The Association for Research in Vision and Ophthalmology (ARVO) annual meeting, held May 1-5, 2011 in Ft. Lauderdale, Fla., provides a significant look at novel research and technology that shapes the future of eye care. Research presented this year will help formulate best practices and help identify additional questions that need to be answered in order to provide appropriate care in the clinical setting.

The topics of biofilm formation, bioburden and the management of adverse events triggered by lens and storage case contamination continue to pique interest. Once bacteria and organisms enter the case from various vectors, they adhere to the case and lens—transformed from a planktonic phenotype to a resistant sessile biofilm phenotyope—making it less susceptible to disinfectants or cleaning agents.

These topics are well represented in the literature and at this year’s ARVO meeting. The 2011 abstracts are teeming with important answers to questions such as the best methods to collect biofilms from contact lenses and cases (lack of standards by FDA and ISO) and how to identify the sequence of events in biofilms to establish a firm foundation and what transpires when they do.

Methicillin Resistance and Biofilm Production in Staphylococcus epidermidis Isolated from Pathological Specimen and Normal Conjunctival Flora (Program #1933)
Researchers in Munich, Germany and Asuncion, Paraguay used 20 isolates of S. epidermidis from pathological clinical infections (study group) and 22 isolates of S. epidermidis from normal conjunctival flora (control group) to investigate the presence of mecA, a methicillin-resistant gene. The isolates from the study and control groups were tested against various antibiotics to determine the resistance rates.

They found that, regardless of the group, biofilm production was detected in 94% of the strains with mecA. The study group—S. epidermidis isolated from a variety of infections—showed a higher degree of antibiotic resistance compared to the control group.

“Because S. epidermidis is a major saprophyte of the eye and often associated with ocular infections, the high frequency of mecA positive, multi-resistance strains in pathological samples, together with their strong association with biofilm production highlights the potential of a pathogenic role of this otherwise saprophytic bacterium.”

Efficacy of Povidone-iodine For Disinfection of Bacteria in The Contact Lens Storage Case (Program # 5838)
Rearchers from Tokushima University and Ophtecs Corp. in Kobe, Japan evaluated the disinfectant efficacy various solutions have against planktonic and adherent bacteria in the contact storage case. The study tested six commercially available multipurpose solutions (MPS), a commercially available hydrogen peroxide and povidone-iodine against 12 clinical isolates. The samples (six strains of Pseudomonas aeruginosa, one strain of Pseudomonas fluorescens, one strain of Stenotrophomonas maltophilia, two strains of Serratia marcescens, one strain of Alcaligenes xylosoxidans and one strain of Chryseobacterum indologenes) were cultivated in a Y culture medium and controlled at 10^7 CFU/ml.

The researchers found that povidone-iodine provided the highest disinfectant efficacy against planktonic bacteria and adherent bacteria inside the contact lens storage case. Most MPSs showed >3log reductions against all strains.

Biocidal Efficacy of Multipurpose Contact Lens Care Solutions Against Stenotrophomonas and Delftia: Resistance and Regrowth (Program # 5847)
Researchers from the Corneal Research and Development Department of Abbott Medical Optics in Santa Ana, Calif., investigated the biocidal efficacy of three MPSs and one-step hydrogen peroxide, particularly their ability to kill strains of Stenotrophomonas and Delftia. Additionally, their research studied the ability of these bacteria to support the growth and replication of Acanthamoeba castellanii.

The commercial MPSs studied contained biocidal agents of: polyquaternium-1 (PQ1) and polyhexamethylene biguanide (PHMB); PHMB alone; PQ1 and alexidine (ALX); PQ1, myristamidopropyl dimethylamine (MAPD), nonanoyl ethylenediaminetriacetic acid (C9ED3A); and 3% hydrogen peroxide with a platinum disk neutralizing system (PEROX). The researchers performed the biocidal testing in accordance with ISO 14729, and viability assessed at six hours, 24 hours and seven to 21 days.

The study found S. maltophilia and D. acidovorans inherently resistant to MPSs composed of PQ1-MAPD-C9ED3A. However, those MPSs consisting of PQ-Alex, PQ1-PHMB, PHMB or PEROX after four hours of exposure time gave a total kill (>4.0 log) with all strains tested. All strains supported the excystment and trophozoite replication of A. castellanni at a rate comparable to E. coli as a food source.

The ability of such bacteria to survive and replicate in MPS holds critical implications for cases involving biofilms. The formation of biofilm within the storage case reduces the disinfection efficacy of MPSs. Biofilm protects bacteria from exposure to the disinfectant by creating an impenetrable barrier within the storage case, in effect allowing Acanthamoeba to grow rampant in its now favorable habitat.

Application of Activated Protein C in Reducing Soft Contact Lens Associated Fungal Biofilms (Program # 6478)
Researchers from the Ophthalmology & Visual Sciences and Dermatology departments at Case Western Reserve University in Cleveland studied the formation activity of fungal biofilms commonly associated with soft-lens wearers. By using an in-vitro established soft contact lens fungal biofilm model, the study tracked the formation of biofilm after exposure to Activated Protein C (APC)—a potent physiologic anticoagulant with profibrinolytic properties.

The fungal biofilms (Fusarium solani 6914 and Fusarium oxysporum 8996) were obtained from fungal keratitis patients and incubated with three different types of worn contact lenses (lotrafilcon A, balafilcon A and etafilcon A).

The researchers assessed the susceptibility of these fungal biofilms under two condition, when the APC solution was added during the adhesion phase of biofilm development and after 48 hours of mature biofilm formation.

The study found APC’s efficacy as an anti-fungal agent to vary across the different trials. Compared to the phosphate buffered saline-soaked worn-lens control group (p<0.05), APC was not effective against F. solani or F. oxysporum biofilm formation when added during the adhesion phase. However, APC significantly reduced F. solani biofilm activity by 69%, 72% and 81% on etafilcon A, lotrafilcon A and balafilcon A lenses, respectively in the post-biofilm formation trials. Further, APC was ineffective at reducing F. oxysporum activity by 26%, 1% and 1% on etafilcon A, lotrafilcon A and balafilcon A lenses, respectively.

Despite this variability, APC’s anti-biofilm activity in this study reveals a potentially powerful solution to the problem of biofilm penetration as seen in MPS’s inability to properly function in the presence of biofilm.

Contact Lens Storage Case Hygiene Practice and Case Contamination (Program # 6480)
Researchers from the School of Optometry and Vision Science at the University of New South Wales in Sydney, Australia tested how effective common contact lens care/cleaning procedures are at removing microbial biofilms. The contact lens storage cases were innoculated with 2mL of 10^6CFU/mL of either P. aeruginosa or S. aureus. Following an incubation period of 48 hours, the cases underwent a cleaning treatment of either a 10 second rinse (hot water or test MPS containing polyhexamethyl biguanide and polyquad), or soaking (MPS or 3% hydrogen peroxide). Subsequently, the cases were left to air dry for six hours or wiped with tissue.

The researchers found that S. aureus was more resistant to hygiene procedures than P. aeruginosa. Further, rinsing (with MPS or hot water) followed by six hours of air-drying was found to be insufficient in removing heavy biofilm.

Comparative Biocompatibility of Contact Lens Multipurpose Disinfecting Solutions with Soft Contact Lenses – Potential Correlations with Lens Preservative Uptake and Release Profiles (Program # 6481)
Researchers at Abbot Medical Optics in Santa Ana, Calif. evaluated the effects of contact lens MPSs by in vitro biocompatability assessment using mouse fibroblast (L929) cells and in relationship to preservative uptake and release profiles with various soft contact lenses.
RevitaLens OcuTec MPS (RO) was evaluated against Opti-Free Replenish (OFR) and Opti-Free Express (OFE). Five soft CLs were examined and in vitro biocompatibility was assessed according to ISO 10993.

RO was found to be less cytotoxic than OFR or OFE in MPS-CLs biocompatibility, as defined by lower in vitro cytotoxicity on L929 cells. Cytotoxicity scores with RO did not correlate with preservative uptake, but a weaker correlation with alexidine dihydrochloride release was observed.

Effects of Multipurpose Disinfecting Solution Excipients on Corneal Cell Physiology and Cytokine Production (Program # 6485)
Researchers at the Brien Holden Vision Institute at the University of New South Wales investigated how excipients nonaoyl EDYA (nEDTA) and propylene glycol (PG) influences cell numbers, cell metabolic activity and cell membrane integrity, as well as the production of cytokines by human corneal limbal epithelial cells (HCLE). They tested human corneal limbal epithelial cells exposed to two different MPSs for two, six and 18 hours.

The MPSs containing nEDTA/PG reduced cell numbers, increased cell membrane permeability and increased cytokine production. However, exposing cells to the individual excipients did not produce similar results. This indicates that further research should be conducted to investigate combinations of excipients.

Galyfilcon A Silicone Hydrogel Lense Infused With Silver Iodide Delay Or Inhibit In-vitro Surface Colonization By Bacteria And Fungi Associated With Adverse Ocular Events (Program # 6503)
Researchers at Georgia State University in Atlanta evaluated the antimicrobial activities of silver salt infused silicone hydrogel lenses in preventing or retarding in-vitro colonization of lenses by bacteria and fungi. Lenses were inoculated and examined daily with light microscopy.

They found that lenses infused with silver-iodide were slower or less likely to be colonized by bacteria and fungi associated with adverse ocular events compared to lenses without silver iodide. “Silver iodide infusion may reduce the risk of the lens serving as a fomite in the transfer of microorganisms from the contact lens case to the eye”

Neutrophil-enhanced P. aeruginosa Biofilms on Silicone Hydrogel Contact Lenses (Program # 6506)
Wearing contact lenses overnight with concurrent bacterial challenge, results in significant accumulation of neutrophils. Biofilms of Pseudomonas are significantly increased in the presence of neutrophils.

Researchers from the University of Texas Southwestern Medical Center in Dallas evaluated the P. aeruginosa (PA) biofilm formation on silicone hydrogel lenses. Their data demonstrates that intense corneal inflammation associated with PA contamination results in enhanced biofilm formation on silicone hydrogel lenses. Further studies are recommended to determine the efficacy of available solution regimens against neutrophil-enhanced biofilms on contact lens surfaces.

Susceptibility Of Ocular Isolates of P. aeruginosa To Contact Lens Multipurpose Solutions (Program # 6507)
Persistent microbial contamination of contact lens storage cases may be a feature of biofilm growth that confers resistance of certain organisms to MPSs.

Researchers at the Brien Holden Institute at the University of New South Wales in Sydney investigated the susceptibility of keratitis isolates of P. aeruginosa to MPSs. Fourteen isolates capable of forming strong biofilms were allowed to adhere for 24 hours in polypropylene CL storage cases. The study confirmed the widespread susceptibility of planktonic keratitis isolates (P. aeruginosa) to contemporary MPSs and MIC was not associated with a cytotoxic phenotype.

Further, the researchers found that the impact of MPS on viable and total biofilm was variable, which “may in part explain persistence of organisms within storage cases despite compliance with manufacturers’ guidelines.”

Biofilm Bacterial Diversity: Association With Disease Severity in Contact Lens Related Keratitis (Program # 6508)
Biofilm formation in contact lens storage cases may predispose wearers to the development of contact lens related keratitis.
Researchers of the Ophthalmology and Microbiology Departments of West Virginia University utilized ribosonal RNA gene sequencing to better understand the composition of contact lens case biofilms. The group collected cases from patients with mild keratitis, keratitis with focal infiltrate and contact lens related corneal ulcers. Cases from asymptomatic lens wearers were also collected as a control group.

The researchers found that bacterial diversity from the contact lens case correlated with the severity of disease and presenting visual acuity, and was greater than asymptomatic controls. Achromobacter and Stenotrophomonas were the predominant residents of storage case biofilms.

Susceptibility of Fungal Biofilms on Worn Soft Contact Lenses to Lens Care Solutions (Program # 6544)
The efficacy of marketed contact lens care products against Fusarium biofilms formed on worn lenses has been questioned. Just how effective are they against fungal biofilms especially Fusarium?

Researchers from Case Western Reserve University in Cleveland investigated the ability of biofilms to form on three types of worn lenses (lotrafilcon A, etafilcon A and balafilcon A) and measured their susceptability to MPSs. The Fusarium strains (Fusarium solani 6914 and Fusarium oxysporum 8996) were from two patients with fungal keratitis.

The Fusarium strains formed biofilms on all three types of lenses and were resistant to the antifungal activity of several soft contact lens care products. Only the hydrogen peroxide care system and one polyquaternium-preserved MPS consistently demonstrated effective antifungal activity against both strains on the three lens types investigated.

We must remember that organisms communicate and survive through biofilm formation. We are just beginning to understand what transpires in the case and on the lens; the abstracts highlighted in this report attempt to shed light on the intriguing aspects of biofilms and bioburden and how they all relate to possible associated adverse events. Hope you enjoy this year’s review and for those who have never attended an ARVO meeting, I  hope you can do so next year!