Table 1 Description of modifications applied to the original ISO22196-2011 protocol
Variable | Original ISO method | Applied modification | Rationale |
|---|---|---|---|
Bacterial strains | Staphylococcus aureus Escherichia coli | Methicillin-resistant Staphylococcus aureus (MRSA) ATCC 33591 Vancomycin-resistant Enterococcus (VRE) ATCC 51299 Pseudomonas aeruginosa ATCC 9027 Acinetobacter baumannii ATCC 19606 Carbapenem-resistant Enterobacterales – Klebsiella pneumoniae ATCC BAA-1705. | Gram-positive and Gram-negative representatives of the ESKAPE bacteria, which are commonly problematic in hospital-acquired infections and associated with multi-drug resistance profiles (7). Among the tested strains, P. aeruginosa and A. baumannii are not carbapenem-resistant. |
Contact time | 24 h | 5 min, 15 min, 30 min. | The OxiLast™ formulation is devised to act prospectively following its application and within a short time. |
Inocula concentration of tests and controls | A target concentration of 1–4 × 105 cells in 0.4 ml used for inoculation onto the substrate surfaces. | A target concentration of 1–4 × 107 or 1–4 × 106 cells in 0.4 ml was used for inoculation onto the substrate surfaces. | Higher CFU counts in the inocula were used to prove efficacy at high inoculation regimens and to measure a desired 4 log10 reduction within the limit of detection of the procedure used. |
Test surface and cover film sizes | Standard size of the cover film shall be a square of (40 ± 2) mm ×(40 ± 2) mm for the 50 mm ×50 mm test specimen. | Cover films used were square of (80 ± 2) mm ×(80 ± 2) mm for the 100 mm ×100 mm test substrate. | A larger surface, with the same volume was chosen to maximize contact between the bacterial cells and the OxiLast™ coating. |
Coating age | Not specified. | Freshly prepared, 24 h, 48 h, 72 h, 5 days, 7 days. | Because the OxiLast™ formulation is devised to exert its antimicrobial properties prospectively, this effect was tested over the indicated coating ages. |
Antibacterial neutralizer solution | Soybean casein digest broth with lecithin and polyoxyethylene sorbitan monooleate broth (SCDLP broth) | Sodium-Thiosulfate 1 mg/ml in 0.85% (w/v) NaCl. | Sodium-thiosulfate is used for chlorine species neutralization in antimicrobial testing (18), and the ISO standard allows the use of a neutralizer of choice upon a provided toxicity test (conducted). |
Neutralizer solution volume used | 10 ml. | 5 ml. | A lower resuspending volume was used to minimize dilution effects due to the lower volume used in plating (i.e. 200 µl instead of 1 ml). |
Recovery of bacteria from test specimens | Mechanical methods such as stomaching, vortexing or sonicating | Scraping of PET covers and PVC substrate slides with sterile scrapers 20 s in two directions after applying the neutralizer solution. | This method allowed for > 99% recovery efficiency of viable cells in control slides. |
Determining the viable bacteria count | Stirring of 1 ml of recovered neutralizer solution (or serial dilutions of it) onto pre-solidified PCA plate together with 15 ml of the same melted culture medium | Plating of 0.2 ml of recovered neutralizer solution (or serial dilutions of it) onto TSA plates or otherwise specified selective agar media. | Because commercial ready-to-use plates were used, the maximum volume that could be practically plated was 200 µl. |