Discovery and whole genome sequencing of a human clinical isolate of the novel species Klebsiella quasivariicola sp. nov

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.

phylogenetically distinct from all currently recognized Klebsiella species. Whole genome 26 sequencing of strain KPN1705 revealed that it was single locus variant of the multilocus 27 sequence type ST-1155. This sequence type was reported only once previously. To further 28 investigate the phylogeny of these two organisms, we sequenced the genome of strain KPN1705 29 to closure and compared its genetic features to Klebsiella reference strains. Results demonstrated 30 strain KPN1705 extensively shares core gene content, antimicrobial resistance genes, and 31 plasmids with K. pneumoniae, K. quasipneumoniae and K. variicola. Since strain KPN1705 and 32 the previously reported novel strain are phylogenetically most closely related to K. variicola, we 33 propose the name K. quasivariicola sp. nov. 34

IMPORTANCE 36
K. pneumoniae, K. quasipnuemoniae and K. variicola are serious human pathogens that are 37 increasingly associated with multidrug resistance and high morbidity and mortality. In a recent 38 study of a large, comprehensive, population-based collection of antibiotic resistant Klebsiella 39 isolates recovered from human patients, we discovered a novel species that is related to but 40 distinct from K. variicola. This clonal group has been reported only once previously. We 41 sequenced the genome of this clinical isolate and compared its genetic features to other 42 Klebsiella strains. We propose the name K. quasivariicola sp. nov. for this new species. 43

OBSERVATION 45
Members of the genus Klebsiella are a common cause of human morbidity and mortality 46 (1, 2). Many community-acquired and healthcare-associated outbreaks of invasive K. 47 pneumoniae disease have been reported (3,4). Over the past two decades, related Klebsiella 48 species have been identified as distinct from K. pneumoniae and classified (5-8). In a recent 49 large, comprehensive, population based study of 1,777 extended-spectrum beta-lactamase 50 (ESBL) producing Klebsiella strains recovered in our clinical microbiology laboratory, we 51 discovered a unique isolate KPN1705 (9, 10). It was genetically related to, but distinct from, K. 52 variicola. We sequenced the genome of this strain, which belongs to a new species herein 53 termed Klebsiella quasivariicola sp. nov., to closure and compared its genetic features to other 54 Klebsiella reference strains. 55

RESULTS 57
Whole genome sequencing reveals Klebsiella quasivariicola sp. nov., a novel Klebsiella 58 pathogenic to humans 59 In a recent study of 1,777 ESBL-producing K. pneumoniae isolates recovered from patients in 60 our health care system, we unexpectedly discovered that 28 strains were phylogenetically allied 61 with K. variicola (13 strains) and K. quasipneumoniae (15 strains)(10). We identified strain 62 KPN1705 as a distinct outlier in the phylogenetic analysis. It shared a common branch with the 63 K. variicola, yet was as distant from the K. pneumoniae, K. quasipneumoniae, and K. variicola 64 reference genomes as they were from each other ( Figure 1). 65 To determine if strains similar to KPN1705 had been previously reported, we determined 66 its multilocus sequence type (MLST). Results revealed that it is a single locus variant of ST-67 1155, with three SNPs in the infB_110 allele. A search of publicly available databases found one 68 previous report of an ST-1155 Klebsiella, which was a description of a novel Klebsiella dubbed 69 Strain 10982 (11). Strain 10982 was recovered from a perianal swab collected on an ICU patient 70 in Maryland in 2005, as part of a study of AmpC-mediated antimicrobial resistance (11). 71 To begin assessing the genetic relationship between strain KPN1705 and other Klebsiella, 72 we sequenced the genome of KPN1705 to closure. The KPN1705 chromosome is 5,540,188 bp, 73 and three plasmids were identified (described below). Strain 10982 was previously sequenced by 74 Hazen et al. and  Next, we characterized the plasmids carried by strain KPN1705. Using our assembled whole 91 genome data, we identified three plasmids, pKPN1705-1 (240,771bp), pKPN1705-2 (97,896bp), 92 and pKPN1705-3 (67,851bp). These plasmids were similar to others found in Klebsiella species 93 and carried a diverse array of replicons and antimicrobial resistance genes. Six intact phage 94 regions were predicted in the core chromosome, consisting of 359 coding sequences in 322.7 kb 95 of core chromosomal sequence (Table S1 Phage BLAST comparing the 4 reference genomes to KPN1705 shows the distance between each is 115 similar, with gaps present in the regions corresponding to 6 predicted phage regions (Figure 2A). 116 A table of the gene presence or absence is included in the supplemental (Table S2 Gene  117   Content). 118

DISCUSSION 120
K. pneumoniae is a well-known cause of human morbidity and mortality. Although less 121 common, the closely related organisms K. variicola and K. quasipneumoniae also cause life-122 threatening infections (5, 7, 10, 15). The difficulty that conventional clinical microbiology 123 laboratories have in distinguishing K. variicola and K. quasipneumoniae from K. pneumoniae 124 may contribute to our underestimation of their potential as human pathogens (10, 16). The 125 discovery of this novel clade of Klebsiella, the K. quasivariicola sp. nov., represents yet another 126 Klebsiella species capable of causing serious human infections. Importantly, when novel strain 127 10982 was first described, the investigators questioned whether it had simply colonized the 128 gastrointestinal tract or if it was potentially pathogenic. Our novel strain KPN1705 was 129 recovered from a wound culture, strongly suggesting a causative role for the abscess. In addition, 130 the detection of multiple antimicrobial resistance genes, including a SHV ESBL enzyme, 131 increases its virulence potential. 132 Our whole genome sequence data provides clues to the relationships between the 133 Klebsiella clades. The core genome content of K. quasivariicola sp. nov., is similar to K. 134 pneumoniae, K. variicola and K. quasipneumoniae, despite the extensive diversity that has been 135 reported to occur within and between clades (6, 9, 10). Also, consistent with previous reports, (6)  136 we observed the plasmids present in KPN1705 to be similar to those found in other Klebsiella 137 species. Importantly, these plasmids carry multiple genes encoding virulence factors and 138 antimicrobial resistance genes (17). 139 These data provide new insight to the natural history and pathogenesis of Klebsiella 140 organisms. Additional strains of Klebsiella quasivariicola sp. nov. are needed to better 141 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 143 of these pathogens. 144

Whole genome sequencing of Klebsiella 147
The genome of strain KPN1705 was previously described using Illumina short read data (9). To 148 obtain long reads to close the genome, we sequenced the genome of strain KPN1705 to closure 149 using the 1D Ligation sequencing kit, R9.4 flow cell, and Oxford Nanopore Technologies 150 MinION Mk-Ib sequencer. 151