Biofilms are complex microbial communities’ adhered to biotic or abiotic surfaces and are embedded in a matrix of extracellular polymeric substance (EPS) . Most bacteria execute certain development stages; a) adherence of cells to a substratum, b) development of micro-colonies, c) maturation of micro-colonies into biofilms and d) detachment of bacteria and acquisition of motile phase, known as biofilm dispersal . Biofilm disassembly/dispersion is believed to play very important role in pathogenicity, environmental distribution and also in phase transition . The dispersal phenomenon can also be triggered by several environmental signals or unfavourable condition. As ...view middle of the document...
There are several reports on role of surface proteins in S. aureus biofilm formation and its stability [10,11]. Among various surface proteins, biofilm-associated-protein (Bap) was first reported as a large, multidomain, cell surface anchored protein which plays a crucial role in S. aureus biofilm development and and pathogenesis of bovine [12,13]. The involvement of Bap in the polysaccharide-intercellular adhesin (PIA) component of the S. aureus biofilm matrix was also studied comprehensively , however, much less is known about biofilm associated proteins (Bap etc.) and their role in stability of biofilm matrix. A recent report shows that repeated domains contain an amyloidogenic peptide motif (-STVTVTF- derived from the C-repeat of the Bap), which is responsible for cell –cell interaction . Therefore, Bap could be an important component to target, pertaining to biofilm dispersal.
Among the natural ways of S. aureus biofilm dispersal, Agr-mediated biofilm dispersal and secretion of major extracellular proteases, SspA, SspB, Aur and Scp as proenzymes were reported . These enzymes although known to theoretically contribute to biofilm detachment, a very little is known about their role in staphylococci. Recently an extracellular serine protease, Esp secreted by a subset of Staphylococcus epidermidis, shown to inhibit biofilm formation and nasal colonization by S. aureus . Lately, it was shown that Esp has proteolytic activity specifically towards biofilm specific proteins which are associated with S. aureus biofilm formation and host-pathogen interaction . In an earlier report, we showed that proteinase K can imitate the naturally produced proteases and proteinase K treatment can be used to enhance the biofilm dispersal through cleavage of surface protein i.e. Bap- dependent S. aureus biofilm establishment . In this additional report, we investigated whether this approach would be useful in general and has wider applicability by using 5 other S. aureus mastitis isolates. To address the problem, 5 S. aureus bovine mastitis isolates were included in the study along with bap–positive S. aureus V329 and a bap-isogenic mutant M556 as positive and negative controls respectively.
2. Materials and Methods
2.1. Microorganisms and culture conditions
A bap–positive S. aureus V329 and its isogenic mutant S. aureus M556 were used in this study along with 5 other isolates of S. aureus viz SA7, SA10 SA33, SA252 and SA392. In M556 was generated by transposon insertion in downstream part of bap gene of S. aureus V329 in such way that Bap protein is synthesized but remains non-functional as cell wall anchoring region is truncated . For each experiment, single colonies were picked from Tryptic Soy Agar (TSA) culture plates and inoculated in Tryptic Soy Broth (TSB) medium supplemented with 0.25% glucose and incubated at 37 oC at 150 rpm. Overnight grown cultures were used for all experiments.
2.2. Quantitative biofilm assay