Abstract
Purpose
This study investigated the efficacy of pre-conditioning lens cases on bacterial biofilm formation and removal.
Methods
Silver impregnated (MicroBlock / ProGuard™ & i-Clean) and control storage cases were pre-conditioned for 24 h with their respective multipurpose solutions (MPDSs). Cases were then inoculated with 2 ml of 10 6 CFU/mL of ocular isolates of either P. aeruginosa or S. aureus and incubated for 48 h. Cases were subsequently disinfected (4−6 hours) as per the manufacturer’s recommended disinfecting time (MRDT) followed by the recommended case hygiene procedures – recapping wet (MicroBlock / ProGuard™ cases only) or rinse and air-dry or rinse, tissue-wipe and air dry (mechanical disruption). Surviving bacteria were enumerated using standard techniques.
Results
Pre-conditioning the MicroBlock / ProGuard™ cases with MPDS significantly reduced biofilm formation (-1.1 log 10 CFU, p < 0.01 for P. aeruginosa & -1.3 log 10 , p < 0.001, CFU for S. aureus ) compared to the i-Clean lens cases. Maintaining the MicroBlock / ProGuard™ lens cases wet after the MRDT resulted in partial removal of bacterial biofilms (-2.9 log 10 CFU, p < 0.001 for P. aeruginosa and -2.6 log 10 CFU, p < 0.001 for S. aureus ). Air-drying of all three types of lens storage cases after MRDT significantly reduced the bacterial biofilm (-5.4 log 10 CFU, p < 0.001 for P. aeruginosa and -3.5 log 10 CFU, p < 0.001 for S. aureus ). Mechanical disruption produced the greatest reduction in the levels of bacterial biofilm in all 3 types of lens cases tested (-6.8 log 10 CFU, p < 0.001 for P. aeruginosa and -4.5 log 10 CFU, p < 0.001 for S. aureus ). Synergi MPDS was significantly better than AQuify MPDS in removing bacterial biofilm from all 3 lens case types for case hygiene treatments with an air-drying step.
Conclusion
Pre-conditioning of silver-impregnated ProGuard™ lens cases inhibited initial bacterial biofilm formation. Synergi MPDS was more effective than AQuify MPDS in removing bacterial biofilm in silver impregnated cases and tissue-wiping significantly improved biofilm removal.
1
Introduction
Microbial contamination of contact lens storage cases has been implicated in corneal infections and inflammation [ ]. Contamination of lens cases is common (30–80%) among lens wearers [ ] and is observed even in the presence of good lens case hygiene practices [ , ]. Silver impregnated lens storage cases have been introduced to reduce microbial contamination by releasing silver ions from the lens case material [ , ]. Silver is a well-known antimicrobial agent affecting bacteria on contact by interference with DNA, cellular respiration, sulfhydryl groups, and enzyme conformation [ ]. Recent in vitro and in vivo studies indicate that these silver impregnated cases reduce but do not completely eliminate microbial contamination [ , ]. Biofilm formation can occur following microbial adhesion to storage case surfaces [ ] and current manufacturer’s recommendations for lens storage case hygiene may be inadequate to remove microbial biofilm from regular as well as silver impregnated lens storage cases [ , , ].
Current recommendations for lens storage case hygiene are inconsistent, with most recommending rinsing of wells (with lens care products or saline) followed by air drying (well and lid orientation unspecified) [ ]. The MicroBlock / ProGuard (Alcon, Fort Worth, TX) case has been shown to release silver when maintained wet [ ] and understandably the manufacturers of the MicroBlock / ProGuard lens cases suggest recapping the lens cases wet following rinsing with the MPDS [ ]. Manufacturers of the i-Clean storage cases (CooperVision, Pleasanton, CA) recommend air drying following rinsing with either MPDS or saline. The lens case hygiene guidelines for silver impregnated lens cases may be confusing for the contact lens wearer. Recent studies have shown that mechanical disruption is most effective in removing bacterial biofilm from silver impregnated [ ] and non-silver contact lens storage cases [ ]. Furthermore, many people do not realise that MPDS and lens cases made by the same manufacturer have often been optimised for use together, and mismatching lens case and disinfecting solution has been shown to be a risk factor for lens case contamination. [ ]
The objectives of the current study were to evaluate the ability of silver impregnated contact lens storage cases to inhibit biofilm formation following pre-conditioning with their complementary and non-complementary lens care products, to evaluate the impact of non-complementary lens care products and to compare the manufacturer’s recommendation for storage case hygiene to the mechanical disruption of robust biofilm. A commercially available non silver lens storage case (CooperVision, Pleasanton, CA) was used as the control.
2
Materials and methods
2.1
Bacterial strains and media
P. aeruginosa 071 or S. aureus 031 isolated from cases of microbial keratitis in Australia were used in this study. Stock cultures of bacteria stored at −80 °C were streaked on a chocolate agar plate (Oxoid Australia, Sydney, NSW, Australia) and incubated at 37 °C for 24 h. The bacterial cells were collected and washed once by centrifugation in phosphate buffered saline (PBS, 8 g /l NaCl, 0.2 g /l KCl, 0.2 g /l KH 2 PO 4 , 1.15 g /l Na 2 HPO 4 , pH 7.4). Bacteria cells were re-suspended in Tryptone Soy Broth (TSB, 17 g/l Casein, 3.0 g/l Soybean Meal, 2.5 g/l Glucose, 5.0 g/l NaCl, 2.5 g/l K 2 HPO 4 ,) diluted with PBS (1:10 for S. aureus and 1:100 for P. aeruginosa ). The concentration of the bacterial suspension was adjusted to 0.1 at 660 nm wavelength using a spectrophotometer (Heliosβ, Unicam Instruments, Cambridge, UK; approximately 1.0 × 10 8 colony forming units per ml; CFU/ml). The suspension was serially diluted in the appropriate media to obtain the final culture concentration of 1.0 × 10 6 CFU/ml.
2.2
Contact lens storage case preconditioning and biofilm formation
Two commercially available silver impregnated contact lens storage cases MicroBlock / ProGuard™ (Alcon, Fort Worth, TX) and i-Clean (CooperVision, Pleasanton, CA), and one non silver contact lens storage case (CooperVision.) were used. Lens cases were preconditioned with one of 2 multipurpose disinfecting solutions (MPDS), one containing polyhexanide (AQuify; Alcon) and the other containing Oxipol™ (Synergi; CooperVision [withdrawn from the market]) as follows; each well of the lens case was filled with 2 ml of the MPDS, recapped and left in a static incubator at 25 °C for 24 h. Following this, the MPDS was discarded and each well was inoculated with 2 ml freshly prepared bacterial suspension, loosely capped and incubated at 37 °C in a digital agitator at 120 rpm for 24 h. After 24 h incubation, bacterial suspension was discarded, and wells gently rinsed with PBS once. All the wells were then refilled with 2 ml of freshly prepared medium (1:10 for S. aureus and 1:100 for P. aeruginosa ) and all the storage cases were re-incubated with agitation for a further 24 h. Following 48 h of incubation, the media was discarded, and wells were gently washed with PBS twice to remove loosely adherent bacterial cells.
Treatment of contact lens storage cases: Following growth of the biofilm, the lens cases were treated as follows:
- 1
Untreated control (n = 54; 22 MicroBlock / ProGuard™, 16 i-Clean & 16 Control cases): No hygiene or disinfection treatment (immediately assayed for numbers of bacteria). MicroBlock / ProGuard™ cases were subject to the manufacturer recommended treatment “Disinfect, Rinse and Recap Wet” and hence required the 6 extra untreated control cases.
- 2
Disinfect with MPDS, rinse and re-cap wet (n = 40 MicroBlock / ProGuard™ cases only; as per manufacturer’s instructions [ ]): Lens case wells were filled with 2 ml of the MPDS and disinfected for 4−6 hours. The solution was then discarded, wells re-filled with 2 ml of the MPDS and cases shaken gently for 10 s. The solution was discarded and cases recapped for 18 h.
- 3
Disinfect with MPDS, rinse and air-dry face down (n = 120; 40 MicroBlock / ProGuard™, 40 i-Clean & 40 non-silver control cases; as per manufacturer’s instructions for i-Clean cases): Lens case wells disinfected as described in treatment 2. Solution then discarded, wells re-filled with 2 ml of the MPDS and shaken gently for 10 s. The solution discarded and cases air dried face down on a clean facial tissue (Kimberly-Clark Australia Pty; Milsons Point, Australia) for 18 h at room temperature.
- 4
Disinfect with MPDS, rub & rinse, tissue wipe and air-dry face down (n = 120; 40 MicroBlock / ProGuard™, 40 i-Clean & 40 non-silver control cases): Lens cases wells were disinfected as described in treatment 2. The solution was then discarded, wells re-filled with 2 ml of the test solution and rubbed clockwise and anti-clockwise for 5 s with a gloved finger (Schiffa powder-free Latex Gloves, Icon Supplies Pty Ltd; Merrylands, Australia), capped and gently shaken for 10 s. The solution was discarded and cases air dried face down on a wire rack (Bel-Art, New Jersey, USA) for 18 h at room temperature.
Biofilm Recovery: Following treatments, 2 ml of PBS was added to each well along with a sterile magnetic stirring bar and vortexed for 1 min to dislodge the bacterial biofilm. Tenfold serial dilutions of the dislodged bacterial biofilm were performed in Dey-Engley neutralizing broth. Aliquots of the dilutions were inoculated in triplicate on nutrient agar plates and enumerated following 18 h of incubation at 37 °C.
Statistical Analysis: Prior to data analysis, log 10 transformation of the bacterial data was performed and values from both wells of a lens case were averaged and used for analysis. Analysis was performed separately for P. aeruginosa and S. aureus . Data was analysed using SPSS version 21 (IBM, Chicago, IL). Data were modelled as multifactorial general linear model (3-way ANOVA). Initially an overall model with the main factors; lens cases, MPDS and case hygiene treatment and all interactions were assessed. If significant interactions were present, the analysis was repeated at each level of the interacting factor. This was performed until there were no more significant interacting factors. The level of significance was set at 5 % and post hoc multiple comparisons were adjusted using Bonferroni correction.
3
Results
Both the test bacterial strains formed robust biofilms in the 2 silver impregnated and the control contact lens storage case after 48 h. S. aureus biofilms were more resistant to the case hygiene treatments compared to the biofilms formed by P. aeruginosa .
3.1
Storage case treatments
Preconditioning Only: Biofilm formation by P. aeruginosa in the MPDS pre-conditioned MicroBlock / ProGuard™ cases was significantly lower (p < 0.01) compared to MPDS pre-conditioned silver impregnated i-Clean (-1.1 log 10 CFU) and non-silver control cases (-1.1 log 10 CFU) ( Fig. 1 ). MicroBlock / ProGuard™ lens cases pre-conditioned with MPDS had significantly lower numbers of S. aureus (p < 0.001) compared to MPDS pre-conditioned silver impregnated i-Clean (-1.4 log 10 CFU) and non-silver control cases (-1.6 log 10 CFU) ( Fig. 1 ).
