Evolution of VIM-1-Producing Klebsiella pneumoniae Isolates from a Hospital Outbreak Reveals the Genetic Bases of the Loss of the Urease-Positive Identification Character

Abstract

Outbreaks of carbapenemase-producing Klebsiella pneumoniae (CPKp) represent a major threat for hospitals. We molecularly characterized the first outbreak of VIM-1-producing K. pneumoniae in Spain, which raised fears about the spread of this strain or of the plasmid carrying bla_VIM-1. Through in-depth genomic analysis of 18 isolates recovered between October 2005 and September 2007, we show that 17 ST39 isolates were clonal, whereas the last isolate had acquired the VIM-1 plasmid from the epidemic clone. The index isolate carried 31 antibiotic resistance genes (ARGs) and was resistant to almost all antibiotics tested. Later isolates further gained mutations in efflux pump regulators ramR and opxR, deletion of mgrB (colistin resistance), and frameshift mutations in ompK36 (β-lactam resistance) likely selected by antibiotic usage. Comparison with publicly available genome sequences and literature review revealed no sign of dissemination of this CPKp strain. However, the VIM-1 plasmid was found in diverse Enterobacterales species, although restricted to Spain. One isolate became urease negative following IS5075 transposition into ureC. Analysis of 9,755 K. pneumoniae genomes showed the same ureC::IS5075 insertion in 14.1% of the isolates and explained why urease activity is a variable identification trait for K. pneumoniae. Transposition into ureC results from the similarity of its 3' end and the terminal inverted repeats of Tn21-like transposons, the targets of IS5075 and related insertion sequences (ISs). As these transposons frequently carry ARGs, this might explain the frequent chromosomal invasion by these ISs and ureC inactivation in multidrug-resistant isolates. IMPORTANCE Evolution of multidrug-resistant bacterial pathogens occurs at multiple scales, in the patient, locally in the hospital, or more globally. Some mutations or gene acquisitions, for instance in response to antibiotic treatment, may be restricted to a single patient due to their high fitness cost. However, some events are more general. By analyzing the evolution of a hospital-acquired multidrug-resistant K. pneumoniae strain producing the carbapenemase VIM-1, we showed a likely environmental source in the hospital and identified mutations contributing to a further decrease in antibiotic susceptibility. By combining the genomic analysis of this outbreak with literature data and genome sequences available in databases, we showed that the VIM-1 plasmid has been acquired by different Enterobacterales but is endemic only in Spain. We also discovered that urease loss in K. pneumoniae results from the specific transposition of an IS element into the ureC gene and was more frequent in fluoroquinolone-resistant isolates and those carrying a carbapenemase gene.

Keywords: Klebsiella pneumoniae; carbapenemase; drug resistance evolution; insertion sequence; mobile genetic elements; urease.

FIG 1

Comparison of pKP1-3, pKP1050-3, and pOXA-48. (A) Comparison of plasmids pKP1-3 and pKP1050-3 (accession no. CP023419.1) carrying bla_VIM-1 and of pOXA-48_1639 carrying bla_OXA-48 (accession no. LR025105.1). pOXA48_1639 was chosen as it was the closest relative to pKP1-3. Gray areas between open reading frames denote nucleotide identities with a gradient representing 99% (light gray) to 100% (dark gray) identity. Identities of an inverted region are represented in red. Genes are indicated by arrows with a color code as in the figure key. Antibiotic resistance genes are numbered as follows: 1, catA1; 2 and 4, msrE_1; 3 and 5, mphE; 6, bla_VIM-1; 7, aacA4_2; 8, dfrB1; 9, ant1_2; 10, cat_2; 11, emrE; 12, folP_4; 13, bla_OXA-48. (B) Analysis of the SNPs detected between pKP1-3 and pOXA-48_1639. Occurrences of SNPs among publicly available IncL/M plasmids ware identified by BLASTN. SNP positions in pKP1-3 are indicated in the first line. Mut. indicates that the mutation is specific to IncL/M VIM-1 plasmids. For other positions, plasmids with the pKP1-3 allele or the pOXA-48_1639 allele are indicated in the second and third line, respectively. pSCH909 carries bla_OXA-10 and bla_TEM-1 but no carbapenemase gene.

FIG 2

Hospital evolution of the K. pneumoniae ST39 VIM-1-producing strain. (A) Phylogeny of the 17 isolates reconstructed by maximum parsimony. Numbers next to branches indicate the number of chromosomal SNPs in the corresponding branch. Presence of plasmids is indicated by colored points, and transposition events are indicated by triangles. IS26 insertion in oqxR occurred in the common ancestor of KP_VIM12 and KP_VIM13. (B) Root-to-tip representation of the number of chromosomal SNPs according to the time (in days) following the isolation of the first isolate, KP_VIM1. The trendline equation and the correlation coefficient are indicated on the graph.

FIG 3

Growth and generation times of isolates with decreased antibiotic susceptibility. (A) Growth of KP_VIM1 and of four isolates mutated in a repressor of efflux pumps (KP_VIM8, KP_VIM12, and KP_VIM14) or in mgrB and ompK36 (KP_VIM17) was followed by using an automatic plate reader. Background was subtracted as described in Materials and Methods. During the first 90 min, the OD₆₀₀ was below 0.0015, and its quantification is noisy. (B) Box plot representation for 10 replicates of the generation times of the five isolates quantified in early exponential phase 2.5 h following the start of the culture (OD₆₀₀ between 0.005 and 0.04). Statistical significances were tested with Student’s t test. ****, P ≤ 0.0001; ns, nonsignificant.

FIG 4

Urease inactivation following IS5075 transposition. (A) Sequence alignment of the sites targeted by IS5075 among KP_VIM isolates. In blue, targets of transposition events occurring during the outbreak: ureC in KP_VIM14 and pKP1-3 Tn21 in KP_VIM5, KP_VIM9, KP_VIM12, and KP_VIM15. The green triangles correspond to IS5075 insertion sites. Conserved bases are indicated in red. Stop and start codons are underlined. (B) Urease activity test of the 17 ST39 isolates. The number of each KP_VIM isolate is indicated on the well. A pink color of the indole reaction reveals a urease-positive phenotype.

FIG 5

Distribution of IS5075 insertions in ureC among K. pneumoniae isolates. Occurrence of IS5075 insertion among the 45 STs with at least 20 isolates among 9,755 K. pneumoniae genome sequences retrieved from the NCBI. Phylogeny was reconstructed using Parsnp (50) and by using a representative isolate from each ST. The tree was rooted according to the work of David et al. (3). Blue bars indicate the percentage of isolates with an insertion in ureC (upper scale), and red dashes indicate the number of isolates in the corresponding ST (lower scale).

FIG 6

Core genome phylogeny of K. pneumoniae ST11 isolates. Phylogeny was obtained by using Parsnp (50) considering 1,603 genomes passing the quality threshold. K-type, mutations in gyrA and parC QRDRs, carbapenemase genes, bla_CTX-M genes, copy number of IS5075 and related ISs, and IS insertion in ureC are annotated by circles from inside to outside as indicated in the figure key (left). The ureC-deficient KL64 lineage is in pink. The KL47 lineage is in blue, and the ureC-deficient sublineage is in red. The two gyrA/parC WT isolates were used as outgroups to root the tree. The tree was visualized by using iTOL (51). ND, no data.