Methicillin-resistant staphylococci biofilms and the possibility of controlling them with chlorhexidine and polyhexanide: prospects for use in real clinical practice

   Introduction. The leading pathogens of implant-associated infection are S. aureus and S. epidermidis, characterized by the ability to biofilm formation, which pose a serious threat to the life and health of patients. Antibiotic therapy is an integral part of the complex treatment of infection, but washing the surgical area after the removal of infected components with antiseptics, including those based on polyhexanide or chlorhexidine, plays a major role.

   Objective. To evaluate the presence of biofilm-forming genes in methicillin-resistant S. aureus and S. epidermidis isolated from patients with orthopedic infection and to perform a comparative analysis of the effect of chlorhexidine and polyhexanide on their biofilms.

   Materials and methods. Cultures were isolated according to the international standards of microbiological research. Identification was performed by MALDI-TOF MS, and antibiotic susceptibility was determined according to EUCAST. DNA was isolated and purified using Auto-Pure S32 and the Magno-Sorb kit. Amplification was performed on a CFX-96 device. Visualization was performed using the ChemiDoc gel documentation system. Biofilms were formed for 48 hours and then treated with polyhexanide or chlorhexidine for 5, 10, 20, 40 minutes. The destructive effect of the antiseptics was determined by staining with gentian violet and subsequent comparison with the control. The presence of live bacterial cells in the biofilm after treatment with the drugs was assessed using a resazurin sodium salt. Statistical analysis was performed in GraphPad Prism 9.0.

   Results. The bap gene was detected, and no ica-operon genes were found. MRSA were characterized by the presence of 3 marker genes, and MRSE strains had one or two marker genes. Polyhexanide was more effective in destroying the biomass of the formed daily biofilms of S. aureus — carriers of the bap, clfA/B genes, in contrast to chlorhexidine. Polyhexanide statistically significantly reduced the biomass with an exposure of more than 10 minutes. A similar pattern was found for the MRSE biofilms. In addition, the drugs almost destroyed the biofilm cells of MRSE, and polyhexanide was more effective against MRSA.

   Conclusion. The study revealed the main genetic determinants of methicillin-resistant S. aureus and S. epidermidis biofilm formation. The established interspecies genetic differences can determine the effectiveness of antiseptics and demonstrate the need for exposure of the studied antiseptic for at least 20 minutes to achieve maximum effect on the biofilm cells of staphylococci.

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