Genetic and virulence characteristics of a Raoultella planticola isolate resistant to carbapenem and tigecycline

Raoultella planticola is an emerging pathogen causing several infections in humans, and its roles in the propagation of antibiotic resistance genes (ARGs) remain uncharacterized. In this study, a carbapenem and tigecycline-resistant R. planticola isolate was recovered from hospital sewage. It carried nine plasmids, bearing 30 ARGs, including one blaKPC-2 and two blaNDM-1. It also contained a plasmid-borne efflux pump gene cluster, tmexCD1-toprJ, conferring resistance to tigecycline. Analysis of plasmid sequences revealed that both blaNDM-1-carrying plasmids were highly similar to those recovered from humans, reinforcing the close relatedness of environmental and clinical isolates. We also identified that plasmid bearing blaNDM-1 or tmexCD1-toprJ1 was transferable, and can be stabilized in the host bacteria, indicating that the R. planticola isolate has a considerable potential in the dissemination of ARGs. Besides, we found that this isolate could produce biofilm and was virulent in a Galleria mellonella infection model. In conclusion, our study shows the convergence of virulence and multidrug resistance in a R. planticola isolate. This potentially virulent superbug may disseminate into its receiving rivers, and finally to humans through cross-contamination during recreation activities or daily use of water, which poses a risk to public health.


Materials and methods
Bacterial isolation. R. planticola SCLZS62 was isolated during a study for the presence of carbapenemresistant Enterobacteriaceae strains in hospital sewage. 5 ml of water sample was collected from the influx mainstream of hospital sewage at the affiliated hospital of Southwest Medical University, Luzhou in western China, in November 2019. As described in our previous study 29 , bacterial cells were concentrated by centrifugation at 5000g for 5 min. The sediment was resuspended in sterile 0.9% NaCl solution and plated onto MacConkey agar containing meropenem (2 μg/ml) and incubated for 24 h at 37 ℃. Pink colonies with various morphologies were picked and repeatedly streaked on new MacConkey agar plates to obtain pure isolates. Initial species identification was performed by PCR amplifying of 16S rRNA gene and Sanger sequencing 30 . The presence of the acquired carbapenemase genes, bla KPC , bla NDM , bla OXA-48 , bla OXA-58 , bla VIM, and bla IMP was screened via PCR assays using primers as previously described 29 .
Antimicrobial susceptibility testing. The minimum inhibitory concentrations (MICs) of amikacin, gentamicin, colistin, meropenem, imipenem, cefoxitin, chloramphenicol, ciprofloxacin, cefotaxime and tigecycline against the target strains were determined using the broth microdilution method according to the Clinical and Laboratory Standards Institute (CLSI) guidelines (CLSI, 2017). Breakpoints for colistin and tigecycline were defined by European Committee on Antimicrobial Susceptibility testing (EUCAST) guidelines (http:// www. eucast. org/ clini cal_ break points/). Escherichia coli ATCC 25922 was used as a quality control for MIC determination. All antibiotics used in this study were obtained from yuanye Bio-Technology Co. (Shanghai, China).
Genome sequencing and analysis. Genomic DNA of R. planticola SCLZS62 was extracted and purified using the QIAamp DNA Mini Kit (Qiagen). The generated DNA was sheared with an average size of 10 kb and submitted for whole genome sequencing using a PacBio RSII sequencer (Pacific Biosciences, Menlo Park, USA). Meanwhile, the genomic DNA was sequenced on a HiSeq 2000 sequencer (Illumina, San Diego, CA, USA), using a paired-end library with an insert size of 150 bp. The de novo assembly of the PacBio reads was carried out with the Link v5.0.1. BWA-MEM was employed for mapping Illumina reads over the PacBio-generated contigs to correct the assembled contigs 31 . Library construction and sequencing was carried out at Beijing Novogene Bioinformatics Technology Co. Ltd. Annotation was carried out using Prokka 32 and BLASTP searches against the UniProtKB/Swiss-Prot database. The species identification was carried out by average nucleotide identity (ANI) analysis with the online software JSpeciesWS (http:// jspec ies. riboh ost. com/ jspec iesws/# analy se). Digital DNA-DNA hybridization (dDDH) values were calculated using GGDC 3.0 server (http:// ggdc. dsmz. de/ distc alc2. php) by means of genome-to-genome sequence comparison 33 . Plasmid incompatibility types were analysed using the PlasmidFinder tool (95%, minimum threshold for identity; 60%, minimum coverage) 34 , and antimicrobial resistance genes were predicted using ResFinder (90%, minimum threshold for identity; 60%, minimum coverage) 35 . Insertion elements (ISs) and integrons were predicted using ISfinder 36 and INTEGRALL 37 . The presence of virulence genes was investigated by searching the virulence factor database (VFDB) with an E value cutoff of 0.0001 38 . The retrieved virulence genes were further screened with a cutoff of > 50% query coverage and > 75% identity. Multiple and pairwise sequence comparisons were carried out using the BRIG tool 39  www.nature.com/scientificreports/ Phylogenetic analysis. Genomes were annotated using Prokka and the generated GFF3 files were used to create a core genome alignment with Roary. Single nucleotide polymorphisms (SNPs) were obtained with snpsites v2.3.2 40 . Based on the SNPs, A maximum-likelihood phylogenetic tree was constructed with FastTree version 2.1.10 41 . The presence of β-lactamase genes was detected by Abricate (https:// github. com/ tseem ann/ abric ate). Carriage of β-lactamase genes and detail information of isolates were annotated on the tree using iTOL 42 .
Plasmid stability. The stability of p2_SCLZS62, p5_SCLZS62, p7_SCLZS62 and p8_SCLZS62 in R. planticola SCLZS62 was studied as previously described with little modification 7 . SCLZS62 was grown overnight in 3 ml LB (antibiotic-free) broth at 37 °C, and 3 µl overnight culture was then incubated into 3 ml fresh LB broth each day, yielding ~ 10 generations of growth per passage. The serial passage was lasted for 21 days. Every three days, cultures were serially diluted and plated on LB agar plates without antibiotics. To determine the percentage of plasmid-containing cells, ~ 100 colonies were screened on LB agar plates containing 4 μg/ml tigecycline or meropenem. The presence of p2_SCLZS62, p5_SCLZS62, p7_SCLZS62 and p8_SCLZS62 was confirmed by PCR assays using primers p2-F/R, p5-F/R, p7-F/R and p8-F/R, respectively, in Table S1. This experiment was performed in triplicate.
In vitro growth assays. Three independent cultures of J53 and transconjugants were grown overnight and diluted to 1:100 in LB broth. Bacteria cultures were incubated while shaking at 37℃. In the total period of 12 h, the value of optical density (OD) at 600 nm (OD600) was consistently recorded at an interval of 1 h with the iMark microplate Reader (Bio-Rad).
Biofilm formation assays. Biofilm formation assays were performed as described previously with minor changes 43 . Overnight cultures of R. planticola SCLZS62 was diluted 1/100 into fresh LB broth, and 200 μl of bacterial suspensions was inoculated into sterile 96-well microplates plates in triplicate. After 24 h at 37 ℃, culture supernatant was removed and the wells were washed with phosphate-buffered saline (PBS) and stained with 200 μl 0.1% (w/v) crystal violet solution at room temperature for 15 min. After that, the wells were washed with PBS for three times to remove excess stain and dry for 1 h. The bound dye was released by adding 100 ul of acetic acid (33%, v/v) and the optical density was measured at 595 nm using a microplate reader. NTUH-K2044, a hypermucoviscous and hypervirulent Klebsiella pneumoniae isolate belonging to the sequence type 23 and capsular type K1 from a patient with primary liver abscess 44 , and MG1655, a non-pathogenic E. coli K-12 strain 45 , were included as the positive and negative control strains, respectively. LB broth without any inoculation serves as a blank control. This experiment was performed in triplicate.
Galleria mellonella infection assays. The virulence potential of the R. planticola SCLZS62 was assessed using wax moth (Galleria mellonella) larvae weighing 250 to 350 mg (Tianjin Huiyude Biotech Company, Tianjin, China) as described previously with minor changes 46 . Overnight culture of R. planticola SCLZS62 from LB agar plates was harvested and adjusted using PBS to final concentrations of 1 × 10 6 CFU/ml, 1 × 10 7 CFU/ml, 1 × 10 8 CFU/ml and 1 × 10 9 CFU/ml. Ten larvae were chosen randomly as a group, and 10 μl aliquots of bacterial suspension was injected into the last left proleg of each larvae using a 50 μl Hamilton syringe. Larvae injected with the K. pneumoniae NTUH-K2044 were used as hypervirulence-positive controls, the E. coli MG1655 as low-virulence controls, and PBS as negative controls. The larvae were then incubated at 37 °C, and the number of live larvae was counted at 12 h intervals for 3 days. In all cases, no dead larvae were observed in the PBS groups. This experiment was performed in triplicate, and a representative result of three independent experiments was used to generate survival curves.
Statistical analysis. Data of biofilm formation assays were analyzed with a student t test performed in the GraphPad Prism version 8.3.0 (GraphPad Software, CA, USA). A two-way analysis of variance (ANOVA) was used to evaluate statistical significance in the bacterial growth assays. The survival rate of the G. mellonella was analyzed using the log-rank test. Differences were considered statistically significant at P < 0.05.

Results and discussion
Genome characterization of R. planticola SCLZS62. SCLZS62 was a MDR strain as it exhibited resistance to all tested antibiotics (≥ 3 classes of antimicrobial agents), except for colistin 47  www.nature.com/scientificreports/ β-lactams (bla KPC-2 , bla NDM-1 , bla CTX-M-14 , bla PLA2a and bla PER-1 ), macrolide (mph(A), mph(E) and msr(E)), fosfomycin (fosA and fosA3), quinolones (aac(6′)-Ib-cr, qnrA1 and qnrS1), rifampicin (ARR-3), sulfonamides (sul1), trimethoprim (dfrA1), and tetracycline (tet(A)). The detection of carbapenemase genes bla KPC-2 and bla NDM-1 explained the resistance to all the cephalosporin and carbapenem drugs tested. It was notable that R. planticola SCLZS62 contained three genes conferring resistance to carbapenems, including two bla NDM-1 and one bla KPC-2 , which were carried by three different plasmids. In addition, it was observed that R. planticola SCLZS62 was resistant to tigecycline, and the recently characterized plasmid-mediated tigecycline resistance genes tet(X) variants were absent. Further analysis of the genome data showed that the newly identified efflux pump gene cluster, tmexCD1-toprJ1, involved in tigecycline resistance was identified on a plasmid in R. planticola SCLZS62, which probably contributed to the resistance of this isolate to tigecycline.
Phylogenetic analysis of R. planticola SCLZS62. To determine a possible clinical relevance of SCLZS62, whole-genome sequences of 57 publicly R. planticola strains retrieved from GenBank (on 2021/09/02) were aligned with that of SCLZS62. A core genome-based phylogeny showed a diverse set of genomes, with a total of 655 core genes and 39,331 SNPs. These R. planticola isolates were mainly recovered from humans and the environment, and also found in animals, plant and insects across various countries. Phylogenetic tree showed that SCLZS62 was present in a cluster with two isolates AS012264 and AS012263 (Accession no. GCA_010598615.1 and GCA_010598665.1) from patients with lung disease in USA in 2016, with 1430 and 1428 SNPs, respectively (Fig. 1, Table S2). This result revealed a potential close clinical relevance of R. planticola SCLZS62. Resistance gene profiles of all R. planticola isolates showed a sporadical acquisitions of carbapenemase genes, with bla KPC-2 being the most frequent one, followed by bla NDM-1 . SCLZS62 and another clinical isolate (GCA_013462275.1), also from China in 2018, represent the only two strains coharboring bla KPC-2 and bla NDM-1 , with 1508 SNPs differences.  . In all of these plasmids, bla NDM was located in a ~ 6.0 kb region bracketed by two copies of Tn3-derived inverted-repeat transposable elements (TIMEs, bases 6434 to 6689 and 23,920 to 24,175 of p2_SCLZS62), which contribute to the mobilization of bla NDM . In addition to bla NDM-1, p2_SCLZS62 also harbored resistance genes rmtC and sul1, which were located in a 11.5-kb region upstream of bla NDM-1 that was also flanked by two copies of TIMEs.

Genetic features of the bla
Genetic features of the bla NDM-1 -harbouring plasmid p5_SCLZS62. Sequence analysis of p5_ SCLZS62 indicated that it was a circular IncC-type plasmid of 173,509 bp with 213 predicted ORFs. Based on the presence or absence of orf1832/orf1847, rhs1/rhs2, i1, and i2, which are key features distinguishing between type 1 and type 2 IncC plasmids, p5_SCLZS62 was recognized as the type 1 IncC plasmid 48 . A BLAST search against the GenBank database showed that p5_SCLZS62 was almost identical (100% coverage and ≥ 99.98% identity) to several plasmids recovered from humans, including pGD31-NDM (Accession no. CP031297) in E. coli from Viet Nam and pSAL-19-0623 (Accession no. MN604267) in Salmonella enterica from Singapore ( Fig. 2A). Compared with the type 1 prototype plasmid pR148, p5_SCLZS62 shared a 128-kb backbone, but the content of antibiotic resistance island, designated ARI, was varied ( Fig. 2A). It is notable that all resistance genes of p5_SCLZS62, including ant(2″)-Ia, qnrA1, sul1 and bla NDM-1 are located in the ARI, with the structure of a novel unit transposons, designated Tn7353, being inserted at a site within a gene named orf3 encoding a putative permease in the plasmid pKPHS3 (Accession no. NC_016839) from clinical K. pneumoniae isolate (Fig. 2B). www.nature.com/scientificreports/ The Tn7353 was a derivative of Tn1696, with identical tnpA-tnpR and 89.57% nucleotide identity of the mer gene cluster, but differed from it mainly by insertion of a complex class 1 integron instead of In4 in Tn1696. The complex class 1 integron consists of one 5′-CS, three 3′-CSs, two ISCR1s, and three VRs bearing resistance genes. bla NDM-1 was located in the ISCR1-1-linked VR-2, with the structure of ΔTn125 consisting of ΔgroL, groS, ΔcutA, dsbD, trpF, ble, bla NDM-1 and ΔISAba125 in order, within In7 p5_SCLZS62 . According to a previous report, ISCR27 may be involved in the initial acquisition and mobilization of bla NDM-1 , and then Tn125 was commonly associated with the wide dissemination of bla NDM-1 in bacteria 49 . The presence of ISCR1 downstream of the truncated Tn125 in this complex class 1 integron suggests an important role of ISCR1 in the evolution and further movement of the bla NDM-1 gene, by mobilizing the ΔTn125 segment in this situation. The construction of the complex class 1 integron harboring multiple resistance genes by the introduction of two ISCR1 elements revealed significant roles of mobile elements like ISCR1 in the creation and evolution of multidrug-and pandrug-resistant regions 47 , which poses a big challenge in fighting against antibiotic resistance. By sequence comparison, the ARI of p5_SCLZS62 showed high similarity (99% query coverage and 99.98% identity) to the corresponding region of pGD31-NDM, which was also identified as a derivative of Tn1696 termed Tn7352, with some deletions: (1) A 3577-bp deletion of the cassette array, bla OXA-4 -aadA2-strA-strB, within the class 1 integron, which probably resulted from homologous recombination via the 5′-CS of the class 1 integron; (2) two deletions at the ISCR1-1-associated VR-2, including a 7856-bp deletion downstream of the ISCR1-1 and a 1780-bp deletion downstream of bla NDM-1 . We speculated that ARI of p5_SCLZS62 might have progressively evolved from the genetic structure like Tn7352 by experiencing multiple genetic events. In addition to the ARI, p5_SCLZS62 also contained an 15-kb region harboring a novel efflux pump gene cluster, designated tmexA-tmexB-toprM ("t" for transferrable 7 ), when it was compared with pR148. The efflux pump region had an ents-toprM-tmexB-tmexA-baeR-baeS-dgcT-rhaS-ywfM-orf-tyrS structure, which was inserted into the uvrD gene, splitting it into two separate parts and meanwhile leaving 6-bp direct repeats (DRs, CTC TAC ) at both ends (Fig. 2C). A BLASTn search in the GenBank database showed that the efflux pump region was also found in plasmids pIncHIB (Accession no. CP036336) in K. pneumoniae from India, and pM216_AC2 (Accession no. AP018145) in E. coli from Japan. Specially, the plasmid-borne efflux pump gene cluster baeS-baeR-tmexAB-toprM was found to be an 88.87% match to a chromosomal DNA fragment of Shewanella baltica strain CW2 (Accession no. CP028355), raising the possibility that this efflux pump gene cluster originates from certain species of Shewanella, such as S. baltica. Despite the presence of perfect DRs, there was no mobile element found in the efflux pump region. The transposition mechanism of this region requires further elucidation.
Sequence analysis showed that this novel efflux pump tMexA-tMexB-tOprM, showed 36.02%, 55.6%, 46.04% amino acid identity to the MexA, MexB, OprM encoded on the chromosome of Pseudomonas aeruginosa, respectively. This gene cluster tmexAB-toprM is adjacent to ORFs annotated as a two-component regulatory system encoded by baeS and baeR, indicating an intact gene cluster encoding an efflux pump. To validate the function of tmexAB-toprM, three recombinant plasmids: pMD19-baeSR-tmexAB-toprM, pMD19-tmexAB-toprM (missing baeSR), pMD19-baeSR-tmexAB (missing toprM) were constructed. Relative to those with the empty vector pUC-19, none of the E. coli DH5α strain carrying recombinant plasmid showed increase in the MICs of the tested antimicrobial agents (Table S3), suggesting that this tmexAB-toprM failed to function as an efflux pump system. Additional studies are needed to fully characterize the biological functions of the tmexAB-toprM gene cluster.
Genetic features of plasmid p7_SCLZS62. p7_SCLZS62 has 334,207-bp circularly closed DNA sequences, and carries 541 predicted ORFs in total. p7_SCLZS62 belongs to IncHI5 group because it contains a replication gene repHI5B, and an additional repFIB-like gene. A BLASTn search against the GenBank database showed the highest 87% query coverage, and 99.93% identity to the plasmid pJNQH579-2 (Accession no. CP078148) in Klebsiella variicola from a clinical sputum specimen in China. Genomic comparison of p7_SCLZS62 with two IncHI5 reference plasmid pKOX_R1 (Accession no. CP003684) and p11219-IMP (Accession no.MF344561) showed that it possess conserved backbones, including repHI5B (together with its iterons) and repFIB-like gene for replication, parAB for partition, and two tra regions (tra1 and tra2) for conjugal transfer 50 (Fig. 3A). This plasmid harbors numerous antibiotic resistance genes (Table 2), as well as a newly identified RND efflux pump gene cluster, tmexCD1-toprJ1, which confers transferable resistance to tigecycline 7 .
p7_SCLZS62 carries three accessory modules, namely the MDR-1 region, MDR-2 region, and efflux pump region. The MDR-1 region was identified as a Tn6381 derivative, which was inserted at a site within an intl1 gene, breaking it into two separate parts Δintl1-5′ and Δintl1-3′ (Fig. 3B). The Tn6381 derivative was generated from insertion of IS26-ΔIn469-IS26 unit into a site downstream of tnpA, leading to a 651-bp deletion in the Tn3-family core transposition module tnpA-res-tnpR, and a truncation of both tnpA and tnpR. The MDR-2 region consists largely of a ΔTn6279-like transposon, and the rest was a tnpA-tnpR module with its terminal 38-bp IRL interrupted by IS5075 and a res remnant res-5′ (Fig. 3C). As a derivative of Tn6279, the ΔTn6279-like transposon had major modifications including an insertion of ISKpn21 and ISAs27, a deletion of ISAba24 and the terminal IS26-aph(3′)-Ia-IS26 unit, and a replacement of In438 by In797. The efflux pump region was identified as a complex chimera structure composed of a Tn6347-like remnant, two IS26-composite transposon-like units (IS26-mph(A)-IS26 and IS26-fosA-IS26), ΔTn1721, ΔTn6361, In268, ΔTn1696, Tn6344 and the efflux pump module (ΔtnpA-int-int-infB-psrP-tnfxB1-tmexC1-tmexD1-toprJ1-ΔtnpA-tnpR) that was most likely to be derived from Tn5393 (Fig. 3D). The efflux pump module in p7_SCLZS62 shared high similarity (95% coverage, 99.97% identity) to that in pHNAH8I-1, the first reported tmexCD1-toprJ1-harboring plasmid 7 . Unlike the strA-strB of Tn5393-3′ retained in the pHNAH8I-1, forming tnpR-strA-strB, a Tn6361 remnant was located downstream of tnpR in p7_SCLZS62, and formed the tnpR-qnrS1-IS26 structure. The acquisition of ΔTn6361 in p7_SCLZS62 was most likely to result from massive recombination events between two copies of tnpR.  Figure S2). This result reinforced the important role of the pCKPC18-1-like untypeable plasmids in the dissemination of bla KPC-2 in Enterobacteriaceae in China [51][52][53] . The backbone of these plasmids include an untypeable replication initiation gene (repA), parA and topB for stability, relEB genes for maintenance, and tviB for conjugation. In the accessory region, bla KPC-2 was carried by a ΔTn6296 genetic platform, in which ΔTn1722-3′ was lost from the prototype Tn6296 (Fig. 4).

Plasmid transferability, stability and host fitness.
Conjugal transfer experiments showed that carbapenem resistance determinants could be transferred into E. coli J53 at the frequency of ~ 10 -6 (transconjugant/ recipient). Among the transconjugants, cells containing p5_SCLZS62 accounted for 73.44%, and p8_SCLZS62 only for 4.69%. Specially, all the transconjugants harbored p2_SCLZS62. This result indicates that these three plasmids carrying carbapenemase genes are mobilizable, with the highest transferability of p2_SCLZS62, and the least efficiency of p8_SCLZS62. MICs of carbapenem (including meropenem and imipenem) and cephalosporin (including cefoxitin and cefotaxime), of these transconjugants, were increased at least 128-fold (Table 1). In addition, p7_SCLZS62 was able to conjugate into E. coli J53 at a frequency of ~ 10 -8 , which is consistent with its intact set of conjugative transfer genes. Antimicrobial susceptibility analysis revealed that the acquisition of p7_SCLZS62 enabled E. coli J53 to become resistant to tigecycline by at least 8-fold (Table 1). To estimate the fitness cost of these resistant plasmids, we compared growth characteristics of the transconjugants harboring resistant plasmids, and the plasmid-free recipient strain J53. Results showed that transconjugants JM-5 and JT-4, which bear plasmids p2_SCLZS62, p5_SCLZS62 and p7_SCLZS62, exhibited a significant growth retardation compared to J53, indicating that the coexistence of these three plasmids confers a fitness cost on the host (Fig. 5A). Stability assays showed that both carbapenem and tigecycline resistance determinants were stable for ≥ 21 d (~ 210 generations) in the R. planticola SCLZS62 in antibiotic-free medium. Among the carbapenem-resistant colonies, plasmids p2_SCLZS62 and p5_SCLZS62 were stably maintained with 100% retention, while p8_SCLZS62 was gradually lost during serial passage with 35.76 ± 5.35% retention after 21 days (Fig. 5B). The transferability and highly stability of plasmids containing bla NDM-1 or efflux pump gene cluster tmexCD1-toprJ1 reinforced the idea that MDR strains like R. planticola SCLZS62 in the aquatic environment serve as a reservoir of ARGs, which has serious public health implications.
Virulence characteristics of R. planticola SCLZS62. Biofilm formation assays showed that the biofilm-forming ability of SCLZS62 was significantly higher than that of E. coli MG1655, and comparable to that of K. pneumoniae NTUH-K2044 (Fig. 6A). The pathogenic potential of SCLZS62 was determined by Galleria mellonella infection testing. Results showed that the survival rates of G. mellonella significantly decreased when infected with strain SCLZS62 or NTUH-K2044 in relative to those with MG1655 under various infection concentrations ( Fig. 6B-E). These findings suggested that SCLZS62 is a biofilm producer, and potentially virulent. In silico analysis detected 38 virulence genes in R. planticola SCLZS62 (Table S4), including gene clusters encoding type 3 fimbriae (mrkABC) and type 1 fimbriae (fimACDEGH), which contribute to biofilm formation, and adherence to host cells 54 . Also, genes encoding aerobactin (iutA) and ent siderophore (entABCES and fepABCG) for iron acquisition, and gnd for serum resistance were identified, which could enhance the ability of the bacteria to survive and colonize within the host 55 . Additionally, several sets of genes encoding type VI secretion system were included, such as dotU/tssL, hcp/tssD, icmF/tssM and impA/tssA, vipB/tssC, vasE/tssK, which play an important role in the invasion and pathogenicity during the infection process of pathogens 56 .

Conclusions
In this study, we report a carbapenem-and tigecycline-resistant R. planticola isolate carrying nine plasmids from hospital sewage. The coexistence of nine plasmids provides great genetic plasticity for further spread of resistance genes, and the fitness of host bacteria to its environment. This isolate contains two types of carbapenemase genes, www.nature.com/scientificreports/ including one bla KPC-2 and two bla NDM-1 genes, which confirms the dissemination of clinically important resistance genes into the environment by sewage discharged from hospitals. The coproduction of multiple determinants for the same resistance is mystifying, while it hints at the important host role of R. planticola in the propagation of ARGs. Finally, we identified that this MDR isolate is potentially virulent, which poses a potential public health risk. The occurrence of virulent superbug in the environment should be closely monitored.