Enhancement of Contact Lens Disinfection by Combin

International Journal of
Environmental Research
and Public Health
Article
Enhancement of Contact Lens Disinfection by
Combining Disinfectant with Visible
Light Irradiation
Katharina Hoenes 1, * , Barbara Spellerberg 2 and Martin Hessling 1
1 Institute of Medical Engineering and Mechatronics, Ulm University of Applied Sciences,
Albert-Einstein-Allee 55, 89081 Ulm, Germany; [email protected]
2 Institute of Medical Microbiology and Hygiene, University of Ulm, Albert-Einstein-Allee 11, 89081 Ulm,
Germany; [email protected]
* Correspondence: [email protected]

Received: 21 July 2020; Accepted: 29 August 2020; Published: 3 September 2020
Abstract: Multiple use contact lenses have to be disinfected overnight to reduce the risk of infections.
However, several studies demonstrated that not only microorganisms are affected by the disinfectants,
but also ocular epithelial cells, which come into contact via residuals at reinsertion of the lens. Visible
light has been demonstrated to achieve an inactivation effect on several bacterial and fungal species.
Combinations with other disinfection methods often showed better results compared to separately
applied methods. We therefore investigated contact lens disinfection solutions combined with 405 nm
irradiation, with the intention to reduce the disinfectant concentration of ReNu Multiplus, OptiFree
Express or AOSept while maintaining adequate disinfection results due to combination benefits.
Pseudomonads, staphylococci and E. coli were studied with disk diffusion assay, colony forming unit
(cfu) determination and growth delay. A log reduction of 4.49 was achieved for P. fluorescens in 2 h for
40% ReNu Multiplus combined with an irradiation intensity of 20 mW/cm2 at 405 nm. For AOSept
the combination effect was so strong that 5% of AOSept in combination with light exhibited the
same result as 100% AOSept alone. Combination of disinfectants with visible violet light is therefore
considered a promising approach, as a reduction of potentially toxic ingredients can be achieved.
Keywords: photoinactivation; 405 nm; visible light; synergy; contact lens disinfection solution;
multipurpose solution; pseudomonas
1. Introduction
With approximately 125 to 140 million contact lens wearers worldwide [1–3] (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 [4–7]. 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 [11,12]. Contact lens wearers thus have an approximately five- to seven-fold higher risk of
microbial keratitis compared to non-contact lens wearers [9,10], 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
Int. J. Environ. Res. Public Health 2020, 17, 6422; doi:10.3390/ijerph17176422 www.mdpi.com/journal/ijerph
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for a species, the disinfection results of commercial solutions are insufficient in some cases [13–15].
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 [19,20], altering of cell shape
and size, loss of mitochondrial enzyme activity, inflammatory response [21], activation of cell death
receptors [22,23] or reduced viability [21,24–26].
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 [27,28]. Endogenous photosensitizers
absorb radiation of distinct wavelengths and induce the formation of reactive oxygen species (ROS),
which attack microbial targets [29–31]. 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 [32,33].
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 [38,39] 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 O2 combined with blue light of 450–490 nm was especially effective in
two independent studies [43,44]. 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 [45,46]. 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 [47–49]. 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 [38,41],
change of color [53] or OD600 (optical density at 600 nm) values after a specified incubation [38,40,54],
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 [55–58]. 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 [32,40].
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 to 106 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 to 106 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:
CI = a/A + b/B, (1)
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.
3. Results
3.1. Disk Diffusion Assay

To assess the antibacterial effect of contact lens disinfectant solution by disk diffusion assay,
we applied ReNu Multiplus and OptiFree Express at concentrations of up to 100% to the agar plates.
However, the multipurpose solutions used for this study did not form clear inhibition zones in any
concentration that was tested. As we assumed this fact to be caused by the molecular structure of the
active components, not being able to pass the cross-linked agar, we tried to decrease the agar concentration
in the plates until the limit of solidity in order to achieve greater pore sizes. However, with 110 mg/10 mL
agar still no enhanced effect on the appearance of inhibition zones formed by the multipurpose solutions
alone or in combination with visible light was identifiable, even at 100%. With unclear inhibition zones,
only visible with background lighting (Figure 1aII), disk diffusion results with multipurpose solutions
were considered not analyzable. Cfu determinations of contact lens disinfection solution, however,
showed a considerable decrease in bacterial count. With the hydrogen peroxide based solution AOSept,
conversely clearly visible inhibition zones were detectable (Figure 1aIII).
Int. J. Environ. Res. Public Health 2020, 17, x 8 of 21
Figure
Figure 1. Demonstration
1. Demonstration ofofdifferent
differentagar agar plate
plate appearances
appearances (a)(a)with
withfragmentary
fragmentarylawn through
lawn through
excessive
excessive lightlight
dosedose of 280
of 280 J/cm
J/cm
2 (I), indefinite inhibition zone with 130 mg/10 mL agar concentration
2 (I), indefinite inhibition zone with 130 mg/10 mL agar concentration
and 100%
and 100% ReNuReNu Multiplus
Multiplus (II),optimal
(II), optimalappearance
appearance with
withdifferent
differentconcentrations of AOSept
concentrations (III).(III).
of AOSept
Diameter of inhibition zones on P. fluorescens lawn achieved with different concentrations of
Diameter of inhibition zones on P. fluorescens lawn achieved with different concentrations of hydrogen
hydrogen peroxide solution AOSept, dependent on the dose of 405 nm applied with 20 mW/cm2 (b).
peroxide solution AOSept, dependent on the dose of 405 nm applied with 20 mW/cm2 (b). Error bars
Error bars indicate the deviation in the three experiments. The red dotted line indicates the size of
indicate the deviation in the three experiments. The red dotted line indicates the size of inhibition
inhibition zones generated with 100% AOSept without irradiation.
zones generated with 100% AOSept without irradiation.
Testing disinfection solution ReNu Multiplus as single treatment with nutrient pads, nearly no
inactivation was observable (Figure 2a). For 100%, which represents the pure commercially available
solution, only 0.48 log reduction was achieved in 2 h at 20 mW/cm2 with a bacterial starting
concentration of 6–8 × 107 CFU/mL. In contrast, a combination treatment with ReNu Multiplus 100%
As can be seen in Figure 1aI, the irradiation dose has to be selected carefully for this technique,
as otherwise a semiconfluent growth of colonies, which is required for the development of clearly
visible inhibition zones, cannot be achieved. The disinfection effect increases with the irradiation
dose as shown in Figure 1b. The highest dose analyzable with a continuous bacterial lawn was
140 J/cm2 at 405 nm. Likewise, the inhibition zones increase with the percentage of hydrogen peroxide
solution. The dotted line on the graph represents the disinfection result when using the disinfectant at
100% concentration, as commercially available. Every data point above this line, leading to greater
inhibition zones, shows the benefit of combining conventional contact lens disinfection techniques
with visible light irradiation. The combination of 140 J/cm2 irradiation at 405 nm with 5% of the
original concentration of the disinfection solution achieves a similar result as a 100% solution without
irradiation. Any higher concentrations leads to even better results in combination with 405 nm.

Testing disinfection solution ReNu Multiplus as single treatment with nutrient pads, nearly
no inactivation was observable (Figure 2a). For 100%, which represents the pure commercially
available solution, only 0.48 log reduction was achieved in 2 h at 20 mW/cm2 with a bacterial starting
concentration of 6–8 × 107 CFU/mL. In contrast, a combination treatment with ReNu Multiplus 100%
and visible light of 405 nm was quite successful with almost complete inactivation of the bacteria.
Even at lower disinfectant concentrations, considerable bacterial reduction was achieved. Plotting
the benefit of combination treatment compared to the sum of single approaches, colloquially called
synergy (Figure 2b), it becomes visible that the relation is not linear. The higher the applied disinfection
solution concentration, the greater is the overall combined bacterial inactivation. However, the increase
of the benefit slows down for higher concentrations. The most distinct difference in the gradient of
achieved log reductions lies between 20 and 40% ReNu Multiplus content with 2.86 and 4.38 log,
respectively. Therefore 40% of ReNu Multiplus in combination with visible light of 405 nm appears
to be the best compromise between reducing the disinfectant concentration and achieving optimal
inactivation results. All other experiments with different methods were therefore carried out with
disinfectant concentrations in this range. Light irradiation with 405 nm alone led to 1.42 log reduction.
Figure
Figure 2. Reduction
2. Reduction results
results with
with nutrient
nutrient pads
pads forfor
allall combinations
combinations testedincluding
tested includingsingle
singleapproaches
approaches as
as reference.
reference. Error
Error bars bars indicate
indicate the deviation
the deviation in the
in the three three experiments
experiments (a), Example
(a), Example of “synergy“
of “synergy“ calculation
calculation asofenhancement
as enhancement of the over
the combination combination
the sumover the sum
of ReNu of ReNu and
Multiplus Multiplus and lightseparately,
light applied applied
separately,
and progress and progress
of these of these
“synergies“ “synergies“
for different ReNu forMultiplus
different concentrations
ReNu Multiplusonconcentrations on P.
P. fluorescens (b).
fluorescens (b).
With nutrient pad analysis we determined that any combination and even light alone produces
better results in a 2 h treatment than application of ReNu Multiplus at 100% for a P. fluorescens
concentration of 6–8 × 107 CFU/mL.
With nutrient pad analysis we determined that any combination and even light alone produces
better results in a 2 h treatment than application of ReNu Multiplus at 100% for a P. fluorescens
concentration of 6–8 × 107 CFU/mL.
3.3. Determination of Bacterial Reduction with Agar Plates

To determine if the results of the nutrient pad analysis can be confirmed with other methods, agar
plate determinations of bacterial inactivation were carried out (Figure 3). In Figure 3, the inactivation
effect tested with cfu determinations on agar plates is depicted for different irradiation intensities.
The bacterial starting concentration was 5 × 105 to 106 CFU/mL. Comparing the different investigations
methods, the combination results correspond relatively well. For a 20 mW/cm2 irradiation in PBS
without disinfectant a 1.42 log decrease of bacterial counts is achieved on nutrient pads (Figure 2a)
while 1.00 log is measured with agar plates (Figure 3b). For a combination of 405 nm irradiation with
5, 20 and 40% of ReNu Multiplus, 2.16, 2.86 and 4.38 log decreases are achieved on nutrient pads,
Int. J. Environ. Res.the
while results
Public on 2020,
Health agar plates
17, x are 1.12, 2.76 and 4.49 log, respectively. 10 of 21
Figure 3. Figure
Log reduction results
3. Log reduction for combinations
results for combinationsofofdifferent concentrations
different concentrations of ReNu
of ReNu Multiplus
Multiplus with with
2 was reached with 10 mW/cm2 in24 h (a),
visible light of 405
visible lightnm on nm
of 405 agaronplates. A dose
agar plates. of 140
A dose J/cm
of 140 2 was
J/cm reached with 10 mW/cm in 4 h (a), 20
20 mW/cm 2 in 2 h (b), 40 mW/cm2 in 1 h (c). Error bars indicate the deviation in the three experiments.
mW/cm2 in 2 h (b), 40 mW/cm2 in 1 h (c). Error bars indicate the deviation in the three experiments.
Comparing different irradiation intensities reaching the same dose of 140 J/cm2 over various
3.4. Loeweexposure
Additivity
times it becomes evident that the highest irradiation intensity is not necessarily the best
choice. At 40 mW/cm2 for 1 h (140 J/cm2 ) the lowest reduction results are achieved with only a 2.01 log
For comparing the combination effects with the single approach results, ReNu Multiplus and
decrease at 40% ReNu Multiplus in combination with 405 nm irradiation. Best combination results
405 nm alone have notat only
were achieved been2 and
20 mW/cm tested
40%atconcentration
analogousofconcentrations/doses
ReNu Multiplus with 4.49 aslog
thereduction.
combinations, but
over the whole concentration range and over an extended dose range (Figure 4). This allows the
calculation of CI values based on Loewe Additivity, which directly gives a benchmark for synergy
categorization. Interestingly, there was no large difference between exposure times of 1, 2 and 4 h for
ReNu Multiplus at any concentration (Figure 4d). There is a tendency towards better efficacy for
longer duration, but the deviation is lower than expected. Only at 80% disinfection solution did the
4 h results exceed the results of shorter durations, with a decrease of 4.60 log compared to 3.49 and
3.38 log at 2 and 1 h, respectively. At 100% ReNu Multiplus there were no colonies visible after any
5 6
3.4. Loewe Additivity

For comparing the combination effects with the single approach results, ReNu Multiplus and
405 nm alone have not only been tested at analogous concentrations/doses as the combinations, but over
the whole concentration range and over an extended dose range (Figure 4). This allows the calculation
of CI values based on Loewe Additivity, which directly gives a benchmark for synergy categorization.
Interestingly, there was no large difference between exposure times of 1, 2 and 4 h for ReNu Multiplus
at any concentration (Figure 4d). There is a tendency towards better efficacy for longer duration, but the
deviation is lower than expected. Only at 80% disinfection solution did the 4 h results exceed the results
of shorter durations, with a decrease of 4.60 log compared to 3.49 and 3.38 log at 2 and 1 h, respectively.
At 100% ReNu Multiplus there were no colonies visible after any exposure time starting from a
concentration of 5 × 105 to 106 CFU/mL. Uniform behavior over disinfection solution concentration
was assumed here, represented by a linear fit. For references at 405 nm irradiation, bacterial solutions
in PBS were irradiated with 10, 20 and 40 mW/cm2 over different intervals reaching a dose of 245 J/cm2 .
The typical behavior for visible light photoinactivation, with a mostly linear slope in a half logarithmic
representation and an additional shoulder at the beginning (non-mono-exponential), becomes evident
here. Four data points within the linear section were achieved for each irradiation intensity. A linear
fit through those points was used as reference for 405 nm irradiation as a single approach to calculate
the doses necessary to reach a certain effect.
FigureFigure 4. Reference
4. Reference experiments
experiments ononagar
agarplates
plates to
to achieve
achievedose-effect
dose-effectcurves forfor
curves single approaches
single on on
approaches
P. fluorescens: 405 nm irradiation in phosphate buffered saline (PBS) with 10 mW/cm 2 (a),
P. fluorescens: 405 nm irradiation in phosphate buffered saline (PBS) with 10 mW/cm (a), 20 mW/cm2 2 20 mW/cm 2
(b) and 40 mW/cm

40 mW/cm2 (c) (c) as as well as ReNu Multiplus exposure for 1, 2, and 4 h at different concentrations
2
(b) and well as ReNu Multiplus exposure for 1, 2, and 4 h at different concentrations
(d). Error bars indicate the deviation in the two experiments.
(d). Error bars indicate the deviation in the two experiments.
In Table 1 the calculated CI values are presented, based on the combination results shown in
In Table 1 the calculated CI values are presented, based on the combination results shown in
Figure 3 and the trendlines from Figure 4, calculated with the Formula (1).
Figure 3 and the trendlines from Figure 4, calculated with the Formula (1).
For 20 mW/cm2 and 2 h irradiation all values lie considerably below 0.85 indicating moderate
For 20 mW/cm 2 and 2 h irradiation all values lie considerably below 0.85 indicating moderate
synergism for 20 and 40% and even synergism for 30%. At this irradiation intensity, the absolute log
synergism for 20
reductions and
also 40%the
reach and even values
highest synergism
with for
4.0930%.
log atAt this
30% irradiation
and 4.49 log atintensity,
40%. It is the absolute log
noteworthy
that at 10 mW/cm2 as well as 20 mW/cm2 the CI values for 30% ReNu Multiplus content are the lowest,
showing the best beneficial effect for the combination. This fits with the decrease of the slope for
higher concentration values and the leap between 20 and 40% noticed at the nutrient pad results
(Figure 2b). None of the combinations examined achieves better results than the samples for ReNu
Multiplus at 100% - at least in the test series with agar plates and low bacterial concentrations - as
reductions also reach the highest values with 4.09 log at 30% and 4.49 log at 40%. It is noteworthy that
at 10 mW/cm2 as well as 20 mW/cm2 the CI values for 30% ReNu Multiplus content are the lowest,
showing the best beneficial effect for the combination. This fits with the decrease of the slope for higher
concentration values and the leap between 20 and 40% noticed at the nutrient pad results (Figure 2b).
None of the combinations examined achieves better results than the samples for ReNu Multiplus at
100% - at least in the test series with agar plates and low bacterial concentrations - as none of the results
in column B shows a percentage over 100%. However, good log results were achieved at 30 and 40%
for 10 mW/cm2 and 20 mW/cm2 . Regardless, CI values for 10 mW/cm2 and also for 40mW/cm2 lie
close to or even above 1, indicating no synergism.
Table 1. Combination Index values calculated based on Loewe Additivity for log reduction results on
agar plates achieved with ReNu Multiplus and 405 nm on P. fluorescens.
Combination a b A B CI
Dose in Dose in
Log % %
J/cm2 J/cm2
−0.82 140 5 137.7 24.8 1.2186
−2.19 140 20 230.6 47.5 1.0278
10 mW/cm2
−3.26 140 30 303.1 65.3 0.9215
−3.86 140 40 344.0 75.3 0.9382
−1.12 140 5 169.3 29.5 0.9963
−2.76 140 20 327.8 60.1 0.7600
20 mW/cm2
−4.09 140 30 457.0 84.9 0.6595
−4.49 140 40 496.5 92.5 0.7142
−0.76 140 5 226.1 28.4 0.7955
−0.73 140 20 223.1 27.8 1.3460
40 mW/cm2
−1.29 140 30 282.5 37.0 1.2841
−2.01 140 40 359.1 51.2 1.1708
Nevertheless, such a combination approach could be employed in practice regarding overall

reductions and taking into account that they have been achieved with clearly lower concentrations.
According to the testing on agar plates the overall result cannot be improved, but the concentration of
antibacterial ingredients in the formulation of contact lens solutions could be reduced when combining
with light to preserve the consumer’s epithelial health, while still achieving acceptable results.
3.5. Effectiveness Dependency of Multipurpose Solution ReNu Multiplus on Bacterial Concentration

Comparing the inactivation results for ReNu Multiplus as a single method achieved with agar
plates (Figure 4d) and nutrient pads (Figure 2a), great differences were observed. While no colonies
were visible after 2 h of exposure to 100% ReNu Multiplus and subsequent distribution on agar plates
(5.5 log reduction), only a 0.48 log reduction was reached with membrane filtration and nutrient
pads. A major difference in the two assays was the bacterial starting concentration with 5 × 105 to
106 CFU/mL for the agar plate assay and 6–8 × 107 CFU/mL for the nutrient pad analysis. We therefore
assumed that the effectiveness of ReNu Multiplus was highly dependent on the bacterial concentration.
Testing this hypothesis, we could show that the log reduction decreases with increased bacterial
concentration (Figure 5a). For a 2 h exposure with a 106 CFU/mL starting concentration, again no
CFU were observable. Yet, at 5 × 107 CFU/mL only 1.9 log reduction, and at 108 CFU/mL only 1.4 log
reduction, were achieved respectively, referring to the specific starting concentration.
In Figure 5b the log results for the combination approach with visible light are presented in direct
comparison between agar plate and nutrient pad assay. At samples where 405 nm was additionally
applied to the different ReNu Multiplus concentrations, the differences in the bacterial inoculum do not
seem to play a pronounced role. This is contrary to the disinfection results with 100% ReNu Multiplus
as single approach, where an increased bacterial load leads to noteworthy loss in effectiveness.
Figure 5. Effectiveness of undiluted ReNu Multiplus disinfection solution (100%) against P. fluorescens
Figure 5. Effectiveness of undiluted ReNu Multiplus disinfection solution (100%) against P. fluorescens
at different bacterial concentrations, tested with agar plates. Error bars indicate the deviation in the
at different bacterial concentrations, tested with agar plates. Error bars indicate the deviation in the
three experiments (a). Comparison of log results with nutrient pads and agar plates for combination of
three experiments (a). Comparison of log results with nutrient pads and agar plates for combination
ReNu Multiplus and visible 405 nm light at 20 mW/cm2 for 140 J/cm2 . Error bars indicate the deviation
of ReNu Multiplus and visible 405 nm light at 20 mW/cm2 for 140 J/cm2. Error bars indicate the
in the three experiments (b).
deviation in the three experiments (b).
In this experimental series the growth of bacteria after treatment with single or combined
In this experimental
disinfection approachesseries the growth
is investigated. of bacteria
In the literatureafter treatment
differences with single
in behaviour or combined
towards disinfection
disinfection approaches is investigated. In the literature differences in behaviour
techniques are often detected between plated results and analytical methods in fluids. The effect of towards disinfection
techniques are often as
ReNu Multiplus detected
a singlebetween
approach plated
againstresults and analytical
P. fluorescens (Figure methods
6a) does innot
fluids.
show The effectimpact
a high of
ReNu andMultiplus
at 5% andas20% a single
growth approach
can occur against
instead P. fluorescens
of a reduction, (Figurewhich6a) was
doesalready
not show a high impact
noticeable in the two
andother
at 5%analysis
and 20%methods.
growth can occur instead of a reduction, which
Log reductions at 40%, the highest ReNu Multiplus concentrationwas already noticeable in the twohere,
tested
other analysis
varied methods.
between 0.35 and Log reductions
0.64, depending at on40%,thethe highesttime.
exposure ReNu Multiplus
Compared concentration
to the results achieved testedwith
here, varied between 0.35 and 0.64, depending on the exposure time.
agar plates with 0.75 (1 h), 1.45 (2 h) and 1.39 (4 h) log reduction at 40%, and those of 0.0 log achieved Compared to the results
achieved with agar
with nutrient plates
pads in 2 with 0.75 values
h at 40%, (1 h), 1.45 (2 h)with
reached and growth
1.39 (4 h) log reduction
analysis lie in theatmiddle.
40%, and Forthose of
irradiation
0.0 with
log achieved
405 nm inwithPBS nutrient
(0%), where pads in (1
0.52 2hh/40at mW/cm
40%, values 2 ), 0.48 reached
(2 h/20 with
mW/cm growth
2 ) andanalysis lie in
0.97 (4 h/10 the 2 )
mW/cm
middle. For irradiation with 405 nm in PBS (0%), where 0.52 (1 h/40
log were measured by growth delay, the values with agar plates reaching 0.30 (1 h/40 mW/cm ), mW/cm 2), 0.48 (2 h/20 mW/cm2) 2
and0.85
0.97(2 (4h/20
h/10mW/cm
mW/cm22)) and log were
0.82 (4measured
h/10 mW/cm by growth delay, thefit
2 ) log reduction values with agar
acceptable. Hereplates reachingpad
the nutrient
0.30value
(1 h/40 mW/cm
with
2
1.42 log), 0.85
(2 h/20(2 h/20
mW/cm mW/cm 2 ) and upwards.
2 ) deviates 0.82 (4 h/10 mW/cm ) log reduction fit acceptable.
2
Here theFor nutrient pad value with 1.42 log (2 h/20

combination results a huge increase occurred )between mW/cm 2 deviates upwards.
30 to 40% disinfectant concentration,
atFor combination
least results a huge
for the experiments at 4increase
and 2 hoccurred
duration, between
i.e., 1030and to 40% disinfectant
20 mW/cm concentration,
2 irradiation intensity.
at least for the experiments at 4 and 2 h duration, i.e., 10 and 20
This was already noticed in the nutrient pad analysis. Log reductions for 40% ReNu Multiplus mW/cm 2 irradiation intensity. This
wascombined
already noticed in the nutrient pad analysis. Log reductions for 40%
with light determined by growth analysis and agar plates are 4.48 and 3.86, respectively, ReNu Multiplus combined
withforlight
10 mW/cm2 and by
determined 3.24,growth
4.49, asanalysis
well as and 4.38 agar
for 20plates
mW/cm are2 at
4.48 and 3.86,
growth, agarrespectively,
plate and nutrientfor 10pad
mW/cm 2 and 3.24, 4.49, as well as 4.38 for 20 mW/cm2 at growth,agar plate and nutrient pad analysis.
analysis. For concentrations of 30% and 20% ReNu Multiplus, log reduction values determined with
For growth
concentrations
analysisofare 30% and 20% ReNu
considerably lowerMultiplus,
than values logachieved
reduction values
with the twodetermined with growth
other methods. Nutrient
analysis are considerably lower than values achieved with the two
pad and agar plate results, however, match well at 20% with a 2.76 log reduction (2 h/20 mW/cm other methods. Nutrient pad and2 ) on
agaragarplate results, however, match well at 20% with a2 2.76 log reduction
plates and a 2.86 log reduction (2h/20 mW/cm ) with nutrient pads. It seems that at disinfectant (2 h/20 mW/cm 2) on agar
plates and a 2.86 log

concentrations belowreduction
40% plating (2h/20 mW/cm2)deliver
techniques with nutrient pads. It results
more optimistic seems thanthat analysis
at disinfectant
in fluid.
concentrations below 40% plating techniques deliver more optimistic results than analysis in fluid.
In the experimental series of growth delay analysis, another multipurpose solution besides
ReNu Multiplus was tested (OptiFree Express) which is known to be rather aggressive to bacteria as
well as to the ocular surface. Results are shown in Figure 6b, where it stands out that, down to 20%
exposure times are more effective in the combination approach than higher irradiation intensities at
shorter durations. For ReNu Multiplus as a single method, Pseudomonas sp. seem to be the
microorganism most difficult to inactivate, which matches literature data [53] and was the reason for
choosing it as principal strain in this study. The addition of irradiation increases the effectiveness of
Int. J. Environ.
ReNu Res. Public
Multiplus. At aHealth
40%2020, 17, 6422
concentration 13 of 20
at least 3 log reduction could be achieved for all bacterial
species tested with the combination approach.
Figure 6. Log reductions determined from growth delay analysis for different intensities of 405 nm light
and different concentrations of disinfection solutions: ReNu Multiplus on P. fluorescens (a), OptiFree
Express on P. fluorescens (b), ReNu Multiplus on E. coli (c) and ReNu Multiplus on S. carnosus (d).
Upper part of diagram presenting results for single approaches, lower part presenting combination
results (+). Error bars indicate the deviation in the three experiments.
In the experimental series of growth delay analysis, another multipurpose solution besides ReNu
Multiplus was tested (OptiFree Express) which is known to be rather aggressive to bacteria as well
as to the ocular surface. Results are shown in Figure 6b, where it stands out that, down to 20% of
OptiFree Express, all bacteria were killed with the solution alone, with the exception of 1 h exposure
with 20%, where 3.14 log reduction was measured. At 5%, however, only reductions lower than one
log were achieved. The combination with light does not seem to have any influence on the results,
neither positive nor negative.
For E. coli (Figure 6c) the inactivation effect of ReNu Multiplus is comparably stronger than for
P. fluorescens. The same applies for irradiation with 405 nm. In each case, the combination approach
achieves higher log reductions for E. coli than the specific concentration of ReNu Multiplus alone.
Comparing the combination with 405 nm alone, 40%, 30% and 20% show better or equal results.
The overall results for the combination of ReNu Multiplus and 405 nm investigated in the growth
analysis are lowest for E. coli compared to P. fluorescens and S. carnosus. Nevertheless, with only 40% of
ReNu Multiplus, 3.05 log can be achieved when combined with 405 nm at 10 mW/cm2 for 4 h.
For S. carnosus (Figure 6d) ReNu Multiplus as a single approach achieves better results than for
P. fluorescens but less inactivation than for E. coli. The impact of light, however, was comparable with
the E. coli results, however, for 10 mW/cm2 even 4.11 log was reached. Similarly, in the combination
approach, it appears that the difference between the irradiation intensity of 10 mW/cm2 and the other
intensities is clearly more pronounced, as it is in experiments with P. fluorescens or E. coli. At the
same time, it has to be noted that error bars are comparably large here. There is no indication of
synergistic behaviour that exceeds the sum of single approaches. Apart from 5% for 1 and 2 h,
the combination clearly exhibits a stronger effect than ReNu Multiplus alone. Compared with light
alone, however, there is only a slight increase of 0.86 log at 20 mW/cm2 /2 h with 40% in the combination,
while most results are similar to light irradiation alone and sometimes even reach fewer log reductions.
The combination with ReNu Multiplus does not seem to lead to an increased outcome compared to
an irradiation in PBS for S. carnosus tested with growth delay, but the results of either light approach
achieve better results than ReNu Multiplus alone at the concentrations examined.
Altogether, the growth analysis seems to indicate that lower irradiation intensities at longer
exposure times are more effective in the combination approach than higher irradiation intensities
at shorter durations. For ReNu Multiplus as a single method, Pseudomonas sp. seem to be the
microorganism most difficult to inactivate, which matches literature data [53] and was the reason for
choosing it as principal strain in this study. The addition of irradiation increases the effectiveness of
ReNu Multiplus. At a 40% concentration at least 3 log reduction could be achieved for all bacterial
species tested with the combination approach.
4. Discussion
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 O2 combined with blue light of 470 nm has previously been reported in S. aureus [44]. 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 [31,40,63–65], 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 (Figure 3) 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 (Figure 6d) 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 and 106 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 5a) when testing ReNu Multiplus as
a single method. This could lead to severe clinical problems as total viable bacterial counts between 106
and 108 /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 [19–26], 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.
5. Conclusions
The combination of contact lens disinfection solutions with the application of visible light
irradiation could provide the same antimicrobial results as commercially available disinfection systems
but with much less toxicity. While only some of the combinations with ReNu Multiplus investigated
in this study were close to the disinfection effect of the pure commercial disinfection solution,
the collectivity of results suggests that with modest increase in concentration or exposure time the
same impact as provided by commercial ReNu Multiplus formulation may be reached. Combination
with light was especially effective against pseudomonas, against which the effectiveness of ReNu
Multiplus alone was problematic. For the hydrogen peroxide solution examined, the combination
effect with visible light was so strong that even 5% of AOSept was sufficient to reach the same result
as the current 100% formulation. It was also shown that an additional disinfection technique might
be advantageous, especially because light inactivation, as well as a combination approach, does not
show limited efficacy at high concentrations of bacteria, which is a problem for ReNu Multiplus’s
effectiveness. Furthermore, light cannot exceed expiration date or lose potency unnoticed, so that its
additional application can cover the role of a double protection. Nevertheless, material properties
of the contact lens after irradiation have to be tested before application of this promising method
is possible.
6. Patents
K. Hoenes, and M. Hessling have filed a German patent application (DE 10 2016 009 175 A1).
Author Contributions: Conceptualization, K.H.; Investigation, K.H.; Methodology, K.H.; Project administration,
M.H. and B.S.; Resources, M.H.; Supervision, M.H. and B.S.; Validation, K.H.; Visualization, K.H.; Writing—original
draft, K.H.; Writing—review & editing, M.H. and B.S. All authors have read and agreed to the published version
of the manuscript.
Funding: This research received no external funding.
Conflicts of Interest: K.H. and M.H. have filed a German patent application (DE 10 2016 009 175 A1).
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© 2020 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access
article distributed under the terms and conditions of the Creative Commons Attribution
(CC BY) license (http://creativecommons.org/licenses/by/4.0/).

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International Journal of

2. Materials and Methods

2.1. Bacterial Strains and Contact Lens Disinfection Solutions

2.2. Irradiation Setup

2.3. Disk Diffusion Assay

2.4. Determination of Bacterial Reduction with Nutrient Pads

2.5. Determination of Bacterial Reduction with Agar Plates

CI = a/A + b/B, (1)

2.6. Determination of Bacterial Reduction via Regrowth Behavior

3.1. Disk Diffusion Assay

3.2. Determination of Bacterial Reduction with Nutrient Pads

3.3. Determination of Bacterial Reduction with Agar Plates

3.4. Loewe Additivity

(b) and 40 mW/cm

Nevertheless, such a combination approach could be employed in practice regarding overall

3.5. Effectiveness Dependency of Multipurpose Solution ReNu Multiplus on Bacterial Concentration

Here theFor nutrient pad value with 1.42 log (2 h/20

plates and a 2.86 log

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