Klebsiella pneumoniae species complex: From wastewater to the environment

Abstract

Klebsiella pneumoniae plays a significant role in nosocomial infections and spreading antibiotic resistance, and therefore forms a major threat to public health. In this study, we investigated the role of the wastewater pathway in the spread of pathogenic bacteria and more specifically, in the spread of antibiotic resistant Klebsiella pneumoniae subspecies. Whole-genome sequencing was performed of 185 K. pneumoniae isolates collected from hospital, nursing home, and community wastewater, the receiving wastewater treatment plant (WWTP), and clinical isolates from the investigated hospital. K. pneumoniae isolates from different sources were not genetically related, except for WWTP influent (46.5%) and effluent (62.5%), revealing survival of bacteria from wastewater treatment. The content of antibiotic resistance (ARGs), virulence, and plasmid replicon genes differed between K. pneumoniae subspecies and their origin. While chromosomal bla genes were specific for each K. pneumoniae subspecies, bla genes predicted in plasmid contigs were found in several K. pneumoniae subspecies, implying possible gene transfer between subspecies. Transferable ARGs were most abundant in patients and hospital isolates (70%), but the average number of plasmid replicon genes per isolate was similar across all sources, showing plasmid content being more relevant than plasmid quantity. Most patient (90%) and hospital wastewater (34%) isolates were K. pneumoniae subsp. pneumoniae, and the yersiniabactin cluster genes ybt, fyuA, and irp12 were only found in this subspecies, as were the IncFII(pECLA), IncHI2A, and IncHI2 plasmid replicon genes, suggesting the clinical origin of these type of plasmids.

Keywords: Antibiotic resistance genes; Hospital; Klebsiella pneumoniae; Plasmid replicon genes; Virulence genes; WWTP; Wastewater; Wastewater pathway; Whole genome sequencing.

Graphical abstract

Fig. 1

CgMLST clustering. A: bar plots showing the total number of isolates per location and the number of clustered isolates (≤ 13 allele differences). B: MST of all isolates coloured by ST, shading shows which isolates cluster together (≤ 15 allele differences). Patient and wastewater locations: only one isolate from community wastewater clustered with another source (nursing home, dark blue). Further, all clusters only contained isolates of the same source. WWTP and surface water locations: clusters 1, 3, 7, 17 and 19 were formed by isolates from both influent and effluent locations. Furthermore, one isolate from the surface water clustered with influent/effluent isolates (cluster 1). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Fig. 2

Average number of ARGs and plasmid replicon genes per location.

Fig. 3

Ordination plots of isolates by their content of resistance genes, virulence genes and plasmid replicons, coloured by subspecies (A) and sources (B). Influent and effluent isolates are shown as WWTP. Plots were made as PCoA plots using the Jaccard method for the distance. Adonis test revealed that species explain 45% of the variation in distance, and 15% is explained by collection sources (p < 0.001).

Fig. 4

Ordination plot of isolates with and without transferable ARGs. Isolates harbouring one or more transferable ARGs are shown in red. Isolates that do not possess any transferable ARGs are shown in grey. Plots were made as PCoA plots using the Jaccard method for the distance. Adonis test revealed that 17% of the variation in distance is explained by transferable ARGs (p < 0.001). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)