Convergence of virulence and multidrug resistance in a single plasmid vector in multidrug-resistant Klebsiella pneumoniae ST15

Background Multidrug resistance (MDR) and hypervirulence (hv) are typically observed in separate Klebsiella pneumoniae populations. However, convergent strains with both properties have been documented and potentially pose a high risk to public health in the form of invasive infections with limited treatment options. Objectives To characterize the genetic determinants of virulence and antimicrobial resistance (AMR) in two ESBL-producing K. pneumoniae isolates belonging to the international MDR clone ST15. Methods The complete genome sequences of both isolates, including their plasmids, were resolved using Illumina and Oxford Nanopore sequencing. Results Both isolates carried large mosaic plasmids in which AMR and virulence loci have converged within the same vector. These closely related mosaic hv-MDR plasmids include sequences typical of the K. pneumoniae virulence plasmid 1 (KpVP-1; including aerobactin synthesis locus iuc) fused with sequences typical of IncFIIK conjugative AMR plasmids. One hv-MDR plasmid carried three MDR elements encoding the ESBL gene blaCTX-M-15 and eight other AMR genes (blaTEM, aac3’-IIa, aph3’-Ia, dfrA1, satA2, blaSHV, sul1, aadA1). The other carried remnants of these elements encoding blaTEM and aac3’-IIa, and blaCTX-M-15 was located in a second plasmid in this isolate. The two isolates originated from patients hospitalized in Norway but have epidemiological and genomic links to Romania. Conclusions The presence of both virulence and AMR determinants on a single vector enables simultaneous transfer in a single event and potentially rapid emergence of hv-MDR K. pneumoniae clones. This highlights the importance of monitoring for such convergence events with stringent genomic surveillance.


INTRODUCTION
The majority of infections caused by Klebsiella pneumoniae (Kp) are typically associated with one of two distinct clinical phenomena caused by non-overlapping Kp populations: healthcare-associated infections caused by MDR Kp strains that also often cause nosocomial outbreaks, and community-acquired, invasive infections caused by hypervirulent (hv) strains 1,2 . However, convergent strains carrying both MDR and hypervirulent genes have been reported [3][4][5][6][7] . Recently, a high-mortality outbreak of ventilator-associated pneumonia caused by a strain of hv carbapenemaseproducing sequence type (ST) 11 Kp was reported in China, demonstrating that the combination of enhanced virulence potential and difficulties in treatment posed by MDR can be fatal. The Chinese report was particularly notable as ST11 is typically associated with MDR, and appears to be the most common cause of carbapenemaseproducing Kp infections reported in China. However, the outbreak strains had additionally acquired a virulence plasmid harbouring iuc (aerobactin siderophore) and rmpA2 (hypermucoidy) loci, which are usually only observed in hypervirulent clones, such as ST23 2,8,9 .
Given that antimicrobial resistance (AMR) and virulence determinants are commonly mobilized on plasmids, their occasional convergence within individual strains is not unexpected.

Ethics statement
The isolates presented here were collected and sequenced as part of a larger national study of Kp in Norwegian hospitals between 2001 and 2015 called NOR-KLEB.
Ethical approval for NOR-KLEB, including the collection and sequencing of Kp isolates and collection of patient data, was provided by the Regional ethics committee: REC west, application ID:2017/1185.

Bacterial isolates
Isolate KP_NORM_BLD_2014_104014 (KP_104014) was cultured from an 86-yearold Romanian male admitted to an Oslo hospital in 2014 with cholangiocarcinoma before developing bacteremia. Isolate KP_NORM_BLD_2015_112126 (KP_112126) was cultured from a 76-year-old female admitted to a Western Norway hospital in 2015 to treat a glioblastoma, and developed neutropenic fever with pneumonia and bacteremia. She had been hospitalized in Romania prior to admission in Norway.
Antimicrobial susceptibility was determined by disk diffusion and broth micro dilution, and hypermucoidy was assessed via the string test.

Whole genome sequencing and analysis
250 and 150 bp paired end reads were generated for n=12 ST15 Norwegian K. pneumoniae isolates on the Illumina MiSeq and HiSeq platforms, respectively, and assembled with Unicycler v0.4.4-beta. In order to resolve the complete plasmid sequences for strains KP_104014 and KP_112126, additional long read sequencing on a MinION R9.4 flow cell (Oxford Nanopore Technologies) was performed, and combined with the Illumina short reads to generate hybrid assemblies, using Unicycler as previously described 11,12 , which were annotated using Prokka v1.11 13 .
Genotyping information including multi-locus sequence type (MLST), capsule type, AMR and virulence gene detection was extracted using Kleborate (https://github.com/katholt/Kleborate) and used to curate the annotation of relevant loci in the plasmids.
To place the hv-MDR strains in context, we performed comparative genomic analyses (described below) with an additional n=10 ST15 strains isolated between 2003 and 2015 from seven hospitals across Norway as part of the NOR-KLEB study (full results to be reported elsewhere), together with publicly available Illumina data identified from papers reporting Kp ST15 genome sequences (genomes and references listed in Supplementary data 1). Illumina read data for Kp genomes collected by the EuSCAPE European survey of carbapenemase-producing Enterobacteriaceae 20 were downloaded and assembled using Unicycler and genotyped using Kleborate to identify ST15 isolates, and the ST15 read sets were included in the comparative analysis.
All read sets were mapped to the genome of KP_104014 using the RedDog v1b 10.

RESULTS AND DISCUSSION
Isolate KP_104014 displayed resistance to cefotaxime, ceftazidime, ciprofloxacin, gentamicin, piperacillin-tazobactam and co-trimoxazole, and susceptibility to meropenem, colistin and tigecycline. The complete genome sequence resolved seven plasmids (Supplementary data 2), including a novel 346 kbp mosaic hv-MDR plasmid pKp104014_1, which harboured bla CTX-M-15 and seven additional AMR genes (Figure 1). Plasmid pKp104014_1 shares regions of homology with typical KpVP-1-type Kp IncFIB K virulence plasmids 9 , such as pK2044 (40% coverage, including iuc and rmpA2), in addition to regions of homology to IncFII K conjugative AMR plasmids (closest match: 246 kbp plasmid pKp_Goe_579-1, accession CP018313.1, from a ST147 Kp isolated in Germany, 59% coverage). The IncFII K regions include genes for conjugative transfer, suggesting the plasmid may be self-transmissible. The Both of the Norwegian hv-MDR isolates belonged to ST15 and carried the siderophore yersiniabactin (in genomic island ICEKp2) and the KL24 locus encoding capsular serotype K24. ST15 is a well-documented international ESBL-producing clone associated with nosocomial outbreaks worldwide, which frequently carries bla CTX-M-15 -encoding IncFII plasmids [16][17][18][19] . To explore the relatedness of the Norwegian isolates to one another and to the wider ST15 Kp population, we constructed a recombination-filtered, core-genome maximum likelihood phylogeny including KP_104014, KP_112126, ten additional ST15 isolates from Norway and 306 publicly available ST15 genomes from 29 other countries (Figure 2,   Supplementary data 1). The tree showed that the two Norwegian hv-MDR isolates were closely related to one another (77 SNPs, 0.001% nucleotide divergence) and a urine isolate from Romania collected in 2013 (110 SNPs), but quite distant (>0.003% divergent) from the other Norwegian and global isolates.
Interestingly, both the Norwegian hv-MDR plasmids were isolated from patients with epidemiological links to Romania (one of Romanian descent, one with recent travel history to Romania), suggesting the convergence of AMR and virulence plasmids may have occurred in that country rather than in Norway. The closely related Romanian isolate genome (ENA accession ERR1415588) carried iuc and rmpA2 and its reads covered 98% of the pKp112126_1 sequence and only 54% of the typical virulence plasmid pK2044. This is consistent with the presence of a mosaic plasmid in this isolate, although the available Illumina reads were not sufficient to resolve the full sequence of the Romanian plasmid containing iuc.
bla CTX-M-15 was present in most (87%) of the ST15 genomes, along with other AMR genes (see Figure 2, Supplementary data 1). There were also multiple independent acquisitions of the ICEKp genomic island encoding yersiniabactin, affecting 48% of all ST15 isolates including 50% of ESBL isolates (Figure 2). The only non-Norwegian ST15 isolates harbouring iuc were 30 isolates from Pakistan and the closely related Romanian isolate, all of which carried iuc and rmpA2 loci in addition to bla CTX-M-15 and multiple other AMR genes. The convergence of AMR and virulence was noted in the original study reporting these genomes from Pakistan 21 , however it is not possible to determine from the draft genomes whether iuc is colocalised on the same plasmid as AMR genes. Mapping of all ST15 read sets to pKp104014_1 showed that iuc+ isolates from Pakistan and iuc-isolates from Nepal (alongside a small number of iuc-isolates from other countries) share many genes with the mosaic plasmid pKp104014_1 (55.7-68.5% coverage for Pakistan isolates; 52.2-70.2% coverage for Nepal isolates) (Figure 2 and Supplementary figure). This confirms that IncFII K and IncFIB K AMR and virulence plasmids circulate in South Asian Kp ST15 populations and could potentially fuse to form hybrid hv-MDR plasmids.
Concerningly, our findings reveal mosaic plasmids carrying both virulence determinants (iuc, rmpA2) and AMR determinants in ESBL-producing isolates of a well-established MDR Kp clone that has been associated with nosocomial infections and outbreaks worldwide. The co-presence of these loci in a single plasmid vector poses a substantial public health threat with the ability to simultaneously spread AMR and virulence, and it highlights the need for surveillance of virulence alongside AMR before such strains become widespread.