The antibacterial activity of contact lens solutions against microbial keratitis.
Investigations have found that prolonged use of contact lens can cause microbial keratitis and corneal ulcers. Although the incidence rates of contact lens-related microbial keratitis are low, this complication can cause poor visual outcome and blindness. [1,2] Contact lens-related microbial keratitis is commonly caused by Staphylococcus aureus and P. aeruginosa. 
P. aeruginosa is a Gram-negative bacterium that is commonly found in many environments, including water. It is a pathogen and resistant to dilute solutions of disinfectants. P. aeruginosa may display resistance to antibiotics if assosiated with contact lens. [4-6] Studies have shown that the use of contact lenses increases adherence of P. aeruginosa to epithelial cells. 
S. aureus is one of pathogens causing keratitis, a visionthreatening disease. [6,7] It is an aerobic Gram-positive bacterium carried by 50-60% of normal population and can readily find access to the eyes. A study has shown that S. aureus is the most common bacteria causing contact lens-related keratitis. 
Different solutions are used for cleaning and disinfecting contact lenses. The antibacterial properties of these contact lens solutions have been studied, but the duration of contact time between the contact lens and the solution has not been determined. Hence, it is necessary to determine the contact time of a contact lens solution.  Besides, most people do not clean the contact lens according to the procedure.
This study determined the antimicrobial activity of three locally available contact lens solutions against P. aeruginosa and S. aureus and determined their contact time. 
MATERIALS AND METHODS
Three bottles of each contact lens solution were evaluated using a single-blind controlled experiment method. These test solutions were categorized based on their identified disinfecting ingredient.  The contact lens solutions tested in this experiment are listed in (Table 1).
Preparation of Microbial Suspension
A bacterial colony was selected from an agar plate culture, incubated at a temperature of 35-37[degrees]C for 24 h, and suspended in a tube containing NaCl 0.9% to achieve turbidity comparable to a 0.5 McFarland standard [1,9] 1 mL of the microbial solution was added to 1 mL of each contact lens solution to achieve a 1:1 concentration. The solutions were termed "biotest solutions."
Positive controls for each challenged organism were created using microbial stock solutions containing NaCl 0.9%. Negative controls were prepared by adding NaCl 0.9% to contact lens solutions to check for possible contaminations.
Test of Antimicrobial Effectiveness
1 mL of the sample was subcultured on an agar recovery medium (Tryptic Soy Broth or Soybean-Casein Digest Medium and Sabouraud Dextrose Broth). The recovery plate was incubated for 24 h at a temperature of 35-37[degrees]C. The number of colonies was determined using total plate count and log reduction.
Figure 1: Average number of colonies of microbial from multipurpose solution Log CFU/mL MPS A MPS B MPS C MPS D * Pseudomonas aeruginosa 3 2 3 3.45 * Staphylococcus aureus 3.54 2.12 3.39 3.55 Note: Table made from bar graph.
The two-way analysis of variance (ANOVA) was used to determine the factors affecting the concentration of challenge organisms (log cfu/mL) with a level of significance (a) of 0.05. Tukey's Honestly Significant Difference test was used to determine the post-hoc differences among the variables presented. 
All products used in this experiment were within their period of expiry. Contact lens solutions were tested by non-probability sampling method. 
The bacterial suspension obtained was determined as 1.0x[10.sup.6] colony-forming units per mililiter using 0.5 McFarland standard, which contained NaCl 0.9% based on the ISO criteria for contact lens solutions (ISO/CD 14729). [11,12] Test organisms were obtained from the Laboratory of Microbiology, Faculty of Pharmacy, Universitas Padjadjaran.
Time recommendations on the label and packaging contact lens solution are listed in (Table 2).
The antibacterial activity of contact lens solution can be seen in (Tables 3 and 4).
The average number of bacterial colonies for each product is presented in (Table 5).
The average number of bacterial colonies based on different contact times is presented in (Table 6). All samples of contact lens solutions tested showed excellent antimicrobial activity. The contact lens solution B had the highest antibacterial activity against S. aureus and P. aeruginosa with a contact time of 6 h.
The effect of each treatment on the microbial load can be seen in (Figure 1).
After ANOVA, the results showed that the contact lens solution B had the most effective antibacterial power than others. Because F = 6.25> [F.sub.[alpha]] = 3.259, [H.sub.0] was rejected, which means there were differences among the three contact lens solutions in terms of their effect on the concentration of P. aeruginosa. For S. aureus, the results showed that the contact lens solution B was more effective than others. Because [F.sub.calc] = 20.39>[F.sub.[alpha]] = 3.259, [H.sub.0] was rejected, which means there were differences among the three contact lens solutions in terms of their effect on the concentration of S. aureus.
The effective contact time for each contact lens solution can be seen in (Figure 2).
Figure 2: Average number of bacteria colonies of microbial Log CFU/mL 1 3 6 24 * Pseudomonas aeruginosa 3.18 2.898 1.536 2.862 * Staphylococcus aureus 4.36 2.778 2.272 2.862 Note: Table made from bar graph.
The figure shows that the average contact time for all contact lens solutions was 6 h. Because [F.sub.calc] = 6.67> [F.sub.[alpha]] = 3.49, [H.sub.0] was rejected, which means varying durations of exposure showed significant effects on the concentration of P. aeruginosa and S. aureus.
This study adopted the standalone criteria from the International Organization for Standardization (ISO/CD 14729) to determine the effectiveness of contact lens solutions against P. aeruginosa and S. aureus. According to this standard, contact lens solutions must be able to reduce the starting concentration of bacteria (P. aeruginosa and S. aureus) by three logs at the least disinfection time. 
Cleaning and rinsing of contact lens can remove more than 90% of microbial contamination, while a non-adherence can lead to microbial keratitis. According to a previous study, 30% of contact lens users do not always clean their lenses using a contact lens solution before use and 44% of them do not wash their hands before handling lenses. This shows that disinfecting contact lens using a contact lens solution before use is essential. 
Our results showed that all the three contact lens solutions have significant antimicrobial effects. The most dominant active substance in those contact lens solutions was polyhexamethylene biguanide, which initiates an attack right at the bacterial surface through to the cytoplasm and cytoplasmic membrane. The effects are higher on Gram-negative bacterium where an action on the membrane acid leads to an increase in fluidity and permeability, causing the release of lipopolysaccharide. [13,14]
The results of this study indicate that all of the contact lens solutions have activity as antimicrobial after exposure for 24 h. Contact lens solutions containing polyhexamethylene biguanid as the active agent are found to have excellent antimicrobial activity and a desirable contact time.
[1.] Iguban EB, Nanagas JP, de Mesa Rodriguez RF. The antimicrobial efficacy of multipurpose contact lens solutions on standard strains of common ocular pathogens. Manila Philipp J Ophthalmol. 2013;38:35-7.
[2.] Wu YT, Zhu H, Harmis NY, Iskandar SY, Willcox M, Stapleton F. Profile and frequency of microbial contamination of contact lens cases. Optom Vis Sci. 2010;87(3):E152-8.
[3.] Mohammadinia M, Rahmani S, Eslami G, Ghassemi-Broumand M, Aghazadh Amiri M, Aghaie G, et al. Contact lens disinfecting solutions antibacterial efficacy: Comparison between clinical isolates and the standard ISO ATCC strains of Pseudomonas aeruginosa and Staphylococcus aureus. Eye (Lond). 2012;26(2):327-30.
[4.] Chalita MR, Hofling-Lima AL, Paranhos A Jr, Schor P, Belfort R Jr. Shifting trends in in vitro antibiotic susceptibilities for common ocular isolates during a period of 15 years. Am J Ophthalmol. 2004;137(1):43-51.
[5.] Garg P, Sharma S, Rao GN. Ciprofloxacin-resistant Pseudomonas keratitis. Ophthalmology. 1999;106(7):1319-23.
[6.] Yeh DL, Stinnett SS, Afshari NA.Analysis of bacterial culturesin infectious keratitis. Am J Ophthalmol. 2006;142(6):1066-8.
[7.] Ong SJ, Huang YC, Tan HY, Ma DH, Lin HC, Yeh LK, et al. Staphylococcus aureus keratitis: A review of hospital cases. PloS One. 2013;8(11):e80119.
[8.] Jalbert I, Willcox MD, Sweeney DF. Isolation of Staphylococcus aureus from a contact lens at the time of a contact lens-induced peripheral ulcer: Case report. Cornea. 2000;19(1):116-20.
[9.] Rosenthal RA, Sutton SV, Schlech BA. Review of standard for evaluating the effectiveness of contact lens disinfectants. PDA J Pharm Sci Technol. 2002;56(1):37-50.
[10.] Kurniawansyah IS, Budiman A, Abdassah M, Mita SR, Farihah RN. Sterile gel propolis Trigona spp as drug for wound burns. Int J Curr Pharm Rev Res. 2016;7(6):416-20.
[11.] Robertson DM, Petroll WM, Jester JV, Cavanagh HD. Current concepts: Contact lens related Pseudomonas keratitis. Cont Lens Anterior Eye. 2007;30(2):94-107.
[12.] Cano-Parra J, Bueno-Gimeno I, Lainez B, Cordoba J, Montes-Mico R. Antibacterial and antifungal effects of soft contact lens disinfection solutions. Cont Lens Anterior Eye. 1999;22(3):83-6.
[13.] Yasuda K, Ohmizo C, Katsu T. Potassium and tetraphenylphosphonium ion-selective electrodes for monitoring changes in the permeability of bacterial outer and cytoplasmic membranes. J Microbiol Methods. 2003;54(1):111-5.
[14.] Alfauziah TQ, Budiman A. Antifungal activity test of essential oil of clove flower against wood mushroom in emulsion dossage form. Farmaka. 2016;14(1):1-10.
Arif Budiman, Haniq Juniswapy Fauzi, Rr. Sulistiyaningsih, Sriwidodo
Department of Science and Technology Pharmacy, Faculty of Pharmacy, Padjadjaran University, Bandung, West Java, Indonesia
Correspondence to: Arif Budiman, E-mail: firstname.lastname@example.org
Received: July 06, 2017; Accepted: July 28, 2017
How to cite this article: Budiman A, Fauzi HJ, Sulistiyaningsih R, Sriwidodo. The antibacterial activity of contact lens solutions against microbial keratitis. Natl J Physiol Pharm Pharmacol 2017;7(11): 1264-1267.
Source of Support: Nil, Conflict of Interest: None declared.
Table 1: Characteristics of contact lens solutions tested Contact lens solution Active substances A Polyaminopropyl biguanide 0.0001% B Polyhexamethylene biguanide C Polyhexanide Table 2: Active substances and recommended contact time of contact lens solutions Contact lens Active subtances Contact time (h) solution A Polyaminopropyl biguanide 6 B Polyhexamethylene biguanide 6 C Polyhexanide 6 Table 3: Results of antibacterial activity against Staphylococcus aureus Contact lens solution The average of colonies (Log CFU/mL) 0 1 A 6.4[+ or -]0.21 5.05[+ or -]0.14 B 6.4[+ or -]0.24 2.6[+ or -]0.21 C 6.4[+ or -]0.19 4.7[+ or -]0.11 Contact lens solution The average of colonies (Log CFU/mL) 3 6 A 3.22[+ or -]0.21 2.53[+ or -]0.15 B 1.72[+ or -]0.19 1.57[+ or -]0.2 C 3.3[+ or -]0.22 2.34[+ or -]0.21 Contact lens solution The average of colonies (Log CFU/mL) 12 A 3.36[+ or -]0.25 B 2.62[+ or -]0.11 C 3.23[+ or -]0.10 Good microbial activity: [less than or equal to]2 Log CFU/mL, fair microbial activity: >Log 2 but [less than or equal to]Log 4 CFU/mL, poor microbial activity: >Log 4 cfu/mL, Staphylococcus aureus: S. aureus Table 4: Results of antibacterial activity against P. aeruginosa Contact lens solution Total of colonies (Log CFU/mL) 0 1 A 6.3[+ or -]0.09 3.7[+ or -]0.15 B 6.3[+ or -]0.11 2.7[+ or -]0.09 C 6.3[+ or -]0.08 3.5[+ or -]0.08 Contact lens solution Total of colonies (Log CFU/mL) 3 6 A 3.7[+ or -]0.12 2.43[+ or -]0.14 B 1.17[+ or -]0.09 0[+ or -]0 C 3.41[+ or -]0.18 1.6[+ or -]0.06 Contact lens solution Total of colonies (Log CFU/mL) 12 A 3.36[+ or -]0.08 B 2.62[+ or -]0.15 C 3.23[+ or -]0.09 Good microbial activity: [less than or equal to]2 Log CFU/mL, fair microbial activity: >Log 2 but [less than or equal to]Log 4 CFU/mL, poor microbial activity: >Log 4 CFU/mL, P. aeruginosa: Pseudomonas aeruginosa Table 5: Average number of bacterial colonies of P. aeruginosa and S. aureus Product P. aeruginosa S. aureus A 3.29 3.54 B 1.62 2.12 C 2.93 3.39 S. aureus: Staphylococcus aureus, P. aeruginosa: Pseudomonas aeruginosa Table 6: Average number of bacterial colonies based on different contact times Contact time (h) P. aeruginosa S. aureus 1 3.18 4.36 3 2.89 2.77 6 1.53 2.27 12 2.86 2.86 S. aureus: Staphylococcus aureus, P. aeruginosa: Pseudomonas aeruginosa
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|Title Annotation:||RESEARCH ARTICLE|
|Author:||Budiman, Arif; Fauzi, Haniq Juniswapy|
|Publication:||National Journal of Physiology, Pharmacy and Pharmacology|
|Date:||Nov 1, 2017|
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