Factors that Influence Biofilm Formation, Desiccation Resistance, and Sanitizer Susceptibility in Listeria monocytogenes at a Simulated Food Processing Plant Temperature of 15°C

Abstract

Listeria monocytogenes is a pathogenic foodborne bacterium whose persistence in food processing environments is in part attributed to its biofilm formation. A library of 11,024 L. monocytogenes 568 (serotype 1/2a) Himar1 insertional mutants was created and screened for altered biofilm formation. Fourteen mutants expressed enhanced biofilm phenotypes, and harboured transposon insertions in genes encoding cell wall biosynthesis, motility, metabolism, stress response, and cell surface associated proteins. Deficient mutants contained interruptions in genes related to peptidoglycan, teichoic acid, or lipoproteins. Enhanced mutants produced significantly (p<0.05) higher cell densities in biofilm formed on stainless steel (SS) coupons at 15°C (48 h) than deficient mutants. Two enhanced biofilm formers were more resistant to enzymatic removal with DNase, proteinase K or pectinase than the parent strain. Scanning electron microscopy of individual biofilms showed formation of biofilm with dense zones by motility mutants, while deficient mutants exhibited sparse growth. In conclusion, biofilm formation of L. monocytogenes at 15°C involved 9 genes not previously linked to biofilm, as well as 10 genes previously associated with its formation at higher temperatures, indicating some temperature driven differences.

In the second research paper, the ability to form biofilm, resist desiccation, and sanitizer treatments was investigated among a panel of 14 L. monocytogenes strains, and in relation to the presence or absence of genetic markers including the stress survival islet (SSI-1), the Listeria genomic island (LGI1), and clonal complex 8, virulence type 59 (CC8 /VT59). SSI-1 alone did not correlate with desiccation survival. Benzalkonium chloride and desiccation resistance was greater in isolates that also carried the LGI1 and CC8/VT59 markers. In conclusion, LGI1 containing CC8/ VT59 isolates may pose a challenge for sanitation efforts and food safety.

The research presented in this thesis will help our understanding of biofilm formation in L. monocytogenes, its response to sanitizer application, and desiccation resistance. This information may lead to better strategies to remove or eradicate L. monocytogenes in food processing environments, and ultimately reduce the risk of foodborne illness.