MODULATING METHICILLIN-RESISTANT STAPHYLOCOCCUS EPIDERMIDIS BIOFILM FORMATION USING EXOGENOUS LACTOFERRIN AND LOW DOSE CARBON MONOXIDE

Abstract

Invasive methods used in the management of hospitalised patients create risks of transferring skin bacteria such as Staphylococcus epidermidis into internal systems. Inside the body, S. epidermidis become pathogenic, causing infections of the wound, blood and medical device-related, through the formation of biofilms. The management of these biofilm-related infections is worsened by strains which are resistant to more than one clinically effective antibiotic, thus, other strategies are needed. Focusing on potential molecules of the human immunity, the natively produced peptide and gas, lactoferrin (LF) and carbon monoxide (CO) has independently shown activity against bacterial biofilms in vitro, in vivo and clinical studies. Hence, the antibiofilm activities of LF against multidrug-resistant S. epidermidis and its ability to work with CO were evaluated in vitro. Bacteria and cell line co-culture techniques were used to examine the ability of LF and CO to enhance the clearance of methicillin-resistant S. epidermidis (MRSE, ATCC 49461) by RAW 264.7 macrophages. The number of surviving bacteria was determined by the spread plate and colony counting method. The abilities of LF to prevent and affect established biomass of MRSE compared to the antibiotic, vancomycin (VN) were determined. Changes to the biomass and metabolic activity post-treatment were determined using crystal violet and resazurin staining methods, respectively. MRSE collected from biofilms treated with LF, were analysed for changes to the expression of the biofilm-associated protein gene (bap) using real-time polymerase chain reaction (q-PCR). Macrophage and MRSE cocultures treated with LF at a dose of 4096 µg/ml and combination of LF and VN produced a significantly lower number of bacteria which survived clearance, in the presence of CO at a low dose of 250 ppm (p<0.05). LF significantly (p<0.05) reduced the biomass and metabolic activity of preformed biofilms from 16 to 24 hours and maintained these effects up to 72 hours. LF also prevented biomass formation for up to 24 hours. LF caused significant upregulation (p<0.05) of the bap gene but maintained a low amount of easily dislodging biomass. A gene implicated for bacterial replication, gyrase subunit B (gyrB) was also upregulated by LF, suggesting the dispersal of bacteria from the treated biofilm. The antibiofilm activities of LF suggest interactions of the peptide with the bacterial cell wall components including biofilmrelated proteins. The enhancement of clearance of planktonic bacteria by macrophages by LF and CO suggest potentiation of antibacterial activity. Additional data are needed to further elucidate LF and CO as innate molecules with potential in developing strategies against bacterial biofilms of clinical significance. Graphical Abstract 1. Exposure of planktonic MRSE to lactoferrin caused inhibition of growth. 2. MRSE was cleared by macrophages better in the presence of lactoferrin and carbon monoxide. 3. Formation of adherent MRSE biomass was prevented by lactoferrin. 4. Preformed MRSE biofilms were eradicated by lactoferrin. 5. Biofilm-associated protein gene was upregulated in MRSE cells collected from biofilms treated with lactoferrin.