will be determined using established protocols. The biofilm forming ability of the samples will be tested in 96-well polystyrene plates and on stainless steel (SS) chips at 7oC (fabrication condition) following established protocols.
Sanitizer evaluation & pathogen survival: Drain samples without Salmonella presence will be enriched and used to form biofilms on SS chips at 7oC. Three types of biofilms will be developed for the study: (1). Biofilms by drain samples only; (2). Biofilms by drain samples with the addition of Salmonella cocktail (mixture of multiple strains of different serovars); (3). Biofilms by Salmonella cocktail only (single-species biofilm).
Biofilms will be treated with sterile water (positive control) or each of the common sanitizers (Decon, Sterilex, DeepClean, bromine, EZ360, Realzyme) following the manufacturer’s instructions. After treatment, samples will be neutralized with Dey/Engley broth and biofilms will be harvested following established protocols. A 1.0 mL aliquot will be removed from each harvested sample, diluted and plated onto TSA plates (to measure total bacteria in mixed biofilms by drain samples) and XLD plates (to measure Salmonella cells in mixed biofilms). The remaining samples will proceed for DNA extraction (see below).
Metagenomic analysis: DNA will be extracted from the above harvested biofilm samples using Qiagen Power-Lyser soil extraction kits. The community structure of each sample will be determined through metagenomic sequencing and analysis of the DNA extracts to identify the pre- and post-sanitization bacterial species and to identify population changes induced by sanitization and Salmonella recruitment.
Data analysis: Biofilm density and bacterial log reduction will be measured and expressed as Log10 CFU/chip. Analysis of variance and comparisons of the logarithmic biofilm cell counts with standard deviations and 95% confidence intervals will be performed with GraphPad Prism using a one-way analysis of variance (ANOVA) with a post Tukey’s or Dunnett’s multiple comparisons test. P<0.05 indicates statistical significance. Analysis of 16S rRNA gene sequencing results will use Quantitative Insights Into Microbial Ecology (QIIME2.0). Taxonomy will be assigned using the SILVA database with the pre-trained classifier based on 99% sequence identity.
