Ozone against Pseudomonas aeruginosa biofilms in contact lenses storage cases

ABSTRACT

Pseudomonas aeruginosa is associated with ocular infections such as keratitis. Multipurpose contact lens solutions can be used for washing, disinfection and storage of contact lenses, however, P. aeruginosa biofilm disinfection by this method is unsatisfactory. The present study aimed to investigate the effectiveness of ozonated water in reducing P. aeruginosa colony count. Lenses kept in storage cases were contaminated with P. aeruginosa and disinfected using ozonized water, chlorhexidine, ultrasound and multipurpose solutions. The multipurpose solutions and ultrasound methods reduced colony count from 1.17 to 1.63 log₁₀ CFU/cm² (92.93% to 97.31%), respectively, of P. aeruginosa biofilm cell viability when compared to the positive control. Both, ozonated water and chlorhexidine showed 7.42 log reduction in the number of viable cells of P. aeruginosa biofilm. As compared to chlorhexidine, ozonized water did not depose any known toxic residues, so that we recommend it as an alternative disinfectant solution for contact lenses storage cases.

KEYWORDS:
Ozonized water; Chlorhexidine; Ultrasound; Multipurpose solutions; Decontamination

INTRODUCTION

Pseudomonas aeruginosa is a pathogenic microorganism with clinical importance, associated with hospital infections and microbial keratitis. This bacterium has the capability to adhere, disseminate and form biofilms on medical devices such as catheters and contact lenses^1–3. It is important that contact lenses users have effective hygiene practices to minimize contamination and biofilm formation in lenses and their storage cases⁴. Multipurpose solutions are commonly used as active agents to decrease the accumulation of proteins on the surface of contact lenses and their storage cases. These solutions should eliminate microorganisms during disinfection and storage of contact lenses, but previous studies have demonstrated that several multipurpose solutions have not been capable of eliminating high concentrations of microorganisms^5,6. Therefore, it is important to search for alternative agents for effectively disinfecting contact lenses and storage cases. In this study, we analyzed the effectiveness of alternative methods, mainly ozonized water, as compared to two multipurpose solutions, for the removal of P. aeruginosa biofilms.

METHODS

Growing of P. aeruginosa biofilm

Pseudomonas aeruginosa ATCC 27853 bacteria were grown in tubes containing Brain Heart Infusion Agar (HiMedia^®, HiMedia Laboratories, India) at 37 °C for 24 h. Cultures were subsequently inoculated in sterile saline solutions (0.85% NaCl, Merck, Germany) to obtain a turbidity equivalent to 1 on the McFarland, 3.0 x 10⁸ colony forming unit (CFU) per mL, according to Zhu et al.⁷ (with some modifications). The bacterial suspension was diluted 1:100 in Mueller Hinton broth (HiMedia^®, HiMedia Laboratories, India) and 1 mL was transferred to each compartment of contact lenses storage cases. The lenses storage cases were maintained 24 h at 25 °C favoring the biofilm formation. The experiments were designed with positive and negative microbial growth controls, performed in triplicate with repetition on two different days.

Disinfection of biofilms

The lenses storage cases were washed three times with 2 mL of saline (0.85% NaCl) and then were disinfected with the following techniques or disinfectant solutions.

Multipurpose solution 1: it is a sterile isotonic solution containing boric acid, EDTA, sodium borate, sodium chloride, DYMED^® (polyaminopropyl biguanide 0.0001%), HYDRANATE^® (hydroxyalkylphosphonate 0.03%) and 1% poloxamine (Renu MultiPlus^® Fresh Lens Comfort™, Bausch & Lomb, Brazil). The disinfection with multipurpose solution 1 was carried out for 4 h following the manufacturer's specifications.

Multipurpose solution 2: it is a sterile, buffered, isotonic, aqueous solution containing sodium citrate, sodium chloride, sodium borate, propylene glycol, TEARGLYDE^® proprietary dual action reconditioning system (TETRONIC^® 1304, nonanoyl ethylenediaminetriacetic acid) with POLYQUAD^® (polyquaternium-1) 0.001% and ALDOX^® (myristamidopropyl dimethylamine) 0.0005% preservatives (Opti-Free^® Replenish^® Multi-Purpose Contact Lens Solution, Alcon, Brazil). The disinfection with multipurpose solution 2 was carried out by incubation for 6 h, following the manufacturer's specifications.

Ultrasound: the ultrasound was applied in a vat with sterile water at frequency of 40 kHz, for 20 min (ALT Sonic Clean, ALT Equipamentos, Brazil).

Ozone: the ozone was produced using medical oxygen (White Martins, Brazil) and an ozone generator (BrazilOzônio, BRO3-3, Brasil Ozônio, Brazil) of 10 g/h. The pressure was set to 0.5 kgf/cm² and flow of 4 mg/L for 20 min. The determination of residual ozone concentration was performed by indirect iodometric method, using 0.005N sodium-thiosulfate (Merck, Germany) as a titrant⁸. The distilled water was saturated with ozone for 20 min before the experiment and saturation maintained during the disinfection process.

Chlorhexidine: the chlorhexidine gluconate solution (University Pharmacy UNIFAL-MG, Brazil) was used at the concentration of 0.12% for 20 min.

Microbial analysis

The biofilm cell viability was evaluated after the attempted disinfection of biofilms. Each compartment of the storage cases was washed with 2 mL of saline solution, three times. A swab was used to sample the surfaces, as follows: surface was scrubbed ten times vertically and horizontally, then the swab was placed in a saline tube and vortexed for 10 s. The bacterial suspension derived from formed biofilms was diluted and inoculated on the surface of cetrimide agar (HiMedia^®, HiMedia Laboratories, India). The plates were incubated at 35 °C for 24 h and the results were expressed as CFU/cm² (area of the cavity of the contact lenses storage cases = 2.9845 cm²). The determination of the logarithmic reduction of growth in each treatment and the control was calculated by the following equation [log reduction = log₁₀(initial CFU/cm²) − log₁₀(final CFU/cm²)]. The results were evaluated by analysis of variance (ANOVA) and the Tukey test (P < 0.001).

RESULTS

The ability of this P. aeruginosa isolate to form biofilms was evaluated prior to the beginning of experiments using crystal violet (data not shown). The results demonstrated that all treatments were effective in disinfecting contact lenses storage cases (P < 0.001), and the treatments with ozone and chlorhexidine can be considered the most efficient because they did not show P. aeruginosa in either of replicates (Table 1). The multipurpose solutions have also decreased the biofilm formation in 1.17 to 1.63 log (92.93% to 97.31%) CFU/cm² (Figure 1), whereas ozone and chlorhexidine inactivated P. aeruginosa biofilm by a 7.34 log (99.99999%) reduction.

Figure 1
Log reduction of Pseudomonas aeruginosa biofilm cell viability after disinfection of contact lenses from experimental cases. Data are expressed as mean ± SD (standard deviation). ***Mean values considered statistically different by the Tukey test when P < 0.001.

Table 1
Pseudomonas aeruginosa biofilm viability in cetrimide agar after the disinfection of contact lenses from the experimental cases.

Treatment	CFU/cm2a ± SD
Positive control	2.63 ± 1.53 × 107
Multipurpose solution 1	2.70 ± 1.83 × 106***
Multipurpose solution 2	6.12 ± 1.94 × 105***
Ultrasound	1.27 ± 0.25 × 106***
Ozone	0.0 ***
Chlorhexidine	0.0 ***

• ^a
  The colony forming units CFU (mean ± standard deviation) derived from formed biofilms on the cavity of contact lens from the case (area = 2.984513 cm²).
• ^***
  Mean values considered statistically different from positive control by the Tukey test when P < 0.001.

There was significant difference in microbial log reduction between positive control and all treatments (Figure 1). There was no difference in microbial reduction percentage between treatments with ultrasound and solutions 1 and 2. However, there were differences between multipurpose solutions or ultrasound when they were compared with ozone or chlorhexidine (P < 0.001).

DISCUSSION

Previous studies have demonstrated that multipurpose solutions are not able to properly disinfect concentrations of bacteria as high as 10⁷ CFU/mL^5,6. These solutions can usually reduce microorganisms by 10^2-10³ CFU/mL^5,6. Similar to this work, previous studies demonstrated around 1 log or 90% of P. aeruginosa biofilm reduction after disinfection with multipurpose solutions^4,6,9. Therefore, these solutions should be used for the cleaning and maintenance of contact lenses before hand washing and manipulation of lenses. They are able to reduce biofilm formation especially in the initial stage of growth, up to 4 or 6 h¹⁰.

One of the alternative treatments used in this study was the ultrasound. It can produce pressure waves with a frequency of 20 kHz or more, causing cavitation and generation of free radicals to inactivate microorganisms. Ultrasound processing is one of the alternative technologies that has shown promise in food industry and to achieve removal of microbial biofilms in medical devices^11,12. Ultrasound at 25 kHz in medical devices for 60 minutes reduces 99.99% of microbial contamination¹².

An alternative treatment, chlorhexidine 0.12%, showed a 7.42 log efficiency. Chlorhexidine is used in antiseptic products due to its broad-spectrum of action against Gram positive and Gram negative bacteria and fungi. It is mainly for use on skin, where it produces low or no irritation¹³. Furthermore, chlorhexidine is able to remain linked in its active form to certain biological surfaces, such as the stratum corneum, acting as a reservoir of the antiseptic with prolonged bactericidal effect¹⁴. Chlorhexidine is also recommended for skin preparation before surgery and insertion of intravascular devices¹⁵. A study using 2% chlorhexidine demonstrated excellent antimicrobial activity for some microorganisms tested in their free form, but it was less effective against biofilms of P. aeruginosa¹⁶. Surfaces of leather and stainless steel cleaned with chlorhexidine in concentrations of 0.5%, 1%, 2%, 3% and 4% showed 100% reduction of P. aeruginosa¹⁷. Although chlorhexidine skin preparation has been shown to provide highly effective antimicrobial pre-surgical skin cleansing, a recent study has demonstrated that there is a significant risk of ocular toxicity when 4% chlorhexidine gluconate is used in periocular areas¹⁸. Further studies are still needed to verify whether the use of chlorhexidine in the disinfection of lenses storage cases would generate some residue and whether washing them with physiological serum could be sufficient to remove these residues.

Notably, ozonized water has also demonstrated a 7.42 log efficiency in the inactivation of P. aeruginosa. Ozone has been used in disinfection of medical devices and food because it is highly reactive and does not leave harmful residues^12,19. The ozone gas is a strong oxidant that promotes oxidation of aminoacids and proteins to alter cellular permeability, resulting in cell lysis^20,21. The lipoprotein and lipopolysaccharide layers of Gram-negative bacteria are the main ozone targets, increasing the microorganism cell permeability, resulting in lysis²⁰. Ozone inactivates P. aeruginosa by the combined results of increased cytoplasmic membrane permeability and cytoplasm coagulation²². The effective concentration of ozone for disinfection of biofilms changes with the stage of biofilm formation²³. Ozone is used for removal of P. aeruginosa on surfaces, which can dramatically reduce the count of living microorganism up to 100%²⁴. Furthermore, the application of ozone gas in medical devices at 33 mg/L for 15 minutes reduces at 5 log (99.999%) of microbial contamination¹². Thus, ozone efficiency in the removal of biofilms may depend on the roughness and composition of the surface to be disinfected, as well as the concentration of ozone and period of time of use.

In conclusion, we found that multipurpose solutions decreased biofilm formation, but did not eliminate it. Multipurpose solutions should be used to clean and preserve the lenses together with effective storage cases hygiene to minimize lenses storage case contamination and biofilm formation. We recommend washing and antisepsis of the hands, before manipulating contact lenses. The most effective treatments for reduction in formation of P. aeruginosa biofilm in contact lenses storage cases were ozonized water and chlorhexidine. Chlorhexidine use is popular, due to its high efficiency and broad availability, but more toxicity tests are necessary to examine residues remaining after treatment of lenses storage cases. As an alternative, ozone is particularly attractive because an ozonated water generator can be purchased for a low cost. Ozonation is an easy, fast and cost effective disinfection technique that can eliminate microbial biofilms due to its oxidative power; furthermore, it does not appear to form toxic residues.

ACKNOWLEDGMENTS

Fundação de Amparo à Pesquisa do Estado de Minas Gerais (FAPEMIG) (APQ01413-12, APQ00507-14), Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) and Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES).

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Publication Dates

• Publication in this collection
  18 Apr 2019
• Date of issue
  2019

History

• Received
  19 Sept 2018
• Accepted
  19 Feb 2019