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Suspension tests not only are inappropriate for the assessment of contact lens disinfectant efficacy but may produce results which give the disinfectant manufacturer and the lens wearer a false sense of security (3). Image analysis and the fluorescent probes 5-cyano-2,3-ditolyl tetrazolium chloride (CTC) and propidium iodide (PI) were used to investigate the disinfection of biofilms of Pseudomonas aeruginosa, an important eye pathogen, on hydrophilic contact lenses using 3% H₂O₂ (the most widely used lens disinfectant). This novel approach offers a rapid and reliable assay representing in-use conditions.

P. aeruginosa (NCIMB 6750) was grown in 10 ml of Trypticase soy broth (TSB), harvested by centrifugation (2,400 × g), and washed three times with phosphate-buffered saline (PBS), and counts were adjusted following spectrophotometry at 660 nm. Eighteen sterile contact lenses (polyhydroxyethyl-methacrylate; water content, 38%) were placed, concavity downwards, in separate bottles containing 10 ml of TSB, inoculated with 0.1 ml of a 37°C overnight TSB culture of P. aeruginosa, and incubated for 4 h at 37°C with gentle agitation.

Lenses were immersed in 10 ml of 3% H₂O₂ for up to 30 min, with six samplings at 5-min intervals, rinsed in 10 ml of PBS, and placed in 5 ml of TSB containing 6,000 U of catalase ml¹ for 1 min (to neutralize the H₂O₂). They were then cut radially to allow them to lie flat on microscope slides and stained with 5 µg of PI (Sigma, St. Louis, Mo.) ml¹ and 10 mM CTC (Polysciences, Inc., Warrington, Pa.) in PBS. After 1 min of incubation with PI at 20°C and 1 h of staining with CTC in the dark at 20°C, the lenses were rinsed in PBS for 5 s and then examined by epifluorescence microscopy with excitation at 510 to 560 nm (using a 580-nm dichroic mirror and a 590-nm barrier filter) in a Nikon Optiphot microscope. Cells that took up PI and fluoresced bright red were deemed to possess compromised cell membranes, while respiring cells reduced CTC to give bright red, fluorescent formazan crystals within the cell. When these stains were used together, there were no significant differences (P > 0.05) between replicate experiments. Stained cells were distinguished from nonspecific reactions by overlaying the fluorescence and phase-contrast images.

The image analysis system comprised a Sony charge-coupled device camera and a Seescan Solitaire image analyzer (Seescan Ltd., Cambridge, United Kingdom) with archiving to hard disk. Random numbers were generated to give coordinates of the sampling sites on the contact lenses. Counts were made from 20 random microscopic fields (each of an area of 33 by 40 µm) in triplicate experiments. Twenty fields of view were chosen as the number required to give a representative record of the colonization of lenses, since above this score the coefficient of variation remained constant. Previous workers have used between 10 and 20 fields per preparation to obtain representative results (1, 5, 6).

Treated lenses were transferred to 10 ml of TSB and incubated at 37°C for 24 h to recover viable cells, and rinse and neutralization suspensions were diluted with equal volumes of double-strength TSB and enriched by incubation at 37°C for up to 48 h. Broths were inoculated onto Trypticase soy agar plates, which were incubated at 37°C for 48 h and then left on the bench overnight to allow pyocyanin production.

There were large decreases of 85.5 to 95.5% in the numbers of bacteria attached to lenses within the first 5 min of exposure to H₂O₂; thereafter, numbers of attached bacteria remained relatively stable (Fig. 1). First-order (i.e., linear) disinfection kinetics were not observed during treatment (Fig. 2): initially, 94.3% of attached cells were PI negative (membrane intact) and 82.7 to 85.4% were CTC positive (respiring); at 5 min, 49.2% of the cells remaining attached were PI negative, and of these 82.9% were CTC positive (i.e., 40.8% of the original attached population); by 10 min, PI-negative cells fell from 49.2 to 26.5% and CTC-positive cells fell from 40.8 to 19.8%; at 15 min, there were slight increases in both PI-negative and CTC-positive cells (from 26.5 to 29.4% and from 19.8 to 25.6%, respectively); by 30 min, PI-negative and CTC-positive cells had fallen to 12.7 and 10.8%, respectively, of the original attached population, and the lenses and neutralization broths yielded viable P. aeruginosa.

View larger version (15K): [in this window] [in a new window]   FIG. 1.   Changes in the number of P. aeruginosa bacteria, per field of view, attached to soft contact lenses during 30 min of treatment with H2O2. Error bars represent standard deviations for 20 fields of view from each of three lenses at each sampling time.

View larger version (15K): [in this window] [in a new window]   FIG. 2.   Changes in the percentages of PI-negative () and CTC-positive () bacteria attached to soft contact lenses during 30 min of treatment with H2O2. Error bars represent standard deviations for 20 fields of view from each of three lenses at each sampling time.

The method described offers an in-use approach to contact lens disinfectant testing which allows the investigation of disinfection on the lens itself. The survival of P. aeruginosa on the lenses and in neutralized rinses, after disinfection for up to 30 min, is almost eight times longer than that achieved by its planktonic counterparts (3, 7). Although it is unlikely that soft contact lenses would be exposed to such high inocula during normal use, our lens colonizations were not confluent; indeed, cells were very often found alone, in pairs, or in microcolonies. Furthermore, our fresh lenses were free of the ocular deposits found on used lenses, and in some commercially available lens care systems, the lenses may be treated with 3% H₂O₂ for as little as 10 min.

In previous work involving suspension testing of contact lens disinfectants (3), our inactivation curves showed plateau regions and increases in counts (the switchback effect) similar to those seen at 15 min in Fig. 2. We suggested the breakup of cell clumps together with the existence of a resistant subpopulation as a possible explanation. The phenomenon was not considered to be owing to H₂O₂ neutralization by endogenous peroxidases alone, as the numbers of both PI-negative and CTC-positive cells continued to fall with prolonged exposure to H₂O₂; also, a similar phenomenon was seen during treatment with chlorhexidine gluconate, a disinfectant not neutralized by peroxidases.

Those members of the bacterial population that survived longer in the disinfection process may also have produced high levels of protective extracellular polysaccharide, which could have protected the cells on the lens surface due to a higher polymer mass. The algC gene (which is important in P. aeruginosa alginate biosynthesis) may be induced in some strains within 30 min of cells becoming sessile (4), and cells attached to a solid surface may show an 85-fold increase in algC gene expression compared with planktonic cells (2).