Comparative genomic analysis of plasmids encoding metallo-β-lactamase NDM-5 in Enterobacterales Korean isolates from companion dogs

Carbapenems are broad-spectrum antibiotics widely used for the treatment of human infections caused by multidrug-resistant (MDR) Gram-negative bacteria. However, emerging carbapenemase-producing Enterobacterales (CPE) are rising as a public threat to human and animal health. We screened clinical bacterial isolates from 241 dogs and 18 cats hospitalized at Veterinary Medical Teaching Hospital, Seoul National University, from 2018 to 2020 for carbapenemase production. In our study, 5 strains of metallo-β-lactamase NDM-5-producing Escherichia coli and Klebsiella pneumoniae were isolated from 4 different dogs. Multilocus sequence typing (MLST) results showed that all E. coli strains were ST410 and all K. pneumoniae strains were ST378. Whole genome analysis of the plasmid showed that blaNDM-5 is carried on a IncX3 plasmid, showing a high concordance rate with plasmids detected worldwide in human and animal isolates. The blaNDM gene was associated with the bleMBL gene and the ISAba125 element, truncated with the IS5 element. The results of this study show that CPE has already become as a threat to both animals and humans in our society, posing the necessity to solve it in terms of "One Health". Therefore, preventive strategies should be developed to prevent the spread of CPE in animal and human societies.

Since the first report of the isolation of a K. pneumoniae NDM-bearing strain from a patient in Sweden in 2009, NDM has spread worldwide due to its location on mobile genetic elements such as plasmids, transposons and integrons 8 . Conjugative plasmids such as Incompatibility group (Inc) F, A/C, L/M, N and X are associated with dissemination of bla NDM via horizontal gene transfer (HGT) 2 . In 2011, NDM-5 was first detected in E. coli from the United Kingdom with mutations of two amino acid positions of 88 (Val → Leu) and 154 (Met → Leu) from NDM-1 that resulted in increased action against carbapenems 9 . NDM-5 is disseminated worldwide and has been detected in various countries including Australia 10 , Denmark 11 , Italy [12][13][14] , Switzerland 15 , the Netherlands 16 , China 17 , India 18 , South Korea 19 , and the USA 20 .
Although prescription of carbapenem in animal medicine is prohibited in any part of the world by the World Organisation for Animal Health (OIE), the bla NDM-5 gene has been detected worldwide not only in companion animals, including dogs and cats 15,21 , but also in domestic animals 17,22,23 . In South Korea, CPE were first reported among E. coli of companion animals in 2018 and reported as New Delhi metallo-β-lactamase-5 (NDM-5) 21 .
Regarding the increasing threat of CPE, continuous surveillance and genetic characterization of CPE isolates have been required to develop the control measures against their spread in human and animal society.
Based on the current situation of CPE, Korean isolates of CPE from companion animals were characterized phenotypically and genotypically. Also, the genetic characteristics of the isolates were revealed by comparison with those from other countries.

Results
Profiles and antimicrobial susceptibility of the isolates. A total of 5 carbapenemase-producing strains were isolated from 520 isolates and identified as 3 E. coli strains and 2 K. pneumoniae strains. Four strains were isolated in 2019 and one (DMCPEC3) in 2020. Three strains were isolated from urine samples, and the others were isolated from ear swab samples (Fig. 1). According to hospital records, the likelihood of physical contact between patients was low. Notably, one E. coli strain (DMCPEC2) and one K. pneumoniae strain (DMCPKP4) were isolated from different urine samples from the same dog. Although the dog was suffering from cystitis and receiving antibiotic treatment with enrofloxacin and amoxicillin/clavulanic acid, it did not show any improvement of clinical signs. The other strain of K. pneumoniae (DMCPKP1) was isolated from urine specimen of a dog patient with increased urine volume and odor. The patient was treated with amoxicillin/clavulanic acid and enrofloxacin but showed no improvement. The other 2 E. coli isolates (DMCPEC3, DMCPEC7) were isolated from ear swab samples of 2 different dogs. Interestingly, the two dogs had previously been treated in the same facility in Seoul, but the timing of each individual's visit was unknown. DMCPEC3 was isolated from a dog with ear pruritus in 2020, but has never been applied with antibiotics in our facility. DMCPEC7 was isolated from ear purulent exudate of other dog patient, which was treated with enrofloxacin.
PCR amplification followed by sequencing revealed that all 5 strains of carbapenemase-harboring strains produced NDM-5, with two amino acid mutations at positions 88 (Val to Leu) and 154 (Met to Leu), regardless of bacterial species or source of isolation. The other four major carbapenemase (bla KPC , bla VIM , bla IMP , and bla OXA-48 ) markers were not detected in all isolates.
Genotypic relatedness according to MLST and PFGE. The genotypic relationships between isolated strains determined with MLST revealed that all E. coli strains belonged to ST410 and all K. pneumoniae strains belonged to ST378. The epidemiological similarity results between PFGE indicated high relatedness of the E. coli strains (> 93%) and the K. pneumoniae strains (99%).  Table 2). All transconjugants were confirmed by PCR identification. In conjugation assay, transferability was proven not only in E. coli but also in the case where K. pneumoniae strains contributed as donors.
Complete sequences of all five NDM-5 harboring plasmids were obtained using the Illumina MiSeq Sequencing System. Plasmid sequencing and PCR replicon typing results identified all isolated plasmids as IncX3, with lengths varying from 45-46 kb, GC contents varying from 46.5 to 46.91% and 60-61 CDSs ( Table 2). Downstream of bla NDM-5 , the ble MBL gene encoding the bleomycin-resistant protein, the trpF gene encoding phosphoribosylanthranilate isomerase, the dsbC gene for oxidoreductase, the cutA gene fragment encoding the periplasmic divalent cation tolerance protein inserted by the IS26 element and the truncated umuD gene encoding a mutagenesis protein were identified. Table 1. Minimum inhibitory concentration level of 5 carbapenemase-producing strains against various antibiotics (μg/mL). Either broth or E-test methods were performed following recommendation of Clinical and Laboratory Standards Institute (CLSI) interpretation criteria 48 . E-test methods were used for 8 antimicrobial agents: ceftazidime, ceftriaxone, cefepime, gentamicin, tobramycin, colistin, aztreonam, tigecycline. The others were tested via broth microdilution method. E. coli strain ATCC 25922 was used as a quality control strain. CPE isolates showed resistance imipenem (> 256 μg/ml), meropenem (32 ~ 128 μg/ml), and ertapenem (32 ~ 64 μg/ml) while seeming to be susceptible against polymyxin B, colistin, and tigecycline.

Antibiotic class Antimicrobial agents
Minimum inhibitory concentration (μg/mL) . pEC2-NDM5 showed high identity and similar gene features with two reference plasmids, pNDM-5_A0917122 and pNDM_MGR194, isolated from our country and India, respectively. pNDM-5_A0917122 was the plasmid isolated from companion animal of South Korea, while pNDM_MGR194 was isolated from human patient of India. On the other hand, the middle parts of transposon IS26 in pEC3-NDM5 and pEC7-NDM5 were lost over a length of 567 base pairs. Plasmids from K. pneumoniae showed higher identity with pNDM5-SCNJ1 from human sputum of China, which was also detected from isolates of K. pneumoniae. The ISAba125 region was previously found to be truncated (166 bp upstream from the bla NDM-5 start codon) by the IS5 element, with 67  www.nature.com/scientificreports/ base pairs of remnants between the bla NDM-5 and IS5 elements 26 . While the ISAba125 region between the IS5 element and IS3000 element was found to be shortened by 112 base pairs in pNDM5-SCNJ1 compared with other IncX3 plasmids, the same gene area of K. pneumoniae in our study was completely lost. Two plasmids from K. pneumoniae strains, pKP1-NDM5 and pKP4-NDM5, showed 100% identity to each other.

Discussion
This investigation identified Enterobacterales bearing NDM-5-producing IncX3 from companion animals. This was the first report in our country of K. pneumoniae as a carbapenemase producer from companion animals, whereas the presence of carbapenemase-producing E. coli was reported previously in our country 21 . Of note, two different isolates, one E. coli (DMCPEC2) and one K. pneumoniae (DMCPKP4) came from different urine samples collected from a single dog at different times. Their identical carbapenemase and plasmid features (99.85% identity) likely indicate the possibility of bacterial interspecies horizontal transfer of genetic elements inside the host, or on medical devices such as urinary catheter. Sporadic dissemination among companion animals or in animal hospitals is suspected, regarding the identical carbapenemase gene profiles, the identical genetic environment structures, the same MLST types and PFGE patterns showing the same pulsotype (> 93% along E. coli strains and > 99% along K. pneumoniae strains), and the genetic environment structure (ISAba125-bla NDM-5 -ble MBL -trpF-dsbC-cutA-umuD). Plasmids can transport multiple antibiotic resistance genes (ARGs) through conjugation between heterogeneous species as well as the identical species, which makes plasmids crucial for bacterial colonization and virulence potential 27 . Incompatibility group IncX3 plays a major role in dissemination of antibiotic resistance genes and is known to harbor bla NDM-1 and bla NDM-5 rather than other bla NDM variants 2 . IncX3 plasmids carrying various carbapenemase genes seem to be disseminated worldwide, mainly in China 28 . A recent investigation performed in China revealed that the IncX3 plasmid harboring bla NDM-5 is disseminated among Enterobacterales in both humans and animals 22,29 . In animals, IncX3 plasmids carrying bla NDM have been detected in E. coli from swine and chickens in various regions in China 17,30 . The identification of IncX3 from China and South Korea is not a www.nature.com/scientificreports/ surprise, considering the global dissemination of these plasmids. Among carbapenemase-harboring plasmids, the IncX3 plasmid is also dominant in South Korea, showing dissemination within diverse bacterial species, followed by IncFII and IncA/C 19 . bla NDM has been suggested to be disseminated from Acinetobacter baumannii to Enterobacterales via the mobile gene element ISAba125 31 . All five isolates in our study support this idea, with association of transposon elements of the upstream ISAba125 element. As described and predicted previously, the ISAba125 element was lost in K. pneumoniae strains examined in our study via horizontal transfer or host response mechanisms 29 . E. coli ST410 is known for interspecies transmission along people, environment, wildlife and companion animals 32,33 . Additional isolation of bla NDM-5 from E. coli ST410 in our country is posing threat of human infection from companion animal or vice versa. A recent study from Denmark suggested E. coli ST410 as a new high-risk clone, causing recurrent infections such as bloodstream infections and carrying carbapenemases such as NDM-5 or OXA-181 34 . E. coli ST410 is reported as NDM-5 producer in various countries worldwide, including China 35 , the United Kingdom 36 and South Korea 21 . In South Korea, ST410 is the 3 rd most dominant NDM-producing clinical E. coli strain, which includes reports from companion animals 19,21,37 . Therefore, detailed investigation to discover the role and dissemination of E. coli ST410 in our country is necessary. To prevent public outbreak, infection control across people, environment, wildlife and companion animal based on One Health Approach is needed.
K. pneumoniae ST378 has never been reported as CPE, even though NDM-5-producing K. pneumoniae has been reported worldwide from various strains 16,23,[38][39][40][41][42] . While the predominant NDM-producing clinical K. pneumoniae strain is ST1061 in South Korea 19 , ST378 was reported as a common sequence type among ESBLand/or AmpC β-lactamase-producing clinical K. pneumoniae isolates in Taiwan 43 . However, information on these clones of ST378 is still scarce, and further monitoring investigations are needed to avoid additional dissemination in our country. Considering that the plasmid of this new strain is similar to the previously described ones, it should be considered as additional evidence of horizontal spread of carbapenemase harboring IncX3 plasmids between Enterobacterales, discovered from companion dog hosts.
While the transmission route of these CPE isolates is still unclear, there are several possible hypotheses. First is circulation inside veterinary teaching hospital environment, considering genetic similarity between CPE strains and their urinary tract infection (possibly urinary catheter infection). Transmission via physical contact of companion animals is also considerable, regarding that MLST types of carbapenemase-harboring E. coli isolates discovered in companion animals in our country are identical, as ST410 21 . Also considering that these isolates were from a tertiary referral hospital in South Korea, it could be indicating unauthorized usage of carbapenems in local veterinary hospitals in our country. Interestingly, two of the host dogs had previously visited the same animal hospital, but it was not known whether they had been in contact, and this information alone could not reach a definite conclusion. Lastly, it could be acquired from contaminated feed or drinking water, or from contact with their human owners.
In this study, the CPE strains were isolated from clinical lesions, and each strain caused ear and urinary tract clinical signs in the host dog. Therefore, the possibility of these strains to be infected and become human clinical pathogens should be considered. Also, bacterial screening of human owner is required to figure out the possibility of human-to-animal transmission, or vice versa.
In conclusion, additional emergence of CPE in this study shows the dissemination of carbapenemase in our society, which is already a public concern considering the forbidden usage of carbapenems in animals. Therefore, further investigation is necessary to unveil the role of IncX3 plasmids carrying bla NDM-5 and the evidence of transmission between human owners and companion animals.

Methods
Bacterial strain identification and carbapenemase gene detection. Specimens, urine and swabs of skin and ears, were collected from suspected of being infected companion dogs and cats hospitalized at Veterinary Medical Teaching Hospital, Seoul National University, from 2018 to 2020. The specimens were collected by professional veterinarians in accordance with the Guide for the Care and Use of Laboratory Animals and the Animal Welfare Act. From the collected specimens, a total of 520 clinical isolates from 241 dogs and 18 cats were isolated using sheep blood agar (Hangang, Gyeonggi, Korea) for the diagnostic isolation and stored in Tryptic Soy Broth (Thermo Fisher Scientific Oxoid Ltd., Basingstoke, UK) with 50% glycerol at − 70 °C for further epidemiological studies. The stored clinical specimens were subject to screening for carbapenem resistance on CHROMagar mSuperCARBA agar (CHROMagar). Total DNA of surviving isolates was purified by a Wizard Genomic DNA Purification Kit (Promega, Madison, WI) and amplified using PCR specific primers detecting 5 widespread carbapenemase genes (bla KPC , bla NDM , bla VIM , bla IMP , and bla OXA-48 ) using previously designed primers [44][45][46][47] . Sequences were identified by the Sanger sequencing method and compared with Gen-Bank data (www. ncbi. nlm. nih. gov/ GenBa nk) with the Basic Local Alignment Search Tool (BLAST) network service. Microbial species were confirmed with matrix-assisted laser desorption ionization-time of flight-mass spectrometry (MALDI-TOF-MS; Bruker Daltonik GmbH, Bremen, Germany) and further confirmed using 16S rRNA sequencing with the primer pair 27F/1492R.

Data availability
Publicly available datasets were analyzed in this study. This data can be found in Table 2 for all accession numbers. All data generated or analyzed during this study have been submitted with this manuscript. All genetic information of the plasmids was deposited in GenBank. Therefore, all data from this study are available publically.