Draft genome sequence of carbapenem-resistant Klebsiella pneumoniae ST6260 isolated from the catheter tip of a female patient in Nepal

TL;DR

This paper reports the draft genome sequence of a drug-resistant Klebsiella pneumoniae strain isolated from a patient in Nepal.

Contribution

The study provides a new draft genome sequence of a carbapenem-resistant K. pneumoniae ST6260 strain from Nepal.

Findings

A carbapenem-resistant Klebsiella pneumoniae ST6260 was isolated from a catheter tip in a female patient.

The genome sequence was generated to better understand its resistance mechanisms and potential for spread.

Abstract

Klebsiella pneumoniae is an opportunistic human pathogen, particularly associated with nosocomial infections and multidrug resistance. Here, we present a draft genome sequence of a carbapenem-resistant K. pneumoniae ST6260 isolated from the catheter tip of a female patient in a referral case received at Kathmandu Model Hospital, Kathmandu, Nepal.

Tables

TABLE 1. Summary of AMR determinants identified in the K. pneumoniae ST6260 genome from Nepala

	Size (bp)	No. of contigs	AMR determinants
Chromosome	5,318,348	91	blaSHV-1, dfrA12, aadA2, fosA, oqxA, oqxB;GyrA-83Y, GyrA-87G, ParC-80I
Plasmid 1 (IncFIB, IncFII, IncR; mobilizable)	148,151	18	blaTEM-1A, blaOXA-9, ant(3″)-Ia, aac(6')-Ib, sul1, blaNDM-5, erm(B), mph(A), rmtB
Plasmid 2 (IncFIB; non-mobilizable)	43,101	2	–b
Plasmid 3 (no rep; mobilizable)	23,345	2	–
Plasmid 4 (no rep; non-mobilizable)	19,072	1	–

Full text

ANNOUNCEMENT

Klebsiella pneumoniae was isolated from the catheter tip of a 27-year-old female. A 5 cm distal tip was received in a sterile container at the hospital (N27°42′9.1656″, E85°19′12.5832″), cultured on MacConkey agar (HiMedia, India) plate using the rolled plate method (1), and then incubated at 37°C for 24 hours. A single colony from the plate was grown in Nutrient broth (HiMedia, India) at 37°C for 4 hours and turbidity matched with 0.5 McFarland for antibiotic susceptibility testing. Antibiotic susceptibility was detected by the disc diffusion method, and carbapenemase production was confirmed by the modified carbapenem inactivation method, as described by CLSI guidelines (2). The isolate was preserved in Tryptic Soy Broth (HiMedia, India) (with 20% glycerol) at −80°C. Later, it was revived onto a Nutrient agar (HiMedia, India) plate for DNA extraction. A single colony from the plate was transferred into Luria-Bertani (LB) (HiMedia, India) broth for overnight incubation at 37°C. The DNA was extracted from LB broth using the CTAB method (3), and quality was determined through 260/280 nm absorbance measures using a NanoDrop spectrophotometer 1000 (Thermo Scientific, USA). Then, the genomic DNA was sent to MicrobesNG for whole-genome sequencing.

Genomic DNA libraries were prepared using the Nextera XT Library Prep Kit (Illumina, USA) following the manufacturer’s protocol with the following modifications: input DNA was increased twofold, and PCR elongation time was increased to 45 seconds. DNA quantification and library preparation were carried out on a Hamilton Microlab STAR automated liquid handling system (Hamilton Bonaduz AG, Switzerland). Libraries were sequenced on an Illumina NovaSeq 6000 (Illumina, USA) using a 250 bp paired end protocol (~890,000 reads per pair, average coverage 70×). Raw reads were checked using FastQC version 0.11.9 (4). Trimmomatic version 0.39 (5) was used to trim reads with the following parameters: LEADING:20 TRAILING:20 SLIDING WINDOW:5:30 MINLEN:50. Species identification was done with Kraken2 version 2.1.43 (6) and Bracken version 2.9 (7). De novo assembly was performed using SPAdes version 3.15.2 (8) with careful mode activated and selecting k-mers up to 127. Bioawk version 1.0 (9) was then used to filter out contigs with size <200 bp and SPAdes coverage <2. Assembly statistics were assessed using QUAST version 5.0.2 (10) and CheckM2 version 1.2.3 (11). PGAP version 6.9 (12) was used for annotation. Kleborate version 2.5 (13) was used to report MLST (13) (BIGSdb database), virulence loci, AMR genes, and K and O locus via Kaptive version 2.0.4 (14). Plasmid reconstruction was performed using MOB-suite version 3.1.8 (15). Default parameters were used except where otherwise stated.

Draft genome assembly stats were as follows: 5,568,701 bp, 117 contigs, 57% GC content, and N50 of 187,282 bp. The draft genome contains a total of 5,585 genes, of which 5,321 were coding DNA sequences, 79 tRNA, 27 rRNA (6 complete and 21 partial), and 149 pseudogenes. K. pneumoniae genotype was ST6260. The predicted capsule type was K17 (locus K17) and O type O1 (locus O1/O2v1). Kleborate predicted a virulence score of 1 with yersiniabactin ybt9 and truncated ICEKp3, and a resistance score of 2, indicating that carbapenemase without colistin resistance was detected, regardless of ESBL genes or OmpK mutations. AMR determinants and plasmids identified are summarized in Table 1.