Abstract:
The growing occurrence of antimicrobial resistance (AMR) is a global cause for concern due to the decreased availability of effective antimicrobial drugs. Therefore, treating resistant infections can become costly or even impossible. Furthermore, the increase in resistant infections leads to longer hospital stays and higher mortality rates. Resistance is prevalent because microorganisms can form biofilms; communities of cells bound to a surface and covered in an extracellular matrix (ECM), that protects cells from the effects of antimicrobial agents. Besides the ECM, biofilm cells further resist antimicrobial drugs by rapidly developing a number of mechanisms. Therefore, development of novel antimicrobial agents is key to overcoming AMR. One potential alternative to conventional antimicrobial drugs are cationic antimicrobial peptides (AMPs) which are short and amphipathic molecules. Some AMPs possess both anti-planktonic and anti-biofilm activity among other beneficial properties which make them a suitable alternative to conventional antimicrobial drugs. In this study, the anti-biofilm activity of Os(11-22)NH2, a short peptide derived from Os, a derivative of a defensin identified in the tick Ornithodoros savignyi, was investigated. For the purpose of this study, the opportunistic fungal pathogen Candida albicans, one of the leading causes of hospital-acquired infections, was used as the model microorganism.
Os was inactive, while the derivative Os(11-22)NH2 was active against planktonic (free-floating) C. albicans with a minimum inhibitory concentration that reduces growth by 50% (MIC50) of 47 μM. The CellTiter Blue (CTB) cell viability assay was used to determine the biofilm inhibiting and eradicating activity. Os(11-22)NH2 inhibited biofilm formation with a minimum concentration of the antifungal agent that reduced biofilm formation by 50% (BIC50) of 81 μM. Inverted light microscopy images confirmed CTB cell viability results and reduced hyphal formation was observed. Treatment of preformed biofilms with Os(11-22)NH2 led to biofilm eradication by Os(11-22)NH2 with a minimum concentration of the antifungal agent that decreased cell viability in a pre-grown biofilm by 50% (BEC50) of 210 μM.
Most AMPs target the cell membrane, therefore, membrane permeabilizing activity was investigated using confocal laser scanning microscopy (CLSM) and the DNA binding dyes propidium iodide (PI) and 4',6-diamidino-2-phenylindole (DAPI). Results indicated that cell membranes were permeabilized by treatment with Os(11-22)NH2 during biofilm inhibition and eradication.
Some AMPs are known to induce the production of reactive oxygen species (ROS) in microorganisms, leading to cell death. Therefore, the fluorescence producing dye 2,7-dichlorodihydrofluorescein diacetate (DCFH-DA) was used to determine whether Os(11-22)NH2 induces ROS production. Os(11-22)NH2 induced ROS production during biofilm inhibition and eradication. In the presence of ascorbic acid, a scavenger of ROS, the production of ROS by the peptide was significantly reduced (p < 0.0001).
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Furthermore, the effect of ascorbic acid on the biofilm inhibiting and eradicating activity of Os(11-22)NH2 was determined using the CTB cell viability assay. For biofilm inhibition, no significant difference was observed between treatments in the presence and absence of ascorbic acid, therefore killing during biofilm inhibition was not due to ROS production. For eradication, a significant increase (p < 0.0001) in biofilm eradicating activity was observed in the presence of ascorbic acid indicating that the biofilm eradicating activity of Os(11-22)NH2 was enhanced by ascorbic acid.
In conclusion, anti-biofilm activity of Os(11-22)NH2 was determined at micromolar concentrations, indicating a potential antifungal application for this peptide. Future research should reveal its mode of action and combination with other antifungal agents.
