Staphylococcus aureus is an important pathogen and biofilm former. Biofilms cause problems in clinics and food production and are highly recalcitrant to antibiotics and sanitizers. Bacteriophage endolysins kill bacteria by degrading their cell wall and are therefore deemed promising antimicrobials and anti-biofilm agents. Depolymerases targeting polysaccharides in the extracellular matrix have been suggested as parts of a multi-enzyme approach to eradicate biofilms. The efficacy of endolysins and depolymerases against S. aureus biofilms in static models has been demonstrated. However, there is a lack of studies evaluating their activity against biofilms grown under more realistic conditions. Here, we investigated the efficacy of the endolysin LysK and the poly-N-acetylglucosamine depolymerase DA7 against staphylococcal biofilms in static and dynamic (flow cell-based) models. LysK showed activity against multiple S. aureus strains, and both LysK and DA7 removed static and dynamic biofilms from polystyrene and glass surfaces at low micromolar and nanomolar concentrations, respectively. When combined, the enzymes acted synergistically, as demonstrated by crystal violet staining of static biofilms, significantly reducing viable cell counts compared to individual enzyme treatment in the dynamic model, and confocal laser scanning microscopy. Overall, our results suggest that LysK and DA7 are potent anti-biofilm agents, alone and in combination.

The objective of this study was to compare the biofilm-forming capabilities of different genotypes of Staphylococcus aureus dairy isolates from Switzerland and northern Italy, including Staph. aureus genotype B (GTB) and methicillin-resistant Staph.aureus (MRSA). We hypothesized that biofilm formation might be more pronounced in the contagious GTB isolates compared with other genotypes affecting individual animals. Twenty-four dairy isolates, including 9 MRSA, were further characterized by genotyping by using ribosomal spacer PCR, spa typing, biofilm formation under static and dynamic conditions, and scanning electron microscopy. The GTB isolates (n = 6) were more able to form biofilms than other genotypes at 37°C and at 20°C after 48 and 72 h of incubation in the static assay using polystyrene microtiter plates. This result was supported by scanning electron micrographs showing a GTB isolate producing strong biofilm with extracellular matrix in contrast to a genotype C isolate. Furthermore, none of the MRSA isolates formed strong biofilms in the static assay. However, some MRSA produced low or moderate amounts of biofilm depending on the applied conditions. Under dynamic conditions, a much more diverse situation was observed. The ability of GTB isolates to be strong biofilm formers was not observed in all cases, emphasizing the importance of growth conditions for the expression of biofilm-related genes. No specific genotype, spa type, or MRSA isolate could be categorized significantly into one level of biofilm formation. Nineteen percent of isolates behaved similarly under static and dynamic conditions. The results of this study expand our knowledge of different dairy-related Staph. aureus subtypes and indicate the benefit of genotyping when biofilms are studied.

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