Novel Enterobacter Lineage as Leading Cause of Nosocomial Outbreak Involving Carbapenemase-Producing Strains

Abstract

We investigated unusual carbapenemase-producing Enterobacter cloacae complex isolates (n = 8) in the novel sequence type (ST) 873, which caused nosocomial infections in 2 hospitals in France. Whole-genome sequence typing showed the 1-year persistence of the epidemic strain, which harbored a bla_VIM-4 ST1-IncHI2 plasmid, in 1 health institution and 2 closely related strains harboring bla_CTX-M-15 in the other. These isolates formed a new subgroup in the E. hormaechei metacluster, according to their hsp60 sequences and phylogenomic analysis. The average nucleotide identities, specific biochemical properties, and pangenomic and functional investigations of isolates suggested isolates of a novel species that had acquired genes associated with adhesion and mobility. The emergence of this novel Enterobacter phylogenetic lineage within hospitals should be closely monitored because of its ability to persist and spread.

Keywords: CPE; Carbapenemase; Enterobacter cloacae complex; Enterobacteriaceae; VIM-4; antimicrobial resistance; bacteria; beta-lactamase; carbapenemase-producing Enterobacteriaceae; next-generation sequencing; nosocomial outbreak.

Figure 1

Epidemic curve of VIM-4–producing Enterobacter cloacae complex isolates (n = 7) in nosocomial outbreak involving carbapenamase-producing Enterobacter strains, Lyon, France, January 12, 2014–December 31, 2015. The attack rate was 0.7/10,000 hospital stays during the study period versus 0.0/10,000 hospital stays during January 11, 2013–November 30, 2014 (p = 0.008). The patients (P1–7) are labeled according to the ST of isolate with which they were infected or colonized. ST, sequence type.

Figure 2

Whole-genome typing of Enterobacter cloacae complex isolates from nosocomial outbreak involving carbapenamase-producing Enterobacter strains, Lyon, France, January 12, 2014–December 31, 2015. A) Dendrogram inferred by the maximum-likelihood method on the basis of core genome SNPs. The node sizes are proportional to the bootstrap values; values >80 are indicated. Scale bar indicates SNPs. The relatedness of the strains was determined by using <15 variant sites as clonality criteria. B) Minimum-spanning tree based on a whole-genome multilocus sequence typing approach, combining the analysis of core genome loci and the presence or absence of accessory genes. Labels on branches indicate the absolute number of variant loci (clonality threshold <10 variant loci). SNP, single-nucleotide polymorphism; ST, sequence type.

Figure 3

Genetic resistance determinants in Enterobacter cloacae complex isolates from nosocomial outbreak involving carbapenamase-producing Enterobacter strains, Lyon, France, January 12, 2014–December 31, 2015. Black cells indicate presence and white cells absence of resistance determinants. The isolates were classified according to the content in resistance determinants by using a binary distance matrix and UPGMA clustering method. Scale bar indicates the dissimilarity in resistance gene content.

Figure 4

Analysis of bla_VIM-4–encoding plasmids from study of nosocomial outbreak involving carbapenamase-producing Enterobacter strains, Lyon, France, January 12, 2014–December 31, 2015. A) Schematic representation of ST1-IncHI2 plasmid pC45-VIM4. The first ring indicates the coordinates of the complete plasmid circle. The 2 outer rings represent the forward and reverse open reading frames, respectively. B) Comparative sequence analysis of ST1-IncHI2 bla_VIM-4–encoding plasmids from this study. The plasmids of isolates C45, C46, C47, C48, C308, C309, C310, E14, and E16 are designated pC45-VIM4, pC46-VIM4, pC47-VIM4, pC48-VIM4, pC308-VIM4, pC310-VIM4, pE14-VIM4, pE16-VIM4, and pC309-VIM4, respectively. C) Comparative sequence analysis of bla_VIM-4–encoding plasmid pC45-VIM4 to the related bla_VIM-1–encoding IncHI2 plasmids pMRVIM0813 (GenBank accession no. KP975077), pRH-R27 (GenBank accession no. LN555650), and pRH-R178 (GenBank accession no. HG530658). Vertical blocks between sequences indicate regions of shared similarity shaded according to blastn (https://blast.ncbi.nlm.nih.gov/Blast.cgi). Blue indicates matches in the same direction; red indicates inverted matches.

Figure 5

Approximately maximum-likelihood phylogenetic trees based on recombination free core single-nucleotide polymorphisms (SNPs) inferred from ST873, ST110 and ST118 genomes and 398 representative genomes of Enterobacter cloacae complex strains in study of nosocomial outbreak involving carbapenamase-producing Enterobacter strains, Lyon, France, January 12, 2014–December 31, 2015. All nodes are supported by Shimodaira-Hasegawa test values >97%. Scale bar indicates SNPs. NA, nonattributed; ST, sequence type.

Figure 6

Average nucleotide identity calculated from BLAST (https://blast.ncbi.nlm.nih.gov/Blast.cgi) pairwise comparisons of ST873 genomes and 398 Enterobacter cloacae complex genomes in study of nosocomial outbreak involving carbapenamase-producing Enterobacter strains, Lyon, France, January 12, 2014–December 31, 2015. NA, nonattributed.

Figure 7

Pangenome analysis of metacluster Enterobacter hormaechei in study of nosocomial outbreak involving carbapenamase-producing Enterobacter strains, Lyon, France, January 12, 2014–December 31, 2015. A) Distribution of COGs); B) functional annotations in the pangenome; C) functional annotations in the variable genome (accessory genome + unique genes); and D) functional annotations for specific genes. Bar charts show the enrichment of COG categories as odds ratios; error bars indicate 95% CIs. Asterisks indicate certain COG categories that are significantly enriched: *p<0.05; **p<0.01;***p<0.001, all by Fisher exact test. Each COG category is identified by a 1-letter abbreviation: C, energy production and conversion; D, cell cycle control and mitosis; E, amino acid metabolism and transport; F, nucleotide metabolism and transport; G, carbohydrate metabolism and transport; H, coenzyme metabolism; I, lipid metabolism; J, translation; K, transcription; L, replication, recombination and repair; M, cell wall/membrane/envelope biogenesis; N, cell motility; O, post-translational modification, protein turnover, and chaperone functions; P, inorganic ion transport and metabolism; Q, secondary metabolism; T, signal transduction; U, intracellular trafficking and secretion; V, defense mechanisms; and X, mobilome. COG, clusters of orthologous groups.