Highly conserved composite transposon harbouring aerobactin iuc3 in Klebsiella pneumoniae from pigs

Abstract

Klebsiella pneumoniae is an important opportunistic pathogen associated with severe invasive disease in humans. Hypervirulent K. pneumoniae, which are K. pneumoniae with several acquired virulence determinants such as the siderophore aerobactin and others, are more prominent in countries in South and South-East Asia compared to European countries. This Klebsiella pathotype is capable of causing liver abscesses in immunocompetent persons in the community. K. pneumoniae has not been extensively studied in non-human niches. In the present study, K. pneumoniae isolated from caecal samples (n=299) from healthy fattening pigs in Norway were characterized with regard to population structure and virulence determinants. These data were compared to data from a previous study on K. pneumoniae from healthy pigs in Thailand. Lastly, an in-depth plasmid study on K. pneumoniae with aerobactin was performed. Culturing and whole-genome sequencing was applied to detect, confirm and characterize K. pneumoniae isolates. Phylogenetic analysis described the evolutionary relationship and diversity of the isolates, while virulence determinants and sequence types were detected with Kleborate. Long-read sequencing was applied to obtain the complete sequence of virulence plasmids harbouring aerobactin. A total of 48.8 % of the investigated Norwegian pig caecal samples (n=299) were positive for K. pneumoniae. Acquired virulence determinants were detected in 72.6 % of the isolates, the most prominent being aerobactin (69.2 %), all of which were iuc3. In contrast, only 4.6 % of the isolates from Thailand harboured aerobactin. The aerobactin operon was located on potentially conjugative IncFIB_K/FII_K plasmids of varying sizes in isolates from both countries. A putative, highly conserved composite transposon with a mean length of 16.2 kb flanked by truncated IS3-family IS407-group insertion sequences was detected on these plasmids, harbouring the aerobactin operon as well as several genes that may confer increased fitness in mammalian hosts. This putative composite transposon was also detected in plasmids harboured by K. pneumoniae from several countries and sources, such as human clinical samples. The high occurrence of K. pneumoniae harbouring aerobactin in Norwegian pigs, taken together with international data, suggest that pigs are a reservoir for K. pneumoniae with iuc3. Truncation of the flanking ISKpn78-element suggest that the putative composite transposon has been permanently integrated into the plasmid, and that it is no longer mobilizable.

Keywords: Klebsiella pneumoniae; Norway; Thailand; aerobactin; composite transposon; pigs.

Fig. 1.

Core-gene phylogeny and virulence determinants of Klebsiella spp. isolates. Phylogenetic tree based on an alignment of 3875 core genes from the 233 Klebsiella pig isolates from Norway (pentagons) and Thailand (stars). Black dots on nodes represent accepted bootstrap values (≥95). Subspecies of K. pneumoniae are represented by colours on the tips. Each ST is presented as a tip label. Presence/absence of specific virulence genes/operons is presented on the four outermost rings: Ybt, yersiniabactin; Abt, aerobactin; Slm, salmochelin; RmpA, rmpA/rmpA2. Model of evolution: GTR+F+R10. Tree scale shows substitutions per site.

Fig. 2.

Comparison of the IncFIB_K/FII_K plasmids harbouring aerobactin from K. pneumoniae from healthy pigs in Norway and Thailand. Reference plasmids from Thailand, Laos and Vietnam are marked with stars. Grey-shaded areas between the plasmids represent the per cent identity. Genes are marked as arrows, and the colours represent the gene functions. MGE = Mobile genetic element.

Fig. 3.

Overview of the aerobactin-harbouring composite transposon identified on IncFIB_K/FII_K plasmids in K. pneumoniae from pigs in Norway and Thailand. To the left is a maximum-likelihood phylogenetic tree showing the evolutionary relationship between the composite transposons. The shapes on the tips represent the host species, and the asterisks the reference sequences. White diamonds on the nodes in the tree represent accepted bootstrap values (≥95). Tree scale shows substitutions per site. To the right is a schematic representation of the genetic content of each composite transposon, where colours represent the different genes.