Preparation of hypochlorous acid and microorganisms
Hypochlorous acid was generated by electrolysis of of 3% hydrochloric acid using an Apia Mini generator (Hokuty Co. Ltd., Kanagawa, Japan). Additional electrolysis cycles were applied to obtain higher concentration HOCl solutions. HOCl solutions with 45-60 ppm total chlorine content were used in all assays unless otherwise specified. The total chlorine content was determined using the UHR chlorine photometer (Hanna Instruments, Inc., Woonsocket, RI, USA) HI96734 and Chlorine Ultra High Range Reagent A and Reagent B (Hanna Instruments). Stored-HOCl was prepared by placing fresh HOCl solution in a plastic bottle and allowing it to stand at room temperature for 1 week. The average total chlorine concentration of stored-HOCl was 26 ppm. HOCl solution passed through DUWL in use (DUWL-HOCl) was obtained at the University of Michigan Graduate Endodontics Clinic as follows. The operating dental unit water was replaced with HOCl solution in dental chairs at the Graduate Endodontics Clinic. After flushing the residual regular water out using a sterilized 3-in-1 syringe, HOCl sample dispensed from DUWL was collected and immediately used. Note that the dental chairs are regularly treated with Citrisil (daily) and Citrisil All-In-One (once a month; both Sterisil, Inc., Palmer Lake, CO, USA). Fusobacterium nucleatum (Fn; American Type Culture Collection (ATCC) 25586, ATCC, Manassas, VA, USA), Prevotella intermedia (Pi; ATCC 25611,), Streptococcus intermedius (Si; ATCC 27335), Parvimonas micra (Pm; ATCC 33270) were grown on Tryptone soya agar (TSA) plate with sheep blood (R01202, 5% Sheep Blood in Tryptic soy agar, Remel, Lenexa, KS, USA) in an anerobic chamber (85% N2, 10% H2, and 5% CO2). Oxyrase (Oxyrase Inc., Mansfield, OH, USA) was used following the manufacturer's instructions in order to reduce the effect of atmospheric oxygen in the suspension preparation. The bacterial suspension was diluted by Oxyrase-PBS solution and adjusted to an optical density (OD) of 1.0 (1.0 x 109 cells/ml) in all bactericidal assays. OD was measured with a spectrophotometer (Thermo Fisher Scientific, Genesys 20, Rochester, NY, USA) with 600 nm wavelength. The bacterial suspensions were freshly prepared on a per-experiment basis.
Evaluation of HOCl efficacy on oral pathogens in vitro
The impact of usage, storage, residual chlorine content, organic matter, and supply route on the microbicidal activity of HOCl solution was evaluated as a change in the "minimum inhibitory volume ratio," which is how much HOCl solution volume is required to completely inactivate a suspension of pathogens. HOCl solution and bacterial suspensions (OD = 1) were mixed at various volume ratios (Range: 0.25 : 1 to 16 : 1 [HOCl: Bacteria by volume]) under ambient air. The mixtures were vortexed, and then chlorine was neutralized with 10 µl of 0.5% Sodium thiosulphate. The entire process took less than 30 seconds to avoid the influence of ambient oxygen as much as possible. Prepared samples (200 µl of each) were anaerobically cultured and triplicate samples were prepared per condition. Oxyrase-treated PBS (no HOCl solution) served as control and applied to bacterial suspension in the same protocol.
In the experiment with stored-HOCl, Pi was used as the representative strain. To evaluate the effect of presence of saliva, commercially available human saliva (991-05-P, Lee Biosolutions, Maryland heights, MO) was employed. Given possible bacteria in the saliva, the saliva was subjected to three freeze-thaw cycles prior to the experiment. After the freeze-thaw cycles, eradication of bacteria was confirmed by anaerobic culture of the saliva on TSA plate with sheep blood for 48 hours. The saliva was mixed with Oxyrase, and Oxyrase-saliva was used for microbial suspensions, instead of Oxyrase-PBS. The saliva itself used in this study was preliminarily confirmed to have no effect on bacterial growth.
Preparation of host cells and virus stock
Mouse hepatitis virus (ATCC VR-764 (MHV-59)) was employed as a SARS-CoV-2 surrogate and propagated in NCTC clone 1469 cells (ATCC CCL-9.1) following ATCC’s instruction. In brief, NCTC clone 1469 cells were cultured in NTCT135 medium (NC1804122, LIFE TECHNOLOGIES, USA) containing 10 % horse serum (H1270 , Millipore Sigma, USA) at 37 °C with 5% CO2 and 100 % relative humidity. Cultured NCTC cells were seed in 96-well at 3.0 x 104 cells/ well as host cells. MHV-59 virus was diluted 1:100 in serum-free NCTC135 medium and inoculated into the host cells. After 1 hour incubation, virus suspension was replaced with NTCT135 medium containing 2% horse serum . The inoculated cells were cultured for 7 days, and the culture supernatants containing the virus were harvested, quickly frozen, and stored as viral stock in liquid nitrogen until use.
Viral titer was evaluated by endpoint dilution assay using NCTC clone 1469 cells. NTCT clone 1469 cells were seeded in 96-well plates with NCTC135 medium at 3.0 x 104 cells/well containing 10% horse serum and incubated for 24 hours at 37°C. The virus solution was diluted with NTCT 135 medium in serial 10-fold dilution and used for inoculation. Cytopathogenic effect (CPE) scores were assessed by inverted phase contrast microscopy on the second day after inoculation. The virus titer (TCID50) was calculated using method of Reed-Muench . Virus stock solutions with a titer of 6.8 x 105 (TCID 50/mL, in a single lot) were used in all virucidal assays.
Evaluation of virucidal effect of HOCl solution
The virus stock was thawed in a 37°C water bath. To determine the minimum inhibitory volume ratio for MHV-59 virus, the virus stock was mixed with HOCl solution at the various volume ratios for 30 seconds at room temperature. Then, effective chloride was neutralized with sodium thiosulfate as described above. The mixtures were diluted 1:100 in NTCT135 medium and inoculated to the host cells. The minimum volume ratio at which CPE was not observed was determined on day 2 after infection as described above. For the effect of saliva, saliva was pretreated with a protease inhibitor (Halt protease inhibitor #1862209, Thermo scientific) for 1 hour at room temperature to reduce the negative effect of the protease on host cells. The volume ratio of saliva to virus suspension was pre-optimized to 9:1, because excess saliva inhibits the viral infection to host cells. A saliva-virus suspension was incubated for 10 minutes at room temperature. The saliva-virus suspension was treated with HOCl at various volume ratios as above. Sample dilution, inoculation, and CPE evaluation are as described above.