Comparison of the Antimicrobial Efficacy of Various Contact Lens Solutions to Inhibit the Growth of Pseudomonas aeruginosa and Staphylococcus aureus

Soft contact lenses provide perfect conditions for the breeding of pathogens. The study is a prospective, experimental study, conducted to know the antimicrobial ability of multipurpose contact lens solution against standard strains of Staphylococcus aureus and Pseudomonas aeruginosa by the stand-alone test. The test method is based on the procedures in the ISO 14729 standard primary stand-alone test. Three multipurpose contact lens care solutions commercially available in Mangalore markets, namely, Biotrue (Bausch & Lomb), Opti-Free Replenish (Alcon), and Aquasoft (Stericon Pharma), were tested for its antimicrobial effect in the microbiology lab at Kasturba Medical College, Mangalore. According to this study, the solutions named “Biotrue” and “Aquasoft” met the primary stand-alone and reached the 3log reduction and 5log reduction criteria in the manufacturer recommended time, respectively. No conclusion could be drawn for Opti-Free Replenish since the minimum recommended disinfection time was overnight, whereas it was noted for 6 hr only, and it should have been experimented further. The effectiveness of multipurpose solutions varies against different bacterial species. We have observed that the antimicrobial activity of different solutions varies with respect to time of incubation, and also there was a marked difference in the activity of some solutions against S. aureus and P. aeruginosa. So, it is necessary for the contact lens users to store their lenses in solutions for longer duration of hours. It is also recommended to use solutions that clear the ISO 14729 standards for better health conditions of the eye.


Introduction
Diseases related to the eye are common and frequently seen in clinical practice. Soft contact lenses provide perfect conditions for the breeding of pathogens. erefore, disinfecting solutions for contact lenses are of utmost importance. e solutions must be effective in inhibiting the growth of pathogens and protecting its users from infections [1].
Different pathogens such as Staphylococcus aureus, Pseudomonas aeruginosa, Escherichia coli, and other organisms commonly cause several eye infections such as bacterial keratitis, bacterial conjunctivitis, and so on [2]. Contact lens solutions may harbour a good number of organisms due to unhygienic practices by the users; climatic changes especially humidity may become one of the contributing factors. Excessive contact lens wearers are more prone to infections and require safety concerns. Because of the emergence of virulence factors, contact lens solutions have a reduced ability to inhibit their growth [1]. Some investigations have shown that contact lens-related keratitis is commonly caused by bacteria, such as Staphylococcus aureus and Pseudomonas aeruginosa [3]. Clinical practitioners expose their patients to contact lens solutions by recommending certain brands for cleaning and disinfecting of their contact lenses. erefore, clinicians need to be aware of how the solutions are susceptible to the growth of microorganisms [1]. Hence, this study will help the clinicians and users to know the efficacy of some of the multipurpose contact lens solutions.

Materials and Methods
e three multipurpose contact lens solutions (MPS) commercially available in the market were tested for their antimicrobial effect in the microbiology lab at KMC, Mangalore. It is a prospective, experimental, comparative study. Standard strains of Staphylococcus aureus ATCC 25923 and Pseudomonas aeruginosa ATCC 27853 were used for this test. ree multipurpose contact lens care solutions commercially available in Mangalore markets, namely, Biotrue (Bausch & Lomb), Opti-Free Replenish (Alcon), and Aquasoft (Stericon Pharma), were evaluated. Normal saline was used as a control with each batch. e products were well within expiry date and tested according to the manufacturer labeled instructions for minimum disinfection time.
e test method was based on the procedures in the ISO 14729 standard primary stand-alone test. e antimicrobial efficacy of the regimen was evaluated according to the ISO 14729 standard required for the primary stand-alone test. A total of 40 test tubes were used for evaluation of three solutions plus one control solution (sterile saline). Two test tubes prepared for each of three solutions were tested for a time interval of 0 min, 10 min, 30 min, 2 hr, 4 hr, and 6 hr. e 40 test tubes were marked as solution A, B, C, and control to prevent bias. e inoculum prepared with 1 ml of the 0.5 McFarland standard preparation (1.5 * 10 8 CFU/ml) was added to 2 ml of sterile saline/BHI (1 : 3), and then 1 ml of this was added to 9 ml of sterile saline to give 1 :10. e final prepared suspension in test tubes was made to 2 ml, consisting of 1900 µl of solution A, B, C, or control solution and 100 µl of the inoculum. After this, the suspensions were incubated at specific time intervals, and 100 µl of each prepared inoculum was plated onto the MacConkey's agar plates and incubated overnight at 35°C in the ambient air and then followed by colony count and calculation of mean for each suspension per time interval. e determination of the logarithmic reduction of the growth in each solution and the control was calculated by the following equation: log reduction � log10(initial CFU ml) − log10(final CFU ml).
(1) e study has received approval from the institutional ethics committee.

Results
According to the primary stand-alone criteria, the tested MPS should reduce the bacterial colonies by 3log units. e positive control had 10 5 colonies on plating onto the media. Colony counts per solution per time interval for S. aureus and P. aeruginosa are shown in Tables 1 and 2. e mean logarithmic reduction in the minimum recommended disinfection time for each of the MPS against the standard S. aureus strain (ATCC 25923) and standard P. aeruginosa strain (ATCC 27853) is shown in Tables 3 and 4, respectively. ere was a great variation in the effects of different MPS on Staphylococcus aureus. Biotrue showed good activity towards S. aureus compared with other solutions (Figure 1). ere was a great variation in the effects of different MPS on Pseudomonas aeruginosa. Biotrue showed good activity towards Pseudomonas compared with other solutions ( Figure 2).
Aquasoft passed the ISO 14729 primary stand-alone test after 4 hr on S. aureus, but in the case of P. aeruginosa, it has passed the test in 6 hr. It is to be noted that there is no use of contact lens in this experiment; hence, the action of the solutions may increase if the contact lens is rubbed or rinsed in the solution. On S. aureus, Aquasoft showed 5log reduction in 4 hr and on P. aeruginosa in 6 hr. Since the solution passed primary stand-alone criteria for both organisms in the recommended time, the requirement for subjecting it to the second test was ruled out. Biotrue passed the ISO 14729 primary stand-alone test within the manufacturer

Discussion
Contact lenses are easily susceptible to the microbial contamination, so it is necessary for the contact lens solutions to meet the ISO 14729 standards for the stand-alone test. Noncompliance is one of the factors that commonly lead to the contact lens-related microbial keratitis [4,5]. In the present study, we tested three different contact lens disinfecting solutions, of which Biotrue chemically contains polyaminopropyl biguanide (0.00013%) and polyquaternium (0.0001%), Opti-Free Replenish contains polyquaternium/Polyquad (0.001%) and myristamidopropyl dimethylamine/Aldox (0.0005%), and Aquasoft contains polyhexamide. Polyhexamide is a disinfectant which carries highly charged active sites that can disrupt microbial cellular membranes by electrostatic interaction which are most effective against a wide range of microorganisms [6,7]. Polyquaternium (Polyquad) is a quaternary ammoniumbased antimicrobial agent and has antibacterial properties, while MAPD showed a wide spectrum of antimicrobial activity on fungi which is not tested in our study. No previous study was done with Biotrue (Bausch & Lomb) for S. aureus and P. aeruginosa, but in one of the studies conducted by Lever and Roya, Renu multipurpose solution which is manufactured by the same manufacturer with similar disinfecting agents, that is, polyaminopropyl biguanide (0.0001%), showed the mean log reduction of >4.6 for S. aureus and >4.2 for P. aeruginosa, and it has achieved the minimum disinfection criteria more quickly over other tested solutions [8]. According to the study done by Mohammadinia et al., Renu multipurpose solution failed to meet the ISO stand-alone criteria for Pseudomonas aeruginosa [3]. In the present study, Biotrue showed a significantly higher disinfecting property against S. aureus, and for P. aeruginosa, the mean reduction of 5 was seen in S. aureus (2 hr) and P. aeruginosa (4 hr), respectively.
In the present study, Opti-Free Replenish which contains 0.001% polyquaternium (Polyquad) and 0.0005% Aldox failed to meet 3log reduction in 6 hr for S. aureus and P. aeruginosa. In 6 hr, S. aureus and P. aeruginosa have shown a mean reduction of 1.75 and 1.26, respectively. However, it should be noted that the product literature says "store lenses in the closed lens case overnight or at least 6 hours." One of the earlier studies conducted by Marsha et al., Opti-Free Express which is similar to Opti-Free Replenish with respect to disinfectants did not show 3log reduction within 6 hr of incubation, where S. aureus showed the mean reduction of 1.252 in 6 hr. It is notable that their studies mention for a reevaluation of the said solution with the secondary regimen test [1]. Similar findings were observed in studies of Lever and Roya too [8].
Aquasoft gained the 3log reduction criteria within 4 hr for S. aureus and 6 hr for P. aeruginosa having the log  reduction of 5. To the best of our knowledge, there are no previous studies which were conducted with this particular solution. One of the previous studies of Iguban et al. using Solocare, where the same polyhexamide was used as the disinfecting agent, also showed good antimicrobial activity against P. aeruginosa and C. albicans [9]. e study conducted by Sakuma et al. observed that none of their MPS was good enough to remove the organism by the primary stand-alone test alone and patients should follow standard instructions carefully, including the cleaning and rinsing of contact lenses.
ere are other factors which can eliminate the number of organisms; it should be kept in mind that complete antimicrobial activity can be attained only when there is a combination of cleaning, rinsing, and disinfecting the lenses [10]. All the study solutions reached 3log reduction, respectively, in the manufacturer recommended time except Opti-Free Replenish because it needs to be tested for overnight incubation (8 hr) to pass the stand-alone criteria. e observed percentage reduction for Opti-Free Replenish at 6 hr for S. aureus and P. aeruginosa was 98.1% and 94.4%, respectively. For 6-hour duration, the solution has not passed the required criteria, which is also one of the recommendations according to the literature. Probably overnight incubation would have passed the stand-alone test which was not done in this study. In comparison to "Unique A," Opti-Free Replenish which is composed of Polyquad similar to the former showed less antibacterial activity, while according to the studies conducted by Kuzman et al., "Unique A" showed excellent activity on S. aureus and P. aeruginosa [11]. According to Hildebrandt et al., Opti-Free Replenish with organic load passed the stand-alone test criteria [12]. It has reached the 3log reduction for S. aureus and P. aeruginosa. But in the absence of organic load, it had shown the mean log reduction less than 3log, and it had failed the stand-alone criteria. ese findings are comparable with our results [10].
According to the study of Mohammadinia et al., the clinically isolated strains are more resistant than standard strains [3]. For virulent strains, MPS takes longer incubation time for their antimicrobial action, so it is recommended that further study is required to determine the effectiveness of MPS against such organisms which is one of the limitations of our study. In the present study, we can analyze that the solutions which containing polyaminopropyl biguanide (0.00013%), polyquaternium (0.0001%) and polyquaternium/ Polyquad (0.001%), and myristamidopropyl dimethylamine/ Aldox (0.0005%) have good disinfecting property against S. aureus and Pseudomonas aeruginosa but polyhexamide has less antibacterial activity.

Conclusion
S aureus and P. aeruginosa cause mild to dreadful eye infections. e findings in our study would help the clinicians as well as the patients use contact lens multipurpose solutions which follow the guidelines of ISO 14729 standards as it reduces the chances of acquiring ocular infections among contact lens wearers. It is recommended that the manufacturers also follow the guidelines for the quality check of the solutions. e effectiveness of MPS varies against different bacterial species. Biotrue has shown good antimicrobial activity and disinfecting property as compared to Aquasoft and Opti-Free Replenish. e current study does not evaluate the efficacy of the solutions in clinical isolates; hence, variations may be observed. We have observed that the antimicrobial activity of different solutions varies with respect to time of incubation, and also there was a marked difference in the activity of some solutions against S. aureus and P. aeruginosa. e solution with better disinfecting action and sufficient hygiene measures is recommended for everyday use for cleaning by contact lens users.

Data Availability
e data used to support the findings of this study are available from the corresponding author upon request.

Conflicts of Interest
e authors of this study have no financial interest nor received any financial support from the companies that manufacture multipurpose contact lens care solutions.