The Antibiofilm efficacy of nitric oxide on soft contact lenses

Bacteria accounts for most cases of the infectious keratitis. If not properly treated, bacterial keratitis can cause sight-threatening complications, such as corneal opacity or perforation [1‐3]. Contact lens wearing significantly increases the risk of bacterial keratitis and it was reported that about 3.5 to 20 soft contact lens wearers developed bacterial keratitis every year [4]. Corneal hypoxia and epithelial damage induced by wearing contact lenses enhances the bacterial adhesion and invasion [4]. Normal host defense mechanism against pathogen, such as tear and blinking can also be significantly interfered by wearing contact lenses [5]. Furthermore, mucins and proteins can attach on the surface of contact lens and promote the pathogen adhesion. The adhesion of specific bacteria to contact lens can form a biofilm [5]. A biofilm is a complex of microbial communities enclosed in an exopolysaccharide matrix adhered to the surface of prosthetics or living organism [6, 7]. Biofilms enable bacteria to survive in unfavorable environments by reducing their metabolic needs, inhibiting the penetration of antimicrobial agents and increasing their inherent resistance to antimicrobial agents [8]. In addition, biofilms formed on contact lenses can play a critical role in developing bacterial keratitis as a depot for continuous bacterial release [8‐10].

Nitric oxide (NO) is a one of well-known anti-bacterial mechanisms of mammalian host [11]. As a small molecular gas, NO can diffuse freely across the cellular membrane [12]. When generated locally with micromolar concentration, NO acts as a cytotoxic antimicrobial agent [11]. In addition, previous studies have shown that NO could act as a key mediator for biofilm dispersal [13, 14]. It was reported that low concentrations of NO induced biofilms dispersal while high concentrations of NO directly killed pathogens [15]. It is known that high concentrations of NO have a broad antibiotic spectrum that can act on both gram positive and negative or both mono-species and multi-species biofilms [16‐20].

Bacteria such as Staphylococcus aureus and Pseudomonas aeruginosa contains nitrite reductase which converts nitrite (NO₂ ⁻) to NO. The gene encoding nitrite reductase is known as nasDE or nirBD in Staphylococcus aureus and nirS in Pseudomonas aeruginosa [21, 22].

Although the evidences of anti-biofilm effect of NO are growing, the study investigating the effect of NO on soft contact lens-associated biofilms is hard to find. Considering the clinical importance of contact lens associated biofilms and resulting bacterial keratitis, the role of NO on soft contact lens-associated biofilm is an interesting research topic.

In the current study, we used sodium nitrite (NaNO₂) as a NO donor and investigated the inhibitory effects of NO on soft contact lens-associated biofilms by Staphylococcus aureus and Pseudomonas aeruginosa, which are two of most common strains of infectious keratitis [23, 24].

In this study, we found that NO showed a dose-dependent suppression of the biofilm formed on soft contact lenses by S. aureus and P. aeruginosa. In addition, the tested concentrations of NaNO₂ showed no significant toxicity on cultured human corneal epithelial cells.

Biofilm formation on the surface of the medical device is closely related to various infectious diseases because it increases continuous pathogen dispersion [7, 8, 25]. It is known that biofilm forms commonly on contact lenses and is one of the major risk factors for infectious keratitis [1, 26‐28]. The characteristics of the contact lenses, such as hydrophobicity or surface irregularity, can also affect bacterial attachment and subsequent biofilm formation [29]. Furthermore, biofilm formation on the anterior surface of contact lens can be somewhat prevented by natural host defense mechanisms such as blinking movement and antimicrobial components within tears [30]. However, the posterior surface of the contact lens touches the cornea and this contact interferes natural defense mechanisms [31]. Therefore, biofilm is found commonly on the posterior surface of contact lens [9]. Bacteria within biofilm are more resistant to antibiotics and host defense mechanisms compared to free planktonic form of bacteria [6, 32].

Previous studies found that NO is a key mediator of biofilm dispersal [13, 15, 16, 33, 34]. Biofilm dispersal needs some molecular triggers which induce a change from biofilm to dispersal phenotype. One of these triggers is a low concentration of NO and another is a decrease of intracellular c-di-GMP [17, 35, 36]. Low concentrations of NO induce biofilm dispersal via signal cascades involving both increase of phosphodiesterase activity and decrease of intracellular c-di-GMP [14, 15]. In addition, planktonic bacteria exposed to low concentrations of NO become even more susceptible to other antibiotics [37, 38].

It is known that high concentrations of NO are broad-spectrum bactericidal agents that can kill both gram positive and negative bacteria [38‐40]. However, NO can also be toxic to host cells at high concentrations. High concentrations of NO can be converted to reactive nitrogen species such as peroxynitrite and reactive nitrogen species can cause cytotoxic effects not only on pathogens but also on host cells.

NO is an unstable free radical gas and has limited solubility in water [12]. This makes it difficult to introduce pure gaseous NO into the culture media. Therefore, various chemical agents that release NO, such as sodium nitroprusside, sodium nitrate (NaNO₃) or sodium nitrite (NaNO₂), have been widely used as NO donor for biological experiment evaluating NO effect [17, 41]. In our study, NaNO₂ was used as a NO donor. NaNO₂ yields nitrite and nitrite can be converted to NO by nitrite reductase. In the previous report, exogenous supply of NaNO₂ successfully decreased the biofilm formation on the culture plate by S. aureus and P. aeruginosa [42].

We tested the safety of NaNO₂ of concentrations (0.1, 1, 10, 100 and 1000 μM) using HCECs. The viability of HCECs was not interrupted by NaNO₂ up to 48 h at all tested concentrations. In addition, the contact lens biofilm formed by S. aureus and P. aeruginosa successfully decreased following addition of NaNO₂. Our results are consistent with previous studies that showed the suppressive effect of NO on biofilm and bacteria on other medical devices [13, 33, 34].

In this study, CFU count tended to increase over a week even with the presence of NO especially in P. aeruginosa. We hypothesize that biofilm formation was inhibited only during the short action period of NO and the residual bacteria then proliferated again. In our study, NaNO₂ was applied once a day to avoid significant change of osmotic pressure of culture media. As a relatively unstable gas, NO has a short half- life [12]. As shown in Fig. 2, the most robust action of NO was expected within 30 min every day in our experimental setting. Therefore, the increase in the CFU count at day 7 might be the cumulative effect of bacterial regrowth during NO-free period of each day. Another possible explanation might be the limited diffusion of NO through the already established biofilm formation by P. aeruginosa at day 7. As previously known, biofilm can protect microorganisms by providing mechanical diffusion barrier to antimicrobial agent [8].

In previous reports, the optimal NO concentration for biofilm suppression was different in two bacteria studied. P. aeruginosa was more susceptible to biofilm dispersal at lower concentrations (0.025 ~ 2500 nM) of NO. [13, 33] On the other hand, relatively high concentrations (124 ~ 1000 μM) of NO was necessary to inhibit biofilm formation by S. aureus [43]. However, our result revealed that both bacteria were susceptible to biofilm dispersal at similar NaNO₂ concentration range (0.1 to 1000 μM). We believe that the difference in the strains and experimental settings (biofilm formation on soft contact lens and different NO donor) may explain the discrepancies between studies.

Although it is one of the first pioneering study to evaluate NO effect on contact lens associated biofilm, this study has several limitations. The first, the interaction of bacteria and host immune system and its modulation by NO was not elucidated in our in vitro study. Secondly, we used the indirect method for the quantification of biofilm, the re-culturing from biofilm, instead of direct quantification of biofilm. We initially tried direct staining and biofilm quantification on contact lenses using crystal violet after complete drying of contact lens. However, there were several drawbacks that made us abandon the direct staining method. We found that the contact lens itself was heavily stained by crystal violet. In addition, a significant amount of biofilm formed on contact lens was not fixed and continuously dropped out during washing, drying and staining procedure. In addition, thermal fixation could not be applied because it would burn the contact lens. Therefore, we chose the indirect quantification method and assumed that only the biofilm attached to the contact lens was the source of bacterial growth in the recultured media. The similar methods were reported to quantify biofilm development on intraocular lens surface previously [44, 45]. In our study, both OD measurement and CFU count were adopted to increase the accuracy of biofilm quantification. Thirdly, NaNO₂ was treated only once a day because osmotic change from the additional sodium in NaNO₂ can affect bacterial growth. NO release from NaNO₂ is expected to be quite concentrated within 30 min of NaNO₂’s application to the culture media, so a more stable and continuous NO donor would be optimal for investigating dose-dependent effect of NO. Furthermore, the exact quantification of NO production from nitrite was not measured in this study. The difference between experimental and physiological conditions should be also considered for interpretation of our results. The dynamic tear clearance and lens movement were absent in our experimental settings and these two factors might change the biofilm kinetics in human ocular surface [46]. Finally, the effect of NO on multispecies biofilms was not investigated even though multispecies-associated keratitis is common with contact lens wear [27].

The clinical significance of our results is the potential use of NO as an active disinfectant in contact lens care solution. NO donors such as NaNO₂ or sodium nitroprusside are already used as intravenous medications for the treatment of cyanide poisoning (NaNO₂) and heart diseases (sodium nitroprusside) [47, 48]. Therefore, if ophthalmic topical safety is ensured through clinical trials, a tablet form of these chemicals can be mixed into daily dispense of contact lens care solution to eradicate bacterial biofilm.

In conclusion, we confirmed that NO can suppress biofilm formation on soft contact lens in a dose dependent manner. Our findings suggest that NO can be developed as a new therapeutic strategy to reduce biofilm-associated contact lens infection with minimal toxicity to corneal epithelium.