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To characterize the CTX-M-15-encoding plasmid in a Klebsiella pneumoniae ST17 strain, responsible for an outbreak at a Norwegian neonatal intensive care unit and subsequent colonization of affected children for up to two years. To identify plasmid-mediated features relevant for the outbreak dynamics, and to investigate the plasmids capability of horizontal transfer, its segregational stability and plasmid-mediated fitness costs. Plasmid profiling was performed by S1-nuclease PFGE, PCR-based replicon typing and Southern blot-hybridization. The complete sequence of the CTX-M-15-encoding plasmid was obtained by 454 sequencing. Plasmid self-transferability was investigated by broth- and filter mating, segregational stability was explored by serial passage, and plasmid-conferred fitness costs were examined in pairwise head-to-head competitions and by growth rate comparisons. CTX-M-15 was encoded by a ~180 kb IncFIIK plasmid in K. pneumoniae ST17. S1-nuclease PFGE profiles of the first and the last CTX-M-15-producing K. pneumoniae isolates, recovered from the four children colonized the longest, suggested that the plasmid was stably maintained during intestinal carriage of up to two years. The DNA sequence of the pKPN3-like plasmid, pKp848CTX, uncovered a Tn3-like antibiotic resistance region and multiple heavy metal- and thermoresistance determinants. Plasmid pKp848CTX could not be transferred to Escherichia coli in vitro and we found no evidence to support horizontal plasmid transfer in vivo. Segregational plasmid loss ranging from 0.83% to 17.5% was demonstrated in evolved populations in vitro, but only minor fitness costs were associated with plasmid-carriage. Plasmid pKp848CTX encodes phenotypic traits, which may have had an impact on the fitness and survival of the K. pneumoniae ST17 strain in the outbreak setting. The antibiotic resistance plasmid pKp848CTX was stably maintained during two years of intestinal colonization, conferring negligible fitness cost to its host, and thus seem well adapted to its K. pneumoniae host.

Sequence-based typing (SBT) of Legionella pneumophila is a valuable tool in epidemiological studies and outbreak investigations of Legionnaires’ disease. In the L. pneumophila SBT scheme, mompS2 is one of seven genes that determine the sequence type (ST). The Legionella genome typically contains two copies of mompS (mompS1 and mompS2). When they are non-identical it can be challenging to determine the mompS2 allele, and subsequently the ST, from Illumina short-reads. In our collection of 233 L. pneumophila genomes, there were 62 STs, 18 of which carried non-identical mompS copies. Using short-reads, the mompS2 allele was misassembled or untypeable in several STs. Genomes belonging to ST154 and ST574, which carried mompS1 allele 7 and mompS2 allele 15, were assigned an incorrect mompS2 allele and/or mompS gene copy number when short-read assembled. For other isolates, mainly those carrying non-identical mompS copies, short-read assemblers occasionally failed to resolve the structure of the mompS- region, also resulting in untypeability from the short-read data. In this study, we wanted to understand the challenges we observed with calling the mompS 2 allele from short-reads, assess if other short-read methods were able to resolve the mompS -region, and investigate the possibility of using long-reads to obtain the mompS alleles, and thereby perform L. pneumophila SBT from long-reads only. We found that the choice of short-read assembler had a major impact on resolving the mompS -region and thus SBT from short-reads, but no method consistently solved the mompS2 allele. By using Oxford Nanopore Technology (ONT) sequencing together with Trycycler and Medaka for long-read assembly and polishing we were able to resolve the mompS copies and correctly identify the mompS2 allele, in accordance with Sanger sequencing/EQA results for all tested isolates (n=35). The remaining six genes of the SBT profile could also be determined from the ONT-only reads. The STs called from ONT-only assemblies were also consistent with hybrid-assemblies of Illumina and ONT reads. We therefore propose ONT sequencing as an alternative method to perform L. pneumophila SBT to overcome the mompS challenge observed with short-reads. To facilitate this, we have developed ONTmompS ( https://github.com/marithetland/ONTmompS ), an in silico approach to determine L. pneumophila ST from long-read or hybrid assemblies.

Following an incidence of Legionnaires disease (LD) in 2007, where a municipal shower system was the likely source of infection, Stavanger municipality initiated a surveillance program for Legionella as part of establishing internal risk evaluation and prevention routines. More than 250 shower systems were examined for cultivatable Legionella pneumophila. The prevalence and diversity of serogroups (sg) and sequence types (STs) of L. pneumophila were mapped using available typing techniques over a period of more than 10 years (2010–2021). The surveillance showed an overall reduction in the L. pneumophila colonisation rate in municipal systems from 11 to 4.5% following prevention measures during the period, with the highest colonisation rate in complex systems (e.g., larger nursing homes and sports complexes). Further, an approximately even distribution between sg1 and 2–14 was seen. Whole genome sequencing (WGS) revealed that only a limited number of STs were detected, and they were consistent at specific locations over time. This study showed that environmental surveillance data in combination with available typing techniques and WGS can give the municipality a better tool for risk management and an overview of ST distributions that can be a valuable asset in future source investigations.

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 iuc 3 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.

Objectives To characterize the CTX-M-15-encoding plasmid in a Klebsiella pneumoniae ST17 strain, responsible for an outbreak at a Norwegian neonatal intensive care unit and subsequent colonization of affected children for up to two years. To identify plasmid-mediated features relevant for the outbreak dynamics, and to investigate the plasmids capability of horizontal transfer, its segregational stability and plasmid-mediated fitness costs. Methods Plasmid profiling was performed by S1-nuclease PFGE, PCR-based replicon typing and Southern blot-hybridization. The complete sequence of the CTX-M-15-encoding plasmid was obtained by 454 sequencing. Plasmid self-transferability was investigated by broth- and filter mating, segregational stability was explored by serial passage, and plasmid-conferred fitness costs were examined in pairwise head-to-head competitions and by growth rate comparisons. Results CTX-M-15 was encoded by a ~180 kb IncFII.sub.K plasmid in K. pneumoniae ST17. S1-nuclease PFGE profiles of the first and the last CTX-M-15-producing K. pneumoniae isolates, recovered from the four children colonized the longest, suggested that the plasmid was stably maintained during intestinal carriage of up to two years. The DNA sequence of the pKPN3-like plasmid, pKp848CTX, uncovered a Tn3-like antibiotic resistance region and multiple heavy metal- and thermoresistance determinants. Plasmid pKp848CTX could not be transferred to Escherichia coli in vitro and we found no evidence to support horizontal plasmid transfer in vivo. Segregational plasmid loss ranging from 0.83% to 17.5% was demonstrated in evolved populations in vitro, but only minor fitness costs were associated with plasmid-carriage. Conclusions Plasmid pKp848CTX encodes phenotypic traits, which may have had an impact on the fitness and survival of the K. pneumoniae ST17 strain in the outbreak setting. The antibiotic resistance plasmid pKp848CTX was stably maintained during two years of intestinal colonization, conferring negligible fitness cost to its host, and thus seem well adapted to its K. pneumoniae host.

Objectives To characterize the CTX-M-15-encoding plasmid in a Klebsiella pneumoniae ST17 strain, responsible for an outbreak at a Norwegian neonatal intensive care unit and subsequent colonization of affected children for up to two years. To identify plasmid-mediated features relevant for the outbreak dynamics, and to investigate the plasmids capability of horizontal transfer, its segregational stability and plasmid-mediated fitness costs. Methods Plasmid profiling was performed by S1-nuclease PFGE, PCR-based replicon typing and Southern blot-hybridization. The complete sequence of the CTX-M-15-encoding plasmid was obtained by 454 sequencing. Plasmid self-transferability was investigated by broth- and filter mating, segregational stability was explored by serial passage, and plasmid-conferred fitness costs were examined in pairwise head-to-head competitions and by growth rate comparisons. Results CTX-M-15 was encoded by a ~180 kb IncFIIK plasmid in K. pneumoniae ST17. S1-nuclease PFGE profiles of the first and the last CTX-M-15-producing K. pneumoniae isolates, recovered from the four children colonized the longest, suggested that the plasmid was stably maintained during intestinal carriage of up to two years. The DNA sequence of the pKPN3-like plasmid, pKp848CTX, uncovered a Tn3-like antibiotic resistance region and multiple heavy metal- and thermoresistance determinants. Plasmid pKp848CTX could not be transferred to Escherichia coli in vitro and we found no evidence to support horizontal plasmid transfer in vivo. Segregational plasmid loss ranging from 0.83% to 17.5% was demonstrated in evolved populations in vitro, but only minor fitness costs were associated with plasmid-carriage. Conclusions Plasmid pKp848CTX encodes phenotypic traits, which may have had an impact on the fitness and survival of the K. pneumoniae ST17 strain in the outbreak setting. The antibiotic resistance plasmid pKp848CTX was stably maintained during two years of intestinal colonization, conferring negligible fitness cost to its host, and thus seem well adapted to its K. pneumoniae host.

Plasmids play a pivotal role in the horizontal gene transfer of antimicrobial resistance (AMR) and virulence determinants among bacteria. Members of the Klebsiella pneumoniae species complex (KpSC) can colonise 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 characterised. Understanding the traits governing successful plasmid transmission within and between ecological niches is critical for developing effective AMR prevention strategies. Here, we characterise the diversity and distribution of KpSC plasmids and identify potential niche associations of AMR, heavy metal resistance, virulence factors, and plasmid clusters. We analysed the plasmidome (i.e. total genetic content attributable to plasmids) of 578 whole-genome sequenced KpSC isolates collected in Norway between 2001-2020 from human (n=453), terrestrial 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 8656 circularised KpSC plasmids. In total, 1415 circularised plasmids were identified and grouped according to rearrangement distance using Pling, resulting in 130 clusters (containing >1 plasmid), of which 36% (n=47) contained plasmids from >1 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 mobilisable plasmids harbouring various transposable elements. The human niche exhibited a significantly higher prevalence of plasmids with AMR genes compared to animal or marine niches (p<0.001), whereas the animal niche displayed a significantly higher prevalence of virulence-encoding plasmids compared to human or marine niches (p<0.001), which was largely due to an enrichment of iuc3 plasmids in pigs. The high diversity of the KpSC plasmids underscores the dynamic nature of plasmid evolution, driven by horizontal gene transfer, 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.

Klebsiella pneumoniae sequence type (ST) 17 is a global problem clone that causes multidrug-resistant (MDR) hospital infections worldwide. In 2008-2009, an outbreak of MDR ST17 occurred at a neonatal intensive care unit (NICU) in Stavanger, Norway. Fifty-seven children were colonised. We observed intestinal persistence of ST17 in all of the children for up to two years after hospital discharge. Here, we investigated the within-host evolution of ST17 in 45 of those children during long-term colonisation and compared the outbreak with 254 global strains. Ninety-two outbreak-related isolates were whole-genome sequenced. They had capsule locus KL25, O locus O5 and carried yersiniabactin. During within-host colonisation ST17 remained stable with few single nucleotide polymorphisms, no acquisition of antimicrobial resistance (AMR) or virulence determinants, and persistent carriage of a blaCTX-M-15-encoding IncFII(K) IncFIB(K) plasmid (pKp2177_1). The global collection included ST17 from 1993-2020 from 34 countries, that were from human infection (41.3%), colonisation (39.3%) and respiratory specimens (7.3%), from animals (9.3%), and from the environment (2.7%). We estimate that ST17 emerged mid-to-late 19th century (1859, 95% HPD 1763-1939) and diversified through recombinations of the K and O loci to form several sublineages, with various AMR genes, virulence loci and plasmids. There was limited evidence of persistence of AMR genes in any of these lineages. A globally disseminated sublineage with KL25/O5 accounted for 52.7% of the genomes. It included a monophyletic subclade that emerged in the mid-1980s, which comprised the Stavanger NICU outbreak and 10 genomes from three other countries, which all carried pKp2177_1. The plasmid was also observed in a KL155/OL101 subclade from the 2000s. Three clonal expansions of ST17 were identified, all were healthcare-associated and carried either yersiniabactin and/or pKp2177_1. To conclude, ST17 is globally disseminated and associated with opportunistic hospital-acquired infections. It contributes to the burden of global MDR infections, but many diverse lineages persist without acquired AMR. We hypothesise that non-human sources and human colonisation may play a crucial role for severe infections in vulnerable patients, such as preterm neonates. Klebsiella pneumoniae is an opportunistic pathogen that frequently causes hospital-associated multidrug-resistant (MDR) infections. Infections with K. pneumoniae strains that are resistant to third generation cephalosporins and/or carbapenems are considered a major public health threat, as there are limited treatment options available. Some MDR K. pneumoniae clones, including ST307 and ST258, are global problem clones because they disproportionately contribute to the burden of MDR infections and are common causes of such infections and/or outbreaks in hospitals around the world. Here we describe another such clone, ST17, which caused an MDR outbreak in our neonatal intensive care unit, affecting 57 children. We found that this clone underwent minor within-host evolution during two years of long-term gastrointestinal colonisation after hospital discharge. We then investigated the evolutionary history of ST17 globally, as it had not previously been studied. When we compared ST17 isolates from around the world with the isolates from our hospital, we discovered that whilst ST17 with antimicrobial resistance has contributed to outbreaks in other neonatal care units, it also frequently causes infections or colonises humans and non-human sources without any antimicrobial resistance. The genome sequences generated in this study have been deposited at the European Nucleotide Archive under BioProject PRJEB36392. The BioSample accession numbers and associated metadata for the genomes are available in Table S1. The completed annotated genome assembly of Kp2177 is available in GenBank under accession numbers CP075591 (chromosome), CP075592 (pKp2177_1), CP075593 (pKp2177_2) and CP075594 (pKp2177_3). The global dataset of 300 ST17 genomes is available for interactive viewing in Microreact at https://microreact.org/project/kpst17.

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.

Objective: We have used the nationwide Norwegian surveillance program on resistant microbes in humans (NORM) to address longitudinal changes in the population structure K. pneumoniae isolates during 2001-15, encompassing the emergence and spread of ESBL-producing Enterobacterales (ESBL-E) in Norway. Material and methods: Among blood (n= 6124) and urinary tract (n=5496) surveillance isolates from 2001-15, we used Illumina technology to whole genome sequence 201 ESBL-producing isolates from blood (n=130) and urine (n=71), and 667 non-ESBL isolates from blood. Complete genomes for four isolates were resolved with Oxford Nanopore sequencing. Results: In a highly diverse collection, Klebsiella variicola ssp. variicola caused a quarter of Klebsiella pneumoniae species complex bacteraemias. ESBL-production was limited to K. pneumoniae sensu stricto (98.5 %). A diverse ESBL population of 57 clonal groups (CGs) were dominated by multidrug resistant CG307 (17%), CG15 (12%), CG70 (6%), CG258 (5%) and CG45 (5%) carrying blaCTX-M-15. Yersiniabactin was significantly more common in ESBL-positive (37.8%) compared to non-ESBL K. pneumoniae sensu stricto isolates (12.7%), indicating convergence of virulence and resistance determinants. Moreover, we found a significant lower prevalence of yersinabactin (3.0 %, 37.8 % and 17.3 %), IncFIB (58.7 %, 87.9 % and 79.4 %) and IncFII plasmid replicons (40.5 %, 82.8 % and 54.2%) in K. variicola ssp. variicola compared to ESBL- and non-ESBL K. pneumoniae sensu stricto, respectively. Conclusion: The increase in Norwegian KpSC ESBLs during 2010-15 was driven by blaCTX-M-15 carrying CG307 and CG15. K. variicola ssp. variicola was a frequent cause of invasive KpSC infection, but rarely carried ESBL. Competing Interest Statement The authors have declared no competing interest. Footnotes * https://microreact.org/project/4dBcaZsZmKoAzvatzPaGds