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  Table of Contents  
ORIGINAL ARTICLE
Year : 2017  |  Volume : 35  |  Issue : 1  |  Page : 80-84
 

Bacterial and fungal biofilm formation on contact lenses and their susceptibility to lens care solutions


1 Department of Microbiology, Kasturba Medical College, Manipal University, Mangalore, Karnataka, India
2 Department of Community Medicine, Kasturba Medical College, Manipal University, Mangalore, Karnataka, India

Date of Web Publication16-Mar-2017

Correspondence Address:
Ethel Suman
Department of Microbiology, Kasturba Medical College, Manipal University, Mangalore, Karnataka
India
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/ijmm.IJMM_16_273

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 ~ Abstract 


Background: Microbial biofilm formation on contact lenses and lens storage cases may be a risk factor for contact lens-associated corneal infections. Various types of contact lens care solutions are used to reduce microbial growths on lenses. Objectives: The present study aimed at comparing the growths of biofilms on the different contact lenses and lens cases. The study also aimed at determining the effect of lens care solutions and bacteriophage on these biofilms. Materials and Methods: One type of hard lens and two types of soft lenses were used for the study. The organisms used were Staphylococcus aureus ATCC 25923, Pseudomonas aeruginosa ATCC 27853, Candida albicans ATCC 60193 and Escherichia coli ATCC 25922. Biofilm production was performed by modified O'Toole and Kolter method and effect of lens cleaning solutions and a crude coliphage on biofilms was also studied. Results were visualised using scanning electron microscopy and quantitated by colony counting method and spectrophotometric measurement of optical density (OD). Statistical analysis was done by SPSS 11.5, Kruskal–Wallis test and Chi-square test. Results: Soft lens cleaning solutions had a significant inhibitory effect (P = 0.020) on biofilm formation on soft lenses and also lens cases (P < 0.001). Soft lens cleaning solution 2 was more efficient than solution 1. However, no such inhibitory effect was observed with regard to hard lens cleaning solution, but for a significant reduction in the OD values (P < 0.001). There was no significant inhibitory effect by bacteriophages. Conclusion: This study showed the importance of selecting the appropriate lens cleaning solution to prevent biofilm production on contact lenses.


Keywords: Anti-biofilm effect, bacteriophage, biofilm, contact lenses, lens cases, lens cleaning solutions


How to cite this article:
Kackar S, Suman E, Kotian M S. Bacterial and fungal biofilm formation on contact lenses and their susceptibility to lens care solutions. Indian J Med Microbiol 2017;35:80-4

How to cite this URL:
Kackar S, Suman E, Kotian M S. Bacterial and fungal biofilm formation on contact lenses and their susceptibility to lens care solutions. Indian J Med Microbiol [serial online] 2017 [cited 2017 Mar 29];35:80-4. Available from: http://www.ijmm.org/text.asp?2017/35/1/80/202339





 ~ Introduction Top


Contact lenses are broadly classified into two categories-rigid or hard lenses and the much more recently developed soft lenses. The two types differ in their composition and the relative advantages and disadvantages they offer. They may also differ in their susceptibility to microbial infections.

Adhesion and colonisation by microorganisms, particularly bacteria and fungi, on contact lenses continues to be implicated in several adverse events. Microorganisms often form biofilms on lens surfaces.

Microbial growth is characterised as a biofilm when the microorganisms attach to a surface and/or to each other. Biofilms offer increased antibiotic resistance to microorganisms as compared to the planktonic/free-living mode of growth.[1],[2]

Microbial biofilm formation on contact lenses and lens storage cases may be a risk factor for contact lens-associated corneal infections.[3] Many ocular infections occur when such prosthetic devices come in contact with or are implanted in the eye, including microbial keratitis (MK),[4] contact lens-related acute red eye,[5] contact lens peripheral ulcer [6] and infiltrative keratitis.[5] MK may result in vision loss as a consequence of corneal scarring.[7] Depending on the study design and location, contact lens wear accounts for approximately 12%–66% of all events of MK.[8],[9],[10],[11],[12],[13],[14] The prevention of persistent contamination and colonisation of contact lenses and lens storage cases may help to reduce the incidence of MK in contact lens users.

Various types of contact lens care solutions are used to reduce microbial growths on lenses. The solutions differ in their relative microbicidal effects.[15] Hence, determining the efficiency of lens care solutions becomes important.

The objective of our study was to determine and compare the growths of biofilms of various bacterial and fungal strains on the different types of contact lenses available by developing in vitro models. We also determined the effect of lens care solution on these biofilms.

We developed in vitro models for contact lens-associated bacterial and fungal biofilms. One type of rigid gas permeable (RGP) hard lens and two types of soft lenses were used in the study, one poly (2-hydroxyethyl methacrylate) (pHEMA) based and the other a silicone hydrogel lens.

The organisms used in the study were Staphylococcus aureus ATCC 25923, Pseudomonas aeruginosa ATCC 27853 and Candida albicans ATCC 60193. These organisms were selected because they were among the most commonly involved organisms in corneal infections in contact lens users.[16],[17],[18],[19],[20]

To test the efficacy of the antimicrobial action of lens-care solutions, one type of solution was used for the hard lenses while two types of solutions were used for the soft lenses.

Bacteriophages are increasingly being used to reduce bacterial infections and also in the treatment of diseases.[21],[22] However, studies conducted to study the interaction of bacteriophages with biofilms have found that while phages reduce bacterial growth, they do not completely eliminate it.[23] Our study also explored this by testing the effect of a crude coliphage on bacterial biofilms. Escherichia coli ATCC 25922 was used to study the effect of the bacteriophage alongside the other test organisms.

Our study attempted to cover the gaps in knowledge that exist in this area due to the lack of research in India on the comparative susceptibility to biofilm formation of various types of lenses and the efficacy of lens cleaning solutions against these biofilms. Although there are a large number of contact lens users in our country, with a high incidence of ocular infections, not much work has been done in this area. Also while there is a large body of research on the use of bacteriophages in reducing hospital-acquired infections and in the treatment of diseases, there is not much work done on the effect of these phages on contact lens infections and their use in preventing or reducing ocular infections.


 ~ Materials and Methods Top


Test organisms used

The test organisms used were P. aeruginosa ATCC 27853, S. aureus ATCC 25923 and C. albicans ATCC 60193. E. coli ATCC 25922 was also used to study the anti-biofilm activity of the phage. Institutional Ethics Committee clearance was obtained for the study. The work was done in the Department of Microbiology, Kasturba Medical College, Mangalore.

Types of lenses and lens care solutions used

One type of hard lens and two types of soft lenses were used for the study. The lenses used were:

  • Hard lens: RGP lens (roflufocon A)
  • Soft lens 1: Silicon hydrogel lens (etafilcon A)
  • Soft lens 2: pHEMA lens.


Commonly used lens care solutions were used for studying the anti-biofilm activity. They included:

  • RGP lens solution: Glucose (0.006% w/v), carboxymethyl cellulose (CMC - 0.02%), disodium ethylenediaminetetraacetic acid (EDTA) (0.1% w/v) in a buffered polymer complex and surfactant
  • Soft lens solution 1: Purified water, hydroxypropyl methylcellulose (HPMC), CMC, EDTA, borax in sodium chloride base
  • Soft lens solution 2: Hydroxyalkylphosphonate, boric acid, edetate disodium poloxamine, sodium borate and sodium chloride preserved with polyaminopropyl biguanide 0.0001%.


A crude coliphage obtained from sewage water was used. This was an in vitro descriptive study.

The sample size consisted of 18 unused RGP contact lenses and 24 unused soft contact lenses of two types each. Each lens was subjected to in vitro biofilm formation by the test organisms in triplicate. In addition, seven soft contact lenses of Type 1 were used to study the effect of phage on the biofilm.

Biofilm formation on contact lenses

This was done by the modified O'Toole and Kolter method.[24] Two hundred microliter of 48 h broth culture in Brain Heart Infusion (BHI) broth of the organism was added to each lens placed in the lens case followed by 200 µl of BHI. The lens cases were incubated at 37°C for 48 h. After incubation, the broth was aspirated into microtitre plates (200 µl of broth in each plate) and optical density 570 (OD570) was measured spectrophotometrically.

The lens and the lens cases were treated with Bouin's fixative for 10 min and then washed with phosphate buffered saline (PBS) of pH 7.4. This was done by adding 200 µl of 0.5M PBS (pH 7.4). It was then aspirated till the lenses and lens cases were dry.

One set of contact lenses were then treated with Bouin's fixative for 10 min and aspirated. The contact lenses were then sputtered, and scanning electron microscopy (SEM) was performed.

The second set of contact lenses were subjected to colony counting. The contact lenses were swabbed and plated on nutrient agar. The plates were incubated at 37°C for 48 h and colony counting was performed. The results were tabulated.

Biofilm formation on contact lens cases

The biofilms that were produced in the contact lens cases were swabbed and colony counting was performed. The results were tabulated.

Effect of lens cleaning solution on biofilms

With each set of biofilm formation on the lenses, the effect of the lens cleaning solutions were also studied. This was done by adding 200 µl of the broth containing the organism and 200 µl of the lens cleaning solution. The cases were incubated at 37°C for 48 h. After incubation, the supernatant was aspirated into microtitre plates, and the OD570 values were recorded spectrophotometrically.

The lenses were subjected to SEM after sputtering. Colony counting of the lenses and lens cases was also performed. The values were tabulated.

Isolation of bacteriophage

Treated sewage sample from sewage water treatment plant was centrifuged at 10,000 g for 10 min.[25] One millilitre of the supernatant was added to 5 ml of 16–18 h old E. coli ATCC 25922 broth culture. The mixture was incubated at 37°C for 18–24 h 1.5 ml of the mixture was centrifuged to pellet out bacterial cells. The supernatant was filtered through 0.45 µm membrane filter. Confirmation of the presence of bacteriophages was done by performing 10-fold dilutions of the filtrate (1:10, 1:100, 1:1000, 1:10,000 up to 10−5). One hundred microliters of the dilutions were added to 1 ml of 18 h old E. coli culture. This was added to 5 ml of molten soft agar (tryptone agar). Pour plating was done, and the plates were incubated in an upright position for 24 h at 37°C. Then the plates were observed for clear plaques, and they were enumerated as plaque forming units per ml per gram (pfu/ml/g). Phages were stored at −20°C.

Effect of bacteriophage on biofilms on contact lenses

The organisms used were S. aureus ATCC 25923, P. aeruginosa ATCC 27853 and E. coli ATCC 25922. Soft lens 1 was used to study the effect of the phage.

The effect of the bacteriophage on biofilms was detected by methods similar to the effect of lens cleaning solution. However, in this case, 200 µl of 1 in 100 dilution of phage suspension was added along with 200 µl of the broth containing organisms. The cases were incubated at 37°C for 48 h. After incubation, the supernatant was aspirated into microtitre plates, and the OD570 values were recorded spectrophotometrically. Colony counting of the lenses and lens cases was performed. The values were tabulated.

Statistical analysis

All experiments were performed in either duplicate or triplicate. Statistical analysis was performed using SPSS 16.0 for Windows (SPSS, Inc., Chicago, IL, USA) and using Kruskal–Wallis and Chi-square test. P < 0.05 was considered statistically significant.


 ~ Results Top


Biofilm formation on contact lenses

There was abundant biofilm formation by the test organisms on both types of contact lenses as measured spectrophotometrically at OD570. The biofilm produced was more in soft lens of Type 1 in comparison to soft lens of Type 2 and hard lens [Table 1]. SEM images also revealed abundant biofilm formation by the test organisms on lens surfaces. However, the difference in the amount of biofilm produced on different types of lenses was not significantly observed.
Table 1: Effect of the lens cleaning solution and bacteriophage on the OD570 values of the supernatant fluid

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Biofilm formation on contact lens cases

Biofilm production was also observed on contact lens cases as revealed by OD570 as well as by colony counts.

Effect of lens cleaning solution on biofilms

There was no significant reduction in the colony forming unit (CFU) counts by lens care solutions, in the case of hard lenses. A significant reduction (P = 0.020) was observed in the case of soft lenses 1 and 2. The bacteriophage produced no significant decrease in any case.

A similar result was observed in case of CFU counts from lens cases with only the soft lens solutions causing a significant decline in CFUs (P < 0.001).

OD570 values of supernatant fluid revealed a significant decrease in biofilms in the presence of lens care solutions in case of hard lenses a well as in Type 1 and Type 2 soft lenses [Table 1]. These findings were similar to the SEM findings [Figure 1],[Figure 2],[Figure 3],[Figure 4],[Figure 5],[Figure 6]. Soft lens cleaning solution 2 was more efficient than solution 1.
Figure 1: Scanning electron microscopy image of biofilm produced by Staphylococcus aureus on soft lens.

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Figure 2: Scanning electron microscopy image of biofilm produced by Staphylococcus aureus on soft lens in presence of solution 1.

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Figure 3: Scanning electron microscopy image of biofilm produced by Pseudomonas aeruginosa on soft lens.

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Figure 4: Scanning electron microscopy image of biofilm formation by Pseudomonas aeruginosa on soft lens in presence of solution 2.

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Figure 5: Scanning electron microscopy image of biofilm formation by Candida albicans on hard lens.

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Figure 6: Scanning electron microscopy image of biofilm formation by Candida albicans on hard lens in presence of solution.

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Effect of bacteriophage on biofilms on contact lenses

The presence of bacteriophage, however, did not cause any decrease in biofilms.


 ~ Discussion Top


Our study has demonstrated that various bacteria as well as fungi like Candida spp. have the ability to form biofilms on contact lenses irrespective of the nature of contact lenses. The study also revealed that besides contact lenses, the lens cases can also facilitate the formation of biofilms by microorganisms. The formation of biofilms on contact lenses may be the cause of MK in prolonged contact lens users.[3] Imamura et al. tested etafilcon A, galyfilcon A, lotrafilcon A, balafilcon A, alphafilcon A and polymacon types of contact lenses and their results showed that clinical isolates of Fusarium and C. albicans form biofilms on all types of lenses. They also showed that the biofilm architecture varied with the lens type used. Furthermore, using the developed in vitro biofilm model, they showed that phylogenetically diverse planktonic fusaria and Candida were susceptible to MoistureLoc and MultiPlus.[26] The nature of the lens did not seem to have any effect on biofilm formation in our study, and this is similar to the study reported by Retuerto et al.[27]

Our results indicate that soft lens cleaning solutions have a significant inhibitory effect on the formation of biofilms on soft type of contact lenses. Soft lens cleaning solution containing hydroxyalkylphosphonate, boric acid, edetate disodium poloxamine, sodium borate (Solution 2) was more efficient than soft lens cleaning solution containing purified water, HPMC, CMC, EDTA and borax in sodium chloride base (solution 1). A previous study has demonstrated that hydrogen peroxide solution and polyquaternium-myristamidopropyl dimethylamine-preserved solution possess antifungal activity against Fusarium strains on contact lenses.[27]

The inhibitory action of lens cleaning solution suggests that by using the appropriate lens cleaning solution biofilms can be prevented on contact lenses as well as in contact lens cases and this can go a long way in preventing ocular infections.

As per our study, the inhibitory effect of bacteriophage on biofilms was not significant. This could be due to the fact that the coliphages that we used in our study were the crude coliphages. Furthermore, these phages were used to inhibit S. aureus and P. aeruginosa biofilms for which they are not specific. Recent studies have suggested the use of bacteriophage endolysins as well as engineered phages expressing anti-biofilm enzymes as promising options for the eradication of bacterial biofilms.[28],[29],[30] Further research in this area may yield promising results.


 ~ Conclusion Top


This study showed the importance of selecting the appropriate lens cleaning solution to prevent biofilm production on contact lenses. Future research into anti-biofilm surface coatings on contact lenses as well as newer strategies involving the possibility of using bacteriophages engineered to express anti-biofilm enzymes may aid in reducing the risk of biofilm-associated contact lens infections.

Financial support and sponsorship

The study was funded by the Indian Council of Medical Research under the Short Term Studentship (STS) – 2013 scheme.

Conflicts of interest

There are no conflicts of interest.[31]



 
 ~ References Top

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Hiti K, Walochnik J, Haller-Schober EM, Faschinger C, Aspöck H. Viability of Acanthamoeba after exposure to a multipurpose disinfecting contact lens solution and two hydrogen peroxide systems. Br J Ophthalmol 2002;86:144-6.  Back to cited text no. 15
    
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Cheng KH, Leung SL, Hoekman HW, Beekhuis WH, Mulder PG, Geerards AJ, et al. Incidence of contact-lens-associated microbial keratitis and its related morbidity. Lancet 1999;354:181-5.  Back to cited text no. 16
    
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Alexandrakis G, Alfonso EC, Miller D. Shifting trends in bacterial keratitis in south Florida and emerging resistance to fluoroquinolones. Ophthalmology 2000;107:1497-502.  Back to cited text no. 20
    
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    Figures

  [Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5], [Figure 6]
 
 
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