A Plasmid Carrying blaIMP-56 in Pseudomonas aeruginosa Belonging to a Novel Resistance Plasmid Family

blaIMP and blaVIM are the most detected plasmid-encoded carbapenemase genes in Pseudomonas aeruginosa. Previous studies have reported plasmid sequences carrying blaIMP variants, except blaIMP-56. In this study, we aimed to characterize a plasmid carrying blaIMP-56 in a P. aeruginosa strain isolated from a Mexican hospital. The whole genome of P. aeruginosa strain PE52 was sequenced using Illumina Miseq 2 × 150 bp, with 5 million paired-end reads. We characterized a 27 kb plasmid (pPE52IMP) that carried blaIMP-56. The phylogenetic analysis of RepA in pPE52IMP and 33 P. aeruginosa plasmids carrying resistance genes reported in the GenBank revealed that pPE52IMP and four plasmids (pMATVIM-7, unnamed (FDAARGOS_570), pD5170990, and pMRVIM0713) were in the same clade. These closely related plasmids belonged to the MOBP11 subfamily and had similar backbones. Another plasmid (p4130-KPC) had a similar backbone to pPE52IMP; however, its RepA was truncated. In these plasmids, the resistance genes blaKPC-2, blaVIM variants, aac(6′)-Ib4, blaOXA variants, and blaIMP-56 were inserted between phd and resolvase genes. This study describes a new family of plasmids carrying resistance genes, with a similar backbone, the same RepA, and belonging to the MOBP11 subfamily in P. aeruginosa. In addition, our characterized plasmid harboring blaIMP-56 (pPE52IMP) belongs to this family.


Introduction
Pseudomonas aeruginosa is an opportunistic pathogen causing nosocomial infections such as ventilator-associated pneumonia, urinary tract infections, blood-associated infections, and skin and soft tissue infections [1][2][3]. Infections with this microorganism are challenging to treat due to its natural resistance and the accelerated emergence of strains resistant to almost all antibiotics, including carbapenems (last-resort treatments) [1]. Therefore, the World Health Organization in 2017 included P. aeruginosa in the critical-level priority pathogens group, along with Acinetobacter baumannii and carbapenem-resistant Enterobacteriaceae [4].

Plasmid Characterization
The MOBScan web application [38] was used to identify relaxases and classify the plasmids into any of the nine MOB families. For in silico classification by the replicon method, we used PlasmidFinder [39].
To determine the phylogenetic relationship of RepA protein in pPE52IMP and plasmids from P. aeruginosa, we analyzed the RepA of plasmids carrying resistance genes and constructed a phylogenetic tree. A total of 164 nucleotide sequences of complete and partial plasmids from the GenBank database were obtained (until October 2021). The plasmid sequences were annotated with Rapid Annotations using Subsystem Technology (RAST) [35] and ResFinder version 4.1 [40] for the detection of antibiotic resistance genes in all plasmids. It is essential to point out that plasmids that did not carry resistance genes were not included. RepA amino acid sequences of the plasmids carrying resistance genes were searched in the annotations using the keywords "replicase", "repA", and "helixturn-helix domain-containing protein", and their replicase A domains were corroborated with Pfam [41]. In addition, RepA with premature stop codons or ORF changes were discarded. Finally, 33 RepA proteins of plasmids (Table S1) (including pPE52IMP) were used to construct the phylogenetic tree. The Molecular Evolutionary Genetics Analysis tool, MEGA version 11.0.10 [42], was used to infer RepA proteins' phylogeny using the UPGMA method (the parameters used were amino acid substitution type, no. of differences method, and 100 bootstrap replicates).

Comparative Analysis of Plasmids Obtained from GenBank and pPE52IMP
For the comparative analysis of plasmids, we selected the complete sequences of the plasmids that shared 100% identity with repA of pPE52IMP and were in the same clade in the phylogenetic tree. To align and compare the sequences, we used MAUVE version 20150226 [43] and CLC Sequence Viewer version 8.0 (CLC bio A/S, Aarhus N, Denmark). To represent the comparison of plasmids, EASYFIG 2.2.5 was used [44].

Structural Features of the pPE52IMP Plasmid
Whole-genome sequencing revealed the presence of a single plasmid, pPE52IMP, that carries the bla IMP-56 variant (GenBank accession no. CP102481.1). The pPE52IMP plasmid had a size of 27,635 bp, 39 open reading frames (ORFs), and guanine-cytosine (G+C) content of 62.2%. Moreover, 32 of the 39 open reading frames had a predicted function: 1 of replication, 6 of stability, 7 of transfer, 13 of adaptation, and 5 transposon-related genes. We could not determine the functional domain of seven hypothetical proteins (Figure 1). and consisted of 113 bp. The stability module involves the partitioning genes parA and parC; however, the parB gene was not found. In addition, the toxin-antitoxin genes phd/doc and krfA gene were identified. The repA was part of the replication module, and no iterons or replication origins close to the repA gene were found ( Figure 1).
The adaptation module contained a class 1 integron carrying blaIMP-56, aadA1, and blaOXA-2 genes. In addition, the Tn3 family transposon carrying a mercury resistance operon (merR, merT, merP, merA, merD, and merE genes) was located ( Figure 1). Figure 1. Structure of the pPE52IMP plasmid of P. aeruginosa strain PE52. Plasmid modules are represented with different colors. Blue: adaptation; yellow: replication; orange: mobilization; purple: stability; green: transposons; gray: hypothetical proteins. GC content, GC skew+ and GC skew-are represented in colors black, purple and green, respectively on the inner map.

Phylogenetic Analysis of RepA
To infer a possible phylogenetic relationship between pPE52IMP and other plasmids from P. aeruginosa, we used RepA to build a phylogenetic tree. For analysis, we included the amino acid sequence of 33 RepA from plasmids carrying antibiotic resistance genes (Table S1) (including RepA of pPE52IMP). The analysis showed a wide diversity of replicases among P. aeruginosa plasmids grouped in 11 clades ( Figure 2). Furthermore, RepA proteins of plasmids with the same incompatibility group were clustered in the same clade such as IncP-2 (pOZ176, pJB37, pPUV-1), IncP-6 (C79, p10265-KPC, pCOL-1), and IncP-7 (p1160-VIM and pNK546b); however, the incompatibility group of one plasmid within the IncP-7 clade (unnnamed1 P8W) was not reported (Figure 2). On the other hand, it is important to note that RepA proteins from pPE52IMP, pMATVIM-7, unnamed1 (FDAARGOS_570), pD5170990, and pMRVIM0713 were in the same clade ( Figure 2). The transfer module consisted of the genes traJ, traK, trbL, trbK, trbJ, virB4, and a relaxase traI belonging to the MOB P11 subfamily. The oriT was located upstream of traK and consisted of 113 bp. The stability module involves the partitioning genes parA and parC; however, the parB gene was not found. In addition, the toxin-antitoxin genes phd/doc and krfA gene were identified. The repA was part of the replication module, and no iterons or replication origins close to the repA gene were found ( Figure 1).

Phylogenetic Analysis of RepA
To infer a possible phylogenetic relationship between pPE52IMP and other plasmids from P. aeruginosa, we used RepA to build a phylogenetic tree. For analysis, we included the amino acid sequence of 33 RepA from plasmids carrying antibiotic resistance genes (Table S1) (including RepA of pPE52IMP). The analysis showed a wide diversity of replicases among P. aeruginosa plasmids grouped in 11 clades ( Figure 2). Furthermore, RepA proteins of plasmids with the same incompatibility group were clustered in the same clade such as IncP-2 (pOZ176, pJB37, pPUV-1), IncP-6 (C79, p10265-KPC, pCOL-1), and IncP-7 (p1160-VIM and pNK546b); however, the incompatibility group of one plasmid within the IncP-7 clade (unnnamed1 P8W) was not reported ( Figure 2). On the other hand, it is important to note that RepA proteins from pPE52IMP, pMATVIM-7, unnamed1 (FDAARGOS_570), pD5170990, and pMRVIM0713 were in the same clade ( Figure 2).

Comparative Analysis of pPE52IMP and Plasmids with Same RepA and Similar Structure
pPE52IMP structure was compared with closed plasmids from the GenBank, which clustered in the same clade of the RepA phylogenetic tree ( Figure 2): pMATVIM-7 (GenBank accession no. AM778842.1), plasmid unnamed (GenBank accession no. CP033834.1), pD5170990 (GenBank accession no. KX169264.1), and pMRVIM0713 (GenBank accession no. KP975076.1). In addition, we found the plasmid p4130-KPC  was not included in the phylogenetic analysis because its RepA was truncated, but it was incorporated in the comparative analysis of the structure (Figure 2). These plasmids ranged from 24 kb to approximately 58 kb, and were isolated in the USA, Brazil, and France. The characteristics of these plasmids are shown in Table S2.
The comparative analysis showed that these six plasmids shared a similar backbone, including genes for replication (repA), partition (parA, parC), and transfer (tra and virB4); however, we found some differences. traJ, traK, and kfrA genes were absent of pD517099. trbJ gene in p4130-KPC was interrupted by a transposon, the N-terminus of TraK in pMAT-VIM-7 is absent, and RepA of p4130-KPC lacks the C-terminus ( Figure 3). however, we found some differences. traJ, traK, and kfrA genes were absent of pD517099. trbJ gene in p4130-KPC was interrupted by a transposon, the N-terminus of TraK in pMAT-VIM-7 is absent, and RepA of p4130-KPC lacks the C-terminus (Figure 3). Figure 3 illustrates that the variable region was found downstream of the phd gene and upstream of the resolvase gene and consisted of genes for adaptation such as carbapenemases type blaIMP-56 (pPE52IMP) and blaVIM-6 (plasmid unnamed and pMRVIM0713) carried by a class 1 integron, blaVIM-7 (pMATVIM-7) carried by a partial class 1 integron, blaKPC-2 (pD5170990) carried by a transposon, and blaOXA-779, blaOXA-732, and blaKPC-2 brought by a class 1 integron and a transposon, respectively (p4130-KPC) ( Figure  3).
As previously mentioned, pPE52IMP was classified into the MOBP11 subfamily [28] but was not classifiable by replicon typing. In silico analysis revealed that plasmids pMATVIM-7, unnamed (FDAARGOS_570), pD5170990, pMRVIM0713, and p4130-KPC were classified into the MOBP11 subfamily but were not classifiable according to the replicon typing scheme [26,45]. These plasmids shared some characteristics, such as having a same replicase and similar backbone, and were classified as MOBP11, but were not classifiable by the incompatibility group.  Figure 3 illustrates that the variable region was found downstream of the phd gene and upstream of the resolvase gene and consisted of genes for adaptation such as carbapenemases type bla IMP-56 (pPE52IMP) and bla VIM-6 (plasmid unnamed and pMRVIM0713) carried by a class 1 integron, bla VIM-7 (pMATVIM-7) carried by a partial class 1 integron, bla KPC-2 (pD5170990) carried by a transposon, and bla OXA-779 , bla OXA-732 , and bla KPC-2 brought by a class 1 integron and a transposon, respectively (p4130-KPC) ( Figure 3).
As previously mentioned, pPE52IMP was classified into the MOB P11 subfamily [28] but was not classifiable by replicon typing. In silico analysis revealed that plasmids pMATVIM-7, unnamed (FDAARGOS_570), pD5170990, pMRVIM0713, and p4130-KPC were classified into the MOB P11 subfamily but were not classifiable according to the replicon typing scheme [26,45]. These plasmids shared some characteristics, such as having a same replicase and similar backbone, and were classified as MOB P11 , but were not classifiable by the incompatibility group.

Plasmids with Similar Backbone as pPE52IMP Present in Other Bacterial Genera
By searching GenBank using the repA gene from pPE52IMP, we found two plasmids with the same repA and similar backbones in Achromobacter ruhlandii (plasmid p138R) and Serratia marcescens (plasmid pSMC1). The sizes of the plasmids were 34 and 41.5 kb and they were isolated from Argentina and Japan, respectively (Table S3). Comparing the complete sequence of the plasmids, we determined that these two plasmids shared a conserved backbone with pPE52IMP. Furthermore, we found that almost all backbone genes shared 100% identity and coverage, except for repA of p138R, which was truncated, and traI of pSMC1, which shared 97.65% nucleotide similarity and 100% coverage. In addition, the variable region of these plasmids carried different carbapenemases (bla IMP-1 , bla CMY-8 ) and other resistance genes such as aac(6 )-Ib4 and aadA2, commonly found in enterobacteria ( Figure S1).

Discussion
The emergence of beta-lactamases with activity against carbapenems has compromised the clinical utility of this class of antibiotics [46]. In P. aeruginosa, class A and B β-lactamases with carbapenemase activity are reported, including VIM, IMP, SPM, NDM, GIM, GES, and KPC [47,48]. IMP, VIM, NDM, and GES types comprise several variants, whereas only one variant for SPM-1 and GIM-1 have been reported [49]. These enzymes are carried in plasmids, integrons, and transposons, which play an important role in their dissemination [49]. Recently, carbapenemases mobilized by mobile genetic elements in Pseudomonas aeruginosa were reviewed and it was found that bla KPC-2 , bla VIM-1 , and bla IMP-45 are carried by plasmids belonging to different incompatibility groups [7]. In addition, other carbapenemases such as bla VIM-2 , bla IMP-6 , and bla IMP-9 are carried by plasmids [18,21,50,51]. Little is known about P. aeruginosa plasmids and their role in resistance gene dissemination; therefore, characterizing plasmids will help better understand this dissemination mechanism.
In this work, we determined the structure of the plasmid pPE52IMP carrying bla IMP-56 (Figure 1), finding that it has lower G+C content (62.2%) than the P. aeruginosa chromosome (approximately 66.6%) [52]; however, it is consistent with the GC content reported in other P. aeruginosa plasmids (from 45.8% to 63.8%) [25]. A previous study revealed that the average GC content of plasmids was 10% lower than their host's chromosome, which suggests that plasmids with very different GC content could not be maintained in their host [53].
The stability module comprises a partitioning system that contributes to the segregation of the plasmid, an addiction system that ensures the killing of plasmid-free cells, and multimer resolution systems that prevent the formation of plasmid multimers [54]. The partitioning system consists of ATPase (parA), centromere-like DNA sequence (parC), and DNA-binding protein (parB) [55]; the latter is composed of a central HTH DNA binding domain flanked by a C-terminal dimer domain and an N-terminal region necessary for protein oligomerization [56]. In the case of pPE52IMP, we found only the parA and parC genes, while the parB gene was absent, and none of the hypothetical proteins present in the plasmid had domains parB-like (Figure 1).
On the other hand, the kfrA gene has been shown to act as a transcriptional autoregulator and participates in plasmid stability [57][58][59], suggesting that this gene could be involved in pPE52IMP stability; however, other studies are necessary to understand how the segregation process is carried out in this plasmid. In addition, the addiction system is composed of the Doc toxin (death on curing) and Phd antitoxin (prevents host death) (Figure 1) that belongs to type II systems, where the toxin is directly blocked by the antitoxin [60]; besides, this toxin/antitoxin system plays an important role in plasmid stability persistence, programmed cell death, and stress response [61].
Conjugative plasmids carry two sets of genes; the first allows DNA processing (DNA transfer and replication (Dtr) genes), and the second is a membrane-associated mating pair formation (Mpf) complex (a form of type 4 secretion system). In contrast, mobilizable plasmids use the Mpf of another genetic element in the same cell [62,63]. The transfer module of pPE52IMP consists of IncP-like plasmid genes traK, traJ, and traI, which are essential for relaxosome formation, and the conjugative transfer genes trbJ, trbK, and trbL are involved in the formation of the Mpf system; however, the genes traH (chaperone activity), traG (coupling protein), traA, traB, traD, and traE (not essential for conjugation) and the genes trbBCDEFGHI (necessary for the formation of the Mpf system) are absent in pPE52IMP [64], suggesting that it could be a mobilizable plasmid. Furthermore, the lack of transconjugants in the conjugation experiment reinforces this analysis (data not shown).
Mercury operons comprise mercury resistance-conferring genes (merEDAPTR) and are commonly located on transposons and integrons carried by plasmids [65]. pPE52IMP carry the mer operon located next to the tn21 and tnpR genes ( Figure 1) that are part of transposable elements of the Tn3 family [66].
On the other hand, some authors have used features of the plasmid backbone to design classification schemes such as PCR-based replicon typing (PBRT) [26] and degenerate primer MOB typing (DPMT) [28] based on plasmid replication and mobility functions, respectively [67]. Plasmids of P. aeruginosa with a similar backbone to pPE52IMP have a MOB P11 subfamily relaxase according to MOB typing [28]; this is consistent with findings reported by Lopez-García [15]. The MOB P11 subfamily belongs to the MOBP superfamily, one of the most abundant in plasmids among gammaproteobacterial (including Pseudomonas) [68].
pPE52IMP and plasmids with similar backbone could not be classified by PBRT [26], which may be related to the fact that this scheme is focused on classifying plasmids from Enterobacteriaceae but not from other bacterial families. pPE52IMP does not belong to any of the 14 incompatibility groups (IncP-1 to IncP-14) described in P. aeruginosa; this is consistent with Shintani et al., 2015 [25], who found that only 21 of 183 Pseudomonadales plasmids analyzed could be classified into the IncP group. The above reflects the need to develop a technique to classify P. aeruginosa plasmids; however, classifying plasmids using MOB typing could help in some cases.
A classification based on replicase sequence homology was designed by Bertini for Acinetobacter baumannii plasmids, identifying 19 homology groups (GRs) [27]. Rep genes that shared at least 74% of identity were in the same group. Other authors have added more groups using the same identity criteria, reporting, to date, 33 GRs [69]. Therefore, we used similar parameters to know the distribution and behavior of RepA in pPE52IMP and plasmids of Pseudomonas aeruginosa reported in the GenBank (Table S1 and Figure 2). It is important to highlight that we included only plasmids carrying resistance genes in the analysis. RepA of plasmids belonging to the same incompatibility group (IncP-2, IncP-7, IncP-6) were clustered in three clades, likely because plasmids belonging to the same incompatibility group have the same or related replication/partitioning system [70]. On the other hand, the RepA of pPE52IMP and plasmids with a similar backbone were clustered together in a separated clade, indicating that they are closely related genetically and are probably a new family of plasmids.
According to the information available in the GenBank, the strains of P. aeruginosa and the other genera that carried plasmids similar to pPE52IMP were isolated from the USA (mainly), Brazil, France, Argentina, and Japan (Tables S2 and S3), which would indicate that these plasmids are circulating in different countries and acting as vehicles for the dissemination of antibiotic resistance genes. Closely related plasmids commonly have a core called the "backbone" associated with plasmid-specific functions such as replication initiation, conjugation, and stability. In addition, the backbone can include virulence genes and antibiotic-and heavy metalresistance genes that confer adaptive advantages to the bacterium [71]. In the analysis of the phylogenetic tree, we found four plasmids of P. aeruginosa strains, and one plasmid of a strain reported in the GenBank with a backbone similar to pPE52IMP. In addition, the plasmids had a variable region with carbapenem resistance genes such as bla VIM-6 , bla VIM-7 , bla KPC-2 , and other beta-lactamase encoding genes such as bla OXA-779 , bla OXA-732 , and bla OXA-10 (Table S2 and Figure 3) carried by class 1 integrons and transposons. Our working group reported that pPE52IMP carries bla IMP-56 in a class 1 integron (GenBank accession no. KY646161) [15]; nevertheless, in this study, we report the structure of the plasmid carrying bla IMP-56 , which belongs to a new family of plasmids.
Plasmids with a conserved backbone carrying resistance genes inserted into hotspot sites have been reported, and the repA gene serves as a hotspot in some of them [72][73][74]. However, in the plasmids analyzed, the resistance genes are inserted between phd and a resolvase gene so that it could be a potential hotspot for integrating the resistance genes in these plasmids, but more studies are necessary.
We also found two plasmids with backbone similar to pPE52IMP in bacteria not closely related to P. aeruginosa, such as p138R from A. ruhlandii, and pSMC1 from S. marcescens (Table S3 and Figure S1). These plasmids carried the aadA1, aac(6 )-lb4 acetylase, bla CMY-8 , and bla IMP-1 genes. These observations could indicate that plasmids of this type could be of a broad host range [73], allowing the dissemination of resistance genes between bacteria different from P. aeruginosa. However, transformation experiments with hosts of other bacterial genera are needed to confirm the host range of this plasmid.

Conclusions
In this study, we described a new family of plasmids carrying resistance genes with the same RepA, a similar backbone, and belonging to the MOB P11 subfamily in P. aeruginosa. In addition, we characterized the first plasmid harboring bla IMP-56 (pPE52IMP), isolated from a Mexican hospital, belonging to this family. This study contributes to understanding how these plasmids encoding carbapenemases spread among bacteria.
Supplementary Materials: The following supporting information can be downloaded at: https: //www.mdpi.com/article/10.3390/microorganisms10091863/s1, Table S1: Characteristics of the plasmids included in the phylogenetic tree of the RepA proteins. Table S2: Characteristics of P. aeruginosa plasmids with similar structure to pPE52IMP. Table S3: Characteristics of plasmids from other bacterial genera with similar structure to pPE52IMP. Figure S1: Comparison of pPE52IMP with plasmids from other bacterial genera with a similar backbone. Funding: This work was supported by 100031833/VIEP2019, VIEP/CONACyT2497/16, and 100031833/VIEP2020. Data Availability Statement: P. aeruginosa PE52 strain was recovered from routine culture and informed patient consent was not required. The protocol to perform this study was approved by the Ethical Committee of Hospital Regional del ISSSTE, Puebla, under number 188-2018.