Convergence and global molecular epidemiology of Klebsiella pneumoniae plasmids harbouring the iuc3 virulence locus: a population genomic analysis

Background: Klebsiella pneumoniae (Kp) is an important pathogen of humans and animals. Recent reports of ‘convergent’ strains that carry both virulence factors and antimicrobial resistance genes (ARGs) have raised serious public health concerns. The plasmid-borne iuc locus, encoding the siderophore aerobactin, is a key virulence factor in Kp; the variant iuc3 is associated with porcine and human clinical isolates and is carried by IncF plasmids that are mostly uncharacterised. The aim of this study is to describe the global diversity of iuc3 plasmids from diverse settings, and their role in the emergence of convergent strains through hybridisation with plasmids carrying ARGs.

Methods: The study was designed to describe both the global and local diversity of iuc3 plasmids from diverse sources, and the co-occurrence of iuc3 with ARGs. We used both long- and short-read sequencing of Klebsiella isolates recovered as part of two large ‘One-Health’ studies in Italy (SpARK) and Thailand (OH-DART) totalling 4148 isolates, including 191 Klebsiella isolates from pigs, 635 from clinical isolates, 1040 from carriage and 2282 from other sources. We included data from NCBI (National Centre for Biotechnology Information), based on the presence of iuc3, using the terms ‘Plasmid’ and ‘Aerobactin’. ARGs and virulence loci were detected using Kleborate v2.3.2, and the sequences were analysed using clustering and phylogenetic methods to ascertain the degree of diversity, evolutionary dynamics and structuring across ecological and geographical axes.

Findings: We identified 48 Kp isolates with iuc3 in the SpARK data and 79 in the OH-DART data. Three of these 127 isolates (2.4%) were from clinical sources, and 73 (57.5%) from pigs. iuc3 isolates corresponded to multiple (n=47) host sequence types (STs), with ST35, ST45, ST881, ST25 harbouring iuc3 in both datasets. We generated hybrid assemblies for 44 (SpARK) and 36 (OH-DART) isolates, plus a single iuc3 isolate from Germany. In total, 53 of these were from pigs (65.4%), 3 from clinical sources (3.7%) and 25 from other sources (30.9%). A single iuc3 K. oxytoca isolate from a pig farm was detected in the SpARK data which was also sequenced.

We identified 388 iuc3 plasmids/isolates from NCBI, of which 83 (21.4%) were from clinical sources, 120 from pigs (30.9%), and 185 from other sources or of unknown provenance (47.7%). All of these were from Kp except two isolates of K. quasipneumoniae subsp. similipneumoniae and one of Enterobacter hormaechei. The combined dataset of 517 iuc3 plasmids ranged in size from 110375-bp to 365580-bp, and mostly corresponded to multiple IncFIB(K) and IncFII rep types.

We found six convergent Kp plasmids from fresh markets in the Thai data, and one from a neighbouring hospital. These emerged through the hybridisation of co-circulating iuc3 plasmids and plasmids encoding extended-spectrum beta-lactamases (ESBLs), although none of these seven plasmids carried genes encoding carbapenemases. As our data was drawn from ‘population snapshots’ of localised populations, we were also able to identify putative iuc3 and ESBL co-circulating parental plasmids.

Clustering and phylogenetic analysis resolved the iuc3 plasmid sequences into three groups. These were consistent using both complete plasmid sequences (n=139) and short read data (n=517). Groups defined by the former consisted of 66 strains (Group 1), 38 strains (Group 2) and 35 strains (Group 3). Group 3 plasmids are mostly carried by isolates circulating in hospitals throughout Asia, with occasional examples in Europe and elsewhere, and carry multiple antimicrobial resistance genes (ARGs) and potential virulence factors. In contrast, group 1 plasmids are commonly carried by porcine isolates in Europe, and group 2 are a heterogenous mixture of geographical and ecological sources.

Interpretation: Plasmid hybridisation occurs frequently outside of the health-care environment and can lead to the convergence of resistance and virulence traits. Generating complete plasmid sequences from regional population-scale samples facilitates the identification of convergent plasmids and their putative parental plasmids. Three robust groups of iuc3 plasmids were resolved, which show both epidemiological and geographical differences; one of these is associated with clinical isolates in Asia and warrants targeted plasmid surveillance.