Cationic Chitosan-Propolis Nanoparticles Alter The Zeta Potential Of S. Epidermidis, Inhibit Biofilm Formation By Modulating Gene Expression And Exhibit Synergism With Antibiotics

Staphylococcus epidermidis, is a common microflora of human body that can cause opportunistic infections associated with indwelling devices. It is resistant to multiple antibiotics necessitating the need for naturally occurring antibacterial agents. Malaysian propolis, a natural product obtained from beehives exhibits antimicrobial and antibiofilm properties. Chitosan-propolis nanoparticles (CPNP) were prepared using Malaysian propolis and tested for their effect against S. epidermidis. The cationic nanoparticles depicted a zeta potential of +40 and increased the net electric charge (zeta potential) of S. epidermidis from -17 to -11 mV in a concentration-dependent manner whereas, ethanol (Eth) and ethyl acetate (EA) extracts of propolis further decreased the zeta potential from -17 to -20 mV. Confocal laser scanning microscopy (CLSM) depicted that CPNP effectively disrupted biofilm formation by S. epidermidis and decreased viability to similar to 25% compared to Eth and EA with viability of similar to 60-70%. CPNP was more effective in reducing the viability of both planktonic as well as biofilm bacteria compared to Eth and EA. At 100 mu g/mL concentration, CPNP decreased the survival of biofilm bacteria by similar to 70% compared to Eth or EA extracts which decreased viability by only 40%-50%. The morphology of bacterial biofilm examined by scanning electron microscopy depicted partial disruption of biofilm by Eth and EA extracts and significant disruption by CPNP reducing bacterial number in the biofilm by similar to 90%. Real time quantitative PCR analysis of gene expression in treated bacteria showed that genes involved in intercellular adhesion such as IcaABCD, embp and other related genes were significantly down-regulated by CPNP. In addition to having a direct inhibitory effect on the survival of S. epidermidis, CPNP showed synergism with the antibiotics rifampicin, ciprofloxacin, vancomycin and doxycycline suggestive of effective treatment regimens. This would help decrease antibiotic treatment dose by at least 4-fold in combination therapies thereby opening up ways of tackling antibiotic resistance in bacteria.