1. Biofilm bacterial diversity: Association with disease severity in contact lens related keratitis L. Wiley1, M. Mcallister1, L.A. Wiley2, T. Elliott2, D. Bridge2, J. Odom1, J. Olson2, West Virginia University Eye Institute1, Department of Microbiology, West Virginia University2 Purpose : Biofilm formation in contact lens cases may predispose to the development of contact lens related keratitis. To better understand the composition of contact lens case biofilm, we used 16S ribosomal RNA (rRNA) gene sequencing to examine cases from patients with mild keratitis, keratitis with focal infiltrate, and contact lens related ulcers, as well as cases from asymptomatic controls. Methods: Contact lens cases were obtained from 5 patients with mild diffuse keratitis, 8 with keratitis and focal infiltrates, and 4 with contact lens related ulcers. Eight cases from asymptomatic soft contact lens wearers were processed as controls. Biofilms were removed from lens cases by scraping and sonication, and DNA was extracted using the Mo Bio Microbial DNA Isolation Kit. Universal primers were used to amplify the bacterial 16S rRNA gene, PCR products were purified, cloned into the pCR 4-TOPO vector (Invitrogen), then re-amplified and sequenced. Sequences were classified by BLAST analysis against GenBank. Each sequence was matched with at least one database entry at the genus level (identity >95%). Results: The number of bacterial types isolated from the case correlated with increasing severity of disease (Spearman rank order correlation  <0.000001). There was a statistically significant difference between the number of bacterial types identified and the four clinical groups: normal, mild keratitis, keratitis with focal infiltrate and corneal ulcer, p=0.0006. All the affected groups exhibited more bacterial types than the controls (Mann-Whitney U test, p=0.0013). Presenting visual acuity was correlated with number of bacteria identified (p=0.011). Achromobacter and Stenotrophomonas were predominant isolates. Heat map: number of bacterial isolates per patient (columns) grouped by disease severity. Seven controls are not included because no bacteria were isolated. References 1.McLaughlin-BorLace L, Stapleton F, Matheson M, Dart JK: Bacterial biofilm on contact lenses and lens storage cases in wearers with microbial keratitis. J AppI Microbiol. 1998 May; 84(5):827-38. 2.Galentine PG, Cohen EJ, Laibson PR, Adams CD, Michaud R, Arentsen JJ: Corneal ulcers associated with contact lens wear. Arch Ophthalmol. 1984; 102(6):891-94. Scanning Electron Micrograph Demonstrating Biofilm on a Contact Lens from a patient with keratitis and a focal infiltrate Discussion: Biofilm formation in contact lens cases can be a potential source of toxin production, and also may harbor organisms which cause contact lens related keratitis.1,2 This involves a developmental cycle in which planktonic organisms aggregate on a surface, and then irreversibly adhere to that substrate. Microcolony formation occurs through cell to cell, quorum-sensing signaling leading to increased bacterial diversity and eventual elaboration of planktonic organisms. While the early biofilm may produce toxins, the mature biofilm evolves to a stage in which it begins to disperse metabolically active planktonic organism, and these bacteria may promote the formation of microabscess or frank corneal ulceration. Microcolonies of the mature biofilm may be released from the main structure, facilitating rapid development of contact lens biofilm. We observed a predominance of gram negative organisms, such as Achromobacter and Stenotrophomonas in early biofilms, whereas advanced biofilms have more gram positive species and a more diverse gram negative population. Our data suggests that Achromobacter and Stenotrophomonas organisms may play an important role in establishing the contact lens case biofilm. Conclusion: Bacterial diversity from contact lens cases was correlated with severity of disease and presenting visual acuity, and was greater than asymptomatic controls. Achromobacter and Stenotrophomonas are prominent residents of contact case biofilms. 20/20 20/23 20/37 20/1900 Mean visual acuity

2. Biofilm bacterial diversity: Association with disease severity in contact lens related keratitis This study provides additional support for the premise that contact lens case biofilm development and progression are associated with the presence and severity of contact lens related anterior segment disease. The practice of testing contact lens solution effectiveness against the planktonic organisms may provide a false sense of security in their ability to prevent infection in actual practice. This process of biofilm evolution is likely to occur in contact lens wearers who are noncompliant with proper cleaning regimen. The practice of “topping” off case solution is an example of a practice certain to invite biofilm formation. –Further study to determine the rate of biofilm formation and composition will be forthcoming Although fungus and protozoa were not evaluated in our study, Stenotrophomonas is a favored nutrient species for Acanthamoeba and may be a precursor to such infections.