Characterization of two novel VIM-type metallo-β-lactamases, VIM-84 and VIM-85, associated with the spread of IncP-2 megaplasmids in Pseudomonas aeruginosa

ABSTRACT This study aimed to characterize two novel VIM-type metallo-β-lactamases, VIM-84 and VIM-85, and reveal the important role of the IncP-2 type megaplasmids in the spread of antimicrobial resistance (AMR) genes. VIM-84 and VIM-85 were encoded by two novel genes bla VIM-84 and bla VIM-85 which showed similarity to bla VIM-24. Both bla VIM-84 and bla VIM-85 are harbored into class 1 integrons embedded into the Tn1403 transposon. The bla VIM-85 gene was identified in a megaplasmid, which was related to 17 megaplasmid sequences with sizes larger than 430 kb, deposited previously in Genbank. A comparative analysis of complete plasmid sequences showed highly similar backbone regions and various AMR genes. A phylogenetic tree revealed that these megaplasmids, which were widely distributed globally, were vehicles for the spread of AMR genes. The bla VIM-24, bla VIM-84, and bla VIM-85 genes were cloned into pGK1900, and the recombinant vectors were further transformed into Escherichia coli DH5α and Pseudomonas aeruginosa PAO1. The antimicrobial susceptibility test of the cloning strains showed high levels of resistance to β-lactams while they remained susceptible to aztreonam. Enzymatic tests revealed that both, VIM-84 and VIM-85, exhibited higher activity in hydrolyzing β-lactams compared to VIM-24. A D117N mutation found in VIM-24 affected binding to the antibiotics. IMPORTANCE The metallo-β-lactamases-producing Pseudomonas aeruginosa strains play an important role in hospital outbreaks and the VIM-type enzyme is the most prevalent in European countries. Two novel VIM-type enzymes in our study, VIM-84 and VIM-85, have higher levels of resistance to β-lactams and greater hydrolytic activities for most β-lactams compared with VIM-24. Both bla VIM-84 and bla VIM-85 are harbored into class 1 integrons embedded into the Tn1403 transposon. Notably, the genes bla VIM-85 are carried by three different IncP-2-type megaplasmids which are distributed locally and appear responsible for the spread of antimicrobial resistance genes in hospital settings.

identified so far (4).Routine amino acid point substitutions are most common in novel variants resulting in distinct functional differences (5).The bla VIM s are commonly embedded in genetic cassettes, often part of a variety of class 1 integrons that are inserted into transposons, mediating widespread dissemination of the genes (6).The genes encoding MBLs are also often found in transferable plasmids which have a narrow host range and are classified by a separate incompatibility typing system.These plasmids are generally categorized as IncP-2 group and have a size of >350 kb, thus aptly called megaplasmids (7).
In this study, we report the identification and characterization of two new variants of VIM-24, VIM-84 and VIM-85, which were identified in P. aeruginosa strains isolated during a multicenter surveillance study in China.Notably, the resistance gene bla VIM-85 was detected in megaplasmids from three clinical CRPA strains.

Bacterial strain collection and identification
Five clinical CRPA isolates named WTJH2, WTJH6, WTJH32, WTJH36, and WTJH43 were recovered from bronchoalveolar lavage samples of patients hospitalized in the vascular surgery department of Wuhan Tongji Hospital (WTJH) in 2018.The strains were cultured on the Pseudomonas Isolation Agar plates (Peptone 20 g/L, MgCl 21.4 g/L, K 2 SO 4 10 g/L, Triclosan 25 mg/L, Agar 13.6 g/L, and Glycerol 20 mL/L) and further confirmed by the matrix-assisted laser desorption ionization-time of flight mass spectrometry (Bruker Daltonik GmbH, Bremen, Germany) and 16S rRNA gene sequencing.The variants of the bla VIM genes were identified by the long-read sequencing (see below) and reconfirmed by PCR amplification.

Cloning
The bla VIM-84 gene with its upstream predicted promoter identified by Softberry (http://www.softberry.com) was amplified from the clinical isolate WTJH2.Since the region upstream of bla VIM-85 was not identical to that of bla VIM-84 , we obtained the entire sequence of bla VIM-85 through site-directed mutagenesis to evaluate the single Asp117Asn substitution from bla VIM-84 .The bla VIM-24 gene was also mutated from bla VIM-84 resulting in amino acid mutation at position 60 from Gln to Arg.The site-direc ted mutagenesis primers are listed in Table S1.The PCR products were recombined and cloned into pGK1900 plasmids as described previously (17), and the resulting expression vectors were subsequently introduced into Escherichia coli DH5α.Then the recombinant plasmids were isolated, and electroporation was used to introduce the episomal elements into P. aeruginosa PAO1.

Antimicrobial susceptibility determination
The minimum inhibitory concentrations (MICs) of antibiotics were detected using the broth microdilution method.Antibiotics included: ampicillin; piperacillin; cefepime; ceftazidime; piperacillin-tazobactam; ceftazidime-avibactam; imipenem; meropenem; aztreonam; amikacin; gentamicin; levofloxacin; and colistin.E. coil ATCC 25922, P. aeruginosa strain ATCC 27853 and Klebsiella pneumoniae ATCC 700603 were used as the quality control.The diluted culture was incubated at 37°C overnight.The results were interpreted according to the 2022 Clinical and Laboratory Standards Institute guidelines (18).

Conjugation and conjugation frequency
Conjugation experiments were performed with clinical isolates as the donors and a rifampin-resistant derivative of P. aeruginosa PAO1 as the recipient.Transconjugants were selected with rifampicin (300 µg/mL) and meropenem (4 µg/mL).The colonies of donor and recipient bacteria were cultured in 2 mL Luria Bertani (LB) medium and shaken at 37°C for 4 h.The donor and recipient strains in LB were mixed at a 1:1 ratio (100 µL, respectively), and 20 µL of the mixture was pipetted onto a sterile and 0.22-μm-poresize Millipore filter on a Mueller-Hinton (MH) agar plate, and then cocultured at 37°C overnight.The bacterial lawn on the filter was harvested and resuspended in 200 µL LB broth, and plated onto the plate containing antibiotics that select for transconjugants at 37°C overnight.The growing colonies on the plate were confirmed via PCR amplification.The conjugation frequency is the number of transconjugants divided by the number of donors.

Plasmid stability
The stability of the megaplasmids pWTJH36 and pWTJH43 was determined by cultur ing the transconjugants PAO1/pWTJH36 and PAO1/pWTJH43 through five successive generations without antibiotics.Briefly, three colonies of the transconjugant were selected as biological replicates and cultured in 3 mL LB broth without antibiotics at 37°C with shaking overnight.About 3 µL of each bacterial fluid was added to 3 mL LB broth (1:1,000 dilution), and then incubated at 37°C in a shaking incubator.The above procedure was repeated until Day 5 and 10 µL of each bacterial fluid was pipetted into 90 µL sterilized 0.9% (wt/vol) NaCl as the 10 −1 dilution.The serial dilution process was repeated until a 10 −8 dilution was obtained.Each clone was diluted three times independently.About 10 µL of the 10 −1 to 10 −8 dilutions were pipetted onto MH agar plates and antibiotics-resistant plates (rifampicin 300 µg/mL and meropenem 4 µg/mL, respectively), and all plates were incubated at 37°C overnight.The number of colonyforming units on MH agar plates and antibiotics-resistant plates was calculated and 30 clones of each transconjugant on antibiotics-resistant plates were selected for PCR verification.The primers for bla VIM-85 and repA are listed in Table S1.

Plasmid fitness cost
A noncompetitive growth kinetics analysis was conducted to evaluate the fitness cost of the megaplasmids pWTJH36 and pWTJH43 in PAO1 compared to plasmid-free PAO1.Three colonies of each strain were selected as biological replicates and cultured independently in 2 mL LB broth overnight.Each bacterial broth was diluted to 1:100 in MH broth and 200 µL diluted culture was pipetted into a 100-well plate in three replicates.A Bioscreen C Analyzer (Oy Growth Curves Ab. Ltd., Finland) was used to record the optical density at 600 nm (OD 600 ) of each culture every 5 min at 37°C for 20 h.The growth rate based on the OD 600 curves was calculated using an R script and the statistical analysis was performed using GraphPad Prism v9.Ordinary one-way analysis of variance multiple comparisons were used to evaluate the differences between the means.P values of <0.05 were considered to be indicative of a statistically significant result.

Expression and purification of proteins
The open reading frames (ORFs) coding for VIM-2, VIM-36, VIM-24, VIM-84, and VIM-85 without signal peptide regions were amplified using the primers listed in Table S1.The bla VIM-2 gene was amplified from WTJH52, a clinical strain carrying bla VIM-2 isolated from this surveillance study.The bla VIM-36 gene was mutated from bla VIM-84 resulting in amino acid mutation at position 205 from Leu to Arg.The site-directed mutagenesis primer is listed in Table S1.The PCR products were then cloned into pET-28a with an N-terminal His6-Tag.The resulting recombinant plasmids were introduced into E. coli BL21 (DE3) by chemical transformation.E. coli BL21 (DE3) carrying the expression vectors were cultured in an LB medium containing 50 mg/L kanamycin until the OD 600 reached 0.6-0.8 and the expression of the cells was induced with 1 mM IPTG overnight at 18°C with shaking, in the presence of 20 mM ZnCl 2 .Cells were harvested and resuspended in 50 mM Tris-HCl (pH 7.5) containing 200 mM NaCl and 50 µM ZnCl 2 and then homogen ized by a continuous flow homogenizer (JNBIO, China).The supernatant was purified using a Nickel-affinity-column and eluted in a linear imidazole gradient (20-300 mM).To remove impurities, the protein was further purified and the purity assessed as described previously (17).

Structural modeling and molecular docking
The inhibitor bounded crystal structure of the VIM-2 metallo-β-lactamase (PDB entry: 2YZ3) was used as the structure template to build the homology model of VIM-24, VIM-84, and VIM-85.Protein Preparation Wizard (20) in Schrödinger Release 2018-1 was used to preprocess and refine the protein structure.OPLS3e force field was used for restrained energy minimization (21).The proteins were further assessed to ensure no steric hindrance issues or other deviations.After that, the centroid of the inhibitor in the enzyme structure was set as the center of grid box, and then prepared ligands were docked there.

Drug-resistance profiles and genomic characterization of the bacterial strains
Antimicrobial susceptibility tests showed that all five clinical isolates, WTJH2, WTJH6, WTJH32, WTJH36, and WTJH43, were resistant to all tested β-lactams as well as gen tamicin, but susceptible to aztreonam, amikacin, levofloxacin, and colistin (Table S2).According to the whole genome sequence analysis of the five isolates, several acquired antibiotic resistance genes were identified.All strains also contained one plasmid each, which contained novel variants of the bla VIM genes, namely bla VIM-84 and bla VIM-85 (Table S2).
The plasmids pWTJH2 and pWTJH32, which contained the bla VIM-84 genes, had a length of ~100 kb and were found to be closely related to each other with 100% nucleotide similarity across the entire sequence.The genetic environment of the bla VIM-84 genes was identical and genes encoding an efflux pump (MexCD-OprJ) were detected in both plasmids.A pairwise comparison of pWTJH2 and pWTJH32 indicated a horizontal flip mediated by transposon Tn5563a, a transposon encoding putative mercuric ion transport proteins (22).The flipped genetic element in pWTJH32 was flanked by two copies of Tn5563a transposons, while pWTJH2 lost one of them (Fig. S1).
The plasmids pWTJH6, pWTJH36, and pWTJH43, the carriers of the bla VIM-85 genes were all much larger with a length of over 420 kb.Due to their size, the episomal elements could be considered megaplasmids.According to BLASTn searches, the complete sequences of pWTJH36 and pWTJH43 were highly similar with 100% coverage and 99.99% nucleotide identity.Similarly, pWTJH6 and pWTJH43 shared 98% query coverage with 99.98% nucleotide identity.Homology searches targeting the complete sequence of pWTJH43 identified 17 megaplasmids, deposited in the NCBI database, ranging in size from 394 to 513 kb with >80% coverage (Table S3).

Genomic features of the pWTJH megaplasmids
A comparative analysis of 17 complete plasmid sequences from the NCBI Genbank and the three pWTJH megaplasmids from our study revealed highly similar genetic structures in the backbone and key elements (Fig. 1).The replication initiator RepA proteins encoded by the three pWTJH megaplasmids were identical to the one encoded in plasmid pOZ176 (located between 109,483 and 120,876 nt), which had been identified to belong to the IncP-2 group using incompatibility testing (23).This indicated that the pWTJH megaplasmids might group in the same IncP-2 incompatibility group.However, a large fragment of the repA-oriV-parAB region of pOZ176 was absent in the pWTJH megaplasmids, as with the IncP-2 plasmid sublineage associated with dissemination of bla IMP-45 (24).Similarly, the genes coding for the putative partitioning proteins ParA and ParB were also identical in these 20 IncP-2 plasmids.Tellurite resistance genes (terABC DEZ) were also identified to be part of the core backbone, shared among the plasmids.Virulence factors were also detected; these included pilB, pilG, pilT, and pilZ genes which encoded a type IV pilus (T4aP) modulating twitching motility and a chemotaxis operon (cheBARZWY) required for pilus assembly (25).The conjugative transfer operon traG gene was found and the traI gene, which might serve as a relaxase, was involved in initiating DNA transfer (26).
The diverse regions of these 20 plasmids were annotated as variable regions 1-3 (VR1-3) and resistance regions 1 and 2 (RR1 and RR2).A BLASTn search of VR1 showed 100% query coverage to eight chromosomes and two plasmids isolated from P. aerugi nosa and the annotation of this unique region suggested that it did not contribute to resistance.VR2 of three pWTJH megaplasmids was almost absent from any of the other plasmids and the homology searches in GenBank showed that no highly related nucleotide sequences were found as coverage was <80%.Conversely, the VR3 flanked by ISPpu29 elements existed in most of the megaplasmids but pWTJH6.The resistance regions RR1 and RR2 were rich in transposases and integrases in proximity to various antimicrobial resistance (AMR) genes.Genes for mercury resistance operons(merRTPCA) were identified in pWTJH megaplasmids in RR2, which are commonly present on plasmids as part of transposons and exhibit a variety of arrangements (27).The βlactamase genes in these plasmids were harbored in RR1.The bla AFM s were associated with the ISCR29 elements which were responsible for dissemination.In addition, the core bla KPC-2 genetic platform ISKpn27-bla KPC-2 -ISKpn6 was associated with the mobile genetic elements Tn1403-like-ΔTn6296, while bla IMP-45 and the novel variant of bla VIM in our study were cassette-borne in class 1 integron (Fig. S2; Fig. 2).
To further understand the relationships between the 20 megaplasmids, we construc ted a phylogenetic tree based on the nucleotide composition of core genes (n = 370) (Fig. 3).The 17 plasmids from Genbank were all carried by strains isolated from China except the plasmid unnamed one from PABCH09 (Genbank accession no.CP056096) which was obtained from the USA, suggesting that the members of IncP-2 megaplasmids were distributed geographically.The megaplasmids could be found in strains from two major groups.Plasmids pWTJH17 and pWTJH12-KPC, which were also isolated from WTJH, clustered close to the pWTJH megaplasmids in our study in the same clade.These observations suggested that nosocomial transmission of IncP-2 megaplas mids might have occurred in WTJH.The plasmid pOZ176 was closely related to the pWTJH megaplasmids.pOZ176 was also harbored by an MDR strain from China, sharing over 70% nucleotide coverages and over 99.8% identities to the pWTJH megaplasmids.The plasmid pOZ176 carried two integrons harboring bla IMP-9 and bla OXA-10 , respec tively, acting as vehicles for the spread of AMR genes regionally as the pWTJH megaplas mids did.Most of the plasmids were hosted by P. aeruginosa isolates while pSY153-MDR (Genbank accession no.KY883660) was obtained from a P. putida isolate, indicating transmission between species had occurred locally.Despite different species, pSY153-MDR carrying bla IMP-45 was closely related to the other plasmids associated with the bla IMP-45 genes.Similarly, a cluster of the megaplasmids harboring bla KPC s was observed, supporting the idea that the carrier megaplasmids acted as vehicles for the dissemina tion of AMR genes (Fig. 3).

Conjugation, stability, and fitness cost of the pWTJH megaplasmids
To examine the transferability of pWTJH megaplasmids, all the bla VIM85 -carrying pWTJH megaplasmids were performed conjugation with rifampin-resistant derivative of P. aeruginosa PAO1 as recipients.Transconjugants were obtained from WTJH36-PAO1 and WTJH43-PAO1 conjugation experiments with high efficiency (10 −2 to 10 −3 transconju gants per donor) but transfer of pWTJH6 from WTJH6 to PAO1 failed in more than three independent conjugation experiments.The stability of pWTJH36 and pWTJH43 in transconjugants was tested after continuous passage culture in non-selective media.We assessed the maintenance of resistance by calculating the ratio of the number of colonies on antibiotics-resistant plates to MH agar plates.No loss of resistance was detected, and plasmid maintenance was confirmed using PCR by sequencing of bla VIM-85 and repA.To determine the fitness cost caused by the reception of megaplasmid, we analyzed the growth curves and found that both pWTJH36-bearing and pWTJH43bearing PAO1 strains demonstrated impaired growth compared with plasmid-free PAO1, but the growth rate between megaplasmid-bearing PAO1 strains showed no difference (Fig. S3).

Genetic characteristics and environment of the bla VIM variants
We reconfirmed the sequences of bla VIM-84 and bla VIM-85 by PCR amplification.Sequenc ing revealed that both bla VIM-84 and bla VIM-85 had a length of 801 bp, and they were highly similar to bla VIM-24 (GenBank accession no.HM855205) with a 99.88% and 99.75% nucleotide identity, respectively, according to BLASTn search.Both of them differed from bla VIM-24 by a nucleotide change (A179G), while bla VIM-85 obtained a further substitution at position 349 from G to A, resulting in one amino acid mutation (Gln60Arg) in VIM-84 and two amino acids replacements (Gln60Arg, Asp117Asn) in VIM-85 compared with VIM-24, respectively.
As expected, both bla VIM-84 and bla VIM-85 genes were embedded into class 1 integrons (Fig. 2).The bla VIM-84 gene was the second gene cassette of a class 1 integron with a fosE gene inserted upstream.The downstream cassettes were aac (6′)-lb4, bla OXA-101 , and ant1 genes successively.This integron was derived from In2021 carrying bla VIM-24 but the qacEΔ1 and sul1 genes in the 3′ conserved segment (3′-CS) were deleted for the insertion of an IS26 element.The genetic environment surrounding bla VIM-85 was intI-bla VIM-85 -aac (6′)-lb4-bla OXA-101 -ant1-qacEΔ1-sul1 and the 3′-CS was inserted by a gene encoding GNAT family N-acetyltransferase which was clipped by the insertion of the transposon Tn5393.The integron carrying bla VIM-85 was embedded in the transposon Tn1403 (GenBank accession no.AF313472.2) which was made up of the backbone containing genes tnpA, tnpR, and orfABCD, a class 1 integron, and Tn5393, the tnpA gene of which was disrupted by the insertion of the IS66 family (28).

Antimicrobial susceptibility of bacteria expressing bla VIM-84 , bla VIM-85 , and bla VIM-24
We first tested the antimicrobial susceptibility of the E. coli strains that we used for cloning.Even when either bla VIM-84 , bla VIM-85 , or bla VIM-24 were expressed in E. coli, the strain DH5α remained susceptible to aztreonam (Table 1).However, the MIC values of piperacillin, ceftazidime, imipenem, and meropenem in the presence of VIM-85 were four-to eightfold higher than VIM-24, while the MICs of cefepime were twofold lower.MICs of VIM-84 and VIM-85 expressions showed that VIM-85 conferred resistance equally well or rendered the strains more resistant to all antibiotics with the exception of aztreonam and ampicillin.
Next, we investigated the effects of the expression of bla VIM in the original host P. aeruginosa, by introducing the plasmids into PAO1.We found that the expression of the  bla VIM proteins in PAO1 resulted in significantly higher resistance levels to almost all antibiotics with the exception of aztreonam.The MICs of β-lactams, especially cefta zidime, piperacillin-tazobactam, and imipenem, were fourfold higher for VIM-84 and VIM-85 compared to VIM-24 when expressed in PAO1.However, the MICs of β-lactams, except piperacillin, were not higher for VIM-85 compared to VIM-84.

Enzymatic activities
The expression of the VIM proteins in the bacteria allowed their survival due to the enzymatic inactivation of the compounds.To demonstrate the activity of the enzymes in vitro, we compared the hydrolytic activity of these two new VIM variants to other variants.Besides VIM-24, two additional VIM variants, VIM-2 and VIM-36, were included, since both VIM-84 and VIM-85 in our report belong to the VIM-2-like subgroup (Fig. S4), and VIM-36 differs from VIM-84 and VIM-85 by only one amino acid change (Leu205Arg) and two amino acids mutations (Leu205Arg and Asn117Asp).All of them were capable of hydrolyzing carbapenem antibiotics, with the highest activity toward imipenem, as demonstrated by the highest k cat /K m values, compared to the other compounds (Table 2).This is consistent with the previous finding that imipenem is a suitable substrate for VIM-type enzymes (5).For both imipenem and meropenem, the enzymatic activities of VIM-84 and VIM-85 were slightly higher compared to VIM-24, VIM-2, and VIM-36.K m and k cat values for VIM-2 and VIM-36 with piperacillin and cefepime could not be measured because the calculated K m values were much higher than the measured substrate concentrations.However, VIM-84 and VIM-85 hydrolyzed piperacillin at slightly higher levels than VIM-24, while hydrolytic efficiencies against cefepime were compara ble among all three enzymes.A comparison of the kinetic parameters of VIM-85 with those of VIM-84 demonstrated that the former enzyme seemed not to be a more efficient β-lactamase for all substrates tested.

Structural modeling of VIM-24, VIM-84, and VIM-85
Having investigated the impact of the enzymes on the cells to inactivate the antibiotics, and after measuring the enzymatic activity of the purified proteins in vitro, we set out to understand the differences between the proteins on the structural level.We therefore obtained homology models of VIM-24, VIM-84, and VIM-85 (Fig. 4).Compared to VIM-24, VIM-84 contained a mutation in position 60 from Gln to Arg.One further amino acid replacement (Asp117Asn) was found in VIM-85 compared with VIM-84.Through in silico simulation, we found that the Q60R mutation was positioned in a hairpin loop linking β2 and β3 sheets.The D117N was located at the R2 loop, while the R205L mutation compared to VIM-2 was located at the Ω loop.According to the modeling result, H116, H240, and Y67 were the key residues that interacted with lactams or inhibitors.The Arg in the Q60R mutation might provide a strong positive charged environment, facilitating the acylation of the cephalosporins by providing a proton thus increasing the hydrolysis capability of the enzyme toward the lactam.The mutations D117N and R205L were positioned outside the binding site of the pocket of the protein.
An asparagine in position 117 would provide an additional amide group that resulted in additional hydrogen bonding possibilities.The formation of such bonds was observed in the docking complexes of the protein with meropenem and ceftazidime.As shown in Fig. 4B and C, the additional hydrogen bond between the carbonyl group of D117 and meropenem (2.21 Å) was longer than that of D117 and ceftazidime (2.86 Å).When R205L took place, the residue became smaller and electrically neutral, which reduced the distance but also charged restraints of the ligands to accommodate entry and binding to the substrate pocket of the enzyme.Similar observations have been reported before (29).

DISCUSSION
Several clinical P. aeruginosa isolates in our study exhibited an extensive drug-resistant phenotype (30).While they remained susceptible to amikacin, resistance to gentamicin was observed, reflecting the existence of resistance mechanisms capable of counteract ing the antibacterial activity of gentamicin without affecting amikacin effectiveness.Plasmid-encoded efflux pumps (MexCD-OprJ) are chromosomal resistance-nodulationdivision (RND) family pumps mediating multidrug resistance in Pseudomonas spp.However, the gene clusters encoding MexCD-OprJ were observed to be encoded on plasmids in our study in pWTJH2 and pWTJH32, while two other groups reported a similar observation on unrelated plasmids in P. putida and P. aeruginosa (31,32).These and our studies show that the plasmid-mediated transmission of these chromosomally located genes encoding RND-type efflux pumps has occurred and these resistance regions can be assembled dynamically in various plasmid backgrounds (7).
With the availability of long-read sequencing, an increasing number of related IncP-2type megaplasmids carrying multiple cassette-borne carbapenemase genes have been investigated (7).The IncP-2 megaplasmids share a common core genetic backbone but are diverse with regard to their AMR gene profiles.Expanding gene content in conserved regions, such as genes involved in replication, segregation, and conjugation, improves the efficiency of vertical and horizontal transmission of megaplasmids (33).The beneficial traits in resistance regions provide adaptive advantages for their host and the associated mobile genetic elements play a role in the acquisition and dissemination of resistance genes.In general, the plasmids incorporate resistance genes via mobile elements and then disseminate within and beyond bacterial species (33).Our findings show that the characteristics of mobile elements highly correspond with the β-lactamase genes they transfer and the megaplasmids harboring the same resistance genes cluster together despite being found in different species.We found that the closely related megaplasmids of pWTJH43 occur mostly in clinical strains in China, and the plasmids from the same clinical setting cluster closely in the same group.Consistent with the phylogenetic analysis, stability and conjugation demonstrated high stability and highefficiency transfer frequency of pWTJH36 and pWTJH43 megaplasmids, indicating that these megaplasmids could be locally distributed and responsible for the nosocomial transmission of AMR genes.However, the transfer of pWTJH6 failed since plasmid properties may vary across host genetic backgrounds (34).Although noncompetitive growth experiments showed the fitness cost of megaplasmid-bearing strains, plasmids could potentially survive at the population level by horizontal transmission if rates of conjugation were sufficiently high to partially offset the loss brought by the fitness costs (34).
A comparison of the MICs of PAO1 expressing bla VIM-24 and bla VIM-84 shows that bla VIM-84 confers similar or even higher resistance to all antibiotics except aztreonam, indicating VIM-84 is stronger or equally efficient in mediating resistance compared to VIM-24.In contrast, the MICs to most β-lactams, except piperacillin, are not higher for VIM-85 than those for VIM-84 expressed in PAO1, despite one more amino acid replacement (Asp117Asn) in VIM-85.All five recombinant VIM-type enzymes hydro lyzed all carbapenem antibiotics tested, but VIM-84 and VIM-85 show slightly greater enzymatic activities.D117N leads to additional hydrogen bonds but creates a weaker network than that of the R205L mutation due to the distance from the binding site within the pocket.
In conclusion, we detected two novel VIM-type MBLs, VIM-84 and VIM-85 which differed from VIM-24 by one amino acid mutation (Gln60Arg) and two amino acids replacements (Gln60Arg and Asp117Asn), respectively.Compared with VIM-24 expres sion, the MICs of bla VIM-84 and bla VIM-85 cloning strains show higher levels of resistance to β-lactams and the enzymatic activities of VIM-84 and VIM-85 demonstrate greater hydrolytic activities for most β-lactams.The IncP-2-type megaplasmids are distributed locally and appear to play an important role in the spread of AMR genes in the hospital setting.

FIG 1
FIG 1 Comparison of the three pWTJH megaplasmids (pWTJH6, pWTIH36, and pWTJH43) and 17 highly related megaplasmids in Genbank.The plasmid pWTJH43 is used as a reference.The first and second circles illustrate the GC content and GC skew.The plasmids are represented by various colorful circles from the inner to the outer and the annotation of pWTJH43 is on the outmost circle.

FIG 2
FIG 2The pairwise comparisons of the genetic environment of the bla VIM .Shaded regions denote nucleotide identity (96-100%).Red arrows denote the genes bla VIM and the other antibiotic resistance genes are denoted by dark blue arrows.Yellow, green and light blue arrows denote structures of mobile elements.

FIG 3
FIG 3The phylogenetic tree based on the concatenated set of core genes (n = 370) displays the relationships of the three pWTJH megaplasmids and their 17 best hits in GenBank.The primary features of their hosts are indicated in various colors and more detailed information of the plasmids identified in GenBank is in

FIG 4
FIG 4 Binding mode analysis of phenylC3SH, meropenem, and ceftazidime bound to VIM.Panels (A-C) show the ribbon representation of ligand bound VIM-85 with nearby interaction residues, the carbon of the inhibitor is green while others are gray.The residues mutated from VIM24 and their corresponding position in the ribbon are colored red (VIM-85) and cyan (VIM-2).Zinc ions are colored gold.Panel (D) shows the superposed binding surface of VIM-85 (colored in residue charges, red for negative, and blue for positive) and VIM-24 (colored in green).