Genomic Insights Into the Mechanism of Carbapenem Resistance Dissemination in Enterobacterales From a Tertiary Public Heath Setting in South Asia

Abstract

Summary: 10.6% patients were CRE positive. Only 27% patients were prescribed at least 1 antibiotic to which infecting pathogen was susceptible. Burn and ICU admission and antibiotics exposures facilitate CRE acquisition. Escherichia coli ST167 was the dominant CRE clone.

Background: Given the high prevalence of multidrug resistance (MDR) across South Asian (SA) hospitals, we documented the epidemiology of carbapenem-resistant Enterobacterales (CRE) infections at Dhaka Medical College Hospital between October 2016 and September 2017.

Methods: We enrolled patients and collected epidemiology and outcome data. All Enterobacterales were characterized phenotypically and by whole-genome sequencing. Risk assessment for the patients with CRE was performed compared with patients with carbapenem-susceptible Enterobacterales (CSE).

Results: 10.6% of all 1831 patients with a clinical specimen collected had CRE. In-hospital 30-day mortality was significantly higher with CRE [50/180 (27.8%)] than CSE [42/312 (13.5%)] (P = .001); however, for bloodstream infections, this was nonsignificant. Of 643 Enterobacterales isolated, 210 were CRE; blaNDM was present in 180 isolates, blaOXA-232 in 26, blaOXA-181 in 24, and blaKPC-2 in 5. Despite this, ceftriaxone was the most commonly prescribed empirical antibiotic and only 27% of patients were prescribed at least 1 antibiotic to which their infecting pathogen was susceptible. Significant risk factors for CRE isolation included burns unit and intensive care unit admission, and prior exposure to levofloxacin, amikacin, clindamycin, and meropenem. Escherichia coli ST167 was the dominant CRE clone. Clustering suggested clonal transmission of Klebsiella pneumoniae ST15 and the MDR hypervirulent clone, ST23. The major trajectories involved in horizontal gene transfer were IncFII and IncX3, IS26, and Tn3.

Conclusions: This is the largest study from an SA public hospital combining outcome, microbiology, and genomics. The findings indicate the urgent implementation of targeted diagnostics, appropriate antibiotic use, and infection-control interventions in SA public institutions.

Keywords: Bangladesh; South Asia; carbapenem-resistant Enterobacterales; outbreak; plasmid-mediated resistance.

Figure 1.

Flowchart diagram of participants included in this study. *Multiple clinical specimens were collected from 61 patients (blood and wound swab, n = 51; blood and urine, n = 3; blood and tracheal aspirate, n = 2; wound swab and urine, n = 2; blood and catheter tip, n = 1; urine and tracheal aspirates, n = 1; blood, urine, and catheter tip, n = 1).**Thirty-five patients had multiple culture-positive samples (blood and wound swab, n = 26; blood and urine, n = 2; blood and tracheal aspirate, n = 2; wound swab and urine, n = 2; blood and catheter tip, n = 1; urine and tracheal aspirates, n = 1; blood, urine, and catheter tip, n = 1). Abbreviations: CRE, carbapenem-resistant Enterobacterales; CSE, carbapenem-susceptible Enterobacterales; DMCH, Dhaka Medical College Hospital; E. coli, Escherichia coli; K. pneumoniae, Klebsiella pneumoniae.

Figure 2.

Antimicrobial susceptibility patterns of different species of Enterobacterales. Data on Salmonella spp. (n = 5), P. anthophila (n = 1), L. adecarboxylata (n = 1), and E. hermannii (n = 1) are not included in this table. *Klebsiella species other than K. pneumoniae. The upper cells corresponding to each species represent the frequency of resistance and the lower cells represent percentage. The heatmap indicates higher (yellow) to lower (green) percentages of resistance. Cells are highlighted in gray if the respective organism is intrinsically resistant to the pertinent antibiotic. Abbreviations: AMC, amoxicillin-clavulanic acid; AMK, amikacin; CAZ, ceftazidime; CIP, ciprofloxacin; CRO, ceftriaxone; CST, colistin; CTX, cefotaxime; E. coli, Escherichia coli; FEP, cefepime; FOF, fosfomycin; GEN, gentamicin; IPM, imipenem; K. pneumoniae, Klebsiella pneumoniae; LVX, levofloxacin; M. morganii, Morganelli morganii; MEM, meropenem; SXT, sulfamethoxazole-trimethoprim; TZP, piperacillin-tazobactam.

Figure 3.

Sankey diagram representing the distribution of carbapenemase alleles among different species of Enterobacterales. Abbreviations: E., Escherichia; K., Klebsiella; KPC, Klebsiella pneumoniae carbapenemase; M., Morganella; NDM, New Delhi metallo-beta-lactamase; OXA, oxacillinases; P., Providencia; S., Serratia.

Figure 4.

ML tree generated from core-genome analysis of Escherichia coli and Klebsiella pneumoniae isolated in this study. (A) ML tree generated from core-genome analysis of E. coli. (B) ML tree generated from core-genome analysis of K. pneumoniae. Core-genome alignment was performed using roary (a tool that rapidly builds large-scale pan genomes, identifying the core and accessory genes) (v3.12.0). The ML trees from the core genome were built with RAxML-ng (v0.9.0.git-mpi) using a GTR evolutionary model and gamma correction with bootstrapping. Isolates retrieved from the NCBI for the phylogenetic analysis in this figure are stated in Supplementary Table 17. Abbreviations: GTR, generalised time reversible; ML, maximum likelihood; NCBI, National Center for Biotechnology Information; ST, sequence type.

Figure 5.

Time-calibrated phylogenetic tree generated from Escherichia coli and Klebsiella pneumoniae genomes. (A) Phylogenetic tree generated from E. coli genomes belonging to ST167. The total number of isolates in this analysis was 97. Closely related isolates from other STs (ST10, ST1702, and novel allele) identified by core-genome phylogeny and pairwise SNPs count (if isolates differed by ≤100 SNPs from any isolate of ST167) were included in this analysis. (B) Phylogenetic tree generated from K. pneumoniae genomes belonging to ST15. The total number of isolates in this analysis was 54. Closely related isolates from a novel allele identified by core-genome phylogeny and pairwise SNP count (if isolates differed by ≤100 SNPs from any isolate of ST15) were included in this analysis. Putative transmission clades (0–10 SNP differences) are highlighted in green. MRCA and clock rate are stated in Supplementary Table 13. Isolates retrieved from the NCBI for the phylogenetic analysis in this figure are shown in Supplementary Table 17. Abbreviations: DMCH, Dhaka Medical College Hospital; ICU, intensive care unit; NCBI, National Center for Biotechnology Information; NDM, New Delhi metallo-beta-lactamase; NICU, neonatal intensive care unit; OPD, outpatient department; PSU, pediatric surgery; SNP, single nucleotide polymorphism; ST, sequence type.

Figure 6.

Spatiotemporal assessment to investigate putative clonal transmission of carbapenem resistance. (A) Putative transmission clusters of Escherichia coli at a ≤10-SNP threshold between the isolates in the respective clusters. (B) Putative transmission clusters of Klebsiella pneumoniae at a ≤10-SNP threshold between the isolates in the respective clusters. The ancestral sequence at each node including the root was inferred using pyjar and the pairwise SNP distance between the roots and each isolate was calculated using pairsnp (v0.0.7). Pairwise SNPs between isolates were generated using pairsnp (v0.0.7). (C) Diagram representing number of linkages among the isolates in the clusters by the 0- to 2-SNP threshold, aligning with epidemiological data. Isolates differed by 0 to 2 SNPs without overlapping of pertinent patients’ hospital stay are represented as a common group in the figure using Tableau (v2020.4). Abbreviations: HDU, high dependency unit; ICU, intensive care unit; NDM, New Delhi metallo-beta-lactamase; NICU, neonatal intensive care unit; SNP, single nucleotide polymorphism; ST, sequence type.

Figure 7.

Schematic layout of genetic context around bla_NDM-5 in different plasmid backgrounds. Arrows represent the position and transcriptional direction of the open reading frames. Accession numbers of specific plasmids’ sequences are stated in Supplementary Table 18. The layout of genetic context has been outlined using Geneious (v11.0.2).

Figure 8.

Schematic layout of genetic context around bla_NDM-1 in different plasmid backgrounds. Arrows represent the position and transcriptional direction of the open reading frames. Truncated genes are denoted by “*”. Accession numbers of specific plasmids’ sequences are stated in Supplementary Table 18. The layout of genetic context has been outlined using Geneious (v11.0.2).