Background: Members of the Klebsiella pneumoniae species complex (KpSC) are opportunistic pathogens that cause severe and difficult-to-treat infections. KpSC are common in non-human niches, but the clinical relevance of these populations is disputed. Methods: In this study, we analysed 3255 whole-genome sequenced isolates from human, animal and marine sources collected in Norway between 2001 and 2020. We used population genomics in a One Health context to assess the diversity of strains, genes and other clinically relevant genetic features within and between sources. We further explored niche-enriched traits using genome-wide association studies and investigated evidence of spillover and connectivity across the KpSC populations from the three niches. Results: We found that the KpSC populations in different niches were distinct but overlapping. Overall, there was high genetic diversity both between and within sources, with nearly half (49%) of the genes in the accessory genome overlapping the ecological niches. Further, several sublineages (SLs) including SL17, SL35, SL37, SL45, SL107 and SL3010 were common across sources. There were few niche-enriched traits, except for aerobactin-encoding plasmids and the bacteriocin colicin a, which were associated with KpSC from animal sources. Human infection isolates showed the greatest connectivity with each other, followed by isolates from human carriage, pigs, and bivalves. Nearly 5% of human infection isolates had close relatives (≤22 substitutions) amongst animal and marine isolates, despite temporally and geographically distant sampling of these sources. There were limited but notable recent spillover events, including the movement of plasmids encoding the virulence locus iuc3 between pigs and humans. Conclusions: Our large One Health genomic study highlights that human-to-human transmission of KpSC is more common than transmission between ecological niches. Still, spillover of clinically relevant strains and genetic features between human and non-human sources does occur and should not be overlooked. Infection prevention measures are essential to limit transmission within human clinical settings and reduce infections. However, preventing transmission that leads to colonisation, e.g. from direct contact with animals or via the food chain, could also play an important role in reducing the KpSC disease burden.

We report 578 hybrid genome assemblies (568 complete) of Klebsiella pneumoniae species complex isolates from human, animal, and marine sources in Norway collected from 2001 to 2020, belonging to five phylogroups including K. pneumoniae (n = 492) and K. variicola (n = 69) and 364 unique sequence types.

We report 579 hybrid genome assemblies (568 complete) of Klebsiella pneumoniae species complex isolates from human, animal and marine sources in Norway collected 2001-2020, belonging to six phylogroups including K. pneumoniae (n=493) and K. variicola (n=69) and 364 unique sequence types.

Plasmids play a pivotal role in the horizontal gene transfer (HGT) of antimicrobial resistance (AMR) and virulence determinants among bacteria. Members of the Klebsiella pneumoniae species complex (KpSC) can colonize humans, animals and various environments and frequently cause nosocomial and community-acquired infections in humans. While plasmid-borne AMR genes are prevalent in clinical strains, the diversity, distribution and association of plasmids encoding AMR and virulence across ecological niches remain poorly characterized. Understanding the traits governing successful plasmid transmission within and between ecological niches is critical for developing effective AMR prevention strategies. Here, we identify ecological and structural factors shaping plasmid persistence and dissemination. We analysed the plasmidome (i.e. total genetic content attributable to plasmids) of 578 whole-genome sequenced KpSC isolates collected in Norway between 2001 and 2020 from human ( n =453), animal ( n =102) and marine ( n =23) sources. Plasmids from complete hybrid assemblies were annotated and clustered to evaluate the plasmid diversity and content across niches. Additionally, the representativeness of this plasmid collection was determined by clustering with a global collection of 8,656 circularized KpSC plasmids. In total, 1,415 circularized plasmids were identified and grouped according to rearrangement distance using Pling, resulting in 130 clusters (≥2 plasmids each), of which 36% ( n =47) contained plasmids from more than one niche. The plasmids exhibited significant diversity, as 37% ( n =524) remained singletons after clustering. AMR and virulence genes existed across diverse clusters and singletons but predominantly resided on 120–250 kbp conjugative or mobilizable plasmids harbouring various transposable elements. Human isolates carried higher overall plasmid burdens and harboured most AMR-encoding plasmids, while animal isolates were significantly enriched for virulence plasmids ( P <0.001), largely due to iuc 3 plasmids in pigs. Plasmids from human, animal and marine isolates formed shared genetic clusters spanning ecological boundaries, revealing the existence of widely distributed backbones already primed for AMR gene acquisition. The extensive diversity of KpSC plasmids highlights the dynamic nature of plasmid evolution, driven by HGT and selective pressures. The presence of variable clusters, marked by high genetic diversity, indicates a dynamic plasmidome capable of rapid adaptation to environmental pressures through the acquisition and rearrangement of accessory genes.

Nasal carriage of Staphylococcus aureus and its antibiotic-resistant derivative, methicillin-resistant S. aureus (MRSA), is a risk factor for nosocomial S. aureus infections. Mupirocin is a topical antibiotic and a key in the decolonization of both methicillin-susceptible S. aureus (MSSA) and MRSA carriage in patients and health care personnel. Recent observations have shown a global increase in the prevalence of mupirocin-resistant MSSA and MRSA, reducing the efficacy of mupirocin in decolonization regimens. LTX-109 is a peptidomimetic synthetic compound that has shown broad-spectrum bactericidal antimicrobial activity in vitro and in animal experiments. However, the development of resistance against LTX-109 in clinical isolates of MRSA and MSSA has not been systematically examined. Background/Objectives: Here, we assess the development of spontaneous and adaptive resistance against LTX-109 in genomically diverse MRSA (n = 3) and MSSA (n = 4) strains. Methods: Adaptive and mutational resistance were examined by serial passaging strains over 60 cycles in a range of LTX-109 and mupirocin concentrations. Spontaneous resistance was examined in high-inoculum agar plates with 2–8 times the concentration above MIC. Results: Throughout serial passage, LTX-109 MICs varied less than 4-fold compared to the initial MIC of 4–8 mg/L, while mupirocin MICs increased in all susceptible strains (n = 5) from 0.25 mg/L to 16–512 mg/L. The spontaneous resistance assay demonstrated no resistance development at 4–8× MIC LTX-109 and an inoculum effect at 2× MIC. Conclusions: Our results demonstrate the novelty of LTX-109 as an antimicrobial agent with no detectable in vitro resistance development in selected clinical strains of MRSA and MSSA.

Members of the Klebsiella pneumoniae species complex (KpSC) are opportunistic pathogens that cause severe and difficult-to-treat infections. KpSC are common in non-human niches, but the clinical relevance of these populations is disputed. Utilising 3,255 whole-genome sequenced isolates from human, animal and marine sources collected during 2001-2020 in Norway, we showed the KpSC populations in different niches were distinct but overlapping. Notably, human infection isolates showed greatest connectivity with each other, followed by isolates from human carriage, pigs, and bivalves. Nearly 5% of human infection isolates had close relatives (≤22 substitutions) amongst animal and marine isolates, despite temporally and geographically distant sampling of these sources. Infection prevention measures are essential to limit transmission within human clinical settings and reduce disease burden. However, as colonisation often precedes infection, preventing transmission that leads to colonisation, e.g. transmission between animals and humans in the community, and in the food chain, could also be beneficial.