Anti-bacterial and anti-biofilm activity of bacteriophages against Klebsiella pneumoniae and Pseudomonas aeruginosa isolated from orthopedic patients

Objective. To investigate the susceptibility of K. pneumoniae and P. aeruginosa to a polyvalent bacteriophage preparation and its effect on biofilm formation and the strain biofilms isolated from orthopedic patients.

Materials and methods. The research sample included 50 clinical isolates of K. pneumoniae and 50 clinical isolates of P. aeruginosa. Identification was performed by MALDI-TOF-MS; antibiotic susceptibility was assessed in accordance with EUCAST v 21. Detection of carbapenemase genes was carried out by real-time PCR. The strain susceptibility to the bacteriophage was determined by a spot test; K. pneumoniae ATCC 33495 and P. aeruginosa ATCC 27853 were determined by assessing their growth curves. Biofilms of strains sensitive to bacteriophages were formed according to the O’Toole method by co-incubation of bacteria with phages. The effect of bacteriophages on 24-hour biofilms was assessed by comparing the optical density of dye extracts of bacteriophage-treated wells and control wells at 570 nm. The data were analyzed using the Statistica environment.

Results. It was found that 7 (14%) of K. pneumoniae and 15 (30%) of P. aeruginosa were resistant to carbapenems. Six strains of K. pneumoniae produced NDM-cabapenemase, while four isolates of P. aeruginosa produced VIM-carbapenemases. The bacteriophage preparation under study was active against 36% and 56% of K. pneumoniae and P. aeruginosa strains, respectively. The majority of the studied strains reduced biofilm production upon co-incubation with a phage; however, a decrease in biomass of greater than 80% was observed only for P. aeruginosa. The effect of the bacteriophage on the already formed biofilms was less pronounced, despite a decrease in the biofilm biomass in 78% and 68% of K.pneumoniae and P. aeruginosa strains, respectively.

Conclusion. The results obtained confirm the need for further research into the action of bacteriophages against pathogens caused by implant-associated infections and the development of bacteriophage therapy for orthopedic patients.

1. Gordillo Altamirano FL, Barr JJ. Phage therapy in the postantibiotic era. Clin Microbiol Rev. 2019;32(2):e00066-18. doi: 10.1128/CMR.00066-18

2. Patey O, McCallin S, Mazure H, Liddle M, Smithyman A, Dublanchet A. Clinical indications and compassionate use of phage therapy: personal experience and literature review with a focus on osteoarticular infections. Viruses. 2018;11(1):18. doi: 10.3390/v11010018

3. Antimicrobial Resistance Collaborators. Global burden of bacterial antimicrobial resistance in 2019: a systematic analysis. Lancet. 2022;399(10325):629–55. doi: 10.1016/S01406736(21)02724-0

4. Landyshev NN, Voronko YG, Timoshina OY, Suslina SN, Akimkin VG, Miroshnikov KA. A review of the regulatory framework for personalized bacteriophages registration. Problems of Virology. 2020;65(5):259–66 (In Russ.). doi: 10.36233/0507-4088-2020-65-5-2

5. Bozhkova S, Tikhilov R, Labutin D, Denisov A, Shubnyakov I, Razorenov V, Artyukh V, Rukina A. Failure of the first step of two-stage revision due to polymicrobial prosthetic joint infection of the hip. J Orthop Traumatol. 2016;17(4):369–76. doi: 10.1007/s10195-016-0417-8

6. Harper DR, Parracho HMRT, Walker J, Sharp R, Hughes G, Werthén M, Lehman S, Morales S. Bacteriophages and biofilms. Antibiotics (Basel). 2014;3(3):270–84. doi: 10.3390/antibiotics3030270

7. Hemmati F, Rezaee MA, Ebrahimzadeh S, Yousefi L, Nouri R, Kafil HS, Gholizadeh P. Novel strategies to combat bacterial bio-films. Mol Biotechnol. 2021;63(7):569–86. doi: 10.1007/s12033-021-00325-8

8. Tapalski DV. Sensitivity of Pseudomonas aeruginosa nosocomial isolates to the preparations for phagotherapy. Vitebsk Medical Journal. 2018;17(2):47–54 (In Russ.). doi: 10.22263/2312-4156.2018.2.47

9. Tapalsky DV, Kozlova AI. Sensitivity of Klebsiella pneumoniae clinical isolates with various levels of antibiotic resistance to bacteriophage preparations. Health and Ecology Issues. 2018;1(55):56–62 (In Russ.). doi: 10.51523/2708-6011.2018-15-1-9

10. Łusiak-Szelachowska M, Weber-Dąbrowska B, Górski A. Bacteriophages and lysins in biofilm control. Virol Sin. 2020;35(2):125–33. doi: 10.1007/s12250-019-00192-3

11. González S, Fernández L, Gutiérrez D, Campelo AB, Rodríguez A, García P. Analysis of different parameters affecting diffusion, propagation and survival of staphylophages in bacterial biofilms. Front Microbiol. 2018;9:2348. doi: 10.3389/fmicb.2018.02348

12. Aslanov BI, Zueva LP, Dolgiy AA. Efficacy of bacteriophages against Pseudomonas aeruginosa biofiolms. Preventive and Clinical Medicine. 2020;4(77):40–5 (In Russ.). doi: 10.47843/2074-9120_2020_4_40

13. Glazunov EA, Zurabov FM, Pavlova, IB, Tolmacheva GS. Effects of the virulent bacteriophages on Klebsiella pneumoniae biofilms. Problems of Veterinary Sanitation, Hygiene and Ecology. 2020;4(36):480–5 (In Russ.). doi: 10.36871/vet.san.hyg.ecol.202004012

14. Fong SA, Drilling A, Morales S, Cornet ME, Woodworth BA, Fokkens WJ, Psaltis AJ, Vreugde S, Wormald PJ. Activity of bacteriophages in removing biofilms of Pseudomonas aeruginosa isolates from chronic rhinosinusitis patients. Front Cell Infect Microbiol. 2017;7:418. doi: 10.3389/fcimb.2017.00418.