Biofilm forming properties and subtype of wild type and rough strains of Listeria monocytogenes in relation to hypochlorous acid tolerance and associated proteins
Folsom, James P.
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This work determines if genetically related strains of L. monocytogenes (n=30) exhibit similar biofilm accumulation, and the effect of nutrient concentration on biofilm formation. Selected strains (n=13) were tested for hypochlorous acid tolerance to determine if planktonic hypochlorous acid tolerance or subtype is associated with biofilm hypochlorous acid tolerance. A variant strain (SBS) exhibiting increased hypochlorous acid tolerance and biofilm production was compared to the wild type using fluorescence 2-D difference gel electrophoresis (DIGE). Biofilms were grown on stainless steel in high or low nutrients. Biofilm accumulation was determined using image analysis after staining the cells. The strains were genetically subtyped by repetitive element sequence-based PCR. Planktonic cells were exposed to 20 through 80 ppm of hypochlorous acid, and biofilms were exposed to 60 ppm. We found that nutrient levels influenced biofilm accumulation, but that the nature of this influence differed with strain. Serotype 4b strains produced more biofilm accumulation in high nutrients than serotype 1/2a strains, while serotype 1/2a strains produced more accumulation in low nutrients. Low nutrient media inhibited biofilm accumulation of serotype 4b strains. There was no correlation between genetic subtype and the amount of biofilm accumulation. Some strains were more tolerant of hypochlorous acid than others were. Biofilm cell density and morphology was not associated with hypochlorous acid tolerance. The 50s ribosomal proteins L7/L12 and L10 were down regulated in biofilm and planktonic SBS respectively. Other proteins down regulated in planktonic SBS were the peroxide resistance protein (Dpr), an unknown protein (LMO1888) and a sugar binding protein (LMO0181). This sugar binding protein was up regulated in biofilm SBS. Regulation of the sugar binding protein indicates that SBS may reserve a carbon source for use during biofilm formation. The results presented show that serotype 1/2a and serotype 4b strains differ in the regulation of their biofilm phenotype, poor biofilm accumulation of serotype 4b strains grown in low nutrient media could be a factor in the predominance of serogroup 1/2 strains in food processing plants. Additionally, hypochlorous acid tolerance mechanisms of planktonic cells and biofilms differ, with planktonic hypochlorous acid tolerance being more affected by inducible traits, and biofilm hypochlorous acid tolerance being more affected by traits not determined in this study.