Genetic Characterization of a blaVIM–24-Carrying IncP-7β Plasmid p1160-VIM and a blaVIM–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 blaVIM–24), In1395 and In1443 (carrying blaVIM–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 blaVIM–24-carrying P. aeruginosa isolate.

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 bla VIM−24carrying IncP-7β plasmid p1160-VIM and a novel bla VIM−4carrying ICE Tn6413 recovered from two different clinical P. aeruginosa isolates.

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. aeruginosaspecific oafA gene (Choi et al., 2013).

Conjugal Transfer
Conjugal transfer experiments were carried out with rifampinresistant P. aeruginosa PAO1 used as recipients and each of the bla VIM -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 cm 2 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 bla VIM .

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.

Overview of Sequenced p1160-VIM and Tn6413
Two bla VIM -positive P. aeruginosa isolates, designated 1160 and 6762, were subjected to high-throughput genome sequencing. The 1160 isolate harbored a bla VIM−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 bla VIM−4 -harboring ICE Tn6413 was found to integrate into tRNA Gly 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).
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 p1160-VIM and Tn6413 were sequenced this work, and all the other elements analyzed were derived from GenBank. $, carrying resistance genes.
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.
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. 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 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%).  Figure S3).
Notably, these five SNPs did not lead to mutations of RepA amino acid sequences.
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 tnpA Tn6346 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.

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 bla VIM -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-bla VIM−24 -aacA4 and bla VIM−4− aadA7-aadA4, respectively. The bla VIM−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 bla VIM−24 -carrying P. aeruginosa isolate and a bla VIMcarrying 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 selftransferable, 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.

FUNDING
This work was supported by the National Key R&D Program (2018YFC1200100) of China.

SUPPLEMENTARY MATERIAL
The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fmicb. 2019.00213/full#supplementary-material FIGURE S1 | Plasmid schematic maps. Three plasmids pCAR1, pDK1 and p1160-VIM are included. Genes are denoted by arrows, and the backbone and accessory module regions are highlighted in black and color, respectively. Innermost circle presents GC-skew [(G−C)/(G+C)], with a window size of 500 bp and a step size of 20 bp. Next-to-innermost circle presents GC content.
FIGURE S2 | ICE schematic maps. Four ICEs Tn6413, Tn6534, Tn6533, and Tn6417 are included. Genes are denoted by arrows, and the backbone and accessory module regions are highlighted in black and color, respectively. Innermost circle presents GC-skew [(G−C)/(G+C)], with a window size of 500 bp and a step size of 20 bp. Next-to-innermost circle presents GC content.
FIGURE S4 | Organization of Tn4676 or Tn4663 comparison with related regions. Genes are denoted by arrows. Genes, mobile elements and other features are colored based on their functional classification. Blue shading denotes regions of homology (nucleotide identity > 95%), and pink shading denotes regions of homology (average nucleotide identity 82%). Numbers in brackets indicate nucleotide positions within corresponding plasmid sequences. Accession number of Tn4651 for reference is AJ344068.