Activity of Specialized Biomolecules against Gram-Positive and Gram-Negative Bacteria

Abstract

The increased resistance of bacteria against conventional pharmaceutical solutions, the antibiotics, has raised serious health concerns. This has stimulated interest in the development of bio-based therapeutics with limited resistance, namely, essential oils (EOs) or antimicrobial peptides (AMPs). This study envisaged the evaluation of the antimicrobial efficacy of selected biomolecules, namely LL37, pexiganan, tea tree oil (TTO), cinnamon leaf oil (CLO) and niaouli oil (NO), against four bacteria commonly associated to nosocomial infections: Staphylococcus aureus, Staphylococcus epidermidis, Escherichia coli and Pseudomonas aeruginosa. The antibiotic vancomycin and silver nanoparticles (AgNPs) were used as control compounds for comparison purposes. The biomolecules were initially screened for their antibacterial efficacy using the agar-diffusion test, followed by the determination of minimal inhibitory concentrations (MICs), kill-time kinetics and the evaluation of the cell morphology upon 24 h exposure. All agents were effective against the selected bacteria. Interestingly, the AgNPs required a higher concentration (4000-1250 µg/mL) to induce the same effects as the AMPs (500-7.8 µg/mL) or EOs (365.2-19.7 µg/mL). Pexiganan and CLO were the most effective biomolecules, requiring lower concentrations to kill both Gram-positive and Gram-negative bacteria (62.5-7.8 µg/mL and 39.3-19.7 µg/mL, respectively), within a short period of time (averaging 2 h 15 min for all bacteria). Most biomolecules apparently disrupted the bacteria membrane stability due to the observed cell morphology deformation and by effecting on the intracellular space. AMPs were observed to induce morphological deformations and cellular content release, while EOs were seen to split and completely envelope bacteria. Data unraveled more of the potential of these new biomolecules as replacements for the conventional antibiotics and allowed us to take a step forward in the understanding of their mechanisms of action against infection-related bacteria.

Keywords: antimicrobial peptides; bactericidal; essential oils; minimum inhibitory concentration; nosocomial.

Figure 1

Silver nanoparticles (AgNPs) colloidal dispersion scanning transmission electron microscopy (STEM) micrographs at magnifications of ×100,000 and ×200,000 with evidence of formation of NPs clusters.

Figure 2

Kill-time curves of AgNPs, vancomycin, LL37, pexiganan, TTO, CLO and NO, at the MICs concentrations, against (a) S. aureus, (b) S. epidermidis, (c) E. coli and (d) P. aeruginosa, up to 24 h culture. Data derived from three repetitions. Positive controls for each bacterium (growth without agent) were also conducted, reaching maximum values of ≈8.0 × 10⁸ colony forming units (CFUs)/mL for S. aureus, ≈1.6 × 10⁹ CFUs/mL for S. epidermidis, ≈1.4 × 10⁹ CFUs/mL for E. coli and ≈8.7 × 10⁸ CFUs/mL for P. aeruginosa after 24 h culture (data not shown).

Figure 3

Micrographs of S. aureus (Sa), S. epidermidis (Se), E. coli (Ec) and P. aeruginosa (Pa) bacteria untreated (control) and treated with the selected antibacterial agents, at the smallest tested concentration just before establishing the MIC value. Concentrations were defined at 2000 μg/mL in Sa, Se and Ec and 625 μg/mL in Pa for AgNPs; 3.9 μg/mL in Sa and Se and 500 μg/mL in Ec and Pa for vancomycin; 250 μg/mL in Sa and Se, 62.5 μg/mL in Ec and 125 μg/mL in Pa for LL37; 15.7 μg/mL in Sa and Pa, 3.9 μg/mL in Se and 31.3 μg/mL in Pa for pexiganan; 33.6 μg/mL in Sa, 89.5 μg/mL in Se, 16.8 μg/mL in Ec and 134.3 μg/mL in Pa for TTO; 15.7 μg/mL in Sa and Se, 9.9 μg/mL in Ec and 19.7 μg/mL in Pa for CLO, and 68.5 μg/mL in Sa and Ec, 91.3 μg/mL in Se and 182.6 μg/mL in Pa for NO. These concentrations allowed for live and dead cells to be observed simultaneously, with morphological differences being more easily identified.