Recent reports indicate the emergence of a new carbapenemase-producing Klebsiella pneumoniae clone, ST307. We sought to better understand the global epidemiology and evolution of this clone and evaluate its association with antimicrobial resistance (AMR) genes. We collated information from the literature and public databases and performed a comparative analysis of 95 ST307 genomes (including 37 that were newly sequenced). We show that ST307 emerged in the mid-1990s (nearly 20 years prior to its first report), is already globally distributed and is intimately associated with a conserved plasmid harbouring the bla_CTX-M-15 ESBL gene and several other AMR determinants. Our findings support the need for enhanced surveillance of this widespread ESBL clone in which carbapenem resistance has occasionally emerged.

Antibiotic resistant Klebsiella pneumoniae is a leading public health threat and gastrointestinal carriage is an established risk factor for subsequent infections during hospitalization. Our study contributes new knowledge of risk factors for gastrointestinal carriage and the genomic population structure of K. pneumoniae colonizing humans in a representative sample of a general population in a community setting. Altogether, 2,975 participants (54% women) >40 y in the population-based Tromsø Study: Tromsø7, Norway (2015–2016) were included. Fecal samples were screened for K. pneumoniae, which were characterized using whole-genome sequencing. Risk factors for carriage were analyzed using multivariable logistic regression on data from questionnaires and the Norwegian Prescription Database. Prevalence of K. pneumoniae gastrointestinal carriage was 16.3% (95% CI 15.0–17.7, no gender difference). Risk factors associated with carriage included age ≥60 y, travel to Greece or Asia past 12 months (adjusted odds ratio 1.49, 95% CI 1.11–2.00), Crohn’s disease/ulcerative colitis (2.26, 1.20–4.27), use of proton pump inhibitors (1.62, 1.18–2.22) and non-steroidal anti-inflammatory drugs past 6 months (1.38, 1.04–1.84), and antibiotic use the last month (1.73, 1.05–2.86). Prevalence was higher among those having used combinations of drug classes and decreased over time with respect to preceding antibiotic use. The K. pneumoniae population was diverse with 300 sequence types among 484 isolates distributed across four phylogroups. Only 5.2% of isolates harbored acquired resistance and 11.6% had virulence factors. Identification of risk factors for gastrointestinal carriage allows for identification of individuals that may have higher risk of extraintestinal infection during hospitalization. The findings that specific diseases and drugs used were associated with carriage show an impact of these possibly through modulating the human gut microbiota promoting colonization. The diverse population structure of carriage isolates reflects the ecologically adaptive capacity of the bacterium and challenges for vaccine prospects and the identification of reservoirs as a potential source for human colonization.

Surface polysaccharides are common antigens in priority pathogens and therefore attractive targets for novel control strategies such as vaccines, monoclonal antibody and phage therapies. Distinct serotypes correspond to diverse polysaccharide structures that are encoded by distinct biosynthesis gene clusters; e.g. the Klebsiella pneumoniae species complex (KpSC) K- and O-loci encode the synthesis machinery for the capsule (K) and outer-lipopolysaccharides (O), respectively. We previously presented Kaptive and Kaptive 2, programmes to identify K- and O-loci directly from KpSC genome assemblies (later adapted for Acinetobacter baumannii), enabling sero-epidemiological analyses to guide vaccine and phage therapy development. However, for some KpSC genome collections, Kaptive (v≤2) was unable to type a high proportion of K-loci. Here, we identify the cause of this issue as assembly fragmentation and present a new version of Kaptive (v3) to circumvent this problem, reduce processing times and simplify output interpretation. We compared the performance of Kaptive v2 and Kaptive v3 for typing genome assemblies generated from subsampled Illumina read sets (decrements of 10× depth), for which a corresponding high-quality completed genome was also available to determine the ‘true’ loci (n=549 KpSC, n=198 A. baumannii). Both versions of Kaptive showed high rates of agreement to the matched true locus amongst ‘typeable’ locus calls (≥96% for ≥20× read depth), but Kaptive v3 was more sensitive, particularly for low-depth assemblies (at 1 order of magnitude faster than Kaptive v2, making it easy to analyse thousands of assemblies on a desktop computer, facilitating broadly accessible in silico serotyping that is both accurate and sensitive. The Kaptive v3 source code is freely available on GitHub (https://github.com/klebgenomics/Kaptive), and has been implemented in Kaptive Web (https://kaptive-web.erc.monash.edu/).

Klebsiella pneumoniae species complex (KpSC) bloodstream infections (BSIs) are associated with considerable morbidity and mortality, particularly in elderly and multimorbid patients. Multidrug resistant (MDR) strains have been associated with poorer outcome. However, the clinical impact of KpSC phylogenetic lineages on BSI outcome is unclear. In an 18-month nationwide Norwegian prospective study of KpSC BSI episodes in adults, we used whole genome sequencing to describe the molecular epidemiology of KpSC, and multivariable Cox regression analysis including clinical data to determine adjusted hazard ratios (aHR) for death associated with specific genomic lineages. We included 1078 BSI episodes and 1082 bacterial isolates from 1055 patients. The overall 30-day case-fatality rate (CFR) was 12.5%. Median patient age was 73.4, 61.7% of patients were male. Median Charlson comorbidity score was 3. Klebsiella pneumoniae sensu stricto (Kp) (79.3%, n=858/1082) and K. variicola (15.7%, n=170/1082) were the dominating phylogroups. Global MDR-associated Kp clonal groups (CGs) were prevalent (25.0%, n=270/1082) but 78.9% (n=213/270) were not MDR, and 53.7% (n=145/270) were community acquired. The major findings were increased risk for death within 30 days in monomicrobial BSIs caused by K. variicola (CFR 16.9%, n=21; aHR 1.86, CI 1.10-3.17, p=0.02), and global MDR-associated Kp CGs (CFR 17.0%, n=36; aHR 1.52, CI 0.98-2.38, p=0.06) compared to Kp CGs not associated with MDR (CFR 10.1%, n=46). Bacterial traits, beyond antimicrobial resistance, have a major impact on the clinical outcome of KpSC BSIs. The global spread of MDR-associated Kp CGs is driven by other mechanisms than antibiotic selection alone. Further insights into virulence determinants, and their association with phylogenetic lineages are needed to better understand the epidemiology of KpSC infection and clinical outcome.

To use the nationwide Norwegian surveillance programme on resistant microbes in humans (NORM) to address longitudinal changes in the population structure of Klebsiella pneumoniae isolates from 2001–15, focusing on the emergence and dissemination of ESBL-producing K. pneumoniae in Norway. 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. In a highly diverse collection, Klebsiella variicola ssp. variicola caused 24.5% of Klebsiella pneumoniae species complex (KpSC) bacteraemias. ESBL production was limited to K. pneumoniae sensu stricto (98.5%). A diverse ESBL population of 57 clonal groups (CGs) were dominated by MDR CG307 (17%), CG15 (12%), CG70 (6%), CG258 (5%) and CG45 (5%) carrying bla_CTX-M-15. Yersiniabactin was significantly more common in ESBL-positive (37.8%) compared with non-ESBL K. pneumoniae sensu stricto isolates (12.7%), indicating convergence of virulence and resistance determinants. Moreover, we found a significantly lower prevalence of yersiniabactin (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 with ESBL- and non-ESBL K. pneumoniae sensu stricto isolates, respectively. The increase in Norwegian ESBL-producing KpSC during 2010–15 was driven by CG307 and CG15 carrying bla_CTX-M-15. K. variicola ssp. variicola was a frequent cause of invasive KpSC infection, but rarely carried ESBLs.

Interpreting the phenotypes of bla _SHV alleles in Klebsiella pneumoniae genomes is complex. Whilst all strains are expected to carry a chromosomal copy conferring resistance to ampicillin, they may also carry mutations in chromosomal bla _SHV alleles or additional plasmid-borne bla _SHV alleles that have extended-spectrum β-lactamase (ESBL) activity and/or β-lactamase inhibitor (BLI) resistance activity. In addition, the role of individual mutations/a changes is not completely documented or understood. This has led to confusion in the literature and in antimicrobial resistance (AMR) gene databases [e.g. the National Center for Biotechnology Information (NCBI) Reference Gene Catalog and the β-lactamase database (BLDB)] over the specific functionality of individual sulfhydryl variable (SHV) protein variants. Therefore, the identification of ESBL-producing strains from K. pneumoniae genome data is complicated. Here, we reviewed the experimental evidence for the expansion of SHV enzyme function associated with specific aa substitutions. We then systematically assigned SHV alleles to functional classes (WT, ESBL and BLI resistant) based on the presence of these mutations. This resulted in the re-classification of 37 SHV alleles compared with the current assignments in the NCBI’s Reference Gene Catalog and/or BLDB (21 to WT, 12 to ESBL and 4 to BLI resistant). Phylogenetic and comparative genomic analyses support that (i) SHV-1 (encoded by bla _SHV-1) is the ancestral chromosomal variant, (ii) ESBL- and BLI-resistant variants have evolved multiple times through parallel substitution mutations, (iii) ESBL variants are mostly mobilized to plasmids and (iv) BLI-resistant variants mostly result from mutations in chromosomal bla _SHV. We used matched genome–phenotype data from the KlebNET-GSP AMR Genotype-Phenotype Group to identify 3999 K. pneumoniae isolates carrying one or more bla _SHV alleles but no other acquired β-lactamases to assess genotype–phenotype relationships for bla _SHV. This collection includes human, animal and environmental isolates collected between 2001 and 2021 from 24 countries. Our analysis supports that mutations at Ambler sites 238 and 179 confer ESBL activity, whilst most omega-loop substitutions do not. Our data also provide support for the WT assignment of 67 protein variants, including 8 that were noted in public databases as ESBL. These eight variants were reclassified as WT because they lack ESBL-associated mutations, and our phenotype data support susceptibility to third-generation cephalosporins (SHV-27, SHV-38, SHV-40, SHV-41, SHV-42, SHV-65, SHV-164 and SHV-187). The approach and results outlined here have been implemented in Kleborate v2.4.1 (a software tool for genotyping K. pneumoniae), whereby known and novel bla _SHV alleles are classified based on causative mutations. Kleborate v2.4.1 was updated to include ten novel protein variants from the KlebNET-GSP dataset and all alleles in public databases as of November 2023. This study demonstrates the power of sharing AMR phenotypes alongside genome data to improve the understanding of resistance mechanisms.