Characterization and Public Health Insights of the New Delhi Metallo-β-Lactamase-Producing Enterobacterales from Laying Hens in China

The New Delhi metallo-β-lactamase (NDM) is a major element for the rapid expansion of the carbapenem-resistant Enterobacterales, which poses a great challenge to public health security. NDM-producing Enterobacterales strains (50 Escherichia coli, 40 Klebsiella pneumoniae, and 5 Enterobacter cloacae) were isolated from laying hens in China for the surveillance of antibiotic-resistant pathogens, and all were found to be multi-drug resistant bacteria. The genomic analysis of these NDM-positive bacteria revealed the ST167, ST617, and ST410 of the fifteen ST-type E. coli clones and ST37 of the four ST-type K. pneumoniae clones to be the same types as the human-derived strains. Among them, some new clone types were also found. Most of the blaNDM genes (blaNDM-1 or blaNDM-5) were on the IncX3 plasmids (n = 80) and were distributed in E. coli, K. pneumoniae, and E. cloacae, while the remaining blaNDM-5 genes were harbored in the E. coli ST167 with IncFII plasmids (n = 15). The typeⅠ1 of the eight IncX3 plasmid subtypes was consistent with the human-derived pNDM5_020001 plasmid (accession no. CP032424). In addition, these two plasmids did not affect the growth of the host bacteria and could be reproduced stably without antibiotics. Our study revealed the high genetic propensity of the NDM-positive Enterobacterales from the laying hens and human commensal Enterobacterales, suggesting the potentially enormous risk of its transmission to humans.


Introduction
The rapid emergence and extensive dissemination of bacterial antibiotic resistance is a nightmare for humans, with extensively drug-resistant Gram-negative bacteria being a particular focus of attention [1]. Carbapenem antibiotics are the latest generation of β-lactam antibiotics that are commonly used in clinics for treating bacterial infections owing to their high potency and ultra-broad-spectrum antibacterial activity [2,3]. However, the emergence and rapid spread of carbapenem-resistant Enterobacterales (CRE), which is in the most serious category of the 12 deadliest drug-resistant bacteria, has emerged as one of the most pressing threats to public health: New Delhi metallo-β-lactamases (NDMs) are the main carbapenem-resistant elements that hydrolyze almost all β-lactam antibiotics and haves been widely distributed worldwide [4,5].
A total of 29 NDM subtypes (NDM-1 to -29) have been identified since NDM1 was first reported in the hospital clinic [5,6]. In addition to NDM-positive bacteria isolated from humans, the animal-derived NDM-carrying bacteria are frequently reported, and traces of NDM have also been found in the retail market as well as in various other

Materials and Methods
The animal experiment operations involved in this study were maintained in compliance with the Animal Ethics Committee (AEC) of the College of Life Sciences, Sichuan University (License: SYXK (Chuan) 2013-185).

Sample Collection and Identification
In this study, 1600 samples (200 cloacal swabs per farm) were collected from eight intensive laying hen farms in six provinces (Sichuan n = 2, Shandong n = 2, Anhui n = 1, Hebei n = 1, Hubei n = 1, Gansu n = 1) in China and assigned corresponding numbers (such as SDCRK for Shandong samples, SCCRK for Sichuan samples). All the samples were stored in a refrigerator at 4 • C for identification. All the isolates were cultured in LB broth (37 • C, 180 r/min, 12 h), inoculated into MacConkey medium with 1 µg/mL meropenem and incubated at 37 • C for 16 h, after which the different morphological colonies were picked up from each sample and identified by 16S rRNA sequencing. As mentioned earlier, the carbapenemase-encoding genes bla NDM were screened in all the confirmed CRE strains by PCR [18].

Whole-Genome Sequencing (WGS) and Identification of the SNPs, STs, and ARGs
The NDM-positive bacteria were inoculated in LB broth and cultured for 8 h at 37 • C with shaking at 180 rpm. The TIANamp Bacteria DNA Kit (Tiangen Biotech Co., Beijing, China, DP302-02) was used to obtain the total DNA according to the instructions. The quality-controlled DNA was processed with the Illumina HiSeq 2000 platform to obtain the original sequence data, and the data were de novo-assembled by SOAPdenovo (http://soap.genomics.org.cn/soapdenovo.html, accessed on 13 November 2019). All the WGS in this study were deposited in the GenBank database under the corresponding accession numbers (Table S3). The WGS of NDM-positive Gram-negative bacteria from the other sources in 2015-2018 were downloaded through the database NCBI. The coregenome SNP-based phylogenetic trees of all the core-genomes were constructed using the software CSI Phylogeny 1.4 and visualized with the software iTOLv5 [19]. MLST was assigned according to the software CGE-MLST 2.0 by mapping data to the alleles using the PubMLST-Public databases and the Institut Pasteur MLST databases. To determine the genetic predisposition of the isolate's bla NDM genes to humans, the genetic correlation between the isolate and the database strain was analyzed. The targeted analysis of all the AMR genes was performed using ResFinder 2.1 [20].

Identification of Plasmids and bla NDM Gene Location and Environment
The determination of the plasmid types was performed using the software Plasmidfinder to analyze the sequencing data [21]. After the splicing and assembly were completed using SPAdes 3.13.0 (University of Manitoba, Manitoba, Canada), NCBI blast and PCR were used to sequence and splice the complete genome sequence of the bla NDMplasmid. The ORF annotation of the plasmid was completed using the Sequin software (version 13.70, Bethesda, MD, USA), and the annotated results were submitted to the GenBank database to obtain the accession numbers (Table S4). The genetic environment of the plasmid and bla NDM genes were analyzed using the BLAST program (http://blast.ncbi.nlm.nih.gov/Blast.cgi, accessed on 30 June 2020). The plasmid map was drawn using the software Artemis (http://sanger-pathogens.github.io/Artemis/Artemis/, accessed on 17 July 2020). The software Easyfig was used to draw comparative maps of the genetic environment of the different types of plasmids and their bla NDM genes.

Conjugation Transfer Experiment
The Rifampicin-resistant E. coli EC600 was used as the recipient to study the conjugative transfection of all the types of bla NDM -IncX3 and bla NDM -IncFIIplasmids by filtering and mating, as described previously [22]. The transconjugants were screened with Mueller-Hinton Agar (MHA) plates supplemented with rifampicin (400 µg/mL) and meropenem (8 µg/mL), and the conjugates were further confirmed using disk diffusion susceptibility testing and PCR. The conjugation frequency was determined based on the relative ratio between the transconjugants and the recipients.

Determination of Growth Curves
Transconjugants of eight types of bla NDM -IncX3 and bla NDM -IncFII plasmids and the recipient bacteria EC600 were streaked on eosin methylene blue (EMB) agar and cultured overnight at 37 • C. Single colonies were picked up from each culture medium and inoculated in the LB medium before the culture was shaken for 12 h (37 • C, 180 r/min). All the bacterial solutions were adjusted to the same OD 600 (0.004) using a spectrophotometer (Shanghai Jinghua Technology Instrument Co., Shanghai, China) with sterile LB medium. The adjusted bacterial solution was inoculated into the LB liquid medium (1:1000) and the culture (37 • C, 180 r/min) was shaken, the appropriate amount of culture solution was transferred to the spectrophotometer to determine the OD 600 every hour. The growth rates were determined from the slope of the growth curve in the logarithmic phase (3-6 h).

Growth Competition Experiment
The paired competitive growth of the transconjugants and recipient bacteria was determined in the LB broth, as mentioned elsewhere [23]. The conjugator bacterial suspension and the recipient bacterial suspension were prepared to the same OD 600 (0.04) according to the scheme description given above and then mixed at a ratio of 1:1. The mixture was transferred to the LB liquid medium (1:1000) for 6 days of culture (37 • C, 180 r/min), 10-fold serial dilutions were carried out, and the mixture was plated on meropenem (8 µg/mL) and antibiotic-free MHA plates for counting. According to the formula S = ln(CI)/ln(d) (S: selection coefficient; CI: competition index, d: dilution factor), the impact of plasmids on bacterial fitness was assessed. A fitness advantage was judged to exist if S > 0.

The bla NDM -Positive Plasmid Stability
The stability of all the types of bla NDM -positive plasmids was evaluated by subculturing the dilutions (1:1000) of the corresponding transconjugants in antibiotic-free LB medium (37 • C, 180 rpm/min) for 30 days. Bacterial suspensions were diluted and spread on meropenem-containing and non-resistant solid media every 5 days for culture and colony counts, and colonies (n = 10) from both plates were randomly isolated and cultured for disk diffusion sensitivity testing and PCR to verify plasmids and bla NDM genes.

Statistical Analysis
Statistical analysis of the growth curves and competition were subjected to a one-way analysis of variance (one-way ANOVA). The homogeneity of the variance was determined using the Levene's test before an ANOVA was conducted using SPSS Statistics 22.0 (SPSS Inc., Chicago, IL, USA). Graphical representations of the conjugation frequency, growth curves, and stability were drawn using GraphPad Prism 6.0 (GraphPad Software Inc., Broomfield, CO, USA).

Genomic Diversity Characteristics of the NDM-Positive Enterobacterales
Isolates of the NDM-positive E. coli were clustered into 15 distinct sequence types (STs) via silico multilocus sequence typing (MLST), including a new type of ST 264 (1/50). The majority of the isolates were concentrated into ST617 (16/50), ST165 (7/50), ST542 (7/50), and ST1286 (5/50) (Figures 1 and S2A). The comparative analysis of the WGS-single nucleotide polymorphisms (SNPs) of E. coli in this experiment and the NDM-positive E. coli from different sources in the database (https://www.ncbi.nlm.nih.gov, accessed on 13 August 2020, n = 39) showed that the bacterial clones ST167, ST617, and ST410 were identified in our samples and humans in different countries or regions, but the SNPs of the same ST-type strains from different sources were far apart ( Figure 2). In this study, the ST167 and ST410 isolates were only distributed in Sichuan, and the ST617 strain was found in Shandong and Sichuan. Although the ST542 strains were isolated from two different regions, they only differed by 0-10 SNPs, the ST165 and ST114 strains were from Sichuan, with differences of 0-10 SNP and 25 SNP, respectively. The ST1286 and ST226 strains were isolated from the same farm in Shandong, with differences of 0 SNP and 25 SNPs, respectively, while the ST48 strain SCCRK-86 from Sichuan differed by only 6 SNPs from the isotype SC516 strain (accession no. NZ_CP025048.1) in the database. The ST156 strains from Sichuan and the ECCRA119 strain (accession no. CP029242) in the database also differed by only 72 SNPs (Figure 2). coli from different sources in the database (https://www.ncbi.nlm.nih.gov, accessed on 13 August 2020, n = 39) showed that the bacterial clones ST167, ST617, and ST410 were identified in our samples and humans in different countries or regions, but the SNPs of the same ST-type strains from different sources were far apart ( Figure 2). In this study, the ST167 and ST410 isolates were only distributed in Sichuan, and the ST617 strain was found in Shandong and Sichuan. Although the ST542 strains were isolated from two different regions, they only differed by 0-10 SNPs, the ST165 and ST114 strains were from Sichuan, with differences of 0-10 SNP and 25 SNP, respectively. The ST1286 and ST226 strains were isolated from the same farm in Shandong, with differences of 0 SNP and 25 SNPs, respectively, while the ST48 strain SCCRK-86 from Sichuan differed by only 6 SNPs from the isotype SC516 strain (accession no. NZ_CP025048.1) in the database. The ST156 strains from Sichuan and the ECCRA119 strain (accession no. CP029242) in the database also differed by only 72 SNPs (Figure 2).  S2B). Although the ST37 and ST4523 strains were distributed across different regions, there were no differences in the SNP of ST4523 strains, and only 0-25 SNPs difference among the ST37 strains ( Figure 3). It is particularly noteworthy that the comparison of the  (Figures 1 and S2B). Although the ST37 and ST4523 strains were distributed across different regions, there were no differences in the SNP of ST4523 strains, and only 0-25 SNPs difference among the ST37 strains ( Figure 3). It is particularly noteworthy that the comparison of the genomes of the strains from this experiment and the bla NDM -carrying K. pneumoniae from the database (https://www.ncbi.nlm.nih.gov, accessed on 16 August 2020, n = 97) revealed that the ST37 isolate from this study differed from the human isotype strain by 156 SNPs. , x FOR PEER REVIEW 7 of 15 genomes of the strains from this experiment and the blaNDM-carrying K. pneumoniae from the database (https://www.ncbi.nlm.nih.gov, accessed on 16 August 2020, n = 97) revealed that the ST37 isolate from this study differed from the human isotype strain by 156 SNPs. All the E. cloacae strains were clustered into ST51 and distributed across the same farm in Shandong (Figure 1). No comparative analysis was conducted due to relatively few strains and single information.
The skeleton structure of all the IncX3 plasmids was highly conserved with the same △UmuD genetic structure (△UmuD-IS26, IS3000-tnpA-△UmuD). Mutations of different subtypes mainly occurred in the region between the two △UmuD ( Figure S3). The plasmid of type I1 (pNDM-IncX3I1, 10/80) was 46,161 bp with a 47% G+C content, and 100% nucleotide identity with pNDM5_020001 (accession no. CP032424), a 46,161bp IncX3 plasmid identified in an E. coli strain from the West China Hospital in China ( Figure 4A,B). All the E. cloacae strains were clustered into ST51 and distributed across the same farm in Shandong (Figure 1). No comparative analysis was conducted due to relatively few strains and single information.

bla NDM -Harboring Plasmids and Genetic Context of bla NDM
All the isolates harbored two or more types of plasmid replicons (range, 2-10): seventeen, eight, and two types of plasmid replicons were detected in the NDM-producing E. coli, K. pneumoniae, and E. cloacae isolates, respectively (Table S2). All the bla NDM-1 (8/95) and most of the bla NDM-5 (72/95) were located on the IncX3 plasmids across 8 subtypes, and the remaining bla NDM-5 (15/95) were located on the IncFII plasmid ( Figure S3 and Table S2).
The skeleton structure of all the IncX3 plasmids was highly conserved with the same UmuD genetic structure ( UmuD-IS26, IS3000-tnpA-UmuD). Mutations of different subtypes mainly occurred in the region between the two UmuD ( Figure S3). The plasmid of type I1 (pNDM-IncX3I1, 10/80) was 46,161 bp with a 47% G+C content, and 100% nucleotide identity with pNDM5_020001 (accession no. CP032424), a 46,161bp IncX3 plasmid identified in an E. coli strain from the West China Hospital in China ( Figure 4A,B).

Fitness and Genetic Stability of the bla NDM -Carrying Plasmids
The conjugation of the bla NDM -IncFII plasmid was not as active as that of the bla NDM -IncX3 plasmids ( Figure S5). No effect on growth was observed between the transconjugants of the bla NDM -carrying plasmids and the recipient strain EC600 ( Figure 7A). The growth trend of the conjugants was better than that of the recipient bacteria, the IncX3-TypeI1 and IncX3-TypeI3-harbouring conjugants had the highest growth rate, and the growth rate of the transconjugants of the IncX3 plasmids was higher than that of the IncFII plasmid ( Figure S6). Competitive assays of the plasmid fitness revealed that the ratios of IncX3 plasmid-carrying bacteria continued to increase in the co-culture of transconjugants and recipient bacteria over a period of 6 days (range, 83.20-83.67%) ( Figure 7B). The relative concentration increased (33.67%), and the growth selection coefficient (0.0244) of the IncX3-TypeI1-harbouring conjugants increased the most, corresponding to the low relative concentration increase in (33.20%), and the selection coefficient of (0.0231), the IncX3-TypeV2-harbouring conjugants (Figures 7B and S7A). The relative concentration of the transconjugant of the IncFII plasmid increased significantly in 0-1 day, and there was no alteration in the later period (about 62%). The growth selection coefficient of the IncFII-carrying conjugants was 0.0032 ( Figures 7C and S7B). The results obtained for the continuous subculture of the plasmid-carrying transconjugants in the antibiotic-free conditions demonstrated that all types of resistant plasmids could stably exist in the host bacteria and that the proportion of all types of plasmid-carrying bacteria remained above 98% in the 30-day antibiotic-free culture process ( Figure 7D). blaNDM gene of the two plasmids was the same, where the plasmid skeleton region of pEC1188-NDM16 which was 130 bp in length was missing from pSDCDK-IncFNDM5 (38710-38849), the fragment was located in the upstream non-coding region of the replication gene repA.

Fitness and Genetic Stability of the blaNDM-Carrying Plasmids
The conjugation of the blaNDM-IncFII plasmid was not as active as that of the blaNDM-IncX3 plasmids ( Figure S5). No effect on growth was observed between the transconjugants of the blaNDM-carrying plasmids and the recipient strain EC600 ( Figure 7A). The growth trend of the conjugants was better than that of the recipient bacteria, the IncX3-TypeI1 and IncX3-TypeI3-harbouring conjugants had the highest growth rate, and the growth rate of the transconjugants of the IncX3 plasmids was higher than that of the IncFⅡ plasmid ( Figure S6). Competitive assays of the plasmid fitness revealed that the ratios of IncX3 plasmid-carrying bacteria continued to increase in the co-culture of transconjugants and recipient bacteria over a period of 6 days (range, 83.20-83.67%) ( Figure 7B). The relative concentration increased (33.67%), and the growth selection coefficient (0.0244) of the IncX3-TypeI1-harbouring conjugants increased the most, corresponding to the low relative concentration increase in (33.20%), and the selection coefficient of (0.0231), the IncX3-TypeV2-harbouring conjugants (Figures 7B and S7A). The relative concentration of the transconjugant of the IncFⅡ plasmid increased significantly in 0-1 day, and there was no alteration in the later period (about 62%). The growth selection coefficient of the IncFⅡ- carrying conjugants was 0.0032 ( Figures 7C and S7B). The results obtained for the continuous subculture of the plasmid-carrying transconjugants in the antibiotic-free conditions demonstrated that all types of resistant plasmids could stably exist in the host bacteria and that the proportion of all types of plasmid-carrying bacteria remained above 98% in the 30-day antibiotic-free culture process ( Figure 7D).

Discussion
Similar to in human hospital clinics, Enterobacterales species are also the major NDMproducing bacteria in layer hen farms [13]. blaNDM-5 was an NDM variant and had an overwhelming epidemic dominance among the tested farms, bearing a resemblance to the previous NDM survey results of the broiler farms [14]. Only a few blaNDM-1-carrying strains were obtained in one farm, which was different from the main prevalence of both blaNDM-

Discussion
Similar to in human hospital clinics, Enterobacterales species are also the major NDMproducing bacteria in layer hen farms [13]. bla NDM-5 was an NDM variant and had an overwhelming epidemic dominance among the tested farms, bearing a resemblance to the previous NDM survey results of the broiler farms [14]. Only a few bla NDM-1 -carrying strains were obtained in one farm, which was different from the main prevalence of both bla NDM-1 and bla NDM-5 seen in human clinics [24]. Although different reports have shown that there are deviations in the isolation rate of various mutants of the bla NDM gene from poultry [9,17], the high prevalence of bla NDM-5 indicated its widespread distribution and spread in the poultry and farm environments. Isolates of the NDM-positive bacteria have been detected to have multi-drug resistance, meaning they were inseparable from the coexistence of multiple-drug-resistant genes. The co-selection of these coexisting resistance genes may be one reason why the bla NDM genes still existed in carbapenem-free farms. Polymyxin is one of the limited means by which to treat serious infections caused by bla NDM -carrying Gram-negative bacteria. Contrary to a previous monitoring report in nationwide clinics [25], in our study, E. coli was found to be co-resistant to polymyxin and carbapenem antibiotics, but not to K. pneumoniae and E. cloacae. The emergence and spread of multi-drug-resistant Enterobacterales seriously threatens the health of humans and animals.
The clonal diversity of the NDM-positive E. coli has been frequently reported, indicating that the prevalence of bla NDM in E. coli was not only caused by clonal propagation [26,27]. The distribution of E. coli clone types differs in different regions or hosts, in this study, the E. coli cloning types ST167, ST167, and ST410 have been reported in humans, sewage, and retail markets [13,28,29]. This suggested that the clonal types of NDM-producing E. coli isolates that were ubiquitous in multiple hosts and in different environments deserve more attention. In this study, the ST206 type cloned strain isolated from Sichuan and the chicken-derived isotype strain in the same region isolated in 2015 only differed by six SNPs. There was a slight difference in the Sichuan ST156 type strain cloned in this study and the poultry-derived isotype strain collected in Zhejiang in 2017 [30]. This suggested that certain clonal types of NDM-positive E. coli had long-term clonal transmission in domestic farms.
Moreover, the result of our study confirmed the previous report that ST37 was the dominant K. pneumoniae clone type in Shandong farms [17]. Simultaneously, ST37 has also been frequently reported as the dominant clone of the NDM-positive K. pneumoniae in human clinics [31,32], which has even been found to persist in the environment and spread between animals and humans [33]. The SNPs of ST37 K. pneumoniae isolated by us and the same type of K. pneumoniae in humans were less different. It is necessary to strengthen standardized management in animal breeding to avoid this type of NDMpositive bacteria spreading between humans and animals. In addition, no SNP differences in ST4523 K. pneumoniae were found between farms in two regions without a direct relation, suggesting that its distribution and spread should be kept under constant surveillance.
The IncX3 plasmid was the most important transmission vector of the bla NDM genes, and frequently found in clinics, farms, food chains, and the environment [17,33,34]. The presence of the IncX3 plasmid in all ST types of the three bacterial species was substantiated by a previous report on the broad-spectrum host range of IncX3 [32]. Although the use of carbapenem in animal feed has been forbidden, its diversified sources and wide host spectrum have been accountable for the large-scale spread of IncX3 plasmid carrying the bla NDM gene in various environments. In this study, pNDM-IncX3I1 was found to have a 100% homology with the human clinical plasmid pNDM5_020001, indicating that the bla NDM -IncX3 plasmid has the great potential for cross-region and cross-species transmission. Seven other subtypes of IncX3 were also identified, and these mutations all occurred in the insertion sequence ISAba125, IS5, and IS3000 between the IS3000 and bla NDM gene, and deletions or missing mutations of these inserted sequences have been reported in the bla NDM -IncX3 plasmids collected from human clinics and retail meat in different regions [35,36]. This suggested that the sequence region between the IS3000 and bla NDM gene was the most common mutation-prone region of bla NDM -IncX3 plasmids.
Another universal bla NDM gene carrier IncFII plasmid was found to have a larger genome than the IncX3 plasmid, and to often carry multiple drug-resistant genes [37,38]. Our observation exhibited that the pSDCDK-IncFNDM5 plasmid contained seven coexisting ARGs except for the bla NDM-5 gene. The structures of the backbone region and multi-drug resistance zone outside the bla NDM gene of the pSDCDK-IncFNDM5 plasmid were highly consistent with those of a pEC1188-NDM16 plasmid obtained from a human clinic [39]. This revealed the potential for the cross-species transmission of the bla NDM -IncFII plasmids. The adhesion of the bla NDM genes of the above two plasmids to the composite transposon "IS26-ISCR1" (IS26-ISAba125-bla NDM -ble MBL -tat-trpF-ISCR1) might be an important reason for transforming bla NDM genes, which was different from that of a previous report which showed that the two ISCR1-entrained NDM genes (ISCR1-dsbc-trpF-ble-bla NDM -qacE 1-sul1-ISCR1) were integrated into the IncHI5 plasmid through circularization and recombination [40]. However, this mechanism deserved further study.
Furthermore, evidence has revealed that plasmids carry bla NDM genes for large-scale and long-term reproduction. The results were found to be consistent with those of previous reports [41,42], and it was found that all the types of bla NDM -IncX3 plasmid identified in this study failed to affect the growth of the host bacteria, even conferring a certain fitness advantage to the host bacteria. Similarly, the bla NDM -IncFII plasmid did not affect the growth of the host bacteria but imparted the host bacteria with a fitness not as intense as that of the IncX3 plasmid. To add to these worries, not only do all types of bla NDM -harboring plasmids have no cost of adaptability, but they can also reproduce stably in the host bacteria without antibiotic selection pressure. This could provide a partial explanation of the reason for the bla NDM genes to distribute and reproduce stably in the carbapenem-free farms with its harboring plasmid.

Conclusions
NDM-positive multi-drug resistant Enterobacterales were isolated from laying hens, leading to the identification of some novel clone types and several clone types that also exist in humans and the environment. Among the eight bla NDM -IncX3 plasmid subtypes and the bla NDM -IncFII plasmid, some were found to be identical or highly homologous to human-derived plasmids, and all types of plasmids were found to confer a certain fitness advantage to the host bacteria enabling them to reproduce stably without antibiotic selection. These results indicated that the high genetic propensity of the NDM-positive plasmids of poultry-derived Enterobacterales for spreading into humans deserved serious attention and also suggested the importance of continuous surveillance for the prevalence of NDM-positive bacteria in poultry to avoid public health incidents.
Supplementary Materials: The following supporting information can be downloaded at: https: //www.mdpi.com/article/10.3390/microorganisms10040800/s1, Figure S1: Distribution of NDMproducing Enterobacterales, sample collection diagram of large-scale chicken farms (A), species identification and distribution results of bla NDM -carrying Enterobacterales (B), type identification and distribution results of bla NDM genes (C). Figure S2: Phylogenetic trees of NDM-producing Enterobacterales, phylogenetic trees of 50 NDM-producing E. coli (A), phylogenetic trees of 40 NDM-producing K. pneumoniae (B). Figure S3: Comparison of genetic structure of bla NDM -IncX3 plasmids. Figure S4: Distribution of 9 kinds of bla NDM -plasmids among 95 Gram-negative bacteria. Figure S5: Transconjugative frequencies of bla NDM -IncX3 and bla NDM -IncFII plasmids. Figure S6: Growth rate of 9 kinds of transconjugants. Figure S7: Selection coefficient of 9 kinds of transconjugants. Table S1: Antibiotic resistance phenotypes of the isolates in this study. Table S2: MLST typing and plasmids of the isolates in this study. Table S3: NCBI accession numbers of 95 bla NDM gene carrying Gram-negative bacteria. Table S4: GeneBank accession numbers of bla NDM gene carrying plasmids. Data Availability Statement: The genome sequences of the isolates in this study can be retrieved and downloaded from the NCBI database (https://www.ncbi.nlm.nih.gov/, accessed on 27 June 2020) according to the accession numbers in Table S3.