Novel insights into genetic characteristics of blaGES-encoding plasmids from hospital sewage

Abstract

Introduction: The prevalence of Guiana extended-spectrum (GES)-type carbapenemase producers is increasing worldwide, and hospital water environments are considered as potential reservoirs. However, the genetic features underlying this resistance are not yet fully understood. This study aimed to characterize bla_GES-encoding plasmids from a single-hospital sewage sample in Japan.

Methods: Carbapenemase producers were screened using carbapenemase-selective agar and polymerase chain reaction. Whole-genome sequencing analyzes were performed on the carbapenemase-producing isolates.

Results: Eleven gram-negative bacteria (four Enterobacter spp., three Klebsiella spp., three Aeromonas spp., and one Serratia spp.) with bla_GES-24 (n = 6), bla_GES-6 (n = 4), and bla_GES-5 (n = 1) were isolated from the sewage sample. Five bla_GES-24 and a bla_GES-5 were localized in IncP-6 plasmids, whereas three bla_GES-6 plasmids were localized in IncC plasmids with IncF-like regions. The remaining bla_GES-6 and bla_GES-24 were, respectively, localized on IncFIB-containing plasmids with IncF-like regions and a plasmid with an IncW-like replication protein. The IncP-6 and IncW-like plasmids had a close genetic relationship with plasmids from Japan, whereas the IncC/IncF-like and IncFIB/IncF-like plasmids were closely related to those from the United States and Europe. All bla_GES genes were located on the class 1 integron cassette of the Tn3 transposon-related region, and the IncC/IncF-like plasmid carried two copies of the integron cassette. Eight of the eleven bla_GES-encoding plasmids contained toxin-antitoxin system genes.

Discussion: The findings on the plasmids and the novel genetic content from a single wastewater sample extend our understanding regarding the diversity of resistance and the associated spread of bla_GES, suggesting their high adaptability to hospital effluents. These findings highlight the need for the continuous monitoring of environmental GES-type carbapenemase producers to control their dissemination.

Keywords: GES; hospital sewage; integron; plasmid; toxin-antitoxin system.

Figure 1

Circular comparison of bla_GES-encoding plasmids with closely related plasmids available in a public database. The reference sequences of AS3_contig8 including IncP-6 (A), AS10_contig3 including IncC/IncF-like (B), AS2_contig2 including IncFIB/IncF-like (C), and CL1_contig4 including IncW-like (D) with their shared regions are visualized. Each plasmid is depicted with a ring of different colors and the color intensity shows the nucleotide homologies. The colored arrows represent the positions and directions of specific plasmid modules. Purple, replicon; red, antimicrobial resistance gene; orange, mobile element; light green, IS family; blue, toxin-antitoxin system gene; navy, conjugal transfer gene.

Figure 2

Evolutionary analysis and genetic environment of bla_GES-encoding plasmids. Maximum likelihood phylogenetic tree was generated with a bootstrapping of 1,000 replicates; and bootstrap values are shown on the major nodes. Plasmid names and Inc types are indicated at the right of the tree. Structures of bla_GES surrounding region into class 1 integron are also represented. The arrows indicate the position and the translation orientation of the coding genes and are colored in accordance with the gene functional classification. The integrase gene, bla_GES, other AMR genes, mobile element, and toxin-antitoxin system gene are highlighted in purple, red, yellow, orange, and blue, respectively. The grey color scale denotes the percentage of sequence identity.