Simultaneous gut colonization by Klebsiella grimontii and Escherichia coli co-possessing the blaKPC-3-carrying pQil plasmid

Abstract

Only two plasmid-mediated carbapenemases (KPC-2 and VIM-1) are reported in Klebsiella grimontii. Here, we report two bla_KPC-3-positive isolates that were identified as K. oxytoca and E. coli by MALDI-TOF MS in the same rectal swab. Whole-genome sequencing indicated that K. oxytoca was actually K. grimontii of ST391, whereas E. coli was of ST10. In both, bla_KPC-3 was carried by a pQil conjugative plasmid. The core-genome analysis identified additional bla_KPC-positive K. grimontii strains from public databases, most of which were misidentified as K. oxytoca. Since K. grimontii represents an emerging reservoir of resistance traits, routine tools should improve their ability to detect this species.

Keywords: Carbapenemase; Conjugation; K. grimontii; K. oxytoca; KPC; Plasmid; pQil.

Fig. 1

Circular BLASTn comparison of the bla_KPC-3-carrying plasmid in LC-1302–2020 and LC-1303–2002 against other deposited plasmids. Plasmids and their similarities are represented by the colored rings. The CDS/genes and IS elements of interest are represented by colored arrows (red: bla_KPC-3; blue: other ARGs; black: IS elements; orange: replicon genes; green: replicon sequence type) with corresponding annotations (red: bla_KPC-3; blue: other ARGs). The approximate regions for the sil, copACBD, and ars operons, as well as tra genes, are indicated by the dashed lines and purple CDS/genes. The approximate region of the transposon associated with bla_KPC-3 (Tn4401a) is shown above with dashed lines. For the plasmid comparison, we show the carbapenemase gene of the reference plasmid (in black), name, main replicon sequence type, size, and the GenBank accession (in blue); on the left, we show the GC content, GC skew, and the metadata corresponding to the plasmids used for the circular BLASTn comparison (GenBank accession [in blue], plasmid name, main replicon sequence type, size, bla_KPC [in red], isolation year, country, isolation source, and host. The IS annotations shown were annotated with ISfinder (https://www-is.biotoul.fr/) using BLASTx search. The circular BLASTn comparison was generated with BLAST Ring Image Generator v0.95 (https://github.com/happykhan/BRIG)

Fig. 2

Core genome phylogeny of LC-1303–2020 and other bla_KPC-2/-3-carrying K. grimontii (n = 12). A total of 8 K. grimontii genomes included from publicly available databases (retrieval date: 16–18/Feb/2022; NCBI Genomes, n = 143; Pathogen Watch, n = 99) were screened for bla_KPC and bla_OXY-6 genes with Kleborate v2.0.4 with default parameters. Sequence type was determined with MLST v2.19.0 using the K. oxytoca scheme. Simultaneously, the NCBI Pathogen Detection web tool (https://www.ncbi.nlm.nih.gov/pathogens) was used to identify K. grimontii genomes deposited under the K. oxytoca organism group (n = 1000; query: “taxgroup_name:Klebsiella oxytoca AND AMR_genotypes:blaOXY-6* AND AMR_genotypes:blaKPC*”), which resulted in 3 nonredundant bla_KPC- and bla_OXY-6-positive K. grimontii genomes. Genomes with no BioSample metadata were excluded (n = 30). Genome assemblies were generated with SPAdes v3.14.0 with read correction (parameter: careful) and used for final species ID with TYGS, ARG and replicon sequence screening with the CGE tools (ResFinder v4.1; PlasmidFinder v2.1). A recombination-free core genome alignment was conducted with Snippy v4.4.5 and Gubbins v2.3.4 with default parameters using the complete genome of LC-1303–2020 as reference. Phylogeny was inferred by maximum likelihood with IQ-TREE v2.1.2 using a GTR nucleotide substitution model with ascertainment bias correction (parameter: GTR + ASC) and 1000 ultrafast bootstrap (UFBoot) (parameter: -bb) and the SH-aLRT test (parameter: -alrt). The tree was visualized and annotated with iTOL v1.6. Countries are represented by the colored circles; strain or isolate name and collection date are highlighted by isolation source (as per BioSample metadata). Delta SNVs (∆SNVs) represent core genome similarities between two or more genomes. Bootstrap support values are shown on branches (SH-aLRT and UFboot, respectively). The tree scale represents the average number of nucleotide substitutions per site. ^aCarbapenemase gene. ^bOther bla genes present; an asterisk corresponds to a variant from the same family. An asterisk in bla_OXA in LC-1303–2020 corresponds to Δbla_OXA-1-like. ^cReplicon sequence types identified by PlasmidFinder at 50% minimum identity.