Optometry & Vision Science:
Efficacy of Contact Lens Multipurpose Solutions Against Serratia Marcescens
HUME, EMMA B. H. PhD; ZHU, HUA PhD; COLE, NERIDA PhD; HUYNH, CAFA BOptom; LAM, SHIRLEY Boptom; WILLCOX, MARK D. P. PhD
Institute for Eye Research, Sydney, New South Wales, Australia (EBHH, HZ, NC, MDPW), School of Optometry and Vision Science, The University of New South Wales, New South Wales, Australia (EBHH, CH, SL), and Vision Cooperative Research Centre, Sydney, New South Wales, Australia (EBHH, HZ, NC, MDPW)
Vision CRC is an Australian Commonwealth funded cooperative research center and under its conditions of funding Vision CRC is required to commercialize its research. As part of that commercialization activity, Vision CRC receives royalty income from the sale of silicon hydrogel contact lenses sold by Bausch & Lomb and CIBA Vision. The Institute for Eye Research (IER) is a not for profit research corporation that is a core participant in Vision CRC and its employees are entitled to benefit from such royalties.
Received August 15, 2006; accepted October 20, 2006.
Purpose. To compare the susceptibilities of clinical isolates of Serratia marcescens and the standard ISO ATCC 13880 strain to five contact lens multipurpose disinfection solutions (MPDSs).
Methods. Five commercially available MPDSs, containing either a polymeric biguanide or polyquaternium, were tested using ISO/CD 14729 stand-alone test for contact lens care products against four ocular isolates of S. marcescens and the strain ATCC 13880. An average log reduction in bacterial numbers at the manufacturer's minimum recommended disinfection time was determined and compared with the criteria for stand-alone disinfection products for each MPDS against each bacterial strain.
Results. All the MPDSs tested met the stand-alone criteria of 3-log reduction of viable bacteria against the ATCC strain of S. marcescens. However, there was more variability in their ability to meet disinfection criteria when tested against the clinical isolates. Two of the clinical isolates were significantly more resistant to disinfection than was the recommended ISO strain (p ≤ 0.034). Two of the polyquaternium-1-based disinfection solutions (solutions D and E, p ≤ 0.005) were less effective overall than the other MPDSs against S. marcescens.
Conclusions. The importance of strain selection for the testing of MPDSs is indicated, and the use of a single laboratory strain may be insufficient to provide assurance that the disinfection solution will be effective against clinical isolates. Furthermore, clinical isolates of S. marcescens may show increased resistance to disinfection with polyquaternium.
Serratia marcescens has been implicated in a range of ocular adverse events1 including endopthalmitis,2 keratoconjunctivitis,3 contact lens related keratitis,3–8 during orthokeratology lens wear,9,10 non-contact lens related keratitis,11–15 and contact lens induced acute red eye (CLARE).16
The frequency of contact lens related corneal infection by S. marcescens has increased significantly over time.17 The incidence rate of S. marcescens related keratitis ranges from 5% to 28%18; the upper range is comparable to the isolation rate of Pseudomonas aeruginosa in contact lens related corneal infections.6 Historically, S. marcescens has shown resistance to contact lens disinfection solutions.19,20 Strains of S. marcescens have been shown to become adapted to chlorhexidine and benzalkonium chloride-based disinfecting solutions,21,22 and to be able to grow in the presence of chlorhexidine.19,21,23 Although hydrogen peroxide-based disinfection solutions are very effective at killing bacteria, S. marcescens was found to persist in contact lens storage cases for up to 6 days after using a neutralized peroxide disinfection system.24,25 This resistance to disinfection may contribute to the increase in frequency of S. marcescens-associated ocular infections.
The prolonged survival of S. marcescens in disinfection solutions was not observed with the newer chemical disinfection formulations based on biguanides or polyquaterniums in studies conducted against a single conjunctivitis isolate.19 However, using standard International Organization for Standardization (ISO) test strains and methodology, it has been demonstrated that a polyquaternium-1-based disinfection solution failed to give a 3-log reduction in viable bacterial numbers and so did not meet the ISO criteria for stand-alone disinfection,26 although these tests used a shorter time than the manufacturer's minimum disinfection time. Recently, six solutions, including those with newer formulations, were tested for their efficacy against the panel of organisms recommended in the ISO.26 They found that ReNu MoistureLoc had marginal efficacy against S. marcescens.26 In view of these findings, along with the lack of information regarding the efficacy of these solutions against clinical strains, there is a need to examine the efficacy against these isolates, using the ISO-recommended protocol. In this study, we have determined the susceptibilities of clinical isolates of S. marcescens in comparison with the standard ISO strain ATCC 13880 to five contact lens multipurpose disinfection solutions (MPDSs).
MATERIALS AND METHODS
Five isolates of S. marcescens were used as described in Table 1. Stock cultures of S. marcescens were stored in 30% glycerol at −70°C. Bacteria were cultured on trypticase soy agar (Becton Dickinson and Company, Franklin Lakes, NJ) at 35°C for 18 to 24 h. Bacteria were suspended in phosphate buffered saline and adjusted turbidometrically to an optical density of 0.1 at 660 nm, which is an equivalent concentration of 1 × 108 colony-forming units per ml (CFU/ml). The inoculum was confirmed retrospectively by viable counts.
Comparison of Susceptibility to Disinfection
The activities of commercially available MDPSs, described in Table 2, were evaluated using the stand-alone method recommended by ISO.27 All solutions were used within the expiry date and discarded 1 month after opening. Briefly, 10 μl of the above bacterial suspensions were inoculated into 1 ml MPDS in eppendorf tubes made of polypropylene, making the final concentration of bacteria 1 × 106 CFU/ml. The initial inoculum always fell with the range of 1 × 105 to 1 × 106 CFU/ml. The inoculated solutions were incubated at 25°C for the manufacturer's minimum recommended disinfection time (MRDT) stated on the product insert (either 4 or 6 h, Table 2), and samples were removed for analysis after this time. The numbers of viable bacteria in the samples were determined by plate counts after serial 1:10 dilutions in Dey Engley neutralization broth (Dey Engley, Difco, Becton Dickinson, North Ryde, Australia), plating in triplicate onto trypticase soy agar plates containing 0.07% asolectin and 0.5% polysorbate 80, and incubation at 37°C for 18 h. Experiments were performed using triplicate samples a minimum of four times with a minimum of three different lot numbers, and the average log reduction for each MPDS and bacterial strain was calculated.
Differences between MPDSs and between strains were determined using repeated measures of analysis of variance (Brown Forsythe). Factors with greater than two categories underwent post hoc testing using the Bonferroni multiple comparisons test. Data were considered significant if p < 0.05.
The mean log reductions at the MRDT for each of the multipurpose solutions against all S. marcescens isolates are reported in Table 1 and Figs. 1 and 2. Overall, two clinical isolates (Smar 5 and Smar 35) were significantly more resistant to MPDS than were the other isolates (p ≤ 0.034).
All five solutions tested achieved at least a 4.5-log reduction in bacterial numbers when tested against the recommended standard S. marcescens strain ATCC 13880 (Fig. 1A). None of the solutions were significantly different in their performance, and therefore, all solutions met the current ISO criterion that stipulates the use of this strain.
Similarly, all five solutions tested achieved at least a 4-log reduction in bacterial numbers when tested against S. marcescens strains Smar 27 (Fig. 1B) and Smar 32 (Fig. 1C), which were the isolates of microbial keratitis and infiltrative keratitis, respectively.
Two clinical strains of S. marcescens (Smar 5 and Smar 35 isolated from a CLARE and microbial keratitis responses, respectively) were more resistant to solutions containing polyquaternium-1 (solution D & E; p = 0.01 and 0.006, respectively) than were the other three strains (Fig. 2). Solution E was the least effective of all solutions (p = 0.006).
Solution C containing (PHMB) showed a more variable ability to achieve reduction in the numbers of viable bacteria for all strains. This is reflected in the relatively large standard deviations for this disinfectant (Figs. 1 and 2). Solution D also displayed variable results with strain Smar 35, which is reflected in the large standard deviation.
We have examined the effect of contact lens MPDSs against five strains of S. marcescens and have demonstrated that the ATCC 13880 strain used in the ISO standard stand-alone disinfection test for contact lens disinfecting solutions was susceptible to all solutions tested at the manufacturer's recommended disinfection time, these results are consistent with the recent findings.26 However, using clinical isolates, we observed a more variable response. Two of the isolates, one from a case of microbial keratitis and the other from an infiltrative keratitis event, were also susceptible to all the solutions tested.
The minimum criterion for a product to be labeled as a stand-alone contact lens disinfection solution in the ISO 14729 standard is a 3-log reduction in the minimum stated disinfection time.27 A strain isolated from a CLARE event showed resistance to two of the MPDSs containing polyquarternium-1. These two solutions failed to meet the 3-log reduction criteria set out in the ISO standard for this CLARE strain. S. marcescens has previously been found to be resistant to contact lens disinfection solutions,19,20 including solutions containing polyquaternium-1.20 One microbial keratitis isolate also demonstrated resistance to one of the solutions tested. These results are similar to those reported by Lakkis and Fleiszig (2001), who found that clinical isolates of Pseudomonas aeruginosa were more resistant to disinfection solutions than was the recommended ATCC strain.28
One variation in our methodology from the ISO was that we used 1 ml instead of 10 ml MPDS. This volume was chosen to allow comparison of several strains in triplicate from a single bottle of MPDS. As some bottles only contained 120 ml, there was insufficient volume to allow the use of 10 ml as described in the ISO. The reduction in sample size may have had implications for the results obtained; therefore, we increased the number of replicates to ensure a good representation of the efficacy of the MPDS. Our results were highly reproducible for the ATCC isolate, and all solutions met the ISO criteria. Triplicates all behaved similarly and were within 3% of each other, suggesting that a 10-fold reduction in the sample size may be sufficient to demonstrate efficacy of an MPDS.
Of the five solutions tested, we found that the solutions containing of biguanides were the most effective. However, although the solution containing PHMB and hydroxypropyl methylcellulose (solution C) met the 3-log reduction criteria for disinfection, the results were more variable when compared with that for other solutions. This variability may be due to variance between lot numbers. However, PHMB is known to partition to glass,29 and this compound may also be partition to the manufacturers' bottles, resulting in variability of performance. In addition, it has been suggested that cellulose-based agents rapidly bind to PHMB reducing its chemical availability and bioactivity.30 The results for solution D and strain Smar 35 were also variable and could be due to the unique interaction of this strain and solution, as this solution did not have variable results for other S. marcescens strains. This is most likely due to phenotypic variation between growths. However, it has been shown that susceptibility to polyquaternium-1 solutions correlate with surface charge of strains of Pseudomonas.31 This strain may clump or have variable regions of surface charge resulting in variability when interacting with this solution.
It must further be considered that, in the ISO standard test, the disinfectants perform under ideal conditions, which do not reflect the “in-use” circumstances. When used in practice, disinfectants need to be active in the presence of organic soil and bacteria adherent to the surface of the contact lens or case, rather than in the planktonic form used in the test. These factors and compliance issues are likely to reduce the efficacy of the MPDS, possibly resulting in higher levels of contamination, as has been demonstrated by Lakkis and Fleiszig (2001) for P. aeruginosa.28 In addition, our findings suggest that the ATCC laboratory strain used in the ISO stand-alone disinfection test, which is grown under ideal conditions, shows a greater susceptibility to disinfection than do some of the clinical isolates tested here, which typically grow in low-nutrient environments. Furthermore, the recent reports on Fusarium keratitis32,33 associated with solution use demonstrates that the ISO standard may not be sufficient for the determining the “in-use” efficacy of MPDS.
The findings presented here highlight the importance of strain selection for the testing of MPDSs for the contact lens market and suggest that the selection of a single laboratory strain is insufficient to provide assurance that the disinfection solution will be effective against clinical isolates. It further suggests that clinical isolates of S. marcescens may show increased resistance to disinfection with MPDSs in which polyquaternium-1 is the main active ingredient in the formulation. This remains to be further investigated.
We thank Simin Masoudi for her technical assistance.
Institute for Eye Research
Rupert Myers Building, The University of New South Wales
Sydney, NSW 2052, Australia
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