The Effects of a Common Contact Solution on Pseudomonas aeruginosa Planktonic and Biofilm Growth Catherine Cooch and Dr. Carolyn Mathur Department of Biology, York College of Pennsylvania Abstract Pseudomonas aeruginosa (PA) is an opportunistic pathogen in humans that can grow freely (planktonic) or as a biofilm. This bacterium can lead to permanent vision loss. Contact lens wearers are at particular risk of infection, and few studies have agreed on the ability of contact lens solutions (CS) to inhibit biofilm formation in contact lens storage cases. To investigate this problem, we measured the planktonic and biofilm growth response of PA to 2 levels of CS (100% and 50%) compared to no treatment. Additionally, we compared the response to CS added pre- and post-biofilm formation. Planktonic cell growth was estimated by measuring increase in absorbance at 585nm. Biofilms were stained, harvested and absorbance measured to compare relative growth. Significant, dose-dependent inhibition (p<0.05) occurred for both planktonic and biofilm cultures. The pre-biofilm cultures were more inhibited than post-biofilm (p<0.05), indicating that the preformed biofilm offered some protection to PA from CS inhibition. Therefore, contact lens wearers should physically remove possible biofilm growth from lens storage cases before adding fresh CS to their lenses during storage, in order to assure maximum lens cleanliness and safety. Introduction Pseudomonas aeruginosa, a gram-negative aerobic rod, is an opportunistic pathogen to humans that usually does not infect healthy tissues 2, 5. Pseudomonas bacteria can be found in either a biofilm or a planktonic form. Environmental signals, such as a nutrient-rich medium, trigger the bacteria in a transformation to the biofilm, where it is attached to some surface 4. When nutrient deprived and not in the biofilm form, Pseudomonas can be found in a free-swimming, planktonic form 5. P. aeruginosa is a common cause of ulcerative keratitis (UK), a serious complication of contact lens wear 3. Permanent visual loss from scarring or perforation of the cornea can be a result of UK 2. It is estimated that 80 million people in the world wear contacts; with 33 million alone living in the United States 3. The increasing numbers of contact wearers demonstrates the importance of determining disinfecting qualities of contact solutions in circulation today. There have been few studies that agree on the ability of contact solutions (CS) to inhibit biofilm growth. Additionally, present guidelines of the International Organization for Standardization (ISO) 1 and the Food and Drug Administration (FDA) 3 mention reducing species of bacteria to certain standards that may not be actual “normal use conditions” such as in contact cases or with common nutrient levels present in the cases. Because I found few previous studies that looked into biofilms and contact solution, with similar conclusions, I chose to look at biofilm and planktonic presence to see how well CS inhibits growth. Objectives 1.To compare pre vs. post biofilm and planktonic response to addition of CS. 2.To compare the dose-dependent responses of biofilm and planktonic cells to CS. 3.To compare planktonic and biofilm response to CS. Hypothesis I hypothesized that CS inhibits planktonic and biofilm growth of Pseudomonas aeruginosa. Also, time of addition of CS to cultures (pre and post biofilm formation) alters responses. Methods Literature Cited 1. Beattie, T.K., Seal, D.V., Tomlinson, A., McFadyen, A.K., and Grimason, A.M Determination of Amoebicidal Activities of Multipurpose Contact Lens Solutions by Using a Most Probable Number Enumeration Technique. Journal of Clinical Microbiology 41.7: Cheng, K.H., Leung, S.L., Hoekman, H.W., Beekhuis, W.H., Mulder, P.G.H., Geerards, A.J.M., and Kijlstra, A Incidence of contact-lens-associated-microbial keratitis and its related morbidity. The Lancet 354: Lakkis, C., and Fleiszig, S.M.J Resistance of Pseudomonas aeruginosa isolates to hydrogel contact lens disinfection correlates with cytotoxic activity. Journal of Clinical Microbiology 39.4: O’Toole, G., Kaplan, H.B., and Kolter, R Biofilm formation as microbial development. Annual Review of Microbiology 54: Pseudomonas aeruginosa. Available from: Accessed 2003 December Future Studies 1.Determine if there are differences among different types of contact solutions. 2.Find out how many days is the optimum growth period for biofilms, and test contact solution inhibition at that time. 3.Find the minimum inhibitory concentration of CS against PA. Conclusions  The addition of CS significantly inhibits the formation and continual growth of biofilm and planktonic cells.  There is no significant difference between 100% CS and 50% CS.  In both experiments, when CS was added the planktonic formation was nearly zero, whereas the biofilm formation was slowed significantly. Results  Addition of CS prior to biofilm formation produced significant inhibition of growth for both planktonic and biofilm cells (P<0.05) – Figure 1.  Addition of CS to preexisting biofilms produced significant inhibition of growth for biofilm cells (P<0.01) but not for planktonic cells – Figure 2.  Growth reduction was increased for the 100% CS compared to the 50% CS for all results except for planktonic growth that occurred when CS was added to pre-formed biofilm – Table 3. Results Cell Growth  Planktonic - Remove supernatant into sterile test tube - Wash dish 1X with 1ml sterile water; add to test tube - Measure absorbance at 585 nm  Biofilm - Add crystal violet (CV) to cover bottom of dish - Incubate 5 minutes - Remove CV by suction and discard - Wash 2X with water and discard - Add 3ml 95% ethanol to each dish - Shake gently for 2 minutes to elute dye - Transfer CV/ethanol solution to sterile test tube - Measure absorbance at 585 nm Acknowledgements I would like to thank Dr. Mathur for her help throughout each experiment and in analyzing the data. Figure 2 Figure 2. Post-biofilm formation growth response to addition of CS. A. Indicates planktonic growth of the different treatments. B. Indicates the biofilm growth at the different treatments. Values were significant for biofilm response (p<0.01), indicated by asterisks (*). Error bars represent standard deviation. A B Figure 1 Figure 1. Pre-biofilm formation growth response to addition of CS. A. Planktonic growth of the different treatments. B. Biofilm growth of the different treatments. Error bars represent standard deviation and asterisk (*) designates significance. P<0.05. A B 50% CS % CS Pre- PlanktonicBiofilm Post- PlanktonicBiofilm Table 3. Percent growth reduction (GR) pre- and post- biofilm formation compared to untreated controls. NB CS Table 1. Experimental design* for CS added at Day 0. CS50/50Control * All volumes added were measured in ml and added to 8ml petri dishes. Tests were run in triplicate. Experiment 1 extended over 8 days. Table 2. Experimental design** for CS added at Day 7. H2OH2O CS CS50/50Control ** Shows volumes (ml) added to 5ml culture of PA after 7 days of growth. All measurements were in ml and added to 8ml petri dishes. Tests were run in triplicate. Experiment 2 spanned 13 days.