Use of whole-genome sequencing to trace, control and characterize the regional expansion of extended-spectrum β-lactamase producing ST15 Klebsiella pneumoniae

Abstract

The study describes the transmission of a CTX-M-15-producing ST15 Klebsiella pneumoniae between patients treated in a single center and the subsequent inter-institutional spread by patient referral occurring between May 2012 and September 2013. A suspected epidemiological link between clinical K. pneumoniae isolates was supported by patient contact tracing and genomic phylogenetic analysis from May to November 2012. By May 2013, a patient treated in three institutions in two cities was involved in an expanding cluster caused by this high-risk clone (HiRiC) (local expansion, CTX-M-15 producing, and containing hypervirulence factors). A clone-specific multiplex PCR was developed for patient screening by which another patient was identified in September 2013. Genomic phylogenetic analysis including published ST15 genomes revealed a close homology with isolates previously found in the USA. Environmental contamination and lack of consistent patient screening were identified as being responsible for the clone dissemination. The investigation addresses the advantages of whole-genome sequencing in the early detection of HiRiC with a high propensity of nosocomial transmission and prolonged circulation in the regional patient population. Our study suggests the necessity for inter-institutional/regional collaboration for infection/outbreak management of K. pneumoniae HiRiCs.

Figure 1. Putative transmission route of the regional outbreak.

The transmission route was reconstructed by epidemiological and genomic data (see Appendix Materials and Methods for details of reconstruction methods). Each node represents a patient, and an arrow indicates a possible transmission event from one patient to another. The blue arrow with solid line represents a direct transmission event supported by both epidemiological data and genetic data, the blue arrow with dash line represents an indirect transmission (e.g. via environment) supported by epidemiological data, and the red arrow indicates the equally parsimonious transmission link which cannot be resolved by neither epidemiological data nor genetic data. The inter-institutional transfer of the patient is shown by dash line, on which the distance between institutions is indicated. The red star represents an outbreak at the secondary hospital, but the isolates were unavailable for our investigation.

Figure 2. Core-genome phylogenetic analysis of K. pneumoniae isolates.

A maximum likelihood tree was constructed based on the alignments of a 4.47 Mb genome, defined as the core genome in this study. The tree was mid-point rooted. The size of node represents the percentage of bootstrap support, and the biggest one is equal to 100. Sequence types are indicated between brackets. The isolation time and resource of all ST15 strains are shown. The cluster of outbreak isolates is simplified as a red triangle. The non-outbreak isolates sequenced in this study are in blue, and the others retrieved from GenBank are in black. The inset shows the close-up unrooted tree of outbreak isolates, in which the patient isolates are shown in red (2012) and purple (2013), and the environment isolates are in green. The number of SNPs is indicated on the branches.

Figure 3. Comparison of capsular polysaccharide synthesis (cps) region identified from ST15 isolates.

The cps regions of K. pneumoniae PMK1 and KP-33P (non-outbreak isolate of this study) were identical. The cps region of a ST15 strain was retrieved from GenBank. The gradients (dark to pale) of the alignment region represent the percentage of sequence identity between samples defined by BLASTn.