Characterization of NDM-1-Producing Carbapenemase in Proteus mirabilis among Broilers in China

Carbapenem-resistant pathogens mediated by metallo-beta-lactamases (MBLs) have spread worldwide, where NDM-1 is a typical and key MBL. Here, we firstly discussed the distribution characterization of NDM-1, which produces multidrug-resistant Proteus mirabilis among broilers in China. From January to April 2019, 40 (18.1%, 40/221) blaNDM-1-carrying P. mirabilis strains were recovered from commercial broilers in slaughterhouse B in China. All the isolates were resistant to imipenem, meropenem and other β-lactams. These isolates belong to five clusters identified via pulsed field gel electrophoresis (PFGE). Further studies on twenty representative strains revealed that seven blaNDM-1 genes were located on plasmids with sizes of 104.5–138.9 kb. Notably, only three strains (PB72, PB96 and PB109) were successfully transferred to Escherichia coli J53, while the other four isolates were located in nontransferable plasmids. The rest were harbored in chromosomes. Ulteriorly, based on whole genome sequencing (WGS), these twenty isolates showed four typical phylogenetic clades according to single nucleotide polymorphisms (SNPs) of a core genome and presented four main genomic backbone profiles, in which type II/III strains shared a similar genetic context. All of the above is evidence of blaNDM-1 transmission and evolution in P. mirabilis, suggesting that the prevalence may be more diverse in broiler farms. Accordingly, as intestinal and environmental symbiotic pathogens, blaNDM-1-positive P. mirabilis will pose greater threats to the environment and public health.


Introduction
Proteus mirabilis is an opportunistic pathogen belonging to the Enterobacteriaceae family, widely distributed in the natural environment and the intestines of humans and animals. Clinically, it can cause diarrhea, sepsis, respiratory issues and urinary tract infections, with a potential risk of zoonosis [1][2][3]. Along with Escherichia coli, Salmonella and methicillinresistant Staphylococcus aureus (MRSA), it is considered to be an important cause of infections in humans and animals [4,5]. P. mirabilis is intrinsically resistant to tetracycline, tigecycline and polymyxins [6]. Carbapenems are potent β-lactam antibiotics are still an crucial choice in the treatment of severe human infections because of their low side effects, they are banned for use in veterinary medicine [7]. However, the reports of carbapenem resistant bacteria in livestock, wild animals, food products and the environment have increased [8][9][10][11], which has gradually aroused high levels of attention globally in the fields of human and veterinary clinical and ecological environments. The anxieties surrounding this threat can be illustrated by the increase in carbapenemases, including New Delhi metallo-β-lactamase (NDM), Klebsiella pneumoniae carbapenemase (KPC) and carbapenem-hydrolyzing oxacillinase 48 type β-lactamases (OXA-48). As a major type of carbapenemase, NDM can impair the efficacy of almost all β-lactams (except aztreonam), and the therapeutic options for infection are mostly limited to polymyxins, tigecycline, fosfomycin and cefiderocol [12,13], the idea that NDM may acquire additional gene resistance in P. mirabilis is worrisome. Hospitals in many countries have widely reported bla NDM-1positive P. mirabilis [14][15][16][17], but only sporadic reports have been published regarding animals used as food sources [18]. Here, we describe a short-term slaughterhouse outbreak caused by bla NDM-1 -positive P. mirabilis and perform microbiological characterization of bla NDM-1 -positive P. mirabilis.

Bacterial Isolation, Species Identification and Detection of β-Lactamases Genes
From January to April 2019, a total of 556 samples were collected from four chicken farms (n = 212, cloacal swab) and two slaughterhouses (n = 344, caecum contents) in Shandong Province, China (Table S1). Cloacal samples were randomly collected from four farms (farm 1:40; 2:72; 3:40 and 4:60) that contained 15,000-20,000 broilers per farm; caecum samples were randomly collected from two slaughterhouses (slaughterhouse A:123; B:221) that contained 20,000-30,000 broilers per slaughterhouse. For the isolation of carbapenem-resistant P. mirabilis (CRP), swabs were cultured in 2 mL of brain heart infusion (BHI) broth at 37 • C for 5-6 h, and the cecum was cut directly in a 10 mL centrifuge tube, followed by streaking on salmonella shigella (SS) agar plates (Luqiao, Beijing, China) containing 1 mg/L of imipenem and 30 mg/L of vancomycin (Baomanbio, Shanghai, China) and incubated at 37 • C for 18-24 h. The morphologically representative clones with black centers were isolated and boiled to extract DNA and were confirmed to be P. mirabilis via 16S rDNA sequencing [19]. All the strains were sub-cultured at least twice prior to further investigation. Common β-lactamase genes, including carbapenemases (bla NDM , bla KPC , bla VIM , bla IMP and bla OXA-48 ) and extended-spectrum β-lactamases (ESBLs) (bla CTX-M , bla TEM , bla SHV , bla OXA-1 , bla OXA-2 and bla OXA-10 ), were amplified via polymerase chain reaction (PCR) for all the strains; specific primers were used with 2 × Taq Master Mix (Dye Plus) (Vazyme Biotech, Nanjing, China), and the carbapenemase-positive products were sequenced (Table S2). The sequences were compared with the reported sequences from the GenBank nucleotide database at http://www.ncbi.nlm.nih.gov/blast/ (accessed on 23 November 2021). Fifty P. mirabilis strains without carbapenemase were randomly selected for use as bla NDM-1 -negative control strains.

Antimicrobial Susceptibility Testing
The agar dilution method was used to determine the MICs of bla NDM-1 -positive P. mirabilis, and their transconjugants were screened with sodium-azide-resistant E. coli J53 against 11 antibiotics. The results were interpreted in accordance with the EUCAST guidelines [20]. Reference strain E. coli ATCC 25922 served as a quality control.

Pulsed Field Gel Electrophoresis (PFGE)
Genotyping of CRP harboring bla NDM-1 was performed via PFGE. Chromosomal DNA was digested with 50U of SmaI (Takara, Dalian, China) for 3 h at 30 • C. Genomic DNA was separated by CHEF DRIII (Bio-Rad, Hercules, CA, USA) gel electrophoresis in a 1% agarose gel in 0.5 × TBE at 14 • C. The operating conditions are as follows: voltage, 6 V/cm; switch angle, 120 • ; switch time, 5-20 s for 19 h. XbaI-digested Salmonella Braenderup H9812 was used as the size ladder. The gels were stained with ethidium bromide (Macklin, Shanghai, China), digitally photographed with Gel Doc TM XR+ (Bio-Rad, Hercules, CA, USA) and normalized as TIFF images. The DNA patterns were analyzed using BioNumerics software v7.6 (Applied Maths, Kortrijk, Belgium) to establish a dendrogram of strain relationships.
UPGMA was used as a clustering algorithm and the Dice correlation coefficient was used with a 1.2% position tolerance. The strains with more than 85% similarity were considered to be related.

S1-PFGE and Southern Hybridization
The localization of bla NDM-1 on the plasmids was verified using S1-PFGE and Southern blotting. the DNA of the strain embedded in Seakem Gold gel with S1 nuclease (TakaRa, Dalian, China) was digested and separated via PFGE, with a conversion time of 2.16-63.8 s for 16.5 h. The DNA fragments were transferred to Hybond N+ (GE AMersham, Pittsburgh, PA, USA) and hybridized with DIG-labeled bla NDM-1 probes. NBT/BCIP was used for color detection (Roche Applied Sciences, Basel, Schweiz).

Conjugation Assay
The transferability of bla NDM-1 was determined using a membrane filter method and E. coli J53 (sodium-azide resistant) as the recipient. Overnight cultures of the donor strain and recipient E. coli J53 were cultured in BHI broth at 37 • C and adjusted to 0.5 McFarland standard. The donor strain (30 µL) was then separately conjugated with E. coli J53 (90 µL) on a microporous membrane. The conjugation mixtures were then diluted and plated onto selective BHI agar plates supplemented with meropenem (2 mg/L) and sodium azide (200 mg/L) to recover transconjugants. PCR and MICs were performed to confirm that transconjugants were derivatives of the recipient strain E. coli J53. The transfer frequencies were calculated as described in a previous report [21].

Whole Genome Sequencing (WGS) and Bioinformatics Analysis
Twenty bla NDM-1 -positive representative strains were randomly selected from the five clonal types shown by PFGE cluster analysis and were further investigated using whole genome sequencing, including clone A (3/8), clone B (3/3), clone C (4/5), clone D (1/1) and clone E (9/23). Genomic DNA were prepared using the TIANamp Bacteria DNA Kit (Tiangen, Beijing, China) and subjected to WGS using the Illumina HiSeq 2500 platform (Novogene Biotech, Beijing, China). Sequencing was performed using a 2 × 150 bp pairedend configuration with a minimal coverage of 50×. After the original sequencing data were obtained, the raw reads were filtered for quality control to remove low-quality reads, adapter and duplication pollution, so as to obtain high-quality clean reads. The reads were assembled utilizing SPAdes v3.10.0 (http://cab.spbu.ru/software/spades/, accessed on 23 November 2021). Initial genome annotations were performed using Rapid Annotation using Subsystem Technology (RAST) (https://rast.nmpdr.org/, accessed on 23 November 2021), Center for Genomic Epidemiology (CGE) services (https://cge.cbs. dtu.dk/services/, accessed on 23 November 2021) and Pathosystems Resource Integration Center (PATRIC) (https://www.patricbrc.org/job/, accessed on 23 November 2021), and the results were manually curated. Insertion elements (IS) and antimicrobial resistance genes were identified using IS Finder and Res Finder 3.2, respectively. Based on this information, the putative coding sequence for the flanking region of the bla NDM-1 gene was obtained for genetic environmental analysis. Whole genome sequences were used to establish a phylogenetic tree using Parsnp in the Harvest package, which was visualized using figuretree.

Antimicrobial Susceptibility
Antimicrobial susceptibility results for the forty P. mirabilis isolates are listed in Table 1. All the bla NDM-1 -positive P. mirabilis exhibited high MICs for imipenem and meropenem (MICs of 128 and 32-64 mg/L, respectively). All isolates were also resistant to amoxicillin, cephalexin, ceftazidime, ceftriaxone, cefotaxime, cefepime and gentamicin ( Figure S1) and sensitive to aztreonam. The aminoglycoside antibiotic gentamicin showed a low resistance level, with an MIC value of 4-8 mg/L, and the resistance rate to the quinolone antibiotic ofloxacin was 22.5%, with an MIC distribution of 1-8 mg/L. Overall, nearly a quarter of the forty strains were found to be pandrug resistant to ten antibiotics. Compared with bla NDM-1 -positive strains, most of the antibiotic resistance rates of bla NDM-1 -negative strains dropped from 88.0% to 8.0%, except for the increase in cefalexin and ofloxacin resistance (22.5%→92.0%), while the resistance rates of imipenem and meropenem decreased to 58.0% and 8.0%, respectively ( Figure 2A). Of note, three aztreonam-resistant (0→6.0%) strains were found. According to the level of resistance, the MIC values were divided into five intervals. In bla NDM-1 -positive strains, the MIC values of the antibiotics imipenem, amoxicillin, cephalexin, ceftazidime and cefotaxime were found to be >64 mg/L, those of meropenem and ceftriaxone were 16-32 mg/L and those of cefepime, gentamycin and ofloxacin were 0.03-8 mg/L. However, in bla NDM-1 -negative strains, the MIC values of the antibiotics amoxicillin, cephalexin, ceftriaxone and cefotaxime were >64 mg/L, and those of the remaining antibiotics ranged from 0.03 to 8 mg/L ( Figure 2B). Most bla NDM-1 -negative strains had lower MIC values and larger distribution spans than bla NDM-1 -positive strains. Of note, the MIC values of ceftriaxone, cefotaxime and ofloxacin were obviously higher than bla NDM-1 -positive P. mirabilis.  USV Symbol Macro(s) Description

PFGE Typing and Phylogenetic Analysis
The results of the PFGE cluster analysis showed that forty CRP isolates belonged to five clones, identified as clone A (n = 8), clone B (n = 3), clone C (n = 5), clone D (n = 1) and clone E (n = 23). Clone E was the dominant clone, with high epidemic and vertical transmission (Figure 3). In addition, CRP isolates of the same cluster were shown to have the same antimicrobial susceptibility patterns. Furthermore, a phylogenetic tree was established based on the core genome of bla NDM-1 -positive P. mirabilis by single nucleotide polymorphism (SNP), including the 20 representative isolates in our study, shown with a purple background in Figure 4, and 278 genomes downloaded from the National Center for Biotechnology Information (NCBI) database and almost all available bla NDM -positive P. mirabilis genome data. Thereinto, data on 54 genomes were from animals, and these included the 20 genomes in this study, which can be seen in Figure 4. Six (A, B, C, D, E and F) main phylogenetic branches were observed in all bla NDM-1 -positive P. mirabilis, including four major clades and two small groups, F and D. In this study, we found that 20 isolates showed diversity and broad phylogeny and belonged to the branches of B, C, D and F. Although the branches of A and E are the most prevalent phylogeny types worldwide, none of the 20 strains belonged to either of these types. Among all clades, nine isolates shared the same group types, even without subtype branches, and belonged to the majorly prevalent types.

Transfer and Location of the bla NDM-1 Gene
Twenty representative CRP strains with different clonal types were selected for S1-PFGE and Southern blotting, including three clone A, three clone B, four clone C, one clone D and nine clone E strains. The results demonstrated that the seven bla NDM-1 strains were located on similar-size plasmids, ranging from 104.5 to 138.9 kb ( Figure 5). Conjugation experiments confirmed that bla NDM-1 can be transferred into E. coli J53. The transconjugants (tPB72, tPB96, tPB109) were successfully recovered from three donor strains with transfer frequencies of 1.3 × 10 −6 -5.8 × 10 −13 (Table S3). PCR detection of resistance genes confirmed that the transconjugants carried bla NDM-1 . All these transconjugants exhibited similar resistance to β-lactam and aminoglycoside antibiotics to donor strains, including imipenem (MICs 16-128 mg/L), meropenem (MICs 4-32 mg/L), amoxicillin and cephalexin (MICs ≥ 256 mg/L), ceftazidime (MICs ≥ 64 mg/L), ceftriaxone (MICs 32-128 mg/L), cefotaxime (MICs 64-128 mg/L) and cefepime (MICs 2-8 mg/L), but they remained susceptible to ofloxacin and aztreonam. The bla NDM-1 genes of most other strains were located on the chromosomes. It is worth noting that all the parental strains that successfully conjugated belonged to clone B, while the MICs in several β-lactam antibiotics of transconjugants were higher than those for parental strains ( Table 2). 1 Figure 4. Phylogenetic tree of 298 bla NDM-1 -positive P. mirabilis by core genome single nucleotide polymorphisms (SNPs). Twenty isolates shaded by violet were from this study, and 278 genomic data were download from the National Center for Biotechnology Information (NCBI). Different clusters were tagged and shaded different colors. The genome labeled with left triangle were collected from animals (54 in total), while others were from humans. Based on phylogenetic results of whole genome sequencing (WGS), 298 bla NDM-1 P. mirabilis were clustered in 6 major clades: A, B, C, D, E and F, labeled by different color backgrounds. In our study, the 20 bla NDM-1 P. mirabilis isolates colored by purple background were clustered into 4 main clades: B, C, D and F, in which clade B was predominant.

Characterization of the bla NDM-1 Genetic Environment
The analysis of whole genomes of strains revealed the distribution of antibiotic resistance genes and replicon types. These twenty strains were mainly divided into four different types of genetic environment (types: I, n = 9; II, n = 3; III, n = 7; IV, n = 1) ( Figure 6). In our study, conjugation and WGS analysis strongly suggested that the bla NDM-1 gene was located on the chromosome in type I strains. Although the genetic environment of type I strains showed certain similarities to that of the other bacterial strains in major and mediate segments, a significant difference was observed in farther boundaries at both ends, as shown in Figure 6. In this context, ISAba125 insertion sequences were identified immediately upstream of the bla NDM-1 genes; notably, uncommon insertion element IS1353, IS1326, pac, sul2 and aphA6 were also upstream of the bla NDM-1 . Downstream of bla NDM-1 , the bla NDM-1 companion gene ble MBL was flanked in front of trpF, followed by sul2, qacE delta 1, aadA1, dfrA1 and lnu(F) and included transfer element IS903B and integron intI2 in the terminal. The type II genetic environment of bla NDM-1 shows extremely similar regions with Type I (pac-sul2-aphA6-ISAba125-bla NDM-1 -ble MBL -trpF) and is extremely similar to clinically isolated P. mirabilis pHFK418-NDM (GenBank accession number MH491967.2). The genetic environment of type III was almost identical to that of type II (99% query coverage and nucleotide identity), but a difference is that the downstream sul2 gene in PB66 was truncated into two segments (not shown). In type IV, we found that the downstream region of the bla NDM-1 flanked an island of multiple resistant genes harboring aadB, CatB8, bla OXA-10 , aadA1, dfrA1, aacA4, qacE delta1 and sul2, followed by intI1, trpF and TnAs2. Regions upstream of bla NDM-1 genes encompass ISAba125, aphA6, pac, TniB and IS26 insertion sequences.

Discussion
As is well known, carbapenem-resistant pathogens are a threat to human public health, especially metallo-β-lactamases including NDM, VIM and IMP. Since NDM-1 was first found in New Delhi, more than 41 subtypes of NDM have become available in GenBank (https://www.ncbi.nlm.nih.gov/genbank/, accessed on 23 November 2021). So far, the presence of NDM has been reported in at least 55 countries and regions, and more than 60 species of bacteria producing NDM-1 or variants have been reported in humans, animals, plants, the environment, etc., with major prevalence in Enterobacteriaceae [22][23][24][25][26]. The livestock animals used as food sources and their environment have the dominant reservoir of carbapenem-resistant Enterobacteriaceae (CRE)-producing NDM, an important NDM spread section, where E. coli and Klebsiella pneumoniae have been dominant epidemic host species in animals used as food sources [27]. Recently, bla NDM-1 -positive P. mirabilis were continuously identified, the population of which is accelerating. Since the first P. mirabilis harboring bla NDM-1 was reported in patients in New Zealand hospitals in 2009 [28], increasing numbers of cases involving P. mirabilis NDM producers have been continuously reported in China [29], Brazil [15], Tunisia [14], Austria [30], India [31], Italy and New Zealand [17,28]. However, all these reports were human, clinical cases of bla NDM-1 -positive P. mirabilis; reports of cases in animals used as food sources are still lacking. Here, our studies revealed the dissemination of NDM-containing P. mirabilis in commercialized broilers, confirming that in poultry, P. mirabilis is a reservoir for bla NDM .
Generally, a reduction in the susceptibility of P. mirabilis to imipenem is mostly due to the intrinsic resistance mechanism loss of outer membrane porins, the decreased expression of PBP1a or the decreased affinity of imipenem to PBP2 [32], which showed low-level resistance. Unfortunately, carbapenemase can confer high-level resistance to β-lactam antibiotics (≥32 µg/mL) by hydrolyzing drugs in comparison to these intrinsic resistance mechanisms. In our study, 40 NDM-producing P. mirabilis strains were isolated from a slaughterhouse rather than a commercial farm. As far as we know, the chickens in this slaughterhouse may be from one or more commercial farms. Accordingly, the 40 NDMproducing P. mirabilis strains may be prevalent in one or more farms. Our study confirmed the phenomenon that all NDM-producing P. mirabilis showed a high carbapenem resistance rate and possessed significantly high resistance levels. As the results showed, the obvious high resistance levels for imipenem and meropenem were observed in bla NDM-1 -positive isolates relative to bla NDM-1 -negative strains (Figure 2). Given this result, a conclusion was drawn that bla NDM-1 takes responsibility for high carbapenem resistance levels in this study. For bla NDM-1 -positive P. mirabilis, in addition to ofloxacin, resistance to all tested antibiotics was demonstrated. In comparison, the resistance spectrum of the non-NDM producer was relatively narrow. Most isolates showed susceptibility to meropenem, ceftazidime and cefepime, which suggested all these antibiotics may be a good option for use in the treatment of infection caused by non-NDM-carrying P. mirabilis. The resistance to other β-lactams may be due to other β-lactamases being carried, such as OXA and TEM. Pertinently, almost all bla NDM -positive P. mirabilis resistance to all β-lactams antibiotic still retained susceptibility to ofloxacin in contrast to negative isolates. According to these results and analysis, catching one strain and losing anther was observed in antimicrobial resistance (AMR), which may be thanks to the cost of fitness caused by bla NDM . Stephan et al. showed a considerable cost of fitness was incurred by bla NDM-1 plasmid [33]. P. mirabilis-encoded β-Lactam resistance genes mainly include bla NDM-1 , bla OXA-1 , bla OXA-10 and bla TEM-1 , which are consistent with the main epidemic types of Salmonella, Enterobacter cloacae and Escherichia coli in livestock (poultry and pigs) reported in China. They have similar resistance gene spectrum, but the resistance phenotype is more serious [34][35][36]. Notably, in our study, the MIC of bla NDM-1 -harboring isolates from animals used as food sources increased 4-8 times more than from the values in human clinical P. mirabilis shown in previous studies [14,37], which were similar to the results shown in investigations regarding wild animals [4]. This suggests that the CRP strains in poultry may be more serious than those in humans, which acts as a warning that the carbapenem-resistant P. mirabilis in food animals should be given more attention.
The prevalence of bla NDM-1 in CRE was mainly mediated by clone transmission and plasmid transfer. However, this depended on the host bacteria and gene. For example, ST258 Klebsiella pneumoniae and ST131 E. coli are considered the major clones associated with carbapenemase genes [38,39], while the spread of bla OXA-48 , bla VIM and bla NDM mainly occurs via plasmids [40]. In China, the IncX3-type plasmid has been a dominant vector in the rapid spread of bla NDM-1 among Enterobacteriaceae [29]. Interestingly, these two mediators were present in our research and were confirmed via PFGE, conjugation and Southern blotting. PFGE analysis showed five major clusters, where type E was most common, suggesting clonal transmission may be present among them. The clonal spread of bla NDM-1 was a common phenomenon in CRE, and evidence is strong that clonal expansion is responsible for a substantial portion of transmitted cases [23,41], while it was rare in P. mirabilis among animals used as food sources. Usually, the clonal transmission of bla NDM was associated with the IS26 element [42], which was also present in the gene scaffold of these type E isolates. Additionally, there were other PFGE classifications showing bla NDM possess broad host bacteria for P. mirabilis and implying bla NDM may possess good adaptability in P. mirabilis [43,44].
Despite the fact that Southern blotting indicated that seven bla NDM-1 genes were located on plasmids in twenty tested isolates, only three plasmids (isolates PB72, PB96 and PB109) were successfully transferred. As early as 2011, Anaïs Potron et al. demonstrated that bla NDM -positive plasmids of different incompatibility groups can transfer into Escherichia coli, Klebsiella pneumoniae, Salmonella typhimurium and Proteus mirabilis [45]. In our test, the bla NDM-1 -positive plasmids were transferable to E. coli from Proteus mirabilis, which serves as further evidence that bla NDM-1 can transfer between E. coli and P. mirabilis by plasmid. Unfortunately, for these examples, the type of plasmid incompatibility was unsuccessfully determined based on the WGS data. However, bla NDM-1 -harboring PB72, PB96, PB109 possessed similar-size plasmids and the same gene environments ( Figure 6). Moreover, these three isolates belonged to the same cluster, which was confirmed via PFGE. Therefore, there was a possibility of clonal spread occurring among these P. mirabilis. Nevertheless, these plasmids in different host bacteria resulted in obvious differences in transfer frequency: 5.8 × 10 −13 , 1.3 × 10 −6 and 6.2 × 10 −8 for PB72, PB96 and PB109, respectively. It is possible that these plasmids in PB72, PB96 and PB109 may have been different or slightly evolutionary, further exhibited at the special mobile level. Given this, we tended to think that plasmids took the bulk of responsibility for bla NDM-1 spread among PB72, PB96 and PB109. Despite another four plasmids that contained bla NDM-1 with the same plasmid size and gene environment as the three abovementioned transferable plasmids, conjunction failed. It is believed that these plasmids may be untransferable among the four P. mirabilis strains. Additionally, these four P. mirabilis that carried untransferable plasmids were classed into two PFGE types (clone C and D), which was an obvious difference from PB72, PB96 and PB109 (clone B).
Subsequently, WGS was performed in an attempt to obtain detailed information; however, no complete plasmid sequences were assembled. Even so, three of the same plasmids carrying bla NDM-1 with sizes of 9430 bp were extracted from the WGS of PB72, PB96 and PB109; they shared an 81.23% homology with the pPrY2001 plasmid recovered from Providencia rettgeri and P. mirabilis ( Figure S2) [6,18]. On the other hand, the S1 results showed that bla NDM-1 seemed to be located on the same size plasmid (about 110 kb), which was similar to the size found in pPrY2001. All of this information indicated that the three bla NDM-1 may be harbored in an analogous plasmid, which needs to be clarified in future research. So, it is possible that the spread of these plasmids may be affected or regulated by host bacteria and emerging diversity in transferability in the case of the same plasmid. The presence of mobile and transferable plasmids that harbored bla NDM , as well as a clonal-spread-associated chromosome encoding bla NDM , was observed in our study. This may imply the evolution of bla NDM transmission from chromosome to transferable plasmid has further developed to mobile plasmids in P. mirabilis; however, all this conjecture requires further confirmation. Based on the results of transferable plasmid mediating bla NDM and broad clonal host P. mirabilis, there is no denying that bla NDM may be adjusting to P. mirabilis and attempting to increase its survival, which would further increase the risk of widespread infection.
The results emphasized the severity of NDM-1-producing P. mirabilis infection in poultry and showed that the animal intestine is an ideal hotbed for Enterobacteriaceae bacteria and resistant genes, suggesting that animal-derived resistant bacteria and genes may be spread to communities through direct contact, food chains or the environment, posing a potential threat to human health [46]. In addition, in view of the importance and interdependence of bacterial resistance with regard to humans, domestic animals, wild animals, plants and the environment, following the concept of "one health" to solve this problem should be considered [47]. Therefore, the timely detection of the resistance distribution of P. mirabilis in animal breeding, meat product processing, clinical patients and the environment can help to control the generation of resistance from the source and curb the spread of resistance in the food chain, which would have a far-reaching impact on the public.

Conclusions
In conclusion, this study reported an outbreak of infection in a chicken slaughterhouse caused by NDM-producing P. mirabilis . The diversity in the mediation of bla NDM via chromosomes with transfer elements, untransferable plasmid and mobile plasmid not only revealed the complexity of the spread of bla NDM but also suggested bla NDM increases the risk of widespread P. mirabilis infection among animals used as food sources. The reinforcement of the surveillance of resistance in the animal origin system is extremely urgent for curbing the transmission and persistence of NDM-producing P. mirabilis.
Funding: This research was funded by the 100 foreign experts and teams plan (WST2017010), and the National Natural Science Foundation of China (32172911).