A Decade-Long Evaluation of Neonatal Septicaemic Escherichia coli: Clonal Lineages, Genomes, and New Delhi Metallo-Beta-Lactamase Variants

Abstract

Longitudinal studies of extraintestinal pathogenic Escherichia coli (ExPEC) and epidemic clones of E. coli in association with New Delhi metallo-β-lactamase (bla_NDM) in septicaemic neonates are rare. This study captured the diversity of 80 E. coli isolates collected from septicaemic neonates in terms of antibiotic susceptibility, resistome, phylogroups, sequence types (ST), virulome, plasmids, and integron types over a decade (2009 to 2019). Most of the isolates were multidrug-resistant and, 44% of them were carbapenem-resistant, primarily due to bla_NDM. NDM-1 was the sole NDM-variant present in conjugative IncFIA/FIB/FII replicons until 2013, and it was subsequently replaced by other variants, such as NDM-5/-7 found in IncX3/FII. A core genome analysis for bla_NDM^+ve isolates showed the heterogeneity of the isolates. Fifty percent of the infections were caused by isolates of phylogroups B2 (34%), D (11.25%), and F (4%), whereas the other half were caused by phylogroups A (25%), B1 (11.25%), and C (14%). The isolates were further distributed in approximately 20 clonal complexes (ST_C), including five epidemic clones (ST131, ST167, ST410, ST648, and ST405). ST167 and ST131 (subclade H30Rx) were dominant, with most of the ST167 being bla_NDM^+ve and bla_CTX-M-15^+ve. In contrast, the majority of ST131 isolates were bla_NDM^-ve but bla_CTX-M-15^+ve, and they possessed more virulence determinants than did ST167. A single nucleotide polymorphism (SNP)-based comparative genome analysis of epidemic clones ST167 and ST131 in a global context revealed that the study isolates were present in close proximity but were distant from global isolates. The presence of antibiotic-resistant epidemic clones causing sepsis calls for a modification of the recommended antibiotics with which to treat neonatal sepsis. IMPORTANCE Multidrug-resistant and virulent ExPEC causing sepsis in neonates is a challenge to neonatal health. The presence of enzymes, such as carbapenemases (bla_NDM) that hydrolyze most β-lactam antibiotic compounds, result in difficulties when treating neonates. The characterization of ExPECs collected over 10 years showed that 44% of ExPECs were carbapenem-resistant, possessing transmissible bla_NDM genes. The isolates belonged to different phylogroups that are considered to be either commensals or virulent. The isolates were distributed in around 20 clonal complexes (ST_C), including two predominant epidemic clones (ST131 and ST167). ST167 possessed few virulence determinants but was bla_NDM^+ve. In contrast, ST131 harbored several virulence determinants but was bla_NDM^-ve. A comparison of the genomes of these epidemic clones in a global context revealed that the study isolates were present in close proximity but were distant from global isolates. The presence of epidemic clones in a vulnerable population with contrasting characteristics and the presence of resistance genes call for strict vigilance.

Keywords: Escherichia coli; India; New Delhi metallo-β-lactamases; antibiotic resistance; epidemic clones; phylogroup; virulence.

FIG 1

The timeline of bla_NDM^+ve (A) and bla_NDM^-ve (B) isolates with phylogroups, sequence types (STs), and prevalence (n). For the bla_NDM^+ve isolates both the Warwick scheme (ST^W) and the Institut Pasteur scheme (ST^IP) are mentioned with the bla_NDM-variants (A). For the bla_NDM^-ve isolates, the phylogroups and the clonal complex in the Warwick scheme (STc^W) with ST^IP are mentioned (B). The prevalence of NDM-1, NDM-5, and NDM-7 over 10 years (C).

FIG 2

The schematic diagram of the regions surrounding bla_NDM in ExPEC isolates. Combination a for EN5076, EN5090, NBE0002, NBE0003, EN5143, NBE0006, NBE0007, EN5177, EN5197, EN5286, EN5308, EN5317, EN5349, and EN5358; combination b for EN5095; combination c for NBE0004; and combination d for EN5132 and NBE0008.

FIG 3

The core genome phylogeny of the bla_NDM^+ve study isolates. 30 out of 35 nonclonal bla_NDM^+ve isolates are incorporated in this analysis. A colored box follows the dendrogram to indicate the source of each isolate. The year-month (Y-M) of sample isolation, phylogroup, ST^W (Warwick scheme), fimH type, C-H (fumC-fimH) type, and serotype are mentioned for all isolates. The heat map indicates the presence/absence of resistance determinants (purple), virulence determinants (orange), plasmid replicon types (light green), and integrons (violet) that follow the NDM-variants.

FIG 4

The SNP-based core genome phylogeny of ST^W131 (A) and ST^W167 (B), including both study and global isolates. The isolates are color-coded at the endpoint by nation. The isolate sample collection year has been externally incorporated into the tree phylogeny along with other resistance determinants. The study isolates are highlighted with a light yellow color. (A) For the ST^W131 contextual SNP analysis, genomes from NCBI (n = 48) and the study isolates (n = 11) were included. The NCBI genomes were primarily blood isolates, although some (n = 11) were urine isolates. In total, ST^W131 genomes from 10 countries were included, and a SNP analysis was carried out against the reference EN5382. The presence/absence of bla_{CTX-M-14/15/27}, bla_IMP-4, and bla_NDM-1/-5 are indicated by a heat map (dark red). (B) For the ST^W167 contextual SNP analysis, genomes from NCBI (n = 33) and the study isolates (n = 14) were included. The NCBI genomes were isolated from blood, urine and rectal swabs, and they were collected from 9 different countries. The presence/absence of bla_{NDM-1/-4/-5/-7/-15}, bla_KPC-2, and bla_OXA-181 are indicated by heat map (dark red).

FIG 4

The SNP-based core genome phylogeny of ST^W131 (A) and ST^W167 (B), including both study and global isolates. The isolates are color-coded at the endpoint by nation. The isolate sample collection year has been externally incorporated into the tree phylogeny along with other resistance determinants. The study isolates are highlighted with a light yellow color. (A) For the ST^W131 contextual SNP analysis, genomes from NCBI (n = 48) and the study isolates (n = 11) were included. The NCBI genomes were primarily blood isolates, although some (n = 11) were urine isolates. In total, ST^W131 genomes from 10 countries were included, and a SNP analysis was carried out against the reference EN5382. The presence/absence of bla_{CTX-M-14/15/27}, bla_IMP-4, and bla_NDM-1/-5 are indicated by a heat map (dark red). (B) For the ST^W167 contextual SNP analysis, genomes from NCBI (n = 33) and the study isolates (n = 14) were included. The NCBI genomes were isolated from blood, urine and rectal swabs, and they were collected from 9 different countries. The presence/absence of bla_{NDM-1/-4/-5/-7/-15}, bla_KPC-2, and bla_OXA-181 are indicated by heat map (dark red).