Properties and chemistry of the OxiLast™ formulation

OxiLast™ (a product of Bio-fence, Israel) is an innovative dilutable water-based formulation designed to form a thin and transparent film that holds a patented compound (patent number WO2021/245,663 A4). The formulation contains the OxiLast™ additive and a film-forming polymer. The additive is the patented new compound obtained in powder form that, due to its chemical structure (rich-nitrogen core), has a high affinity to attract a negatively-charge molecule such as OCl− (originated from dissolving the Sodium Dichloroisocyanurate, NaDCC in water). In that, OxiLast™ is not a standalone disinfectant but rather an adjuvant to chlorine. It is worth noting that OxiLast™ does not alter the oxidative power or efficient killing properties of active chlorine. This is because the stabilization of chlorine by OxiLast™ is solely accomplished through hydrogen bonds and electrostatic interactions, which delay the release of chlorine, overcoming the limitation of its short lifespan. When added to water and chlorine, no new chemical compounds are formed, and the fundamental chemistry of chlorine remains unaltered.

The film formation is achieved by including a distinctive water-soluble polymer that possesses both aliphatic and polar groups, enabling it to effectively adhere to a wide range of surfaces, such as plastic, metal, wood and resin-coated materials. Consequently, this formulation can be applied to nearly any surface found in dry environments, particularly in patient rooms. The adhesion of the resulting film to each surface is sufficiently robust to provide protection, yet it remains easily removable with water and soap. OxiLast™ is metal-free and therefore, no metal salts are released. The additive and polymer are classified as non-hazardous according to the Globally Harmonized System of Classification and Labelling of Chemicals (GHS). Risk assessment concerning dermal, oral and inhalational exposure to the chlorine available while using the product concluded that no excess risk is foreseen to professional users applying the product and patients and general public present.

Study plan

The overall efficacy of the OxiLast™ formulation was evaluated by inoculating coated and non-coated surfaces with standardized inocula (defined amounts of cells) of selected model Gram-positive and Gram-negative bacterial pathogens relevant to HAIs under different conditions and contact times (CT) and performing live counts on solid media. Initially, a laboratory-based experiment was devised and carried out to test the general antimicrobial efficacy of the formulation, for which most of the procedures were based on the ISO 22196:2011: ‘Measurement of antibacterial activity on plastics and other non-porous surfaces’ [12] with certain modifications, where the formulation was tested as a function of CT with defined target bacterial inocula. Three additional testing conditions were evaluated in the laboratory setting (i.e. challenges). These included inoculation with organic load (simulating contaminated body fluids), mechanical abrasion of the coating (simulating abrasion during everyday activities), and successive loadings (simulating repeated hospital exposures); the first two were based on the BSI-PAS-2424 2014 guidelines [17], with reported modifications. Subsequently, a modeled environmental experiment was conducted in a real unoccupied hospital room to test the antimicrobial effectiveness when applied onto surfaces considered hot spots for pathogen circulation in hospitals while comparing with the hospital routine disinfection procedure and the presence of organic load simulants. All modifications to standard methods are listed in Tables 1 and 2. A schematic view of the study plan is provided in the Supplementary Material (Figure S1).

Table 1 Description of modifications applied to the original ISO22196-2011 protocolTable 2 Description of modifications applied to the original BSI-PAS-2424-2014 protocolBacterial strains used

The following ATCC reference strains were used and tested, representing both Gram-positive and Gram-negative pathogens of medical importance: methicillin-resistant Staphylococcus aureus (MRSA) ATCC 33591, vancomycin-resistant Enterococcus faecalis (VRE) ATCC 51299, carbapenem-resistant Klebsiella pneumoniae ATCC BAA-1705, Pseudomonas aeruginosa ATCC 9027, Acinetobacter baumannii ATCC 19606. The MRSA, VRE and Klebsiella strains represent multi-resistant isolates while P. aeruginosa and A. baumannii strains used were relatively susceptible and not carbapenem-resistant. For testing steps in which only two species were included, a Gram-positive and a Gram-negative species were always included.

Additional carbapenem-resistant K. pneumoniae (blaNDM-producing) and MRSA isolates recovered at the Clinical Micrbiology Laboratory of the Hadassah Hebrew University Medical Center from routine cultures were used in the modeled environmental experiment; these strains were identified by MALDI-TOF MS (VITEK MS, bioMeriuex, Marcy l’Etoile, France) and their susceptibility was confirmed using the VITEK2 (bioMerieux) and in house PCR testing per institutional microbiology protocols.

Formulation preparation and application

The OxiLast™ ready-to-use (RTU) formulation was prepared by dissolving 46.7 g of the concentrated formulation into 286.3 g of double distilled water and subsequently adding one tablet of sodium dichloroisocyanurate (NaDCC) (Klorkleen®, effervescent tablet; Medentech, Ireland), for a final concentration of 3000 ppm (i.e. 3 g/l) of active chlorine. For the laboratory phase experiments, the formulation was applied onto PVC slides with two cycles of spraying three times with a nebulizer bottle and subsequent dispersion of the liquid layer with a clean cloth moisturized with the same formulation to obtain a visibly uniform coating. Dry-coated slides were then stored at room temperature until use and transferred to sterile 12 × 12 × 1.7 cm3 petri dishes (Greiner Bio One, Kremsmünster, Austria) prior to use. For the modeled environmental experiment, a dry microfiber wipe (29 × 29 cm2) was fully immersed and moisturized within 60 g of the formulation and applied slowly in an S-shape motion, covering an area of 0.4 m2. In the same modeled environmental experiment, the routine KlorKleen® suspension was made by dissolving 1 tabled of the product to 1 L of water and used as above. Finally, the coated or disinfected surfaces described above were left to dry and remained untouched for a minimum one hour or until use (Table 3).

Bacterial inocula preparation

Bacterial suspensions for inocula were freshly prepared before use according to ISO 22196 with minor modifications by resuspending a loopful of 16–24 h old bacterial culture from tryptic-soy agar (TSA) (Hylabs, Rehovot, Israel) in a small volume of sterile diluted nutrient broth (NB) (Merck, Darmstadt, Germany) (made with a 1:500 dilution of NB in sterile water). Subsequently, more of such diluted NB was added to achieve a McFarland 0.5 density measured using a Densicheck Plus reader (BioMerieux, Marcy-l’Étoile, France), equivalent to a ~ 108 colony forming units (CFU) per ml. The initial 0.5 McFarland stock was further diluted (using the diluted NB solution) to meet the final target inocula described below. Bacterial stock suspensions were confirmed by live counts, by plating aliquots of appropriate serial dilutions made in 0.85% (w/v) sterile NaCl solution on TSA plates and incubating for 24 h at 37 °C.

Laboratory phase experiment

For the laboratory phase experiments, clean 10 × 10 cm2 non-smooth PVC slides and 8 × 8 cm2 PET cover films were disinfected by immersion in 70% (v/v) ethanol overnight and air-dried before use. Four experimental tests were conducted in the laboratory phase experiment, as described below. Different arrays of coating ages, CTs, or CFU loads were used for each test, as summarized in Table 3. Coating ages spanned from freshly prepared (i.e. 1 h before testing) to one, two, three, five, and seven days-old coatings. The CTs of the tested inocula applied onto OxiLast-coated surfaces varied between 5, 15, or 30 min. The chosen testing inocula were in a range that is equivalent to 0.6 log10 range in line with the ISO 22,196, being 1-4 × 107 and 1-4 × 106 CFU for most cases (see below). For each of the below-described experiments, the sterility of the tested PVC slides and films from every batch, as well as the procedure itself, were ensured by testing a negative control (i.e. no bacteria used in the procedure).

(i)

General antimicrobial efficacy testing

The basic antimicrobial activity was evaluated based on ISO 22196 and involved all five ATCC reference bacterial strains. For each defined CT, 1-4 × 107 CFU were inoculated in triplicate onto OxiLast™ coated and single control (non-coated) PVC slides by transferring 400 µl of fresh 0.5 McFarland suspension and covering them with the PET cover slides for uniform distribution over an 8 × 8 cm2 area. Where a 1-4 × 106 CFU inoculum was used, 400 µl from a 1:10 dilution of the 0.5 McFarland suspension was used.

(ii)

Organic load challenge

Based on BSI-PAS2424, a suspension of 1-4 × 107 CFU in a 3 mg/ml solution was prepared by mixing equal volumes of a sterile 6 mg/l bovine serum albumin (BSA) fraction V (Merck, Darmstadt, Germany) solution and a 1:5 dilution of the 0.5 McFarland suspension. For each defined CT, 1-4 × 106 CFU were inoculated by transferring 400 µl of such suspension in triplicate onto OxiLast™ coated and single control (non-coated) PVC slides and covering them with the PET cover slides as above. Tested species included the above described reference strains MRSA ATCC 33591 and P. aeruginosa ATCC 9027 as representative species of biofilm-producing Gram-positive and Gram-negative bacteria.

(iii)

Successive loading test

Two different CFU load regimes were used to inoculate the same surface at a defined CT of 15 min each. Triplicate OxiLast-coated test PVC slides and one control were initially inoculated with 360 µl of a 1:10 dilution of the 0.5 McFarland suspension (equivalent to 1-4 × 106 CFU); subsequently, 40 µl of the same suspension (equivalent to 1-4 × 105 CFU) were used to re-inoculate the same surface. This approach would ensure a compromise between a tolerable deviation of 10% from the established 1-4 × 106 CFU inoculation, and the final volume tested on a single PVC slide. The reference strains MRSA ATCC 33591 and P. aeruginosa ATCC 9027 were tested in this experiment, as above.

(iv)

Abrasion test

OxiLast-coated PVC slides were challenged with an adapted mechanical abrasion test based on the BSI-PAS2424. An apparatus was assembled with a flat base of 10 × 10 cm topped with a weight such that the neat apparatus’ weight was 2,185 g and the exerted weight per cm2 at the base was 21.85 g. A polypropylene wipe was wrapped at the apparatus’ base and OxiLast-coated and control slides were subjected to three cycles of a forward and backward motion application of the polypropylene wipe with neat pressure applied only by the apparatus’ weight onto the whole area of the coated surfaces, as per BSI-PAS2424. Finally, triplicate OxiLast-coated abrased slides were tested along with single non-coated abrased controls by inoculation with a 1-4 × 106 CFU per surface and processed as per the general testing and with 30 min CT only. The reference strains MRSA ATCC 33591 and P. aeruginosa ATCC 9027 were tested in this experiment, as above.

Bacterial cell recovery and enumeration in the laboratory phase

At the end of each defined CT, the PET cover films were lifted, and the inocula were washed with 5 ml of sterile 1 mg/ml sodium thiosulfate solution (Merck, Darmstadt, Germany) to neutralize and deactivate the active chlorine [18]. Inoculated surfaces and PET covers were then thoroughly scratched with 20 backward and forward motions in four directions with a sterile cell spreader to resuspend cells. Aliquots (200 µl) of resuspended cells and related serial 10-fold dilutions in 0.85% NaCl were then plated in duplicates on TSA plates and incubated for 24 h at 37 °C and colonies enumerated; plates were then reincubated for 24 additional hours, and colonies recounted for variations check. A toxicity test was conducted as per ISO 22196 to exclude adverse effects of the thiosulfate solution on the tested bacteria strains (data not shown).

Modeled environmental experiment

In the modeled environmental experiment, a non-occupied standard hospital room made available for the experiment at the Hadassah Medical Center was used. The setting included two patient beds (with the semi-smooth PVC bedrail surfaces being tested), two cabinets with food trays (also PVC and semi-smooth), and a long windowsill (smooth resin-coated surface), representing three types of ‘high touch’ surfaces. The surfaces were inoculated with defined amounts of CFU of different strains, simulating accidental contamination of cleaned surfaces treated with the routine hospital disinfectant as the comparator, or OxiLast™, and further tested with or without an organic matrix simulant.

Tested surfaces were initially cleaned of any dust and dirt by employing a commonly used detergent-based product, and then chlorine-based disinfectant used at the hospital (Klorkleen®, 1000 ppm active chlorine) as per the hospital’s standard procedures and finally wiped with 70% (v/v) ethanol to ensure maximized disinfection, prior to use. Surfaces were then tested as such (controls), or after disinfection with the standard chlorine-based disinfectant or coating with OxiLast™. The tested surfaces in the modeled environmental experiment included selected areas of 4 × 5 cm2. Fresh bacterial suspensions of 1-4 × 107 CFU/ml in 5 ml volume were prepared using 0.5 ml of a McFarland 0.5 suspension prepared as above, 3.5 ml of sterile NaCl 0.85%, and 1 ml tryptic-soy broth (TSB) (Merck, Darmstadt, Germany). Selected areas from all tested conditions (Table 4) were inoculated with 1-4 × 106 CFU applied as 20 droplets of 5 µl from the above suspension, as previously described [19], and let dry at room temperature for precisely 1 h. Tested species included the reference strains MRSA ATCC 33591 and K. pneumoniae ATCC BAA-1705, for all tests, and the MRSA and K. pneumoniae clinical isolates in indicated tests (see below); the K. pneumoniae strains were chosen because carbapenemase-producing Enterobacterales (CPE), including K. pneumoniae species, are endemic in globally as well as in Israeli hospitals [9] and preventing the spread of these species are of particular interest for the infection and prevention control units of hospitals. For each tested condition described below, a comparison was made between two test surfaces and one negative control (no coating or treatment).

Four experimental conditions were tested in this modeled environmental experiment, as described below and detailed in Table 4.

(i)

General antimicrobial efficacy testing

The antimicrobial efficacy of the formulation was tested on bedrails, cabinets, and windowsill for both ATCC reference strains on OxiLast™ coatings up to seven days old.

(ii)

Comparison with hospital routine disinfectant

The possible residual disinfection capacity of the routine hospital disinfectant solution was evaluated on bedrails, cabinets, and windowsills for both ATCC reference strains on surfaces freshly disinfected, as per hospital routine, or after one day only. No residual effect was expected.

(iii)

Extended testing on clinical isolates

To reinforce the results from (i) the antimicrobial efficacy of the OxiLast™ coating was tested with two routine hospital isolates of MRSA and K. pneumoniae, which were tested on fresh (day 0), and one and three-day-old coatings; tested surfaces included bedrails and cabinets.

(iv)

Organic compounds

The OxiLast™ formulation was further tested in the presence of an organic compound mixture to assess any potential interference of organic material with the chlorine-based antimicrobial activity of the formulation. In this case, the bacterial suspension as above was spiked with additional artificial test soil (ATS) (Healthmark Industries Company, Inc., Frasier MI) to a final concentration of 3 mg/ml to simulate organic content from body fluids (e.g. proteins, hemoglobin, carbohydrates, mucin, cellulose and lipids, not including defibrinated blood) as previously described [20]. The tested coating ages included fresh (day 0), and one and three days old coatings; tested surfaces included bed rails and cabinets.

Viable bacterial cell recovery and enumeration in the modeled environmental experiment

Dried inoculated areas were processed as previously described [19] by using a combination of a wet and a dry flocked nylon swabs to retrieve bacterial cells; swabs were then stored in 2.5 ml of commercial bacterial storing Swab Rinse Kit solution (SRK, Copan Diagnostic, Murrieta, USA) and kept at 4 °C for 2–3 h until processed. Sample tubes with swabs were thoroughly vortexed for 15 s and direct and serial 10-fold dilutions 0.85% NaCl aliquots (100 µl) were plated. For each species and each dilution, plating onto two agar plates was done in parallel: a rich non-selective medium (TSA) for maximizing bacterial growth from the OxiLast-coated surfaces and a selective or differential medium to assist with colony counts in case of substantial contamination. These included mannitol salt agar for MRSA, and CHROMagar orientation for K. pneumoniae (all from Hylabs, Rehovot, Israel). Plates were incubated as for the laboratory phase experiment and colonies were enumerated after 24 and 48 h. Growth was evaluated for comparison and differentiation of sporadic off-target colonies on the cultures. Whenever a substantial discrepancy arose (i.e. >20% CFU counts between the two technical replicates), this was always to the detriment of the TSA plate (non-selective) and the count on that plate was discarded.

Viable bacterial cell estimation, recovery efficiency, and antimicrobial efficacy

The CFU on all inoculated surfaces at each endpoint of CT for both tests and controls were estimated by means of the liquid bacterial resuspensions in the neutralizer solution after testing, according to the formulas below:

$$Nt=\left(\frac\right)\times Vf$$

Where ‘Nt’ is the total number of cells on the tested surface, ‘Np’ is the average colony number on two replicate plates from a given dilution, ‘df’ is the dilution factor, ‘Vp’ is the volume plated in ml, and ‘Vf’ is the final suspension volume in ml (5 ml for the laboratory phase experiment and 2.5 ml for the modeled environmental experiment). To maximize stringency during the laboratory phase testing, in compliance with ISO 22196, whenever no colonies were observed from direct plating of the neutralizer solution, the reported number per ml of Np was 5, corresponding to the inferred limit of quantification (LOQ) of 1 CFU per volume plated of the resuspension in the neutralizer solution (0.2 ml).

The recovery efficiency of cells with the two described methods was calibrated on the controls as the ratio between: (i) the Nt recovered from the control surfaces and (ii) the CFU number per inoculum on the same controls; the latter was calculated (based on volume used) from the actual cell count per ml of the McFarland 0.5 suspensions used in each experiment to generate the inocula.

The antimicrobial efficacy was estimated in each tested condition as:

$$R=\frac_^\left(Log Nt Control-Log Nt Test n\right)}$$

Where ‘R’ is the antimicrobial efficacy expressed as the average log10 reduction of the inocula from n test surfaces with respect their respective single controls.

A summary of all CFU counts retrieved from all controls in all the described experiments is provided in Supplementary Table S1.

Endpoints

The antimicrobial efficacy of the OxiLast™ formulation was considered adequate when a ≥ 4 log10 reduction was evident. The minimum expected activity was set at ≥ 3 log10 reduction.

Table 3 Conditions used for the laboratory phase experiment with OxiLast™ coatingTable 4 Conditions used in the modeled environmental experiment testing