A nationwide genomic study of clinical Klebsiella pneumoniae in Norway 2001-15: introduction and spread of ESBLs facilitated by clonal groups CG15 and CG307

Abstract

Objectives: 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.

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 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 blaCTX-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.

Conclusions: The increase in Norwegian ESBL-producing KpSC during 2010-15 was driven by CG307 and CG15 carrying blaCTX-M-15. K. variicola ssp. variicola was a frequent cause of invasive KpSC infection, but rarely carried ESBLs.

Figure 1.

Maximum likelihood tree of the 868 Klebsiella pneumoniae species complex genomes. Tips are coloured according to the Klebsiella species. Prevalent CGs representing >5% of isolates, either in the ESBL or the non-ESBL group, are highlighted in green and grey, respectively. CGs commonly associated with hypervirulence are highlighted without colour. The highlighted areas include all genomes on the most recent common ancestor node of the CG indicated. The circles from inner to outer show genomes with the predominant sequence type (ST, blue bars) within the CG, presence of ESBL-encoding genes (black bars) and isolates meeting the study definition of hypervirulence (red bars).

Figure 2.

Temporal distribution of Klebsiella pneumoniae species complex (KpSC) ESBL clonal groups (CGs) in blood (a) and urine (b) as proportion of the surveillance for each year. Distribution of CGs in blood and urine separated by most prevalent CGs and other CGs. *The surveillance starts in January each year.

Figure 3.

Epidemiology and phylogeny of CG307. (a) Map of Norway with surveillance regions as defined by the Norwegian surveillance programme on resistant microbes (NORM), with bar plot showing number of CG307 isolates per region per year. (b) CG307 core genome phylogeny with metadata, distribution of AMR determinants, virulence determinants and replicon families. Red and blue boxes mark proposed clonal expansions. A study isolate from blood was used as reference isolate (Genbank accessionCP073627).

Figure 4.

Distribution of resistance determinants, virulence factors and plasmid replicons among clonal groups (CGs). For data see Table S3. (a) Distribution of CGs in the ESBL and non-ESBL groups, separated by most prevalent CGs (each representing ≥5% of isolates in group) and other CGs. In the non-ESBL group, <5% CGs which were the (i) most prevalent CGs from the ESBL group and the (ii) hypervirulence-associated CG23 and CG86 are shown separately. (b) The intensity of the box shading indicates the percentage of genomes harbouring the respective determinant. White shading with a black dot indicates that there are no determinants present. For each antibiotic class, the presence of resistant phenotype and resistance determinants are indicated. GEN, gentamicin, CIP, ciprofloxacin, SXT, trimethoprim/sulfamethoxazole, Agly, acquired aminoglycoside resistance genes; *, does not confer resistance to gentamicin; **, chromosomal mutation position; ***, may reduce susceptibility to both aminoglycosides and fluoroquinolones. (c) Total number of genomes in the ESBL and non-ESBL groups.