Corneal infection is a serious ocular complication associated with contact lens wear. An almost constant rate of microbial keratitis has been associated with contact lens wear from 1989 to 2004.1–3 Risk factors for developing microbial keratitis include male gender, smoking, no or infrequent lens disinfection, use of chlorine- or heat-based disinfection systems, having diabetes, no surfactant in the disinfecting solution or no use of rub/rinse step during lens cleaning/disinfection, noncompliance with the hygiene regime including “topping off” old used solution in cases with new solution, poor case hygiene, and solutions that encourage encystment of Acanthamoeba sp.4–10 Thus, there is a need to investigate lens care solutions and perhaps introduce newer disinfecting agents to improve the rate of contact lens–related microbial keratitis.
Currently, millions of people worldwide use multipurpose disinfecting solutions (MPDS) or a single solution for disinfecting, cleaning, rinsing, and storing contact lenses to minimize the risk of contamination of contact lenses during wear and overnight storage. The rate of use of MPDS has gradually increased over the last 10 years to account for >90% of disinfection types in the UK or Australia.11,12 Typically, there are a number of cationic disinfectants such as biguanides (alexidine, chlorhexidine, hexamethylene biguanides, and polymeric biguanides, i.e. polyhexamethylene biguanide [PHMB]), quaternary ammonium compounds (polyquaternium-1 [POLYQUAD] and cetylpyridinium chloride [CPC]), and myristamidopropyl dimethylamine (ALDOX) used due to their high binding affinity to the membranes of microorganisms. Interaction of the cationic compounds with the membrane of microorganisms causes irreversible loss of essential cellular components by displacing divalent cations such as calcium and magnesium.13,14 Simple chelating agents such as ethylenediamine tetra-acetic acid (EDTA) and its salts found in most MPDS products chelate cations in cell membranes and enhance the antimicrobial effectiveness of the primary disinfectant. In addition, a suitable buffer system supplemented with isotonic agents, stabilizers, and surfactants is used in the formulations of MPDS to optimally clean the lens surface and to enhance the comfort of the eye.
Performance of MPDS is specified by various national and international standards. According to the International Organization for Standardization (ISO) standard for contact lens care solutions (14729), a disinfecting solution should meet the primary stand-alone criteria in which starting concentrations of specified bacteria and fungi are reduced by mean values of not less than 3 and 1 logs, respectively, within a specified disinfection time, or the solution must meet the regimen criteria (reduction in numbers of microbes adherent to contact lenses). However, it has been found that many available MPDS do not perform optimally under field conditions where different types of organisms are encountered. Moreover, the literature shows that several commercially available products do not meet the ISO standards for all strains of bacteria, or other types of microbes, even if they do for the American Type Culture Collection strains outlined in the standards.15–20
A naturally occurring polycationic peptide, protamine, is found in spermatic cells of many animal species.21,22 Protamine has broad spectrum of antimicrobial activity, being active against a range of Gram-positive and Gram-negative bacteria and fungi.23–25 Protamine, along with other cationic substances, disrupts the cell wall of microorganisms resulting in their lysis14 and expulsion of intracellular components of both Gram-positive and Gram-negative bacteria.26,27 Protamine also inhibits phosphorylase activity28 and affects cellular functions of bacteria.29 Moreover, protamine has been used safely in humans as an anti-heparin agent in blood products.30 Therefore, the broad antimicrobial spectrum properties of protamine and its safety in humans make it a promising antimicrobial agent to be used in formulating MPDS. It has been shown that a protamine concentration of 228 μM, in combination with EDTA and PGMB, can produce >2 log reductions in viable trophozoites and >0.5 log reduction in numbers of viable cysts of Acanthamoeba strains.31
The purpose of this study was to evaluate the antimicrobial efficacy of protamine using the stand-alone method recommended by the ISO 14729 standard for Contact Lens Care Products. The synergistic effect of protamine in combination with PHMB and EDTA, and in vitro cytotoxicity of protamine were determined. Additionally, the effect of cation concentration in the solution on efficacy of protamine against microorganisms was investigated.
MATERIALS AND METHODS
Challenge Microorganisms and Growth Conditions
The ISO recommended five panel microorganisms and a range of clinical isolates (Table 1) were grown and prepared as described by the ISO 14729 for testing the efficacy of multipurpose disinfectants. Briefly, the bacterial strains were grown on Trypticase Soy Agar (TSA; Oxoid Sydney, Australia) at 37 °C for 18 to 24 hours. Candida and Fusarium strains were grown on Sabouraud’s Dextrose Agar (SDA; Oxoid) for 18 to 24 hours at 37 °C and Potato Dextrose Agar (PDA; Oxoid) for 10 to 14 days at 25 °C, respectively. The microorganisms were harvested in sterile phosphate buffered saline (PBS; 8 g/l NaCl (0.8%), 0.2 g/l KCl, 1.15 g/l Na2HPO4, 0.2 g/l KH2PO4, pH 7.3) containing 0.05% v/v polysorbate (PBST). The suspensions of Candida and Fusarium were filtered through sterile 40 and 70 μm cell strainers (BD-Falcon, Erembodegem, Belgium), respectively, to produce single cell dispersions and remove hyphae. The final inoculums of all cell suspensions were adjusted to a concentration of 1.0 × 108 colony forming units per milliliter (CFU/ml; OD of 0.1 at 660 nm) in sterile PBST.
Preparation of Test Solutions
Protamine isolated from salmon spermatozoa (molecular weight 4381 g/mol; Sigma St. Louis, MO), ethylenediamine tetra-acetic acid (EDTA; Sigma), and polyhexamethylene biguanide (PHMB; Dayang Chemicals Co, Hangzhou, China) were prepared as 10% (w/v), 0.05% (w/v), or 0.01% (w/v) stock solutions, respectively, in distilled water and filter sterilized using 0.02 μm polycarbonate filters (Millipore Australia Pty Ltd., North Ryde, NSW, Australia). Protamine, EDTA, and PHMB stock solutions were stored in sterile glass bottles at 4 °C (for protamine) or ambient temperature (for PHMB and EDTA). Test solutions included (1) PBS containing various concentrations (22.8, 57.1, 144.1, 171.2, and 228.3 μM) of protamine, (2) PBS containing 144.1 μM of protamine and 1.5 mM EDTA, or (3) PBS containing 144.1 μM of protamine and 1.5 mM EDTA with 0.0001% (w/v) PHMB. Controls included PBS alone, PBS containing 0.0001% (w/v) PHMB, or PBS containing 1.5 mM EDTA. A nonionic surfactant Pluronic F87 (BASF, Mt. Olive, NJ; 0.05% v/v) was used in all the solutions except the PBS-alone control. The influence of cations on antimicrobial efficacy of protamine was also evaluated using PBS but containing different concentrations of NaCl (0.8, 0.4, or 0.2% (w/v)) alone or in combination with protamine/EDTA/PHMB.
Antimicrobial Efficacy of Test Solutions
The antimicrobial performance of the various solutions was evaluated using the stand-alone test described by the ISO 14729. Briefly, 100 μl of each challenge microorganism were inoculated into 1 ml test solutions (final concentrations for bacteria and fungi: 1 × 106 CFU/ml) in polypropylene tubes. After incubation at 25 °C for 6 hours, the numbers of surviving bacteria or fungi in the solutions were determined by plating onto TSA containing 0.07% lecithin and 0.5% polysorbate 80 (TSAN; Sigma) for bacteria or PDA plates for fungi after serial 1:10 dilution in Dey-Engley neutralizing broth (DE; Becton Dickinson, Franklin, Lakes, NJ). The average reduction in log colony forming units in test solution versus control (PBS) for each strain was calculated after incubation at 37 °C for 18 hours for bacteria and 25 °C for 48 hours for fungi. All the experiments were repeated at least three times and the average log reduction for each test strain was calculated.
Evaluation of Cytotoxicity of Protamine Solution
Potential cytotoxicity of test solutions was assessed by agar overlay and direct contact cytotoxicity assays as described in ISO 10993-5 1999 standard8 for in vitro cytotoxicity assessment for medical devices. The agar overlay assay, which is designed to determine the cytotoxicity of diffusible compounds, was performed using filter discs which were saturated with combinations of PBS, protamine, PHMB, and EDTA (Table 2). After 24 hours of exposure, in vitro responses of murine fibroblast cells (American Type Culture Collection L-929) were analyzed according to a standard key, in which the zonal extent of cell damage is graded on a scale of 0 to 3. Reactivity grades above 1 indicate cytotoxicity under the conditions stipulated. PBS alone (negative control) and 5% v/v ethanol in PBS (positive control) were also included.
Before direct contact assay, three lenses of each balafilcon A (Bausch & Lomb, Rochester, NY) or etafilcon A (Johnson & Johnson Vision Care, Jacksonville, FL) materials were washed twice in 3 ml PBS and then soaked with 3 ml of each test solution (Table 2) for 72 hours at 25 °C. Lenses were chosen as these are silicone hydrogel lens and hydrogel lens, both of which are negatively charged and therefore likely to interact substantially with the positively charged protamine. The lenses were then washed in 2 ml PBS and transferred into a sterile glass vial containing 2 ml of new tests solutions. The direct contact cytotoxicity was tested by placing the test lenses directly on the Murine L929 cell monolayer for a 24-hour period. Cultures were stained with a vital stain (Trypan Blue) and cytotoxicity assessed using bright-field and phase-contrast microscopy. Cytotoxic responses were graded according to a standard key, which quantifies the zonal extent of cell damage (0 to 4 maximum). Controls used were silastic medical grade tubing (Dow Corning Corporation, Midland, MI) as a negative control and samples of surgical latex gloves (Ansell Medical, Victoria, Australia) as positive control. All tests were performed three times, and the median values are reported.
For statistical comparison, a log transformation was applied to the colony forming units (CFU) to produce data appropriate for a parametric analysis. Univariate analysis of variance (UNIANOVA) was performed to assess the interaction between the test products, test strains, and test types. Post hoc testing via Tukey equal variance comparison was performed to assess the multiple differences. Statistical significance was set at p <0.05.
For all microorganisms, protamine produced a progressive dose-dependent killing effect on test strains. In comparison to control (PBS), the mean log reduction for all ISO 14729 panel isolates increased with increasing concentrations of protamine (p < 0.05). Further, ≥6 log decrease in the number of cells of Serratia marcescens ATCC13880, Pseudomonas aeruginosa ATCC9027, and Staphylococcus aureus ATCC6538 occurred at a protamine concentration of 228.3 μM. The antimicrobial activities of protamine at 144.1 μM or below were higher against P. aeruginosa than S. aureus and S. marcescens (p < 0.05). A solution of 144.1 μM protamine showed complete killing of P. aeruginosa and 3.0 ± 0.5, 2.4 ± 0.75, 1.5 ± 0.01, and 1.0 ± 0.05 log reductions against S. aureus, S. marcescens, Candida albicans, and Fusarium solani, respectively, after 6 h of disinfection (Fig. 1). At concentrations of 171.2 μM protamine and above, there was greater kill of bacteria than fungi (p < 0.05). Error bars are not given in the figure for ease of view. For all data, the standard deviation was ≤0.5 log.
The average log reductions in response to protamine either alone or in combination with EDTA and PHMB are shown in Fig. 2. In these experiments, a constant concentration of protamine (144.1 μM) was used to be able to detect an increase or decrease in microbial killing. The inhibitory effect of 144.1 μM protamine solution containing 1.5 mM EDTA against most microorganisms, with the exception of P. aeruginosa, was significantly (p = 0.02) higher than the solution without EDTA and displayed 4 ± 0.09, 4 ± 0.47, 2.4 ± 0.41, and 1.9 ± 0.14 log reductions against S. aureus, S. marcescens, C. albicans, and F. solani, respectively, within 6 hours of disinfection time. Protamine in combination with 0.0001% PHMB and 1.5 mM EDTA showed the highest microbicidal effect for all microbes except P. aeruginosa. Adding PHMB and EDTA to protamine showed complete inhibition of growth of all three ISO 14729 panel bacterial strains tested. The synergistic effect of EDTA with quaternary ammonium disinfectants (similar to PHMB) has been described previously.32 The bactericidal activity of 0.0001% PHMB alone ranged from 3 ± 1 logs for S. aureus to 1 ± 0.5 log for S. marcescens, and the bactericidal activity of 1.5 mM EDTA ranged from 0.1 ± 0.5 log for S. marcescens to no effect on S. aureus. The combination of protamine/EDTA/PHMB displayed a significant synergy in comparison to protamine alone (p < 0.05) for F. solani ATCC 36031 and C. albicans ATCC 10231 and achieved >2 log reductions in cell numbers of these fungi. The fungicidal activity of PHMB alone was relatively low, giving log reductions ranging from 0.7 to 0.95 ± 0.25. EDTA alone showed little or no antimicrobial activity against the test strains, giving average of log reductions ranging from 0 to 0.35 ± 0.13.
The efficacy of 144.1 μM protamine alone and in combination with 0.0001% PHMB and 1.5 mM EDTA was further evaluated against seven strains of clinical isolates (Fig. 3). The data showed that the combination (protamine/EDTA/PHMB) completely killed the clinical strains of S. marcescens, Stenotrophomonas maltophilia, and Delftia acidovorans, and the activity was significantly superior to the solution of protamine alone (p < 0.05). Similarly, the same combination also showed excellent and significantly higher efficacy against the clinical strains of C. albicans (001 and 002) compared with the protamine alone solution, displaying >5 and >3 log reductions after 6 hours of disinfection, respectively (Fig. 3; p < 0.01). The protamine/EDTA/PHMB combination did not increase the >3 log kill of the methicillin-resistant S. aureus strain 060.
The effect of Na+ on the activity of protamine was investigated by adding various concentrations of NaCl to PBS, and the results are shown in Fig. 4. The antimicrobial activity of protamine against tested microorganisms was increased in the presence of the low concentration of NaCl (0.2% w/v). There was no growth of challenge stains of S. aureus, S. marcescens, and P. aeruginosa in the solution containing 144.1 μM protamine in the presence of 0.4% NaCl. The solution reduced the level of viable C. albicans and F. solani by 3.0 ± 0.09 and 1.5 ± 0.47 log units, respectively, within 6 hours of disinfection time. The controls of the NaCl concentrations alone indicated that there was no influence of the salt concentration on the growth of microorganisms.
Cytotoxicity of the Protamine Formulations
The cytotoxicity of each formulation was determined by agar overlay and direct contact assays in vitro. Both assays displayed no evidence of cytotoxicity as a result of exposure to the each test solutions compared with the negative control, whereas the positive control demonstrated high cytotoxicity (Table 2).
In this study, both the antimicrobial activity and cytotoxicity of solutions containing protamine were evaluated according to the ISO 14729 and ISO 10993-5 guidelines. The results demonstrated that protamine was effective against a range of microorganisms including the panel microorganisms recommended in the ISO 14729 and clinical isolates. The solution containing 144.1 μM protamine in combination with 1.5 mM EDTA and 0.0001% PHMB was found to meet or exceed the stand-alone test criteria, giving at least 3 log and 1 log reduction of bacteria and fungi, respectively, as recommended in the ISO 14729 guidelines within 6 hours of disinfection time. A disinfection time of 6 hours was chosen in the current study as this time or less is recommended for most multipurpose disinfecting solutions.15–20 This combination showed higher antimicrobial activity than that of protamine alone. The activity of the formulation containing protamine/EDTA/PHMB was increased when the concentration of NaCl was decreased. In addition, protamine had a minimal toxicity.
As the current study used the standard ISO 14729 test and the recommended microbes along with that test, it is possible to compare the results obtained to those reported for commercially available multipurpose disinfecting solutions. The single disinfectant containing solutions ReNu MultiPlus (Bausch + Lomb, Rochester, NY, now called ReNu fresh, containing 0.0001% PHMB and 3 mM EDTA), Complete Moisture Plus (Advance Medical Optics, Santa Ana, CA; 0.0001% PHMB, EDTA (concentration not given)), Solo-Care Aqua (CIBA Vision, Atlanta, GA; 0.0001% PHMB, 0.7 mM EDTA), and MeniCare Soft (Menicon, Nagoya, Japan; 0.0001% PHMB, EDTA (concentration not given)) gave >4.5 log reductions against the ISO strains of S. marcescens, P. aeruginosa, and S. aureus 17,33–36 after 4 to 6 hours (depending on the manufacturer’s recommendation) of disinfection time. These data compare favorably with the current data on the protamine/EDTA/PHMB solution. Similarly, the dual disinfectant containing multipurpose solutions such as Opti-Free Express (Alcon laboratories, Fort Worth, TX; 0.001% polyquaternium, 0.0005% myristamidopropyl dimethylamine, EDTA) and Opti-Free Replenish (Alcon laboratories; 0.001% polyquaternium-1, 0.0005% myristamidopropyl dimethylamine, nonanoyl EDTA) also show high disinfectant activity (>4.5 log) against the ISO bacterial strains, similar to the current protamine/EDTA/PHMB solution.35,36 Note that the concentrations of excipients other than disinfectants in multipurpose disinfecting solutions are often not disclosed by manufacturers.
Differences in antimicrobial activity of multipurpose disinfecting solutions are usually most evident when their activity is assessed against fungal strains and clinical isolates of bacteria or fungi. Kilvington et al.35 demonstrated that the antifungal activity of various single or dual disinfectant multipurpose solutions against the ISO strains of C. albicans and F. solani varied from 1.5 log (Solo-Care Aqua; C. albicans) to 4.4 log (Biotrue Bausch + Lomb; 0.0001% polyquaternium, 0.00013% PHMB, EDTA; C. albicans), whereas Santodomingo-Rubido et al.36 found for the same ISO fungal strains that the least active MPDS was Complete Moisture Plus (<1.0 log against F. solani) and the most active was Opti-free Express (>4 log against C. albicans). Similarly, Hume et al.20 found that Complete Moisture Plus was the least active (0.4 log), but ReNu MoistureLoc (Bausch + Lomb; 0.00045% alexidine, EDTA) was the most active (5.1 log) against the ISO F. solani strain. The protamine/EDTA/PHMB multipurpose disinfecting solution of the current study showed >2 log reductions for both the ISO fungal strains, indicating an acceptable level of disinfection according to the ISO standard. Several studies have found that microbial isolates more recently isolated than the standard ATCC strains and from clinical infections are often more resistant to the action of multipurpose disinfecting solutions.17,20,33,37,38 The current study demonstrated that the protamine containing disinfectant solution gave at least 3 log reductions (and usually >4 log; Fig. 3) when tested against clinical isolates of several bacteria and fungi.
Cationic antibacterial agents bind with high affinity to the negatively charged cell membranes of bacteria and displace divalent cations in the membranes causing the loss of essential cellular components.13 The effectiveness of cationic antibacterial agents may be affected by the presence of excess cations. Previous data have shown that the antimicrobial activity of human β-defensin 2 against ocular pathogens is sensitive to the presence of salt content in human tears.39 In the present study, the results also demonstrated that the antimicrobial activity of protamine decreased when the concentration of salt (NaCl) in the medium increased, but remained adequate, according to ISO standards, even at 0.8% NaCl. Unlike the naturally occurring defensins and other cationic peptides in tears, protamine in an MPDS does not necessarily need to be formulated in the presence of low salt, as long as the formulation is made at an osmolality that does not affect the contact lens itself (e.g. its size or shape) or have any adverse effect on the ocular surface.
Protamine, a naturally occurring cationic peptide, has a long history of use and general safety in humans.30 PHMB is very well known for its antimicrobial properties and is used as a disinfectant for contact lens solution. The protamine/EDTA/PHMB solution is effective as an anti-Acanthamoeba solution and does not induce encystment of these protozoa.31 This study highlights the potential for protamine to be used for development of effective and nontoxic disinfection solution for soft contact lenses as it also demonstrates effective antibacterial and antifungal activity, while showing no cytotoxicity to mammalian cells. However, further investigations such as stability, compatibility with contact lenses, and in vivo toxicity are warranted in future studies of novel MPDS for contact lenses.
Mark D. P. Willcox
School of Optometry and Vision Science
University of New South Wales
Sydney, NSW 2052
Received December 15, 2015; accepted April 28, 2016.
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