Genomics and Virulence of Klebsiella pneumoniae Kpnu95 ST1412 Harboring a Novel Incf Plasmid Encoding Bla ctx-M-15 and Qnrs1 Causing Community Urinary Tract Infection

Abstract

The emergence of extended-spectrum β-lactamase (ESBL)-producing multidrug resistant Klebsiella pneumoniae causing community urinary tract infections (CA-UTI) in healthy women undermines effective treatment and poses a public health concern. We performed a comprehensive genomic analysis (Illumina and MinION) and virulence studies using Caenorhabditis elegans nematodes to evaluate KpnU95, a bla_CTX-M-15-producing CA-UTI K. pneumoniae strain. Whole genome sequencing identified KpnU95 as sequence type 1412 and revealed the chromosomal and plasmid-encoding resistome, virulome and persistence features. KpnU95 possess a wide virulome and caused complete C. elegans killing. The strain harbored a single novel 180.3Kb IncFIB(K) plasmid (pKpnU95), which encodes ten antibiotic resistance genes, including bla_CTX-M-15 and qnrS1 alongside a wide persistome encoding heavy metal and UV resistance. Plasmid curing and reconstitution were used for loss and gain studies to evaluate its role on bacterial resistance, fitness and virulence. Plasmid curing abolished the ESBL phenotype, decreased ciprofloxacin MIC and improved bacterial fitness in artificial urine accompanied with enhanced copper tolerance, without affecting bacterial virulence. Meta-analysis supported the uniqueness of pKpnU95 and revealed plasmid-ST1412 lineage adaptation. Overall, our findings provide translational data on a CA-UTI K. pneumoniae ST1412 strain and demonstrates that ESBL-encoding plasmids play key roles in multidrug resistance and in bacterial fitness and persistence.

Keywords: Caenorhabditis elegans; ESBL; Klebsiella pneumoniae; blaCTX-M-15; community urinary tract infection; genomics; persistome; qnrS1; sequence type 1412 (ST1412).

Figure 1

The effect of pKpnU95 on the ESBL phenotype and ciprofloxacin (CIP) MIC testing. ESBL confirmation assay using ceftazidime (CAZ); upper-left disc, ceftazidime (CAZ); lower-left CAZ + clavulanic acid (CLA); upper-right, cefotaxime (CTX); and lower-right CTX + CLA (A–E); and Etest MIC testing of ciprofloxacin (CIP) (F–J). The clinical KpnU95 (A,F), the cured strain lacking the plasmid, KpnΔpKpnU95 (B,G), the cured strain transformed with the plasmid, KpnΔpKpnU95/pKpnU95 (C,H), E. coli DH10B (D,I) and E. coli DH10B/pKpnU95 (E,J). Images are representative of three biological replicates.

Figure 2

Growth kinetics, copper tolerance and virulence assessment of KpnU95 strains. Growth curves of KpnU95 strains in rich media LB, minimal BM2 medium and on artificial urine were performed in 96-multiwell plates by monitoring OD600 nm during 14 h. Each growth curve represents the average of three or five independent experiments (A); Copper tolerance in MH in different concentrations of CuSO₄. The doubling time of each growth curve represents the average of three independent growth experiments. * p value ≤ 0.05 and ** p value ≤ 0.01 (B); The survival curves of C. elegans nematodes fed on KpnU95 strains or E. coli OP50 as a control strain in BM2 (C) or on artificial urine media (D). The nematode survival curves were plotted according to the average survival counts of two replicate experiments performed in four replicate wells. Statistical analysis was performed using the Log-rank (Mantel–Cox) test (C,D).

Figure 3

Comparison of pKpnU95 and homologous K. pneumoniae plasmids. A BRIG diagram shows pKpnU95 (black) and alignments of the 18 related plasmids, designated in different colors according to their STs. pKpnU95 backbone region is presented on the inner circle (black segment). The annotation of pKpnU95 is shown on the two outer rings representing the genes and the mobile elements as strand-oriented arrows. IS elements and transposons labeled with ‘#’ showed >90% identity and >30% coverage of the sequences from the ISfinder database. Truncated transposons may not encode all expected ORFs, but only part of them. ** Computationally predicted only.

Figure 4

Phylogeny of pKpnU95-related plasmids. The maximum-likelihood tree shows the relationships between concatenated SNPs extracted from pKpnU95 and its related plasmids (n = 18). Bootstrap support values (>50% out of 1000 replicates) are presented at nodes. Plasmid groups carried by K. pneumoniae isolates of the same ST are highlighted with colors. Plasmid characteristics such as the presence of specific resistance cassettes and pKpnU95 coverage together with host STs, sources and years of isolations are presented on the right.