Effect of povidone-iodine and propanol-based mecetronium ethyl sulphate on antimicrobial resistance and virulence in Staphylococcus aureus

Abstract

Background: Reports are available on cross-resistance between antibiotics and biocides. We evaluated the effect of povidone-iodine (PVP-I) and propanol-based mecetronium ethyl sulphate (PBM) on resistance development, antibiotics cross-resistance, and virulence in Staphylococcus aureus.

Methods: The minimum inhibitory concentration (MIC) of PVP-I and PBM were determined against S. aureus ATCC 25923 using the agar-dilution method. Staphylococcus aureus ATCC 25923 was subjected to subinhibitory concentrations of the tested biocides in ten consecutive passages followed by five passages in a biocide-free medium; MIC was determined after each passage and after the fifth passage in the biocide-free medium. The developed resistant mutant was tested for cross-resistance to different antibiotics using Kirby-Bauer disk diffusion method. Antibiotic susceptibility profiles as well as biocides' MIC were determined for 97 clinical S. aureus isolates. Isolates were categorized into susceptible and resistant to the tested biocides based on MIC distribution pattern. The virulence of the biocide-resistant mutant and the effect of subinhibitory concentrations of biocides on virulence (biofilm formation, hemolysin activity, and expression of virulence-related genes) were tested.

Results: PVP-I and PBM MIC were 5000 μg/mL and 664 μg/mL. No resistance developed to PVP-I but a 128-fold increase in PBM MIC was recorded, by repeated exposure. The developed PBM-resistant mutant acquired resistance to penicillin, cefoxitin, and ciprofloxacin. No clinical isolates were PVP-I-resistant while 48.5% were PBM-resistant. PBM-resistant isolates were more significantly detected among multidrug-resistant isolates. PVP-I subinhibitory concentrations (¼ and ½ of MIC) completely inhibited biofilm formation and significantly reduced hemolysin activity (7% and 0.28%, respectively). However, subinhibitory concentrations of PBM caused moderate reduction in biofilm activity and non-significant reduction in hemolysin activity. The ½ MIC of PVP-I significantly reduced the expression of hla, ebps, eno, fib, icaA, and icaD genes. The virulence of the biocide-resistant mutant was similar to that of parent strain.

Conclusion: PVP-I is a highly recommended antiseptic for use in healthcare settings to control the evolution of high-risk clones. Exposure to PVP-I causes no resistance-development risk in S. aureus, with virulence inhibition by subinhibitory concentrations. Also, special protocols need to be followed during PBM use in hospitals to avoid the selection of resistant strains.

Keywords: Antibiotics; Biocides; Biofilm; Hemolysin; Povidone-iodine; Propanol-based mecetronium ethyl sulphate; Resistance; Staphylococcus aureus; Virulence.

Fig. 1

Development of biocide-resistant mutants. Fold change in the minimum inhibitory concentration (MIC) of Staphylococcus aureus ATCC 25923 (P0) after passage in subinhibitory concentrations of the tested biocides (Povidone-iodine and propanol-based mecetronium ethyl sulphate) for ten consecutive passages (P1–P10) and after five passages in biocide-free medium (P15)

Fig. 2

Distribution of biocides minimum inhibitory concentrations in the clinical isolates of Staphylococcus aureus. a povidone-iodine and b propanol-based mecetronium ethyl sulphate

Fig. 3

Number of clinical Staphylococcus aureus isolates with different antimicrobial susceptibility patterns to tested antibiotics. C chloramphenicol, CIP ciprofloxacin, CN gentamicin, DA clindamycin, E erythromycin, FOX cefoxitin, LZD linezolid, P penicillin, RD rifampicin, SXT sulfamethoxazole/trimethoprim, TE tetracycline, VA vancomycin

Fig. 4

Distribution of isolates with different propanol-based mecetronium ethyl sulphate (PBM) resistance patterns. a among methicillin susceptible Staphylococcus aureus (MSSA) and methicillin resistant Staphylococcus aureus (MRSA), b among multi drug reistant (MDR) and non-MDR isolates, (c) among isolates from hospitalized and non-hospitalized patients. The statistical analyses were performed using Chi-square test. ^*p < 0.05

Fig. 5

Growth patterns of parent Staphylococcus aureus ATCC 25923 under different conditions and of biocide-resistant mutant. Culture optical density measured hourly for the propanol based mecetronium ethyl sulphate (PBM)-resistant mutant and for the parent S. aureus ATCC 25923 in the absence (control) and presence of ¼ or ½ of the minimum inhibitory concentration (MIC) of either of povidone-iodine (PVP-I) or PBM. The data are represented as mean of three independent experiments. Error bars represent the standard deviation

Fig. 6

Virulence of the biocide-resistant mutant and the parent Staphylococcus aureus ATCC 25923 under different conditions. a Biofilm activity; and b Hemolytic activity of S. aureus ATCC 25923 in the absence (control) and presence of ¼ and ½ of the minimum inhibitory concentration (MIC) of povidone-iodine (PVP-I) and propanol based mecetronium ethyl sulphate (PBM) and of the PBM-resistant mutant. Error bars represent standard deviation. ***p < 0.0005, ****p < 0.0001

Fig. 7

Relative expression of virulence-related genes in Staphylococcus aureus ATCC 25923. The relative expression of hla, icaA, icaD, eno, epbs and fib genes in S. aureus ATCC 25923 treated with ½ of minimum inhibitory concentration (MIC) of povidone-iodine (PVP-I) compared to that of untreated S. aureus ATCC 25923. Error bars represent the standard deviation ***p < 0.0005, ****p < 0.0001