The Emergence and Molecular Characteristics of New Delhi Metallo β-Lactamase-Producing Escherichia coli From Ducks in Guangdong, China

This study aimed to determine the prevalence and transmission characteristics of New Delhi metallo β-lactamase (NDM)-producing Escherichia coli from ducks in Guangdong, China. In this study, a total of 28 NDM-producing E. coli isolates were recovered from 88 unduplicated diseased duck samples (31.8%) from veterinary clinics in Guangzhou, Foshan, Qingyuan, and Huizhou. Two variants, blaNDM−1 and blaNDM−5, were detected and the latter was present in 89.6% of the isolates (25/28). Multilocus sequence typing (MLST) analysis indicated that these E. coli isolates possessed six distinct STs, and ST156 was the most prevalent followed by ST648, ST746, ST354, ST10, and ST162. In addition, phylogenomic analysis found that two of the isolates that were recovered from a single sample possessed different genomes, and the blaNDM-carrying IncX3 plasmids may be horizontal transfer between E. coli isolates in the intestinal tracts of ducks. Whole-genome sequencing (WGS) analysis further revealed that blaNDM co-existed with other 25 types of antimicrobial resistance genes (ARGs), of which 16 ARGs were highly prevalent with detection rates >50%, and a high incidence of coproducing blaNDM and mcr-1 E. coli isolates (22/88, 25.0%) was detected in ducks. This study underscores the importance of surveillance for blaNDM-harboring microbes in ducks.


INTRODUCTION
Carbapenems are critically important for the treatment of infections caused by multidrug-resistant Gram-negative bacteria. However, carbapenemase-producing Enterobacteriaceae have become a major global public health threat . In particular, the New Delhi metallo β-lactamase (NDM) was initially found in Escherichia coli and Klebsiella pneumoniae isolates in India in 2018 (Yong et al., 2009). Since then, bla NDM -positive isolates have been found globally (Wu et al., 2019). bla NDM genes have been found in species belonging to 11 bacterial families, and the Enterobacteriaceae are the major hosts of bla NDM (Wu et al., 2019;Zhai et al., 2020).
Carbapenem antibiotics have never been licensed for veterinary use in any country worldwide; however, there have been sporadic reports of bla NDM -positive isolates from a variety of animal hosts. bla NDM -positive E. coli isolates were frequently detected from swine in multiple geographic areas in China (Ho et al., 2019). Similar to the detected rates of bla NDM gene in swine, bla NDM -positive E. coli isolates were highly prevalent in commercial broiler farms . In addition, several bla NDM -positive E. coli isolates from backyard animals shared closely related core single nucleotide polymorphisms (SNP) with human isolates (Li et al., 2019). A recent study has reported that bla NDM -positive Enterobacteriaceae were detected from migratory birds in China (Liao et al., 2019). Therefore, continued monitoring for bla NDM -positive Enterobacteriaceae in food-producing animals is urgently required.
Duck production has the potential to play a major role in the agricultural economy, and Asian countries alone contribute 84.2% of total duck meat produced in the world (Biswas et al., 2019). According to Food and Agriculture Organization (FAO) data (2019), duck meat production rose from 2.64 to 3.02 million tons from 2015 to 2019 in Asia, while in China, duck meat production rose from 2.19 to 2.50 million tons from 2015 to 2019 in China (FAO, 2019). The data show that China is the largest producer and consumer of cultivated duck in the world (Chen et al., 2020). bla NDM -positive E. coli isolates were highly prevalent along the Chinese poultry production chain, including commercial broiler farms, slaughterhouses, and supermarkets . Therefore, the prevalence of bla NDMpositive E. coli isolates from duck should be addressed through continued monitoring.
Although we previously reported that the rate of bla NDMpositive E. coli isolates at three duck farms in Guangdong was significantly higher than four other provinces in China, the sample collected was limited in western Guangdong province (Wang et al., 2021). Thus, in this study, we furthermore examined the epidemiology and molecular characterization of bla NDMpositive E. coli isolates recovered from ducks in representative areas for breeding ducks of Guangdong, China.

Ethics Statement
The Institutional Review Board of South China Agricultural University (SCAU-IRB) approved the protocols. All animals were sampled under authorization from the Animal Research Committees of South China Agricultural University (SCAU-IACUC).

Sampling Information
A total of 88 unduplicated samples, including 42 liver samples and 46 caecum samples, were collected from 88 diseased ducks, these diseased ducks were sent to the veterinary clinical diagnostics laboratory in Foshan University from duck farms in Guangzhou, Huizhou, Foshan, and Qingyuan of Guangdong province (Figure 1). Briefly, all sample was added to 1 ml of LB Broth and incubated for 16-18 h in 37 • C, followed by inoculating on MacConkey plates containing 2 mg/L meropenem for 12 h. Multiple red clones were selected for identification using MALDI-TOF MS Axima TM (Shimadzu-Biotech Corp., Kyoto, Japan) and 16S rRNA sequencing. For carbapenem-resistant E. coli isolates, five major carbapenem resistance genes, namely, bla KPC , bla NDM , bla IMP , bla OXA−48 , and bla VIM , were detected by PCR using previously described primers . Samples were collected after obtaining consent from farms and veterinarians.

Antimicrobial Susceptibility Testing
Antibiotic susceptibility testing was performed by the agar dilution method and interpreted according to the Clinical and Laboratory Standards Institute guidelines (CLSI M100-S28) for the following antimicrobials: gentamicin, amikacin, meropenem, imipenem, aztreonam, cefotaxime, ceftazidime, cefoxitin, florfenicol, ciprofloxacin, fosfomycin, trimethoprimsulfamethoxazole, and tetracycline (CLSI, 2018). Susceptibility to colistin and tigecycline were assessed by broth microdilution as recommended by the European Committee on Antimicrobial Susceptibility Testing (EUCAST Version 6.0) (EUCAST, 2016). E. coli ATCC 25922 was used as the quality control strain.

Conjugation Assay and Southern Blotting
To investigate the transferability of the resistance genes, a conjugation assay was performed for all bla NDM -positive E. coli isolates with the streptomycin-resistant E. coli C600 as the recipient strain. Donor strains and E. coli C600 were mixed and applied to a 0.22 µm filter in LB agar plates for 16-18 h. The mixture culture was then diluted and spread on selective MacConkey agar plates containing both 1 mg/L of meropenem and 2,000 mg/L of streptomycin to recover transconjugants. Transconjugants were confirmed by PCR and Pulsed Field Gel Electrophoresis (PFGE) patterns. S1-PFGE and Southern blotting were performed to obtain plasmid size, and the Salmonella enterica serotype, Braenderup H9812, was used as the standard size marker.

Whole-Genome Sequencing (WGS)
Total DNA was extracted from bla NDM -producing E. coli isolates using a Genomic DNA Purification Kit (TIANGEN, Beijing, China) as per the instructions of the manufacturer. WGS was performed with the Illumina Hiseq 2500 System (Novogene Guangzhou, China) using the paired-end 2 × 150-bp sequencing protocol. The draft genome was de novo assembled using the SPAdes version 3.9.0 (Bankevich et al., 2012). All genome assemblies of 28 E. coli isolates were deposited in GenBank and are registered with BioProject number PRJNA669620. Then, the sequence types, replicon types, and antibiotic resistance genes of all the isolates were identified by the Center for Genomic Epidemiology (http:// www.genomicepidemiology.org/). The sequence comparison of bla NDM -carrying plasmids was performed using Mauve and Brig (Darling et al., 2004;Alikhan et al., 2011). In silico phylotyping of E. coli was carried out using the ClermonTyping method (http://clermontyping.iame-research.center/). Phylogenetic trees for the bla NDM -producing E. coli isolates were structured using CSI Phylogeny (v1.4), and E. coli (18FS1-1) was used as the reference genome (Kaas et al., 2014). The corresponding characteristics of each isolate were visualized using the online tool iTOL (https://itol.embl.de/). The population structure of each phylogenetic tree was defined using rhierbaps (Tonkin-Hill et al., 2018). The genome assemblies were analyzed using a gene-by-gene approach and the allelic distance from the core genome multilocus sequence typing (MLST) (cgMLST) was visualized in a minimum-spanning tree using BacWGSTdb 2.0 (Feng et al., 2021).

Plasmid Analysis
Conjugation experiments were performed using the bla NDMpositive E. coli isolates collected from ducks, and all the bla NDMcarrying plasmids were successfully transferred to the recipient strain E. coli C600 str . S1-PFGE and hybridization analyses confirmed that bla NDM genes from 25 isolates were located on ∼ 50 kb plasmids, and the others genes were located on ∼ 140 kb (n = 2) and ∼ 200 kb (n = 1) plasmids (Supplementary Figure 2). As shown in Supplementary Figure 3, the ∼ 50 kb plasmids carrying bla NDM were the IncX3 incompatibility group and were similar to bla NDM−5 -carrying IncX3 plasmid from an ST25 K. pneumoniae isolated from human peritoneal fluid in China (Acc. No. KU761328). In addition, we found that IncX3 plasmids were carried by two different E. coli isolates that were recovered from the same sample in four out of five cases ( Table 1). This provides evidence for the horizontal transfer of bla NDM -carrying IncX3 plasmids in the intestines of ducks. WGS analysis demonstrated that 14 different Inc types were present on plasmids in the 28 bla NDM -positive E. coli isolates (Figure 2). Except for the IncX3 plasmid, IncFIB (17/28, 60.7%), IncHI2 (15/28, 53.6%), and IncI1 (10/28, 35.7%) plasmids were highly prevalent in these isolates (Figure 2).

DISCUSSION
In this study, a total of 28 (28/88, 31.8%) bla NDM -producing E. coli isolates were recovered from ducks; this result is similar to our previous report that indicated ahigh prevalence of bla NDM -positive E. coli isolates at duck farms in western Guangdong province. Although there was an overwhelming dominance of bla NDM−1 and bla NDM−5 in the clinical and livestock isolates (Shen et al., 2018;Zhai et al., 2020), bla NDM−5 was more prevalent than bla NDM−1 among the tested duck farms. This finding is similar to the previous report of the high prevalence of bla NDM−5 in the chicken production chains . The 28 bla NDM -positive E. coli isolates belonged to six distinct STs (ST156, ST648, ST746, ST354, ST10, and ST162) discussed in the current study, of which, ST156, ST648, and ST746 were the most prevalent. ST156 and ST648 have been associated with the dissemination of bla NDM−5 and mcr-1producing E. coli isolates (Yang et al., 2016). The bla NDM -positive ST746 and ST354 prevalent in ducks and poultry have become a primary reservoir for bla NDM -positive ST746 E. coli isolates in China (Wang et al., 2021). Phylogenomic analysis found that two of the isolates that were recovered from a single sample possessed different genomes, which indicates the development of diversity in the population of bla NDM -positive E. coli isolates from ducks.
In the current study, all bla NDM−5 genes identified were carried by IncX3 plasmids. IncX3 plasmids may serve as one of the major platforms on which bla NDM genes are evolving with the generation of new NDM variants, such as bla NDM−1/4/5/6/7/13/17/19/20/21 (Wu et al., 2019;Zhai et al., 2020). However, a high prevalence of bla NDM−5 -carrying IncX3 plasmid from bacteria of animal farms in China (Zhai et al., 2020). The bla NDM -carrying IncX3 plasmids have a narrow host range and have been mainly found in Enterobacteriaceae worldwide, which may be an association to highly conjugatable and stable and exert no fitness costs on their bacterial hosts (Johnson et al., 2012). In addition, this study found that IncX3 plasmids were carried with two different E. coli isolates recovered from a single sample in four out of five samples. This provides evidence for the horizontal transfer of bla NDM -carrying IncX3 plasmids between E. coli isolates in the intestine of ducks.

CONCLUSIONS
In conclusion, we identified 28 bla NDM -positive E. coli isolates from diseased ducks in Guangdong, China. Notably, this is the first study to report the development of diversity in the population of bla NDM -positive E. coli isolates from ducks. WGS analysis further determined that bla NDM coexisted with other ARGs, including mcr-1, and bla NDM -carrying IncX3 plasmids were most likely horizontally transferred between E. coli isolates in the duck intestinal tract. This study underscores the importance of surveillance for bla NDM -harboring microbes in ducks and indicates a high likelihood for the spread of carbapenem resistance from the poultry production chain to humans.

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 at: https://www.ncbi.nlm.nih. gov/genbank/, PRJNA669620.

AUTHOR CONTRIBUTIONS
M-GW wrote the first draft of the manuscript. X-PL, YY, and L-XF contributed to conception and design of the study. DW, R-SY, LJ, D-TC, and S-LZ performed the statistical analysis. JS and Y-HL wrote sections of the manuscript. All authors contributed to manuscript revision, read, and approved the submitted version.