Molecular genetic characterization of three new Klebsiella pneumoniae bacteriophages suitable for phage therapy

The Klebsiella pneumoniae bacterium is capable of causing the broad range of human nosocomial infections associated with antibiotic resistance and high mortality. Virulent bacteriophage therapy is one of the promising alternatives to antibiotic treatment of such infections. The study was aimed to isolate virulent bacteriophages effective against the relevant clinical K. pneumoniae strains, and to perform the molecular genetic characterization of these phages. Bacteriophages were isolated from the river water samples using the enrichment method. The whole-genome sequencing was performed on the MiSeq platform (Illumina). Three novel K. pneumoniae bacteriophages belonging to families Autographiviridae (vB_KpnP_NER40, GenBank MZ602146) and Myoviridae (vB_KpnM_VIK251, GenBank MZ602147; vB_KpnM_FRZ284, GenBank MZ602148) have been isolated and characterized. On the collection of 105 K. pneumoniae clinical strains, it has been found that bacteriophages vB_KpnP_NER40 and vB_KpnM_VIK251 have a narrow lytic spectrum (22% and 11%), which is limited to strains of the capsular types К2 and К20 respectively. In contrast, bacteriophage vB_KpnM_FRZ284 has a broad lytic spectrum (37%), causing the lysis of strains with different types of capsular polysaccharide. The phages are strictly virulent and have no genes encoding integrases, toxins or pathogenicity factors in their genomes. Genes of depolymerases, encoding the potential receptor binding proteins, have been found in the genomes of the capsular-specific bacteriophages vB_KpnP_NER40 and vB_KpnM_VIK251. The cocktail of three bacteriophages has lysed about 65% of the studied collection of K. рneumoniae strain and is potentially applicable for therapeutic purposes.

1. Paczosa MK, Mecsas J. Klebsiella pneumoniae: going on the offense with a strong defense. Microbiology and Molecular Biology Reviews. 2016; 80 (3): 629–61.

2. Podschun R, Ullmann U. Klebsiella spp. as nosocomial pathogens: Epidemiology, taxonomy, typing methods, and pathogenicity factors. Clin Microbiol Rev. 1998; 11 (4): 589–603.

3. Lee CR, et al. Global dissemination of carbapenemase-producing Klebsiella pneumoniae: epidemiology, genetic context, treatment options, and detection methods. Frontiers in microbiology. 2016; 7: 895.

4. Kuzmenkov AY, et al. AMRmap: an interactive web platform for analysis of antimicrobial resistance surveillance data in Russia. Front Microbiol. 2021; 12: 377.

5. Górski A, et al. Phage therapy: current status and perspectives. Med Res Rev. 2020; 40 (1): 459–63.

6. Payne RJH, Jansen VAA. Phage therapy: the peculiar kinetics of self-replicating pharmaceuticals. Clin Pharmacol Ther. 2000; 68 (3): 225–230.

7. Schooley RT, et al. Development and use of personalized bacteriophage-based therapeutic cocktails to treat a patient with a disseminated resistant Acinetobacter baumannii infection. Antimicrob Agents Chemother. 2017; 61 (10): e00954–17.

8. Dedrick RM, et al. Engineered bacteriophages for treatment of a patient with a disseminated drug-resistant Mycobacterium abscessus. Nat Med. 2019; 25 (5): 730–3.

9. Clark JR, March JB. Bacteriophages and biotechnology: vaccines, gene therapy and antibacterials. Trends Biotechnol. 2006; 24 (5): 212–8.

10. Pires DP, et al. Bacteriophage-encoded depolymerases: their diversity and biotechnological applications. Appl Microbiol Biotechnol. 2016; 100 (5): 2141–51.

11. Wyres KL, et al. Identification of Klebsiella capsule synthesis loci from whole genome data. Microb genomics. 2016; 2 (12).

12. Sobirk SK, Struve C, Jacobsson SG. Primary Klebsiella pneumoniae liver abscess with metastatic spread to lung and eye, a North-European Case Report of an emerging syndrome. Open Microbiol. 2010; 4 (1): 5–7.

13. Knecht LE, Veljkovic M, Fieseler L. Diversity and function of phage encoded depolymerases. Front Microbiol. 2020; 10: 2949.

14. Solovieva EV, et al. Comparative genome analysis of novel Podoviruses lytic for hypermucoviscous Klebsiella pneumoniae of K1, K2, and K57 capsular types. Virus Res. 2018; 243: 10–18.

15. Volozhantsev N, et al. Characterization and therapeutic potential of Bacteriophage-encoded polysaccharide depolymerases with — galactosidase activity against Klebsiella pneumoniae K57 capsular type. Antibiot. 2020; 9 (11): 1–16.

16. Scorpio A, et al. Treatment of experimental anthrax with recombinant capsule depolymerase. Antimicrob Agents Chemother. 2008; 52 (3): 1014.

17. Kornienko M, et al. Analysis of nosocomial Staphylococcus haemolyticus by MLST and MALDI-TOF mass spectrometry. Infect Genet Evol. 2016; 39: 99–105.

18. M100 Performance Standards for Antimicrobial Susceptibility Testing An informational supplement for global application developed through the Clinical and Laboratory Standards Institute consensus process. 29th ed. Clinical and Laboratory Standards Institute, Wayne, Pennsylvania, 2019. Available from: https://clsi.org/media/2663/m100ed29_sample.pdf.

19. Diancourt L, et al. Multilocus sequence typing of Klebsiella pneumoniae nosocomial isolates. J Clin Microbiol. 2005; 43 (8): 4178–82.

20. Brisse S, et al. Wzi gene sequencing, a rapid method for determination of capsular type for Klebsiella strains. J Clin Microbiol. 2013; 51 (12): 4073–8.

21. Van Twest R, Kropinski AM. Bacteriophage enrichment from water and soil. Methods Mol Biol. 2009; 501: 15–21.

22. Mazzocco A, et al. Enumeration of bacteriophages using the small drop plaque assay system. Methods Mol Biol. 2009; 501: 81–85.

23. Sambrook J, Fritsch EF, Maniatis T. Molecular cloning: a laboratory manual. 1989; 2.

24. Liu B, et al. VFDB 2019: a comparative pathogenomic platform with an interactive web interface. Nucleic Acids Res. 2019; 47 (D1): D687–D692.

25. Liu B, Pop M. ARDB — Antibiotic Resistance Genes Database. Nucleic Acids Res. 2009; 37.

26. Wang C, et al. Protective and therapeutic application of the depolymerase derived from a novel KN1 genotype of Klebsiella pneumoniae bacteriophage in mice. Res Microbiol. 2019; 170 (3): 156–64.

27. D’Andrea MM, et al. φbO1E, a newly discovered lytic bacteriophage targeting carbapenemase-producing Klebsiella pneumoniae of the pandemic Clonal Group 258 clade II lineage. Sci Rep. 2017; 7 (1): 1–8.

28. Yang J, et al. A nosocomial outbreak of KPC-2-producing Klebsiella pneumoniae in a Chinese hospital: dissemination of ST11 and emergence of ST37, ST392 and ST395. Clin Microbiol Infect. 2013; 19 (11): E509–E515.

29. Muggeo A, et al. Spread of Klebsiella pneumoniae ST395 nonsusceptible to carbapenems and resistant to fluoroquinolones in North-Eastern France. J Glob. 2018; 13: 98–103.

30. Kuptsov NS, Kornienko MA, Gorodnichev RB, Danilov DI, Malakhova MV, Parfenova TV, et al. Efficacy of commercial bacteriophage products against ESKAPE pathogens. Bulletin of RSMU. 2020; (3): 18–24.