Genomic epidemiology and longitudinal sampling of ward wastewater environments and patients reveals complexity of the transmission dynamics of bla KPC-carbapenemase-producing Enterobacterales in a hospital setting

Abstract

Background: Healthcare-associated wastewater and asymptomatic patient reservoirs colonized by carbapenemase-producing Enterobacterales (CPE) contribute to nosocomial CPE dissemination, but the characteristics and dynamics of this remain unclear.

Methods: We systematically sampled wastewater sites (n = 4488 samples; 349 sites) and patients (n = 1247) across six wards over 6-12 months to understand bla_KPC-associated CPE (KPC-E) diversity within these reservoirs and transmission in a healthcare setting. Up to five KPC-E-positive isolates per sample were sequenced (Illumina). Recombination-adjusted phylogenies were used to define genetically related strains; assembly and mapping-based approaches were used to characterize antimicrobial resistance genes, insertion sequences (ISs) and Tn4401 types/target site sequences. The accessory genome was evaluated in some of the largest clusters, and those crossing reservoirs.

Results: Wastewater site KPC-E-positivity was substantial [101/349 sites (28.9%); 228/5601 (4.1%) patients cultured]. Thirteen KPC-E species and 109 strains were identified using genomics, and 24% of wastewater and 26% of patient KPC-E-positive samples harboured one or more strains. Most diversity was explained by the individual niche, suggesting localized factors are important in selection and spread. Tn4401 + flanking target site sequence diversity was greater in wastewater sites (P < 0.001), which might favour Tn4401-associated transposition/evolution. Shower/bath- and sluice/mop-associated sites were more likely to be KPC-E-positive (adjusted OR = 2.69; 95% CI: 1.44-5.01; P = 0.0019; and adjusted OR = 2.60; 95% CI: 1.04-6.52; P = 0.0410, respectively). Different strains had different bla_KPC dissemination dynamics.

Conclusions: We identified substantial and diverse KPC-E colonization of wastewater sites and patients in this hospital setting. Reservoir and niche-specific factors (e.g. microbial interactions, selection pressures), and different strains and mobile genetic elements likely affect transmission dynamics. This should be considered in surveillance and control strategies.

Figure 1.

Sampling and sequencing flowchart for the study (created using www.biorender.com).

Figure 2.

bla_KPC-Enterobacterales (KPC-E)-positive environmental sites by Week in 2016 and hospital unit. The number of environmental site samples performed and KPC-E-positive samples by week of sampling, stratified by unit (top three panels), and the proportion of KPC-E-positive sites by week of sampling, again stratified by unit (bottom panel). Plumbing replacement on the cardiac unit (wards 3 and 4) was carried out at Week 0.

Figure 3.

Carbapenem-susceptible Enterobacterales (CSE) and carbapenem-resistant-Enterobacterales (CRE) patient cultures by week in 2016 and hospital unit. Top three panels show counts of Enterobacterales culture-positive samples stratified by unit and carbapenem susceptibility; bottom panel shows the proportion of all cultures taken that were CRE, again stratified by unit.

Figure 4.

Number of unique patient/environmental niches colonized by common bla_KPC-2 Enterobacterales species/strains and the total number of isolates for each cluster. Includes species/strains if >50 isolates of a species were identified (see Methods). Stars denote patient niches, circles wastewater niches; larger shape size denotes a larger number of isolates sequenced. Strains observed in both niches are either overlapping shapes (same number of niches), or joined by a line (different numbers of niches).

Figure 5.

Schematic of within-reservoir and KPC-E sample diversity using genomics (generated in www.biorender.com).

Figure 6.

Enterobacter cloacae strain 10: features of population structure and transmission dynamics—niche restriction, persistence and genetic turnover. Summaries of (a) chromosomal SNP distances for E. cloacae strain 10 across the three sites it colonized; (bi) AMR and (bii) IS profiles; (c) plasmid replicon profiles; and (d) deletions in Tn4401a in isolates. Each sequenced isolate is plotted as a dot reflecting within-sample diversity at given timepoints and longitudinally, with colour in panels (bi) and b(ii) reflecting distinct AMR and IS profiles respectively and numbers within the dots representing the numeric identifier for profiles listed in Supplementary dataset 1.

Figure 7.

Klebsiella pneumoniae strain 9: features of population structure and transmission dynamics—rapid patient-patient transmission. Putative transmission network for sub-lineages of K. pneumoniae strain 9, defined by accessory genome clustering (clusters 1, 2, 3a–h). Horizontal bars represent admission episodes to study wards for each patient (pale blue = W45, geratology; pale pink = AM1, acute medicine; pale purple = AM2, acute medicine; A13 represents the only environmental (shower) site involved. Empty black triangles denote CPE-negative rectal screens on patients. Arrows denote possible transmission links (dashed arrows denote events that have no geographical overlap or could be associated with two links based on negative screen, geographical location and timing). For clarity, arrows are not drawn for clusters 3g and 3h, which affected the most patients—these are shown in detail as an inset panel at the bottom right. In some cases patients were colonized by other species-strains contemporaneously (not shown here). (Figure generated in ggplot and www.biorender.com).

Figure 8.

Klebsiella pneumoniae strain 11 cluster 1: features of population structure and transmission dynamics—multiple Tn4401 transposition events. Examples of possible transmission/genetic events are annotated on the figure. Each isolate sequenced is represented as a dot, with colour denoting the TSS profiles observed. Plasmid replicon profiles are annotated as text. (Figure generated in ggplot and www.biorender.com).