- Listeria monocytogenes (Lm) is a pathogenic bacterium associated with the foodborne disease listeriosis in humans. Listeriosis affects thousands of people in the U.S. yearly, with neonates, elderly, pregnant women, and immunocompromised persons disproportionately affected. Lm has a high mortality rate in these populations (20 - 30%) with symptoms including septicemia, meningitis, encephalitis, and, in pregnant women, stillbirth and miscarriage. Due to the severity of the disease it causes, the U.S. has a zero-tolerance policy for Lm in ready-to-eat (RTE) food products. Lm is ubiquitous and often associated with food processing facilities, and the food processing environment itself is the main source of contamination in RTE products. Cleaning and sanitation are vital in reducing the presence of Lm in these areas. There are a variety of sanitizers available for use in the food industry. Among these are a widely used class of sanitizers called quaternary ammonium compounds (QUATs). Additionally, the U.S. Environmental Protection Agency (EPA) recently approved seven active ingredients for use in eco-friendly alternatives to traditional sanitizers. Despite these and other control methods, recalls and outbreaks due to Lm continue to occur. The objectives of this thesis were: (i) to determine the minimum bactericidal concentrations (MBCs) of two traditional (TR) sanitizers, including benzalkonium chloride (BAC) and a commercial QUAT (CQAC), and two eco-friendly (EF) sanitizers, including an alcohol-based (ALB) and a citric acid-based (CAB) sanitizer, against Listeria spp. (n=22) at temperatures ranging from 4 – 30oC; (ii) to evaluate the behavior of Lm (n=8) in sub-lethal concentrations of these sanitizers, mimicking unintentional dilution that may occur in processing facilities; and (iii) to assess the efficacy of sanitizers against Lm (n=5) attached to stainless steel (SS) and plastic at 4oC and 30oC for 24 h.
Minimum bactericidal concentrations (MBCs), based on the percentage of the manufacturer recommended concentration (% MRC), were significantly lower for TR sanitizers compared to those of EF sanitizers (P < 0.001; ANOVA). Temperature had a significant impact on MBCs of all sanitizers, with MBCs decreasing as temperature increased (P < 0.05; ANOVA). Similar growth behavior of isolates exposed to sub-lethal EF sanitizers was observed. In contrast, growth in sub-lethal concentrations of TR sanitizers revealed two isolates that were severely inhibited, confirming that differences in the ability of isolates to tolerate QUATs exist in the tested population.
Attachment to surfaces affected tolerance to sanitizers, with attached bacteria more tolerant than those in the planktonic state. All Lm isolates tested had survival rates above 50% when attached to SS and plastic at 30oC and treated with BAC and CAB at their MRC/ET. Incidence of inactivation for all sanitizers tested increased when both Lm attachment and sanitizer treatment occurred at 4oC. Differences in survival on two surface materials was also observed, with higher survival rates occurring with attachment on SS compared to plastic coupons. Scanning electron micrographs revealed greater population of bacteria on coupons when attached at 30oC compared to 4oC. Additionally, micrographs of plastic coupons showed they had a smoother surface compared to SS coupons. Notably, addition of a cleaning step, by scrubbing inoculated coupons (30oC, 24h) with cotton tips soaked in detergent, prior to treatment with sanitizers, decreased the incidence of survival on SS and plastic coupons treated with ALB and CAB; however, it did not significantly reduce survival of Lm on SS or plastic treated with QUATs.
Overall, the response of Lm isolates to sub-lethal concentrations varied the most with QUAT sanitizers, indicating that genetic variation between Lm strains may have a greater effect on the efficacy of these sanitizers. Attachment of Lm isolates on commonly used food contact surface materials greatly reduced the efficacy of all sanitizers tested. Food processing industry should consider cleaning and sanitizing more often than once every 24 h to reduce the risk of surface adherent Lm surviving cleaning and sanitation procedures. Collectively, these findings highlight important variables, such as sanitizer types, temperatures, and surfaces, that need to be carefully considered by food processing facilities when designing and implementing their cleaning and sanitation regimes.