Abstract
Abstract
Purpose:
The aim of this study was to examine the antimicrobial activity and the preservative efficacy of a novel preservative solution containing sodium hydroxymethyl glycinate (SHMG) and edetate disodium (EDTA), which is used for preservation of some commercial ophthalmic formulations.
Methods:
In vitro susceptibility assays were performed against several gram-positive (Staphylococcus aureus, Staphylococcus epidermidis, and Bacillus cereus) and gram-negative (Escherichia coli and Pseudomonas aeruginosa) bacteria representative of the microbial flora of epithelial surfaces or colonizing the conjunctiva, as well as against Candida albicans and Aspergillus niger. Using different concentrations of SHMG alone or in combination with EDTA, the minimal inhibitory and microbicidal concentrations against these organisms were assessed. In addition, 8 brands of multidose eye drops containing 0.002% SHMG and 0.1% EDTA as preservative were tested for antimicrobial activity using the antimicrobial effectiveness test recommended by the international pharmacopoeias.
Results:
The minimal inhibitory and bactericidal/fungicidal concentration values of SHMG ranged from 0.0025% to 0.0125% for bacteria and from 0.125% to 0.50% for mold and yeast. Susceptibility testing demonstrated that the addition of EDTA substantially increased the SHMG activity against all bacterial and fungal strains. The preservative effectiveness test was applied to commercial eye drops. All the drop solutions met the criteria reported by the U.S. Pharmacopeia for parenteral and ophthalmic preparations. All products also satisfied the major acceptance criteria of the European Pharmacopeia with respect to the antifungal activity. With regard to the antibacterial activity, the less-stringent criteria of the European Pharmacopeia were fulfilled.
C
Introduction
The glycine derivative, sodium hydroxymethyl glycinate (SHMG; Suttocide A, International Specialty Products), is frequently used as a preservative for personal-care products, such as cosmetics and liquid detergents for human use. SHMG was placed on the list of permanently approved preservatives by the European Union at levels up to 0.5% without restrictions of use (Directive 76/768/EEC amended by directive 96/41/EC).
Recently, multidose artificial tears containing the mixture of 0.002% SHMG and 0.1% EDTA as a novel preservative have been marketed in Italy. Since these solutions are claimed to have a mild impact on the ocular surface (they are not cytotoxic, irritant, or allergenic in rabbits' eyes: data on file), we set out to evaluate whether different concentrations of SHMG alone or in combination with EDTA could be an efficient preservative for different ophthalmic preparations. These solutions were evaluated for their ocular tolerability and for their antimicrobial activity against a variety of bacterial and fungal strains. In addition, the preservative efficacy of the selected solution of 0.002% SHMG and 0.1% EDTA (SHMG/EDTA) was assayed by recording the death curves of reference strains in the commercial eye drop solutions containing SHMG/EDTA as a preservative.
Methods
Strains and preparation of inocula
The bacterial and fungal strains used in this study were obtained from the American Type Culture Collection (ATCC) or were isolated from clinical samples. The following strains were used: Staphylococcus aureus ATCC 6538, Staphylococcus epidermidis BL2, Bacillus cereus ATCC 14579, Escherichia coli ATCC 8739, Pseudomonas aeruginosa ATCC 9027, Candida albicans ATCC 10231, and Aspergillus niger ATCC 16404.
Bacterial strains were routinely grown either in the Mueller–Hinton broth (MHb; Oxoid) or on Mueller–Hinton agar (MHa; Oxoid) containing Ca2+ (100 μg/mL) and Mg2+ (35 μg/mL) at 37°C. Bacterial inocula were prepared from log-phase cultures in MHb by diluting bacteria in phosphate-buffered saline (pH 7.4). Fungal strains were grown either in the Sabouraud broth (BioMeriéux) or on Sabouraud dextrose agar (SDA; BioMeriéux) at 30°C. For the preparation of the inocula, C. albicans and A. niger were grown at 30°C on SDA for 48 h and 1 week, respectively. Sterile saline (0.85%) was added to the plates, and A. niger cultures were gently swabbed with a Pasteur pipette to dislodge the spores from the hyphal mat. The fungal suspensions were then transferred to a sterile tube.
Active compounds and commercial products
SHMG (Suttocide A®; International Specialty Products) as a 50% aqueous solution and a 20% solution of EDTA (Sigma) in sterile distilled water were the stock solutions used. The following commercial eye drops containing 0.002% SHMG and 0.1% EDTA as a preservative were used: BLUgel®, BLUsal®, BLUyal®, IRIDIUM®, TRIUM® (all from Sooft Italia srl); NEXT 300®, NEXT 500®, and TIORETIN® (all from Bioos Italia srl).
Susceptibility testing
For in vitro susceptibility testing, all microbial suspensions were adjusted to 0.5 MacFarland standard for 80%–85% transmission using a Vitek colorimeter (BioMerieux) and diluted appropriately as requested by the assay. The inocula were retrospectively quantitated by seeding serial dilutions of bacterial suspensions on MHa or dilutions of fungal suspensions on SDA and counting the CFUs after incubation at 37°C or 30°C. To assess the antimicrobial activity of SHMG alone or in association with 0.1% EDTA, the minimal inhibitory concentrations (MICs) against bacterial strains were determined using the CLSI broth-microdilution method with MHb. 3 For fungal testing, a 3-(N-morpholino)propane sulfonic acid-buffered RPMI 1640 medium was used.4,5 MIC endpoints were read as the lowest SHMG concentration totally inhibiting macroscopically visible growth of the inoculum. The minimal bactericidal (MBC) and fungicidal concentration (MFC) were determined by the methods published by the CLSI. 6 All susceptibility tests were performed in triplicate.
Preservative effectiveness test
The antimicrobial effectiveness test was performed by inoculating commercial eye drop solutions with microbial suspensions to obtain the initial concentration of 105–106 CFU/mL of each organism. These mixtures were stored without shaking at 21°C±2°C, and the samples were withdrawn from the containers at specified time intervals for enumeration of the test organisms. The organisms used for testing were P. aeruginosa, E. coli, S. aureus, C. albicans, and A. niger as described in the U.S. Pharmacopeia 7 (USP) and European Pharmacopoeia (EP) 8 . Immediately after inoculation and at different time intervals (24 h, 7, 14, 21, or 28 days), 0.1 mL of serially diluted test samples was plated in duplicate on tryptic soy agar (TSA; Becton Dickinson) for the bacteria and on SDA for the yeast and mold. The TSA plates were incubated at 37°C for 24–48 h, and the SDA plates were incubated at 30°C for 3 to 7 days. After incubation of the plates, the CFUs were counted, and the base 10 logarithm (log10) for the average of the 2 plates was determined.
Results
Antimicrobial activity of SHMG
Selected bacterial and fungal strains frequently causing eye infections were screened for their sensitivity to SHMG. For this, in vitro susceptibility testing of S. aureus, S. epidermidis, B. cereus, E. coli, P. aeruginosa, C. albicans, and A. niger was performed using SHMG concentrations ranging from 5% to 0.0005%. The MIC and MBC/MFC values were identical and amounted to 0.0025% SHMG for S. aureus, S. epidermidis, E. coli, and P. aeruginosa. Against B. cereus, the MIC and MBC values were 0.0125% and 0.025%, respectively. C. albicans and A. niger were much less sensitive to the SHMG activity, with thMICs for SHMG of 0.25% and 0.125% and the MFC values of 0.5% and 0.25%, respectively.
To evaluate whether the antimicrobial activity of SHMG could be increased by the addition of EDTA, all the above-mentioned strains were tested for susceptibility using different concentrations of SHMG supplemented with 0.1% EDTA. As shown in Fig. 1, the addition of EDTA substantially increased the SHMG activity against all bacterial and fungal strains, both those required by the pharmacopeias, and 2 more strains (S. epidermidis and B. cereus) known to be pathogenic for the ocular surface. In fact, the concentrations of SHMG required to inhibit microbial replication were 0.0015% for all gram-positive bacteria (B. cereus, S. aureus, and S. epidermidis), 0.003% for C. albicans, and 0.005% for all gram-negative bacteria (E. coli and P. aeruginosa) and A. niger, and a 0.025% concentration was sufficient to kill all the tested organisms.
Antimicrobial activity of sodium hydroxymethyl glycinate (SHMG) added with 0.1% edetate disodium. Gray bars: minimal inhibitory concentration; white bars: minimal bactericidal or fungicidal concentration.
We also tried to decrease the EDTA concentration to 0.05%, increasing the SHMG concentration to either 0.007 or 0.01% to maintain an efficient antimicrobial activity. However, preliminary testing on healthy human volunteers showed that these latter associations were poorly tolerated after topical administration, most likely because of the increased SHMG concentration. Therefore, we decided to pursue further studies only with the association containing EDTA 0.1% and SHMG 0.002%.
Preserving efficacy of the SHMG/EDTA mixture
To assess whether the mixture containing 0.002% SHMG and 0.1% EDTA (SHMG/EDTA) was effective in preserving commercial eye drop formulations, 8 commercial multidose eye drops containing SHMG/EDTA were tested for their antimicrobial activity by the preservative effectiveness test. Tables 1 and 2 show how the microbial concentration (expressed as log10 CFU/mL) changes with time for all the commercial products deliberately inoculated with each of the test organisms at a concentration of 105–106 CFUs/mL. All eye drop solutions met the criteria reported by the USP 7 for parenteral and ophthalmic preparations, as more than a 1-log and 3-log reduction in the counts of all bacterial strains was registered after 7 and 14 days of incubation, and no bacteria were recovered from day 14 to day 28 (Table 1). In addition, no increase in the fungal concentration was ever found in all tested solutions, and no residual fungi were recovered at day 21 and 28 (Table 2). All products also satisfied the major acceptance criteria (A criteria) of the EP 8 with respect to the antifungal activity (Table 2). With regard to the antibacterial activity, the less-stringent B criteria, but not the A criteria, of the EP 8 were fulfilled (Table 1). In fact, more than a 1-log and 3-log reduction in bacterial counts was found for all test bacteria at day 1 and 7, and no bacteria were present in all solutions from day 14 to day 28 (Table 1).
USP, U.S. Pharmacopoeia; EP-A, European Pharmacopoeia A criteria; EP-B, European Pharmacopoeia B criteria; nr, no recovery; ni, no increase.
EDTA, edetate disodium.
Discussion
The microbiological safety of a pharmaceutical product has always been of special interest for industries, as microbial contamination can lead to product degradation or constitute a risk for the health of the consumer and potentially spread the infection. Regarding eye drops, it has been shown that preservative-free preparations in multiple application containers are at risk of contamination by microbial pathogens, thus potentially placing some patients at an increasing risk of developing serious ocular infections. 9 Although preservatives added to ocular products are aimed at reducing microbial contamination at the levels demanded by regulatory bodies, several reports have shown that the incidence of microbial contamination for preserved eye drops may vary from 5.6% to 37%.10–13 This is probably due to poor availability of data regarding the activity and suitability of preservatives currently used in ophthalmology. 14
Starting from the observation that some multidose artificial tears that are marketed in Italy contain a novel preservative made up of a mixture of 0.002% SHMG and 0.1% EDTA (SHMG/EDTA), we systematically evaluated for the first time the antimicrobial activity of SHMG alone or in combination with 0.1% EDTA (decreasing EDTA and increasing the SHMG concentration led to poor tolerability of the solution) against bacteria and fungi frequently found in the periocular area and most commonly involved in the ocular infections. As described by the CLSI, 6 in vitro susceptibility testing was performed in a nutrient-rich culture medium, a condition mimicking the addition of the compounds to drug solutions containing the factors favoring microbial growth. We demonstrate that SHMG shows a wide bacteriostatic activity at concentrations ranging from 0.0025% to 0.0125% and that higher concentrations of SHMG (0.25% or 0.5%) are needed for the fungistatic activity against C. albicans and A. niger. The addition of 0.1% EDTA to SHMG substantially reduces the minimal concentration of SHMG required for both bacteriostatic/fungistatic and bactericidal/fungicidal activity in vitro. In fact, in this condition, 0.005% and 0.025% concentrations of SHMG were, respectively, sufficient to inhibit growth or to kill all tested organisms.
The efficiency of SHMG/EDTA in protecting selected commercial artificial tears against contamination with accidentally introduced microorganisms was evaluated by the preservative effectiveness test (challenge test) outlined in the United States and EPs.7,8 The antimicrobial activity of SHMG/EDTA was shown to depend on the composition of the artificial tears and was different for the diverse organisms tested. However, all the tested eye drops met the USP and EP-B criteria, but not the more-stringent EP-A. Previous studies document that eye preparations containing a wide range of preservatives can fail to meet the major EP-A criteria for antimicrobial efficacy. 14 In fact, in addition to the preservative activity, other factors are considered in the choice of a preservative for ophthalmic use. Preservatives can produce adverse effects on eye tissues, such as toxic or allergic reactions. 2 For this reason, monographs of the EP allow the use of the less-stringent B criteria for antimicrobial efficacy, where these can be justified. The manufacturer's choice of using SHMG/EDTA for preservation of the tested eye drops was probably driven by the balance between the preservative activity and ocular toxicity. In fact, among different preservatives, EDTA has shown the least toxic activity on ocular surface epithelial cells.15,16 Therefore, based on the results obtained by the susceptibility tests in a liquid medium, we can suggest that 0.005% SHMG and 0.1% EDTA could be safely used as a preservative for pharmaceutical products, including ophthalmic solutions, containing components favoring bacterial or fungal growth.
Footnotes
Acknowledgments
Special thanks to Dr. Giulio Luciani for his expert scientific assistance. This study was supported by the research grants from the University of Pisa and Sooft Italia srl, Italy. Critical reading of the manuscript by Dr. Dario Rusciano and Dr. Antony Bridgewood is also acknowledged.
Author Disclosure Statement
No competing financial interests exist.