Emergence and transmission of New Delhi metallo‐beta‐lactamase‐5‐producing Escherichia coli Sequence Type 361 in a Tertiary Hospital in South Korea

Abstract Background The emergence of carbapenem‐resistant Escherichia coli (E coli) is a serious global health threat, but little is known about carbapenemase‐producing E coli in Daejeon, South Korea. The aim of this study was to investigate characteristics of thirteen carbapenem‐resistant E coli isolates in a tertiary hospital. Methods Thirteen non‐duplicate carbapenem‐resistant E coli strains were collected from October 2017 to January 2018. Antimicrobial susceptibility was determined with the E test or disk diffusion method. The carbapenem minimum inhibitory concentrations (MICs) were determined by the agar dilution method. The colistin and tigecycline MICs were determined by broth microdilution. The resistance genes, including carbapenemase genes, were evaluated by polymerase chain reaction, and DNA sequencing was performed to characterize the genes. Pulsed‐field gel electrophoresis and multilocus sequence typing (MLST) were performed to evaluate the clonal relatedness of isolates. The clinical data of patients were retrospectively reviewed. Results All the E coli isolates harbored blaNDM‐5 gene and were resistant to most of the antimicrobial agents, such as carbapenem, cephalosporins, ciprofloxacin, and chloramphenicol, excluding amikacin and colistin. Other resistant genes, such as blaTEM‐1, blaCTX‐M‐15, blaCMY‐2, aac(6')‐Ib‐cr, and qepA, were detected. The E coli isolates harboring bla NDM‐5 belonged to ST361 (n = 11), ST12 (n = 1), ST410 (n = 1), and PFGE types A (n = 11), B (n = 1), and C (n = 1). Conclusions This study reports on an outbreak of a predominant epidemic clone, the NDM‐5 producing, multidrug‐resistant E coli ST361 isolate. These findings suggest that we should pay attention to infection control measures to limit the spread of NDM‐5‐producing pathogens.


| INTRODUC TI ON
The Enterobacteriaceae family are leading causes of infections, such as urinary tract infections, hospital-and healthcare-associated pneumonia, and bloodstream infections. 1 Carbapenems that exhibit broad antibacterial activity among beta-lactams are used as effective antibiotics against Enterobacteriaceae-producing extended-spectrum beta-lactamases (ESBL) and plasmidic AmpC (pAmpC). 2 However, as the emergence of carbapenemase-producing Enterobacteriaceae (CPE) is increasingly reported worldwide, bacterial resistance to antibiotics has become a major source of concern for public health in recent years. [3][4][5][6] According to the report of the European Survey on carbapenemase-producing Among them, New Delhi metallo-beta-lactamase (NDM) belongs to Ambler class B and can hydrolyze almost all beta-lactam antibiotics, including penicillins, cephalosporins, and carbapenems, except monobactams, such as aztreonam. 8,9 Since NDM-1 producing Klebsiella pneumonia isolates were initially described in 2008, 9 to date, 21 NDM subtype variants have been reported worldwide. 10 The NDM subtypes contain one to five amino-acid substitutions that confer different levels of hydrolyzing activity against carbapenems and other β-lactam substrates. 3 Among NDM variants, the first NDM-5 was identified in 2011 from a multidrug-resistant E coli ST648 isolate in the United Kingdom, from a patient previously hospitalized in India, and differed from NDM-1 with two amino acid substitutions at positions 88 (Val → Leu) and 54 (Met → Leu). 11 In South Korea, the bla NDM-5 gene was first detected in 2015, in Klebsiella pneumoniae clinical isolates co-producing oxacillinase 181, 12 and has been reported intermittently since then. However, an outbreak of NDM-5-producing isolates, especially E coli ST 361, has not yet been described.
In this study, we report an outbreak of NDM-5-producing E coli in a tertiary hospital in South Korea and characterize the molecular epidemiology and antibiotic resistance profiles of the isolates.

| Bacterial strains
Chungnam National University Hospital is a 1300-bed tertiary care hospital located in the Daejeon of South Korea, a city of about 1 490 000 residents. Prior to this outbreak, carbapenem-resistant E coli isolates were rarely detected and no case involving carbapenemase-producing E coli had been detected in this hospital.
CR-ECO1, which is resistant to carbapenem, including ertapenem and imipenem, was isolated from a urine specimen, obtained from a patient in the neurosurgery ward on October 25, 2017. On October 31, 2017, another carbapenem-resistant CR-ECO2 was isolated from a urine sample of a patient hospitalized in the same ward, within the same period. Until January 23, 2018, a total of thirteen non-duplicate carbapenem-resistant E coli isolates were collected in the hospital. The identification of isolates was performed using the VITEK 2 ID-GNB cards (bioMérieux SA) according to the manufacturer instructions. The modified Hodge test, using ertapenem disks and Carba NP (bioMérieux SA), was conducted to confirm the phenotypic identification of carbapenemase production. 13 Both E coli ATCC 25922 and Pseudomonas aeruginosa ATCC 27853 were used as quality control strains for antimicrobial susceptibility testing. Salmonella enterica serovar Braenderup strain H9812 (ATCC BAA 664) was used as a reference marker for pulsedfield gel electrophoresis (PFGE). The clinical data for each patient were retrospectively reviewed.

| Antimicrobial susceptibility testing
The minimum inhibitory concentrations (MICs) of the carbapenems, such as ertapenem and imipenem, were determined by the agar dilution method, according to the Clinical and Laboratory Standards Institute (CLSI) guideline. 14 Antimicrobial susceptibility to nine drugs (piperacillin/tazobactam, cefepime, cefotaxime, ceftazidime, gentamicin, amikacin, ciprofloxacin, trimethoprim/sulfamethoxazole, and chloramphenicol) was evaluated using the E test (bioMérieux) on Mueller-Hinton (MH) agar (Difco Laboratories) in accordance with CLSI guidelines. 15 Antimicrobial susceptibility to three drugs (ampicillin, cefazolin, and aztreonam) was evaluated using the disk diffusion method on MH agar (Difco Laboratories) in accordance with CLSI guidelines. 15 The MICs for colistin and tigecycline were assessed by the broth microdilution method with MH broth (Difco Laboratories) in accordance with the recommendations of the joint CLSI-EUCAST (2016) 16 and European Committee on Antimicrobial Susceptibility Testing (EUCAST) criteria. 17 Table 1.

| Clinical characteristics of the Escherichia coli isolates
All thirteen carbapenem-resistant E coli isolates showed positive phenotypic screening results for both the Modified Hodge test and the Carba-NP test. These isolates were obtained from stool (n = 6), urine (n = 5), bile fluid (n = 1), and pus (n = 1) from hospitalized patients aged from 46 to 83, with mean age of 69 years ( Table 2). The stool samples were obtained using rectal swabs from patients admitted to an intensive care unit for CPE screening test. The outbreak timeline on the admission date and date of isolation of each patient is shown in Figure 1.

| D ISCUSS I ON
The CR-ECO 13 isolate, which is E coli ST410, is an extensively drug-resistant strain, resistant to almost all antibiotics except for amikacin and colistin. This strain co-harbored bla CTX-M-15 , bla CMY-2 , and bla TEM-1 , which encodes an ESBL, conferring resistance to aztreonam and aac(6')-Ib-cr, which mediates high-level resistance to aminoglycosides and fluoroquinolones. Escherichia coli ST410 has been reported worldwide as a potential high-risk pathogen associated with resistance to fluoroquinolones, third generation cephalosporins, and carbapenems. 27 Although ST410 is not a main strain of this outbreak, it poses a significant public health risk. These findings suggest that strict infection control is essential to prevent a dissemination of these high-risk clones. The E coli ST12 (CR-ECO 7) was susceptible to various antimicrobial agents (eg, aztreonam, gentamicin, amikacin, ciprofloxacin, trimethoprim/sulfamethoxazole, chloramphenicol, and colistin) and negative for other resistant genes tested, except for bla  .
We carried out retrospective investigation with limited patient information. So, the entry and transmission route of bla NDM-5 -producing E coli in this hospital is unclear. Environmental culture tests were carried out to investigate the possibility of dissemination through medical equipment and the surrounding environment, but carbapenemaseproducing E coli were not detected. Other patients in contact with known NDM-5 carriers were also screened, but no NDM-5 producers Considering that E coli is one of the major causes of communityacquired infections, and the spread of these strains is possible not only in the hospital, but also in the surrounding environment, the severity of dissemination of E coli carrying the bla NDM-5 gene will be even greater. 2 In conclusion, the rapid dissemination of NDM-5-producing E coli emphasizes that stringent infection control measures and active surveillance play important roles to prevent the spread of these pathogens.

CO N FLI C T S O F I NTE R E S T
The authors declare that there is no conflict of interest regarding the publication of this article. F I G U R E 2 Dendrogram based on pulsed-field gel electrophoresis patterns, multilocus sequence typing, antibiogram, and distribution of resistance genes of thirteen NDM-5-producing Escherichia coli isolates. The red and blue squares indicate resistance and susceptibility to each antibiotic, respectively