ABSTRACT
The emergence of convergent Klebsiella pneumoniae strains showing multiresistance, characteristic of nosocomial pathotypes and hypervirulent traits typical of community-acquired isolates, makes them important models for studying K. pneumoniae pathogenesis. Here, we describe the convergent, multidrug-resistant KLEB-33 strain harboring several hypervirulence genes and make its genome available to the scientific community.
KEYWORDS: Klebsiella pneumoniae, multidrug resistance, hypervirulence, convergent, OXA-48, ST16, biofilm formation, hypermucoviscous
ANNOUNCEMENT
The emergence of convergent strains of Klebsiella pneumoniae (1, 2), showing multidrug resistance typical of the classic pathotype (cKp) and hypervirulence traits (hvKp pathotype) (3, 4), prompted us to make public the draft genome of KLEB-33 (LREC_296), a multiresistant, hypermucoviscous, biofilm-producing convergent strain (5), harboring multiple hypervirulence genes, belonging to the sequence type ST16 (cgMLST 1705), an emerging high-risk lineage similar to other high-risk lineages spread worldwide, such as ST11 (6). The phenotypic and genotypic characteristics of this strain (Table 1) make it a strong candidate for use as a model for convergent multidrug-resistant strains of K. pneumoniae, and therefore, the availability of its genomic sequence will be a valuable resource for the scientific community.
TABLE 1.
General genomic features of the K. pneumoniae KLEB-33 isolate
| Acquired resistance determinants | Gene | Identity (%) | Reference coverage (nt) |
|---|---|---|---|
| Beta-lactam | bla OXA-48 | 100.00 | 798/798 |
| bla SHV-1 | 99.88 | 861/861 | |
| Fosfomycin | fosA5 | 95.00 | 420/420 |
| Disinfectant | oqxB | 98.89 | 3,153/3,153 |
| oqxA | 99.15 | 1,176/1,176 |
The KLEB-33 isolate was recovered from a biliary drainage of a patient diagnosed with cholangitis at the Hospital Universitario Central de Asturias (Spain) in October 2018 as part of a study approved by the Institutional Ethics Committee (CEImPA 03/2018). The strain was recovered with a swab and isolated on Columbia agar 24 h/37°C in aerobiosis. Bacterial identification was performed by Matrix-Assisted Laser Desorption/Ionization - Time of Flight (MALDI-TOF) mass spectrometry. This OXA-48 carbapenemase-producing strain is resistant to ertapenem, ampicillin, amoxicillin-clavulanic acid, piperacillin-tazobactam, ciprofloxacin, and nitrofurantoin, as assessed by the broth microdilution susceptibility method with the MicroScan Neg MIC 57 panel (Walwaway Plus, Beckman Coulter) and confirmed by Etest (bioMérieux). European Committee on Antimicrobial Susceptibility Testing v13.0 clinical breakpoints and guidelines for Enterobacteriaceae were used for interpretation.
Genomic DNA (gDNA) was extracted from 1 mL Luria-Bertani (LB) culture (~109 CFU/mL) incubated in aerobiosis, 180 rpm, 37°C/16–20 h, using the Purelink genomic DNA mini kit (Thermo Fisher Scientific, Waltham, USA). The quantity and quality of gDNA were assessed using Qubit (v4.0) and Nanodrop ND-1000 (Thermo Fisher Scientific), respectively. Whole Genome Sequencing (WGS) libraries were prepared using the Nextera DNA Flex library preparation kit, followed by paired-end sequencing (2 × 150 bp) on a NextSeq 500 device (Illumina, Inc., San Diego, CA, USA). Sequencing quality assessment and genome assembly of the raw reads were performed using the “Aquamis” pipeline (https://gitlab.com/bfr_bioinformatics/AQUAMIS/; settings: default; accession: October 2022) (7). The genome sequence comprises 5,466,901 nt assigned to 73 contigs (sequencing depth ≥ 80 per consensus base; N50: 273,927 nt; GC content: 57.24%; number of raw reads: 1,947,940). In the first version of the genome, the sequence is not closed (still segmented into contigs) and is not circular.
The “BakCharak” pipeline was run for initial in silico typing (https://gitlab.com/bfr_bioinformatics/bakcharak; settings: default; accession: October 2022), and the automated prokaryotic genome annotation pipeline (v6.3, National Center for Biotechnology Information) was used for genome annotation (8). Surface polysaccharide locus prediction (capsid and O-type/variant) and cgST determination were performed with Pathogenwatch (https://pathogen.watch). The presence of virulence and hypervirulence determinants in the genomic assemblies was predicted using the Klebsiella locus/sequence definitions database and the Bacterial Isolate Genome Sequence Database software of the Institut Pasteur (https://bigsdb.pasteur.fr/klebsiella/) (9). The presence of hypervirulence genes was confirmed using the BLASTn tool. General genomic characteristics and in silico analysis are summarized in Table 1.
ACKNOWLEDGMENTS
This study was supported by the project PID2019-104439RB-C21/AEI/10.13039/501100011033 from the Agencia Estatal de Investigación (AEI, Spain), and grants ED431C 2021/11 and ED431B2023/41 from the Consellería de Cultura, Educación e Ordenación Universitaria (Xunta de Galicia). S.S.-B. acknowledges the Spanish Ministry of Sience, Innovation and Universities for his pre-doctoral program FPU21/01147. M.R. was supported by the Maria Zambrano program and Research Consolidation grant (CNS2023-145299) from the Spanish Ministry of Sience, Innovation and Universities. I.G.-M. acknowledges the Consellería de Cultura, Educación e Ordenación Universitaria, Xunta de Galicia, for their post-doctoral grant (ED481B-2021-006).
Contributor Information
Ana Otero, Email: anamaria.otero@usc.es.
Catherine Putonti, Loyola University Chicago, Chicago, Illinois, USA.
DATA AVAILABILITY
The Whole Genome Shotgun project of K. pneumoniae KLEB-33 (LREC_296) was deposited in GenBank under the accession no. JAPJTV000000000.1. The raw sequencing data are accessible in the SRA database with the following link (https://www.ncbi.nlm.nih.gov/sra/PRJNA891646), under the BioProject PRJNA891646. Sequencing raw reads and assembly data are linked to BioSample SAMN31340319. The SRA records can also be retrieved from the European Nucleotide Archive (ENA) (https://www.ebi.ac.uk/ena/browser/view/PRJNA891646). The version described in this paper is the first version.
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Associated Data
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Data Availability Statement
The Whole Genome Shotgun project of K. pneumoniae KLEB-33 (LREC_296) was deposited in GenBank under the accession no. JAPJTV000000000.1. The raw sequencing data are accessible in the SRA database with the following link (https://www.ncbi.nlm.nih.gov/sra/PRJNA891646), under the BioProject PRJNA891646. Sequencing raw reads and assembly data are linked to BioSample SAMN31340319. The SRA records can also be retrieved from the European Nucleotide Archive (ENA) (https://www.ebi.ac.uk/ena/browser/view/PRJNA891646). The version described in this paper is the first version.