With approximately 125 to 140 million contact lens wearers worldwide [1
] (numbers from 2004 and 2010) the prevention of lens-related infection is a serious healthcare issue. Several ocular diseases are associated with contact lens wear, such as contact lens acute red eye (CLARE), contact lens peripheral ulcer (CLPU) and infiltrative keratitis [4
]. Due to the high numbers of contact lens users, even complications with a rare occurrence will concern a considerable number of patients.
The incidence of contact lens related microbial keratitis is 1.9 per 10,000 for daily wear of soft contact lenses in Australia [8
] and 1.8–2.44 per 10,000 in Scotland for all types [9
], reaching up to 3.09 per 10,000 in Hongkong [10
]. Estimates of risk appear stable over time as quantified over a 20 year period [11
]. Contact lens wearers thus have an approximately five- to seven-fold higher risk of microbial keratitis compared to non-contact lens wearers [9
], with increasing risk for extended or overnight wear.
One of the problems might be the partially insufficient effectiveness of contact lens disinfection solutions. When testing other isolates than the given microbial test strains in the normative standard for a species, the disinfection results of commercial solutions are insufficient in some cases [13
]. Nevertheless, it is not recommendable to enhance the antimicrobial impact of contact lens disinfection systems by increasing the concentration of the solutions. The reason for this is the potential toxicity to epithelial structures of some contact lens solution ingredients. It is reported [16
] that the use of preserved lens care solutions led to an increased P. aeruginosa
binding, presumably by an up-regulation of receptors on corneal epithelial cells, while at the same time a disruption in epithelial homeostasis occurred. Another study [17
] found a 12-fold increase of P. aeruginosa
uptake into the corneal epithelium of rabbits following the wear of multipurpose solution-soaked lenses. Uptake of preservatives into different types of polymeric lens materials was demonstrated [18
], as well as the release of disinfectant, occurring after reinsertion of the lens to the ocular surface.
Several studies testing chemical disinfection solutions on epithelial cells demonstrated that already the limit of health compatibility has sometimes been reached. Various epithelial cell cultures showed cell membrane damage [19
], loss or damage of tight junctions [19
], altering of cell shape and size, loss of mitochondrial enzyme activity, inflammatory response [21
], activation of cell death receptors [22
] or reduced viability [21
The inactivation of microorganisms by irradiation with visible light, especially in the violet and blue spectral range, has been a recent topic in disinfection research [27
]. Endogenous photosensitizers absorb radiation of distinct wavelengths and induce the formation of reactive oxygen species (ROS), which attack microbial targets [29
]. As most bacterial and fungal species harbor porphyrins and flavins, which are considered as relevant responsible photosensitizers, the sensitivity of over 40 different microbial species, including bacteria and fungi, towards visible light has been demonstrated [32
]. Even viruses have successfully been inactivated by exposure to 405 nm in phosphate buffered saline [34
] or nutrient broth [35
] with doses of 2804 J/cm2
for a 3.9 log reduction and 510 J/cm2
for a 5.4 log reduction, respectively.
Previous work suggested reducing microbial burden in contact lens care by applying a light dose, destructive of relevant microorganisms and fungi [36
]. This could be achieved by using transparent contact lens cases in combination with a LED-equipped base irradiating the inside from beyond [37
As there seem to be synergistic or at least combined effects of irradiation techniques such as photodynamic therapy (PDT) and antibiotics [38
] we investigate whether similar effects occur when combining contact lens disinfection solutions and LED-based irradiation at 405 nm. Few investigations of visible light, without the addition of external photosensitizers, in combination with other antimicrobial approaches have been performed. Fila et al. [40
] examined irradiation at 405 nm in combination with antibiotics on different Pseudomonas strains by checkerboard assay without using external dyes. Another strategy was applied by Moorhead et al. [41
] combining 405 nm irradiation with chlorinated disinfectants against Clostridium difficile
spores. 460 nm led to an antibacterial effect in a triple combination together with ineffective antibiotics and non-effective silver nanoparticles [42
]. Pure H2
combined with blue light of 450–490 nm was especially effective in two independent studies [43
]. All of these studies noticed an increased effect of the combination compared to single methods, which were sometimes used in sub-lethal concentrations, but not all of them tested for synergy.
When examining the combination of two different techniques an analysis procedure for quantification of effectiveness has to be specified. The term “synergy” is often used, which is colloquially defined as an effect exceeding the sum of the single effects when performing both techniques simultaneously [45
]. However, there is a lack of definition for this term in normative standards [45
]. In many research works entitled with the term “synergy” there is often no detailed analysis carried out concerning this phenomenon as long as the effect of the two combined methods exhibits an enhanced impact [47
]. Other studies define specific decision criteria, such as a reduction increase of 2 log for the combination compared to the most effective single component, as a definition of synergy [50
The American Society for Microbiology conscientiously defined experimental procedures for determining synergistic effects, which are disk diffusion assays, E-tests for antibiotic susceptibility, checkerboard assays, post-antibiotic effects (PAE) and the Bliss model for biofilm testing [38
]. Others claim that, because a synergism is a physiochemical mass-action law issue, it has to be calculated with Combination Index (CI) values [51
], based on Loewe Additivity.
Foucquier et al. [45
] deliver an overview of the mathematical background for calculations of combination effects. The authors divide approaches into effect-based and dose-effect based. “Response Additivity” is defined as the improvement when comparing the combined effect with the additive effect of both single agents, which would be the colloquial understanding of synergy. This definition belongs to the effect-based group of strategies, which inherit some limitations like, in this case, assumed linear dose-effect curves for both agents. Dose-effect-based strategies, however, rely on the mathematical framework of Loewe Additivity [52
] considering non-linear dose-effect curves, determining which concentration of each drug alone produces the same effect as the combination, rather than comparing effects of given concentrations. This approach requires a certain amount of data and can rapidly become demanding. Generally, any defined effect level can be used for comparison [46
]. Measurement variable can be any parameter giving knowledge about bacterial condition, such as colony forming units [38
], change of color [53
] or OD600
(optical density at 600 nm) values after a specified incubation [38
], as terms in the equation are dimensionless quantities [46
]. From the results, the Combination Index (CI), also called Fractional Inhibition Concentration (FIC), can be calculated for several concentration/dose combinations, which is considered to be the most suitable analysis for synergy testing [45
Several slightly variant categorizations of CI values and their meanings exist. In this study, one of the earliest definitions from Chou is applied, which he later refined [46
] in the categories defining synergism: slight synergism (0.85–0.9), moderate synergism (0.7–0.85), synergism (0.3–0.7) and strong synergism (0.1–0.3). CI values exceeding 1 are called nearly additive (0.9–1.10), slight antagonism (1.10–1.20), moderate antagonism (1.2–1.45), antagonism (1.45–3.3), and strong antagonism (3.3–10).
In cases of microbial keratitis associated with contact lens wear, predominantly environmental organisms were isolated as causative agents, with P. aeruginosa
being the most frequently recovered organism [55
]. The strong association between P. aeruginosa
and ocular infections might also be caused by a suitable environment for Pseudomonads in the system of lens and storage cases. Microbial keratitis in contact lens wear is frequently associated with the presence of biofilm in the contact lens case [59
]. Pseudomonas species are known to be biofilm builders [7
] and the storage case gives a good environment for proliferation [59
]. In a study of various Pseudomonas aeruginosa
isolates some demonstrated the ability to grow to levels above the initial inoculum in one of the chemical disinfectants examined [15
For this reason, we chose a Pseudomonas strain for most of our experiments. Since we are not allowed to cultivate pathogenic strains in our facilities, experiments were carried out with Pseudomonas fluorescens
. In regard to visible light irradiation, it seems that relatives of the same species act similarly [32
In this study we applied a disk diffusion assay, cfu (colony forming unit) determinations on agar plates and nutrient pads, including different procedures for the post-exposure elimination of the disinfection solution. For analysis on agar plates the calculation of Combination Index values based on Loewe Additivity was performed. Furthermore, the monitoring of growth delay, similar to post-antibiotic effect studies (PAE), was applied as method to investigate combination effects of contact lens disinfection solutions and visible light irradiation at 405 nm.
2. Materials and Methods
2.1. Bacterial Strains and Contact Lens Disinfection Solutions
Pseudomonas fluorescens (DSM4358), E. coli (DSM1607) and S. carnosus (DSM20501) were obtained from DSMZ (Deutsche Sammlung für Mikroorganismen und Zellkulturen, Braunschweig, Germany). Pseudomonads were cultivated in 535 medium (30 g tryptic soy broth (Sigma-Aldrich Chemie GmbH, München, Germany) per liter) in an overnight culture of 3 mL at 30 °C and 170 rpm. 200 µL of this pre-culture was cultivated in 30 mL fresh medium at 30 °C and 170 rpm until an optical density of 0.35 in mid-exponential phase was reached. For E. coli and S. carnosus the same procedure at 37 °C was applied with M92 medium (30 g tryptic soy broth (Sigma-Aldrich Chemie GmbH, München, Germany), 3 g yeast extract (Merck KGaA, Darmstadt, Germany) per liter) for S. carnosus and LB medium (10 g tryptone (VWR international, Leuven Belgium), 5 g yeast extract (Merck KGaA, Darmstadt, Germany), 10 g sodium chloride (VWR international, Leuven Belgium) per liter) for E. coli. Bacterial cultures were centrifuged at 7000× g for 5 min and the resultant pellet resuspended in phosphate buffered saline (PBS). After a further washing step in PBS the suspension was diluted to the desired population density for experimental use in PBS.
For disk diffusion assays the bacterial solution was diluted to 0.5 McFarland standard, which was approximately 108
CFU/mL. Instead of Müller-Hinton-Broth commonly applied for this assay type, 535 medium was used and poured in equally filled dishes with 10 mL per 90 mm diameter dish. For nutrient pad analysis, the solution was adjusted to 6–8 × 107
CFU/mL, as the detection limit for the reduction lies one log beyond the used starting concentration and an approximately 6 log reduction was pre-determined for 100% ReNu Multiplus combined with light. For agar plate assays a concentration of 5 × 105
CFU/mL was adjusted, referring to the recommendation of the normative standard for contact lens solution testing [60
]. Likewise, samples for growth delay analysis were adjusted to a concentration of 5 × 105
CFU/mL for the irradiation/disinfection solution exposure treatment. As medium was added for incubation in the microplate reader in a proportion of 1:10, the final concentration for incubation was diluted by one log. The bacterial concentrations indicated represent the concentration in the well already mixed with different concentrations of contact lens solutions. Bacteria were plated on the same media as applied in the fluid culture. Dey-Engley neutralization broth (DEB, Thermo Fisher Scientific, Waltham, MA, USA) was used to eliminate the effect of disinfection solutions after treatment for agar plate and growth delay assays. For nutrient pad assays pseudomonads were incubated on cetrimide pads 14075–47-N (Sartorius, Göttingen, Germany) after membrane filtration.
Untreated controls were analyzed for each assay type to exclude unintended bacterial reduction by environmental factors. In cases where log reductions of sample results had the same algebraic sign as the control, the absolute value of the control was subtracted, otherwise it was ignored. By this means, reductions caused by environmental factors were taken into account, in a manner not to improve inactivation results.
Contact lens disinfection solutions examined in this study were ReNu Multiplus (Bausch+Lomb, Rochester, NY, USA), OptiFree Express (Alcon, Fort Worth, TX, USA) and AOSept Plus (Alcon, Fort Worth, TX, USA). All solutions were used within expiration date.
2.2. Irradiation Setup
For irradiation a LED light source of 405 nm was applied (LZ4–40UB00–00U8 (LED Engin, Inc., San Jose, CA, USA). The emission was measured with a spectrometer (SensLine AvaSpec-2048 XL, Avantes, Appelsdorn, The Netherlands), after a pre-heating interval. The measured peak emission was determined at 405.9 nm with a bandwidth of 19 nm. The LED was mounted to a heat sink, which was actively cooled with a fan during experiments to avoid heating the sample. This package was placed on top of a truncated hollow pyramid with a high reflective inside, which ensured that the sample area was irradiated homogenously (described earlier in [61
]). Experiments were performed in 48 well plates placed on a black underground to avoid unintentional potentiation of irradiation by light reflection from the white laboratory table. 1 mL of sample was transferred into several wells of a 48 well microtiter plate and the pyramid placed on top of the plate, covering 3 × 5 wells. The average sample temperature measured with an infrared thermometer (Raytek Fluke Process Instruments GmbH, Berlin, Germany) was 23.8 °C, with a maximum of 26.2 °C. Irradiation intensity depended on the experimental series and was adjusted by means of an optical power meter OPM150 (Qioptiq, Göttingen, Germany).
2.3. Disk Diffusion Assay
Disk diffusion assays are a technique anchored in routine clinical microbiology, especially in antibiotic susceptibility testing. The measurement parameter is the formation of circular growth inhibition zones, which are caused by diffusion of the applied drug from impregnated disks through the agar medium. No detailed definition of synergy is given for this method in official guidelines, although Wozniak et al. [38
] defined synergy in a disk diffusion assay as an increase of the inhibition zone by 2 mm in combined treatment compared to the single treatment values.
Dilutions of the examined contact lens disinfection solutions in PBS were prepared directly before use to concentrations of 100, 80, 60, 40, 20 and 5%, respectively. 100% refers to the formulation of the specific disinfection solution that is commercially available. Bacterial solutions were irradiated as described above and plated on 535 agar plates of defined thickness. Irradiation doses used for disk diffusion assays have been 0 J/cm2 as control, and 35 J/cm2, 70 J/cm2 and 140 J/cm2, achieved in different time intervals with an intensity of 20 mW/cm2. For the plating technique a volume of 1 mL was distributed by rotary movement of the dish, letting plates air dry afterwards. As the large volume would increase the applied bacterial concentration designed for a 100 µL application, the suspensions were diluted in PBS by 1 log before plating. Soaked disks were placed manually with flamed forceps. After incubation for 24 h at 30 °C, inhibition zones were determined manually by fitting circles to a photograph of the plates in an image processing program. All plates were prepared in duplicates and each experiment was repeated three times. P. fluorescens and all three contact lens solution types were investigated in this assay.
2.4. Determination of Bacterial Reduction with Nutrient Pads
Determinations of cfu were performed on P. fluorescens for combinations of ReNu Multiplus multipurpose solution and 405 nm visible light at a dose of 140 J/cm2. This dose was chosen as it is easily reachable within an overnight disinfection, even when considering a low-cost LED as a potential irradiation product instead of the high-power LED used in the test setup. On the other hand, this dose exhibits a moderate effect when applied alone so that a combination treatment will still result in bacterial concentrations above the detection limit. Concentrations of 5, 20, 40, 60, 80 and 100% of ReNu Multiplus were tested on P. fluorescens as single treatment and in combination with 405 nm irradiation at 20 mW/cm2 in a time interval of 2 h, as well as the effect of light alone in PBS (0% ReNu Multiplus). The bacterial starting concentration was 6–8 × 107 CFU/mL. 100 µL sample volume was diluted serially in PBS. A volume of 500 µL of the desired dilution was then immediately subjected to membrane filtration to eliminate the disinfection solution. Bacteria remained on the filters with a pore size of 0.45 µm, which were placed on moistened nutrient pads. After incubation at 30 °C for 30 h, disks were photographed and colonies enumerated manually. The resultant count was converted to CFU/mL, and in log reduction referring to the plated starting concentration. Each experiment was performed in triplicates and repeated three times.
2.5. Determination of Bacterial Reduction with Agar Plates
Just as for cfu determinations on nutrient pads, an irradiation dose of 140 J/cm2 was chosen. The bacterial starting concentration was 5 × 105 to 106 CFU/mL, as recommended in the normative standard for contact lens solution testing. In this test series three different irradiation intensities were selected to reach this dose within different time intervals. With 10, 20 and 40 mW/cm2 the defined dose was reached within 4, 2 and 1 h irradiation time respectively. This will automatically lead to different residence times for the disinfection solution, whereas 4 h is the minimum disinfection time given by the contact lens solution manufacturer. Each experiment for the combination effect was performed in triplicate and repeated three times.
To be able to calculate the CI value, reference experiments for the disinfection procedures applied separately were carried out in triplicates and repeated twice. Irradiations with 405 nm at 10, 20 and 40 mW/cm2 on bacteria in PBS as well as the effect of ReNu Multiplus without irradiation over intervals of 4, 2 and 1 h at concentrations of 0, 5, 20, 30, 40, 50, 70, 80 and 100% serve as reference for the combined experiments.
100 µL of each sample was transferred to 900 µL Dey-Engley neutralizing broth (DEB) and incubated for at least 15 min at room temperature. DEB samples were diluted to proper bacterial concentrations in PBS and plated manually with a glass spatula. After incubation for 30 h at 30 °C agar plates were photographed and enumerated manually. The resultant count was converted to CFU/mL, and in log reduction referring to the plated starting concentration. Each experiment was performed in triplicate and repeated at least three times.
Based on Loewe Additivity, CI values are then calculated as follows:
where a and b are the concentrations of each agent used in the combination, while A and B are the concentrations of the agents that are necessary to reach the same effect when used separately.
Combination Indexes are generally reported without any assessment of the degree of certainty [45
], but as investigations of biological systems inevitably contain experimental errors, we used the definition from Chou [46
] in a conservative way and only categorized results of “moderate synergism” or more as enhanced outcome.
2.6. Determination of Bacterial Reduction via Regrowth Behavior
In antibiotic testing, where combined testing is frequently performed, post antibiotic effects (PAE) indicate the delay of the regrowth after the exposure to a drug over a certain period and can likewise be used to monitor the differences between single drugs and their combination. The difference to checkerboard assays is that the exposure time is limited, and the drug is removed or eliminated thereafter. As continuous irradiation is not possible inside a microplate reader during incubation, the effect of the disinfection solution equally has to be stopped to achieve comparable results. As this scenario would also represent a realistic application for contact lens care, this method was chosen in place of a checkerboard assay in this study.
The exposure time was set to 4 h as this is the smallest time interval given in manufacturer instructions for contact lens disinfection solutions. This leads to an irradiation intensity of 10 mW/cm2
to reach a dose of 140 J/cm2
. Furthermore, higher irradiation intensities of 20 and 40 mW/cm2
were tested with exposure times of 2 and 1 h, respectively. Contact lens disinfection solution concentrations were tested at 40, 30, 20 and 5% of the commercially available formulation. Besides ReNu Multiplus, another multipurpose solution was examined against P. fluorescens
. OptiFree Express has often been reported to achieve high bacterial impact, but at the same time is aggressive to human ocular epithelium [62
]. Therefore, it would be desirable to reduce concentration of ingredients through a combined use with light. Besides another multipurpose solution, further strains (S. carnosus
and E. coli
) were tested with this technique together with ReNu Multiplus and visible light.
After exposure, samples of 100 µL were immediately transferred to 900 µL of DEB to neutralize the effect of the disinfection solution. This was also performed with samples that have only been irradiated in PBS. After incubation for at least 15 min at room temperature 20 µL of each sample was transferred into a 96 well plate and mixed with 180 µL of specific growth medium. The violet color of DEB thereby was diluted by factor 1:10 so that the sample was translucent enough to monitor increasing turbidity through growth in a microplate reader. Microtiter plates were incubated in a Clariostar Plus (BMG Labtech, Ortenberg, Germany) at 30 °C for P. fluorescens and at 37 °C for all other strains for at least 30 h with measurement of OD600 in 5 min intervals and shaking for 30 s before each measurement, ensuring almost continuous rotary growth conditions. Additionally, sequential ten-fold dilutions of each strain in untreated condition were measured with the same protocol. Each experiment was repeated three times. Depending on how many bacteria were inactivated during exposure of light and/or disinfection solution, the regrowth will be delayed. Based on the untreated dilutions, a calibration curve could be prepared, putting into context the measured OD value at a certain time towards the underlying log reduction.
In this study we tested the combination of contact lens disinfection solutions with visible violet light of 405 nm. Combining different approaches has a long history not only for disinfection techniques, but also for medical therapies. For just as long, experts have been discussing how to quantify these results. Dose-effect-based strategies seem advantageous as is explained in detail in [45
], because they do not have limitations through assumptions such as linearity. Furthermore, it is recommended to use several different methods to come to a conclusion. In our investigations, we often achieved varying results for the same parameters, when testing with different analytical methods. Nevertheless, related tendencies are obvious in all test methods.
The combination effect is assumed to increase with light dose. This was observed at disk diffusion testing. With all other test methods, a fixed dose of irradiation (140 J/cm2
) was used. Synergism of pure H2
combined with blue light of 470 nm has previously been reported in S. aureus
]. Unfortunately, ReNu Multiplus and OptiFree Express solutions did not form clear inhibition zones on agar plates even with reduced agar concentrations. A positive combination effect could be observed for the hydrogen peroxide solution AOSept, while it is only a presumption that this would also be valid for multipurpose solutions. As high concentrations of bacteria (approximately 108
CFU/mL) are applied for disk diffusion assays to produce a dense bacterial lawn, bacterial concentration dependency of multipurpose solutions, as observed in Figure 5
for colony counts, could be the reason for the absence of clearly visible inhibition zones.
As the effect of light irradiation alone increases with the dose [31
], a similar dose dependency for a combined application appears likely. However, an important fact in combination testing is that it is not possible to predict the results, as some drugs have several targets or independent antimicrobial mechanisms [51
]. A combination of photodynamic therapy and various antibiotics, for example, showed a decrease in development of resistance for some drugs while for other antibiotics resistance was acquired through the combination with PDT [66
Therefore, any combination of two methods has to be investigated separately and it is not possible to test for general statements about synergy [46
]. Combinations at varying doses/concentration levels can lead to very different results with the same two approaches in combination [39
]. Similarly, in Table 1
, where CI values are determined, 20 mW/cm2
and 30% ReNu Multiplus lead to explicit synergy with a CI of 0.66, while 10 mW/cm2
at the same concentration of ReNu Multiplus is only additive with a CI of 0.92. At a light intensity of 40 mW/cm2
even moderate antagonism with a CI of 1.28 occurs. At the same time, it is important to clarify that for practical considerations it is not the most important issue to attain mechanistic synergy, but to achieve a high antibacterial impact. The occurrence of synergy does not necessarily arrive at the best overall results, because the highest antimicrobial effect can occur in the absence of synergy. For practical considerations, this shows that synergy is not necessarily relevant for product design, where overall reduction is the relevant measurand, while the best synergistic grade is achieved at the highest increase of the combination’s benefit.
Concerning the irradiation intensity in combination with the multipurpose solution ReNu Multiplus, our results indicate that lower intensities used over a longer exposure period will lead to higher inactivation results, than higher irradiation intensities at shorter durations reaching the same dose. The results achieved with agar plates show that 40 mW/cm2
irradiation does not compete with the inactivation effect of 10 or 20 mW/cm2
) in combination with ReNu Multiplus. The same tendencies are observable at growth delay analysis for P. fluorescens
, E. coli
and S. carnosus
. Combining this knowledge with the assumption of an increasing effect at higher doses, applications with long irradiation intervals seem to be advantageous.
OptiFree Express, another multipurpose solution with a very potent antibacterial effect, but likewise unhealthy for the consumer [62
], shows a totally different reaction pattern than ReNu Multiplus. The solution, which kills all bacteria at a concentration of just 20%, rapidly loses activity at a concentration of 5%, while the effect of ReNu Multiplus decreases continuously with gradual dilution. For OptiFree Express the addition of light does not seem to improve effectiveness. An opposite effect can be observed for ReNu Multiplus against S. carnosus
d) where the irradiation with light delivers the main impact. Only for 40% at 20 and 40 mW/cm2
does the addition of ReNu Multiplus lead to an increase higher than the effect of 405 nm alone. It is therefore recommended to further investigate other contact lens disinfection solutions.
A noticeable fact in this study is the huge differences of ReNu Multiplus effectiveness examined with nutrient pads compared to agar plates. The effectiveness of multipurpose solutions under different experimental conditions may depend on the bacterial inoculum. The normative standard for testing contact lens solutions [60
] suggests a starting concentration between 105
CFU/mL. The nutrient pad experiments, however, were carried out with an inoculum of 6–8 × 107
CFU/mL. In fact, the log reduction considerably decreased with rising bacterial load (Figure 5
a) when testing ReNu Multiplus as a single method. This could lead to severe clinical problems as total viable bacterial counts between 106
/mL were found in 13 out of 18 contact lens cases of patients with corneal infiltrative infections using multipurpose solutions [67
]. Testing the combination of ReNu Multiplus with 405 nm irradiation, the bacterial concentration did not seem to play a pronounced role. Irradiation procedures with visible light are less dependent on the bacterial inoculum as they are based on endogenous photosensitizers, which increase in parallel to the bacterial concentration. Only absorption and scattering issues seem to limit the effectiveness for high bacterial concentrations [31
]. It is remarkable that in spite of the marginal single impact of ReNu Multiplus at high bacterial loads the combination effect in the presence of ReNu Multiplus increases to values well exceeding the effect of light alone.
Concerning the plausibility of the investigation of a non-pathogenic surrogate, we evaluated the literature data available for photoinactivation of Pseudomonads. P. aeruginosa
strains are among the most often examined microorganisms regarding visible light inactivation [33
], but results for other representatives of the genus are scarce. Applying 400 nm at a dose of 100 J/cm2
on P. fluorescens,
Angarano et al. [68
] achieved a 0.5 log reduction, which is in good accordance with our results.
Maclean et al. [31
] achieved a 1 log reduction of P. aeruginosa
(NCTC 9009) at a dose of 42.9 J/cm2
with 405 nm of 10 mW/cm2
irradiation intensity. Fila et al. [40
] examined a broad range of P. aeruginosa
strains including wild-type strains, drug-sensitive clinical isolates and multi-drug-resistant clinical isolates with very similar behaviors of 7 log reduction at around 50 J/cm2
. Dependent on whether the average dose is considered or if the shoulder is taken into account, this leads to a result of 7–12 J/cm2
for 1 log reduction. Gupta et al. [69
] isolated a P. aeruginosa
strain from patients with arthroplasties for which an averaged dose of 133 J/cm2
was needed for a 1 log reduction of 405 nm at 123 J/cm2
To our knowledge, these are the most extreme examples showing the upper and lower values for P. aeruginosa
eradication measured to date, the variation probably caused by differences in the setup or test protocol, as different strains examined with the same protocol react similarly [40
]. Still, with 154 J/cm2
for 1 log reduction of P. fluorescens
at 20 mW/cm2,
our results are overshooting those values. It seems that P. fluroescens
is less susceptible to 405 nm than its pathogenic relative.
The choice of an appropriate surrogate concerning the performance of a disinfection method should rather be more conservative and survive longer than the target organism [70
]. As this seems to be the case with our choice of a Pseudomonad representative, we believe that the results can be considered meaningful. Nevertheless, this technique has to be tested with the pathogenic variant P. aeruginosa
according to the standard DIN EN ISO 14729 for contact lens disinfection equipment prior to routine usage.
The reasons for combination testing can be various with different favorable outcomes, such as increasing the effectiveness or decreasing the dosage while increasing or maintaining the same efficacy to avoid toxicity [46
]. Minimizing or slowing down the development of drug resistance can also be a motivation [46
]. This study was designed mainly to address the second aspect. Meyer et al. [71
] describe the reduction of a concentration/dose to reach the same effect as before when adding a second component as “synergistic potency“, which is useful to apply in applications with side effects, in comparison to “synergistic efficacy“ where the aim is to enhance the final result by use of the same drug-concentrations as before.
As mentioned before a considerable volume of aggressive ingredients is stored in the polymeric material of contact lenses after disinfection [18
]. As contact lens solutions are known to have adverse effects on the patient’s eye [19
], a reduced concentration will decrease the patient’s risk of epithelial damage. Since some frequently used contact lens solutions are already at the limit of efficacy, a reduction of the formulation is often not possible. For several multipurpose solutions, including ReNu Multiplus and OptiFree Express, it was shown, however, that diluting the original concentration in PBS led to higher cell viability and integrin expression [72
], using concentrations between 1% and 10%, compared to 100%. Another study that investigated dilutions of three different multipurpose solutions on mouse fibroblasts reported that a 25% dilution of all solutions tested could be considered non-toxic [73
]. Therefore, reducing contact lens solution concentrations seems favorable. We tried to achieve this by a combination with visible violet light of 405 nm.
It was already proven that light as a single method is usable to meet the criteria of contact lens disinfection and a prototype of an applicable, as well as commercially suitable, system has been developed [37
]. The combination of disinfection solution and visible light might not only overcome the problems of efficacy limitation of disinfectants due to biocompatibility issues; the existence of a second technique may also prevent complete failure of a specific lens care product as happened in 2006 with an outbreak of Fusarium solani
keratitis in several parts of the world [74
]. Maintaining a system with two different disinfection strategies would not so easily lead to complete ineffectiveness.
Following on from the disinfection properties of visible light irradiation, the possible impact on the material characteristics of contact lenses has to be investigated prior to the consideration of translation into routine usage. It has to be ensured that not only the required microbiological parameters are met, but simultaneously the lens itself would not be influenced detrimentally. Preliminary tests, applying irradiation doses simulating cumulative exposure over monthly use, revealed only slight changes concerning transmission, still well within the limits defined by the standard DIN EN ISO 18369–2 (data not shown). However, differences to this test protocol can occur in routine use due to rubbing of the lens, so it is recommended to perform further tests concerning material compatibility, including examination of mechanical stability.