Research Article: Cationic chitosan-propolis nanoparticles alter the zeta potential of S. epidermidis, inhibit biofilm formation by modulating gene expression and exhibit synergism with antibiotics

Date Published: February 28, 2019

Publisher: Public Library of Science

Author(s): Teik Hwa Ong, Ebenezer Chitra, Srinivasan Ramamurthy, Catherine Chong Sze Ling, Stephen Periathamby Ambu, Fabian Davamani, Esaki M. Shankar.


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 ~25% compared to Eth and EA with viability of ~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 μg/mL concentration, CPNP decreased the survival of biofilm bacteria by ~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 ~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 downregulated 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.

Partial Text

Staphylococcus epidermidis survives on the skin as normal flora under the epithelium and is recognized as an opportunistic pathogen commonly encountered in hospital-acquired infections without critical implications. The bacteria attach to solid surfaces forming biofilms; this process is characterized by distinct phases–initiation of establishment and colonization leading to infectious stage, primary reversible adhesion developing into secondary irreversible adhesion, and biofilm formation [1]. S. epidermidis can adhere to both biotic and abiotic surfaces of indwelling or implanted medical devices or tissues and form biofilms, leading to treatment failure and relapse of infections [2]. It is implicated in cardiac prosthetic valve infections causing endocarditis, which might lead to intra-cardiac abscesses and mortality. S. epidermidis exhibits resistance to β-lactam antibiotics, which are commonly used in clinical settings [3]. Its antimicrobial resistance is mainly linked to its capability to colonize and produce biofilms.

Net surface charge of bacteria is crucial for their survival, and alteration in the surface charge can have physiological consequences. Surface charge neutralization has been explored as an antibacterial activity employed by antimicrobial agents acting on bacterial surface. Zinc oxide nanoparticles with positive zeta potential were reported to have high antimicrobial activity against both Gram-positive and Gram-negative bacteria compared to those with negative zeta potential [20]. Exposure of P. aeruginosa to high concentrations of benzalkonium chloride, a cationic surfactant led to a reduction in the membrane negative charge caused by alteration in gene expression, thereby causing a major adaptative feature in the bacteria to withstand the surfactant effect [21]. Nanoparticles with positive surface charge are known to interact with bacteria with negative surface potential, thereby resulting in membrane depolarization and inhibition of bacterial growth [20]. The surface charges of antimicrobial agents also determine their binding efficacy. While propolis extracts lower the membrane potential of bacteria, our cationic nanoparticles have the opposing effect. They easily bind to the anionic bacteria and increase their zeta potential. Propolis extracts with a negative surface charge resulted in weaker interaction between the surfaces due to the repulsive force. Changes in zeta potential of bacteria affect their cell surface permeability; a change in zeta potential affects bacterial cellular physiology leading to mortality and/or inhibition of growth kinetics.




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