Sutton, Lucy (2026) Response of Listeria monocytogenes biofilms to oxidative sanitisers in the fresh food supply chain of leafy greens. University of Southampton, Doctoral Thesis, 233pp.
Abstract
Salads and herbs are an important part of a healthy diet, however during their processing and packaging they may be exposed to contamination from foodborne pathogens. L. monocytogenes, a Gram-positive, facultative anaerobe with the highest mortality rate among major foodborne pathogens, is of particular concern due to its ubiquity, ability to grow at refrigeration temperatures, and tendency to form biofilms in food processing facilities. Biofilms are complex 3D communities, encapsulated in extracellular polymeric substances (EPS), which protects cells from environmental stress and antimicrobials. Subsequently, once established, biofilms are very hard to remove and can become a recurrent source of contamination events in the fresh food supply chain. To mitigate this risk, chlorine and peracetic acid (PAA) are widely used oxidative sanitisers, however their efficacy in treating mature Listeria biofilms remains poorly investigated.
This study investigated the response of L. monocytogenes Scott A biofilms to chlorine and PAA treatment, considering growth temperature (4°C and 20°C), and biofilm maturity. Sanitiser efficacy was evaluated using culturable counts, episcopic differential interference contrast microscopy with epifluorescence (EDIC/EF), Raman spectroscopy, and transcriptomics to provide a multi-level analysis of biofilm viability, structure, chemistry, and gene expression.
L. monocytogenes Scott A formed mature biofilms at both 4°C and 20°C, with architecture and EPS strongly influenced by temperature. Biofilms grown at cold temperature were taller, dense in EPS and chemically enriched in lipids, eDNA and polysaccharides, whereas room temperature biofilms consisted of proteins and eDNA. Biofilms at both temperatures displayed tolerance to sanitiser treatment, though those grown at 4°C showed slightly greater susceptibility. PAA was consistently more effective than chlorine at disrupting biofilm, showing deeper EPS penetration and induction of dispersal via motility gene upregulation. Transcriptomic analyses revealed stress adaptation mechanisms including shifts in metabolism (ethanolamine utilisation), acid resistance, pH homeostasis and efflux systems, and suppression of growth functions. PAA triggered a broader but less significant changes in gene expression, whereas chlorine treatment elicited fewer but more statistically robust alterations.
Overall, this work demonstrates that L. monocytogenes Scott A biofilms exhibit temperature-dependent and biochemical adaptations that enhance sanitiser tolerance and survival in the fresh food supply chain. PAA was more effective than chlorine at disrupting the biofilm, but neither sanitiser significantly reduced culturable or viable cell numbers, and both induced a stress response and increased tolerance. These findings provide new insights into Listeria tolerance, supporting the advancement of current sanitisation procedures to reduce the risk of foodborne listeriosis outbreaks.
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