Venuti, Iolanda (2024) Pathogens Persistence in the Food Chain: Innovative Approaches in Detection, Food Safety, and Control. [Tesi di dottorato]

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Abstract

Foodborne pathogens have become a growing public health threat, driven by globalization, climate change, and changing consumer behaviors, particularly the rising demand for ready-to-eat products and the use of innovative distribution channels. These shifts not only facilitate pathogen persistence and dissemination but also introduce complex challenges for ensuring food safety within increasingly diversified production and distribution systems. Despite stringent regulatory standards and advanced hygiene protocols, bacterial and viral pathogens possess adaptive mechanisms—such as biofilm formation and resilience to adverse environmental conditions—that enhance their survival, allowing them to evade standard control and detection measures. These adaptations are particularly problematic in high-risk areas, where persistent contamination or challenges in pathogen eradication can significantly compromise food safety. Addressing these issues necessitates the development of advanced monitoring and control strategies to mitigate pathogen survival mechanisms. This thesis tackles these challenges through a multifaceted approach, combining conventional methodologies with biotechnological innovations to improve pathogen detection, assess persistence, and inhibit biofilm formation. The chapters collectively aim to bridge critical knowledge gaps and offer practical solutions that strengthen existing regulatory frameworks, ultimately contributing to enhanced food safety and consumer protection. Chapter 1 investigates the microbiological safety of vending machines (VMs), widely used yet under-researched sources of food and beverages. This study performed a comprehensive microbiological assessment of VMs in the Campania region, Italy, integrating traditional microbiological methods with molecular analyses. Results revealed significant microbial contamination, with Total Viable Counts (TVC) frequently surpassing acceptable limits, particularly on the internal surfaces of beverage dispensing nozzles and within hot-drink products. Coffee and ginseng powders showed comparatively lower TVC values, while no contamination was observed in chocolate samples. Bacillus cereus was prevalent in over 80% of certain components (e.g., water intake pipe, mixer bowl), and Staphylococcus aureus was frequently found in milk powder and cappuccino samples. The study underscores the need for enhanced hygiene protocols and stricter regulatory oversight to mitigate contamination risks in vending machines, contributing to safer food service practices and enhanced consumer protection. Chapter 2 shifts focus to biofilm formation by Salmonella enterica serovar Typhimurium, focusing on the inhibitory effects of two long-chain fatty acids (LCFAs), arachidonic acid (AA) and linoleic acid (LA). The study demonstrates that both fatty acids reduce biofilm formation in a concentration-dependent manner, with AA showing particularly strong efficacy. Confocal microscopy confirmed significant reductions in biofilm thickness and density. Reporter assays revealed that both AA and LA downregulated curli-related genes, undermining biofilm structure. Gene expression analyses demonstrated that AA and LA downregulated curli-related genes, weakening biofilm structure, while AA specifically increased fliC expression, promoting a shift toward bacterial motility. The involvement of the fadR gene in AA-induced motility further suggests a regulatory link between fatty acid metabolism and bacterial behavior. These findings underscore the potential of LCFAs, particularly arachidonic acid, as targeted and natural biofilm control agents to reduce Salmonella persistence, enhancing sanitation and mitigating health risks in the food industry. Chapter 3 provides an in-depth analysis of viral contamination and antibiotic resistance genes (ARGs) in retail-sourced mussels in Campania, Italy. High prevalences of Human Noroviruses GI (77%) and GII (40%), Rotaviruses (63%), and Astroviruses (23.33%) were observed. Fecal contamination indicators, including somatic coliphages and crAssphage, correlated strongly with Norovirus, Rotavirus, and Astrovirus presence. Capsid-integrity RT-qPCR assay confirmed the presence of potentially infectious enteric viruses, indicating consumer health risks. ARGs for beta-lactams, quinolones, and chloramphenicol were also detected, with phages potentially acting as ARG vectors within shellfish microbiota. These findings emphasize the importance of implementing advanced surveillance measures to monitor and mitigate viral contamination and antibiotic resistance in mussels to safeguard consumer health and to enhance current control and management strategies in high-risk sectors. By addressing critical gaps in monitoring, sanitation, and regulatory practices, this work advances our understanding of microbial contamination in complex food systems. It provides a foundation for enhanced regulatory standards and control strategies that align with the evolving landscape of food production and distribution. Through the integration of biotechnological innovations for precise pathogen detection and the use of natural compounds to disrupt biofilms, this thesis equips food safety authorities with essential tools for effective risk management for strengthening public health protections in an increasingly complex food environment.

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