ABSTRACT
The genome sequences of 10 lytic bacteriophages targeting multidrug-resistant Klebsiella pneumoniae are reported. This group of bacteriophages, isolated from canal water and hospital effluent in Thailand, is composed of seven members of genus Drulisvirus and three unclassified Caudoviricetes.
KEYWORDS: Klebsiella pneumoniae, bacteriophages, Drulisvirus, unclassified Caudoviricetes, complete genomes
ANNOUNCEMENT
Klebsiella pneumoniae (Kp) infections have become increasingly difficult to treat due to the emergence of multidrug resistance (MDR), particularly carbapenem-resistant strains (1, 2). Bacteriophage (phage) therapy has become one of the potential alternative therapeutic treatments to combat MDR-Kp (1, 3). We report 10 lytic Kp phages: 3 unclassified Caudoviricetes and 7 belonging to genus Drulisvirus.
These Kp phages were isolated as part of a phage-hunting effort from various geographical areas in Asia. Local MDR-Kp clinical isolates were used as isolation hosts for respective phages (Table 1) (4). Collected water was filtered through 0.45 and 0.22 µm filters consecutively (5). The resulting filtrate (8 mL) was mixed with 2 mL Heart Infusion Broth (HIB, BD DIFCO) and 50 µL of fresh overnight culture of respective host (Table 1) at 37°C overnight (18–24 h). Enriched filtrates were titrated by spot assays on HIB double-layer agar, and plaques were isolated by overlay of respective host (Table 1) (6). Distinct plaques were selected, resuspended in SM buffer (100mM NaCl, 50mM Tris-HCl, pH 7.5, 10mM MgSO4, and 0.01% gelatin), and purified at least twice to obtain pure phage isolates. Purified phage was propagated in propagation media (HIB, 0.5 mM CaCl2, 2 mM MgCl2, and 0.1% glucose) and titrated to titer >1010 PFU/mL for nucleic acid extraction and then extracted with QIAamp DNA Mini Kit (Qiagen). DNA was quantified to ensure a minimum concentration of 1,000 ng/µL with Qubit dsDNA HS (High Sensitivity) Assay Kit (Thermo Fisher Scientific). Library preparation, quality control, and sequencing were performed commercially by BIONICS (Korea) using Enzymatic Preparation kit (EP-kit, Celemics, Inc., Korea) and MiSeq v2 (300 cycles) on Illumina MiSeq (Illumina). Analysis involved using the Bacterial and Viral Bioinformatics Resource Center (BV-BRC) for quality control (FastQC v0.12.0), trimming of raw reads (Trim Galore v0.6.5dev), de novo assembly (Unicycler), and genome polishing (Pilon v1.23) (7–11). All 10 Kp phages are linear double-stranded DNA viruses (12). All genomes were completed at >98% with high quality as evaluated by CheckV 1.0.1 on CheckV database V1.5 (13). Taxonomic classification was conducted using BLASTn against the National Center for Biotechnology Information (NCBI) nucleotide database in conjunction with VIRIDIC’s intergenomic similarity calculation between Kp phages and their closely related genomes for genus-level classification with >70% nucleotide identity as defined by the International Committee on Taxomony of Viruses (ICTV) (14, 15). Pharokka v1.7.3 did not identify lysogeny-related genes (16). Genomes were visualized using LoVis4u version 0.1.2 (17). All bioinformatics analyses were conducted using default parameters.
TABLE 1.
Genomic characteristics of seven Drulisvirus and three unclassified Kp bacteriophagesa
| Parameter | THK_08_1 | THK_18 | THQ_03 | THK_01 | THK_05 | THK_16 | THQ_05 | THQ_12 | THQ_16 | THQ_17 |
|---|---|---|---|---|---|---|---|---|---|---|
| Location | Bangkok | Bangkok | Chonburi | Bangkok | Bangkok | Bangkok | Chonburi | Chonburi | Chonburi | Chonburi |
| Sample source | Canal water | Canal water | Hospital effluent (pre-treated water) | Canal water | Canal water | Canal water | Hospital effluent (pre-treated water) | Hospital effluent (pre-treated water) | Hospital effluent (pre-treated water) | Hospital effluent (pre-treated water) |
| Genome size (bp) | 49,039 | 49,153 | 60,402 | 43,224 | 44,166 | 44,189 | 43,461 | 44,657 | 44,140 | 44,157 |
| Genome coverage (×) | 146.9 | 147.34 | 152.73 | 146.55 | 158.31 | 145.16 | 150.07 | 152.12 | 144.5 | 148.55 |
| GC content (%) | 46.91 | 47.23 | 45.46 | 52.07 | 52.91 | 52.81 | 53.08 | 53.22 | 52.96 | 52.73 |
| CheckV completeness (%) | 100 | 100 | 100 | 98.46 | 100 | 100 | 98.99 | 100 | 100 | 100 |
| CheckV quality | High-quality | High-quality | Complete | High-quality | High-quality | High-quality | High-quality | High-quality | High-quality | High-quality |
| No. of coding sequences (CDs) | 93 | 100 | 117 | 64 | 65 | 68 | 64 | 64 | 69 | 68 |
| No. of coding sequences (CDs) with predicted function | 28 | 31 | 39 | 29 | 28 | 26 | 26 | 26 | 27 | 26 |
| No. of hypothetical proteins | 65 | 69 | 78 | 35 | 37 | 42 | 38 | 38 | 42 | 42 |
| Lifestyle prediction | Lytic | Lytic | Lytic | Lytic | Lytic | Lytic | Lytic | Lytic | Lytic | Lytic |
| Closest related phage (accession number) | Klebsiella phage ZCKP8 (MZ440881) | Klebsiella phage RCIP0103 (OR532897) | Klebsiella phage phi_KPN_H2 (OQ267593) | Klebsiella phage phi1_146051 (PP889500) | Klebsiella phage vB_Kpn_K51PH129C1 (OY978783) | Klebsiella phagevB_KpnP_ IME323-1 (PQ998983) | Klebsiella phage PWKp1 (MZ634338) | Klebsiella phage KA (OR809313) | Klebsiella phagevB_KpnP_ IME323-1 (PQ998983) | Klebsiella phagevB_KpnP_ IME323-1 (PQ998983) |
| BLAST coverage (%) | 62 | 66 | 94 | 90 | 91 | 91 | 90 | 95 | 92 | 92 |
| BLAST identity (%) | 91.69 | 93.26 | 97.08 | 93.35 | 93.89 | 93.70 | 91.84 | 94.56 | 95.81 | 93.49 |
| Family | Unclassified | Unclassified | Unclassified | Autoscriptoviridae | Autoscriptoviridae | Autoscriptoviridae | Autoscriptoviridae | Autoscriptoviridae | Autoscriptoviridae | Autoscriptoviridae |
| Genus | Unclassified | Unclassified | Unclassified | Drulisvirus | Drulisvirus | Drulisvirus | Drulisvirus | Drulisvirus | Drulisvirus | Drulisvirus |
| GenBank accession no. | PZ103645 | PZ103646 | PZ103647 | PZ103638 | PZ103639 | PZ103640 | PZ103641 | PZ103642 | PZ103643 | PZ103644 |
| Biosample | SAMN55149949 | SAMN55149951 | SAMN55149952 | SAMN55149947 | SAMN55149948 | SAMN55149950 | SAMN55149953 | SAMN55149954 | SAMN55149955 | SAMN55149956 |
| Sequence Read Archive accession no. | SRR37136277 | SRR37136275 | SRR37136274 | SRR37136279 | SRR37136278 | SRR37136276 | SRR37136273 | SRR37136272 | SRR37136271 | SRR37136270 |
| No. of raw reads | 98,137 | 80,282 | 95,485 | 100,811 | 109,389 | 104,701 | 84,235 | 102,371 | 92,731 | 94,589 |
| No. of spots | 98,137 | 80,282 | 95,485 | 100,811 | 109,389 | 68,707 | 84,235 | 102,371 | 92,731 | 94,589 |
| Host: Klebsiella pneumoniae | AMR-0258 | AMR-1872 | AMR-0139 | AMR-0123 | AMR-0135 | AMR-0999 | AMR-0135 | AMR-0327 | AMR-0999 | AMR-1400 |
GPS locations and collection dates: Canal water: 13°43′11.9″N, 100°33′38.6″E, 13.719961, 100.560729, 22 April 2024; Hospital effluent: 12°41′02.9″N, 100°59′14.3″E, 12.684136, 100.987304, 9 April 2024.
The three unclassified Kp phages (THK_08_1, THK_18, and THQ_03) belong to class Caudoviricetes. THK_08_01 and THK_18 share >90% nucleotide identity across >50% genome length. THQ_03 had no detectable sequence similarity to either THK_08_01 or THK_18 and had a larger genome (Table 1 and Fig. 1A), indicating that it belongs in a different subclass (15).
Fig 1.
Comparative genomic analysis of 10 Klebsiella phages reported in this study. (A) Visualization of Drulisvirus, unclassified phages, and their closest relatives by LoVis version 0.1.6. The predicted coding sequences (CDSs) are depicted as arrows, and the direction indicates orientation. The color code for gene function is provided at the bottom of the figure. (B) Intergenomic similarity heatmap generated by VIRIDIC (15) based on BLASTn, visualizing percent nucleotide identity, genome alignment fractions, and length ratios. Reference genomes retrieved from NCBI GenBank for comparison are comprised of Klebsiella phages ZCKP8 (MZ440881), RCIP0103 (OR532897), phi_KPN_H2 (OQ267593), phi1_146051 (PP889500), vB_Kpn_K51PH129C1 (OY978783), vB_KpnP_IME323-1 (PQ998983), PWKp1 (MZ634338), vB_KpnP_SU552A (NC_0028870), vB_KpnP_KpV41 (NC_028670), and KA (OR809313).
The genome sizes of the Drulisvirus phages (THK_01, THK_05, THK_16, THQ_05, THQ_12, THQ_16, and THQ_17) range from 43,224 to 44,657 bp with 52.07% to 53.22% G + C content (Table 1). They all share a nucleotide similarity of >70% with Drulisvirus genus from the most closely related genomes from the NCBI database, as illustrated by the VIRIDIC heatmap (Fig. 1B) (15). Genomic comparison revealed that THK_16, THQ_16, and THQ_17 have nearly identical sequences, indicating that they are closely related isolates of the same lineage.
ACKNOWLEDGMENTS
This project was funded by the Military Infectious Diseases Research Program under grant MI240071.
The authors would like to thank the staff of the Queen Sirikit Naval Hospital, Chonburi, Thailand, for providing water sources.
The views expressed in this publication reflect the results of research conducted by the authors and do not necessarily reflect the official policy or position of the Defense Health Agency, U.S. Army, Department of War, nor the U.S. Government.
Contributor Information
Paphavee Lertsethtakarn, Email: PaphaveeL.fsn@afrims.org.
Kenneth M. Stedman, Portland State University, Portland, Oregon, USA
DATA AVAILABILITY
https://www.ncbi.nlm.nih.gov/bioproject/PRJNA814829/The genomic sequences of Kp hosts are available under BioProject PRJNA814829. The sequences of Kp phages are available under BioProject PRJNA1287271. The accession numbers for GenBank, Sequence Read Archive, and Biosample are listed in Table 1.
REFERENCES
- 1. Macesic N, Uhlemann A-C, Peleg AY. 2025. Multidrug-resistant gram-negative bacterial infections. The Lancet 405:257–272. doi: 10.1016/S0140-6736(24)02081-6 [DOI] [PubMed] [Google Scholar]
- 2. Qala Nou MS, Amirian Z, Dehghani F, Vejdan A-K, Rooin R, Dehghanmehr S. 2025. Systematic review and meta-analysis on the carbapenem-resistant hypervirulent Klebsiella pneumoniae isolates. BMC Pharmacol Toxicol 26:25. doi: 10.1186/s40360-025-00857-8 [DOI] [PMC free article] [PubMed] [Google Scholar]
- 3. Xing J, Han R, Zhao J, Zhang Y, Zhang M, Zhang Y, Zhang H, Nang SC, Zhai Y, Yuan LY, Wang S, Wu H. 2025. Revisiting therapeutic options against resistant Klebsiella pneumoniae infection: phage therapy is key. Microbiol Res 293:128083. doi: 10.1016/j.micres.2025.128083 [DOI] [PubMed] [Google Scholar]
- 4. Ruekit S, Srijan A, Serichantalergs O, Margulieux KR, Mc Gann P, Mills EG, Stribling WC, Pimsawat T, Kormanee R, Nakornchai S, Sakdinava C, Sukhchat P, Wojnarski M, Demons ST, Crawford JM, Lertsethtakarn P, Swierczewski BE. 2022. Molecular characterization of multidrug-resistant ESKAPEE pathogens from clinical samples in Chonburi, Thailand (2017-2018). BMC Infect Dis 22:695. doi: 10.1186/s12879-022-07678-8 [DOI] [PMC free article] [PubMed] [Google Scholar]
- 5. Lertsethtakarn P, Chaisatit C, Gonwong S, Nobthai P, Silapong S, Thaloengsok S, Ruamsap N, Nikolich M, Boudreaux D. 2025. Complete genome sequences of two Klebsiella pneumoniae bacteriophages in genus Marfavirus. Microbiol Resour Announc 14:e00823-25. doi: 10.1128/mra.00823-25 [DOI] [PMC free article] [PubMed] [Google Scholar]
- 6. Sergueev KV, Filippov AA, Farlow J, Su W, Kvachadze L, Balarjishvili N, Kutateladze M, Nikolich MP. 2019. Correlation of host range expansion of therapeutic bacteriophage Sb-1 with allele state at a hypervariable repeat locus. Appl Environ Microbiol 85:e01209-19. doi: 10.1128/AEM.01209-19 [DOI] [PMC free article] [PubMed] [Google Scholar]
- 7. Olson RD, Assaf R, Brettin T, Conrad N, Cucinell C, Davis JJ, Dempsey DM, Dickerman A, Dietrich EM, Kenyon RW, et al. 2023. Introducing the Bacterial and Viral Bioinformatics Resource Center (BV-BRC): a resource combining PATRIC, IRD and ViPR. Nucleic Acids Res 51:D678–D689. doi: 10.1093/nar/gkac1003 [DOI] [PMC free article] [PubMed] [Google Scholar]
- 8. Andrews S. 2010. A quality control tool for high throughput sequence data. https://www.bioinformatics.babraham.ac.uk/projects/fastqc.
- 9. Krueger F. 2012. A wrapper tool around Cutadapt and FastQC to consistently apply quality and adapter trimming to FastQ files, with some extra functionality for MspI-Digested RRBS-Type (Reduced Representation Bisufite-Seq) libraries. Available from: http://www.bioinformatics.babraham.ac.uk/projects/trim_galore
- 10. Wick RR, Judd LM, Gorrie CL, Holt KE. 2017. Unicycler: resolving bacterial genome assemblies from short and long sequencing reads. PLoS Comput Biol 13:e1005595. doi: 10.1371/journal.pcbi.1005595 [DOI] [PMC free article] [PubMed] [Google Scholar]
- 11. Walker BJ, Abeel T, Shea T, Priest M, Abouelliel A, Sakthikumar S, Cuomo CA, Zeng Q, Wortman J, Young SK, Earl AM. 2014. Pilon: an integrated tool for comprehensive microbial variant detection and genome assembly improvement. PLoS One 9:e112963. doi: 10.1371/journal.pone.0112963 [DOI] [PMC free article] [PubMed] [Google Scholar]
- 12. Adriaenssens EM, Cook R, Dolja V, Koonin EV, Krupovic M, Kuhn JH, Lood C, Reyes Muñoz A, Turner D, Varsani A, Vaz PK, Waltzek T, Wolf YI, Yutin N, Zerbini FM. 2025. ICTV virus taxonomy profile: duplodnaviria 2025. J Gen Virol 106:1–2. doi: 10.1099/jgv.0.002139 [DOI] [PMC free article] [PubMed] [Google Scholar]
- 13. Nayfach S, Camargo AP, Schulz F, Eloe-Fadrosh E, Roux S, Kyrpides NC. 2021. CheckV assesses the quality and completeness of metagenome-assembled viral genomes. Nat Biotechnol 39:578–585. doi: 10.1038/s41587-020-00774-7 [DOI] [PMC free article] [PubMed] [Google Scholar]
- 14. Altschul SF, Gish W, Miller W, Myers EW, Lipman DJ. 1990. Basic local alignment search tool. J Mol Biol 215:403–410. doi: 10.1016/S0022-2836(05)80360-2 [DOI] [PubMed] [Google Scholar]
- 15. Moraru C, Varsani A, Kropinski AM. 2020. VIRIDIC—a novel tool to calculate the intergenomic similarities of prokaryote-infecting viruses. Viruses 12:1268. doi: 10.3390/v12111268 [DOI] [PMC free article] [PubMed] [Google Scholar]
- 16. Bouras G, Nepal R, Houtak G, Psaltis AJ, Wormald PJ, Vreugde S. 2023. Pharokka: a fast scalable bacteriophage annotation tool. Bioinformatics 39:btac776. doi: 10.1093/bioinformatics/btac776 [DOI] [PMC free article] [PubMed] [Google Scholar]
- 17. Egorov AA, Atkinson GC. 2025. LoVis4u: a locus visualization tool for comparative genomics and coverage profiles. NAR Genom Bioinform 7:lqaf009. doi: 10.1093/nargab/lqaf009 [DOI] [PMC free article] [PubMed] [Google Scholar]
Associated Data
This section collects any data citations, data availability statements, or supplementary materials included in this article.
Data Availability Statement
https://www.ncbi.nlm.nih.gov/bioproject/PRJNA814829/The genomic sequences of Kp hosts are available under BioProject PRJNA814829. The sequences of Kp phages are available under BioProject PRJNA1287271. The accession numbers for GenBank, Sequence Read Archive, and Biosample are listed in Table 1.