S pneumoniae

The following four bacterial standard strains from the American Type Culture Collection (ATCC, Manassas, VA, USA) were used: H. influenzae ATCC 49247, S. aureus ATCC 29213, S. pneumoniae ATCC 49619, and S. pyogenes ATCC 19615. Cultivation and assay media (broth/agar) were Mueller–Hinton broth (MHB) complemented by Haemophilus test medium (H. influenzae), MHB (S. aureus), and brain–heart infusion (S. pneumoniae and S. pyogenes). The pH of the broths was adjusted to a final value of 7.6 using Trizma base (Sigma-Aldrich). All microbial strains and cultivation media were purchased from Oxoid (Basingstoke, UK). Stock cultures of bacterial strains were cultivated in broth medium at 37 °C for 24 h prior to testing in the incubator (Memmert GmbH & Co. KG, Buchenbach, Germany). For the preparation of inoculum, the turbidity of the bacterial suspension was adjusted to 0.5 McFarland standard using a Densi-La-Meter II (Lachema, Brno, Czech Republic) to obtain a final concentration of 10⁸ colony-forming units per mL.

AV-PVP-TCA-I₂ was tested against a selection of 10 microbial strains (C. albicans WDCM 00054 Vitroids, S. aureus ATCC 25923, S. pneumoniae ATCC 49619, E. faecalis ATCC 29212, S. pyogenes ATCC 19615, Bacillus subtilis WDCM 0003 Vitroids, K. pneumoniae WDCM 00097 Vitroids, E. coli WDCM 00013 Vitroids, P. aeruginosa WDCM 00026 Vitroids, and P. mirabilis ATCC 29906). The positive controls were the common antibiotics gentamicin and nystatin (for C. albicans WDCM 00054 Vitroids only). Pure ethanol and ultrapure water were the negative controls and did not inhibit anything. All the tests were repeated three times, and the average was presented in this study. The formulation was impregnated and also tested on sterile discs, sterile PGA sutures, cotton gauze bandages, surgical facemasks, and KN95 masks against the same reference strains.

The three samples AV-PVP-I₂, AV-PVP-I₂-NaI and AV-PVP-NaI impregnated on sterile, multifilamented, uncoated PGA sutures were dip-coated separately. The coating was done on 10 suture fragments of approximately 2.5 cm. These sutures were prepared by treating them with acetone and drying at room temperature. They were immersed into each 50 mL of AV-PVP-I₂, AV-PVP-I₂-NaI and AV-PVP-NaI solutions (1 mM) 25 °C, and stirred at 130 rpm for 18 h. The color of the sutures changed from blue to brownish-blue. The coated sutures were removed from the solutions, dried for 24 h under ambient conditions and tested in vitro by the zone of inhibition assay against the reference strains S. pneumoniae ATCC 49619, S. aureus ATCC 25923, E. faecalis ATCC 29212, S. pyogenes ATCC 19615, Bacillus subtilis WDCM 00003, E. coli WDCM 00013, P. aeruginosa WDCM 00026, P. mirabilis ATCC 29906, K. pneumoniae WDCM 00097, and C. albicans WDCM 00054. The AV-PVP-I₂-NaI coated sutures and the uncoated PGA were analyzed by SEM and compared.

The reference strains S. pneumoniae ATCC 49619, S. aureus ATCC 25923, E. faecalis ATCC 29212, S. pyogenes ATCC 19615, E. coli WDCM 00013 Vitroids, P. aeruginosa WDCM 00026 Vitroids, K. pneumoniae WDCM00097 Vitroids, and C. albicans WDCM 00054 Vitroids were used for the antimicrobial testing. These strains were stored at −20 °C and inoculated in MHB by adding the selected fresh bacteria and fungi into 10 mL Mueller Hinton broth. These prepared suspensions were kept at 4 °C until use.

The compounds AV, AV-PVP, AV-PVP-I₂, AV-PVP-I₂-NaI, and AV-PVP-NaI were tested against the Gram-positive S. pneumoniae ATCC 49619, S. aureus ATCC 25923, S. pyogenes ATCC 19615, E. faecalis ATCC 29212, B. subtilis WDCM 00003, Gram-negative bacteria P. mirabilis ATCC 29906, P. aeruginosa WDCM 00026, E. coli WDCM 00013, and K. pneumoniae WDCM 00097. The antifungal activities were tested against C. albicans WDCM 00054. The batches of 36 and 54 h showed a trend of steadily decreasing antimicrobial activities compared to the 18 h stirring batches and were not specifically mentioned further.

AV-PVP-Thymol-I₂ underwent testing against a panel of 10 reference strains, including C. albicans WDCM 00054 Vitroids, S. aureus ATCC 25923, S. pneumoniae ATCC 49619, E. faecalis ATCC 29212, S. pyogenes ATCC 19615, B. subtilis WDCM 0003 Vitroids, K. pneumoniae WDCM 00097 Vitroids, E. coli WDCM 00013 Vitroids, P. aeruginosa WDCM 00026 Vitroids, and P. mirabilis ATCC 29906. Gentamicin and nystatin served as positive controls, the latter specifically for C. albicans WDCM 00054 Vitroids. Negative controls included pure ethanol and ultrapure water, neither of which exhibited inhibitory effects. Each test was conducted thrice, and the average results were reported. Additionally, the formulation was applied to sterile discs, cotton gauze bandages, sterile PGA sutures, KN95 masks, and surgical facemasks, to be tested against the same reference strains.