Emergence of High Level Carbapenem and Extensively Drug Resistant Escherichia coli ST746 Producing NDM-5 in Influent of Wastewater Treatment Plant, Seoul, South Korea

High level carbapenem and extensively drug resistant (XDR) Escherichia coli strain N7, which produces a variant of New Delhi metallo-β-lactamase (NDM-5), was isolated from the influent of the Jungnang wastewater treatment plant located on Han River, Seoul, South Korea. Phenotypic and genotypic resistances to carbapenem were tested using agar and broth dilution methods, and polymerase chain reaction. Whole-genome sequencing was performed to characterize the genetic structure of strain N7. E. coli strain N7, which harbors the blaNDM–5 gene, showed high level of carbapenem resistance at concentrations of doripenem (512 mg/L) and meropenem (256 mg/L), and XDR to 15 antibiotics. Based on the genomic sequence analysis, two plasmids, a hybrid IncHI2/N-type and an IncX3 type, were present. The former contains a cluster (blaNDM–5-bleMBL-trpF-dsbD) bracketed by multi-insertional sequences, IS3000, ISAba125, IS5, and IS26. The latter carries the following resistance genes: blaCTX–14, aac(3)-IV, aadA1, aadA2, aph(3′)-Ia, aph(4)-Ia, sul1, sul2, sul3, dfrA12, fosA3, oqxA, oqxB, mph(A), and floR, and cmlA1. The chromosome, contig3, and contig5 also carry blaCTX–64 and mdf(A), tet(A), and erm(B), tet(M) and aadA22, respectively. Strain N7 also harbors virulence factors such as fimH, flu, ecpABCDE, sfmA, hlyE, and gadA. This study demonstrates the emergence of high level carbapenem resistant XDR E. coli strain N7 containing blaNDM–5 in aquatic environment, Seoul, South Korea. Due to the presence of mobile genetic elements, this strain could horizontally transfer resistance genes, including blaNDM–5 to environmental bacteria. Thus, it is necessary to conduct continuous surveillance for carbapenem resistance in various aquatic environments.


INTRODUCTION
Carbapenem-resistant Enterobacteriaceae (CRE) is one of the most critical pathogens, together with carbapenem-resistant Acinetobacter baumannii and Pseudomonas aeruginosa, and has been clinically issued with growing concerns in need of new antibiotics (Tacconelli et al., 2017). CRE can produce several enzymes belonging to the class of New Delhi metallo-β-lactamase (NDM) to hydrolyze carbapenems (Doi and Paterson, 2015). Since the first report of NDM (Yong et al., 2009), a series of NDM variants, which possess distinct hydrolytic activity against β-lactams (bla NDM ) from NDM-1 to NDM-29, have been identified with the clinical evolution of NDM (Cheng et al., 2018). In particular, NDM-5 producing Escherichia coli shows higher level of resistance to carbapenems compared to previously reported NDM-1 producing bacteria (Hornsey et al., 2011).
The first occurrence of NDM-5 producing E. coli EC405 was reported in a patient in the United Kingdom in 2011, and it showed a high level of resistance to cephalosporins, carbapenems, aminoglycosides, and quinolones, while being susceptible to colistin and tigecycline (Hornsey et al., 2011). Following this discovery, two carbapenemase-producing Enterobacteriaceae (NDM-5 producing E. coli and NDM-1 producing Klebsiella pneumoniae) showing distinct hydrolytic activity against imipenem were isolated from a traveler from Bangladesh in 2013 and Indonesia in 2014, respectively (Nakano et al., 2014). Subsequently, in South Korea, NDM-9 and NDM-5 producing Klebsiella variicola and E. coli strains were recovered from a river in 2017 (Di et al., 2017) and patients in 2018 (Jhang et al., 2018), respectively, suggesting that environmental and clinical NDM-producing bacteria are in circulation.
The bla NDM genes have been predominantly found in opportunistic pathogenic bacteria displaying resistance to multiple antimicrobials, particularly, Enterobacteriaceae, such as E. coli, Klebsiella sp., and Enterobacter sp. (Bush, 2010). Since the isolation of clinical NDM-1 producing Acinetobacter spp. and Pseudomonas spp. in 2012 (Bharadwaj, 2012), the occurrence of NDM-producing bacteria has been on the rise in various aquatic environments including river stream, wastewater treatment plants (WWTPs), and tap water Luo et al., 2013;Di et al., 2017). WWTPs have been suggested as potential hot spots for antibiotic resistance (Karkman et al., 2018). Contamination determinants from households, hospitals, farms, and other non-point source pollutions may play a role in selective pressure for the increase in antibiotic resistance, escalating antibiotic resistance that enables the development of multi-drug resistant (MDR), extensively drug resistant (XDR), and/or pandrug resistant (PDR) bacteria, which make it increasingly difficult to treat infections.
In this study, we report the emergence of pathogenic, and highly carbapenem-resistant and XDR E. coli strain N7, isolated from the urban influent of Jungnang WWTP on the Han River located in Seoul, the capital city of South Korea. Wholegenome sequencing analyses of E. coli strain N7 indicated that 23 antibiotic resistance genes (ARGs) including bla NDM−5 , a variant of NDM, were present in chromosome, plasmids, and contigs. Among them, seventeen were carried on two plasmids, which were formulated structurally in a manner of well-known conserved clusters with either class 1 integron and/or insertional sequences (ISs), suggesting that E. coli strain N7 can act as a carrier of ARGs in the aquatic environment.

Isolation and Identification of Carbapenem-Resistant Bacteria From a WWTP
The influent sample was collected from the Jungnang (JN) WWTP on the Han River, Seoul, South Korea in May of 2018 by using sterile bottles. After collection, the samples were immediately shipped to the laboratory under cool conditions (4 • C) and filtered through a 0.22 µm pore size membrane filter (Advantec, Tokyo, Japan). The membranes were suspended in 10 mL of Mueller-Hinton (MH) broth (MBCell, Seoul, South Korea), thoroughly vortexed, and then processed with a serial dilution up to 10 −3 times (10 0 , 10 −1 , 10 −2 , and 10 −3 ). A 100 µL of sample of the MH broth was spread on mSuperCARBA (CHROMagar, France) agar plates and the plates were incubated at 37 • C for 48 h. After incubation, the colonies on the plates were streaked on new MH agar plates containing 8 mg/L of meropenem to obtain a single colony of presumptive carbapenemase-producing bacteria. The isolate grown on the plates were taxonomically identified using 16S rDNA gene sequencing (Macrogen, Seoul, South Korea).

Whole Genome Sequencing
The genome was constructed de novo using PacBio sequencing data (Pacific Biosciences, Menlo Park, CA, United States). Sequencing analysis was performed at Chunlab Inc. (Seoul, South Korea). PacBio sequencing data were assembled with PacBio SMRT Analysis 2.3.0 using the HGAP2 protocol (Pacific Biosciences). The resulting contigs from PacBio sequencing data were circularized using Circulator 1.4.0 (Sanger Institute, Hinxton, Cambridgeshire, United Kingdom) (Yoon et al., 2017). Circular maps for plasmid structures and linear maps generated by Circulator 1.4.0 and geneCo (Jung et al., 2019), respectively, were manually modified. The chromosomal and plasmid origins of replication were identified using DoriC 5.0 and the plasmid types were determined by PlasmidFinder 1.3, using FASTA file (Carattoli et al., 2014). ARGs were identified using ResFinder (Zankari et al., 2012). Multi-locus sequence type (MLST) was determined by sequences of seven housekeeping genes (adk, fumC, gyrB, icd, mdh, purA, and recA) according to a previous description (Clermont et al., 2000). The WGS data were deposited in GenBank under the accession JABWPS000000000.

Serotyping and Virulence Determinants
Carbapenemase-producing E. coli strain was serotyped with four O-antisera (O26, O111, O146, and O157) (SSI Diagnostica, Hillerød, Denmark) by incubation in MH broth for 16 h, boiling at 95 • C for 15 min. Equal volume of the lysate and antisera were mixed in a 96-well culture plate, and then incubated at 52 • C overnight. The agglutination of O-antigen and O-antisera was visually checked according to a previously described protocol (SSI Diagnostica). Virulence genes and serotype were determined from WGS data using VirulenceFinder and SerotypeFinder 2.0 (Carattoli et al., 2014;Joensen et al., 2015).

Isolation and Identification of Carbapenem-Resistant Bacteria
Among the 50 isolates from the influent of JN WWTP, 24 isolates were presumptive carbapenem-resistant bacteria. The PCR detection of 11 carbapenemase genes (bla IMP , bla VIM , bla NDM , bla SPM , bla AIM , bla DIM , bla GIM , bla SIM , bla KPC , bla BIC , and bla OXA−48 ) revealed that only one isolate was positive for the bla NDM . This isolate, N7, was taxonomically identified as E. coli by 16S rDNA gene sequencing. MLST revealed that E. coli strain N7 belonged to ST746.  (Table 1). Compared to the CLSI clinical breakpoint, E. coli strain N7 exhibited high level of resistance to eight classes of the antibiotics tested, except for trimethoprim-sulfamethoxazole. Regarding the extent of the antibiotic resistance up to 15 of 16 antibiotics tested, strain N7 is likely to be an XDR bacterium.
In addition, E. coli strain N7 carries the following eight virulence factors: adhesion-associated molecules (fimH, flu,

DISCUSSION
In the present study, we report on the emergence of XDR E. coli strain N7 which is positive for bla NDM−5 and characterization of the genetic context of ARGs, including bla NDM−5 . Since the discovery of NDM in a Swedish patient who traveled to India, its variants have grown to 28 different types from diverse bacteria, mostly isolated from clinical samples. In South Korea, NDM-5 producing Enterobacteriaceae have been reported only in clinical environments (Park et al., 2016(Park et al., , 2019Kim et al., 2020), and NDM-9 producing K. variicola were found in river (Di et al., 2017). Escherichia coli strain N7 belonging to ST746 isolated from the urban influent of JN WWTP shows a variant of the NDM, NDM-5 type. From the WGS, we identified  two plasmids such as a narrow host range plasmid IncX3 (Johnson et al., 2012) and a hybrid IncHI2/N. The narrow host range plasmid IncX3 carries a cluster structure of 5 -IS3000-DISAba125-IS5-bla NDM−5 -ble MBL -trpF-dsbD-IS26-3 containing the bla NDM−5 gene. Figure 1A shows the composition of ISs, which cassettes structural genes of 5 -bla NDM−5 -ble MBL -trpF-dsbD-3 (Liu et al., 2013;Nakano et al., 2014;Zhu et al., 2016;Yuan et al., 2019) with a minor change in the presence and absence of IS5 and the extent of truncated ISAba125 among the analyzed E. coli and K. pneumoniae strains. The question is still remained why the structural genes of 5 -ble MBL -trpF-dsbD-3 with bla NDM−5 are always clustered together. In addition, the IncX3 type plasmid in E. coli strain N7 also contains a type IV secretion system (virD2-virB1-virB4-virB5-virB6-virB8-virB9-virB10-virB11-virD4) located at a site opposite that of bla NDM−5 . It should be noted that the type IV secretion system has also been hypothesized to be involved in horizontal gene transfer between other bacteria (Juhas et al., 2008). Taken together, E. coli strain N7 is likely to have a system to transfer recently emerged bla NDM−5 gene to other bacteria due to multiple ISs and type IV secretion system, although it contains the narrow host range vector system (Liakopoul et al., 2018).
It is known that E. coli ST746 carries extended-spectrum β-lactamase (ESBL) genes from fishes (Sellera et al., 2018) and human patients (Wu et al., 2018). In this study, E. coli strain N7 harbored ESBL and eight virulence factors. Surprisingly, E. coli strain N7, which showed MIC of meropenem at 256 mg/L, was also resistant to several antibiotics with very high MIC values for the tested antimicrobials (Table 1), compared to other NDM-5 producing E. coli strains (Hornsey et al., 2011;Rahman et al., 2014;Zhu et al., 2016;Jhang et al., 2018). This XDR pattern can be explained by the presence of several resistance genes located on the broad host range plasmid (Figure 2; Zhao et al., 2018). Therefore, the presence of XDR E. coli, isolated from the influent of WWTP located in a city, along with the carbapenem-resistance gene raises public health concerns due to the possible dissemination of ARGs to other pathogenic bacteria, and difficulty in treatment of infections. Indeed, XDR pathogenic E. coli strains have been reported from human patients (harboring bla KPC−2 ) (Jeong et al., 2018) and from chickens (co-producing bla NDM and mcr-1) (Lv et al., 2018), increasing the likelihood of infectious disease outbreaks. The characteristics of XDR E. coli strain N7 can be attributed to the presence of corresponding resistance genes located on two plasmids of an IncX3 and a hybrid IncHI2/N. The occurrence of the IncHI2 plasmid has been frequently reported in Salmonella strains with multiple ARGs (Chen et al., 2016). In our experiments, most of the resistance genes were found on the hybrid plasmid IncHI2/N of E. coli strain N7, containing diverse resistance determinants, including aminoglycoside [aac(3)-IV, aadA1, aadA2, aph(3 )-Ia, and aph(4)-Ia], β-lactam (bla CTX−64 , bla CTX−14 , and bla NDM−5 ), fosfomycin (fosA5), macrolide [mdf(A) and mph(A)], phenicol (floR and cmlA1), quinolone (oqxA and oqxB), sulfonamide (sul1, sul2, and sul3), and trimethoprim (dfrA12).

CONCLUSION
In conclusion, NDM-5 producing E. coli strain N7, which shows a high level of carbapenem resistance and an XDR pattern, was found in the megacity influent of Jungnang WWTP, Seoul, South Korea. Our findings suggest that pathogenic XDR E. coli originating from urban activities may be disseminated into the river from WWTP and is a potential carrier or spreader of ARGs, including emerging carbapenemase genes. Thus, we need to focus on the continuous surveillance of carbapenemase-producing bacteria in diverse environments.

DATA AVAILABILITY STATEMENT
The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found in the article/ supplementary material.

AUTHOR CONTRIBUTIONS
HS: experiment, data analysis, and manuscript writing. YK: methodology. DH: revision of manuscript. H-GH: overall revision, methodology, and data analysis. All authors contributed to the article and approved the submitted version.

FUNDING
This work was supported by the Korea Ministry of Environment (MOE) as "the Environmental Health Action Program" (2016001350006).