Abstract
Biofilms enable the persistence of pathogens in food processing environments. Sanitizing agents are needed that are effective against pathogens entrapped in biofilms that are more difficult to inactivate than planktonic cells that are displaced and found on equipment surfaces. We examined conditions to develop, analyze, and enumerate the enhanced biofilms of three different foodborne pathogens assisted by fluorescence adherence assay and enzymatic detachment. We compared three different isomeric forms of fluorescent substrates that are readily taken up by bacterial cells based on carboxy-fluorescein diacetate (5-CFDA, 5,6-CFDA, 5,6-CFDA, SE). Biofilm-forming strains of Escherichia coli O157:H7 F4546 and Salmonella Montevideo FSIS 051 were identified using a microplate fluorescence assay defined previously for L. monocytogenes. Adherence levels were determined by differences in relative fluorescence units (RFU) as well as recovered bacterial cells. Multiple hydrolytic enzymes were examined for each representative pathogen for the most suitable enzyme for detachment and enumeration to confirm adherence data obtained by fluorescence assay. Cultures were grown overnight in microplates, incubated, washed and replenished with fresh sterile growth medium; this cycle was repeated for seven consecutive days to enrich for robust biofilms. Treatments were performed in triplicate and compared by one-way analysis of variance (ANOVA) to determine significant differences (p < 0.05).
Highlights
The development of a biofilm usually involves a cellular adherence event that develops into irreversible attachment followed by development of a 3-dimensional biofilm that progresses into a mature and intricate biofilm [1,2]
Because of the repeated occurrence of in-plant contamination problems with L. monocytogenes, E. coli O157:H7, and Salmonella serovars, we examined improvements to a method to create robust biofilms with hardy biofilm formers to facilitate biofilm research
A variety of E. coli O157:H7, L. monocytogenes, and Salmonella serovars and strains from our culture collection were screened by a microplate fluorescence adherence assay to confirm or identify high level adherence
Summary
The development of a biofilm usually involves a cellular adherence event that develops into irreversible attachment followed by development of a 3-dimensional biofilm that progresses into a mature and intricate biofilm [1,2]. During this progression, individual cells or portions of biofilm may slough off that are distributed elsewhere [3]. Micro-colonies are rapidly formed and the secretion of extracellular polysaccharides (EPS) start to develop, becoming the ‘glue’ of the biofilm architecture. Higher densities of EPS, channels, and pores results in the positioning of bacteria away from the substrate surface and facilitates
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