In nature and man-made environments, microorganisms reside in mixed-species biofilm where behavior is modified compared to the single-species biofilms. Pathogenic microorganisms may be protected against adverse treatments in mixed-species biofilms leading to health risk for humans. Here, we developed two mixed-five-species biofilms that included the foodborne pathogens Listeria monocytogenes or Staphylococcus aureus, respectively. The five species, including the pathogen, were isolated from a single food-processing environmental sample thus mimicking the environmental community. In mature mixed five-species biofilms on stainless steel, the two pathogens remained at a constant level of ∼10⁵ CFU/cm². The mixed-five-species biofilms as well as the pathogens in mono-species biofilms were exposed to biocides to determine any pathogen-protective effect of the mixed biofilm. Both pathogens and their associate microbial communities were reduced by peracetic acid treatments. S. aureus decreased 4.6 log cycles in mono-species biofilm, but the pathogen was protected in the five-species biofilm and decreased only 1.1 log cycles. Sessile cells of L. monocytogenes were affected equally as a mono-biofilm or as a member in the mixed-species biofilm; decreasing by three log cycles when exposed to 0.0375 % peracetic acid. When the pathogen was exchanged in each associate microbial community, S. aureus was eradicated while there was no significant effect of the biocide on L. monocytogenes or the mixed community. This indicates that particular members or associations in the community offered the protective effect. Further studies are needed to clarify the mechanisms of biocide protection, and the species playing the protective role in microbial communities of biofilms. Importance This study demonstrates that foodborne pathogens can be established in mixed species biofilms and that this can protect them from biocide action. The protection is not due to specific characteristics of the pathogen, here S. aureus and L. monocytogenes, but likely caused by specific members or associations in the mixed species biofilm. Biocide treatment and resistance is a challenge for many industries and biocide efficacy should be tested on microorganisms growing in biofilms, preferably mixed systems, mimicking the application environment.

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.

Listeria monocytogenes is an opportunistic pathogen that affects specific groups of individuals, with a high mortality rate. The control of L. monocytogenes in dairy industries presents particular challenges, as this bacterium is capable of adhering and forming biofilms, as well as thriving at refrigerated temperatures, which enables it to persist in harsh environments. The consumption of dairy products has been linked to sporadic cases and outbreaks of listeriosis, and L. monocytogenes is frequently detected in these products in retail stores. Moreover, the bacterium has been shown to persist in dairy-processing environments. In this work, we review the main characteristics of L. monocytogenes and listeriosis, and highlight the factors that support its persistence in processing environments and dairy products. We also discuss the main dairy products involved in outbreaks of listeriosis since the early 1980s, and present control measures that can help to prevent the occurrence of this pathogen in foods and food-processing environments.

Staphylococcus aureus, a major food-poisoning pathogen, is a common contaminant in dairy industries worldwide, including in Brazil. We determined the occurrence of S. aureus in five dairies in Brazil over eight months. Of 421 samples, thirty-one (7.4%) were positive for S. aureus and prevalence varied from 0 to 63.3% between dairies. Sixty-six isolates from the 31 samples were typed by Multi-Locus Sequence Typing to determine if these isolates were persistent or continuously reintroduced. Seven known Sequence Types (ST), ST1, ST5, ST30, ST97, ST126, ST188 and ST398, and four new ST were identified, ST3531, ST3540, ST3562 and ST3534. Clonal Complex (CC) 1 (including the three new ST), known as an epidemic clone, was the dominant CC. However, there were no indications of persistence of particular ST. The resistance towards eleven antibiotic compounds was assessed. Twelve profiles were generated with 75.8% of strains being sensitive to all antibiotic classes and no Methicillin-resistant S. aureus (MRSA) strains were found. The enterotoxin-encoding genes involved in food-poisoning, e.g. sea, sed, see and seg were targeted by PCR. The two toxin-encoding genes, sed and see, were not detected. Only three strains (4.5%) harbored seg and two of these also harbored sea. Despite the isolates being Methicillin-sensitive S. aureus (MSSA), the presence of CC1 clones in the processing environment, including some harboring enterotoxin encoding genes, is of concern and hygiene must have high priority to reduce contamination.

Bacteriocins of lactic acid bacteria (LAB) are proteinaceous compounds that may present antimicrobial activity towards important foodborne pathogens and spoilage-related microflora. Due to these properties, bacteriocin-producing strains or purified bacteriocins have a great potential of use in biologically based food preservation systems. Despite the growing number of articles describing the isolation of bacteriocinogenic strains, genetic determinants for production, as well as the purification and biochemical characterization of these inhibitory substances, there are only limited reports of successful application of bacteriocins to meats. This paper presents a critical review of the methods available for screening of bacteriocin-producing LAB strains from meats and also discusses the proposed mechanisms of action for LAB bacteriocins. Additionally, an overview of the Brazilian experience in the application of LAB bacteriocins to meats and meat products is given.