The Susceptibility of Klebsiella Pneumoniae Strains Isolated from COVID-19 Patients to Commercially Available Bacteriophage Medications

Background. Superinfection caused by Klebsiella pneumoniae occupies a leading position in the structure of bacterial complications in COVID-19 patients. The intensive circulation of Klebsiella in specialised hospitals has contributed to the consolidation of the most clinically and epidemiologically important strains of this pathogen, in particular, representatives of hypervirulent and carbapenem-resistant clonal lines, which have not lost their relevance even in the post-pandemic period. The use of bacteriophages as therapeutic and anti-epidemic agents seems justified given the widespread use of multidrugresistant strains of K. pneumoniae.

Aim of the study. To evaluate the susceptibility of K. pneumoniae strains associated with nosocomial infections in patients with COVID-19 to polyvalent bacteriophage medications.

Materials and methods. The study included 96 non-repeating K. pneumoniae strains isolated from clinical material of patients admitted to a major hospital in St. Petersburg with severe and moderate forms of COVID-19 from May 2020 to January 2021. The susceptibility of clinical strains to bacteriophages was assessed using the spot test analysis. Commercially available bacteriophage preparations used for testing included the following: purified polyvalent pyobacteriophage, sextaphage, and purified polyvalent Klebsiella pyobacteriophage. In order to identify the probable mechanisms of resistance of hospital strains of K. pneumoniae, the nucleotide sequences of the genomes of 6 strains of this pathogen belonging to the dominant hospital genetic lines ST3, ST39, ST307, ST395, ST874 were studied.

Results. Negative results of spot tests were observed in 32.29% (95% CI=23.8–42.2) of cases; in general, the proportion of patients eligible for treatment with phage therapy was 49% (95% CI=39.2–58.8). Loci of class 1 subtypes IV-A3 and I-E, potentially associated with resistance to CRISPR-Cas, were identified in the genome structure of the studied strains, as well as a number of prophage sequences potentially associated with resistance to bacteriophages.

Conclusion. The study demonstrated low activity of polyvalent bacteriophage medications against K. pneumoniae strains causing nosocomial infections in patients with COVID-19. Increasing the diversity of bacteriophage strains active against epidemiologically relevant K. pneumoniae clones can expand the possibilities of phage therapy for Klebsiella infections. The rational use of medications containing these bacteriophages is possible within the paradigm of personalised phage therapy.

1. Zhou F., Yu T., Du R., Fan G., Liu Y., Liu Z., Xiang J., Wang Y., Song B., Gu X., Guan L., Wei Y., Li H., Wu X., Xu J., Tu S., Zhang Y., Chen H., Cao B. Clinical course and risk factors for mortality of adult inpatients with COVID-19 in Wuhan, China: a retrospective cohort study. Lancet. 2020; 395 (10229): 1054–1062. doi: 10.1016/S0140-6736(20)30566-3.

2. Sang L., Xi Y., Lin Z., Pan Y., Song B., Li C. A. Zheng X., Zhong M., Jiang L., Pan C., Zhang W., Lv Z., Xia J., Chen N., Wu W., Xu Y., Chen S., Liu D., Liang W., Liu X., Liu X., Li S., Zhong N., Ye D., Xu Y., Zhang N., Zhang D., Li Y. Secondary infection in severe and critical COVID-19 patients in China: a multicenter retrospective study. Annals of Palliative Medicine. 2021; 10 (8): 8557–8570. doi: 10.21037/apm-21-833.

3. Goncharov A.E., Zueva L.P., Mokhov A.S., Kolodzhieva V.V., Meltser A.A., Smirnova M.V., Khavlina T.V., Orishak E.A. Spread of multi-antibiotic-resistant health-care pathogens in hospitals to treat COVID-19 patients. Epidemiology and Vaccinal Prevention. 2021; 20 (2): 68–73. doi: https://doi.org/10.31631/2073-3046-2021-20-2-68-73. (in Russian)

4. Mashigo B., Parker A., Lalla U., Allwood B. W. Moolla M. S. Lovelock T., Koegelenberg CFN. An outbreak within an outbreak: the impact of infection prevention and control strategies on hospital-acquired infections and the occurrence of multi-drug resistant organisms during the COVID-19 pandemic. S Afr Med J. 2023 Dec 4; 113 (12): 42. doi: 10.7196/SAMJ.2023.v113i12.97.

5. Loconsole D., Sallustio A., Sacco D., Santantonio M., Casulli D., Gatti D., Accogli M., Parisi A., Zagaria R., Colella V., Centrone F., Chironna M. Genomic surveillance of carbapenem-resistant Klebsiella pneumonia reveals a prolonged outbreak of extensively drug-resistant ST147 NDM1 during the COVID-19 pandemic in the Apulia region (Southern Italy). J Glob Antimicrob Resist. 2024 Mar; 36: 260–266. doi: 10.1016/j.jgar.2024.01.015.

6. Sandfort M., Hans J. B. Fischer M. A. Reichert F., Cremanns M., Eisfeld J., Pfeifer Y., Heck A., Eckmanns T., Werner G., Gatermann S., Haller S., Pfennigwerth N. Increase in NDM-1 and NDM-1/OXA-48-producing Klebsiella pneumoniae in Germany associated with the war in Ukraine, 2022. Euro Surveill. 2022 Dec; 27 (50): 2200926. doi: 10.2807/1560-7917.ES.2022.27.50.2200926.

7. Samoilova A.A., Kraeva L.A., Mikhailov N.V., Saitova A.T., Polev D.E.,Vashukova M.A., Gordeeva S.A., Smirnova E.V., Beljatich L.I., Dolgova A.S., Shabalina A.V. Genomic analysis of Klebsiella pneumoniae strains virulence and antibiotic resistance. Russian Journal of Infection and Immunity = Infektsiya i Immunitet. 2024; 14 (2): 339–350 doi: https://doi.org/10.15789/2220-7619GAO-15645 (in Russian)]

8. Mohammadi M., Saffari M., Siadat S. D. Hejazi S. H. Shayestehpour M., Motallebi M., Eidi M. Isolation, characterization, therapeutic potency, and genomic analysis of a novel bacteriophage vB_KshKPC-M against carbapenemase-producing Klebsiella pneumoniae strains (CRKP) isolated from Ventilator-associated pneumoniae (VAP) infection of COVID-19 patients. Ann Clin Microbiol Antimicrob. 2023 Feb 24; 22 (1): 18. doi: 10.1186/s12941-023-00567-1.

9. Рациональное применение бактериофагов в лечебной и противоэпидемической практике. Методические рекомендации. М.: 2022; 32. [Racional'noe primenenie bakteriofagov v lechebnoj i protivoepi-demicheskoj praktike. Metodicheskie rekomendacii. Moscow: 2022; 32. (in Russian)

10. Goncharov A. E., Zueva L. P., Mokhov A. S., Kolodzhieva V. V., Meltser A. A., Smirnova M. V., Khavlina T. V., Orishak E. A. Spread of Multi-Antibiotic-Resistant health-care pathogens in hospitals to treat COVID-19 patients. Epidemiology and Vaccinal Prevention. 2021; 20 (2): 68–73. doi: https://doi.org/10.31631/2073-3046-2021-20-2-68-73. (in Russian)

11. Goncharov A. E., Azarov D. V., Mokhov A. S., Pochtovyi A. A., Kustova D. D., Gushchin V. A., Lebedeva E. A., Kolodzhieva V. V., Kireeva A. G., Kraeva L. A., Dolinny S. V., Burgasova O. A., Goncharova A. R., Belkova E. I., Dmitriev A. V. Characteristics of hypervirulent multidrug resistant Klebsiella pneumoniae strains in inpatients with severe COVID-19. Infekc Bolezni (Infectious Diseases). 2022; 20 (2): 33–40. doi: https://doi.org/10.20953/1729-9225-2022-2-33-40. (in Russian)

12. Russel J., Pinilla-Redondo R., Mayo-Muñoz D., Shah S. A., Sørensen S. J. CRISPRCasTyper: automated identification, annotation, and classification of CRISPR-Cas Loci. CRISPR J. 2020 Dec; 3 (6): 462–469. doi: 10.1089/crispr.2020.0059.

13. Shang J., Tang X., Guo R., Sun Y. Accurate identification of bacteriophages from metagenomic data using Transformer. Brief Bioinform. 2022 Jul 18; 23 (4): bbac258. doi: 10.1093/bib/bbac258.

14. Shang J., Tang X., Sun Y. PhaTYP: predicting the lifestyle for bacteriophages using BERT. Brief Bioinform. 2023 Jan 19; 24 (1): bbac487. doi: 10.1093/bib/bbac487.

15. Zueva L. P. Aslanov B. I., Akimkin V. G. Contemporary view on the role of bacteriophages in evolution of nosocomial strains and prophylaxis of healthcare associated infections. Zh Mikrobiol Epidemiol Immunobiol. 2014 May-Jun; (3): 100–107. (in Russian)

16. Villarroel J., Larsen M. V. Kilstrup M., Nielsen M. Metagenomic Analysis of Therapeutic PYO Phage Cocktails from 1997 to 2014. Viruses. 2017 Nov 3; 9 (11): 328. doi: 10.3390/v9110328.

17. Bayazitova L. T., Tyupkina O. F., CHazova T. A., Rodionova M. S., Anamov R. I., Popcov O. I., Valiullina I. R., Nasybullova Z. Z. Ocenka liticheskoj aktivnosti bakteriofagov v otnoshenii Klebsiella pneumoniae, vydelennyh ot detej s pnevmokokkovym nositel'stvom. Prakticheskaya Medicina. 2023; 21 (5): 80–85. (in Russian)

18. Kozlova A. I., Tapal'skij D.V. Chuvstvitel'nost' k antibiotikam i bakteriofagam klinicheskih izolyatov Klebsiella pneumoniae s klassicheskim i gipermukoidnym fenotipami. Voennaya Medicina. 2019; 1 (50): 44–48. (in Russian)

19. Makarova K. S. Zhang F., Koonin E. V. SnapShot: Class 1 CRISPR-Cas Systems. Cell. 2017 Feb 23; 168 (5): 946–946.e1. doi: 10.1016/j.cell.2017.02.018.

20. Xue C., Sashital D. G. Mechanisms of type I-E and I-F CRISPR-Cas systems in Enterobacteriaceae. EcoSal Plus. 2019 Feb; 8 (2): 10.1128/ecosalplus.ESP-0008-2018. doi: 10.1128/ecosalplus.ESP-0008-2018.

21. Benz F., Camara-Wilpert S., Russel J., Wandera K. G. Čepaitė R., Ares-Arroyo M., Gomes-Filho J. V. Englert F., Kuehn J. A. Gloor S., Mestre M. R. Cuénod A., Aguilà-Sans M., Maccario L., Egli A., Randau L., Pausch P., Rocha EPC, Beisel C. L. Madsen J. S. Bikard D., Hall A. R. Sørensen S. J. Pinilla-Redondo R. Type IV-A3 CRISPR-Cas systems drive inter-plasmid conflicts by acquiring spacers in trans. Cell Host Microbe. 2024 Jun 12; 32 (6): 875–886.e9. doi: 10.1016/j.chom.2024.04.016.

22. Wang J., Feng Y., Zong Z. The Origins of ST11 KL64 Klebsiella pneumoniae: a Genome-Based Study. Microbiol Spectr. 2023 Mar 27; 11 (2): e0416522. doi: 10.1128/spectrum.04165-22.

23. Hu F., Pan Y., Li H., Han R., Liu X., Ma R., Wu Y., Lun H., Qin X., Li J., Wang A., Zhou M., Liu B., Zhou Z., He P. Carbapenem-resistant Klebsiella pneumoniae capsular types, antibiotic resistance and virulence factors in China: a longitudinal, multi-centre study. Nat Microbiol. 2024 Mar; 9 (3): 814–829. doi: 10.1038/s41564-024-01612-1.

24. Eckstein S., Stender J., Mzoughi S., Vogele K., Kühn J., Friese D., Bugert C., Handrick S., Ferjani M., Wölfel R., Millard A., Ben Moussa M., Bugert J. J. Isolation and characterization of lytic phage TUN1 specific for Klebsiella pneumoniae K64 clinical isolates from Tunisia. BMC Microbiol. 2021 Jun 21; 21 (1): 186. doi: 10.1186/s12866-021-02251-w.

25. Hallal Ferreira Raro O., Nordmann P., Dominguez Pino M., Findlay J., Poirel L. Emergence of carbapenemase-producing hypervirulent Klebsiella pneumoniae in Switzerland. Antimicrob Agents Chemother. 2023 Mar 16; 67 (3): e0142422. doi: 10.1128/aac.01424-22.

26. Chen H., Liu H., Gong Y., Dunstan R. A. Ma Z., Zhou C., Zhao D., Tang M., Lithgow T., Zhou T. A Klebsiella-phage cocktail to broaden the host range and delay bacteriophage resistance both in vitro and in vivo. NPJ Biofilms Microbiomes. 2024 Nov 14; 10 (1): 127. doi: 10.1038/s41522-024-00603-8.

27. Chen H., Liu H., Gong Y., Dunstan R. A. Ma Z., Zhou C., Zhao D., Tang M., Lithgow T., Zhou T. A Klebsiella-phage cocktail to broaden the host range and delay bacteriophage resistance both in vitro and in vivo. NPJ Biofilms Microbiomes. 2024 Nov 14; 10 (1): 127. doi: 10.1038/s41522-024-00603-8.