Whole-Genome Analysis of blaNDM-Bearing Proteus mirabilis Isolates and mcr-1-Positive Escherichia coli Isolates Carrying blaNDM from the Same Fresh Vegetables in China

The global spread of colistin or carbapenem-resistant Enterobacteriaceae (CRE) has been a pressing threat to public health. Members of Enterobacteriaceae, especially Proteus mirabilis and Escherichia coli, have been prevalent foodborne pathogens and such pathogens from fresh vegetables have triggered foodborne illness in China. However, reports about CRE, especially P. mirabilis from fresh vegetables, are still lacking. In this study, we identified five blaNDM-positive P. mirabilis and five blaNDM-positive generic E. coli concurrently from five fresh vegetables in two markets from China, and four of the five E. coli also carried mcr-1. The 10 isolates were characterized with methods including antimicrobial susceptibility testing, conjugation, whole-genome sequencing and phylogenetic analysis. All 10 isolates were multidrug-resistant (MDR). blaNDM-5 in five E. coli isolates and one P. mirabilis carrying blaNDM-5 was located on similarly transferable IncX3 plasmids, while transferably untypable plasmids were the carriers of blaNDM-1 in four P. mirabilis isolates from different types of vegetables/markets. mcr-1 in the four blaNDM-5-positive E. coli was located on similarly non-conjugative IncHI2 MDR plasmids lacking transfer region. Notably, ISCR1 complex class 1 integron capable of capturing blaNDM-1 was found on all untypable plasmids from P. mirabilis, and five copies of ISCR1 complex class 1 integron containing blaNDM-1 even occurred in one P. mirabilis, which showed high-level carbapenem resistance. Plasmid and phylogenetic analysis revealed that the blaNDM-positive P. mirabilis and E. coli from fresh vegetables might be derived from animals and transmitted to humans via the food chain. The concurrence of blaNDM-positive P. mirabilis and E. coli carrying both mcr-1 and blaNDM in different types of fresh vegetables eaten raw is alarming and threatens food safety. Sustained surveillance of these foodborne pathogens among fresh vegetables is urgent to ensure the health of food consumers. We report for the first time the concurrence of blaNDM-positive P. mirabilis and mcr-1-bearing E. coli carrying blaNDM from the same fresh vegetables.


Introduction
The global spread of carbapenem-resistant Enterobacteriaceae (CRE) has been a pressing threat to public health [1], and such pathogens have been disseminated widely in clinical settings in many counties, including China [2]. New Delhi metallo-β-lactamase (NDM) has been the main type of carbapenemases conferring resistance to almost all βlactams, and CRE is the most common NDM carriers [3]. NDM-producing CRE isolates have been frequently found in humans [4], hospital wastewater [5] and animals [3] around the world. With the rapid increase in CRE, colistin has been re-used as the "last line of defense" for the treatment of CRE [6]. However, several mcr variants have been identified in various Enterobacteriaceae species [7], challenging the efficacy of colistin. The concurrence of colistin resistance in CRE isolates has been a great clinical concern, challenging the and enteroaggregative E. coli (EAEC) were identified using the previously described PCR method [28]. The criteria used for determining pathotypes were as follows: isolates carrying eaeA and escV and possible additional genes ent and bfpB were EPEC; isolates carrying elt and/or estla or estlb were ETEC; isolates carrying stx1 and/or stx2 and possible additional eaeA were STEC; isolates carrying invE and ipaH were EIEC; isolates carrying pic and/or aggR were EAEC. Diffusely adherent E. coli (DAEC) was identified by specific PCR for afa/dr as previously reported [29]. For P. mirabilis, the detection of eight virulence genes (ptA, zapA, ucaA, ireA, hpmA, mrpA, pmfA and atfA) that are often found in isolates from urinary tract infection was performed by using PCR, as previously described [30].

Antimicrobial Susceptibilities
Antimicrobial susceptibilities to 13 antimicrobials were determined for the obtained meropenem-resistant E. coli isolates using the broth microdilution method [31]. The 13 antimicrobial agents included cefotaxime, ceftazidime, ampicillin, meropenem, ciprofloxacin, levofloxacin, nalidixic acid, kanamycin, amikacin, streptomycin, tigecycline, tetracycline and colistin. Except for tigecycline and colistin, the susceptibilities to the remaining 11 antimicrobials were also measured for P. mirabilis isolates using the broth microdilution method. The resistant criteria recommended by the 2019 EUCAST were used for tigecycline and colistin [32], and the results for the remaining 11 drugs were interpreted according to the CLSI breakpoints [31].

Plasmid Conjugation and Replicon Typing
To investigate the transferability of bla NDM and mcr-1, conjugation experiment was carried out using P. mirabilis isolates harboring carbapenemase genes and mcr-positive E. coli carrying carbapenemase genes as the donors. E. coli C600 resistant to streptomycin was used as a recipient, and the broth mating method was performed as previously reported [34]. Eosin methylene blue (EMB) agar containing both colistin (2.5 µg/mL) and streptomycin (2000 µg/mL) was used to screen mcr-positive transconjugants, while Mac-Conkey agar plates containing both meropenem (1 µg/mL) and streptomycin (2000 µg/mL) were used to isolate transconjugants harboring carbapenemase genes. Transconjugants were confirmed using the PCRs mentioned above, and antimicrobial susceptibilities for transconjugants were also determined. Plasmid replicon types within transconjugants were detected using a PCR method [35], and the IncI2 and IncX3 replicons were also screened [36,37].
To analyze the phylogenetic relationships of the mcr-1-positive E. coli isolates carrying bla NDM in this study and those reported from humans and animals, we used our 3 E. coli genomes from fresh vegetables and 57 assembled genomes carrying mcr-1 or bla NDM from different countries and sources in the pathogen database of NCBI (https: //www.ncbi.nlm.nih.gov/pathogens (accessed on 15 July 2022)) (Table S1). Furthermore, the 2 bla NDM -positive P. mirabilis (M15061B and M15101B) from fresh lettuces in our study and 15 genomes of bla NDM -positive P. mirabilis from the NCBI database (1 from humans in Italy, 2 from humans in Czech, 4 from humans in China and 8 from animals in China) were used to trace the origins of foodborne P. mirabilis carrying bla NDM . The CSI Phylogeny 1.4 (https://cge.food.dtu.dk/services/CSIPhylogeny/ (accessed on 20 July 2022)) was used to obtain SNPs, and the phylogenetic tree was further visualized using iTOL v6 (https://itol.embl.de (accessed on 21 July 2022)).

Characterization of Plasmids Carrying bla NDM or mcr
For the nanopore-sequenced E. coli M15061H and P. mirabilis M15061B, the complete plasmid sequences carrying bla NDM were obtained, while the plasmid SPAdes tool (http://spades.bioinf.spbau.ru/plasmidSPAdes/ (accessed on 22 Jul 2022)) was used to extract contigs of plasmids carrying bla NDM or mcr in the Illumina-sequenced isolates. For the sequenced transconjugants, contigs of plasmid were obtained after filtering the chromosomal DNA data of E. coli C600. The contigs of the reconstructed plasmid were aligned against the NCBI and the complete plasmids in this study to select the best match. The circular comparison of bla NDM or mcr-1-positive plasmids was performed using the BRIG version 0.95 [39], and the plasmid linear alignment was analyzed with Easyfig version 2.1 [40].

Data Availability
The two mcr-1-bearing E. coli isolates carrying bla NDM (M15071H, M15081H) and bla NDM -bearing E. coli M15061H have been deposited in BioProject PRJNA869497, which also contains the three bla NDM -positive transconjugants from P. mirabilis. The sequences of bla NDM -positive P. mirabilis M15061B and M15101B have been submitted to NCBI under the BioProject PRJNA869497.

Virulence Genes and Concurrence of bla NDM -Positive P. mirabilis and E. coli from the Same Fresh Vegetables
In this study, carbapenem-resistant P. mirabilis isolates were found in five of the eight vegetable samples from one farmer's market and one supermarket in Zhejiang Province. The five P. mirabilis isolates were from one tomato, two lettuce and two cucumber samples (Table 1). Notably, five carbapenem-resistant E. coli isolates were also isolated from the five fresh vegetables, respectively. All P. mirabilis isolates carried bla NDM , and bla NDM-1 was the most prevalent type (n = 4), while bla NDM-5 was found in all E. coli isolates (Table 1). Worryingly, except M15061H, the remaining four E. coli isolates co-harbored mcr-1 and bla NDM-5 . Notably, four vegetable samples carried both bla NDM-1 and bla NDM-5 . All 15 virulence genes used for identifying diarrheagenic E. coli pathotypes were not found in our E. coli isolates, indicating that they were generic E. coli. Of the eight virulence genes detected, seven, including hpmA, mrpA, ptA, ireA, zapA, pmfA and atfA, were found in all the five bla NDM -positive P. mirabilis (Table 1).

Antimicrobial Resistance Patterns of bla NDM -Positive P. mirabilis and generic E. coli Isolates
The five bla NDM -positive E. coli isolates showed multidrug resistances, including resistance to β-lactams, aminoglycosides, tetracyclines and fluoroquinolones. Notably, the four E. coli isolates carrying both mcr-1 and bla NDM were also resistant to colistin (≥4 g/L) ( Table 2). In the bla NDM -positive P. mirabilis isolates, M15061B was susceptible to fluoroquinolones, while the remaining isolates showed resistance to fluoroquinolones. All P. mirabilis isolates were also multidrug-resistant, including resistances to all β-lactams tested. In this study, only the P. mirabilis strain M15092B was resistant to amikacin. Luckily, all five bla NDM -positive E. coli strains remained susceptible to both amikacin and tigecycline.

MLST Typing and Transfer of bla NDM or mcr-1
MLST analysis showed that all four E. coli isolates harboring both mcr-1 and bla NDM-5 from three types of vegetables in two markets belonged to the ST6050 type, while isolate M15061H harboring only bla NDM-5 was the ST533 type (Table 1). Transconjugants containing bla NDM were obtained in all 10 isolates in this study, and the carriage of bla NDM resulted in all transconjugants being resistant to ampicillin, cefotaxime, meropenem and ceftazidime ( Table 2). Only IncX3 replicon was found in the five bla NDM -positive transconjugants from E. coli, while the replicons in three P. mirabilis-derived transconjugants were untypable (Table 2). Notably, two plasmid replicons were found in transconjugants M15092BT. The mcr-1 in the four ST6050 E. coli isolates could not be transferred into the recipient E. coli C600, although the conjugation experiment was performed three times.
Based on the phylogenetic results of WGS, a total of 41,286 SNPs was obtained from these 60 mcr-1 and/or bla NDM -positive E. coli isolates from different origins and counties. These 60 E. coli isolates were clustered into 5 clades ( Figure 1A). The two mcr-1-positive NDM-producing ST6050 isolates M15071H and M15081H in this study belonged to clade I and had a limited number of variations (7 SNPs), although they were from different types of vegetables in the same market. The bla NDM-5 -positive M15061H belonged to clade II and had a large number of variations from M15071H and M15081H (19,597 to 19,604 SNPs). Notably, both the E. coli isolates in our study, M15071H and M15081H, were closely clustered together with previously reported bla NDM /mcr-1-positive isolates (e.g., isolates A20, L935) from humans in different countries, especially China. The bla NDM /mcr-1-positive E. coli isolates from animals (isolates 50080, 1003p and 51008369SK1) and environments (isolates HD6415, ICBEC3AM and ME2L-20-113) from different countries, including China, were also clustered together with our two foodborne E. coli isolates (M15071H and M15081H) ( Figure 1A). M15061H, carrying bla NDM-5 in this study, was also clustered together with pre- viously reported bla NDM /mcr-1-bearing isolates from animals, humans and environments in different countries, including China.
II and had a large number of variations from M15071H and M15081H (19,597 to 19,604 SNPs). Notably, both the E. coli isolates in our study, M15071H and M15081H, were closely clustered together with previously reported blaNDM/mcr-1-positive isolates (e.g., isolates A20, L935) from humans in different countries, especially China. The blaNDM/mcr-1-positive E. coli isolates from animals (isolates 50080, 1003p and 51008369SK1) and environments (isolates HD6415, ICBEC3AM and ME2L-20-113) from different countries, including China, were also clustered together with our two foodborne E. coli isolates (M15071H and M15081H) ( Figure 1A). M15061H, carrying blaNDM-5 in this study, was also clustered together with previously reported blaNDM/mcr-1-bearing isolates from animals, humans and environments in different countries, including China. A total of four phylogenetic clades were observed among the 17 NDM-producing P. mirabilis isolates, and 20,792 SNPs were obtained. Two blaNDM-positive P. mirabilis isolates (M15061B and M15101B) from different vegetable samples and different markets in this study were clustered together and had no SNP variation (0 SNPs) ( Figure 1B). It is worth noting that the two vegetable-sourced blaNDM-positive P. mirabilis isolates in this study were clustered together with animal-sourced isolates from China, which were mainly located in clade II. All NDM-producing P. mirabilis isolates from humans were located in clades I and III, including clinical isolates from humans in China ( Figure 1B).

Sequences of Plasmids Harboring mcr-1
The two Illumina-sequenced E. coli isolates carrying both mcr-1 and blaNDM (M15071H and M15081H) harbored IncHI2 plasmids with mcr-1. Both mcr-1-harboring IncHI2 plasmids pmcr_M15071H and pmcr_M15081H were about 177 kb in size and carried 13 types of resistance genes, which included colistin (mcr-1), β-lactams (blaOXA-1), trimethoprim A total of four phylogenetic clades were observed among the 17 NDM-producing P. mirabilis isolates, and 20,792 SNPs were obtained. Two bla NDM -positive P. mirabilis isolates (M15061B and M15101B) from different vegetable samples and different markets in this study were clustered together and had no SNP variation (0 SNPs) ( Figure 1B). It is worth noting that the two vegetable-sourced bla NDM -positive P. mirabilis isolates in this study were clustered together with animal-sourced isolates from China, which were mainly located in clade II. All NDM-producing P. mirabilis isolates from humans were located in clades I and III, including clinical isolates from humans in China ( Figure 1B).
In order to investigate whether the backbone structures of mcr-1-bearing IncHI2 plasmids in unsequenced E. coli isolates (M15092H and M15101H) were similar to pmcr_M15071H, seven pairs of primers were designed. The seven pairs of primers were designed according to transfer region 2 (trhE-trhK, trhV-trhC and traU-traN), domain China, respectively. The yellow, green, red and dark green rings represent pNDM5_M15061H, pNDM5_M15071H, pNDM5_M15081H and pNDM5_M15092B in this study. The outer circle with black arrows represents annotation of the reference plasmids; among them, the red, blue and orange represent resistance genes, transfer-related genes and transposase genes, respectively.
In order to investigate whether the backbone structures of mcr-1-bearing IncHI2 plasmids in unsequenced E. coli isolates (M15092H and M15101H) were similar to pmcr_M15071H, seven pairs of primers were designed. The seven pairs of primers were designed according to transfer region 2 (trhE-trhK, trhV-trhC and traU-traN), domain protein (DUF and VWA), heavy metal resistance (terZ-terD), tetracycline resistance (tetR(A)-tet(A)) and partial transfer region 1 (traI-traG) of pHNSHP45-2(KU341381) ( Table 5 and Figure 2A). Except for transfer region 1 (traI-traG), the remaining six regions detected were all found in M15092H and M15101H, confirming that the backbone structure of the mcr-1-bearing plasmids in the two unsequenced E. coli as highly similar to pmcr_M15071H. Thus, all four IncHI2 plasmids carrying mcr-1 in the current study were pmcr_M15071H-like plasmids and lacked transfer region 1.

Discussion
It is well known that the mcr-carrying isolates or CRE pose a great threat to public health. To date, more than 40 subtypes of NDM have been reported in more than 60 species of bacteria from humans, animals and environments, with a high prevalence of NDM-producing Enterobacteriaceae, especially E. coli [3,41]. P. mirabilis, a member of Enterobacteriaceae, is an opportunistic pathogen for humans and animals. P. mirabilis is also a foodborne pathogen, and P. mirabilis from vegetables has been linked with foodborne illness [24]. However, there has been no report about bla NDM -positive P. mirabilis isolates from fresh vegetables, especially in China. Currently, studies about vegetable-sourced E. coli isolates co-carrying mcr and carbapenemases are still lacking. Here, we identified, for the first time, five bla NDM -positive P. mirabilis and five bla NDM -bearing E. coli concurrently from the same five fresh vegetables in China, and four of the five E. coli also carried mcr-1, confirming that fresh vegetables have been an important reservoir for bla NDM -positive Enterobacteriaceae, including P. mirabilis.
In this study, five fresh vegetable samples co-harbored bla NDM -positive P. mirabilis and E. coli carrying bla NDM , including two lettuces, two cucumbers and one tomato. The two cucumber samples from one market and one supermarket, respectively, harbored P. mirabilis with different subtypes of NDM. The two lettuces of different sampling origins also carried different ST types of bla NDM -positive E. coli. These results indicate that these five samples may be from different vegetable farms. In this study, ST6050 was the prevalent type of E. coli harboring both mcr-1 and bla NDM from fresh vegetables, different from that in our previous report [12], in which the two E. coli carrying both mcr-1 and bla NDM belonged to ST2847 and ST156, respectively. To our knowledge, this is the first report of ST6050 type of E. coli co-carrying mcr-1 and bla NDM , especially in food. Carbapenemase-producing ST533 E. coli has appeared in patients [42]. Therefore, the ST533 E. coli carrying bla NDM-5 in lettuce will be a threat to human health. Luckily, all bla NDM -positive E. coli isolates in this study were not diarrheagenic E. coli. However, the bla NDM and mcr-1 within the generic E. coli isolate from vegetables eaten raw in the current study may be transferred to other foodborne pathogens or clinical pathogens. Carbapenemases can confer high-level resistance to β-lactams, including carbapenems. For example, all 10 bla NDM -positive E. coli and P. mirabilis isolates in our study showed high-level resistance to meropenem (≥128 µg/mL). All five bla NDM -positive E. coli isolates from fresh vegetables in this study showed multidrug resistance. Luckily, all these five vegetable-sourced bla NDM -positive E. coli isolates were susceptible to tigecycline and amikacin, similar to our previously reported two E. coli isolates carrying both bla NDM and mcr-1 from vegetables [12]. These results suggest that amikacin and tigecycline may be good options for treating human infection caused by such bacteria. All P. mirabilis isolates in this study also showed multidrug resistances, but four isolates were susceptible to amikacin, suggesting that amikacin might be a good option for human infection caused by bla NDM -positive P. mirabilis. This finding was similar to that for clinical P. mirabilis carrying bla NDM-1 in China [21]. Clinical NDM-producing P. mirabilis has been reported in China [21], Tunisia [43], Portugal [44] and Austria [45]. The treatment of infections caused by P. mirabilis represents a particular challenge because of its intrinsic resistance to colistin and tetracyclines, including tigecycline. P. mirabilis is generally associated with food spoilage and can also cause foodborne illness [22,23]. In China, vegetables contaminated with P. mirabilis have been linked with foodborne illness [24]. All five bla NDM -positive P. mirabilis from fresh vegetables in this study possessed seven of the eight virulence genes, which were all often found in clinical P. mirabilis linked with urinary tract infection [30], indicating a potential threat to humans. Furthermore, P. mirabilis is notorious for its ability to actively disseminate antimicrobial resistance genes, including bla NDM [21]. Thus, the concurrence of bla NDM -positive MDR P. mirabilis and mcr-1-positive E. coli producing NDM in fresh vegetables that are often eaten raw, poses a threat to human health.
IncX4 and IncI2 are the two major types of mcr-1-positive plasmids in E. coli from animals [46] and humans [47]. IncHI2 plasmids often carry multiple resistance genes [12], and IncHI2 plasmids possessing mcr-1 have also been found in E. coli from cooked retail meat in China in recent years [48]. In our previous study, which only investigated E. coli carrying mcr-1 among vegetables, IncX4 and IncI2 plasmids carrying mcr-1 were also the two major plasmid types [49]. However, mcr-1 was located on IncHI2 plasmids in all four vegetable-source bla NDM -positive E. coli isolates in this study. These data suggest that IncHI2-type plasmids play an important role in spreading mcr-1 among vegetables in China, indicating further surveillance of IncHI2 plasmids carrying mcr-1 is needed. Most previously reported mcr-1-harboring IncHI2 plasmids range from 210 to 260 kb in size and contain two transfer regions, as shown in [12], resulting in the transferability of these IncHI2 plasmids. However, the four mcr-1-positive IncHI2 plasmids in the current study were about 177 kb in size and did not harbor transfer region 1 (traJ~trhG genes), which might lead to the failure of conjugation for these plasmids. mcr-1 has often been linked with one or two copies of ISApl1, which plays an important role in spreading mcr-1 [50]. One copy of ISApl1 was linked to mcr-1 on the IncHI2 plasmids in our study, and Tn6330, an ISApl1-flanked composite transposon was not found. Besides mcr-1, IncHI2 plasmids in the current study also carried additional twelve types of resistance genes, including βlactams (bla OXA-1 ), trimethoprim (dfrA12), fluoroquinolones (aac(6 )-Ib-cr), aminoglycosides (aph(4)-Ia, aph(3")-Ib, aac(3)-IV, aph(6)-Id and aadA2), tetracyclines (tet(A)), rifamycin (arr-3) and amphenicol (catB3). Most antimicrobials mentioned above are used for both humans and animals. Thus the adverse effects of these drugs on the spread of mcr-1 should be paid more attention to.
IncX3 plasmids have been the main vector for the spread of bla NDM among Enterobacteriaceae [51]. Notably, almost all IncX3 plasmids carrying bla NDM carried the bla NDM-5 gene, while the E. coli isolates carrying other subtypes of bla NDM from animals harbored other replicon types rather than IncX3 in a previous study [3], consistent with the findings in our study that all IncX3 plasmids in the six isolates (E. coli and P. mirabilis) from fresh vegetables carried bla NDM-5 and P. mirabilis isolates with bla NDM-1 harbored no IncX3 replicon. The environment surrounding bla NDM-5 contained the mobile element IS5, suggesting that bla NDM-5 was recombined into IncX3 plasmids by insertion or transposition. A retrospective analysis of IncX3 plasmids in China showed that the backbone of IncX3 plasmids has been highly conserved [52]. The bla NDM-5 -positive IncX3 plasmids in this study were transferable and also had a highly similar backbone structure, although these plasmids were from bacteria of different genera, different ST types or different types of vegetables. These results suggest that the horizontal transfer of similar IncX3 plasmids might be responsible for the spread of bla NDM-5 among vegetables in China. All IncX3 plasmids from fresh vegetables in our study were similar to the bla NDM -positive IncX3 plasmids pKW53T (KX214669, 46,161 bp) from the urinary tract infection patient in Kuwait, pCREC-591_4 (CP024825, 46,161 bp) from patient ascites in Korea and pNDM-SCCRK18-72 (MN565271, 46,161 bp) from swine in China. These results suggest that the transferable bla NDM -bearing IncX3 plasmids in fresh vegetables might be derived from animals and then transmitted to humans through the food chain.
Unlike E. coli and Klebsiella pneumoniae, in which IncX3 and IncFII plasmids were the two main vectors for spreading bla NDM , the bla NDM-1 -bearing plasmids in P. mirabilis isolates were often assigned to an unknown incompatibility group [53]. In China, bla NDM-1 was also located on plasmids with unknown replicon type in P. mirabilis isolates from chicken [54]. Similarly, we obtained four untypable plasmids carrying bla NDM-1 from P. mirabilis isolates in vegetables, further confirming the association of untypable plasmids and bla NDM-1 in P. mirabilis isolates. The four untypable bla NDM-1 -bearing plasmids in P. mirabilis isolates from different types of vegetables or markets in our study were highly similar to untypable bla NDM-1 -positive pSNYG35 from cloacal swabs of broilers in China, further confirming the previous finding that the family of bla NDM-1 -carrying untypable plasmids in P. mirabilis shares high homologous backbones [53]. This result indicates that bla NDM-1 -positive plasmids in P. mirabilis from vegetables may come from isolates from animals. Insertion sequence common region (ISCR) is an IS91-like element that could mobilize adjacent sequences through the mechanism "rolling-circle replication" [55], and ISCR1 is a well-established gene capture system. bla NDM−1 could be disseminated by a circular ISCR1-bla NDM−1 element [56], and in this study, the untypable plasmids in P. mirabilis contained ISCR1 complex class 1 integron (sul1-qacE-arr-3-bla NDM-1 -ble MBL -ISCR1). The ISCR1 complex class 1 integron containing sul1-qacE-arr-3-bla NDM-1 -ble MBL -ISCR1/IS91 was also found on plasmid pSNYG35 in P. mirabilis isolated from cloacal swabs of broilers in China [53]. These results deepen our conjecture that the plasmids or bla NDM-1 in P. mirabilis from vegetables may have come from animals via plasmid transfer or gene capture. Notably, the nanopore-sequenced data confirmed that plasmid pNDM1_M15061B in P. mirabilis contained five copies of the ISCR1 complex class 1 integron (sul1-qacEarr-3-bla NDM-1 -ble MBL -ISCR1) in this study. We speculate that the ISCR1 element captures multiple antimicrobial resistance genes, including sul1-qacE-arr-3-bla NDM-1 -ble MBL , by several steps, resulting in the formation of pNDM1_M15061B. The meropenem MICs of previously reported bla NDM-1 -positive P. mirabilis from animals ranged from 32 to 64 µg/mL [54], and those from humans were from 2 to 64 µg/mL [21]. P. mirabilis (64 µg/mL) from humans was confirmed to possess two copies of bla NDM-1 . The meropenem MICs of M15061 and its transconjugant M15061BT in this study were ≥128 µg/mL, which might be attributed to the fact that both M15061 and its transconjugant M15061BT carried five copies of ISCR1 complex class 1 integron-containing bla NDM-1 . The meropenem MICs of the remaining four P. mirabilis and their transconjugants in our study were also ≥128 µg/mL, indicating that the remaining four P. mirabilis may also harbor at least one copy of ISCR1 complex class 1 integron-containing bla NDM-1 , although the structure of multiple copies of ISCR1 complex class 1 integron in these isolates were not obtained only from Illumina-sequenced data.
The sequenced mcr-1-positive NDM-producing ST6050 isolates (M15071H and M15081H) in this study were highly similar (7 SNPs), although they were isolated from different types of vegetables in the same market, indicating a very close genetic relationship between these two isolates. Notably, the NDM-producing E. coli isolates from fresh vegetables were clustered together with previously reported bla NDM /mcr-1-positive isolates from animals, humans and environments in different countries, including China. These results suggest that the NDM-producing E. coli isolates with mcr-1 from fresh vegetables in our study may be derived from animals through fecal fertilization and transferred to humans. The bla NDM -positive P. mirabilis isolates obtained from vegetables in this study had no SNP variation and were also clustered with animal-sourced isolates from China. These results suggest that bla NDM -positive P. mirabilis isolates in vegetables may be derived from animals because a relatively high prevalence of P. mirabilis isolates carrying bla NDM-1 has already been found in chickens from China [54].

Conclusions
In conclusion, we reported, for the first time, five bla NDM -positive P. mirabilis and five bla NDM -bearing generic E. coli concurrently from the same five fresh vegetables in China, and four of the five E. coli also carried mcr-1. bla NDM-5 in all E. coli isolates, and P. mirabilis carrying bla NDM-5 from fresh vegetables was located on similar IncX3 transferable plasmids, while similarly untypable transferable plasmids were the carriers of bla NDM-1 in P. mirabilis isolates from different types of vegetables or markets. mcr-1 in all bla NDM -5 -positive E. coli was located on similarly non-conjugative IncHI2 MDR plasmids lacking a transfer region. Notably, ISCR1 complex class 1 integron capable of capturing bla NDM-1 was found on transferable untypable plasmids from P. mirabilis in this study, and five copies of ISCR1 complex class 1 integron were even found in one P. mirabilis. Plasmid comparison and phylogenetic analysis revealed that the bla NDM -positive P. mirabilis and bla NDM -positive E. coli in fresh vegetables might be derived from animals by fecal fertilization and could be transmitted to humans through the food chain. Fresh retail vegetables might have been underestimated vehicles of E. coli and P. mirabilis in spreading resistance genes, including both bla NDM and mcr-1. The concurrence of bla NDM -positive P. mirabilis and E. coli possessing both mcr-1 and bla NDM in different types of fresh vegetables eaten raw is alarming and threatens food safety. Sustained surveillance of resistance in foodborne pathogens in the food chain, especially fresh vegetables, is urgent for preventing the transmission of MCR-producing and/or NDM-producing Enterobacteriaceae to ensure the health of food consumers.
Supplementary Materials: The following supporting information can be downloaded at: https: //www.mdpi.com/article/10.3390/foods12030492/s1, Table S1: Information about strains used for Escherichia coli evolutionary tree from NCBI database.