Molecular Basis for the Development of Sanitizer Tolerance in Listeria Monocytogenes

NON-TECHNICAL SUMMARY: Numerous product recalls and disease outbreaks have been caused by the contamination of ready-to-eat foods with L. monocytogenes. In many of these cases the same genotype of L. monocytogenes was isolated from the processing plant environment that was found in the food, leading to the conclusion that the processing environment was the source of the contamination. The most common chemical sanitizing agents used in food processing environments are oxidizing compounds or quaternary ammonium compounds. This project would be the first exploration of the molecular basis of stress adaptation by biofilm cells of L. monocytogenes. This project will identify genes regulated in response to biofilm growth and stress adaptation. Based on this information we will propose a molecular basis for survival of L. monocytogenes growing on food processing plant surfaces treated with chemical sanitizers. Understanding the means by which biofilm cells of L. monocytogenes adapt to sublethal stresses associated with incomplete sanitization will provide a knowledge base that can lead to improved pathogen control practices. <P>
APPROACH: This research will use L. monocytogenes strains ATCC 15313 and 19115. Each strain will be grown as a biofilm on glass wool. Cells will be harvested from the glass wool. Cells will be lysed by adding RLT buffer and ziconia/silica beads to the frozen bead beater tubes containing cell pellets. RNA will be quantified by measuring OD at 260 nm. RNA samples will be shipped to the Minion laboratory on dry ice. Fluorescently labeled cDNA targets will be generated. A set of ORF-specific primers will be assembled from the primer set used to generate the PCR products on the array and used to prime the cDNA reaction for mRNA species in the total RNA preparations. Target cDNAs will be labeled. Following purification of the labeled cDNA, samples will be dried. Targets will be denatured. Targets from a control and treated culture will be combined and hybridized. Each array will be scanned. The natural logarithm of the background-corrected signals from a single scan will be adjusted. The median of these adjusted-log-background-corrected signals across multiple scans will then be computed. A separate mixed linear model analysis will be conducted for each probe sequence using the normalized data. Each mixed model will include fixed effects for treatment and dye as well as to account for variation across slides. The p-values from these t-tests will be converted to q-values. These q-values will be used to obtain approximate control of the False Discovery Rate (FDR) at a specified value. Along with q-values, estimates of fold-change will be computed for each probe by taking the inverse natural log of the mean treatment difference estimated as part of our mixed linear model analyses. This is essential for identifying the genes to be studied in Part 2 by mutational analysis. Part 2. The mutants will be prepared. PCR primers will be designed based on genomic sequence data to yield a pair of products representing the upstream and downstream fragments of the gene. The PCR primers will be designed with restriction sites so that the fragments will be ligated with a DNA fragment carrying aphA-3 from Enterococcus faecalis inserted between them. The resultant fragment will be cloned into the temperature sensitive plasmid pAUL-A, which will be introduced into the wild-type strain of L. monocytogenes by electroporation and grown on plates with kanamicin at a non-permissive temperature. Proper insertion of the mutation will be evaluated by PCR. Complementation controls will be constructed with the site-specific integration phage vector pPL2. The amount of EPS and biofilm growth produced by mutants and wild type strains will be determined. After removal of unattached cells the biofilms will be stained with conconavalin A conjugated with Texas Red (EPS labelling) and Hoescht 33258 (cell stain). Stained biofilms will be viewed at 40X using an epifluorescent microscope and images analyzed for labeled area using ImagePro software. Gene expression of the mutants will be determined using microarray analysis of the biofilm and planktonic cells. This analysis was previously described.

<P>PROGRESS: 2005/09 TO 2008/09<BR>
OUTPUTS: Our goal, to perform microarray studies with Listeria monocytogenes treated with sanitizers, was completed. Our results were transmitted to the PI on the project. <BR> PARTICIPANTS: Michael Carruthers, PhD student, received training in microarray techniques and trouble shooting with this project. Melissa Madsen, Assistant Scientist, also received training on this project. <BR> <BR>
IMPACT: 2005/09 TO 2008/09<BR>
The outcome of the project was not positive. Sanitizer treatment of Listeria monocytogenes resulted in severe degradation of the RNA and prevented suitable results on the microarrrays. One positive result, however, was the training of students and staff on microarray troubleshooting. By carefully controlling hybridization and cDNA generation, we were able to get excellent images using RNA isolated from fresh bacteria, in contrast to the results from sanitizer treated cells.