The pathogenic bacterium Listeria monocytogenes can persist in food processing plants for many years, even when appropriate hygienic measures are in place, with potential for contaminating ready-to-eat products and, its ability to form biofilms on abiotic surfaces certainly contributes for the environmental persistence. In this research, L. monocytogenes was grown in biofilms up 8 days attached to stainless steel and glass surfaces, contributing for advancing the knowledge on architecture of mature biofilms, since many literature studies carried out on this topic considered only early stages of cell adhesion. In this study, biofilm populations of two strains of L. monocytogenes (serotypes 1/2a and 4b) on stainless steel coupons and glass were examined using regular fluorescence microscopy, confocal laser scanning microscopy and classic culture method. The biofilms formed were not very dense and microscopic observations revealed uneven biofilm structures, with presence of exopolymeric matrix surrounding single cells, small aggregates and microcolonies, in a honeycomb-like arrangement. Moreover, planktonic population of L. monocytogenes (present in broth media covering the abiotic surface) remained stable throughout the incubation time, which indicates an efficient dispersal mechanism, since the culture medium was replaced daily. In conclusion, even if these strains of L. monocytogenes were not able to form thick multilayer biofilms, it was noticeable their high persistence on abiotic surfaces, reinforcing the need to focus on measures to avoid biofilm formation, instead of trying to eradicate mature biofilms.

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Listeria monocytogenes, a foodborne pathogen, is hard to eliminate from food processing environments because it can persist in biofilms. Searching for natural agents that can effectively act against L. monocytogenes biofilms is important to prevent food contamination, especially in the case of ready-to-eat foods. Here, we determine the key components of the lemongrass and ginger essential oils (EOs) and evaluate the activity of each EO against L. monocytogenes biofilms.Biofilms grown on stainless steel and glass coupons were monitored by culture method and confocal laser scanning microscopy (CLSM). These techniques revealed the presence of live and dead cells. Monoterpenes were the main components in the lemongrass EO; monoterpenes and sesquiterpenes were the main constituents of the ginger EO. Although the lemongrass EO affected L. monocytogenes biofilms significantly, none of the tested treatments inhibited the pathogen completely. Therefore, controlling the presence of L. monocytogenes in food processing areas demands preventive measures. Practical applicationMicrobial biofilms constitute a challenge for effective sanitation in the food industry. Synthetic products are commonly used as sanitizers, but consumers have demanded more natural food produced in eco-friendly environments. This scenario has motivated the search for natural antimicrobials that can be applied in food facilities to eliminate microorganisms from biofilms. Plant-derived compounds represent an alternative source of antimicrobials due to their potential acceptance by the consumers and wide availability. This study shows that treatment with lemongrass essential oil significantly affects biofilms formed by the foodborne pathogen Listeria monocytogenes, but the treatment does not eradicate the biofilms. Our results demonstrate that it is possible to identify natural antimicrobials with potential use as alternative sanitizers in the food industry.

Studying biofilm dispersal is important to prevent Listeria monocytogenes persistence in food processing plants and to avoid finished product contamination. Reactive oxygen and nitrogen intermediates (ROI and RNI, respectively) may trigger cell detachment from many bacterial species biofilms, but their roles in L. monocytogenes biofilms have not been fully investigated. This study reports on ROI and RNI quantification in Listeria monocytogenes biofilms formed on stainless steel and glass surfaces; bacterial culture and microscopy combined with fluorescent staining were employed. Nitric oxide (NO) donor and inhibitor putative effects on L. monocytogenes dispersal from biofilms were evaluated, and transcription of genes (prfA, lmo 0990, lmo 0807, and lmo1485) involved in ROI and RNI stress responses were quantified by real-time PCR (qPCR). Microscopy detected the reactive intermediates NO, peroxynitrite, H 2 O 2 , and superoxide in L. monocytogenes biofilms. Neither NO donor nor inhibitors interfered in L. monocytogenes growth and gene expression, except for lmo0990, which was downregulated. In conclusion, ROI and RNI did not exert dispersive effects on L. monocytogenes biofilms, indicating that this pathogen has a tight control for protection against oxidative and nitrosative stresses.