Microscopic analysis of temperature effects on surface colonization and biofilm morphology of Salmonella enterica

Authors: VICE, ZACHARIAH - Texas A&M University; ZHOU, YOU - University Of Nebraska; CHITLAPILLY DASS, SAPNA - Texas A&M University; Wang, Rong

Submitted to: Foods
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 1/8/2025
Publication Date: 1/15/2025
Citation: Vice, Z., Zhou, Y., Chitlapilly Dass, S., Wang, R. 2025. Microscopic analysis of temperature effects on surface colonization and biofilm morphology of Salmonella enterica. Foods. 14(2). Article 268. https://doi.org/10.3390/foods14020268.
DOI: https://doi.org/10.3390/foods14020268

Interpretive Summary: Salmonella is a type of foodborne pathogen that can cause meat contamination. Despite thorough cleaning at meat processing plants, these pathogens can sometimes survive by forming protective layers called biofilms on contact surfaces that can help the bacteria resist cleaning and sanitization. Since biofilm formation can be influenced by environmental temperature, we used special microscopes to investigate how different temperatures would affect the attachment and structure of these biofilms on stainless steel surface. We tested this using various Salmonella strains found in contaminated meat at temperatures of 7, 15, and 37°C. Our microscope analysis showed that as the temperature increased, the biofilms formed by the Salmonella strains became more structured and contained more protective cell substances. However, even at a low temperature of 7°C, we still detected significant bacterial attachment. Our study suggests that mature biofilms formed in warmer conditions can protect bacteria from routine cleaning. Additionally, the low temperature typically used in meat processing plants aren’t sufficient to prevent Salmonella from forming biofilms, which can still contaminate meat product. Therefore, it’s important to consider the effect of temperature on bacterial attachment and biofilm development when setting hygiene standards and cleaning protocols in these facilities.

Technical Abstract: Salmonella enterica represents a diverse group of pathogens commonly associated with food contamination including red meat. Even though pre- and post-harvest cleaning and sanitization procedures are widely implemented at meat processing plants to mitigate the hazard, S. enterica cells may escape the process by colonizing, on contact, surfaces in the form of a biofilm that functions as an aggregated microbial community to facilitate mutual protection, antimicrobial resistance, proliferation and dissemination. Biofilm development is a complex process that can be affected by a variety of factors including environmental temperature. We developed methods using scanning electron microscopy and confocal microscopy with a novel image analysis software tool to investigate the temperature in fluence on S. enterica cell colonization and biofilm formation by directly visualizing and comparing the biofilm matrix’s morphological differences under various temperatures. Cocktails of S. enterica strains belonging to serovars, commonly isolated from meat samples, were applied to develop biofilms on a stainless steel surface at 7, 15, or 37 'C. Results of the microscopy analysis showed that as temperature increased, better-defined biofilm structures with extracellular polymeric structures (EPS) could be identified. However, S. enterica colonization and aggregated bacterial biomass were clearly observed at the low temperature (7 'C) as well. These results demonstrate that the environmental temperature significantly contributes to S. enterica biofilm formation as the higher temperatures encour age bacterial active proliferation and biofilm maturation leading to the development of well-pronounced structures, while the lower temperature may promote cell attachment but, meanwhile, limit the EPS biosynthesis and biofilm maturation. Our study indicates that the mature S. enterica biofilms formed under favorable conditions may protect the pathogens with the well-developed 3-demensional (3D) structure against routine treatment. Furthermore, the low temperatures commonly maintained at meat plants are not able to effectively prevent S. enterica colonization and biofilm formation since at such temperatures there could still be colonized biomass that can contaminate the products. Therefore, the temperature effect on pathogen colonization and biofilm development should be taken into consideration while evaluating hygiene standards and sanitization procedures at the processing facilities.