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

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 blaGES-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 blaGES-24 (n = 6), blaGES-6 (n = 4), and blaGES-5 (n = 1) were isolated from the sewage sample. Five blaGES-24 and a blaGES-5 were localized in IncP-6 plasmids, whereas three blaGES-6 plasmids were localized in IncC plasmids with IncF-like regions. The remaining blaGES-6 and blaGES-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 blaGES 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 blaGES-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 blaGES, 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.


Introduction
The emergence of carbapenemase producers threatens the effective treatment of gram-negative bacterial infections, owing to their resistance to the most β-lactams, which are commonly used antibiotics, thus limiting therapeutic options (Gasink et al., 2009;Otter et al., 2017;Bonomo et al., 2018).Guiana extended-spectrum (GES)-type carbapenemase producers are prevalent in both clinical and environmental settings and are known to cause nosocomial outbreaks (Boyd et al., 2015;Naas et al., 2016;Yamasaki et al., 2017;Ellington et al., 2020;Literacka et al., 2020;Nakanishi et al., 2022;Yoo et al., 2023).The bla GES genes are essentially linked to mobile genetic elements, which promote the spread of resistance genes to clinically relevant pathogenic bacteria and have been observed within integron gene cassettes on plasmids with diverse types of replicons (Naas et al., 2016).Although bla GES genes have a high diffusion capacity in the medical environment (Nordmann and Poirel, 2014;Ellington et al., 2020), the genetic contexts of these resistance genes have not been extensively explored.
Hospital wastewater, which connects hospitals with the public health system, is a high-risk interface for the accumulation and effective dissemination of antimicrobial resistance (AMR) genes, increasing their burden on the environment, since the residual antibiotic levels of water may increase, allowing the selection for resistant microorganisms (Rodriguez-Mozaz et al., 2015;Bengtsson-Palme and Larsson, 2016;Mutuku et al., 2022).In addition, the aquatic environment serves as a potential hotspot for gene transfer and bacterial toxin-antitoxin systems, which are essential for the maintenance of these AMR genes, as well as for facilitating gene transfer among different bacterial species (Lee et al., 2015;An et al., 2018).Thus, the microbiological surveillance of hospital sewage is crucial to represent these risks and identify the route of dissemination of the resistant factors outside of hospital settings.Recently, the emergence of bla GES has become an increasing environmental concern with potentially serious public health implications (Halat and Moubareck, 2020).However, the role of environmental transmission in GES-type carbapenemase producers has not been broadly researched, and the genetic features associated with environmental spread remain poorly understood.
This study aimed to investigate bla GES -harboring plasmids isolated from single-hospital sewage using whole-genome sequencing and demonstrate their genomic diversity.Our results provide further insights into the genetic context and impact on the evolution and expansion mechanism of bla GES in hospital wastewater, elucidating the role of the healthcare water environment as a prospective reservoir of AMR.

Wastewater collection and detection of carbapenemase producers
We collected untreated hospital sewage in a sterile bottle from one site of the Tokyo Medical and Dental University Hospital on May 30, 2022, and processed it within 2 h.The sample was concentrated 10×, and aliquots were plated on bromothymol blue (BTB) agar without antibiotics, BTB agar supplemented with ampicillin (32 μg/mL) and sulbactam (16 μg/mL), CHROMagar mSuperCARBA carbapenemaseselective agar (Kanto Chemical, Tokyo, Japan), and BTB agar containing colistin (4 μg/mL), which is reportedly associated with carbapenemase production (Bradford et al., 2015).The agar plates were incubated in ambient air conditions at 37°C.Bacterial colonies with distinct morphologies were subcultured on deoxycholate hydrogen sulfide lactose agar and gram-negative isolates were identified using 16S rRNA sequencing.The sequences were queried against the list of the Ribosomal Database Project, 1 and the species were identified based on the closest relation (Cole et al., 2014).All gram-negative bacteria were transferred to CHROMagar mSuperCARBA agar for carbapenemase screening, and the selected colonies were tested for the presence of carbapenemase-encoding genes (bla IMP , bla VIM , bla KPC , bla GES , bla OXA-48-like , and bla NDM ) using standard polymerase chain reaction (PCR), as described previously (Dallenne et al., 2010;Wachino et al., 2011).

Whole-genome sequencing and analysis
Bacterial DNA was extracted from the resulting colonies of each isolate using a NucleoBond HMW DNA Kit (Macherey-Nagel, Düren, Germany).Library preparation was conducted using the Nextera DNA Flex Library Prep Kit (Illumina, San Diego, CA, United states) for Illumina short reads, and paired-end wholegenome sequencing was performed using the Illumina MiniSeq platform, following the manufacturer's protocol.A DNA library for long reads was prepared using a Ligation Sequencing Kit and then sequenced using a Nanopore MinION sequencer and R9.4.1 flow cell (Oxford Nanopore Technologies), according to the manufacturer's instructions.Filtering and trimming of raw short reads were performed using Fastp v0.22.0 (An et al., 2018) and long reads were quality-filtered using Porechop v0.2.4 (Wick et al., 2017a) De Coster et al., 2018).The filtered reads were de novo hybrids assembled using Unicycler v0.5.0 (Wick et al., 2017b).Circularization of the assembled contigs was confirmed using Bandage v.0.8.1 (Wick et al., 2015).Assembled contigs were annotated using Rapid Annotation using Subsystem Technology v2.0 (Aziz et al., 2008).Bacterial species were identified based on the average nucleotide identity values using the GTDB Toolkit Classify v1.6.0 (Chaumeil et al., 2020).Sequence type (ST) was determined using multi-locus sequence typing (MLST) v2.0 tool 2 and was assigned to isolates using the MLST database 3 (Larsen et al., 2012).The STs of Klebsiella variicola were determined using a previously reported database 4 ( Barrios-Camacho et al., 2019).Acquired AMR genes were detected using ResFinder v4.1 (Zankari et al., 2012) and Basic Local Alignment Search Tool (BLAST) (Altschul et al., 1990).PlasmidFinder v2.0 was used to identify the incompatibility (Inc) type of the plasmids (Carattoli et al., 2014).Integrons were detected using IntegronFinder v2.0 (Neron et al., 2022).ISfinder was used to identify transposon and insertion sequences (Siguier et al., 2006).The BLAST Ring Image Generator v0.95 was used to align assembled reads containing bla GES with closely related plasmids available in the GenBank genome database (Alikhan et al., 2011).EasyFig v2.2.2 was used for a linear comparison of the genetic context of bla GES (Sullivan et al., 2011).A maximum likelihood phylogenetic tree of bla GES -encoding plasmids and the high-homology plasmid from GenBank was constructed using the MEGA X software (Kumar et al., 2018).

Transferability of the bla GES -encoding plasmids
We examined the transferability of plasmids carrying bla GES using bacterial conjugation and transformation methods as described previously, with some modifications (Ota et al., 2022).Based on the Inc. type and the phylogenetic tree of bla GES -encoding plasmids, Enterobacter kobei AS2, K. variicola AS3, K. variicola AS10, and Aeromonas hydrophila CL1 were selected as donor strains.Bacterial conjugation was performed using the agar mating method with sodium azide-resistant Escherichia coli J53 and rifampicin-resistant Escherichia coli C600 as recipient strains.Exponential-phase Luria-Bertani (LB) broth cultures of donor and recipient strains were mixed in equal proportions and incubated on LB agar plates at 37°C.The conjugation mixture was plated on BTB agar plates with 2 μg/mL meropenem or 2 μg/mL cefotaxime plus 50 μg/mL rifampicin for Escherichia coli C600 recipients or 100 μg/mL sodium azide for Escherichia coli J53 recipients, to select for potential transconjugants.For transformation, plasmid DNA was extracted from the donor strains using the NucleoBond Xtra Midi Kit (Takara Bio, Shiga, Japan) and then electroporated into Escherichia coli HST08 Premium Electro-Cells (Takara Bio).The probable transformants were selected on LB agar plates supplemented with either 2 μg/mL meropenem or 2 μg/mL cefotaxime.The bla GES transconjugants and transformants were screened using PCR and antimicrobial susceptibility testing (Dallenne et al., 2010).To confirm 2 http://genomicepidemiology.org/ 3 http://pubmlst.org/4 https://mlstkv.insp.mx/ the localization of bla GES , short-read whole-genome sequencing was conducted using a MiniSeq sequencing platform, and plasmid-derived reads were assembled using PlasmidSPAdes (Galaxy Version 3.15.4+galaxy2) (Giardine et al., 2005;Antipov et al., 2016).Conjugation frequency was determined as the number of transconjugants obtained per recipient cell.

Isolation of GES-type carbapenemase producers from hospital sewage
A total of 38 gram-negative bacteria were recovered from hospital sewage and identified using 16S rRNA gene sequencing (Supplementary Table S1).Among them, 11 isolates (four Enterobacter spp., three Klebsiella spp., three Aeromonas spp., and one Serratia spp.) were identified as GES-type-β-lactamase producers using standard PCR, but no carbapenemase genes other than bla GES were detected.Hence, these isolates were used for short-and long-read sequencing (Table 1).Each isolate was classified as a phylogenetically distinct strain with an average nucleotide identity value of more than 95.0%, based on whole-genome species identification and MLST.The bla GES genes of all 11 isolates were detected on circular plasmids and identified as bla GES-24 (n = 6), bla GES-6 (n = 4), and bla GES-5 (n = 1).The bla GES -encoding plasmids AS12 and CL1 also contained the OXA-17 and PAC-1 β-lactamase genes, respectively.

Structural characterization of bla GES -harboring plasmids
The genetic structure of the bla GES -encoding plasmids was identified in all GES-type carbapenemase-producing isolates (Figure 1; Table 1).Five of the six bla GES-24 and bla GES-5 were localized on IncP-6 plasmids, whereas three of the four bla GES-6 plasmids were located on IncC plasmids with IncF-like conjugal transfer regions.The remaining bla GES-6 and bla GES-24 were localized on IncFIB-containing plasmids with IncF-like regions and a plasmid with an IncW-like replication protein, respectively.These circular closed plasmids ranged in length from 15,748 bp to 170,503 bp.All bla GES -containing plasmids were located on the class 1 integron cassette of the Tn3 transposonrelated region and contained genes resistant to several antibiotics, including β-lactams, aminoglycosides, quinolones, sulfamides, and chloramphenicol.The bla GES -containing plasmid of AS10 strain carried two copies of the class 1 integron cassette, each with bla GES-6 .Four of the six IncP-6 plasmids, two of the three IncC/IncF-like plasmids, and one IncW-like plasmid carried a VapBC toxin-antitoxin module.The HigBA toxin-antitoxin system was located on all three IncC/IncF-like plasmids.Partial toxin-antitoxin components, BrnA, VbhA, and BrnT, were detected in the IncP-6, IncC/IncF-like, and IncW-like plasmids, respectively.No toxin-antitoxin region was observed in two IncP-6 and IncFIB/IncF-like plasmids.BLAST analysis of the whole sequence of the IncP-6 plasmid of AS3_contig8 indicated more than 98% identity with the plasmid pKAM644_7 (98% query coverage, AP026414) harbored by environmental Klebsiella quasipneumoniae from Japan (Figure 1A).The IncC/IncF-like plasmid AS10_contig3 shared 100% homology and 90% coverage with the bla GES -noncoding Escherichia coli plasmid pECO-dc1b (CP026207) isolated from wastewater and sludge in the United States of America (Figure 1B).The AS2_contig2 plasmid harboring IncFIB/IncF-like regions showed more than 99% identity with Raoultella ornithinolytica strain 23 plasmid p23_C (35% query, CP048352) from the environment in Switzerland, Enterobacter cloacae strain EN3600 plasmid unnamed5 (34% query, CP035637) from a clinical blood sample in China, and R. ornithinolytica strain MQB_Silv_108 plasmid pIncFII_Silv108 (34% query, CP104453) from wastewater in Spain (Figure 1C).The IncW-like CL1_contig4 plasmid displayed similarities to Japanese isolates of R. ornithinolytica plasmid pWP3-W18-ESBL-06_2 (AP021985) with 78% coverage and 99% identity from wastewater treatment plant effluent, Enterobacter roggenkampii 6 BC1 plasmid p6BC1_GES-24 (LC735980) with 57% coverage and 98% identity from municipal wastewater, and Enterobacter roggenkampii OIPH-N260 plasmid pN260-3 (AP023450) with 55% coverage and 99% identity from human bile (Figure 1D).A maximum likelihood phylogenetic tree and the genetic context of the bla GES -encoding plasmids are shown in Figure 2. Four of the six IncP-6 plasmids showed a closer phylogenetic relationship with the bla IMP-1 -and bla GES-5 -encoding IncP-6 plasmid pN260-3 (AP023450) from Japan.The remaining two IncP-6 plasmids and all IncC/IncF-like plasmids clustered differently.Alignment analysis of the genetic environment of these plasmids showed relatively high background similarity around the bla GES -containing region.In particular, the bla GES genetic structures of the three IncP-6 plasmids (AS3_contig8, CA1_ contig5, and CA6_contig7) and IncC/IncF-like plasmids were very similar, whereas the AS10_contig3 plasmid had two structural copies.

Transferability of bla GES -harboring plasmids
Four isolates with different replicon types of bla GES -harboring plasmids were evaluated for their ability to transfer bla GES to Escherichia coli recipient strains.In the conjugation experiment, bla GES of AS10 was successfully transferred to the Escherichia coli J53 recipient strain (AS10_transconjugant), whereas transconjugants of AS2, AS3, and CL1 were not recovered.The transfer frequency of bla GES -encoding plasmids in AS10 was 7.8 × 10 −6 .Electrotransformation experiments failed to produce bla GES -containing transformants from AS2, AS3, and CL1 as confirmed using PCR.We similarly confirmed transferability for isolates with the same IncP-6 plasmid (AS1, AS8, AS12, CA1, and CA6) as AS3, but no transconjugants or transformants were obtained.A scaffold sequence containing bla GES was obtained from the assembled short-read data of the AS10_transconjugant.The scaffold data of the AS10_transconjugant showed high homology with AS10_contig3 (Supplementary Figure S1).

Susceptibility profile of GES-type carbapenemase-producing isolates
The antimicrobial susceptibility profiles of the isolates with bla GES are described in Table 2. Eight isolates were non-susceptible to carbapenems (imipenem, meropenem, and doripenem), while three were susceptible to them.Regarding non-β-lactam antibiotics, more than half of the isolates were resistant to tobramycin (4/8; 50%),

Discussion
The incidence of carbapenemase-producing infections significantly increases mortality and medical costs (Gasink et al., 2009;Otter et al., 2017).GES-type carbapenemase producers are present in hospitals for prolonged time, causing outbreaks within hospitals (Boyd et al., 2015;Yamasaki et al., 2017;Ellington et al., 2020;Literacka et al., 2020;Nakanishi et al., 2022;Yoo et al., 2023).However, the genetic features of resistant isolates are not well understood, and their diffusion capabilities could be underestimated.In this study, we characterized diverse plasmids carrying GES-type carbapenemase genes in a sample of hospital sewage in Japan.The existence of a wide variety of GES-type carbapenemase producers within the same sample raises concerns about their potential to promote genetic variability in the environment.Furthermore, these bla GES -containing plasmids have a novel genetic context with two integron cassettes and several toxinantitoxin-related regions involved in plasmid maintenance (Yang and Walsh, 2017), indicating their contribution to the evolution and dissemination of resistance.These results have key implications for hygiene because carbapenems are terminal use antibiotics whose resistance may disseminate to the environment through hospital effluents (Mills and Lee, 2019).
Hospital sewage, which is abundant in pathogens and residual antimicrobials, can serve as a persistent reservoir for AMR (Zhang et al., 2020).Antimicrobial regimens are dependent on the patients being treated at each hospital; hospital drug usage influences the bacterial composition and AMR gene abundance in hospital wastewater (Guo et al., 2021;Perry et al., 2021).In routine wastewaterbased monitoring, AMR gene profiles are distinct among hospitals using varying amounts of antibiotics, thus highlighting the prevalence of AMR in hospitals (Majlander et al., 2021).Therefore, hospital effluents with selective pressure from environmentally persistent antibiotics can act as major sources of AMR, thereby increasing the risk of AMR dissemination and infection caused by AMR isolates.
A study analyzing samples from hospital wastewater treatment plants identified bla GES as one of the dominant β-lactamase genes (Hubeny et al., 2021).Indeed, GES-type carbapenemase producers have been identified in hospital wastewater samples from various countries (Gomi et al., 2018;Suzuki Y. et al., 2020;Maehana et al., 2021;Conte et al., 2022;Takizawa et al., 2022;Zagui et al., 2023), which is consistent with our results.Additionally, bla GES -harboring isolates have been widely detected in wastewater treatment plants (Girlich et al., 2012;Urase et al., 2022), suggesting that the resistance genes from sewage may also be disseminated into communities.Hence, hospital sewage is a potential hazard for transferring resistant organisms to the environment, which is an important public health issue.GES-type-β-lactamases have been increasingly reported in gramnegative bacteria; bla GES-5 , bla GES-6 , and bla GES-24 display carbapenemase activities (Naas et al., 2016;Uechi et al., 2018).The MICs to meropenem in these carbapenemase producers vary widely from ≤0.5 to >32 μg/mL, consistent with the low-level resistance to meropenem reported for GES-type carbapenemases (Ellington et al., 2020).In addition to bla GES-24 , Aeromonas hydrophila CL1 contains a novel class C β-lactamase, PAC-1 (Bour et al., 2019) derived from Pseudomonas aeruginosa in the plasmid but is still sensitive to meropenem.The 11 GES-type bacteria isolated in this study were classified as genetically distinct strains using whole-genome sequencing-based species identification and MLST analysis.Among the identified genera, Aeromonas spp.are ubiquitous bacteria primarily found in most water environments and have recently gained attention as an arsenal of AMR genes and mobile genetic elements in natural environments (Usui et al., 2016;Fernandez-Bravo and Figueras, 2020;Gomes et al., 2021;Maehana et al., 2021;Canellas et al., 2023).Previous studies have reported a higher prevalence of diverse AMR genes and integron cassettes in Aeromonas spp.isolates (Fernandez-Bravo and Figueras, 2020;Canellas et al., 2023).Maehana et al. isolated Aeromonas spp.carrying four tandem copies of bla GES-24 from sewage water at a medical institution (Maehana et al., 2021).These observations, coupled with our results, suggest that Aeromonas spp.could be a possible bacterial indicator for predicting the dissemination of AMR in hospital sewage.
PlasmidFinder and BLAST analyzes showed that the bla GEScontaining plasmids can be classified into the following four groups: IncP-6, IncC/IncF-like, IncFIB/IncF-like, and IncW-like.While these various types of GES producers have been isolated from patients worldwide (Literacka et al., 2020;Umeda et al., 2021), similar bla GEScontaining plasmids were not obtained from clinical isolates in our hospital.Although phylogenetic tree analysis revealed genetic relatedness in some IncP-6 or IncC plasmids in this study, determining high-resolution plasmid relationships using phylogenetic analysis is still challenging (Orlek et al., 2017;Suzuki M. et al., 2020).In the circular comparison of bla GES -encoding plasmids, the whole sequences of the IncP-6 and IncW-like plasmids showed high homology with 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.Ota et al. 10.3389/fmicb.2023.1209195Frontiers in Microbiology 07 frontiersin.orgthose of bla GES -encoding plasmids isolated in Japan (Umeda et al., 2021).bla GES with the IncP-6 plasmid backbone has also been isolated from both clinical and environmental samples in Poland (Literacka et al., 2020) and Brazil (Conte et al., 2022), suggesting that the plasmid may play an important role in the global dissemination of these genes.BLAST analysis revealed that the full sequences of the IncC/IncF-like and IncFIB/IncF-like plasmids were highly similar to plasmids discovered in various bacterial species in both clinical and environmental samples.The IncF-like conjugative transfer proteins in these plasmids are involved in horizontal gene transfer and may expand their ability to disseminate AMR genes (Lang and Zechner, 2012;Rozwandowicz et al., 2018).The plasmids encoding bla GES containing IncC (Takashima et al., 2022) and IncFIB (Teixeira et al., 2022) have been reported globally.We simultaneously detected four plasmid types related to sporadic isolates worldwide in wastewater from a single hospital.Notably, the AS3, CA1, and CA4 strains had nearly identical bla GES -harboring plasmids, despite belonging to different bacterial species.Thus, bla GES may spread by coordinating changes in the diverse genetic structures of plasmids for long-term persistence and could be a major cause of hospital wastewater contamination.
The bla GES gene of all plasmids was located on the class 1 integron cassette, along with a Tn3 transposon-derived sequence.The plasmid backbone was deposited in various bacterial species databases (Umeda et al., 2021).Comparison of the context around bla GES revealed that the genetic environment was relatively conserved among the plasmid incompatibility groups.However, AS10_contig3 harbored two class 1 integron cassettes with bla GES .Tandem copies of bla GES exist within an integron cassette (Xu et al., 2018;Maehana et al., 2021;Takizawa et al., 2022); however, multiple copies of the integron cassette with bla GES have not been observed.The expression level of AMR genes within integron cassettes was the highest when the gene was in the first cassette (Collis and Hall, 1995;Souque et al., 2021), indicating that the novel bla GES -surrounding region of AS10_contig3 could potentially increase the total amount of integron cassette expression and phenotypic resistance.The horizontal transmission of plasmids is critical in the spread of AMR genes.Our conjugation experiment showed that the IncC/IncFlike plasmid was successfully transferred to the recipient strain, whereas the IncP-6, IncFIB/IncF-like, and IncW-like plasmids were not transferred to Escherichia coli J53 and C600 recipient strains.The different profiles of conjugal transfer genes in each plasmid may affect their transferability (Zatyka and Thomas, 1998).The transconjugant of bla GES-6 -containing IncC/IncF-like plasmid acquired resistance to most β-lactam antibiotics, including carbapenems, indicating that bla GES-6 was functional in the Escherichia coli parent strain.None of the transformants were obtained from IncP-6, IncFIB/IncF-like, or IncWlike plasmids via electroporation using the Escherichia coli HST08 strain.These failures in both conjugation and transformation are consistent with previous findings and may be due to the exclusion of Escherichia coli from the host replication range in bla GES -containing plasmids (Bonnin et al., 2011;Chudejova et al., 2018;Conte et al., 2022).Moreover, the transfer mechanisms responsible for AMR gene acquisition include integron-mediated mobilization of gene cassettes (Fluit and Schmitz, 1999).Thus, our results indicate that integrons, instead of plasmids, may mainly be responsible for the dissemination of bla GES among bacteria.
Toxin-antitoxin systems are distributed in environmental plasmids with AMR genes and are known to allow stable plasmid persistence in bacteria (Lee et al., 2015;An et al., 2018;Takashima et al., 2022).Wozniak et al. reported that these systems advance the maintenance of a conjugative element (Wozniak and Waldor, 2009).We showed that most bla GES -encoding plasmids contained multiple toxin-antitoxin systems and their components.Seven of the 11 plasmids carried the VapBC toxinantitoxin system, which is associated with growth inhibition to resist environmental stress (Eroshenko et al., 2020).The IncC/IncF-like plasmid, which was successfully conjugated, notably encoded the HigBA toxin-antitoxin system and demonstrated plasmid stability in the environment (Qi et al., 2021).
In summary, we provided insights into the genomic structures of plasmids harboring bla GES from different bacterial species in hospital sewage.Our results indicate considerable genetic diversity of bla GESencoding plasmids in the same water sample, suggesting their high adaptability to aquatic environments.These findings revealed the importance of healthcare wastewater as a potential reservoir of bla GES , highlighting the need for serial monitoring of environmentally emerging GES-type carbapenemase producers.

FIGURE 1
FIGURE 1Circular 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.

TABLE 1
Bacterial species, sequence type, and genome characteristics of bla GES -encoding plasmids.

TABLE 2
Susceptibility profile of bla GES -producing isolates, Escherichia coli J53 recipient strain, and the transconjugant.