VIM-encoding IncpSTY plasmids and chromosome-borne integrative and mobilizable elements (IMEs) and integrative and conjugative elements (ICEs) in Pseudomonas

The carbapenem-resistance genes blaVIM are widely disseminated in Pseudomonas, and frequently harbored within class 1 integrons that reside within various mobile genetic elements (MGEs). However, there are few reports on detailed genetic dissection of blaVIM-carrying MGEs in Pseudomonas. This study presented the complete sequences of five blaVIM-2/-4-carrying MGEs, including two plasmids, two chromosomal integrative and mobilizable elements (IMEs), and one chromosomal integrative and conjugative element (ICE) from five different Pseudomonas isolates. The two plasmids were assigned to a novel incompatibility (Inc) group IncpSTY, which included only seven available plasmids with determined complete sequences and could be further divided into three subgroups IncpSTY-1/2/3. A detailed sequence comparison was then applied to a collection of 15 MGEs belonging to four different groups: three representative IncpSTY plasmids, two Tn6916-related IMEs, two Tn6918-related IMEs, and eight Tn6417-related ICEs and ten of these 15 MGEs were first time identified. At least 22 genes involving resistance to seven different categories of antibiotics and heavy metals were identified within these 15 MGEs, and most of these resistance genes were located within the accessory modules integrated as exogenous DNA regions into these MGEs. Especially, eleven of these 15 MGEs carried the blaVIM genes, which were located within 11 different concise class 1 integrons. These blaVIM-carrying integrons were further integrated into the above plasmids, IMEs/ICEs with intercellular mobility. These MGEs could transfer between Pseudomonas isolates, which resulted in the accumulation and spread of blaVIM among Pseudomonas and thus was helpful for the bacteria to survival from the stress of antibiotics. Data presented here provided a deeper insight into the genetic diversification and evolution of VIM-encoding MGEs in Pseudomonas.


Introduction
VIM hydrolyzes nearly all β-lactam except for aztreonam and currently consists of 76 variants (https:// www. ncbi. nlm. nih. gov/ patho gens/ refge ne) [1]. The bla VIM genes are mostly found in Pseudomonas and Enterobacteriaceae and occur frequently within class 1 integrons with distinct gene cassette arrays (GCAs). The bla VIM -carrying integrons are always presented in various mobile genetic elements (MGEs) such as plasmids, integrative and conjugative elements (ICEs), and integrative and mobilizable elements (IMEs), enhancing the mobility and dissemination of bla VIM genes [2].There are a wealth of sequenced bla VIM -carrying plasmids, which can be assigned to various incompatibility (Inc) groups such as IncA, IncC, IncN, IncP and IncL/M [3][4][5][6][7]. IMEs/ICEs are frequently located in bacterial chromosomes: ICEs encode self-conjugation modules and thus are able to transfer between cells [8], whereas IMEs needs a helper conjugative element to achieve intercellular mobility [9].
Our previous studies showed the detailed genetic characteristics of two novel carbapenemase-encoding MGEs: SIM-encoding plasmid pHN39-SIM [10] and bla VIM-4 -containing ICE Tn6413 [7]. This follow-up study presented the complete sequences of five novel bla VIM-2/-4 -carrying MGEs, including two plasmids pJ20133-VIM and p716811-VIM, and two chromosomal IMEs Tn6917 and Tn6918, and one chromosomal ICE Tn6953 from Pseudomonas isolates. These two plasmids were assigned to a novel Inc group Inc pSTY . Comprehensive sequence comparisons were then applied to three representative Inc pSTY plasmids and 12 chromosome-borne IMEs and ICEs (including the above five ones), providing a deeper understanding of the genetic diversification and evolution of VIMencoding MGEs in Pseudomonas.

Bacterial strains and identification
Five clinical isolates P. aeruginosa SE5443, P. putida 716811 and 159349, and P. monteilii J20133 and 918607 were collected from four different Chinese public hospitals (Additional file 1: Table S1). The 16S rRNA genes and the bla VIM genes were detected as described previously [7].

Genomic DNA extraction, sequencing, and sequence assembly
Bacterial genomic DNA was isolated using the Ultra-Clean Microbial Kit (Qiagen, NW, Germany), and sequenced from a sheared DNA library with average size of 15 kb (ranged from 10 to 20 kb) on a PacBio RSII sequencer (Pacific Biosciences, CA, USA), as well as a paired-end library with an average insert size of 350 bp (ranged from 150 to 600 kb) on a HiSeq sequencer (Illumina, CA, USA) [11]. The paired-end short Illumina reads were used to correct the long PacBio reads utilizing proovread [12], and then the corrected PacBio reads were assembled de novo utilizing SMARdenovo (https:// github. com/ ruanj ue/ smart denovo).

Phylogenetic analysis
Indicated nucleotide sequences were aligned using Clustal Omega 1.2.2 [21], and then maximum-likelihood phylogenetic trees were constructed from aligned sequences using MEGA X 10.1.8 [22] with a bootstrap iteration of 1000.

Conjugal transfer
Each indicated bla VIM -carrying MGE was transformed from its wild-type isolate into rifampin-resistant P. aeruginosa ATCC 27853 or PAO1, through conjugal transfer or electroporation experiments. Three milliliters of overnight cultures of each donor and recipient bacteria were mixed together, harvested, and resuspended in 80 mL of Brain Heart Infusion (BHI) broth (BD Biosciences). The mixture was spotted on a 1 cm 2 hydrophilic nylon Data presented here provided a deeper insight into the genetic diversification and evolution of VIM-encoding MGEs in Pseudomonas.

Phenotypic assays and multi-locus sequence typing (MLST)
Bacterial antimicrobial susceptibility was tested by Bio-Mérieux VITEK 2, and interpreted as per the 2020 Clinical and Laboratory Standards Institute (CLSI) guidelines [23]. Activity of Amber class A/B/D carbapenemases was determined with a modified CarbaNP test [24]. The sequence types (STs) of Pseudomonas isolates were identified according to the online Pseudomonas MLST schemes (https:// pubml st. org/ organ isms).

Identification of three STs for Pseudomonas isolates
Three different STs, namely ST129, ST17, and ST639, were identified from the five Pseudomonas isolates (Additional file 1: Table S1), whose complete genome sequences were determined in this work. P. putida 716811 and 159349 belonged to ST129 and ST17, respectively. P. aeruginosa SE5443 belonged to ST639. ST129 was a novel ST of P. putida. The two P. monteilii isolates J20133 and 918607 could not be assigned with any ST types due to lack of P. monteilii MLST scheme.

Proposal of a novel group of Inc pSTY plasmids
Two bla VIM -carrying plasmids pJ20133-VIM and p716811-VIM were identified from the complete genome sequences of the two Pseudomonas isolates J20133 and 716811 (Additional file 1: Table S1). A novel Inc pSTY group was proposed from a total of seven available fully sequenced single-replicon plasmids (Additional file 1: Table S2; last accessed August 10th 2020) that included the above two plasmids together with five additional ones from GenBank, because these seven plasmids harbored not only homologous repA (replication initiation protein) genes together with its iterons ( Fig. 1) but also similar backbone gene organizations (Fig. 2). Since the Inc group of plasmids could be divided according to the homology of repA genes [25][26][27], a phylogenetic tree ( Fig. 1) was constructed based on the repA sequences of these seven plasmids, showing that these seven plasmids  could be divided into three separately clustering subgroups Inc pSTY -1/2/3. As shown by pairwise comparison of repA sequences, plasmids within each subgroup showed ≥ 95% identity, while those from different subgroups displayed ≤ 95% but ≥ 82% identity (Additional file 1: Table S3). pSTY [28], p716811-VIM, and pHN39-SIM [10] were the first sequenced plasmids of Inc pSTY -1, Inc pSTY -2, and Inc pSTY -3, respectively, and thus were identified as the references of the corresponding Inc pSTY subgroups.
The integration of the 59.7-kb paa region into pSTY led to truncation of orf246-to-orf846 region (as originally observed in pJ20133-VIM) into a 138-bp orf846 remnant (Fig. 2). A presumed prototype paa region was terminally bracketed by two copies of Tn5563 [29]; the right copy was intact while the left copy was truncated due to insertion of Tn6481 in the 59.7-kb paa region from pSTY (Additional file 1: Fig. S1). This 59.7-kb paa region still contained a complete set of phenylacetic acid degradation genes (Additional file 1: Fig. S1). Tn6734 was an IME containing a phenol degradation gene locus (Additional file 1: Fig. S2a). Tn6603a was a Tn3-family unit transposon containing an oxidative stress defense gene osmC (Additional file 1: Fig. S2b). The 67.2-kb MDR Table 1 Major features of the three Inc pSTY plasmids analyzed The Inc pSTY -1 reference plasmid pSTY [28] from GenBank, and the Inc pSTY -1 plasmid pJ20133-VIM plus the Inc pSTY -2 reference plasmid p716811-VIM sequenced in this study were included in a detailed sequence comparison region from pJ20133-VIM contained In58, Tn5046 and ΔTn512, a 3.6-kb Tn4662a remnant, and several IS elements (Additional file 1: Fig. S3). According to the previous grouping scheme of Tn7family transposons [30], the phylogenetic tree was constructed based on the nucleotide sequences of tnsA, which encoded the endonuclease responsible for excision of Tn7-family transposons. A phylogenetic analysis of Tn6735, the 11.5-kb Tn7-family transposon remnant, Tn6922, and Tn6921, together with additional 12 representative sequenced Tn7-family transposons from Gen-Bank based on the tnsA genes, indicated that the first two belonged to a novel Tn6735 subfamily and an unnamable subfamily (it had no intact sequenced transposon), respectively (Fig. 3). Similar to Tn7 [31], both Tn6735 (carrying pyrimidine biosynthesis genes) and the 11.5-kb Tn7-family transposon remnant encoded the core transposition determinants (endonuclease TnsA, transposase TnsB, regulator TnsC, target-site selection protein TnsD, and TnsB-binding sites), and the later one still encoded an additional target-site selection protein TnsE (Additional file 1: Fig. S4).

Collection of 12 chromosome-borne IMEs/ICEs for sequence comparison
Three bla VIM -carrying chromosomal MGEs Tn6917, Tn6918 and Tn6953 were identified from the complete genome sequences of the three Pseudomonas isolates 918607, 159349, and SE5443 (Additional file 1: Table S1). A detailed sequence comparison was applied to a total of 12 chromosomal MGEs belonging to three groups: Fig. 3 Evolutionary relationships of the Tn7-family transposons. A maximum likelihood phylogenetic tree was constructed from aligned tnsA sequences. Degree of support (percentage) for each cluster of associated taxa, as determined by bootstrap analysis, is shown next to each branch. Bar corresponds to scale of sequence divergence. Squares denote Tn7-family transposons or transposon remnants sequenced in this study Tn6917 was the only bla VIM -carrying one belonging to Tn6916-related IMEs in GenBank. Tn6919 was the only one (in addition to Tn6918) belonging to Tn6918related IMEs in GenBank, and it did not carry bla VIM . The seven Tn6417-related ICEs were selected from Gen-Bank because Tn6417 was used as reference and the rest six ones Tn6413, Tn6954, Tn6955, Tn6956, Tn6957 and Tn6958 carried bla VIM genes. Similar to the above Inc pSTY plasmids, the modular structure of each IME/ICE was divided into the backbone and the accessory modules.

Comparison of two related IMEs Tn6916 and Tn6917
Tn6916 (a 51.0-kb prototype IME initially identified in P. monteilii FDAARGOS_171 [33]) and Tn6917 were integrated at the same site downstream of the P. monteilii chromosomal gene dinG (ATP-dependent DNA helicase), and they shared the core backbone markers attL (attachment site at the left end), int (integrase), and attR (attachment site at the right end); in addition, the β-lactam resistance gene ampC was considered as a backbone component of these two IMEs because no associated MGEs were identified for this resistance gene (Fig. 4). Tn6916 thereby had no accessory modules, but bla VIM-2 -carrying In528 (see below) was integrated at a site upstream of eamA (transcription regulator) and identified as the sole accessory module in Tn6917 (Fig. 4).

Comparison of two related IMEs Tn6918 and Tn6919
Tn6918 (a 70.49-kb prototype IME initially found in P. putida 159,349) and Tn6919 were integrated at a site downstream of the P. putida chromosomal gene tRNA Thr , and they shared the core backbone markers attL, int1, int2, and attR (Fig. 5). There were at least three major modular differences in the backbone of Tn6919 relative to Tn6918: i) inversion of orf1965, ii) interruption of orf549 and orf2007 due to insertion of ΔISPpu13 and ISPen2, respectively, and iii) replacement of 3'-termimal 22.8-kb orf270-to-orf213 region by 3.4kb orf255-to-orf282 region (Fig. 5). The ISPa122-mer region was integrated at the same site of the backbones of Tn6918 and Tn6919 (Fig. 5), and it was composed of ISPa122 and a mer-carrying ΔTn5041-like element (Additional file 1: Fig. S6). Tn6918 and Tn6919 acquired bla VIM-2 -carrying In1770 (see below) and strAB region, respectively, which were inserted at the same site upstream of orf555 (Fig. 5). The strAB region contained a cryptic unit transposon Tn6920, a strABcarrying unit transposon Tn6921, a 3.6-kb Tn4662a remnant, and several IS elements (Additional file 1: Fig.  S7). Tn6920 and Tn6921 had the core transposition tniABQ− res −tniR modules with the highest sequence identities to Tn5053 [34] and Tn5058a [32], respectively, and thus they belonged to the Tn5053 subfamily of Tn7 family (Fig. 3).

Comparison of eight Tn6417-related ICEs
Tn6417 was a 108.2-kb reference ICE [7] initially found in P. aeruginosa DHS01 [35]. The eight related ICEs Tn6417, Tn6413, Tn6953, Tn6954 (GZAF3_GI) [36], Tn6955 (ICE6440) [3], Tn6956 (ICE6441) [3], Tn6957, and Tn6958 [37] shared the core backbone markers attL/ attR (these sequences showed somewhat differences but shared a core motif 'CCT TCG CCC GCT CCA'), int, cpl (coupling protein), rlx (relaxase), and a F (TivF)-type type IV secretion system gene set (mating pair formation). However, the backbones of these eight ICEs varied in size from 67.8 to 95.1 kb and had at least four major modular differences: (i) presence of unique orf672, orf306, piL1-to-orf381 region, and orf3336-to-orf2514 region in Tn6953; (ii) presence of two highly similar regions lysR2-to-orf1068 and orf348-to-orf1188 in Tn6417 and Tn6413, respectively; (iii) absence of smc-to-orf3006 region from Tn6955; and (iv) interruption of orf2280 owing to insertion of intron E.c.I5 in Tn6954 and Tn6955 (Fig. 6). The first six ICEs were integrated into the P. aeruginosa or A. faecalis chromosomal tRNA Gly gene, while the last two ones into the P. aeruginosa chromosomal gene mhpC (methyl ester carboxylesterase) and orf1203 (putative DNA-binding protein), respectively. Each ICE carried a single accessory module, and thus there were the resulting eight different accessory modules bla VIM-2 -carrying In1779 (see below), Tn6532, Tn6403, Tn6959, a 15.2-kb Tn6346-related region, Tn6960, Tn6961, and Tn6962 from the eight ICEs; the first one was inserted into ftsk (cell division protein), while all the other seven ones were integrated at a site upstream of orf582 and identified as the derivatives of Tn6346 (Fig. 7). Tn6346 was a prototype Tn3-family unit transposon originally identified in Achromobacter spp. AO22 [38] and manifested as a hybrid of the core transposition module tnpAR-res from Tn5051 and the mer region from Tn501 (Fig. 7). The seven Tn6346 derivatives differed from Tn6346 in two major aspects: (i) insertion of IS1071 at the same position within tnpA in all these seven Tn6346 derivatives, and furthermore insertion of ISPa91 at another position within tnpA in only Tn6960; and (ii) insertion of different concise class 1 integrons In159, In127, In1365, In56, In1879, In1853, and In58 into urf2 in these seven Tn6346 derivatives, sometimes leading to truncation of surrounding regions (Fig. 7). The bla VIM genes were found the last five integrons (see below) rather than the first two (Additional file 1: Fig. S8).

Transferability and antimicrobial susceptibility
pJ20133-VIM, p716811-VIM, and Tn6953, which were selected to represent the Inc pSTY -1 and Inc pSTY -2 plasmids and the Tn6417-related ICEs, respectively, could be transferred from the relevant wild-type isolates into ATCC 27853 or PAO1 through conjugation, generating the transconjugants PAO1/pJ20133-VIM, PAO1/p716811-VIM, and ATCC 27853/Tn6953 respectively. All these wild-type and transconjugant strains had the Ambler class B carbapenemase activity (data not shown) and were resistant to imipenem and meropenem with minimal inhibit concentration (MIC) values ≥ 16 mg/L (Additional file 1: Table S5), owing to production of VIM enzyme.

Discussion
The present work identifies a novel group of Inc pSTY plasmids, which can be further divided into the three subgroups Inc pSTY -1/2/3. A detailed genetic dissection analysis is applied to a collection of 15 Pseudomonas MGEs, including three Inc pSTY plasmids, two Tn6916-related IMEs, two Tn6918-related IMEs, and eight Tn6417-related ICEs. All these IMEs/ICEs were located within the bacterial chromosomes. At least 22 genes for resistance to seven different categories of antibiotics and heavy metals are identified within these 15 MGEs (Additional file 1: Table S6). Eleven of these 15 MGEs carry the bla VIM genes, which are all located within GCAs of concise class 1 integrons. For 10 of these integrons, the bla VIM genes were presented together with other resistance genes, especially including those for resistance to β-lactams, aminoglycosides, trimethoprim, and rifampicin. These bla VIM -carrying integrons were further integrated into the above plasmids, IMEs and ICEs. Confirmed by conjugal transfer experiments herein, Inc pSTY plasmids and Tn6417-related ICEs have the ability to transfer from one cell to another cell. These two groups MGEs could transfer autonomously by utilizing self-encoded conjugation gene sets [39,40]. However, the conjugal transfer of Tn6916-and Tn6918-related IMEs are nonautonomous due to lack of essential conjugation genes encoding Cpl and type IV secretion system, and thereby its intercellular transfer is relied on the help of other conjugative elements [40].

Conclusion
The four groups of bla VIM -carrying MGEs characterized in this study have a wide range of hosts including Pseudomonas, and they are able to transfer across different bacterial species. Integration of bla VIM genes into these MGEs contributes to the accumulation and distribution of bla VIM genes and enhances the ability of bacteria to survive under selection pressure of carbapenems especially in hospital settings. Moreover, these four groups of MGEs display a high-level diversification in modular structures, which have complex mosaic natures and carry a large number of drug resistance genes (particularly in the MDR regions in these MGEs). This study provides a deeper insight into the genetic diversification and evolution of VIM-encoding MGEs in Pseudomonas.