Complete genome sequences of two Klebsiella pneumoniae bacteriophages in genus Marfavirus

ABSTRACT

The complete genomes of two Klebsiella phages isolated in Thailand, THK_07 and THQ_07, belonging to genus Marfavirus are reported. The genome sizes are 169,304 and 169,451 bp for THK_07 and THQ_07, respectively, with 40% GC content and seven tRNAs identified in both phages.

ANNOUNCEMENT

Klebsiella pneumoniae (KP) has become an increasing global health threat because of its ability to acquire exogenous antimicrobial resistance genes, contributing to an emergence of carbapenem-resistant and extended-spectrum β-lactamase-producing strains (1–4). Bacteriophage therapy is a potential alternative or adjunctive antimicrobial as empirical antibiotic therapy becomes less effective. We describe the genomes of two Klebsiella phages from genus Marfavirus, THK_07 and THQ_07.

Water from a canal in Bangkok and hospital effluent from Thailand was screened for bacteriophages using a panel of local clinical KP isolates. Both of the phages were isolated from KP strain AMR-0197 (BioProject: PRJNA814829). Collected water (250 mL) was centrifuged, filtered, (0.45 and 0.22 µm), enriched by mixing 8 mL of filtrate with 2 mL of 5× Heart Infusion Broth (HIB) (Becton Dickinson) and 50 µL of an overnight AMR-0197, followed by an overnight incubation at 37°C with shaking. Enriched filtrates were titrated by spot assays on HIB double-layer agar. An overnight AMR-0197 (100 µL) was mixed with 100 µL of serially diluted enriched filtrate (approximately 100-fold below the pre-determined titer) in SM buffer, and 100 µL of the mixture was mixed with 0.7% HIB soft agar, then overlaid on 1.5% HIB agar plates. Distinct plaques were picked, resuspended in SM buffer, and filtered, at least two times or up to three times to obtain pure phage isolates.

Bacteriophages were propagated to titer above 1 × 10¹⁰ PFU/mL, and genomic DNA was extracted with QIAamp DNA Mini Kit (Qiagen). DNA was quantified with Qubit dsDNA HS (High Sensitivity) Assay Kit (Invitrogen by ThermoFisher Scientific) to ensure that the concentration was at a minimum of 1,000 ng/µL. Library preparation, quality control, and sequencing were performed commercially by BIONICS (Seoul, Korea) using Enzymatic Preparation kit (EP-kit, Celemics, Inc., Korea) and MiSeq v2 (300 cycles) for sequencing on Illumina MiSeq instrument (Illumina, San Diego, CA, USA). An analysis was performed using an assembly service tool on Bacterial and Viral Bioinformatics Resource Center server (5). The protein coding sequences (CDSs) were annotated using Pharokka v1.7.3 and the corresponding database v1.4.0 (6). PhageTerm and CheckV were used to analyze genome termini and completeness, respectively (7, 8). All software’s default parameters were used.

A summary of genome sequences is in Table 1. Genomic analysis revealed that no lysogenic-related genes were present (Fig. 1A). Both genomes were predicted to be circularly permuted and terminally redundant. For taxonomic classification, closely related genomes were obtained from the NCBI database and the intergenomic similarity was analyzed using VIRIDIC (9), showing >88% intergenomic similarity to reference Marfavirus genomes (Fig. 1B).

TABLE 1.

A complete genome sequencing results for THK_07 and THQ_07

	THK_07	THQ_07
Location (Province)	Bangkok	Chonburi
Sample source	Canal water	Hospital effluent (pre-treated water)
No. of raw reads	101,154	108,207
No. of spots	68,537	108,207
Total length of complete genome (bp)	169,304	169,451
Genome coverage (×)	142.89	141.82
CheckV completeness	99.21%	99.05%
CheckV quality	High-quality	High-quality
GC content (%)	40.84%	40.95%
No. of CDSs	303	302
No. of genes with predicted function	134	135
No. of tRNAs	7	7
Taxonomic identification (genus)	Marfavirus	Marfavirus
GenBank accession no.	PV963843	PV963844
Biosample	SAMN49815975	SAMN49815976
SRA accession no.	SRR34390307	SRR34390306

Fig 1.

Genomic analysis THK_07 and THQ_07. (A). Visualization of genomic comparative analysis of THK_07, THQ_07, and closely related phages’ genomes using EasyFig version 2.2.2 (https://mjsull.github.io/Easyfig/). Functional gene groups are indicated in different colors (see legend). BLASTn hits are represented by gray bars, with darker shades indicating higher percent identity. Functional categories of phage proteins in Klebsiella phages vB_Kpn_F48 (GenBank accession no. MG746602) and Klebsiella phage marfa (GenBank accession no. MN044033) were extracted from the PHROG database. (B). VIRIDIC heatmap demonstrating intergenomic similarity between THK_07 and THQ_07 to closely related references: Klebsiella phages marfa and Klebsiella phage vB_Kpn_F48 (9). The darker color with percent similarities (%) indicates high intergenomic similarities between genomes and alignment genome fractions (0.9 and 1.0) are presented underneath.

Testing of these phages on an expanded panel of locally isolated multidrug resistance KP (38) showed that both phages were able to target additional 29 and 26 of KP hosts, respectively. These preliminary findings suggest that they could be considered as candidates in the development of bacteriophage cocktail therapy.

DATA AVAILABILITY

The genomic sequences of KP host AMR-0197 are available at https://www.ncbi.nlm.nih.gov/biosample/SAMN28097302, BioProject: PRJNA814829, BioSample: SAMN28097302; Sample name: AMR-0197; SRA: SRS12903102. The sequences of THK_07 and THQ_07_02 are available at https://www.ncbi.nlm.nih.gov/bioproject/?term=PRJNA1287271 under BioProject: PRJNA1287271.