Abstract
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.
Highlights
Staphylococcus aureus is an opportunistic bacterial pathogen which can cause a broad variety of infectious diseases in both humans and animals
To determine the activity of LysK against biofilms of multiple S. aureus strains, a selection of strains from our laboratory collection (Table 1) was first assessed for their ability to form biofilms on polystyrene surfaces in a static 96-well plate-based model. These included SA113, a reportedly strong biofilm former and producer of PNAG [32,43], which served as a positive control in this experiment; three bovine mastitis isolates from different geographic regions; multiple food isolates; and RN6911, a mutant strain deficient of the accessory gene regulator [33]
RN6911 demonstrated the best biofilm forming ability of all tested strains, as revealed by crystal violet (CV) staining of biofilms grown for 24 h at 30 ◦ C (Figure 1A)
Summary
Staphylococcus aureus is an opportunistic bacterial pathogen which can cause a broad variety of infectious diseases in both humans and animals. S. aureus is frequently responsible for food poisoning via production of heat-stable enterotoxins [6] Besides their important role as human pathogens, staphylococci (and S. aureus in particular) are a major cause of bovine mastitis, an infection of the mammary gland in cows. This leads to severe losses in milk production and quality and increased costs due to veterinary treatment and culling of animals, making it the most costly disease for the dairy industry [7]. In both human and veterinary clinics, the increasing prevalence of antibiotic-resistant strains such as methicillin-resistant S. aureus (MRSA) as well as the ability of staphylococci to form biofilms result in prolonged therapies and increased treatment costs [8,9]
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