Isolation and Characterization of Novel Bacteriophages Targeting Carbapenem-Resistant Klebsiella pneumoniae
DOI:
https://doi.org/10.70749/ijbr.v4i5.3254Keywords:
Carbapenem-resistant Klebsiella pneumoniae, Bacteriophage therapy, Transmission electron microscopy, Multidrug resistance, and Lytic bacteriophagesAbstract
The rapid emergence of carbapenem-resistant Klebsiella pneumoniae (CRKP) has become a major global health concern due to its association with multidrug resistance and limited therapeutic options. This study aimed to isolate and characterize novel bacteriophages with lytic activity against clinical CRKP isolates as potential alternatives to conventional antibiotics. A total of 50 clinical isolates were collected from various specimens, of which 42 were confirmed as K. pneumoniae. Among these, 30 isolates (71.4%) were identified as carbapenem-resistant and selected for bacteriophage screening. Environmental samples, including sewage wastewater, hospital effluents, and drainage water, were used for phage isolation. Three lytic bacteriophages, designated vB_KpnP-1, vB_KpnP-2, and vB_KpnP-3, were successfully isolated and purified. Host range analysis revealed that the phages infected 80%, 70%, and 60% of CRKP isolates, respectively. Transmission electron microscopy demonstrated that the isolated phages belonged to distinct morphological groups resembling Myoviridae, Siphoviridae, and Podoviridae. Stability assays showed that the phages remained highly active between 4°C and 50°C and across a pH range of 5–9. One-step growth curve analysis revealed latent periods of 20–30 min and burst sizes ranging from 102 ± 9 to 145 ± 12 PFU/cell, indicating efficient replication and strong lytic potential. Among the isolated phages, vB_KpnP-1 exhibited the broadest host range, highest environmental stability, and greatest replication efficiency. Overall, the findings demonstrate that these bacteriophages possess favorable biological characteristics and represent promising candidates for the development of phage-based therapeutic strategies against multidrug-resistant K. pneumoniae infections.
Downloads
References
1. Ahmad, J., & Ahmad, S. (2023). Simultaneous application of non-antibiotics with antibiotics for enhanced activity against multidrug resistant pseudomonas aeruginosa.
2. Ahmad, J., & Pervez, H. (2021). Antimicrobial Activities of Medicinal Plant Rhamnus Virgata (Roxb.) Batsch from Abbottabad, Nathia Gali, KPK, Pakistan. Annals of the Romanian Society for Cell Biology, 25(7), 1502-1511.
3. Ali Syed, I., Alvi, I. A., Fiaz, M., Ahmad, J., Butt, S., Ullah, A., . . . Hayat, S. (2024). Synthesis of silver nanoparticles from Ganoderma species and their activity against multi drug resistant pathogens. Chemistry & Biodiversity, 21(4), e202301304.
https://doi.org/10.1002/cbdv.202301304
4. Anand, T., Virmani, N., Kumar, S., Mohanty, A. K., Pavulraj, S., Bera, B. C., . . . Tripathi, B. (2020). Phage therapy for treatment of virulent Klebsiella pneumoniae infection in a mouse model. Journal of Global Antimicrobial Resistance, 21, 34-41.
https://doi.org/10.1016/j.jgar.2019.09.018
5. Askoura, M., Saed, N., Enan, G., & Askora, A. (2021). Characterization of polyvalent bacteriophages targeting multidrug-resistant Klebsiella pneumonia with enhanced anti-biofilm activity. Applied Biochemistry and Microbiology, 57(1), 117-126.
https://doi.org/10.1134/s000368382101004x
6. Aziz, A., Ahmad, B., Ateeq, M., Gulfam, N., Bangash, S. A., Ullah, H., & Ahmad, J. (2022). Isolation and Identification of MDR Solmonella Typhi from Different Food Products in District Peshawar, KPK, Pakistan. Tobacco Regulatory Science (TRS), 555-572.
7. Cheetham, M. J., Huo, Y., Stroyakovski, M., Cheng, L., Wan, D., Dell, A., & Santini, J. M. (2024). Specificity and diversity of Klebsiella pneumoniae phage-encoded capsule depolymerases. Essays in Biochemistry, 68(5), 661-677.
https://doi.org/10.1042/ebc20240015
8. Chen, H., Liu, H., Gong, Y., Dunstan, R. A., Ma, Z., Zhou, C., . . . Zhou, T. (2024). A Klebsiella-phage cocktail to broaden the host range and delay bacteriophage resistance both in vitro and in vivo. npj Biofilms and Microbiomes, 10(1), 127.
https://doi.org/10.1038/s41522-024-00603-8
9. Fayez, M. S., Hakim, T. A., Agwa, M. M., Abdelmoteleb, M., Aly, R. G., Montaser, N. N., . . . El-Shibiny, A. (2021). Topically applied bacteriophage to control multi-drug resistant Klebsiella pneumoniae infected wound in a rat model. Antibiotics, 10(9), 1048.
https://doi.org/10.3390/antibiotics10091048
10. Gholizadeh, O., Ghaleh, H. E. G., Tat, M., Ranjbar, R., & Dorostkar, R. (2024). The potential use of bacteriophages as antibacterial agents against Klebsiella pneumoniae. Virology Journal, 21(1), 191.
https://doi.org/10.1186/s12985-024-02450-7
11. Gorodnichev, R. B., Volozhantsev, N. V., Krasilnikova, V. M., Bodoev, I. N., Kornienko, M. A., Kuptsov, N. S., . . . Slukin, P. V. (2021). Novel Klebsiella pneumoniae K23-specific bacteriophages from different families: similarity of depolymerases and their therapeutic potential. Frontiers in microbiology, 12, 669618.
https://doi.org/10.3389/fmicb.2021.669618
12. Hao, G., Shu, R., Ding, L., Chen, X., Miao, Y., Wu, J., . . . Wang, H. (2021). Bacteriophage SRD2021 recognizing capsular polysaccharide shows therapeutic potential in serotype K47 Klebsiella pneumoniae infections. Antibiotics, 10(8), 894.
https://doi.org/10.3390/antibiotics10080894
13. Hari, M. B., Sreelekshmi, A., Babu, P., Jaykumar, A. P., Barath, S., Magar, S., . . . Haripriyan, J. (2026). Novel bacteriophages effectively target multidrug-resistant clinical isolates of Klebsiella pneumoniae. European Journal of Clinical Microbiology & Infectious Diseases, 45(1), 271-286.
https://doi.org/10.1007/s10096-025-05300-8
14. Hayat, S., Ahmad, J., Khan, H. A., Tara, T., Ali, H., Sohail, M., & Ali, M. (2022). Antimicrobial Activity of Rhizome of Christella dentata.(forsk.) Brownsey & Jermy Against Selected Microorganisms. Tobacco Regulatory Science,[s. l].
15. Herridge, W. P., Shibu, P., O’Shea, J., Brook, T. C., & Hoyles, L. (2020). Bacteriophages of Klebsiella spp., their diversity and potential therapeutic uses. Journal of medical microbiology, 69(2), 176-194.
https://doi.org/10.7287/peerj.preprints.27890v1
16. Ichikawa, M., Nakamoto, N., Kredo-Russo, S., Weinstock, E., Weiner, I. N., Khabra, E., . . . Mordoch, R. (2023). Bacteriophage therapy against pathological Klebsiella pneumoniae ameliorates the course of primary sclerosing cholangitis. Nature communications, 14(1), 3261.
https://doi.org/10.1038/s41467-023-39029-9
17. Javed, S., Ahmad, J., Zareen, Z., Iqbal, Z., Hubab, M., Rehman, M. U., . . . Ahmed, S. (2023). Study on awareness, knowledge, and practices towards antibiotic use among the educated and uneducated people of Khyber Pakhtunkhwa Province, Pakistan. ABCS Health Sciences, 48, e023218-e023218.
https://doi.org/10.7322/abcshs.2021168.1881
18. Khan, S., Fiaz, M., Alvi, I. A., Ikram, M., Yasmin, H., Ahmad, J., . Ahmad, A. (2023). Molecular profiling, characterization and antimicrobial efficacy of silver nanoparticles synthesized from Calvatia gigantea and Mycena leaiana against multidrug-resistant pathogens. Molecules, 28(17), 6291.
https://doi.org/10.3390/molecules28176291
19. Kou, X., Yang, X., & Zheng, R. (2024). Challenges and opportunities of phage therapy for Klebsiella pneumoniae infections. Applied and environmental microbiology, 90(10), e01353-01324.
https://doi.org/10.1128/aem.01353-24
20. Laraib, S., Lutfullah, G., Nain Taara Bukhari, J. A., Almuhayawi, M. S., Ullah, M., Ullah, A., & Ashique10, S. (2023). Exploring the Antibacterial, Antifungal, and Anti-Termite Efficacy of Undoped and Copper-Doped ZnO Nanoparticles: Insights into Mutagenesis and Cytotoxicity in 3T3 Cell Line. JOURNAL OF BIOLOGICAL REGULATORS AND HOMEOSTATIC AGENTS, 37(12), 6731-6741.
https://doi.org/10.23812/j.biol.regul.homeost.agents.20233712.637
21. Latka, A., Lemire, S., Grimon, D., Dams, D., Maciejewska, B., Lu, T., .Briers, Y. (2021). Engineering the modular receptor-binding proteins of Klebsiella phages switches their capsule serotype specificity. MBio, 12(3), 10.1128/mbio. 00455-00421.
https://doi.org/10.1128/mbio.00455-21
22. Li, N., Zeng, Y., Bao, R., Zhu, T., Tan, D., & Hu, B. (2021). Isolation and characterization of novel phages targeting pathogenic Klebsiella pneumoniae. Frontiers in Cellular and Infection Microbiology, 11, 792305.
https://doi.org/10.3389/fcimb.2021.792305
23. Li, P., Guo, G., Zheng, X., Xu, S., Zhou, Y., Qin, X., . . . Ma, J. (2024). Therapeutic efficacy of a K5-specific phage and depolymerase against Klebsiella pneumoniae in a mouse model of infection. Veterinary Research, 55(1), 59.
https://doi.org/10.1186/s13567-024-01311-z
24. Munir, S., Amanat, T., Raja, M. A., Mohammed, K., Rasheed, R. A., Hussein, D. S., & Hayat, S. (2023). Antimicrobial efficacy of phyto-synthesized silver nanoparticles using aqueous leaves extract of Rosamarinus officinalis L. Pakistan journal of pharmaceutical sciences, 36(3), 941-946.
25. Poerio, N., Olimpieri, T., Henrici De Angelis, L., De Santis, F., Thaller, M. C., D’Andrea, M. M., & Fraziano, M. (2022). Fighting MDR-Klebsiella pneumoniae infections by a combined host-and pathogen-directed therapeutic approach. Frontiers in Immunology, 13, 835417.
https://doi.org/10.3389/fimmu.2022.835417
26. Rehman, A., Bashir, K., Farooq, S., Khan, A., Hassan, N., Sheraz, M., Ullah, A. (2023). Molecular analysis of aminoglycosides and β-lactams resistant genes among urinary tract infections. Bulletin of Biological and Allied Sciences Research, 2023(1), 56-56.
https://doi.org/10.54112/bbasr.v2023i1.56
27. Shah, R., Sarosh, I., Shaukat, R., Alarjani, K. M., Rasheed, R. A., Hussein, D. S., . . . Khan, M. K. (2023). Antimicrobial activity of AgNO 3 nanoparticles synthesized using Valeriana wallichii against ESKAPE pathogens. Pakistan journal of pharmaceutical sciences, 36.
28. Singh, A. N., Singh, A., Singh, S. K., & Nath, G. (2024). Klebsiella pneumoniae infections and phage therapy. Indian Journal of Medical Microbiology, 52, 100736.
https://doi.org/10.1016/j.ijmmb.2024.100736
29. Wang, C., Wang, S., Jing, S., Zeng, Y., Yang, L., Mu, Y., . . . Zhang, G. (2024). Data‐driven engineering of phages with tunable capsule tropism for Klebsiella pneumoniae. Advanced Science, 11(33), 2309972.
https://doi.org/10.1002/advs.202309972
30. Zhao, M., Li, H., Gan, D., Wang, M., Deng, H., & Yang, Q. E. (2024). Antibacterial effect of phage cocktails and phage-antibiotic synergy against pathogenic Klebsiella pneumoniae. Msystems, 9(9), e00607-00624.
Downloads
Published
Issue
Section
License
Copyright (c) 2026 Indus Journal of Bioscience Research
This work is licensed under a Creative Commons Attribution 4.0 International License.
