ABSTRACT
Originally thought to be a single species, Klebsiella pneumoniae has been divided into three distinct species: K. pneumoniae, K. quasipneumoniae and K. variicola. In a recent study of 1,777 extended-spectrum beta-lactamase (ESBL)-producing Klebsiella strains recovered from human infections in Houston, we discovered one strain (KPN1705) causing a wound infection that was phylogenetically distinct from all currently recognized Klebsiella species. Whole genome sequencing of strain KPN1705 revealed that it was single locus variant of the multilocus sequence type ST-1155. This sequence type was reported only once previously. To further investigate the phylogeny of these two organisms, we sequenced the genome of strain KPN1705 to closure and compared its genetic features to Klebsiella reference strains. Results demonstrated strain KPN1705 extensively shares core gene content, antimicrobial resistance genes, and plasmids with K. pneumoniae, K. quasipneumoniae and K. variicola. Since strain KPN1705 and the previously reported novel strain are phylogenetically most closely related to K. variicola, we propose the name K. quasivariicola sp. nov.
IMPORTANCE
K. pneumoniae, K. quasipnuemoniae and K. variicola are serious human pathogens that are increasingly associated with multidrug resistance and high morbidity and mortality. In a recent study of a large, comprehensive, population-based collection of antibiotic resistant Klebsiella isolates recovered from human patients, we discovered a novel species that is related to but distinct from K. variicola. This clonal group has been reported only once previously. We sequenced the genome of this clinical isolate and compared its genetic features to other Klebsiella strains. We propose the name K. quasivariicola sp. nov. for this new species.
OBSERVATION
Members of the genus Klebsiella are a common cause of human morbidity and mortality (1, 2). Many community-acquired and healthcare-associated outbreaks of invasive K. pneumoniae disease have been reported (3, 4). Over the past two decades, related Klebsiella species have been identified as distinct from K. pneumoniae and classified (5-8). In a recent large, comprehensive, population based study of 1,777 extended-spectrum beta-lactamase (ESBL) producing Klebsiella strains recovered in our clinical microbiology laboratory, we discovered a unique isolate KPN1705 (9, 10). It was genetically related to, but distinct from, K. variicola. We sequenced the genome of this strain, which belongs to a new species herein termed Klebsiella quasivariicola sp. nov., to closure and compared its genetic features to other Klebsiella reference strains.
RESULTS
Whole genome sequencing reveals Klebsiella quasivariicola sp. nov., a novel Klebsiella pathogenic to humans
In a recent study of 1,777 ESBL-producing K. pneumoniae isolates recovered from patients in our health care system, we unexpectedly discovered that 28 strains were phylogenetically allied with K. variicola (13 strains) and K. quasipneumoniae (15 strains)(10). We identified strain KPN1705 as a distinct outlier in the phylogenetic analysis. It shared a common branch with the K. variicola, yet was as distant from the K. pneumoniae, K. quasipneumoniae, and K. variicola reference genomes as they were from each other (Figure 1).
Genetic relationships among Klebsiella quasivariicola sp. nov. strains KPN1705 and Strain 10982 and reference strains of K. pneumoniae (NJST258_2), K. variicola (At-22), and K. quasipneumoniae (700603). Phylogenetic relationships were defined by the neighbor-joining method in FastTreeMP with double precision using the closed KPN1705 genome as a reference. The core genome was defined as the chromosomal sequence with the 6 predicted phage sequence regions excluded.
To determine if strains similar to KPN1705 had been previously reported, we determined its multilocus sequence type (MLST). Results revealed that it is a single locus variant of ST-1155, with three SNPs in the infB_110 allele. A search of publicly available databases found one previous report of an ST-1155 Klebsiella, which was a description of a novel Klebsiella dubbed Strain 10982 (11). Strain 10982 was recovered from a perianal swab collected on an ICU patient in Maryland in 2005, as part of a study of AmpC-mediated antimicrobial resistance (11).
To begin assessing the genetic relationship between strain KPN1705 and other Klebsiella, we sequenced the genome of KPN1705 to closure. The KPN1705 chromosome is 5,540,188 bp, and three plasmids were identified (described below). Strain 10982 was previously sequenced by Hazen et al. and the assembled 218 contigs are published (11). SNPs were called for reference genomes of K. pneumoniae (NJST258_2), K. quasipneumoniae (700603), K. variicola (At-22), and Strain 10982, using our closed KPN1705 as a reference. The pairwise distance between K. pneumoniae and K. variicola compared to KPN1705 was 250,000 and 251,939 SNPs, respectively. Similarly, the pairwise distance between K. pneumoniae and K. variicola and Strain 10982 was 253,227 and 253,864 SNPs. This level of difference between the novel strains and other Klebsiella clades is similar to the distance separating the K. pneumoniae, K. variicola and K. quasipneumoniae from one another (mean: 269,799 SNPs, range: 247,050-287,991 SNPs) (Figure 2A). In comparison, KPN1705 and Strain 10982 were closely related, differing from one another by only 34,455 SNPs (Figure 1). This level of difference is similar to the average pairwise distance between any two K. variicola strains (average: 38,056 SNPs, range: 31,777 – 45,299) (10). Together, these whole genome sequence data suggest that KPN1705 and Strain 10982 represent a novel Klebsiella species, and we propose the name Klebsiella quasivariicola sp. nov.
Gene content differences between the reference genomes Klebsiella quasivariicola sp. nov. (KPN1705), K. pneumoniae (NJST258_2), K. variicola (At-22), and K. quasipneumoniae (700603). A. Bidirectional BLAST was performed by using the PATRIC resource to illustrate the differences in gene content between these two reference genomes. The color indicates the percent identity of the BLAST hit for each gene, with darker shading indicating a bidirectional hit and lighter shading indicating a unidirectional hit. Outer-most ring is the KPN1705 chromosome reference, followed by K. pneumoniae NJST258_2, K. quasipneumoniae 700603, and K. variicola At-22 on the innermost ring. B. Venn diagram showing shared gene content between the K. pneumoniae NJST258_2 (Kp, red), K. quasipneumoniae 700603 (Kqp, yellow), K. variicola At-22 (Kv, blue), and K. quasivariicola sp. nov. (Kqv, green) as determined by Roary.
Plasmid and phage content in Klebsiella quasivariicola sp. nov. strain KPN1705
Next, we characterized the plasmids carried by strain KPN1705. Using our assembled whole genome data, we identified three plasmids, pKPN1705-1 (240,771bp), pKPN1705-2 (97,896bp), and pKPN1705-3 (67,851bp). These plasmids were similar to others found in Klebsiella species and carried a diverse array of replicons and antimicrobial resistance genes. Six intact phage regions were predicted in the core chromosome, consisting of 359 coding sequences in 322.7 kb of core chromosomal sequence (Table S1 Phage).
Antimicrobial gene content in Klebsiella quasivariicola sp. nov
The SHV-LEN-OKP beta-lactamases are core chromosomal genes of Klebsiella that are usually segregated by Klebsiella species: K. pneumoniae (SHV restricted), K. quasipneumoniae (OKP restricted), and K. variicola (LEN restricted) (8, 12, 13). SHV beta-lactamase genes can also be carried on plasmids (14). We assessed the antimicrobial gene content of KPN1705 and determined it carries the LEN-24 beta-lactamase on its chromosome, similar to what is commonly found in K. variicola. This further contributed to our suggestion to call this novel species K. quasivariicola sp. nov. KPN1705 also carried the gene encoding the SHV-30 ESBL enzyme on plasmid pKPN1705-3. Genes encoding KPC, OXA, CTX-M, TEM and NDM-1 were not detected.
Gene content comparison between K. pneumoniae, K. variicola, K. quasipneumoniae and K. quasivariicola
We compared the gene content between our ESBL-producing K. pneumoniae, K. variicola, K. quasipneumoniae and K. quasivariicola sp. nov.. We identified a total of 8,184 unique genes present in the pangenome of all four species (Figure 2B). A Klebsiella core genome consisted of 3,357 unique genes that were present in the reference genome of each clade. A bidirectional BLAST comparing the 4 reference genomes to KPN1705 shows the distance between each is similar, with gaps present in the regions corresponding to 6 predicted phage regions (Figure 2A). A table of the gene presence or absence is included in the supplemental (Table S2 Gene Content).
DISCUSSION
K. pneumoniae is a well-known cause of human morbidity and mortality. Although less common, the closely related organisms K. variicola and K. quasipneumoniae also cause life-threatening infections (5, 7, 10, 15). The difficulty that conventional clinical microbiology laboratories have in distinguishing K. variicola and K. quasipneumoniae from K. pneumoniae may contribute to our underestimation of their potential as human pathogens (10, 16). The discovery of this novel clade of Klebsiella, the K. quasivariicola sp. nov., represents yet another Klebsiella species capable of causing serious human infections. Importantly, when novel strain 10982 was first described, the investigators questioned whether it had simply colonized the gastrointestinal tract or if it was potentially pathogenic. Our novel strain KPN1705 was recovered from a wound culture, strongly suggesting a causative role for the abscess. In addition, the detection of multiple antimicrobial resistance genes, including a SHV ESBL enzyme, increases its virulence potential.
Our whole genome sequence data provides clues to the relationships between the Klebsiella clades. The core genome content of K. quasivariicola sp. nov., is similar to K. pneumoniae, K. variicola and K. quasipneumoniae, despite the extensive diversity that has been reported to occur within and between clades (6, 9, 10). Also, consistent with previous reports, (6) we observed the plasmids present in KPN1705 to be similar to those found in other Klebsiella species. Importantly, these plasmids carry multiple genes encoding virulence factors and antimicrobial resistance genes (17).
These data provide new insight to the natural history and pathogenesis of Klebsiella organisms. Additional strains of Klebsiella quasivariicola sp. nov. are needed to better characterize this new species. Improved diagnostic methods or widespread use of whole genome sequencing of clinical isolates may be necessary to ensure timely and appropriate identification of these pathogens.
MATERIALS AND METHODS
Whole genome sequencing of Klebsiella
The genome of strain KPN1705 was previously described using Illumina short read data (9). To obtain long reads to close the genome, we sequenced the genome of strain KPN1705 to closure using the 1D Ligation sequencing kit, R9.4 flow cell, and Oxford Nanopore Technologies MinION Mk-Ib sequencer.
Bioinformatics analysis of strains
The single nucleotide polymorphism calling pipeline and additional bioinformatics pipelines were described previously (9). BLAST was performed using the NCBI BLAST toolkit and CLC Genomics Workbench v.10.1. Visualization of SNP distribution was performed using CLC Genomics Workbench v.10.1. FASTQ files were assembled into contigs using Spades v3.10.1, and contigs were annotated using Prokka v1.12 (18, 19). Unicycler v0.4.0 was used for hybrid assembly and polishing of short reads and long reads into a closed genome for KPN1705 (20). Gene content analysis was performed using Roary v3.6.1 (21). Bidirectional BLAST and circos visualization were performed using PATRIC (www.patricbrc.org). Assembly of SNPs into phylogenetic trees was accomplished with the scripts prephix v3.3.0, phrecon v4.6.0, and FastTreeMP v2.1 (22). Phage regions were predicted using PHASTER (23). Prephix and phrecon are available from https://github.com/codinghedgehog. The Venn diagram was made in RStudio 1.0.136 using R 3.3.2 and the VennDiagram package v1.6.17 (24).
MALDI-TOF Identification
KPN1705 was isolated by the Houston Methodist Diagnostic Microbiology Laboratory as described previously (25).
Accession Numbers
The genomes of the strain sequenced for this study have been deposited in the NCBI database under BioProject PRJNA376414 and BioSample SAMN06438648. The accession numbers for the KPN1705 closed genome and plasmids are CP022823-CP022826. Reference genome Genbank accession numbers are as follows: NJST258_2 (CP006918.1), 700603 (CP014696.2), At-22 (CP001891.1), and Strain 10982 (GCA_000523395.1).
ACKNOWLEDGEMENTS
This work was supported by funds from the National Institute of Allergy and Infectious Diseases, National Institutes of Health, Department of Health and Human Service (contract HHSN272201400027C). We acknowledge Dr. J. Kristie Johnson for helpful discussions regarding Strain 10982, and Kathryn Stockbauer for help in preparing this manuscript.
REFERENCES
