Emergence of a Novel Lineage and Wide Spread of a blaCTX-M-15/IncHI2/ST1 Plasmid among Nosocomial Enterobacter in Guadeloupe

Abstract

Between April 2018 and August 2019, a total of 135 strains of Enterobacter cloacae complex (ECC) were randomly collected at the University Hospital Center of Guadeloupe to investigate the structure and diversity of the local bacterial population. These nosocomial isolates were initially identified genetically by the hsp60 typing method, which revealed the clinical relevance of E. xiangfangensis (n = 69). Overall, 57/94 of the third cephalosporin-resistant strains were characterized as extended-spectrum-β-lactamase (ESBL) producers, and their whole-genome was sequenced using Illumina technology to determine the clonal relatedness and diffusion of resistance genes. We found limited genetic diversity among sequence types (STs). ST114 (n = 13), ST1503 (n = 9), ST53 (n = 5) and ST113 (n = 4), which belong to three different Enterobacter species, were the most prevalent among the 57 ESBL producers. The bla_CTXM-15 gene was the most prevalent ESBL determinant (56/57) and was in most cases associated with IncHI2/ST1 plasmid replicon carriage (36/57). To fully characterize this predominant bla_CTXM-15/IncHI2/ST1 plasmid, four isolates from different lineages were also sequenced using Oxford Nanopore sequencing technology to generate long-reads. Hybrid sequence analyses confirmed the circulation of a well-conserved plasmid among ECC members. In addition, the novel ST1503 and its associated species (ECC taxon 4) were analyzed, in view of its high prevalence in nosocomial infections. These genetic observations confirmed the overall incidence of nosocomial ESBL Enterobacteriaceae infections acquired in this hospital during the study period, which was clearly higher in Guadeloupe (1.59/1000 hospitalization days) than in mainland France (0.52/1,000 hospitalization days). This project revealed issues and future challenges for the management and surveillance of nosocomial and multidrug-resistant Enterobacter in the Caribbean.

Keywords: Caribbean; ESBL; Enterobacter cloacae complex; Nanopore; ST114; ST1503; healthcare; hsp60; molecular sequencing; plasmid.

Figure 1

Distribution of 135 E. cloacae complex (ECC) members isolated from clinical samples according to the hps60 approach. For each hsp60 cluster recovered in this bacterial collection, nosocomial strains were grouped according to their resistance profile to β-lactam antibiotics: wild-type (WT), extended-spectrum-β-lactamase (ESBL), cephalosporinase overproduction without ESBL gene (CoP), or carbapenemase production (CP). The distributions in Figure S3 and the hsp60ECCtool output were identical (for details see Supplementary Data Set S1). Cluster XIV was defined according to Beyrouthy et al. and corresponds to E quasihormaechei [2,3,6]. * UD refers to “undefined hsp60 cluster” [18]. They were not identified in the original paper of Hoffman et al. [4].

Figure 2

Maximum likelihood phylogenetic tree of clinical ESBL-producing E. cloacae complex (ECC) isolated between 2018 and 2019 in Guadeloupe. The core-genome phylogenetic tree was drawn from 57 genomes with iTOL. The tree was rooted at the mid-point and only bootstrap values equal to 100% are indicated by a black circle. The following metadata are indicated: the current species name (PSID) on the inner colored ring and the related ECC Sutton’s clade and hsp60 cluster (CC) on the second one [3,4,6]. The type of hospital department (THD) is indicated on the last circle. Strains with a bla_CTX-M-15 gene with a location predicted on a plasmid and an IncHI2/ST1 plasmid replicon are in red. * New sequence types (STs).

Figure 3

Syntenic analysis of five bla_CTX-M-15/IncHI2/ST1 plasmids from the UHCG. This figure was developed with BRIG software. The first black ring represents the pGENC414 plasmid, used as the reference, and the second ring indicates the GC skew. This first plasmid was isolated from the E. cloacae complex taxon 4 ST1503 (GenBank accession number OL331020, recovered in July 2019). The inner to the outer rings correspond to the following pairwise comparison with IncHI2/ST1 plasmids from other ECC STs isolated at the same hospital: pGENC423 (E. cloacae, ST167, OL331021, August 2019), pGENC405 (E. asburiae, ST53, OL331019, May 2019), pGENC213 (E. xiangfangensis, ST113, OL331018, November 2018) and pGENC200 (E. xiangfangensis, ST114, CP061495, October 2018). The last rings represent the genetic map of pGENC414, with the first hypothetical proteins shown as grey arrows, followed by coding sequences in black arrows, except for genes associated with antibiotic resistance (red), conjugation (green), formaldehyde detoxification (fuchsia), heavy-metal resistance (orange), Fe(III) transporter system (yellow), lac operon (aqua) and plasmid replication, partition or maintenance (blue). The genetic details and metadata of the plasmids are shown in Supplementary Data Set S3.

Figure 4

Maximum likelihood phylogenetic tree of E. cloacae complex (ECC) taxon 4 isolated in Guadeloupe (n = 18) and internationally (n = 20). The core-genome phylogenetic tree was drawn with iTOL, from 38 genomes recovered worldwide between 2006 and 2021 [24]. The tree was rooted with E. chengduensis reference (in bold: CP043318) [5,26]. ECC taxon 4 reference is also presented in bold (JZXZ00000000) [5], and only bootstrap values equal to 100% are indicated by a black circle. The following metadata are indicated: date of isolation, sequence type (ST) and antibiotic resistance genes in black squares. Only acquired genes that confer resistance to β-lactam antibiotics were included. Strains with a bla_CTX-M-15 gene with a location predicted on a plasmid and an IncHI2/ST1 plasmid replicon are shown in red. Metadata are shown in Supplementary Data Set S4.