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A Novel Antibiofilm Technologyfor Contact Lens Solutions
Bruce F. Farber, MD, Hsi-Chia Hsieh, PhD, Eric D. Donnenfeld, MD,Henry D. Perry, MD, Arthur Epstein, OD, Arlene Wolff, BS
Purpose: Nonsteroidal anti-inflammatory drugs, including sodium salicylate, inhibitextracellular bacterial biofilm production . The authors studied the effect of the additionof sodium salicylate on bacterial adherence and biofilm formation on contact lenses andcases and commonly used medical polymers,
Methods: The study was done in vitro with bacterial adherence and biofilm measuredon lenses and cases that were exposed to saline contaminated with Staphylococcusepidermidis and Pseudomonas aeruginosa with and without 1 and 3 mm sodium salicylate. Bacterial adherence to contact lenses was quantitated by a vortex assay and byscanning electron microscopy. Biofilm formation on contact lens cases and other polymers was measured by an optical density assay and a radiolabeling assay .
Results: Inhibition of biofilm formation was demonstrated on plastic contact lenscases in a dose-related manner with 1 and 3 mm sodium salicylate. A dose-relateddecrease in bacterial adherence also was noted. Assays with contact lenses also demonstrated less adherence in the presence of sodium salicylate. Electron micrographs ofthe contact lens showed less biofilm, most noticeable with 3 mm salicylate. Other studiesdemonstrated decreased adherence of S. epidermidis to polyethylene and polystyrene.Sodium salicylate also decreased biofilm on plastic tissue culture wells, but sorbic acidparadoxically increased deposition.
Conclusion: The authors found that the addition of low-dose sodium salicylate tosaline decreased the adherence of S. epidermidis and P. aeruginosa to contact lensesand lens cases. Biofilm production also was decreased on the lens cases and on medicalpolymers used to make plastic cases. These studies suggest that sodium salicylatedeserves additional study to determine its use in contact lens solutions.Ophthalmology 1995;102:831-836
Infectious keratitis remains a serious complication ofcontact lens use. Poggio et all estimated an annual incidence of infectious keratitis of 20.9 per 10,000 patientswearing cosmetic extended-wear contact lenses and 4.1per 10,000 patients wearing daily wear cosmetic soft contact lenses. A conservative estimate based on approximately 20 million Americans wearing only daily wear softcontact lenses would create an annual incidence of 8200
Originally received: lune 10, 1994.Revision accepted: November 23, 1994.
From the Departments of Medicine and Ophth almology, North ShoreUniversity Hospital-Cornell University Medical College, Manhasset.
Presented in part at the ARVO Annual Meeting, Sarasota , May 1994.Dr. Farber holds patent rights to th is techn ology.
Reprint requests to Bruce F. Farber , MD, North Shore Uni versity Hospital, 300 Community Dr, Manhasset , NY ) 1030.
cases of infectious keratitis. The majority of contact lensrelated corneal ulcers are due to Pseudomonas aeruginosa.r" Many factors have been postulated to be important influences on the risk of infection. These factors include the lens material, wearing schedule, disinfectiontechniques, adherence of bacteria to the lens, and contamination of the lens care system and solutions. l"
Contact lens deposits or spoilage creates an undesirableinterface between the contact lens and the conjunctivaand corneal surface. Contact lens deposit symptomatologyincludes decreased visual acuity , foreign body sensation,mucous discharge, tearing , redness, photophobia, and abnormal lens fit.8.
9 Severe deposits may result in decreasedoxygen permeability of the contact lens with the formationof superficial and deep cornea) neovascularization, intrastromallipid, and intracorneal hemorrhage. 10-14 Proteindeposits play an important role in the development of
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Ophthalmology Volume 102, Number 5, May 1995
contact lens-related allergy, including giant papillary conjunctivitis. Contact lens deposits harbor infectious organisms and increase the risk of infectious keratitis." Thesedeposits include tear film proteins, sebaceous secretions,exogenous materials, mineral deposits, and bacterial biofilm."
Recent data have demonstrated that the attachment ofmicroorganisms to medical devices is mediated by biofilm,or capsular polysaccharides which form a protective matrix around a device. Biofilm produces a scaffold whichfacilitates bacterial adhesion and colonization.P In addition, biofilm protects and insulates bacteria from antiinfectives and disinfectant systems. Biofilm formation hasbeen demonstrated on both contaminated lenses and inlens cases, even after appropriate sterilization.v" Thesebacteria likely serve as an inoculum for contact lens-related bacterial keratitis. Several authors have documentedculturing organisms from the contact lens and contactlens case that were identical to the patients' infectiouskeratitis.t-'
Nonsteroidal anti-inflammatory drugs, including sodium salicylate, inhibit extracellular bacterial biofilmproduction.P'?" These compounds inhibit bacterial attachment to a number of medical polymers, includingsilastic and polyurethane catheters. We investigated theaddition oflow-dose sodium salicylate to contact lens solutions. We attempted to determine whether the additionofsodium salicylate to contact lens solutions can decreasebiofilm formation on contact lenses and their cases andbacterial attachment to contact lenses. We chose to studyP. aeruginosa because it is the most common cause ofcontact lens-related infectious keratitis and Staphylococcusepidermidis because it is the organism most commonlycultured from contact lenses.i"
Methods
Strain
S epidermidis strain 91 was used in these experiments.This is a slime-positive strain that has been described previously.!" A clinical isolate ofP. aeruginosa also was used.
Lens Cases
Studies on biofilm formation were performed in plasticcontact lens cases (Allergan, Irvine, CA). An overnightculture of the test organism (108 colony-forming units[CFU]/ml) in trypticase soy broth (TSB) was added tosaline with and without sodium salicylate in a 1:1 dilution.The bacterial suspension was incubated for 5 hours at 37°C, and the suspension was removed. In the experimentswith P. aeruginosa, nonadherent film floating on the culture was removed with a cotton swab. The remaining biofilm was washed gently two times with 5 to 10 ml salineto remove nonadherent bacteria and debris. The adherentfilm was removed mechanically with a pipette and dissolved in 3 ml saline. The optical density was read on aBausch and Lomb spectronic 601 at 686 nm (Rochester,
832
NY). In addition, bacterial counts were done on the samples by serial dilution and plating onto Mueller-Hintonagar plates, which were incubated at 37° C for 18 hours.
Low-density Polyethylene
Low-density polyethylene was used as a primary referencematerial. It had been prepared under controlled conditionsand has been used as a standard in experimental studies.'The material was obtained from Abiomed, Inc (Danvers,MA), and has been described in detail. The sheets werecut into 10 X 26-mm rectangular sections. A bacterialsuspension similar to that described previously was used.The polyethylene sheets were placed in the suspensionswith and without sodium salicylate for 24 hours at 37°C. They were removed, and the white biofilm was mechanically removed with a no. 1014 brush and placed in5 ml saline. The optical density then was read as previouslydescribed.
Polystyrene
Bacterial suspensions were prepared as previously notedand incubated with and without sodium salicylate at 3rC for 24 hours in 50 ml clear polystyrene tubes (Corning,NY). The supernatant was removed, and the white filmon the walls of the tube was inspected. The biofilm thenwas dissolved in 5 ml saline by mechanically removing itwith a plastic transfer pipette. The preparation was readat an optical density of 686 JLm. In addition, similar experiments were performed, but 14C-sodium acetate (4 JLCi)(1_14C) was added to all tubes before incubation. Thesupernatant was removed from the tubes, and 2 ml Aquasol was added. The biofilm was removed mechanicallyfrom the sides of the tube, resuspended in Aquasol andwas counted in scintillation vials.
Contact Lenses
An overnight TSB culture was standardized to one-halfoptical density at 686 JLm and diluted 1:100 in broth. Thebacterial suspension then was added in a 1:1 dilution tosaline with and without sodium salicylate. Acuvue (Johnson and Johnson, Raritan, NJ) contact lenses were incubated in the contaminated saline for 20 hours at 3rc. The lenses were removed and placed in 5 ml salinewith gentle washing. They then were removed and placedin 5 ml fresh saline, vortexed at number 7 for 1 minutefive times. The optical density of the saline was measuredat 686 JLm, and colony counts were performed by serialdilution. Lenses also were incubated with P. aeruginosa(107 CFU/ml) for 2 hours in saline alone or supplementedwith sodium salicylate. The lenses were removed andwashed in saline three times. They were incubated with3H-leucine (2 JLCi/ml) for 30 minutes and then washedand counted with Aquasol in scintillation vials. The resultsare expressed in counts per minute.
The effect of the addition of sodium salicylate to H 20 2
during the disinfection of contact lenses was studied.Lenses were placed in lens cases with H20 2 supplemented
Farber et al . Antibiofilm Technology for Contact Lens
Figure 1. Electron micrographs of contact lenses that were incubated in saline and 3 mm sodium salicylate in the presence of Staphylococcus epidermidis.The amount of biofilm seen on the 3-mm lens was less than that seen in the control lens (original magnification, X2000).
with 1 and 3 mmolj(mm) salicylic acid. The HzOz wasneutralized with catalase. The lens were rinsed in salineand incubated in P. aeruginosa for 2 hours. They thenwere washed in saline three times, and incubated in radiolabeled leucine (2 l1Cijml) for 30 minutes at 20° C.They were washed three times, suspended in Aquasol,and counted in scintillation vials.
Electron Microscopy
Lens were incubated in saline or saline with 3 mm sodiumsalicylate (Fig 1). They then were contaminated with anS. epidermidis bacterial suspension as previously describedfor 4 hours. The soft contact lenses were fixed in 2% buffered glutaraldehyde. A rapid dehydration was performedwith graded alcohols. They then were processed by criticalpoint drying. A palladium gold carbonation was used forplating by sputter coating, and the lenses were viewedwith a scanning Jeol electron microscope (Mexford, MA).
Keuls comparison test was used to detect differences between the salicylate and sorbic acid group.
Results
Inhibition ofS. epidermidis biofilm was demonstrated onthe sides of plastic contact lens cases with 1 and 3 mmsodium salicylate as seen in Figure 2. There was 39% inhibition with 1 mm and 95% inhibition with 3 mm. Thedifference was significant with P < 0.0001. A dose-relateddecrease in corresponding bacterial counts also was observed. The control lens cases contained 1.2 X 107 CFUjml in the dissolved biofilm. There were 9.6 X 106 CFUjml in 1 mm and 2.4 X 106 CFU/ml in 3 mm sodiumsalicylate. Results of a similar assay performed with P.aeruginosa are demonstrated in Figure 3. A dose-relateddecrease in adherent biofilm was noted. A corresponding
<,r-,
-,-,
"~
Studies Using Salicylate and Sorbic Acid
Contact lenses were incubated overnight in 24 well platescontaining S. epidermidis in TSB diluted 1:1 with sterilesaline with or without sodium salicylate (3 mm) or sorbicacid (0.015%). They then were rinsed gently with salineand transferred to a I5-ml centrifuge tube and vortexedwith 4 ml NaCl vigorously fivetimes for 1 minute. Colonyforming units then were done. In addition, overnight cultures were placed in the wells without lens. The mediawere removed, and the remaining biofilm was scrapedout of the wells and suspended in 1 ml saline. Opticaldensities were read at 686 11m.
0.12
0.1
0.08
Optical 0.06
density0.04
0.02
0o Imm 3mm
Statistical Analysis
Simple linear regression analysis was used to detect doserelated differences between the control, the 1-, and 3-mmgroups. One way analysis of variance with a Newman-
Concentrationsalicylate (mm).
Figure 2. Biofilm formation measured in optical density versus concentration of salicylate (millimolar) with Staphylococcus epidermidis.
833
Ophthalmology Volume 102, Number 5, May 1995
95,53563,23546,784
Counts/min(%)
(100)(89)(47)
0.6180.5540.296
Optical Density"(um)
Table 2. Inhibition of Biofilm to Polystyrene UsingRadiolabeled Staphylococcus epidermidis
• Mean of three assays.
Controls1mm3mm
"" '-.0" " .....
!lA<,
........~.oa
.0"
01
Il
o
007
o
o.
Optical 0
Density 0
o
Table 1. Inhibition of Biofilm on Polyethylene
Figure 3. Biofilm formation measured in optical density versus concentration of salicylate (millimolar) with Pseudomonas aeruginosa.
Concentrationsalicylate (mm)
decrease in the number of CFU/ml bacteria also was observed.
The results of the studies using S. epidermidis withstandardized polyethylene are presented in Table I. Thestudy was performed in triplicate. There was minimal inhibition noted at I mm and 21% at 3 mm (P < 0.04).Studies using polystyrene are presented in Table 2. Therewas a dose-related linear inhibition ofbiofilm as measuredby both optical density (P < 0.00 I) and uptake of radiolabeled sodium acetate. Table 3 gives the results of thestudies using contact lenses and S. epidermidis. In thesestudies, the number ofbacteria adhering to contact lensesincubated in contaminated saline was quantitativelymeasured in two ways. Both the optical density and number of colony-forming units per milliliter bacteria fell ina dose-related manner with increasing concentrations ofsodium salicylate. Although there was a dose-related decrease in adherence, it did not achieve statistical significance.
Table 4 lists the results ofan assay in which adherenceto lenses was studied with P. aeruginosa. Both assaysdemonstrate a dose-related inhibition of growth (colonyforming units per milliliter); however, it was not statisti-
Despite adequate disinfection, corneal infection continuesto be a problem associated with soft contact lens use. Thepathophysiology of these infections is complex and probably multifactorial. It seems logical that contaminationof lens care cases and solutions is a major risk factor ofinfection. In addition, recent data from studies of othermedical devices and lenses suggest that adherence ofbacteria is significantly dependent on biofilm formation. Microorganisms produce a protective polysaccharide filmthat is resistant to disinfectants and antimicrobial agents. 17
Our previous work has demonstrated that the salicylates and other nonsteroidal anti-inflammatory agents interfere with bacterial attachment to a number of medicalpolymers. 17
, 19,20 In addition, we have demonstrated that
cally significant. The radiolabeling method produced asimilar degree of inhibition. In Table 5, adherence of P.aeruginosa to lenses was studied after exposure in contaminated neutralized H202 . A dose-related decrease inadherence again was noted (P < 0.003).
Optical densities ofthe biofilm scraped out of the plasticwells were 0.788 nm for controls, 0.194 Jl-m for salicylate,and 1.435 Jl-m for sorbic acid (mean trials, 7). There wasa highly significant difference among the groups (P <0.00 I). Of note was the paradoxical increase in biofilmfound with sorbic acid. Colony counts done on lenses thatwere incubated demonstrated 6 X 105 CFU/rnl in thecontrol saline, 1.3 X 104 CFU/ml with salicylate, and 2.5X 105 CFU/ml with sorbic acid. These differences werenot significant.
Discussion
3mmlmmo
Controls
1mm
3mm
Optical Density(~m)
0.1320,1670,1830.1660.1530.1460.1120.1350.136
Mean
0.160
0.155
0,127
(%) Table 3. Adherence of Staphylococcus epidermidisto Contact Lenses
(100)Optical Density (%) CFU/ml
Controls 0.044 (100) 5.2 X 107
(96) 1mm 0.026 (59) 4.4 X 107
3mm 0.008 (18) 2.8 X 107
6mm 0.004 (9) 1.2 X 107
(79)CFU = colony-forming units.
834
Farber et al . Antibiofilm Technology for Contact Lens
Table 4. Adherence of Pseudomonas aeruginosa toContact Lenses
the salicylates decrease the amount of S. epidermidis extracellular slime. 16 Although experience with biofilm hasbeen most extensive with P. aeruginosa, the observed effect has been demonstrated with a variety of other organisms.
In this study, we demonstrated that the addition oflow-dose sodium salicylate to saline decreases biofilm formation on contact lens cases. In addition, there was reduced bacterial adherence to lenses exposed to saline contaminated with P. aeruginosa and S. epidermidis compared with control saline. The concentrations of sodiumsalicylate used were 0.013% to 0.039%.The concentrationof other nonsteroidal anti-inflammatory drugs used incommercially available topical ophthalmic drops is 0.1%to 1.0% or approximately 2.5 to 25 times greater than thehighest concentration ofsodium salicylatewe used. Hence,we are using concentrations that would be otherwise considered subtherapeutic. It is unclear whether other nonsteroidal anti-inflammatory drugs have this property.
Sorbic acid currently is used as a preservative in contactlens solutions at a concentration of 0.1%, which is higherthan the concentrations that we used. Our studies suggestthat it paradoxically may increase biofilm deposition onlenses, opposite of the effect with sodium salicylate.
Previous research has shown that P. aeruginosa preferably adheres to focal deposits on contact lenses." Thestrong association of Pseudomonas to contact lens-relatedbacterial keratitis may be explained by the production oflarge amount of extracellular slime. This may allow Pseudomonas to adhere to the surface of the contact lens. Itis unclear whether the addition of a nonsteroidal antiinflammatory drug to the lens solutions would decreasethe rate of corneal infection. However, by decreasing bacterial adhesion to the contact lens, we postulate a decreased risk of inoculation into the cornea. This hopefullywould decrease the incidence of bacterial keratitis. It isalso possible that decreasing biofilm might decrease theincidence oflens intolerance due to allergic problems. Wesuggest that further studies on biofilm formation andnonsteroidal anti-inflammatory drugs are indicated, aswell as toxicity studies of the chronic effect of low dosenonsteroidal anti-inflammatories on the ocular surface.
Vortex Method
Controls1 mm3mm
Radiolabel Method
ControlSalicylate (1 mm)
CFU = colony-forming units.
Triall
CFU/ml
4.0 X 106
1.4 X 1068.5 X 105
Counts/min
209,434119,046
Trial 2
CFU/ml
1.8 X 106
1.2 X 1068.4 X lOS
Table 5. Adherence of Pseudomonas aeruginosa toContact Lenses after HzOz Disinfection
Counts/min Final pH
HzOz 490,524 7.0HzOz + 1 mm 398, 149 6.8HzOz + 3 mm 215,831 6.4
References
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2. Alfonso E, Mandelbaum S, Fox MJ, Forster RK. Ulcerativekeratitis associated with contact lens wear. Am J Ophthalmol1986;10I :429-33.
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4. Wilhelmus KR. Review ofclinical experience with microbialkeratitis associated with contact lenses. CLAO J 1987;13:211-14.
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6. Donzis PB, Mondino BJ, Weissman BA, Bruckner DA. Microbial contamination of contact lens care systems. Am JOphthalmol 1987; I04:325-33.
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8. Nesburn AB. Symposium: contact lenses. Complicationsassociated with therapeutic soft contact lenses. Ophthalmology 1979;86:1130-7.
9. Tripathi RC, Tripathi BJ, Millard CB. Physiochemicalchanges in contact lenses and their interactions with thecornea and tears: a review and personal observations. CLAOJ 1988; 14:23-32.
10. Donnenfeld ED, Ingraham H, Perry HD, et al. Contact lensrelated deep stromal intracorneal hemorrhage. Ophthalmology 1991;98:1793-6.
II. Rozenman Y, Donnenfeld ED, Cohen EJ, et al. Contactlens-related deep stromal neovascularization. Am JOphthalmol 1989;107:27-32.
12. Bloomfield SE, Jakobiec FA, Theodore FH. Contact lensinduced keratopathy: a severe complication extending thespectrum of keratoconjunctivitis in contact lens wearers.Ophthalmology 1984;91:290-4.
13. Richard NR, Anderson JA, Tasevska ZG , Binder PS. Evaluation of tear protein deposits on contact lenses from patients with and without giant papillary conjunctivitis. CLAOJ 1992;18:143-7.
14. Allansmith MR, Korb DR , Greiner JV, et al. Giant papillaryconjunctivitis in contact lens wearers . Am J Ophthalmol1977;83:697-708.
15. Tojo M, Yamashita N, Goldmann DA, Pier GB. Isolationand characterization of a capsular polysaccharide adhesionfrom Staphylococcus epidermidis. J Infect Dis 1988;157:713-22.
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Ophthalmology Volume 102, Nur.lber 5, May 1995
16. Teichberg S, Farber BF, Wolff AG, Roberts B. Salicylic aciddecreases extracellular biofilm production by Staphylococcusepidermidis: electron microscopic analysis [letter]. J InfectDis 1993;167:1501-3.
17. Farber BF, Wolff AG. The use of nonsteroidal antiinflammatory drugs to prevent adherence of Staphylococcus epidermidis to medical polymers. J Infect Dis 1992;166:861-5.
18. Wang I, Anderson JM, Marchant RE. Staphylococcus epi-
836
dermidis adhesion to hydrophobic biomedical polymer ismediated by platelets. J Infect Dis 1993;167:329-36.
19. Farber BF, Wolff AG. The use of salicylic acid to preventthe adherence of Escherichia coli to silastic catheters. J Urol1993;149:667-70.
20. Farber BF, Wolff AG. Salicylic acid prevents the adherenceof bacteria and yeast to silastic catheters. J Biomed MaterRes 1993;27:599-602.