Exploring microwave irradiation as a method to disinfect contact lens cases

Abstract

Purpose:

Biofilm formation caused by infrequent contact lens case replacement and the ineffectiveness of multi-purpose solutions (MPS) on biofilm removal is associated with high rates of bacterial keratitis infections. This study demonstrated biofilm elimination from the contact lens case by microwave irradiation.

Methods:

Staphylococcus aureus biofilms indicative of 3 to 9 months of contact wear were cultured in contact lens cases and visualized with crystal violet (CV) staining. Biofilms in contact cases were then exposed to four treatment regimens: No treatment (n = 8), 45 seconds microwave irradiation (n = 8), tap water (n = 6), and MPS (n = 9). Bacterial survival was assessed by colony forming unit (CFU) assay using streak dilutions.

Results:

Visualization of the biofilms through CV staining revealed that biofilms coalesce between ribs of the contact case. In 5/8 cases no CFU were cultivated from the case after treatment with microwave irradiation. In tap water and MPS the first dilution averaged 6±2 and 31±13 CFUs per plate, respectively, while microwave irradiation averaged < 1 CFU per plate. In Dilution 2, the average reduced to 0.7±0.7 and 6±5 CFUs per plate for tap water and MPS, respectively, while microwave irradiation had 0 CFUs in Dilution 2.

Conclusion:

Biofilms that coalesce between the ribs of the contact case pose a threat because this area is difficult to thoroughly scrub and could act as a basis for infection through fouling of contact lenses. Of the four treatment regimens, microwave irradiation displayed the most consistent and highest rate of bacterial eradication. Tap water was less effective compared to microwave irradiation, and poses other side effects, but greatly reduced CFU count compared to no treatment. MPS displayed the poorest bacterial eradication of the treatments. Thus, microwave irradiation is worth further investigation as a viable in-home disinfecting option.

Introduction

The popularization of soft contact lenses has corresponded to a dramatic increase in infectious keratitis, in some cases by 95% (1). Keratitis is estimated to cost the US healthcare system $175 million annually (2). There is a strong association between patient lens case contamination and keratitis (3–5). Yet, many contact wearers regularly fail to follow their contact lens care guidelines. Bui et al. found that education alone does not sufficiently improve behavior regarding proper lens and case care (6). Over 40% of adult users do not replace their case within the recommended 3 months (7–10). It is common for contact lens wearers to order 6 months to a year supply of lenses and this may be the only time in which they consider replacing their case, instead of the recommended 3 months. If not replaced regularly, bacteria can coalesce within the case forming biofilms known to play a significant role in infection (11–14), which is illustrated in FIG 1. Also, because bacteria inside the case are not easily visible, contact lens wearers may not be motivated to purchase or replace their case. It has been well established that multi-purpose contact solutions are not potent enough to disinfect the contact lens case once biofilms have formed (11, 15–17). Possibly, because the physical attributes of biofilm that confer resistance to antibiotics may play a similar role in reducing the effectiveness of MPS (18–20). Further, there is concern that MPS is not strong enough to address real world applications where there exists synergistic multi-species biofilms, and more invasive bacterial genotypes than those tested in International Organization for Standardization (ISO) guidelines (21–24). The majority of keratitis infections are bacteria based, particularly from the Staphylococcus aureus and Pseudomonas aeruginosa species (8, 12, 25). Of these two species, S. aureus is more associated with recurrent infection and a higher rate of corneal transplantation (26). Published work has already highlighted ISO testing standards for not encompassing realistic conditions for MPS biofilm eradication (21, 22, 27, 28). Inadequate disinfection by MPS and improper patient compliance with cleanliness standards means that an additional at-home technique would be beneficial to reduce biofilm formation in contact cases.

FIG. 1.

Enlarged illustration of proposed bacterial infection cycle in the contact case. 1.) Regular lens case use introduces bacteria. 2.) Due to improper lens case care, bacteria secrete extracellular polymeric substances that coalesce into biofilm between the ribs of the case. 3.) Biofilm acts as a basis for future infection.

The microwave is a common household appliance and the majority of contact wearers will have regular access to one. Previous work has proven the use of microwave irradiation effective against Candida albicans on dentures and Acanthamoeba in the contact lens case (29, 30). This work seeks to further investigate the effects of microwave irradiation on S. aureus survival in comparison to three prevailing methods: no treatment, water-based treatment, and MPS treatment. At-home microwave irradiation may provide a route to disinfection of contact cases more favorable to patients than complete case replacement, as well as a safer alternative to the use of tap water which has been shown to greatly increase a wearers risk for infection (31).

Materials and Methods

Preparation of contact lens case and multi-purpose solution

The contact lens cases (Alcon) were cleaned prior to biofilm growth. Cases are made from polypropylene, the same material as microwavable kitchenware. MPS (Alcon OPTI-FREE Puremoist HydraGlyde®) was used as-received without a neutralizing agent to simulate consumer use of the product. The following active ingredients are listed for MPS: POLYQUAD 0.001%, ALDOX 0.0006%, HydraGlyde, sodium citrate, Tetronic® 1304, and boric acid. Contact lens cases were cleaned by soaking the case and unattached lids in 10% bleach for 1 hour followed by another hour in 70% methanol (32). The cases and lids were then transferred with sterile gloves upside down to dry for an hour on fresh paper towels. Once dry the cases were recapped and transferred to a sterile container only opened in a sterile biosafety cabinet to avoid cross contamination. Specimens were disposed of after experimentation by mixing with chlorine bleach and disposing via biohazardous waste protocol.

S. aureus strain selection and culture

For this study, S. aureus (ATCC 6538) was selected. This strain is used for the testing of contact lens care products under ISO standards and readily forms biofilms (15, 33). S. aureus was stored at −80°C in glycerol stock as received. To start a culture, colonies were transferred via inoculation loop into 5 mL of Todd Hewitt Yeast broth (Sigma-Aldrich) and allowed to incubate at 37°C for 24 hours. After incubation, the culture was diluted to reach an optical density of 0.1 at OD₆₆₀ (approximately 6 × 10⁸ CFU/mL) on a Genesys 30 spectrophotometer (Thermo Fisher Scientific). This protocol was adapted from Wu et al (33).

Formation of a three to nine-month biofilm analog

In a sterile biosafety cabinet, clean contact cases were uncapped, and each lens case well was filled with 3 mL of THY containing a final concentration of 2.5 × 10⁶ CFU/mL diluted from the overnight stock. Carefully, the case was loosely recapped and transferred to a 37°C incubator with 5% CO₂ for 24 hours of growth.

After overnight incubation, cases were carefully transferred back into the sterile biosafety cabinet. These cases were then opened, and serologically pipetted to aspirate the liquid media, making sure not to disturb the cultured biofilms. Cases were then rinsed with 3 mL of PBS to remove remaining media as well as planktonic bacteria unassociated with the biofilm. This PBS rinse step was performed twice per well. Additionally, a paper towel lightly moistened with 70% methanol is used to wipe down the lids of each case as needed when drops of media contact the lids during transport. Cases were allowed to dry for 3 minutes before the treatment regimens were performed. Each step of the biofilm formation process is illustrated in FIG 2.

FIG. 2.

Schematic describing the biofilm formation protocol for a 3 to 9-month-old analog (blue), four treatment regimens protocol (red), colony forming unit assay (green)

Treatment regimens

After biofilm formation, each case was treated with one of four treatment regimens shown in FIG 2. Cases without treatment and cases receiving microwave irradiation were recapped and set for 24 hours at room temperature undisturbed. After 24 hours, no treatment cases were rinsed two times with PBS to remove planktonic bacteria and ensure similar hydration to the other treatments before performing the CFU assay. Similarly, microwave treatment cases were rinsed once with PBS, microwaved for 45 seconds and then rinsed once more with PBS before performing the CFU assay. While in the microwave, the caps are unscrewed and placed outside of the microwave. The microwave (Westinghouse model WCM11100SSB) was listed to have 120 VAC, 60 Hz, and 12.5 amp. Forty-five seconds in the microwave was sufficient to reduce bacterial viability considerably (34). For the MPS and tap water treatments, each case was rinsed with either room temperature MPS or tap water and then filled with 3 mL in each lens case well of their respective solution before being left to sit for 24 hours in an undisturbed location. After 24 hours, they were rinsed two times with either room temperature MPS or tap water and then plated for CFU assay. The MPS came from a 150 mL squeeze bottle that was lightly sprayed to rinse the case without applying a shear force on the biofilm. Similarly, for tap water a light stream was generated by slightly turning the unfiltered laboratory faucet. Tap water was received from PWSID: KY0340250, a full water quality report was published in 2020 and stated full compliance with federal regulations (35).

Crystal violet staining

Crystal violet dye (Sigma Aldrich) was used to visualize the presence of biofilm in the contact lens case. Deionized (DI) water was added to generate a 30% dye to DI water mixture and 3 mL was added to each lens case well to incubate for 15 minutes at room temperature. After incubation, the case was rinsed 5 times with DI water, with caution not to disturb the stained biofilm. Excess stain was then removed for 15 minutes with 3 mL of 70% ethyl alcohol in each lens case well. Lastly, the ethyl alcohol is aspirated and left to dry for 5 minutes before optical imaging with a 12-megapixel camera. This protocol is adapted from Wu et al (33).

Colony forming unit assay

Each treatment is quantified by counting CFUs. An 5 uL inoculation loop (VWR) was used to comprehensively swab the interior of a single contact lens case well and then to streak a Tryptic Soy Agar plate (us.vwr.com) four times. A fresh inoculation loop was then used to dilute this first set of streaks by crossing over the previous set with another four streaks. This process is then repeated to generate the second dilution, which is visualized in FIG 2. The procedure was repeated for the second lens case well on a fresh agar plate. After streaking, each plate was labeled and left to incubate at 37°C for 48–72 hours until CFUs are large enough to easily enumerate. Plates were imaged one at a time and counted by eye marking each colony as it was counted. Counts for each dilution are completed independently of one another and a maximum of 200 CFUs are counted for a single dilution. A CFU is considered to belong to the highest concentration dilution it contacts. CFUs are averaged for each group and reported as average ± standard error.

Statistical analysis

Pairwise comparisons between CFUs among the twelve groups (four treatment regimens, three dilutions) were performed through the Wilcoxon rank sum test. Analyses were performed using MATLAB software (MathWorks, Inc., version R2020a) with the command ranksum, which provides a non-parametric comparison. This test was used to generate comparisons between each treatment and dilution level apart from a group that contains more than 50% CFUs that are saturated (CFU = 200).

Results

Microwave irradiation and tap water stunt bacterial growth more than multi-purpose solution

Optical images of one representative agar plate from each of the treatments appear in FIG 3. The average number of CFUs per treatment is shown in FIG 4 and are 200±0, 6±4, 120±38, and 144±29 at Dilution 0 for no treatment, microwave irradiation, tap water, and MPS, respectively. Dilution 0 refers to the original set of streaks while 1 and 2 refer to the consecutive streak dilutions of this original. The average number of CFUs per treatment at Dilution 1 are 143±22, 0.13±0.13, 6±2, 31±13 and the averages at Dilution 2 are 60±23, 0±0, 0.67±0.67, 6±5 for no treatment, microwave irradiation, tap water, and MPS, respectively. All averages are given alongside the standard error. All p-values from pairwise significance testing are included in Table 1. Each dilution within a given treatment is statistically significant from other dilutions within that same treatment for no treatment, tap water, and MPS. All cases that received no treatment (n = 8) are fully saturated in their initial streak (over 200 CFUs) and by Dilution 2 the average is still over 60 CFUs. In contrast, cases treated with microwave irradiation (n = 8) show very little bacterial presence. The first streaking of bacteria after microwave treatment typically resulted in little to no bacterial growth. No growth is seen by the second dilution for microwave irradiation. Thirdly, tap water (n = 6) seems to reduce the presence of bacteria in comparison to receiving no treatment, but unlike microwave irradiation, there is still a consistent presence of CFUs on the original streaks. However, subsequent dilutions for tap water quickly reduced the CFU count. MPS treatment (n = 9) appeared to have an effect similar to that of tap water. Though there is a slightly higher average of bacterial presence with MPS than with tap water at the same dilution, there is no statistically significant difference. All treatments were found to be significant in comparison to no treatment of the same dilution. Further, microwave irradiation was found to be statistically significant in its ability to reduce bacterial presence in comparison to both tap water and MPS. These results indicate that microwave irradiation has a stronger effect than MPS or tap water on reducing bacteria count in the contact case once a biofilm has formed.

FIG. 3.

Optical images of agar plates used to count CFUs per dilution for (A) no treatment, (B) microwave irradiation treatment, (C) tap water treatment, and (D) MPS treatment. Each agar plate contains three sets of four streaks. Each set of four streaks is one dilution. Plates are 100 mm in diameter and 15 mm in height.

FIG. 4.

Colony forming units per dilution for each treatment type. No treatment (orange, n = 8), Microwave irradiation (red, n = 8), Tap Water (blue, n = 6), MPS (green, n = 9). The Maximum enumerated value is 200 CFUs. Error bars show standard error. Significance determined by Wilcoxon rank sum test *p < 0.05.

TABLE 1.

P-values for pairwise significance testing using Wilcoxon rank-sum test.

		No treatment			Microwaved Irradiation			Tap water			MPS
		Dilution 0	Dilution 1	Dilution 2	Dilution 0	Dilution 1	Dilution 2	Dilution 0	Dilution 1	Dilution 2	Dilution 0	Dilution 1	Dilution 2
No Treatment	Dilution 0	x	†	†	†	†	†	†	†	†	†	†	†
	Dilution 1	x	x	0.021	1.554E-04	1.554E-04	1.554E-04	0.695	6.660E-04	6.660E-04	†	0.002	8.227E-05
	Dilution 2	x	x	x	0.015	0.001	0.001	0.261	0.031	0.005	†	0.356	0.009
Microwave Irradiation	Dilution 0	x	x	x	x	0.323	0.200	0.008	0.268	0.476	†	0.031	0.664
	Dilution 1	x	x	x	x	x	1.000	0.001	0.006	0.857	†	0.001	0.095
	Dilution 2	x	x	x	x	x	x	6.660E-04	0.006	0.857	†	0.001	0.041
Tap water	Dilution 0	x	x	x	x	x	x	x	0.041	0.004	†	0.092	0.007
	Dilution 1	x	x	x	x	x	x	x	x	0.041	†	0.260	0.163
	Dilution 2	x	x	x	x	x	x	x	x	x	†	0.008	0.235
MPS	Dilution 0	x	x	x	x	x	x	x	x	x	x	†	†
	Dilution 1	x	x	x	x	x	x	x	x	x	x	x	0.027
	Dilution 2	x	x	x	x	x	x	x	x	x	x	x	x
Average CFUs		200	143	60	6	< 1	0	120	6	< 1	144	31	6
Standard Error		--	22	23	4	< 1	--	38	2	< 1	29	13	5
No. of plates		8	8	8	8	8	8	6	6	6	9	9	9

Dark red boxes represent p < 0.01, Light red boxes represent p < 0.05 and blue boxes represent not significant, p > 0.05.

^†sample sets with greater than 50% saturation were not used for significance testing.

Biofilm formation coalesces in interior ribs of contact lens cases and is not visible to the contact lens wearer

Alcon cases have 16 interior ribs, between which, the highest level of staining was observed. CV staining Images appearing in FIG 5 were in good agreement with published work in which college age wearers cases were CV stained at a period of three to nine-months (36). A clean case (FIG 5a) was indistinguishable from the unstained biofilm case (FIG 5b), which is shown after CV staining in FIG 5c.

FIG. 5.

Optical images of (A) sterile Alcon contact lens case, (B) a contact lens case containing unstained biofilm, and (C) the same contact lens case after CV staining.

Discussion

This study evaluated the effectiveness of microwave irradiation compared to tap water and MPS to eliminate biofilm in contact lens cases. Buildup of bacteria in contact lens cases leads to a high risk of infection; however, this buildup is difficult to see with the naked eye (15). Because S. aureus is a gram-positive bacterium, CV dye was able to stain the outer peptidoglycan cell wall a dark purple. Thus, the use of CV allowed us to determine that the biofilm formation protocol generates an analog similar to what a contact lens wearer may experience between 3 to 9 months of use (33, 36). This time period is significant because 3 months is the recommended case replacement lifetime; yet, 40% of adults exceed this timeframe (7, 9–10, 37).

While the use of CV to visualize biofilm is not novel, many studies employ flat bottom well plates as a substrate. This type of dish increases the speed of testing but does not replicate the environment of the ribbed, smoothly curved contact case. As illustrated in FIG 5, biofilms tended to coalesce in the space between ribs where there was both greater surface area for biofilm adhesion and protection from shearing forces. These ‘hotspots’ between ribs are also where cellular debris and nutrients may settle and assist biofilm growth (18–20, 36). One downfall of the microwave irradiation method are lysed cellular debris remaining in the case after treatment, which could be overcome by the use of a ‘rub and rinse’ step (28, 33). MPS results may be similar to those of tap water because biofilms pose an innate resistance to infiltrating substances, like antibiotics, due to the layering of extracellular polymeric substances (EPS), it is suspected that MPS may face similar difficulty infiltrating the EPS network (18–20).

The number of CFUs enumerated was capped at 200, as greater values became too densely populated to accurately count. This saturation is important to consider for plates that had over 200 CFUs present, which was recorded for at least one agar plate in the original streaking (Dilution 0) for tap water, MPS, and no treatment, as well as the first and second dilution under no treatment. These values should be considered an underestimate of CFUs. In particular, every agar plate under no treatment initially plated over 200 CFUs and was thus expected to be much higher. To keep rigorous in our statistics, any sample sets with greater than 50% saturation during significance testing where excluded. The excluded sample sets were no treatment Dilution 0 and MPS Dilution 0. Therefore, in Fig. 5 we visualized statistical comparisons to the no treatment Dilution 1 group.

In five out of eight samples, microwave irradiation completely disinfected the contact lens case and of the remaining three samples, the highest CFU value was 26. Variables not considered by this study include analysis of case integrity after multiple uses and testing of other brands and materials. Heat distribution is also variable from one microwave model to another along with displaying inconsistent effects based on placement inside the microwave (38, 39). Despite these variables not yet explored, results were found to be repeatable and promising for the potential use of microwave irradiation as a convenient way to disinfect the contact lens case. Patient health could be greatly impacted by extending the life of the contact lens case. Overdue case replacement is one of the three most frequently ignored lens care guidelines (7). If further investigation determined that a case can last for 6 or 12 months when paired with the microwave, as opposed to the 3-month FDA recommendation violated by 40% of adult users (7, 9–10, 37), this method would be a novel way of improving patient compliance. Additionally, a longer timeframe may sync well with the purchase of new contact lens solution, new lenses, or a routine optometrist visit. Lastly, the reduced presence of biofilm in the contact lens case as a result of microwave irradiation could theoretically lead to a reduction in the current rates of infection (2, 12).

Although one might conclude from this data that tap water is more effective than no treatment, there is an abundance of data that suggests tap water poses other risks such as acanthamoeba keratitis (31, 40). Tap water is highly variable by location and time. This variability may be due to varying pH values and the presence of many added compounds that are irritating to the eye. Thus, while these factors may contribute to the reduction of bacteria in biofilm, it is perhaps due to the variable nature of tap water and should not be used as an alternative to MPS that is developed with longevity and comfort of the eye in mind (41). Future studies may hypothesize that in the event a medically necessary contact wearer runs out of solution the microwave could be paired with a sterile saline solution when seeking a short-term replacement.

One strength of this experimental design was the ability to compare drastically different results against one another by placing three streak dilutions on one plate. The original streaking for no treatment and MPS are likely underestimates but Dilutions 1 and 2 can confidently be compared to one another. From this comparison, MPS appears to yield about 1/6 the number of bacteria at each dilution when compared to no treatment, but still pales in comparison to microwave irradiation. Thus, while MPS does reduce the number of bacteria compared to receiving no treatment, it does not appear to completely disinfect the contact case. However, for a product that is constantly exposed to the cornea there must be limitations to prevent irritation or long-term damage. FIG 4 gives a strong visual representation of how much more potent MPS (green) would need to be to achieve the same results as microwave irradiation (red).

In conclusion, a biofilm analogous to what may exist in a contact lens wearers case was developed using CV staining techniques. Microwave irradiation was able to kill all bacteria in this case five out of eight times while no other treatment, including MPS, was able replicate this effect to the same statistical significance. These techniques also revealed that biofilm typically coalesces in between the ribs of the case. However, a contact lens wearer will not see biofilm present in their case, discouraging proper patient compliance. Therefore, the use of microwave irradiation to supplement contact lens case replacement is a promising technique to improve patient compliance and investigation should be continued. Further, work should be done to determine how long a case can be used when supplemented with microwave irradiation and how frequently this process must be performed. The results of this study along with other researchers’ discoveries suggest that microwave irradiation could provide a new disinfection technique capable of reducing bacterial keratitis infections in contact lens wearers (29, 30).