Polyhexanide and Trigexylon® against biofilms of carbapenemase-producing K. pneumoniae and P. aeruginosa

Objective. To compare the effect of polyhexanide and Trihexylon® on the biofilms formed by carbapenemase-producing K. pneumoniae and P. aeruginosa isolated from patients with orthopedic infection. Materials and methods. Isolation of K. pneumoniae and P. aeruginosa was performed in 2025 in accordance with the internationally-accepted standards of microbiological research. Species identification was performed by MALDI-TOF MS. Susceptibility to antibacterial drugs was determined by EUCAST v.15.0. Carbapenemase genes were detected by real-time PCR. Biofilms of carbapenem-resistant K. pneumoniae and P. aeruginosa were cultivated for 48 h. Subsequently, the wells were treated with 200 μl of 0.2% polyhexanide solution or Trihexylon® for 5, 10, 20, 40 min. The destructive effect of antiseptic compositions was determined by staining the biofilms with 0.1% gentian violet solution followed by their comparison with the control. To determine the effect of the studied antiseptic compositions on sessile bacterial cells, 190 μl of LB medium and 10 μl of an aqueous solution of resazurin were added to each well. Statistical analysis was performed in the GraphPad Prism 9.0 environment. Results. K. pneumoniae and P. aeruginosa produced various types of carbapenemases. All strains included in the study were sensitive to polyhexanide and Trihexylon®, and formed biofilms. Both antiseptic compositions effectively reduced the biomass of formed biofilms of P. aeruginosa and K. pneumoniae. Exposure to Trihexylon® for 5 min had no effective destructive effect on biofilms of the IMP-producing isolate of P. aeruginosa. In comparison, the polyhexanide solution was active following 5 min of exposure. Both compositions demonstrated antibacterial activity against the sessile forms of P. aeruginosa and K. pneumoniae. Conclusion. The results obtained indicate the potential antibiofilm activity of polyhexanide for clinical use. This is of particular importance for orthopedic surgery, where the prevention and treatment of biofilm-related infections are critical. Further studies should focus on optimizing the exposure duration and developing improved irrigation strategies to ensure sufficient antibacterial activity, cytocompatibility, and economic efficiency considering the cost of the preparations.

1. Tubb CC, Polkowksi GG., Krause B. Diagnosis and prevention of periprosthetic joint infections. J Am Acad Orthop Surg. 2020;28(8):e340-e348. doi: 10.5435/JAAOS-D-19-00405

2. Kasimova AR, Tufanova OS, Gordina EM, Gvozdetsky AN, Radaeva KS, Rukina AN, Bozhkova SA, Tikhilov RM. Twelveyear dynamics of leading pathogens spectrum causing orthopedic infections from 2011 to 2022: A retrospective study. Traumatology and Orthopedics of Russia. 2024;30(1):66–75 (In Russ.). doi: 10.17816/2311-2905-16720

3. Tufanova OS, Kasimova AR, Astakhov DI, Rukina AN, Bozhkova SA. Factors affecting the course and prognosis of implant-associated infection caused by Klebsiella spp. Traumatology and Orthopedics of Russia. 2024;30(2):40–53. (In Russ.). doi: 10.17816/2311-2905-16719

4. Pfang BG, García-Cañete J, García-Lasheras J, Blanco A, Auñón Á, Parron-Cambero R, Macías-Valcayo A, Esteban J. Orthopedic implant-associated infection by multidrug resistant Enterobacteriaceae. J Clin Med. 2019;8(2):220. doi: 10.3390/jcm8020220

5. Almatroudi A. Biofilm resilience: molecular mechanisms driving antibiotic resistance in clinical contexts. Biology (Basel). 2025;14(2):165. doi: 10.3390/biology14020165

6. Macias-Valcayo A, Aguilera-Correa JJ, Broncano A, Parron R, Auñon A, Garcia-Cañete J, Blanco A, Esteban J. Comparative in vitro study of biofilm formation and antimicrobial susceptibility in Gram-negative bacilli isolated from prosthetic joint infections. Microbiol Spectr. 2022;10(4):e0085122. doi: 10.1128/spectrum.00851-22

7. Alves PJ, Barreto RT, Barrois BM, Gryson LG, Meaume S, Monstrey SJ. Update on the role of antiseptics in the management of chronic wounds with critical colonisation and/or biofilm. Int Wound J. 2021;18(3):342–58. doi: 10.1111/iwj.13537

8. Van den Poel B, Saegeman V, Schuermans A. Increasing usage of chlorhexidine in health care settings: blessing or curse? A narrative review of the risk of chlorhexidine resistance and the implications for infection prevention and control. Eur J Clin Microbiol Infect Dis. 2022;41(3):349–62. doi: 10.1007/s10096-022-04403-w

9. Lasko MJ, Nicolau DP. Carbapenem-resistant Еnterobacterales: considerations for treatment in the era of new antimicrobials and evolving enzymology. Curr Infect Dis Rep. 2020;22:6. doi: 10.1007/s11908-020-0716-3

10. Mendes G, Santos ML, Ramalho JF, Duarte A, Caneiras C. Virulence factors in carbapenem-resistant hypervirulent Klebsiella pneumoniae. Front Microbiol. 2023;14:1325077. doi: 10.3389/fmicb.2023.1325077

11. Dudek B, Brożyna M, Karoluk M, Frankiewicz M, Migdał P, Szustakiewicz K, Matys T, Wiater A, Junka A. In vitro and in vivo translational insights into the intraoperative use of antiseptics and lavage solutions against microorganisms causing orthopedic infections. Int J Mol Sci. 2024;25(23):12720. doi: 10.3390/ijms252312720

12. Kadirvelu L, Sivaramalingam SS, Jothivel D, Chithiraiselvan DD, Karaiyagowder Govindarajan D, Kandaswamy K. A review on antimicrobial strategies in mitigating biofilm-associated infections on medical implants. Curr. Res. Microb. Sci. 2024;6:100231. doi: 10.1016/j.crmicr.2024.100231

13. Algammal A, Hetta H.F, Mabrok M, Behzadi P. Editorial: Emerging multidrug-resistant bacterial pathogens "superbugs": A rising public health threat. Front Microbiol. 2023;14:1135614. doi: 10.3389/fmicb.2023.1135614

14. Barrigah-Benissan K, Ory J, Dunyach-Remy C, Pouget C, Lavigne JP, Sotto A. Antibiofilm properties of antiseptic agents used on Pseudomonas aeruginosa isolated from diabetic foot ulcers. Int J Mol Sci. 2022;23(19):11270. doi: 10.3390/ijms231911270