Genetic Characterization of a bla VIM-24-Carrying IncP-7β Plasmid p1160-VIM and a bla VIM-4-Harboring Integrative and Conjugative Element Tn6413 From Clinical Pseudomonas aeruginosa.

This study presents three novel integrons In1394, In1395, and In1443, three novel unit transposons Tn6392, Tn6393, and Tn6403, one novel conjugative element (ICE) Tn6413, and the first sequenced IncP-7 resistance plasmid p1160-VIM from clinical Pseudomonas aeruginosa. Detailed sequence comparison of p1160-VIM (carrying Tn6392 and Tn6393) and Tn6413 (carrying Tn6403) with related elements were performed. Tn6392, Tn6393, and Tn6403 were generated from integration of In1394 (carrying bla VIM-24), In1395 and In1443 (carrying bla VIM-4) into prototype Tn3-family unit transposons Tn5563, Tn1403, and Tn6346, respectively. To the best of our knowledge, this is the first report of a bla VIM-24-carrying P. aeruginosa isolate.

Introduction

Plasmids of thirteen incompatibility groups in Pseudomonas (IncP-1 to IncP-7 and IncP-9 to IncP-14) have been recognized, varying in genetic structure, size and host range. IncP-7 plasmids, with a narrow host range, are of particular interest in environmental biodegradative potentials. Most sequenced members of this group, such as pCAR1 (Maeda et al., 2003), pND6_1 (Li et al., 2004), pWW53 (Pickup and Williams, 1985), pDK1 (Kunz and Chapman, 1981), and pHE24 (Supplementary Table S1), belong to toluene catabolic or degradation plasmids (D-plasmids) rather than resistance plasmids (R-plasmids).

Integrative and conjugative elements (ICEs), also known as conjugative transposons, are typically found integrated into host bacterial chromosomes and encode integrase (Int), excisionase (Xis) and type IV secretion system responsible for integration, excision, interbacterial transfer, respectively. ICEs confer antibiotic resistance (such as Tn916) (Franke and Clewell, 1981), heavy metal resistance (such as R391) (Peters et al., 1991), and carbon utilization (such as ICEclc) (Gaillard et al., 2006).

Verona integron-encoded metallo-β-lactamase (VIM) is one of the most predominant families among class B carbapenemases and can hydrolyze nearly all β-lactams including carbapenems, except aztreonam (Queenan and Bush, 2007). This study dealt with a detailed genetic characterization of a novel blaVIM–24-carrying IncP-7β plasmid p1160-VIM and a novel blaVIM–4-carrying ICE Tn6413 recovered from two different clinical P. aeruginosa isolates.

Materials and Methods

Bacterial Isolates

Pseudomonas aeruginosa 1160 was isolated in 2015 from a sputum specimen of an elderly patient in a teaching hospital in Hebei Province, China. P. aeruginosa 6762 was recovered in 2016 from a sputum specimen of an elderly patient in a public hospital in Lanzhou Province, China. Bacterial species was identified by 16S rRNA gene sequencing and PCR detection of P. aeruginosa-specific oafA gene (Choi et al., 2013).

Conjugal Transfer

Conjugal transfer experiments were carried out with rifampin-resistant P. aeruginosa PAO1 used as recipients and each of the blaVIM-positive 1160 or 6762 isolate as donor. Three milliliters of overnight cultures of each of donor and recipient bacteria were mixed together, harvested and resuspended in 80 μl of Brain Heart Infusion (BHI) broth (BD Biosciences). The mixture was spotted on a 1 cm2 hydrophilic nylon membrane filter with a 0.45 μm pore size (Millipore) that was placed on BHI agar (BD Biosciences) plate and then incubated for mating at 30°C for 12 to 18 h. Bacteria were washed from filter membrane and spread on Muller-Hinton (MH) agar (BD Biosciences) plates containing 1000 μg/ml rifampin together with 2 μg/ml meropenem for selecting an P. aeruginosa transconjugant carrying blaVIM.

Sequencing and Annotation

The genomic DNA of strain 6762 or the plasmid DNA of strain 1160 was isolated using an UltraClean Microbial Kit or a Large Construct Kit (Qiagen, NW, Germany), respectively, and then sequenced from a mate-pair library with average insert size of 5 kb (ranged from 2 to 10 kb) using a MiSeq sequencer (Illumina, CA, United States). DNA contigs were assembled based on their contig coverages using Newbler 2.6 (Nederbragt, 2014). Open reading frames and pseudogenes were predicted using RAST 2.0 (Brettin et al., 2015) combined with BLASTP/BLASTN (Boratyn et al., 2013) searches against the UniProtKB/Swiss-Prot database (Boutet et al., 2016) and the RefSeq database (O’Leary et al., 2016). Annotation of resistance genes, mobile elements, and other features was carried out using the online databases including CARD (Liang et al., 2017), ResFinder (Zankari et al., 2012), ISfinder (Siguier et al., 2006), INTEGRALL (Moura et al., 2009), and the Tn Number Registry (Roberts et al., 2008). Multiple and pairwise sequence comparisons were performed using MUSCLE 3.8.31 (Edgar, 2004) and BLASTN, respectively. Gene organization diagrams were drawn in Inkscape 0.48.1[1].

Phylogenetic Analysis

The nucleotide sequences of repA coding regions of indicative plasmids were aligned using MUSCLE 3.8.31 (Edgar, 2004). The unrooted neighbor-joining trees were generated from the aligned repA sequences using MEGA7 (Kumar et al., 2016), and evolutionary distances were estimated using the maximum composite likelihood method, with a bootstrap iteration of 1000.

Phenotypic Assays

Activity of Ambler class A/B/D carbapenemases in bacterial cell extracts was determined by a modified CarbaNP test (Wei et al., 2016). Bacterial antimicrobial susceptibility was tested by BioMérieux VITEK 2 and interpreted as per the 2017 Clinical and Laboratory Standards Institute (CLSI) guidelines (Wayne, 2017).

Nucleotide Sequence Accession Numbers

The sequence of p1160-VIM and that of the 6762 chromosome were submitted to GenBank under accession numbers MF144194 and CP030075, respectively.

Results and Discussion

Overview of Sequenced p1160-VIM and Tn6413

Two blaVIM-positive P. aeruginosa isolates, designated 1160 and 6762, were subjected to high-throughput genome sequencing. The 1160 isolate harbored a blaVIM−24-carrying plasmid p1160-VIM, which had a circular DNA sequence of 205.4 kb in length, with an average G+C content of 56.3%. p1160-VIM belonged to the IncP-7 group because it had a IncP-7 repA gene responsible for plasmid replication initiation.

A 114.1-kb blaVIM–4-harboring ICE Tn6413 was found to integrate into tRNA gene in the 6762 chromosome. The modular structure of each of p1160-VIM and Tn6413 was divided into the backbone (responsible for replication, maintenance and conjugal transfer) and separate accessory modules (defined as acquired DNA regions associated with mobile elements) integrated at different sites of the backbone (Supplementary Figures S1, S2 and Table 1).

Table 1

Major features of plasmids and ICEs analyzed.

Category	Plasmids			Chromosomally integrated ICEs
	pCAR1	pDK1	p1160-VIM	Tn6413	Tn6533	Tn6534	Tn6417
Accession number	AB088420	AB434906	MF144194	CP030075	AP014651	KX196168	CP013993
Group	IncP-7α	IncP-7β	IncP-7β	Tn6417	Tn6417	Tn6417	Tn6417
Reference of the relevant group	Yes	Yes					Yes
Total length (bp)	199,035	128,921	205,426	114,067	109,026	118,715	108,186
Total number of ORFs	217	117	237	157	112	104	107
Mean G+C content, %	56.3	56	56.2	60.5	61.3	61.3	61.3
Length of the backbone (bp)	115,716	76,947	135,455	84,038	84,181	83,215	85,992
Accessory modules	Tn4676, ISpa73, a Tn3-family transposon remnant, ISPre3, and ISPre4	Tn4662, Tn4663, IS1162, and ISpa81	Tn6392$, Tn6393$, ISpa75, ISpa79, ISpa80, ISpa81, ISpa83, and ISPre2	Tn6403$	Tn6531$	Tn6530$	Tn6532$
Host bacterium	P. resinovorans CA10	P. putida HS1	P. aeruginosa 1160	P. aeruginosa 6762	P. aeruginosa NCGM257	P. aeruginosa RI_IH-2	P. aeruginosa DHS01
Nucleotide positions in the chromosome	–	–	–	337873.. 451939	5233626.. 5342651	1..118715	5365108.. 5473293

p1160-VIM could be transferred from the 1160 isolate into P. aeruginosa PAO1 through conjugation, generating the transconjugant 1160-VIM-PAO1. The self-transmissible nature of p1160-VIM was consistent with the presence of complete conjugal transfer regions in this plasmid. Strains 1160 and 1160-VIM-PAO1 had class B carbapenemase activity, and they were resistant to cefuroxime, ceftazidime, ceftriaxone and cefepime (with minimal inhibitory concentration values ≥ 64), and imipenem and meropenem (with minimal inhibitory concentration values ≥ 4), which were resulted from production of VIM enzymes in these strains. Repeated conjugation attempts failed to transfer Tn6413 from the 6762 isolate to PAO1.

Subgrouping of IncP-7 Plasmids Including p1160-VIM

A group of ten completely or partially sequenced plasmids (Supplementary Table S1; including p1160-VIM) with IncP-7 repA genes (≥ 95% nucleotide identity to that of p1160-VIM), were collected, and two phylogenetic trees (Figure 1) were constructed based on repA nucleotide and amino acid sequences, respectively. These ten plasmids could be divided into two separately clustering subgroups designated IncP-7α and IncP-7β. As shown by pairwise comparison of repA nucleotide sequences, plasmids within each of these two subgroups showed ≥ 99% nucleotide identity, while plasmids from these two different subgroups displayed ≤ 96% nucleotide identity (Supplementary Table S2a). Considerable genetic diversity was found between the repA genes of IncP-7α and IncP-7β, representing two separated lineages.

FIGURE 1

Neighbor-joining phylogenetic trees of sequenced IncP-7 plasmids. Phylogenetic trees are constructed based on repA nucleotide (A) and amino acid sequences (B), respectively. Degree of support (percentage) for each cluster of associated taxa, as determined by bootstrap analysis, is shown next to each branch. Triangles indicate IncP-7α and IncP-7β reference plasmids, while squares denote p1160-VIM sequenced in this work.

Predicted iterons (RepA-binding sites) were found within the oriV region downstream of repA, and plasmids from both subgroups shared a conserved iteron motif and an identical iteron copy number (Figure 1 and Supplementary Table S1).

pCAR1 (Maeda et al., 2003) and pDK1 (Kunz and Chapman, 1981) were identified as IncP-7α and IncP-7β reference plasmids, respectively, because they were the first sequenced plasmids harboring complete conjugal transfer regions. In the phylogenetic tree based on nucleotide sequences, p1160-VIM displayed a long branch, which resulted from presence of five single nucleotide polymorphisms (SNPs) in p1160-VIM, while all other plasmids had identical repA sequences (Supplementary Figure S3). Notably, these five SNPs did not lead to mutations of RepA amino acid sequences.

Comparison of p1160-VIM With pCAR1 and pDK1

pCAR1, pDK1 and p1160-VIM were included in a genomic comparison. These three plasmids had > 92% nucleotide identity across > 52% of their backbone sequences (Supplementary Table S2b), and their conserved backbone was composed of gene or gene loci responsible for replication initiation (repA), partitioning (parABCW), and conjugal transfer (rlx, cpl, tivF3, and tivF6). There were three major modular differences within their backbones (Figure 2): (i) a terABC region could be found in p1160-VIM and pCAR1 rather than pDK1; (ii) a 23.9-kb orf324–to–orf891 region was found in only p1160-VIM; and (iii) the endA gene was intact in pCAR1 but was interrupted or truncated in p1160-VIM and pDK1. All these modular differences were resulted from integration of relevant accessory modules.

FIGURE 2

Linear comparison of p1160-VIM with related plasmids. A linear comparison is carried out for complete DNA sequences of pCAR1 (accession number AB088420), p1160-VIM (this study), and pDK1 (accession number AB434906). Genes are denoted by arrows. Genes, mobile elements and other features are colored based on function classification. Blue shading denotes regions of homology (> 95% nucleotide identity), and pink shading denotes regions of homology (nucleotide identity between 90 and 95%).

pCAR1, pDK1, and p1160-VIM carried totally different profiles of accessory modules (Table 1), which were composed of 10 distinct IS elements (ISPre2, ISPre3, ISPre4, IS1162, ISpa73, ISpa75, ISpa79, ISpa80, ISpa81, and ISpa83), 5 different intact Tn3-family unit transposons (Tn4676 from pCAR1, Tn4662, and Tn4663 from pDK1, and Tn6392 and Tn6393 from p1160-VIM; a typical unit transposon encodes a transposase and a site-specific recombinase or resolvase as core transposition determinants, and also carries one or several accessory genes), and one Tn3-family transposon remnant. Only Tn6392 and Tn6393 of the above accessory modules (Table 2). Tn4676 (Supplementary Figure S4a) carried core transposition genes (tnpAC and tnpST) genetically related to Tn4651 (Maeda et al., 2003), and also an ant (two-component anthranilate 1,2-dioxygenase) operon (Urata et al., 2004) interrupted by insertion of ISPre1 and a car (carbazole/dioxin degradation) operon (Nojiri et al., 2001). Tn4662 encoded a RelBE toxin-antitoxin system involved in plasmid maintenance. Tn4663 (Supplementary Figure S4b) was derived from Tn4659 (Yano et al., 2007) and harbored a toluene-catabolic xyl gene cluster (Yano et al., 2010).

Table 2

Drug resistance genes in mobile elements sequenced this study.

Mobile element	Resistance marker	Resistance phenotype	Nucleotide position	Region located
p1160-VIM	strAB	Aminoglycoside resistance	8845..10484	Tn6393
	sul1	Sulphonamide resistance	18181..19020 40901..41740
	qacED1	Quaternary ammonium compound resistance	19014..19361 41734..42081
	folA	Trimethoprim resistance	19489..20022
	qnrVC	Quinolone resistance	26051..26707
	mph(E)	Macrolide resistance	34491..35375
	msr(E)	Macrolide resistance	35431..36906
	aadA1a	Streptomycin resistance	43627..44418
	catB3q	Chloramphenicol resistance	46142..46774
	ereA1c	Erythromycin resistance	46884..48104
	aacA4	Aminoglycoside resistance	188445..188963 189993..190511	Tn6392
	blaVIM–24	Carbapenem resistance	189102..189902
	mer	Mercuric resistance	195914..197025
Tn6413	aadA2	Streptomycin resistance	398558..399337	—
	qacED1	Quaternary ammonium compound resistance	399501..399848 411418..411765
	sul1	Sulphonamide resistance	399842..400681 411759..412598
	msr(E)	Macrolide resistance	402951..404426
	mph(E)	Macrolide resistance	404482..405366
	blaVIM–4	Carbapenem resistance	409112..409996
	aacA7	Aminoglycoside resistance	410090..410548
	aacA4	Aminoglycoside resistance	410731..411249
	mer	Mercuric resistance	414543..418060

Comparison of Tn6392 With Tn5563

Tn6392 (Figure 3) from p1160-VIM was a novel derivative of Tn5563, which was originally characterized in P. alcaligenes and had the structure IRL (inverted repeat left)–tnpR (resolvase)–res (resolution site)–orf2 (hypothetical protein)–pliT (pilT domain-containing protein)–tnpA (transposase)–mer (mercuric resistance gene locus)–IRR (inverted repeat right), bracketed by 5-bp or 7-bp direct repeats (DRs; target site duplication signals) at both ends (Yeo et al., 1998). Tn6392 differed from Tn5563 by insertion of a novel class 1 integron In1394 into res. The prototype Tn402-associated class 1 integron was typically organized as IRi (inverted repeat at the integrase end), 5’-CS [5’-conserved segment: intI1 (integrase)-attI1 (a specific recombination site)], GCA (gene cassette array), 3’-CS [3’-conserved segment: qacED1–sul1–orf5–orf6], a Tn402 tni module [tniA (transposase)–tniB (ATP-binding protein)–tniQ (transposition auxiliary protein)–res–tniR (serine resolvase)], and IRt (inverted repeat at the tni end) (Gillings et al., 2008). In1394, bracketed by 5 bp DRs at both ends, contained all the above core integron structures except 3’-CS. The GCA of In1394 consisted of a blaVIM–24 gene and two copies of aacA4.

FIGURE 3

Organization of Tn6392 and comparison to related regions. Genes are denoted by arrows. Genes, mobile elements and other features are colored based on their functional classification. Shading denotes regions of homology (nucleotide identity > 95%). Numbers in brackets indicate nucleotide positions within corresponding plasmid sequences. Accession number of Tn5563 for reference is U88088.

Comparison of Tn6393 With Tn1403

Tn6393 (Figure 4) was a novel derivative from Tn1403 after insertion of a novel class 1 integron In1395 instead of In28 at the same position within res. Tn1403 was initially identified in P. aeruginosa and displayed a backbone structure IRL–tnpAR–res–sup–uspA–dksA–yjiK–IRR, with integration of accessory modules In28 and Tn5393c into res and dksA, respectively (Stokes et al., 2007). In1395 belonged to complex class 1 integron, which was typically organized as IRi–5’-CS–VR1 (variable region 1)–3’-CS1 (the first copy of 3’-CS1: qacED1–sul1)–ISCR1 (comment region)–VR2 (variable region 2)–3’-CS2 (a second 3’-CS: qacED1–sul1–orf5–orf6)–tni–IRt. In1395, bracketed by 5-bp DRs at both ends, was composed of IRi, 5’-CS, VR1 [GCA: gcu104–aacA1–catB3q:ISpa62– gcu161–ereA1c:ISpa62], 3’-CS1, ISCR1 (further interrupted by ΔTn4662b–ΔIScs605–msr(E)–mph(E)–Tn4662b), VR2 [containing qnr, ΔISCR1, folA and other genes], 3’-CS2, IS6100 (replacing tni) and IRt.

FIGURE 4

Organization of Tn6393 and comparison to related regions. Genes are denoted by arrows. Genes, mobile elements and other features are colored based on their functional classification. Shading denotes regions of homology (nucleotide identity > 95%). Numbers in brackets indicate nucleotide positions within corresponding plasmid sequences. Accession number of Tn1403 for reference is AF313472.

Comparison of Tn6413 With Tn6534, Tn6533, and Tn6417

Tn6413 (Supplementary Figure S2) was a novel ICE that could be divided into a single 30-kb accessory module Tn6403 (Figure 6) and the remaining backbone regions. Tn6413 belonged to a collection of 31 ICE or ICE-like sequences (Supplementary Table S3, including Tn6417, Tn6534, and Tn6533) with > 95% nucleotide identity across > 59% of Tn6413 backbone. Tn6417 was the first sequenced one and identified as the reference of these 31 Tn6417-family ICE sequences. A genomic comparison (Figure 5) was subjected to Tn6413, Tn6534, Tn6533, and Tn6417 because they shared mostly highly similar backbones with 99% nucleotide identity and > 94% query coverage. These four Tn6417-family ICEs, which genetically differed from the two existing ICE families in P. aeruginosa (Kung et al., 2010) shared conserved DNA processing and conjugation genes. Three major modular differences were fund within the backbones of these four ICEs: (i) presence of orf348 in only Tn6417; (ii) presence of orf645 in only Tn6417; and iii) 3’-terminal regions (orf432–to–orf1188, orf693–to–orf866, orf798–to–orf468, and orf693–to–orf1068 from Tn6413, Tn6534, Tn6533 and Tn6417, respectively) differed from one another.

FIGURE 6

Organization of Tn6403 and comparison with related regions. Genes are denoted by arrows. Genes, mobile elements and other features are colored based on their functional classification. Shading denotes regions of homology (nucleotide identity > 95%). Numbers in brackets indicate nucleotide positions within corresponding chromosome sequences. Accession number of Tn6346 for reference is EU696790.

FIGURE 5

Linear comparison of Tn6413 with related ICEs. A linear comparison is carried out for DNA sequences of Tn6413 (this study), Tn6534 (accession number KX196168), Tn6533 (accession number AP014651), and Tn6417 (accession number CP013993). Genes are denoted by arrows. Genes, mobile elements and other features are colored based on function classification. Shading denotes regions of homology (> 95% nucleotide identity).

Each of these four Tn6417-family ICEs carried a single accessory module: Tn6403, Tn6531, Tn6530, and Tn6532 (Figure 6) from Tn6413, Tn6533, Tn6534, and Tn6417, respectively; all these accessory modules were integrated at the same site of the ICE backbones and identified as Tn6346 derivatives. The Tn3-family unit transposon Tn6346, originally found in heavy metal-tolerant Achromobacter spp., was a hybrid of the core transposition module tnpAR–res of Tn5051 and the mer region of Tn501 (Ng et al., 2009). Tn6403, Tn6531, Tn6530, and Tn6532 differed from Tn6346 by (i) interruption of original tnpATn due to insertion of IS1071, and (ii) insertion of four different class 1 integrons at the same position within the urf2 gene of mer. Tn6403, Tn6530 and Tn6532, rather than Tn6531, were bracketed by 5-bp DRs.

In127, In1328, In1155, and In159 found in Tn6403, Tn6531, Tn6530, and Tn6532, respectively, were intact integrons because all of them had paired terminal 25-bp repeats. Except In1155, all the other three were bracketed by 5-bp DRs. Notably, these integrons captured additional elements beside GCAs: IS26–msr(E)–mph(E)–IS26 unit and a novel blaVIM–4-carrying class 1 integron In1443, cmlA9–tetRA(G)–Tn5393c–ISCR3 and cmx-carrying In0, cmlA9–tetRA(G)–ISCR3 and empty In0, and empty In0 in In127, In1328, In1155, and In159, respectively. In1443 was organized as IRi–5’-CS (interrupted by insertion of ISPa82)–GCA (blaVIM–4–aadA7–aadA4)–Δ3’-CS–IS6100 (replacing tni)–IRt.

Conclusion

IncP-7 R-plasmids are not commonly found in natural isolates, and p1160-VIM represents the first fully sequenced IncP-7 R-plasmid. Based on repA sequences, IncP-7 plasmids can be further divided into two separately clustering subgroups IncP-7α and IncP-7β. The two novel blaVIM-carrying transposons Tn6392 and Tn6413, which are integrated into the IncP-7β plasmid p1160-VIM and the P. aeruginosa chromosome, respectively, represent two different categories of transposons: Tn3-family unit transposon and Tn6417-family ICE. Tn6392 and Tn6413 contain novel class 1 integrons In1394 and In1443, which harbor the two GCAs aacA4–blaVIM–24–aacA4 and blaVIM–4-aadA7–aadA4, respectively. The blaVIM–24 gene was initially discovered from a Klebsiella pneumoniae isolate in Colombia in 2011 (Montealegre et al., 2011). This study presents the first report of a blaVIM–24-carrying P. aeruginosa isolate and a blaVIM-carrying IncP-7 plasmid. Both p1160-VIM and Tn6413 are conjugative (self-transmissible) mobile elements, promoting horizontal transfer of resistance genes carried. Presence of IRi/IRt and a complete tni module would ensure In1394 self-transferable, while replacement of tni by IS6100 would impair mobility of In1443. Class 1 integrons (e.g., In1394 and In1443) could be integrated into a transposon (e.g., Tn6392 and Tn6413) to restore or enhance their mobility.

Ethics Statement

The use of human specimens and all related experimental protocols were approved by the Committee on Human Research of the First Affiliated Hospital of Hebei North University and that of the General Hospital of Xinjiang Military Region, and carried out in accordance with the approved guidelines. The research involving biohazards and all related procedures were approved by the Biosafety Committee of the Beijing Institute of Microbiology and Epidemiology.

Author Contributions

DZ and ZY conceived the study and designed experimental procedures. LZ, ZZ, LH, XJ, and YJZ performed the experiments. LZ, ZZ, YJZ, JF, BG, YEZ, and WY analyzed the data. LZ, ZZ, and HY contributed reagents and materials. DZ, ZY, LZ, and ZZ wrote this manuscript.

Conflict of Interest Statement

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.