Activity and biocompatibility of antimicrobial contact lenses

Contact lens related infection and inflammation are major problems for contact lens wearers and practitioners alike. Colonisation by a variety of microorganisms such as bacteria, fungi and Acanthamoeba on contact lenses is implicated in the development of these adverse events. A contact lens with high antimicrobial activity may inhibit the ability of microorganisms to grow on the surface and consequently reduce these adverse events. Antimicrobial peptides (AMPs) are small proteins which have been shown to have activity against a wide spectrum of microorganisms. This thesis investigated the spectrum of antimicrobial activity of an AMP covalently attached to contact lenses and its subsequent safety and biocompatibility in animal and human studies. The AMPs melimine and LL-37 were covalently attached onto contact lenses via EDC coupling, which was able to bind high concentrations of melimine but not LL-37. Further, melimine coated lenses demonstrated high antimicrobial activity including activity against multidrug-resistant bacteria, fungal and Acanthamoeba strains. However, the lenses were associated with apparently high levels of dead bacterial cells (evidenced by no differences in radio-labelled RNA or bacterial endotoxin from Gram negative bacteria on melimine-coated and uncoated lenses). The melimine coating was readily heat sterilisable, non-toxic to mammalian cells in vitro, did not change contact lens parameters, and formed a wettable lens surface. The melimine coating was compatible with the lens care disinfectants Biotrue™ and RevitaLens OcuTec®. In vivo investigation of the safety of the melimine coating in a rabbit model of contact lens wear following ISO guidelines revealed that these lenses were not associated with any toxic or hypersensitive responses. A one day clinical trial showed that melimine-coated lenses could be safely worn by humans without any major side effects and any delayed toxic reactions. However, melimine lenses were associated with higher corneal straining. This corneal staining was similar to solution induced corneal staining seen during contact lens wear. Following human wear, lenses retained more than 1.5 log inhibition against Pseudomonas aeruginosa and Staphylococcus aureus. Melimine coatings on contact lenses have excellent potential for further development of biocompatible and effective broad spectrum antimicrobial contact lenses

Factors influencing bacterial adhesion to contact lenses

The process of any contact lens related keratitis generally starts with the adhesion of opportunistic pathogens to contact lens surface. This article focuses on identifying the factors which have been reported to affect bacterial adhesion to contact lenses. Adhesion to lenses differs between various genera/species/strains of bacteria. Pseudomonas aeruginosa, which is the predominant causative organism, adheres in the highest numbers to both hydrogel and silicone hydrogel lenses in vitro. The adhesion of this strain reaches maximum numbers within 1h in most in vitro studies and a biofilm has generally formed within 24 h of cells adhering to the lens surface. Physical and chemical properties of contact lens material affect bacterial adhesion. The water content of hydroxyethylmethacrylate (HEMA)-based lenses and their iconicity affect the ability of bacteria to adhere. The higher hydrophobicity of silicone hydrogel lenses compared to HEMA-based lenses has been implicated in the higher numbers of bacteria that can adhere to their surfaces. Lens wear has different effects on bacterial adhesion, partly due to differences between wearers, responses of bacterial strains and the ability of certain tear film proteins when bound to a lens surface to kill certain types of bacteria

A Pilot Study of the Synergy between Two Antimicrobial Peptides and Two Common Antibiotics

Background: Frequent and unrestricted use of antibiotics has been associated with the development of antibiotic resistance by microorganisms. Thus, there is a need to find novel antibacterial agents or a combination of agents as the first line of treatment for various infections. This study aimed to investigate the synergy between antimicrobial peptide (AMP) combinations or between AMP‐antibiotics combinations using two common pathogens, Pseudomonas aeruginosa and Staphylococcus aureus. Methods: The AMPs melimine, Mel4 and protamine, and antibiotics cefepime and ciprofloxacin were used in this study. The minimum inhibitory concentration (MIC) of each were evaluated against P. aeruginosa and S. aureus strains by a microtiter broth dilution. Based on the MIC of each antimicrobial agent, a checkerboard assay was performed to investigate the synergy between them, which was expressed as the fractional inhibitory concentration (FIC). Results: The combination of melimine and ciprofloxacin showed synergistic activity against antibiotic sensitive or resistant strains of P. aeruginosa and with FIC values ≤0.5. Conclusion: Combinations of AMPs and the fluoroquinolone ciprofloxacin is a promising method for reducing resistance to the fluoroquinolone of P. aeruginosa

Comparative mode of action of the antimicrobial peptide melimine and its derivative Mel4 against Pseudomonas aeruginosa

Melimine and Mel4 are chimeric cationic peptides with broad-spectrum antimicrobial activity. They have been shown to be highly biocompatible in animal models and human clinical trials. The current study examined the mechanism of action of these two antimicrobial peptides against P. aeruginosa. The effect of the peptides of endotoxin neutralization, and their interactions with cytoplasmic membranes using DiSC(3)-5 and Sytox green, Syto-9 and PI dyes were analysed. Release of ATP and DNA/RNA were determined using ATP luminescence and increase in OD 260 nm. The bacteriolytic ability of the peptides was determined by measuring decreases in OD 620 nm. Both the peptides neutralized LPS suggesting their interaction with lipid A. Cytoplasmic membrane was disrupted within 30 seconds, which correlated with reductions in cellular viability. At 2 minutes melimine or Mel4, released 75% and 36% cellular ATP respectively (P < 0.001). Membrane permeabilization started 5 minutes with simultaneous release of DNA/RNA. Flow cytometry demonstrated 52% and 18% bacteria were stained with PI after 30 minutes. Overall, melimine showed higher capacity for membrane disruption compared to Mel4 (P < 0.001). The findings of this study have been summarized as a timeline of bactericidal activity, suggesting that the peptides permeabilized P. aeruginosa within 5 minutes, started lysis within 2 hours of exposure

Author Correction:Comparative mode of action of the antimicrobial peptide melimine and its derivative Mel4 against Pseudomonas aeruginosa (Scientific Reports, (2019), 9, 1, (7063), 10.1038/s41598-019-42440-2)

An amendment to this paper has been published and can be accessed via a link at the top of the paper

Mode of action of the antimicrobial peptide Mel4 is independent of Staphylococcus aureus cell membrane permeability

Mel4 is a novel cationic peptide with potent activity against Gram-positive bacteria. The current study examined the anti-staphylococcal mechanism of action of Mel4 and its precursor peptide melimine. The interaction of peptides with lipoteichoic acid (LTA) and with the cytoplasmic membrane using DiSC(3)-5, Sytox green, Syto-9 and PI dyes were studied. Release of ATP and DNA/RNA from cells exposed to the peptides were determined. Bacteriolysis and autolysin-activated cell death were determined by measuring decreases in OD620nm and killing of Micrococcus lysodeikticus cells by cell-free media. Both peptides bound to LTA and rapidly dissipated the membrane potential (within 30 seconds) without affecting bacterial viability. Disturbance of the membrane potential was followed by the release of ATP (50% of total cellular ATP) by melimine and by Mel4 (20%) after 2 minutes exposure (p<0.001). Mel4 resulted in staphylococcal cells taking up PI with 3.9% cells predominantly stained after 150 min exposure, whereas melimine showed 34% staining. Unlike melimine, Mel4 did not release DNA/RNA. Cell-free media from Mel4 treated cells hydrolysed peptidoglycan and produced greater zones of inhibition against M. lysodeikticus lawn than melimine treated samples. These findings suggest that pore formation is unlikely to be involved in Mel4-mediated membrane destabilization for staphylococci, since there was no significant Mel4-induced PI staining and DNA/RNA leakage. It is likely that the S. aureus killing mechanism of Mel4 involves the release of autolysins followed by cell death. Whereas, membrane interaction is the primary bactericidal activity of melimine, which includes membrane depolarization, pore formation, release of cellular contents leading to cell death

Action of antimicrobial peptides against bacterial biofilms

Microbes are known to colonize surfaces and form biofilms. These biofilms are communities of microbes encased in a self-produced matrix that often contains polysaccharides, DNA and proteins. Antimicrobial peptides (AMPs) have been used to control the formation and to eradicate mature biofilms. Naturally occurring or synthetic antimicrobial peptides have been shown to prevent microbial colonization of surfaces, to kill bacteria in biofilms and to disrupt the biofilm structure. This review systemically analyzed published data since 1970 to summarize the possible anti-biofilm mechanisms of AMPs. One hundred and sixty-two published reports were initially selected for this review following searches using the criteria 'antimicrobial peptide' OR 'peptide' AND 'mechanism of action' AND 'biofilm' OR 'antibiofilm' in the databases PubMed; Scopus; Web of Science; MEDLINE; and Cochrane Library. Studies that investigated anti-biofilm activities without describing the possible mechanisms were removed from the analysis. A total of 17 original reports were included which have articulated the mechanism of antimicrobial action of AMPs against biofilms. The major anti-biofilm mechanisms of antimicrobial peptides are: (1) disruption or degradation of the membrane potential of biofilm embedded cells; (2) interruption of bacterial cell signaling systems; (3) degradation of the polysaccharide and biofilm matrix; (4) inhibition of the alarmone system to avoid the bacterial stringent response; (5) downregulation of genes responsible for biofilm formation and transportation of binding proteins

Effect of large diameter and plasma coating on the initial adaptation of gas permeable contact lens fitting for neophytes

Purpose To determine whether the adaption of neophytes to rigid gas permeable lenses (GPs) could be accelerated by changing their diameter or surface lubricity. Methods This was a 2 part prospective double-masked randomised wear study. An GP lens (Roflufocon D material, 9.6 mm diameter) with and without a Hydrapeg plasma coating surface was worn by 119 neophytes (21.1 ± 3.5 years; 77% female) on separate occasions. A further 114 neophytes (average age 20.1 ± 1.0 years; 72% female) wore the uncoated lens with a 9.6 mm diameter lens in one randomly allocated eye and 10.1 mm (n = 51) or 10.6 mm (n = 63) in the other with a basecurve compensation keeping the fit equivalent. Lens fit and corneal staining was assessed after 20 min, and comfort and bulbar redness were assessed at the time of lens application and after 5, 10, 15, and 20 min. Participants also reported their ease of application and removal on a 5 point scale. Results Plasma coated GP lenses or increasing diameter did not improve comfort (p = 0.673, p > 0.05) or bulbar redness (p = 0.805, p > 0.05) during a 20 min adaptation period in neophytes. In both cohorts, comfort improved and bulbar redness reduced with time (p 0.05). Conclusion Changing GP diameter or surface lubricity is not beneficial to the adaption of healthy neophyte

Broad spectrum antimicrobial activity of melimine covalently bound to contact lenses

PURPOSE. To develop a stable antimicrobial contact lens, which is effective against the International Organization for Standardization (ISO) panel microorganisms, Acanthamoeba castellanii and drug resistant strains of Pseudomonas aeruginosa and Staphylococcus aureus. METHODS. Melimine was covalently incorporated into etafilcon A lenses. The amount of peptide present on the lens surface was quantified using amino acid analysis. After coating, the heat stability (121°C), lens surface hydrophobicity (by captive bubble), and in vitro cytotoxicity to mouse L929 cells of the lenses were investigated. Antimicrobial activity against the micro-organisms was evaluated by viable plate count and fluorescence microscopy, measuring the proportion of cell death compared with control lenses with no melimine. RESULTS. The most effective concentration was determined to be 152 ± 44 lg lens-1 melimine on the lens surface. After coating, lenses were relatively hydrophilic and were nontoxic to mammalian cells. The activity remained high after autoclaving (e.g., 3.1, 3.9, 1.2, and 1.0 log inhibition against P. aeruginosa, S. aureus, A. castellanii, and Fusarium solani, respectively). Fluorescence microscopy confirmed significantly reduced (P < 0.001) adhesion of viable bacteria to melimine contact lenses. Viable count confirmed that lenses were active against all the bacteria and fungi from the ISO panel, Acanthamoeba and gave at least 2 log inhibition against all the multidrug resistant S. aureus and P. aeruginosa strains. CONCLUSIONS. Melimine may offer excellent potential for development as a broad spectrum antimicrobial coating for contact lenses, showing activity against all the bacterial and fungal ISO panel microorganisms, Acanthamoeba, and antibiotic resistant strains of P. aeruginosa and S. aureus

Confronting global 21-cm signal with Z3\mathbb{Z}_3 symmetric dark matter models

While the $\mathbb{Z}_3$ symmetric dark matter models have shown tremendous prospects in addressing a number of (astro-)particle physics problems, they can leave interesting imprints on cosmological observations as well. We consider two such promising models: semi-annihilating dark matter (SADM) and Co-SIMP $2\rightarrow 3$ interaction, and investigate their effects on the global 21-cm signal. SADM alone cannot address the EDGES dip but can perform better with the aid of an excess radio background, whereas Co-SIMP can naturally explain the EDGES absorption feature by virtue of an intrinsic cooling effect without invoking any such excess radiation. Hence, the latter model turns out to be a rare model within the domain of CDM, that uses leptophilic interaction to achieve the EDGES dip. Further, keeping in mind the ongoing debate between EDGES and SARAS 3 on the global 21-cm signal, we demonstrate that our chosen models can still remain viable in this context, even if the EDGES data requires reassessment in future. We then extend our investigation to possible reflections on the Dark Ages, followed by a consistency check with the CMB and BAO observations via Planck 2018(+BAO) datasets. This work thus presents a compelling case of exploring these interesting particle physics models in the light of different cosmological observations.Comment: 23 pages, 13 sets of figures, 4 tables. Major update: analysis with Neff added. Accepted in JCA