A high-resolution genomic analysis of multidrug-resistant hospital outbreaks of Klebsiella pneumoniae

Abstract

Multidrug-resistant (MDR) Klebsiella pneumoniae has become a leading cause of nosocomial infections worldwide. Despite its prominence, little is known about the genetic diversity of K. pneumoniae in resource-poor hospital settings. Through whole-genome sequencing (WGS), we reconstructed an outbreak of MDR K. pneumoniae occurring on high-dependency wards in a hospital in Kathmandu during 2012 with a case-fatality rate of 75%. The WGS analysis permitted the identification of two MDR K. pneumoniae lineages causing distinct outbreaks within the complex endemic K. pneumoniae. Using phylogenetic reconstruction and lineage-specific PCR, our data predicted a scenario in which K. pneumoniae, circulating for 6 months before the outbreak, underwent a series of ward-specific clonal expansions after the acquisition of genes facilitating virulence and MDR. We suggest that the early detection of a specific NDM-1 containing lineage in 2011 would have alerted the high-dependency ward staff to intervene. We argue that some form of real-time genetic characterisation, alongside clade-specific PCR during an outbreak, should be factored into future healthcare infection control practices in both high- and low-income settings.

Keywords: Klebsiella pneumoniae; antimicrobial resistance; bloodstream infections; carbapenemases; nosocomial infections.

Figure 1

Hospital outbreaks of Klebsiella pneumoniae in Kathmandu, Nepal, in 2012

1. A Graphical representation of the layout of the wards at Patan Hospital affected by the 2012 K. pneumoniae outbreaks. The affected wards on the first and second floor (directly above) are highlighted in colour, which correspond with the sub-lineages indicated in Fig 2A; nurseries and neonatal intensive care unit (NICU), orange; paediatric intensive care unit (PICU), purple; adult intensive care unit (AICU), dark green; medical ward, bright green.

2. B Plot (red line) shows the monthly ratio of Klebsiella spp. isolated from blood at Patan Hospital in 2012 with respect to Klebsiella spp. isolated from all other samples (primary y-axis). Broken line corresponds to the mean ratio of Klebsiella spp. isolated from blood in 2012. Histogram shows the number of Klebsiella spp. isolated from blood samples at Patan Hospital per month in 2012 (secondary y-axis).

3. C Maximum likelihood phylogeny based on core genome SNPs of the 89 K. pneumoniae isolates with the corresponding metadata: month of isolation (MON) in 2012 (see key), ward (NICU, PICU and nurseries (NICU) or other (OTH)) and specimen type from which the organism was isolated (red, blood; pink, others), antimicrobial susceptibility profile (susceptible, light grey; non-susceptible, dark grey; and missing data, white) by disc diffusion against MEM (meropenem), IMP (imipenem), CTX (cefotaxime), CIP (ciprofloxacin), OFX (ofloxacin), GEN (gentamicin), AMK (amikacin), CHL (chloramphenicol) and SXT (trimethoprim/sulfamethoxazole). The presence (dark blue) or absence (light blue) of the pNDM-MAR-like plasmid and the bla_NDM-1 gene. The black arrow distinguishes the lineage with three mutations in genes associated with fluoroquinolone susceptibility (GyrA-S83F, GyrA-S87A, ParC-S80I). The grey arrows identify lineages with two mutations in genes associated with fluoroquinolone susceptibility (GyrA-S83I, ParC-S80I). The scale bar indicates the number of nucleotide changes per site (see Materials and Methods).

Figure 2

Temporal phylogenetic reconstruction and retrospective PCR detection of Klebsiella pneumoniae Outbreak Lineage 1

1. A Bayesian phylogenetic reconstruction of Outbreak Lineage 1 using the core genome SNPs of 24 isolates with a corresponding temporal timescale. The black filled circles indicate a posterior probability > 0.8 on internal nodes. The red filled circles indicate K. pneumoniae isolates cultured from blood. The ward from which each specimen was collected: orange (NICU and nurseries A, B, C), dark green (AICU), light green (medical ward), light blue (OPD, outpatient department) and purple (PICU). The presence (dark blue) and absence (light blue) of specific genetic determinants for each of the 24 isolates, including bla_NDM-1, are highlighted.

2. B Linear regression plot of the date (x-axis) against the root-to-tip divergence (y-axis) used to indicate correspondence between phylogenetic and temporal signal. Estimation of temporal signal was performed in Path-O-Gen v1.4 for the core genome SNP-based maximum likelihood phylogeny of 24 Outbreak Lineage 1 K. pneumoniae. Additional estimated parameters are inset.

3. C The temporal distribution of 70 PCR-confirmed Outbreak Lineage 1 Klebsiella pneumoniae infections by their ward of isolation. First row: outbreak isolates originating from the NICU, PICU and the nurseries. Second row: isolates originating from other wards, including the AICU, gynaecology ward, maternity ward, medical ward, OPD and surgical ward. The red bars correspond to isolates originating from blood; the black bars correspond to isolates originating from all other specimen types. Third row: the distribution of PCR-confirmed Outbreak Lineage 1 strains containing bla_NDM-1. Arrows signify the first and last occasions on which bla_NDM-1 positive blood-borne Klebsiella pneumoniae Outbreak Lineage 1 strains were isolated.

Figure 3

The architecture of the novel capsule biosynthesis cluster (cps) in the ST15 Outbreak Lineage 1 Klebsiella pneumoniae Graphical representation showing the composition of the cps gene cluster within the Outbreak Lineage 1 K. pneumoniae isolates. The genes are identified by either their common name (e.g. wbaP) or their predicted function (e.g. beta-xylosidase). Genes that are shared by other strains of K. pneumoniae are coloured orange, and genes specific to this cps cluster are coloured blue and red. The beta-xylosidase gene (red) was selected as the target for lineage-specific PCR amplification.

Figure 4

The resistome of the 89 hospital isolates of Klebsiella pneumoniae Maximum likelihood phylogeny based on core genome SNPs of the 89 Klebsiella pneumoniae isolates adapted from Fig1 set against the resistome of 15 antimicrobial resistance genes (named at the top of the figure). The dark and light grey boxes show the presence and absence of the specific resistance genes in the corresponding isolates, respectively. bla, beta-lactamase; qnr,aac(6’)-Ib-cr, quinolone resistance genes; strAB, streptomycin resistance genes; sul, sulphonamide resistance gene; cat, chloramphenicol resistance gene; tet, tetracycline resistance gene; armA,aadA,aac(3)-IIa, aminoglycoside resistance genes; and msr(E), mph, macrolide resistance genes.