2.1. Chemicals and solution preparation
KBr was purchased by CARLO ERBA Reagents, H3PO4 (85%) was from Merck-Sigma Aldrich, while the NaClO (3%) was a commercial hypochlorite used for water intended for human consumption (in compliance with UNI EN 901:2013).
The albumin, used for the dirty test, was purchased by VWR.
Tryptone soya agar (TSA) from Microbiol was used as culture media.
The present solution, named MDI-102, consists in active free bromine solution (Br2, HBrO/BrO-) in a concentration ranged between 50 and 550 ppm. MDI-102 was prepared as follows. KBr (0.1-1%), phosphoric acid solution (H3PO4 85%) 0.05-0.5% and sodium hypochlorite (NaClO al 3%) 0.005-0.05% were mixed in ultra-pure water solution (90-99%). The exact concentration of the components depends on the final MDI-102 solution concentration (80 or 500 ppm). The reactions occurring in the solution during the preparation are outlined in Figure 1. Briefly, KBr dissolved in water, forms Br- and K+ (r. 1). After the addiction of hypochlorite, and the chlorine formation in water (r. 2-3), the bromide reacts with the chlorine forming Br-, HBrO, Br2 and Cl- (r. 4-5), which are the main components of the solution MDI-102. Furthermore, as previously described, due to the excess of KBr compared to the hypochlorite, all the ClO- can be considered converted to Cl-; it follows that the only antimicrobial effect is due to Br-based species.
Due to the low NaClO stability, the hypochlorite concentration is always analysed before the solution preparation. The free halogen concentration has been assessed using Hach Lange kit. The kit measures the free halogen concentration as follow. The N,N-diethyl-p-phenylenediamine, in the kit, is oxidized by the free halogen making a reddish compound which absorbs at 510 nm. The concentration is measured using Hach DR6000 spectrophotometer (cuvette with 2.5 cm optical path length). The NaClO concentration in the experiments was ranged from 2.9 and 3%.
2.2. Time kill assays
Time kill assay test (kinetic tests) was performed in order to test the bactericidal activity (BA) of MDI-102, using the bacterial strains reported by:
- Standard UNI EN ISO 11930:2019 (Evaluation of the antimicrobial protection of cosmetics);
- Standard UNI EN 1276:2019 (Quantitative suspension test for the evaluation of bactericidal activity of chemical disinfectants and antiseptics used in food, industrial, domestic and institutional areas);
- Standard UNI EN ISO 14729:2010 (Contact lens care products - Microbiological requirements and test methods for products and regimens for hygienic management of contact lenses);
- Stroman et al.21 (blepharitis treatments products) and Groden et al.10 (blepharitis flora).
The tested microorganisms were:
- Pseudomonas aeruginosa ATCC: 9027 (UNI EN ISO 11930:2019),
- Enterococcus hirae ATCC: 10541 (UNI EN 1276:2019),
- Serratia marcescens ATCC: 13880 (UNI EN ISO 14729:2010),
- Staphylococcus aureus ATCC: 6538 (UNI EN 1276:2019),
- Pseudomonas aeruginosa ATCC: 15442 (UNI EN 1276:2019),
- Escherichia coli ATCC: 8739 (UNI EN ISO 11930:2019),
- Staphylococcus epidermidis ATCC:12228 (Stroman et al. (2017) DOI:10.214/OPTH.5132851).
MDI-102 bactericidal activity was tested at 500 and 80 ppm (active bromine concentration). The tests were carried out following the Standard UNI EN 1276:2019 with slight modification. Briefly, the solution was composed by 8 mL of testing solution (MDI-102), 1 mL of inoculum (microorganisms solution) and 1 mL of water. Microrganisms were inoculated at concentration ranged between 2x105 and 3x107 colony-forming unit (CFU) mL-1 21. Microbial concentration was monitored at different times: 0.5, 1, 2, 5, 20, 30 minutes after the addition of the bactericidal solution (MDI-102). After the selected time a neutraliser (Dey/Engley Neutralizing broth) was added as inactivator. A blank test (control) without the bactericidal solution was carried out before each test in order to assess the initial microbial concentration, thus to monitor the microbial concentration over time. Tests were performed at 25 ± 1 °C, room temperature. After the fixed time, a solution aliquot was sampled, inoculated on a Petri dish and incubated at 36 ± 1 °C for 48 h. These tests are following referred as “clean tests”. All the tests were carried out in triplicates; the standard error for all the tests was ranged between zero and 1.5%.
Time kill assay tests were also carried out in “dirty condition” in order to assess the solution’s BA under real conditions. Dirty tests were performed adding albumin (3 g L-1) as interfering substance (UNI EN 1276:2019).
The tests were carried out as previously described, thus with same microorganisms’ strains and concentration, times, temperatures, etc. The only difference between the clean and the dirty condition was the presence of albumin in the dirty condition tests, 1 mL of albumin solution (3 g L-1) was used instead of 1 mL of water. The dirty condition tests were carried out using the only MDI-102 at 80 ppm (MDI-10280ppm) solution because it has been considered sufficient to prove the bactericidal activity of the solution. In fact, if MDI-10280ppm is effective in dirty condition, the solution at higher active bromine concentration (MDI-102500ppm) must be effective as well.