Efficacy of a Topical Wound Agent Methanesulfonic Acid and Dimethylsulfoxide on In Vitro Biofilms

A topical desiccating wound agent containing methanesulfonic acid, dimethylsulfoxide and amorphous silica was evaluated in three in vitro models for its efficacy against biofilms produced by Pseudomonas aeruginosa (ATCC-15442) and Staphylococcus aureus (ATCC-6538). The in vitro biofilm models used were; the MBEC Assay®, Centre for Disease Control (CDC) Biofilm Reactor® and a Semi-solid biofilm model. A 30-s exposure of a topical wound desiccating agent was used in each model. A complete eradication of viable cells was demonstrated in all models for both strains (p < 0.0001). Imaging with scanning electron microscopy (SEM) was performed where possible. All three models demonstrated complete eradication of viable cells with a 30 s application of a topical wound desiccating agent.


Introduction
Chronic non-healing wounds of the lower extremity represent a global burden to the healthcare system and are a significant contributor to reduced quality of life and morbidity in affected persons. Chronic wounds have typically been classified based on their underlying aetiology with the most commonly reported in the literature being diabetic foot ulcers, venous leg ulcers, Pressure Injuries and non-healing surgical wounds [1]. Despite the underlying pathophysiology differing between these types of chronic wounds they share some common features; most notably prolonged or excessive inflammation [2], persistent infections [3,4], and the inability of dermal and/or epidermal cells to respond to reparative stimuli [5].
Over the last decade there has been significant focus on the role of biofilms as causes of chronic infections and in contributing to delayed wound healing. Biofilms have collectively been described as "aggregates of microorganisms which may be embedded in a protective matrix, may attach to host tissue or in-dwelling medical devices or exist as aggregates in fluids adjacent to those surfaces. In contrast to planktonic microorganisms, biofilm associated microorganisms demonstrate aggregation, reduced growth rates and altered gene expression" [6]. These changes may help to explain behavioural changes and why biofilms can exhibit enhanced tolerance to antimicrobials and the host immune response. In chronic non-healing wounds, evidence suggests that biofilms may be present in up to 80% of cases [7]. Once pathogenic biofilms become established in host tissue, they can drive chronic and persistent infections, which may delay ulcer healing [8].
A significant shift in clinical paradigms has been proposed for the treatment and management of wounds with chronic biofilm infections [9,10]. Physical removal and/or disruption of biofilms by surgical or conservative sharp debridement form the pillar of treatment. However, the spatial distribution of microbial aggregates in tissue [11][12][13] present challenges in ensuring removal, therefore augmenting debridement through the use of topical agents (antimicrobial, non-antimicrobial) has been advocated. A limitation with using topical agents is the limited robust data on their efficacy in vivo. A recent systematic review identified that 90% of all topical wound agents tested for efficacy against biofilm were conducted in vitro, using 16 different models [14]. Although there are acknowledged limitations of in vitro models, such as the absence of a model which truly mimics a human wound, in vitro analysis forms an integral component in screening for potentially beneficial agents.
In this study, we report on a novel topical wound desiccating agent containing metanesulfonic acid (MSA)-dimethylsulfoxide (DMSO) and amorphous silica (SiO 2 ) and its efficacy against in vitro biofilms. The use of this gel has been reported in a small case study of lower limb ulcers. The manufacturers of the gel solution report that the use of a desiccating acid as a wound treatment was investigated based on its antibacterial and denaturation effects, and efficacy in other medical areas [15]. DMSO has historically been used in the treatment of ischemic ulcers and scleroderma [16]. We undertook a systematic approach to testing this agent using three separate in vitro biofilm models. The MBEC Assay ® is a standardised model for allowing rapid screening of agents for antimicrobial inhibition and efficacy. The CDC Biofilm Reactor ® is a standardised in vitro model capable of reproducible biofilms on individual coupons under flow, and the semi-solid model has been designed to simulate a wound environment through the growth of bacteria encapsulated in agar [17].

CDC Biofilm Reactor ®
Polycarbonate discs with P. aeruginosa and S. aureus biofilms were exposed to a topical MSA-DMSO gel solution for 30 s. When compared against untreated controls, coupons exposed to topical MSA-DMSO gel solution demonstrated complete eradication of viable cells for both P. aeruginosa and S. aureus ( Figure 3). This equated to a reduction of greater than 8.68 ± 0.1 Log10 (p < 0.0001, 95% CI 8.5 to 8.3) for P. aeruginosa and 6.7 ± 0.1 Log10 (p < 0.0001, 95% CI 6.5 to 6.8) for S. aureus. SEM images of P. aeruginosa and S. aureusuntreated coupons show dense aggregates surrounded by extracellular matrix (observed in P. aeruginosa only) ( Figure 4). No microbial aggregates are seen in treated coupons of P. aeruginosa biofilm and small aggregates of <10 bacterial cells are seen in S. aureus-treated coupons.

Discussion
In vitro biofilms are commonly (but not always) embedded in a protective, hydrated, self-produced exopolymeric matrix that comprises lipids, proteins, eDNA and exopolysaccharides [18,19]. Topical agents that induce desiccation through hygroscopy may be beneficial as therapies in managing chronic biofilm infections in humans [9] given their ability to effectively dehydrate components of biofilm architecture and the microbial cells themselves. The agent tested in this series of in vitro models is a novel formulation containing methanesulfonic acid, dimethylsulfoxide and amorphous silica in a gel formulation. The principal action of MSA is its hygroscopicity, a general term used to describe materials that readily take up water from their surroundings. This action, when in proximity to planktonic microbial cells, leads to desiccation of the cell membrane lipid bilayer. This is one of the primary cellular components affected by variations in hydration level, causing changes in lipid packing that may have damaging effects on cell viability [20].
There is some limited preliminary research suggesting that desiccation agents may be an option to consider in managing chronic wounds with biofilm driven infections [21]. Traditional wound care already uses various weak acidic formulations as treatments [22,23] as well as honey which possesses hygroscopic abilities resulting in dehydration. [21,24,25]. These agents exist at low concentrations and are generally well tolerated with low toxicity. There is also evidence to suggest that weak acids could be developed into treatments for biofilm infections, with preliminary studies suggesting that these acids may eradicate biofilm through penetration of the matrix and cell membrane, which may be a similar mechanism to MSA [26].
MSA is classified as a strong acid and through its potent hygroscopic action demonstrated an ability to eradicate all viable cells of both S. aureus and P. aeruginosa in all three models. Although it has demonstrated effects against bacterial cells, there are likely also collateral affects to host cells. This has not been investigated as part of this work, and safety studies to determine toxicity against host cells are required moving forward. In this study, we have made efforts to ensure that the results are meaningful by testing the product for the duration it should be used for [27], and testing in a range of models from a simple screening tool to a model designed to try and emulate a wound environment.
This study has several limitations to acknowledge. In vitro studies are not reflective of the wound environment [14] and can at best provide indications to progress with further research. The models were performed in triplicate and run independently, but were not performed in multiple laboratories by independent researchers [28]. These models were run as single species, which is not indicative of the polymicrobial nature of chronic wounds [29,30] and clinical isolates were not examined. Safety testing for toxicity to host cells is required.
Although these preliminary investigations have produced promising results, significant further investigation and research is required. The in vitro results indicate that the progression of testing and development may be warranted, however extensive safety testing is required to ensure the response on host cells and surrounding tissue is fully understood and the risks identified, particularly with the known cytotoxic actions of MSA [31,32].

Bacteria
ATCC strains of P. aeruginosa (ATCC-15442) (Manassas, VA, USA) and S. aureus (ATCC-6538) (Manassas, VA, USA) were used independently in all three models. All broth and agar plates were manufactured in the laboratory from raw materials purchased from Sigma-Aldrich (St. Louis, MI, USA). For the standardised MBEC Assay ® and CDC biofilm reactor (BioSurface Technologies Corporation, Bozeman, MT, USA) the strains were streaked on Tryptone Soy Agar (TSA) plates for 24 h at 37 • C and isolated colonies were grown overnight in Tryptone Soy Broth (TSB) at 35 • C and 110 ± 10 rpm. For the semi-solid biofilm model, the strains were streaked on Luria-Bertani (LB) Agar plates was made as described previously and incubated for 24 h at 37 • C. A single colony was then used to inoculate in LB broth which was then incubated overnight at 37 • C and 250 rpm.

Test Agent
Debrichem ® (DebX Medical, Amsterdam, The Netherlands) is a topical gel solution with the principal agent being methanesulfonic acid (MSA). MSA acidity is buffered by the use of dimethylsulfoxide (DMSO) and amorphous silica (SiO 2 ). The final concentration is 83% MSA and 13.8% DMSO. The gel contains a red dye (1-(p-Nitrophenylazo)-2-naphthol, 1-(4-Nitrophenylazo)-2-naphthol, also known as Para Red) in order to better define the product application on the wound and the surrounding skin. Debrichem is applied topically to the wound bed and the recommended time of application by the manufacturer is 60 s, after which the area is flushed with saline, to cease the action of the acid. For these in vitro experiments, an exposure time of 30 s was selected to 1; ensure application that was less than the maximum application time and 2; to determine if a shorter period of exposure then the currently recommended time provide efficacious. All products used in this study were prepared by DebX Medical, Netherlands.

In Vitro Biofilm Models
All testing was performed in triplicate on two independent occasions.

MBEC Assay ®
MBEC Assay ® testing was completed according to the ASTM International Standard Test Method for Testing Disinfectant Efficacy against P. aeruginosa Biofilm using the MBEC Assay ® [33]. Briefly, an overnight suspension of each test strain was diluted to 2 × 10 7 CFU/mL and 1 × 10 7 CFU/mL for P. aeruginosa and S. aureus, respectively. A total of 150 µL of each inoculum was added to a 96-well plate (MBEC Assay ® , Innovotech, Edmonton, AB, Canada), and the 96-peg lid was then inserted onto the plate and incubated for 24 h at 35 ± 2 • C with continuous shaking (110 ± 10 rpm).
Biofilm pegs were washed in sterile saline (NaCl 0.9%) for 10 s, then transferred to a new 96-well plate containing 200 µL of either MSA-DMSO solution or sterile saline (control) for a contact-exposure time of 30 s. Following treatment, the pegs were rinsed twice in saline for 10 s each. At this point, pegs for imaging were removed and fixed in a 4% Glutaraldehyde solution overnight at 4 • C. To facilitate the disaggregation of the biofilm, the recovery plate was sonicated in an ultrasonic water bath (SoniClean™ 160TD, Stepney Australia) on high for 30 min. The recovery solution was then serially diluted, and spot plated in triplicate onto TSA plates and incubated at 35 ± 2 • C for 18-24 h for plate counts and quantitative analysis. In total, nine pegs on each occasion were treated with MSA-DMSO solution for quantification analysis (6 pegs) and SEM imaging (3 pegs), with the same number of control pegs treated with sterile saline (NaCl 0.9%).

CDC Biofilm Reactor ® Model
The CDC Biofilm reactor ® (BioSurface Technologies Corporation, Bozeman, MT, USA) was prepared using the ASTM Method E3161-18 'Standard practice for preparing a P. aeruginosa or S. aureus Biofilm using the CDC Biofilm Reactor' [34], and the effect of MSA-DMSO was determined using the ASTM Method E2871-19 'Standard Test Method for Determining Disinfectant Efficacy Against Biofilm Grown in the CDC Biofilm Reactor Using the Single Tube Method'20. Briefly, an overnight culture of P. aeruginosa was diluted to 2 × 10 7 CFU/mL, then 1 mL was added to the CDC reactor containing 500 mL sterile 300 mg/L TSB. The reactor was placed on a magnetic stir plate (Talboys™ 4 × 4 Ceramic Stirrer 230 V, USA) at 125 ± 5 r/min at room temperature for a 24-h batch phase growth. A growth medium (100 mg/L TSB) was then added during the continuously stirred tank reactor (CSTR) phase with a residence time of 30 ± 2 min, using a Digital Pump (Masterflex Digital Drive and Pump Head, Vernon Hills, IL, USA) for a further 24 h. For S. aureus, an overnight culture was diluted to 1.2 × 10 7 CFU/mL before being added to the reactor containing 500 mL of sterile 3 g/L TSB solution. The reactor was placed on the magnetic stir plate at 60 ± 5 r/min and incubated at 36 ± 2 • C for 24 h of batch phase growth. The CSTR phase was then run for an additional 24 h at 36 ± 2 • C using 1 g/L TSB, with a residence time of 30 ± 2 min.
Biofilm coupons of both strains were rinsed in a 50 mL conical centrifuge tube containing 30 mL 0.9% saline. Four coupons were dropped into individual centrifuge tubes containing 4 mL of MSA-DMSO gel solution for 30 s ensuring even coating of the coupons. Control coupons underwent the same treatment process with sterile saline. The coupons stored for imaging were rinsed in 0.9% saline and fixed in 4% Glutaraldehyde solution overnight. Coupons for quantification were then rinsed in a tube containing 10 mL of 0.9% saline and dropped into a new tube containing 36 mL of 0.9% saline. Each tube was then vortexed on the highest setting for 30 ± 5 s at room temperature and then placed in an ultrasonic water bath (SoniClean™, Stepney, South Australia, Australia) at 45 ± 5 kHz for 30 ± 5 s at room temperature. This process was repeated for a total of 3 × rounds of vortexing and 2 × rounds of sonication. For the treated coupons, the entire recovery solution was processed through a Nalgene Reusable Filter Unit with a 0.2 µm filter, with the filter membrane then being placed directly onto a TSA plate. The control coupon tubes were briefly vortexed and diluted from 10 0 to 10 6 and plated in duplicate using the spread plate method on TSA. The plates were then incubated at 35 • C for 48 h and 72 h for the control and treated plates, respectively.

Semi Solid Biofilm Model
The semi-solid model has been previously described by Cone et al. [17]. It simulates a wound environment through the growth of bacteria encapsulated in agar.
Briefly, Wound Simulation Medium (WSM) was prepared for a final concentration of; 5 mL Bolton Broth with 1% agar, mixed with 2.5 mL of both bovine fetal serum (Sigma-Aldrich, St Louis, MI, USA) and defibrinated horse blood (Australian Ethical Biologicals, VIC, Australia). Overnight cultures of each strain were grown in LBB and diluted to a final dilution of 10 −4 in WSM. The semi solid model was assembled by adding WSM to the centre of a gene frame (Thermofisher Scientific, Waltham, MA, USA) adhered to a glass microscope slide (26 mm × 76 mm). The medium was allowed to set before 2 µL of the P. aeruginosa and 4 µL of the S. aureus inoculum were added to the centre of the medium. Additional WSM was added to encapsulate the inoculum and allowed to set. The slides were then placed in a sterile container with moist tissue paper before being sealed with Parafilm and incubated at 37 • C for 24 h.
Following incubation, 150 µL of MSA-DMSO gel solution was pipetted over the middle of the slide for 30 s, before being rinsed off with approximately 3 mL of sterile saline. Control slides were rinsed with sterile saline. Each gel was then aseptically transferred to a 2 mL Eppendorf tube containing 1.5 mL of sterile saline and a 5 mm stainless steel bead. The tubes then underwent bead beating in a TissueLyser II (Qiagen, Hilden, Germany) at 20 Hz for 15 s to break up the agar. Following bead beating, the contents of each tube were transferred to 45 mL of 0.9% saline and centrifuged at maximum speed (6000× g) for 10 min. Following centrifuging, the supernatant was carefully decanted, and the process repeated. All but 2 mL of the supernatant was removed, which was used to resuspend the pellet through vortexing the tube at a low speed. The tubes were then placed in the ultrasonic water bath (SoniClean™, Stepney, South Australia) and degassed for 5 min, followed by sonication on high for 5 min. Each solution was then serially diluted from 10 0 to 10 6 in saline, and spot plated in triplicate on LBA plates. The plates were then incubated at 37 ± 2 • C for 18-24 h and colony counted.

Confirmation of Biofilm Growth through Scanning Electron Microscopy (SEM)
SEMwas performed with the LSM 880 confocal microscope (Carl Zeiss, Oberkochen, Germany) to visualise and confirm the growth of bacterial biofilms on the MBEC Assay ® pegs and CDC biofilm reactor ® polycarbonate discs. Bacterial biofilms were sampled at 5-200 µm with a random field selection. Fixed specimens were dehydrated through serial dilutions of ethanol and hexamethyldisilazane (HDMS) (Polysciences, Inc., Warrington PA, USA). Coupons were rinsed twice in phosphate buffered saline (PBS) then underwent a 50% ethanol and saline wash, 70% ethanol, 95% and two 100% ethanol washes. They were then washed in 2:1 ethanol and HMDS, 1:1, 1:2 ethanol and HMDS and finally two 100% HMDS washes. Coupons were then air dried in the fume hood for at least 48 h. They were then mounted, gold coated for 60 s at a current of 60 mA and examined.

Statistics
Unpaired two-tailed t tests were used to compare individual treatments with the control. Data are given as mean, median, and standard deviation (±). Data were analysed through Statistical Package for Social Sciences Version 25 (IBM Corp., Armonk, NY, USA). Data were Log transformed before statistical analyses were performed.

Conflicts of Interest:
The authors declare no conflict of interest.