Dua, H. S. | Search bioRxiv

bioRxiv2022

Camellia sinensis solvent extract confers trophocidal and cysticidal effects against Acanthamoeba castellanii

AimWe examined the anti-acanthamoebic efficacy of solvent extract of C. sinensis) and its chemical constituents against trophozoites and cysts of A. castellanii. Materials and methodsThe effects of C. sinensis solvent extract on A. castellanii was investigated by using anti-trophozoite, anti-encystation, and anti-excystation assays. The solvent extract was also fractionated using Gas Chromatography and the chemical constituents of C. sinensis were tested, individually or combined, against the trophozoites. ResultsTrophozoite replication was inhibited within 24-72 h with exposure to 625-5000 {micro}g/mL of C. sinensis solvent extract. C. sinensis also exhibited a dose-dependent inhibition of encystation, with a marked cysticidal activity at 2500-5000 {micro}g/mL concentrations. Two constituents of C. sinensis, namely epigallocatechin-3-gallate and caffeine, significantly inhibited trophozoite replication and encystation at 100 M and 200 M, respectively. Cytotoxicity analysis showed that 156.25-2500 {micro}g/mL of solvent extract was not toxic to human corneal epithelial cells, while up to 625 {micro}g/mL was not toxic to Madin-Darby Canine Kidney cells. ConclusionsThis study shows the anti-acanthamoebic potential of C. sinensis solvent extract against trophozoites and cysts. Further pre-clinical studies are required to elucidate the in vivo efficacy and safety of C. sinensis solvent extract. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=152 HEIGHT=200 SRC="FIGDIR/small/507025v1_ufig1.gif" ALT="Figure 1"> View larger version (29K): org.highwire.dtl.DTLVardef@9850forg.highwire.dtl.DTLVardef@feb98dorg.highwire.dtl.DTLVardef@147fc64org.highwire.dtl.DTLVardef@10b497e_HPS_FORMAT_FIGEXP M_FIG C_FIG

microbiology↗

bioRxiv2021

Evaluation of Host Defense Peptide (CaD23)-Antibiotic Interaction and Mechanism of Action: Insights from Experimental and Molecular Dynamics Simulations Studies

Background/aimHost defense peptides (HDPs) have the potential to provide a novel solution to antimicrobial resistance (AMR) in view of their unique and broad-spectrum antimicrobial activities. We had recently developed a novel hybrid HDP based on LL-37 and human beta-defensin-2, named CaD23, which was shown to exhibit good in vivo antimicrobial efficacy against Staphylococcus aureus in a bacterial keratitis murine model. This study aimed to examine the potential CaD23-antibiotic synergism and to evaluate the underlying mechanism of action of CaD23. MethodsAntimicrobial efficacy was determined using minimum inhibitory concentration (MIC) assay with broth microdilution method. Peptide-antibiotic interaction was evaluated against S. aureus, methicillin-resistant S. aureus (MRSA), and Pseudomonas aeruginosa using established checkerboard assay and time-kill kinetics assay. Fractional inhibitory concentration index (FICI) was calculated and interpreted as synergistic (FICI<0.5), additive (FICI between 0.5-1.0), indifferent (FICI between >1.0 and [≤]4), or antagonistic (FICI>4). SYTOX green uptake assay was performed to determine the membrane-permeabilising action of CaD23. Molecular dynamics (MD) simulations were performed to evaluate the interaction of CaD23 with bacterial and mammalian mimetic membranes. ResultsCaD23-amikacin and CaD23-levofloxacin combination treatment exhibited a strong additive effect against S. aureus SH1000 (FICI=0.56) and MRSA43300 (FICI=0.56) but a borderline additive-to-indifferent effect against P. aeruginosa (FIC=1.0-2.0). CaD23 (at 25 g/ml; 2x MIC) was able to achieve complete killing of S. aureus within 30 mins. When used at sub-MIC concentration (3.1 g/ml; 0.25x MIC), it was able to expedite the antimicrobial action of amikacin against S. aureus by 50%. The rapid antimicrobial action of CaD23 was attributed to the underlying membrane-permeabilising mechanism of action, evidenced by the SYTOX green uptake assay and MD simulations studies. MD simulations revealed that cationicity, alpha-helicity, amphiphilicity and hydrophobicity (related to the Trp residue at C-terminal) play important roles in the antimicrobial action of CaD23. ConclusionsCaD23 is a novel membrane-active synthetic HDP that can enhance and expedite the antimicrobial action of antibiotics against Gram-positive bacteria when used in combination. MD simulation serves as a useful tool in dissecting the mechanism of action and guiding the design and optimisation of HDPs.

microbiology↗