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 8 months. Of 421 samples, 31 (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 (STs), 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 four new ST), known as an epidemic clone, was the dominant CC. However, there were no indications of persistence of particular ST. The resistance toward 11 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.

In nature and man-made environments, microorganisms reside in mixedspecies biofilms, in which the growth and metabolism of an organism are different from these behaviors in 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 one or the other of the foodborne pathogens Listeria monocytogenes and Staphylococcus aureus. The five species, including the pathogen, were isolated from a single food-processing environmental sample, thus mimicking the environmental community. In mature mixed fivespecies biofilms on stainless steel, the two pathogens remained at a constant level of ϳ10 5 CFU/cm 2 . The mixed five-species biofilms as well as the pathogens in monospecies 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 by 4.6 log cycles in monospecies biofilms, but the pathogen was protected in the fivespecies biofilm and decreased by only 1.1 log cycles. Sessile cells of L. monocytogenes were affected to the same extent when in a monobiofilm or as a member of the mixed-species biofilm, decreasing by 3 log cycles when exposed to 0.0375% peracetic acid. When the pathogen was exchanged in each associated 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 to identify 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 are a challenge for many industries, and biocide efficacy should be tested on microorganisms growing in biofilms, preferably mixed systems, mimicking the application environment.

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The development of more efficient sanitizers for reducing the biofilm produced by pathogenic bacteria is of great importance, especially in the food industry. The aim of this study was to conduct a preliminary evaluation of the efficiency of oleuropein (OLE), extracted from olive leaves, and peracetic acid (PAA), alone or in combination, to inactivate biofilms formed by a strong biofilm-producer strain of Listeria monocytogenes (ATCC 7644). A disk diffusion method was also used to assess the susceptibility of the ATCC 7644 strain cells to OLE and/or PAA. Triplicate biofilm assays were prepared on stainless steel coupons (1x1 cm 2) during 48 h without stirring. After incubation, the stainless steel was washed (NaCl 0.85%) and immersed in solutions of OLE (5.0 mg/mL) and/or PAA (2.0%) for 1 min. After 1-min treatment, the disinfectant solutions were removed from the tube. Two mL of sodium thiosulfate 0.1 M was placed in the tube to stop reaction. The inactivation of biofilms was assessed by confocal laser scanning microscopy. L. monocytogenes cells tested in the disk diffusion method was not susceptible to OLE (inhibition zone (IZ) = 6 mm), although its susceptibility was intermediate for PAA (IZ = 8.67 mm) and higher for PAA+OLE (IZ = 14 mm). Compared with PAA, OLE alone in contact with biofilms had also lower bactericidal activity on the biofilms. However, the treatment of OLE in combination with PAA resulted in greater inactivation of L. monocytogenes cells in biofilms. Results indicate a potential application of OLE for enhancing the bactericidal effect of PAA against L. monocytogenes biofilms, although further studies are necessary to understand the mechanisms of action of OLE in combination with commercial chemical sanitizers.