Development of Salmonella enterica serovar Typhimurium, Listeria monocytogenes, and Pseudomonas aeruginosa multi-species in vitro dry surface biofilm models: Insights into resilience and persistence in low-moisture environments

Abstract

Salmonella enterica serovar Typhimurium, Listeria monocytogenes, and Pseudomonas aeruginosa can persist as dry surface biofilms (DSB) and survive desiccation for prolonged periods of time. This could serve as potential sources of contamination in low-moisture food (LMF) processing environments. The objectives of this study were to develop in vitro mono-and mix-culture DSB models to better understand pathogen survival under dehydrated conditions as DSB. Mono-and mix-culture wet surface biofilms (WSB) of S. enterica ser. Typhimurium ATCC 14028, L. monocytogenes FSL R8-5318, and P. aeruginosa ATCC 15442 were developed following EPA MLB SOP MB-19 protocol on borosilicate glass coupons using a CDC® biofilm reactor. WSB were harvested and dried for 24, 48, and 72 h at 21 °C to form DSB. Mean log10 CFU/cm2 were calculated at each desiccation time point and compared to control (WSB) using LSM of PROC GLM to fit linear models (P = 0.05). Approximately 8.8 ± 1.7, 7.1 ± 1.1, 6.9 ± 0.6, and 6.3 ± 0.8 mean log10 CFU/coupon were recovered from mono-culture Salmonella DSB at 0, 24, 48, and 72 h, respectively. Overall, Salmonella mean log10 densities were limited when co-cultured with P. aeruginosa although there were no significant differences in Salmonella density over desiccation time points. Mean log10 CFU/coupon for L. monocytogenes monoculture DSB were comparatively lower i.e., 5.7 ± 0.4 (0 h), 3.8 ± 0.1 (24 h), and 3.4 ± 0.6 (48 h), and 2.6 ± 0.3 (72 h). However, when co-cultured with P. aeruginosa, L. monocytogenes exhibited consistently higher cell densities 6.7 ± 0.4, 6.0 ± 0.9, 5.6 ± 0.04, and 4.9 ± 0.6, at 0, 24, 48, and 72 h, respectively. On average, P. aeruginosa consistently exceeded six log10 at each desiccation time point in co-culture. Confocal laser scanning microscopy (CLSM) images confirmed biofilm development and persistence over time in mono and mixed cultures. This work provides foundational evidence toward better understanding pathogen survival under dehydrated conditions, interactions between different pathogens within desiccated biofilms, and their respective survival rates. Additionally, these data provide quantitative assessment of bacterial load within DSB under high-risk scenarios for LMFs. These models provide opportunities to explore targeted mitigation strategies, including efficacy of dry sanitation practices, to prevent the formation or remove DSB in LMF processing environments.