Human tear fluid modulates the Pseudomonas aeruginosa transcriptome to alter antibiotic susceptibility.

PURPOSE: Previously, we showed that tear fluid protects corneal epithelial cells against Pseudomonas aeruginosa without suppressing bacterial viability. Here, we studied how tear fluid affects bacterial gene expression. METHODS: RNA-sequencing was used to study the P. aeruginosa transcriptome after tear fluid exposure (5 h, 37 oC). Outcomes were further investigated by biochemical and physiological perturbations to tear fluid and tear-like fluid (TLF) and assessment of bacterial viability following tear/TLF pretreatment and antibiotic exposure. RESULTS: Tear fluid deregulated ~180 P. aeruginosa genes ≥8 fold versus PBS including downregulating lasI, rhlI, qscR (quorum sensing/virulence), oprH, phoP, phoQ (antimicrobial resistance) and arnBCADTEF (polymyxin B resistance). Upregulated genes included algF (biofilm formation) and hemO (iron acquisition). qPCR confirmed tear down-regulation of oprH, phoP and phoQ. Tear fluid pre-treatment increased P. aeruginosa resistance to meropenem ~5-fold (4 µg/ml), but enhanced polymyxin B susceptibility ~180-fold (1 µg/ml), the latter activity reduced by dilution in PBS. Media containing a subset of tear components (TLF) also sensitized bacteria to polymyxin B, but only ~22.5-fold, correlating with TLF/tear fluid Ca2+ and Mg2+ concentrations. Accordingly, phoQ mutants were not sensitized by TLF or tear fluid. Superior activity of tear fluid versus TLF against wild-type P. aeruginosa was heat resistant but proteinase K sensitive. CONCLUSION: P. aeruginosa responds to human tear fluid by upregulating genes associated with bacterial survival and adaptation. Meanwhile, tear fluid down-regulates multiple virulence-associated genes. Tears also utilize divalent cations and heat resistant/proteinase K sensitive component(s) to enhance P. aeruginosa sensitivity to polymyxin B.

Impact of topical corticosteroid pretreatment on susceptibility of the injured murine cornea to Pseudomonas aeruginosa colonization and infection.

Research with animal models of Pseudomonas aeruginosa keratitis has shown that use of a topical corticosteroid alone against an established infection can significantly increase the number of colonizing bacteria or worsen clinical disease. Moreover, retrospective analysis has suggested that corticosteroid use in humans is associated with an increased risk of keratitis in eyes with pre-existing disease. Thus, while corticosteroids are often used to reduce ocular inflammation in the absence of infection, the risk of opportunistic infection remains a concern. However, the effect of corticosteroids on the intrinsic barrier function of uninfected corneas is unknown. Here, we tested if short-term topical corticosteroid treatment of an uninfected murine cornea would increase susceptibility to P. aeruginosa colonization or infection after epithelial injury. Topical prednisolone acetate (1%) was administered to one eye of C57BL/6 mice three times a day for 3 days; control eyes were treated with sterile PBS. Prior to inoculation with a cytotoxic P. aeruginosa corneal isolate strain 6206, corneas were subject to superficial-injury by tissue paper blotting, or scratch-injured followed by 12 h of healing. Previously we have shown that blotting renders mouse corneas susceptible to P. aeruginosa adhesion, but not infection, while 12 h healing reduces susceptibility to infection after scratching. Corneas were evaluated at 48 h for bacterial colonization and microbial keratitis (MK). To monitor impact on wound healing, corneal integrity was examined by fluorescein staining immediately after scarification and after 12 h healing. For both the tissue paper blotting and scratch-injury models, there was no significant difference in P. aeruginosa colonization at 48 h between corticosteroid-pretreated eyes and controls. With the blotting model, one case of MK was observed in a control (PBS-pretreated) cornea; none in corticosteroid-pretreated corneas. With the 12 h healing model, MK occurred in 6 of 17 corticosteroid-pretreated eyes versus 2 of 17 controls, a difference not statistically significant. Corticosteroid-pretreated eyes showed greater fluorescein staining 12 h after scarification injury, but this did not coincide with increased colonization or MK. Together, these data show that short-term topical corticosteroid therapy on an uninfected murine cornea does not necessarily enhance its susceptibility to P. aeruginosa colonization or infection after injury, even when it induces fluorescein staining.

Human Tear Fluid Reduces Culturability of Contact Lens-Associated Pseudomonas aeruginosa Biofilms but Induces Expression of the Virulence-Associated Type III Secretion System.

PURPOSE: The type III secretion system (T3SS) is a significant virulence determinant for Pseudomonas aeruginosa. Using a rodent model, we found that contact lens (CL)-related corneal infections were associated with lens surface biofilms. Here, we studied the impact of human tear fluid on CL-associated biofilm growth and T3SS expression. METHODS: P. aeruginosa biofilms were formed on contact lenses for up to 7 days with or without human tear fluid, then exposed to tear fluid for 5 or 24 h. Biofilms were imaged using confocal microscopy. Bacterial culturability was quantified by viable counts, and T3SS gene expression measured by RT-qPCR. Controls included trypticase soy broth, PBS and planktonic bacteria. RESULTS: With or without tear fluid, biofilms grew to ∼108 CFU viable bacteria by 24 h. Exposing biofilms to tear fluid after they had formed without it on lenses reduced bacterial culturability ∼180-fold (P<.001). CL growth increased T3SS gene expression versus planktonic bacteria [5.46 ± 0.24-fold for T3SS transcriptional activitor exsA (P=.02), and 3.76 ± 0.36-fold for T3SS effector toxin exoS (P=.01)]. Tear fluid further enhanced exsA and exoS expression in CL-grown biofilms, but not planktonic bacteria, by 2.09 ± 0.38-fold (P=.04) and 1.89 ± 0.26-fold (P<.001), respectively. CONCLUSIONS: Considering the pivitol role of the T3SS in P. aeruginosa infections, its induction in CL-grown P. aeruginosa biofilms by tear fluid might contribute to the pathogenesis of CL-related P. aeruginosa keratitis.

The effect of contact lens hygiene behavior on lens case contamination.

PURPOSE: To assess the association between the levels of lens case contamination and lens wearers' hygiene behaviors. METHODS: Contact lens wearers were surveyed for information on wearer demographics and contact lens hygiene behavior. Microbial analysis of lens cases was performed. Multivariate analysis was used to identify factors associated with contact lens storage case contamination. RESULTS: One hundred nineteen contact lens wearers responded. The mean (±SD) age of the participant was 32 (±1) years (range, 18 to 69 years). Seventy percent of the participants were female. Sixty-six percent of lens cases were contaminated (median, 25; range, 0 to 107 colony-forming units [CFUs]). In the multivariate analysis, washing hands with soap and water (1.8 ± 2.0 log10 CFU/case) rather than just water or no washing was associated with lower levels of lens case contamination (2.4 ± 2.1 log10 CFU/case; p = 0.005). Lens cases received wet had higher levels of contamination than dry cases (1.5 ± 1.5 vs. 2.4 ± 2.5 log10 CFU/case; p = 0.015). Mismatching lens case and disinfecting solution was a risk factor for lens case contamination (p = 0.019). Lens wearers who had more than 2 years of wearing experience had higher levels of contamination than those who had less than 2 years of wearing experience (2.1 ± 2.1 vs. 1.1 ± 1.5 log10 CFU/case; p = 0.04). CONCLUSIONS: Major factors that can reduce lens case contamination were washing hands with soap and water, air-drying lens cases, and matching of the disinfecting solution with lens case (i.e., from the same manufacturer). This information is beneficial when advising lens wearers in clinical practice.

Pseudomonas aeruginosa Survival at Posterior Contact Lens Surfaces after Daily Wear.

PURPOSE: Pseudomonas aeruginosa keratitis is a sight-threatening complication of contact lens wear, yet mechanisms by which lenses predispose to infection remain unclear. Here, we tested the hypothesis that tear fluid at the posterior contact lens surface can lose antimicrobial activity over time during lens wear. METHODS: Daily disposable lenses were worn for 1, 2, 4, 6, or 8 hours immediately after removal from their packaging or after presoaking in sterile saline for 2 days to remove packaging solution. Unworn lenses were also tested, some coated in tears "aged" in vitro for 1 or 8 hours. Lenses were placed anterior surface down into tryptic soy agar cradles containing gentamicin (100 µg/mL) to kill bacteria already on the lens and posterior surfaces inoculated with gentamicin-resistant P. aeruginosa for 3 hours. Surviving bacteria were enumerated by viable counts of lens homogenates. RESULTS: Posterior surfaces of lenses worn by patients for 8 hours supported more P. aeruginosa growth than lenses worn for only 1 hour, if lenses were presoaked before wear (∼ 2.4-fold, p = 0.01). This increase was offset if lenses were not presoaked to remove packaging solution (p = 0.04 at 2 and 4 hours). Irrespective of presoaking, lenses worn for 8 hours showed more growth on their posterior surface than unworn lenses coated with tear fluid that was aged for 8 hours in vitro (∼ 8.6-fold, presoaked, p = 0.003; ∼ 5.4-fold from packaging solution, p = 0.004). Indeed, in vitro incubation did not impact tear antimicrobial activity. CONCLUSIONS: This study shows that postlens tear fluid can lose antimicrobial activity over time during contact lens wear, supporting the idea that efficient tear exchange under a lens is critical for homeostasis. Additional studies are needed to determine applicability to other lens types, wearing modalities, and relevance to contact lens-related infections.

Impact of cleaning regimens in silver-impregnated and hydrogen peroxide lens cases.

PURPOSE: Lens storage case hygiene practices are important for safe contact lens wear. However, the effectiveness of the manufacturer's direction for use and various cleaning regimens in reducing biofilm load is yet to be evaluated and compared. This in vitro study compared the effectiveness of several cleaning methods using silver-impregnated lens case and hydrogen peroxide disinfection systems. METHODS: Biofilms of Pseudomonas aeruginosa 122 and Staphylococcus aureus ATCC 6538 were grown in silver-impregnated and hydrogen peroxide lens cases. After the establishment of the biofilms, the silver-impregnated case was subjected to one of four cleaning regimens: "manufacturer's directions for use--rinsed and recapped"; "rubbed, rinsed, and recapped"; "rubbed, rinsed, and air dried"; or "rubbed, rinsed, tissue wiped, and air dried." Hydrogen peroxide cases underwent one of two regimens: "manufacturer's directions for use--rinsed with saline and air dried" or "soaked in solution for 6 hrs." The level of residual bacteria was quantified. The efficacy of each cleaning regimen was then compared. RESULTS: Mechanical rubbing and wiping of silver-impregnated cases and soaking hydrogen peroxide cases in hydrogen peroxide were the most effective treatments for reducing biofilms. Once the silver-impregnated cases were rubbed, air drying or recapping the cases did not have any significant effect on the level of the biofilm. The level of initial biofilm formation of silver-impregnated cases was significantly lower than those of polypropylene lens cases (P<0.0001). CONCLUSIONS: Rubbing and rinsing with disinfecting solution and wiping with a tissue can be considered to be effective in removing biofilms from silver-impregnated lens cases. Resoaking the basket-type lens case in hydrogen peroxide between use was found to be effective in removing biofilms from these cases.

Impact of lens case hygiene guidelines on contact lens case contamination.

PURPOSE: Lens case contamination is a risk factor for microbial keratitis. The effectiveness of manufacturers' lens case cleaning guidelines in limiting microbial contamination has not been evaluated in vivo. This study compared the effectiveness of manufacturers' guidelines and an alternative cleaning regimen. METHODS: A randomized cross-over clinical trial with two phases (n = 40) was performed. Participants used the lens types of their choice in conjunction with the provided multipurpose solution (containing polyhexamethylene biguanide) for daily wear. In the manufacturers' guideline phase, cases were rinsed with multipurpose solution and air dried. In the alternative regimen phase, cases were rubbed, rinsed with solution, tissue wiped, and air-dried face down. The duration of each phase was 1 month. Lens cases were collected at the end of each phase for microbiological investigation. The levels of microbial contamination were compared, and compliance to both regimens was assessed. RESULTS: The case contamination rate was 82% (32/39) in the manufacturers' guideline group, compared with 72% (28/39) in the alternative regimen group. There were significantly fewer (p = 0.004) colony forming units (CFU) of bacteria from cases used by following the alternative regimen (CFU range of 0 to 10, and median of 12 CFU per well) compared with that of the manufacturer's guidelines (CFU range of 0 to 10, and median of 28 CFU per well). The compliance level between both guidelines was not significantly different (p > 0.05). CONCLUSIONS: The alternative guidelines are more effective in eliminating microbial contamination from lens cases than that of the current manufacturer's guideline. Simply incorporating rubbing and tissue-wiping steps in daily case hygiene reduces viable organism contamination.

The effectiveness of various cleaning regimens and current guidelines in contact lens case biofilm removal.

PURPOSE: Lens case hygiene is important for safe contact lens wear. However, there are no evidence-based data to suggest optimum hygiene regimens. This in vitro study aimed to evaluate and compare the effectiveness of manufacturers' guidelines and several other regimens in removing biofilm using various types of contact lens cases and disinfecting agents. METHODS: Biofilms of Staphylococcus aureus ATCC 6538 and Pseudomonas aeruginosa 122 were formed on two types of unused contact lens cases. Subsequently, each contact lens case was subjected to one of four cleaning regimens using two different multipurpose disinfecting solutions or distilled water: "rinse and air-dry (guidelines)," "rub, rinse and air-dry," "tissue-wipe and air-dry," and "rub, rinse, tissue-wipe, and air-dry." The levels of residual biofilm were then quantified using viable counts and compared. RESULTS: The manufacturers' guidelines resulted in 1 to 2 log CFU reduction of either biofilm. "Rub, rinse, tissue-wipe, and air-dry" was the most effective cleaning regimen (P < 0.001), capable of removing 4 to 6 log CFUs of bacteria; higher levels of biofilm were removed by mechanical friction from nonridged cases than that of ridged cases (P < 0.001). Biofilm removal varied with multipurpose solution tested. CONCLUSIONS: Current manufacturers' guidelines are not adequate in eliminating microbial contamination. Simply incorporating a rubbing/wiping step in daily case hygiene reduces viable organism recovery. Factors such as the cleaning regimen, antimicrobial potency of multipurpose solution, and the interior topography of a lens case may impact the surface detachment of biofilm during the cleaning process.

Do swimming goggles limit microbial contamination of contact lenses?

PURPOSE: Wearing goggles over contact lenses while swimming is often recommended by eye care professionals. Limited data are available to assess this recommendation. The purpose of this study was to examine whether wearing goggles while swimming limits bacterial colonization on contact lenses and whether the type of lens worn affects contamination rates. METHODS: Twenty-three subjects underwent two swimming sessions at an ocean (salt water) pool (Maroubra beach Rock Pool, Sydney, Australia). Silicone hydrogel (Ciba Focus Night and Day) or hydrogel lenses (Ciba Focus Daily) were inserted into subjects' eyes before 30 min of swimming sessions, and subjects used modified goggles to mimic goggled and non-goggled conditions. At the end of each session, lenses were collected for microbial investigation. Viable bacterial colonies were classified as gram positive and gram negative and enumerated. The level of bacterial colonization on contact lenses between goggled and non-goggled conditions and between the two lens materials were compared. RESULTS: The range of colony forming units recovered from goggled lenses were 0 to 930 compared with 0 to 1210 on non-goggled lenses. The majority of subjects (16/23) had more microorganisms in the non-goggled condition than when wearing goggles (p = 0.03). Gram negative organisms were found in three non-goggled lenses. No significant difference was shown in the number of bacteria isolated from silicone hydrogel and hydrogel lenses (p > 0.6) irrespective of wearing goggles. Water samples had consistently higher numbers of bacterial counts than those adhered to the lenses; however, no association was found between the number of bacteria in the water sample and those found on the contact lenses. CONCLUSIONS: Consistently, fewer bacterial colonies were found on the goggled contact lens, thus suggesting goggles offer some protection against bacterial colonization of contact lenses while swimming. These data would support the recommendation encouraging lens wearers to use goggles while swimming.

Removal of biofilm from contact lens storage cases.

PURPOSE: Lens case hygiene practices are important in maintaining safe contact lens wear. However, the effectiveness of various lens case cleaning practices have not been evaluated and compared. This in vitro study aimed to evaluate and compare the efficacy of cleaning practices that are most commonly carried out by lens wearers and recommended by practitioners. METHODS: Pseudomonas aeruginosa 122, Serratia marcescens ATCC 13880, and Staphylococcus aureus ATCC 6538 were the challenge bacteria for biofilm formation on unused lens cases from two different manufacturers. After establishment of the biofilm, each lens case was subjected to one of the six cleaning regimens: "rinsed," "rubbed and rinsed," "air-dried," "soaked in a multipurpose contact lens solution," "tissue-wiped," and "lids recapped." The level of residual biofilm was quantified at the end of each cleaning regimen. The efficacy of each cleaning regimen was then compared. RESULTS: Mechanical rubbing and wiping of lens cases were the most effective cleaning regimen tested in reducing biofilm. Soaking lenses in disinfecting solution for 6 hours removed the majority of biofilm from lens cases. Rinsing lens cases alone provided only minimal efficacy in reducing biofilm. Air-drying or recapping the cases with the lid without any other additional cleaning methods were the least efficient at removing biofilm. CONCLUSIONS: Based on this study, digital rubbing and rinsing and/or wiping the lens cases with tissue is recommended. Air-drying or recapping the lens case lids after use without any additional cleaning methods should be discouraged with non-antimicrobial lens cases.

Impact of air-drying lens cases in various locations and positions.

PURPOSE: To determine the rate and type of microbial contamination when contact lens cases are air-dried in two different positions (face up and face down) and in four different locations (toilet, bathroom, office, and bedroom). METHODS: Unused contact lens cases (n = 97) were rinsed with 2-ml sterile phosphate buffered saline and then placed on facial tissue paper in different locations: humid (toilet and bathroom) and non-humid (office and bedroom) and air-dried at room temperature. After 24 h, the contact lens cases were collected and sampled for microbial numbers and microbial types identified using standard techniques. The microbial profile and the rate of contamination between different locations and positions were compared. RESULTS: Irrespective of the air-drying location, contact lens cases positioned face up had a significantly higher contamination rate (34/48, 71%) compared with contact lens cases air-dried face down (6/49, 12%) (p < 0.001). For those contact lens cases air-dried face up, there was more contamination when placed in humid environments (toilet and bathroom) than in the non-humid environments (office and bedroom) (p = 0.01). However, the contact lens case contamination rate among various locations was similar when contact lens cases were air-dried face down. Total microorganisms recovered from contact lens cases ranged from 0 to 275 colony forming unit per well. The most frequently recovered microorganisms from the contaminated contact lens cases were coagulase-negative Staphylococci, fungi, and Bacillus spp. Thirty-three percent (13/40) of contact lens cases were contaminated with multiple species. CONCLUSIONS: Small numbers of microorganisms from the environment may contaminate contact lens cases while cases are air-dried face up. Cases air-dried in humid environments have higher levels of microbial contamination; this is particularly true when contact lens cases are positioned face up. On the basis of this limited study, we would recommend contact lens cases be air-dried face down.

Profile and frequency of microbial contamination of contact lens cases.

PURPOSE: To evaluate the frequency level and profile of contact lens storage case contamination in asymptomatic contact lens wearers and to examine whether different areas of the same lens case may show a different rate and profile of contamination. The relationship between lens storage case contamination and the age of the lens storage case was also examined. METHODS: Sixty-four lens cases and case age information were collected from asymptomatic contact lens wearers. Lens cases were sampled at two locations, the upper rim and the lower base. The samples underwent microbiological investigation for recovery of bacteria and fungi. Contamination rate between the two sampling locations and the relationship between the contamination levels and the age of the lens case were analyzed. RESULTS: Contamination occurred in 58% (37 of 64) of lens cases. The most frequently recovered microorganisms were coagulase-negative Staphylococci (51%, 19 of 37), Bacillus spp. (43%,16 of 37), and fungi (27%,10 of 37). For flat-well lens cases, higher numbers of microorganisms were recovered from the upper rim than that from the lower base (p = 0.02), and a greater variety of Gram-negative bacteria were recovered from the upper rim. A higher recovery rate of Micrococcus spp. (p = 0.02; in flat cases) and coagulase-negative Staphylococci (p = 0.01; for both flat and basket type cases) was found from the base of the case well compared with the upper rim. For stand-up cases, higher numbers of microorganisms were recovered from the lens basket compared to the upper hinge (p = 0.047). Lens cases that were <9 months of age had lower levels of contamination (p = 0.013) than older cases. CONCLUSIONS: Frequent replacement of lens cases may reduce microbial contamination. Future studies should specify the areas swabbed in the lens case. Better lens storage case design and additional hygiene attention need to be introduced to reduce contamination in these "risky" areas.