The carbapenem resistance gene blaOXA-23 is disseminated by a conjugative plasmid containing the novel transposon Tn6681 in Acinetobacter johnsonii M19

Carbapenem resistant Acinetobacter species have caused great difficulties in clinical therapy in the worldwide. Here we describe an Acinetobacter johnsonii M19 with a novel blaOXA-23 containing transposon Tn6681 on the conjugative plasmid pFM-M19 and the ability to transferand carbapenem resistance. A. johnsonii M19 was isolated under selection with 8 mg/L meropenem from hospital sewage, and the minimum inhibitory concentrations (MICs) for the representative carbapenems imipenem, meropenem and ertapenem were determined. The genome of A. johnsonii M19 was sequenced by PacBio RS II and Illumina HiSeq 4000 platforms. A homologous model of OXA-23 was generated, and molecular docking models with imipenem, meropenem and ertapenem were constructed by Discovery Studio 2.0. Type IV secretion system and conjugation elements were identified by the Pathosystems Resource Integration Center (PATRIC) server and the oriTfinder. Mating experiments were performed to evaluate transfer of OXA-23 to Escherichia coli 25DN. MICs of A. johnsonii M19 for imipenem, meropenem and ertapenem were 128 mg/L, 48 mg/L and 24 mg/L, respectively. Genome sequencing identified plasmid pFM-M19, which harbours the carbapenem resistance gene blaOXA-23 within the novel transposon Tn6681. Molecular docking analysis indicated that the elongated hydrophobic tunnel of OXA-23 provides a hydrophobic environment and that Lys-216, Thr-217, Met-221 and Arg-259 were the conserved amino acids bound to imipenem, meropenem and ertapenem. Furthermore, pFM-M19 could transfer blaOXA-23 to E. coli 25DN by conjugation, resulting in carbapenem-resistant transconjugants. Our investigation showed that A. johnsonii M19 is a source and disseminator of blaOXA-23 and carbapenem resistance. The ability to transfer blaOXA-23 to other species by the conjugative plasmid pFM-M19 raises the risk of spread of carbapenem resistance. The carbapenem resistance gene blaOXA-23 is disseminated by a conjugative plasmid containing the novel transposon Tn6681 in Acinetobacter johnsonii M19.


Introduction
Carbapenems are considered to be reliable and effective antibiotic agents against most pathogenic bacteria because of their broad antibacterial spectrum [1] and are used in the treatment of serious nosocomial infections caused by cephalosporin-resistant bacteria [1]. Species of the Acinetobacter genus are extremely well adapted to the hospital environment and can easily become resistant to available antimicrobial agents; therefore, the isolation of carbapenem-resistant Acinetobacter species has raised increasing concerns [2][3][4][5][6]. Acinetobacter johnsonii is an opportunistic human pathogen that colonizes humans but rarely causes clinical infections. Nevertheless, verification of a carbapenem-resistant strain of A. johnsonii encoding an extended-spectrum β-lactamase raises concern [7,8].
β-lactamases are common mediators of β-lactam resistance and have been divided into four classes: A, B, C and D [2]. Members of class D, which are also referred to oxacillinases (OXAs), are notable contributors to carbapenem resistance and have been frequently observed in Acinetobacter species [9]. OXAs with carbapenemase activity were classified into 12 subgroups based on their amino acid sequences [10], and OXA-23 is the major source of carbapenem resistance in Acinetobacter [11].
From the bacterial perspective, conjugative plasmids are an ideal vehicle for transferring resistance genes among species. Fortunately, only a few types of plasmids in Acinetobacter species are conjugative and able to transfer resistance genes into new hosts [12]. However, numerous transposons, such as Tn2006, Tn2007, Tn2008 and Tn2009, have frequently been found to be associated with OXA genes [13]. The migration of OXA genes onto transposons has allowed them to become transmissible factors [14]. In this study, we isolated the high-level carbapenem-resistant strain A. johnsonii M19 from hospital sewage and discovered that it contained a novel transposon in a conjugative plasmid, thus allowing us to explore the potential for dissemination of carbapenem resistance by this species. These results provide new insights into the mechanisms of dissemination of carbapenem resistance.

Isolation and identification of the carbapenem-resistant strain M19
Hospital sewage was obtained from the influx of the wastewater treatment facility in Shandong province, China. The sewage samples were diluted and spread onto Luria-Bertani (LB) agar plates containing 8 mg/L meropenem (Sigma Co. Shanghai, China) and then incubated at 30 °C for 24 h. A single clone, named M19, was isolated and cultured in LB medium containing meropenem at 30 °C overnight and stored in 15% glycerin at − 20 °C.
A partial fragment of the 16S rRNA gene of M19 was amplified with the universal primers 27F (5′-agagtttgatcctggctcag-3′) and 1492R (5′-ggttaccttgttacgactt-3′) and sequenced. Similarity analyses of the 16S rRNA sequences were conducted using BLASTn (https :// blast .ncbi.nlm.nih.gov/Blast .cgi). A phylogenetic tree was produced by using the neighbor-joining algorithms with the Molecular Evolutionary Genetics Analysis 7 (MEGA 7) software based on BLAST results of the 16S rRNA sequence [15]. Antimicrobial susceptibility tests were performed to determine the MICs for carbapenems based on the breakpoints defined by the Clinical and Laboratory Standards Institute [4].

Whole-genome sequencing, annotation and analysis
The M19 genome was sequenced by PacBio RS II and Illumina HiSeq 4000 platforms at BGI Co., Ltd. (Wuhan, China). Gene prediction was performed on the M19 genome assembly by glimmer3 (https ://www.cbcb.umd. edu/softw are/glimm er/) with Hidden Markov models [16]. Genome annotation was performed using the Prokaryotic Genome Annotation Pipeline on NCBI (https ://ncbi.nlm.nih.gov/genom e/annot ation _prok/). Virulence factors and pathogenicity analysis were identified based on the core dataset in the Virulence Factors of Pathogenic Bacteria database (VFDB) [17] and the Pathogen Host Interactions (PHI) database [18].

Bioinformatics analyses of resistance genes, transposon and conjugation system
Antibiotic resistance genes (ARGs) were analyzed by RAST and BLASTp based on the core dataset in the Antibiotic resistance genes database (ARDB) [19]. Multisequence comparison was carried out by Clustal Omega [20] and ESPript [21]. Homologous model construction was operated by Discovery Studio 2.0 [22]. Molecular docking was performed by the CDOCKER protocol of Discovery Studio 2.0 [22]. IS transposases were detected by IS-Finder [23]. The new transposon was denominated and registered as Tn6681, according to the Transposon Registry (https ://trans poson .lstme d.ac.uk/). The genetic context of Tn6681 was compared with Tn2008 and Tn2008B using BLASTn. The conjugation system was identified by PATRIC server [24] and oriTfinder [25].

Mating experiments
Broth-based mating experiments were carried out using M19 as the donor and Escherichia coli 25DN as the recipient as described previously [26]. M19 and 25DN were cultivated overnight in LB medium containing 8 mg/L meropenem and 220 mg/L sodium azide. The mixture was incubated at 37 °C for 30 min, and transconjugants were selected on plates containing 8 mg/L meropenem, 220 mg/L sodium azide and 0.1 mg/L 5-bromo-4-chloro-3-indolyl-beta-D-glucuronic acid. The conjugal transfer efficiency was calculated, and six transconjugants, named MAT-1 to MAT-6, were isolated and purified. The MICs of carbapenems for these transconjugants were determined as above. To determine whether the plasmid pFM-M19 and bla OXA-23 were transferred to transconjugants, DNA fragments of the plasmid in transconjugants MAT-1 to MAT-6 were extracted and used as templates. The transconjugants were analyzed by PCR with a primer pair (Plasmid-For: 5′-tgtataggtgtgatgccttgta-3′; Plasmid-Rev: 5′-agaaacacagtgatgggagata-3′) for pFM-M19 or a primer pair (OXA23-For: 5′-ctgtcaagctcttaaataatattcagc-3′; OXA23-Rev: 5′-tattcgtcgttagaaaaacaattattg-3′) for the bla OXA-23 gene, and DNA sequencing was performed to confirm the presence of bla OXA-23 and plasmid-related genes.
The whole genome of M19 was sequenced, and the assembled genome contained one 3.75 Mb circular chromosome with 41.4% GC content, and one 55 kb circular plasmid, here named pFM-M19, with 35.8% GC content. The general features of the complete genome sequence are included in Additional file 1: Table S1. Overall, 197 genes (5.24% of the total genes) could be assigned to a VFDB number, and 228 genes (6.07% of the total genes) to a PHI number, indicating that M19 has a high pathogenic potential for humans or other hosts.  Table S2). Three classes of β-lactamaseencoding genes (class B, class C and class D) were identified, including genes encoding six metallo-β-lactamases (MBLs), two AmpCs and two OXAs (OXA-23 and OXA-211). Furthermore, other antibiotic resistance genes, including efflux pumps, a porin and an aminoglycosidemodifying enzyme gene, were also identified.

M19 harbours two oxacillinases genes, bla OXA-23 and bla OXA-211
Genome annotation of A. johnsonii M19 revealed the presence of two OXA-encoding genes, which are responsible for carbapenem resistance, bla OXA-211 in the chromosome and bla OXA-23 in plasmid pFM-M19. In addition, bla OXA-211 in M19 has the same genetic context conserved in other A. johnsonii strains (Additional file 1: Fig.  S1) and which appears to be ubiquitous in this species [28].
The tertiary structure of M19 OXA-23 was modelled based on the crystal structure of 4JF4, which is an OXA-23 from A. baumannii (GenBank accession number CAB69042.1) and which had the highest amino acid similarity with M19 OXA-23 in the Protein Data Bank (Fig. 2). In this model, the hydrophobic tunnel was formed by Phe-110 and Met-221, and had an elongated shape. Meropenem, imipenem and ertapenem were able to traverse the hydrophobic tunnel and bound to similar positions in the tunnel (Fig. 2). Additionally, Phe-110, Lys-216, Thr-217, Met-221 and Arg-259 were the conserved reactive amino acids (Additional file 1: Fig. S3).

bla OXA-23 is located in the novel transposon Tn6681 in pFM-M19
To evaluate the potential for horizontal transfer of bla OXA-23 , the genetic context of bla OXA-23 was investigated. Notably, sequence analysis found that the region containing bla OXA-23 formed a composite transposon with the components ISAba14-HP-ATPase-bla OXA-23 -ΔISAba1-ISAba14 (Fig. 3); this novel transposon has been named Tn6681 in the Transposon Registry and GenBank (Accession number: MN081614). Further alignment analysis showed that Tn6681 was highly similar to a chromosome fragment of A. baumannii CBA7, which was isolated in Korea (Accession number: CP020586.1) [35]. However, the bla OXA-23 context region of CBA7 is ISAba10-HP-ATPase-bla OXA-23 -ISAba1-ISAba15, which differs somewhat from Tn6681. In addition, two ISAba14 genes, marked as ISAba14 L and ISAba14 R , were found upstream (3,63,408 bp) of the ISAba10 gene and downstream (78,188 bp) of the ISAba15 gene in the CBA7 chromosome and share 99.91% identity with ISAba14 in Tn6681 (Fig. 3). Given their overall similarity, we propose that Tn6681 and this region of the CBA7 chromosome have the same ancestor.
ISAba14 genes belong to the IS3 family and have been previously identified as part of the active composite transposon Tn2114 in A. baumannii RAB [36]. Analysis of the inverted repeats (IRs) of ISAba1 showed that the right inverted repeat (IRR) of ISAba1 remained only 9 bp and the direct repeat and inverted repeat sequences vanished, but the left inverted repeat (IRL) of ISAba14 L shared sequence similarity with IRR of ISAba14 R (17/26) in particular the motif TATTT(TG/AT)GCG in their extremities (Fig. 4a). The direct repeat sequences (ATC ACT T) of 7 bp were also identified (Fig. 3). This overall structure formed a composite transposon Tn6681, which is a novel bla OXA-23 containing transposon.
Additionally, the arrangement ATPase-bla OXA-23 -ISAba1 constitutes a classic genomic organisation found in Tn2008 of A. pittii (GenBank accession number MF078634) and Tn2008B from A. baumannii (GenBank accession number LN877214.1) [13]. In Tn2008, the promoter of bla OXA-23 was overlapped by ISAba1 upstream of the start codon of OXA-23, and both the -10 and -35 regions of this promoter are within the sequence of the ISAba1 gene [37][38][39]. In Tn6681, the insertion of ISAba14 into ISAba1 generated two ΔISAba1, but the complete -10 and -35 regions of the bla OXA-23 promoter were fully maintained (Fig. 4b), indicating that bla OXA-23 should be expressed normally in M19.

Conjugative plasmid pFM-M19 disseminates bla OXA-23 and carbapenem resistance
To evaluate the ability to transfer bla OXA-23 and carbapenem resistance, the conjugation systems of pFM-M19 were analyzed. Components of conjugative machinery were identified in pFM-M19, such as a relaxase; the type IV coupling protein (T4CP) gene (traG) for initiation of conjugation; type IV secretion system (T4SS)-related genes, including the translocation channel protein genes (trbD, trbL, trbF, trbG and trbI); the pilus protein genes (trbC and trbJ) and the ATPase genes (trbE, trbB and traG), indicating that pFM-M19 was a conjugative plasmid (Fig. 5a). Mating experiments between M19 and E. coli 25DN were carried out. The results revealed that the conjugal transfer efficiency of pFM-M19 from M19 to E. coli 25DN was approximately 1.6 × 10 -4 CFU/donor when 8 mg/L meropenem was used as the selective pressure. The MICs of carbapenems in six transconjugants were 20 mg/L (imipenem), 16 mg/L (meropenem) and 4 mg/L(ertapenem), which were weaker than that of the donor strain M19 but much higher than that of strain 25DN ( Table 2). PCR analysis was performed to confirm the dissemination of carbapenem resistance via plasmid pFM-M19 and bla OXA-23 . The results showed that both pFM-M19 and bla OXA-23 were detected in all transconjugants ( Fig. 5b and c) and suggested that E. coli 25DN obtained carbapenem resistance due to the acquisition of bla OXA-23 along with pFM-M19.
Class D β-lactamases, commonly referred to as OXA, are responsible for carbapenem resistance, and their encoding genes are conserved and widespread in Acinetobacter species [44,45]. M19 harbours bla OXA-211 in the chromosome and bla OXA-23 in plasmid pFM-M19. OXA-23 was the first reported class D β-lactamase and was originally detected in a patient isolate of A. baumannii in Scotland in 1993 [46]. Twenty years later, the structure of OXA-23 was resolved and revealed that the elongated pocket of OXA-23 provides a hydrophobic environment for high reaction efficiency with carbapenems [11]. OXA-23 is considered to be the major β-lactamase for carbapenem resistance in Acinetobacter [11], suggesting that the OXA-23 encoded by plasmid pFM-M19 plays a key role in carbapenem resistance in M19. Interestingly, the bla OXA-23 found in plasmid pFM-M19 has not been previously reported in A. johnsonii strains, indicating that M19 obtained this gene from other bacterial species and further increasing concern over the ability of bla OXA-23 to spread among species.
Mobile elements are considered to be responsible for the movement and dissemination of bla OXA-23 , and all of the reported genetic structures that contain bla  have been classified as transposons [13]. Previously, bla OXA-23 had been found in five transposons, with ISAba1 upstream of the start codon of bla OXA-23 in four of these (Tn2006 [47], Tn2008 [48], Tn2008B [49], Tn2009 [50]) and with ISAba4 preceding bla OXA-23 in Tn2007 [47]. In plasmid pFM-M19, we found that bla OXA-23 was located in the new transposon Tn6681, which has the genetic context ISAba14-HP-ATPase-bla OXA-23 -ΔISAba1-ISAba14-ΔISAba1 and which was likely formed as two copies of ISAba14 were inserted into the ISAba1 of Tn2008. The structure of Tn6681 has some differences from that of the other transposons previously reported to contain bla OXA-23 [13]. In addition, in Tn2008, a high level of expression of the bla OXA-23 gene is associated with significant resistance to carbapenems in A. baumannii, and this expression is controlled by promoter elements within ISAba1 [37][38][39]. However, although sequence analysis revealed that ISAba14 inserted into ISAba1 in Tn6681, the promoter of bla OXA-23 appears to be intact, indicating that the bla OXA-23 gene may still be highly expressed and responsible for the striking carbapenem resistance in M19.
The acquisition of novel genes by plasmids, especially conjugative plasmids [51], along with mobile genetic elements such as transposons or insertion sequences, makes them perfect vehicles for the spread of antibiotic resistance [52]. The conjugative DNA transfer mechanism is well conserved and depends on a T4SS [53,54]. Genes of T4SS and T4CP modules, including genes for a translocation channel protein, pilus protein and ATPase, were identified in pFM-M19, indicating that pFM-M19 is a conjugative plasmid, which has also been confirmed by mating experiments in this study. However, it should be pointed out that the typical origin of transfer site (oriT) was not detected on pFM-M19 by oriT finder, suggesting that pFM-M19 might initiate the transfer from a cryptic oriT. Moreover, the combination of transposon Tn6681 and the conjugative plasmid pFM-M19 may provide a robust means for bla OXA-23 transfer, with the potential for Tn6681 to shift bla OXA-23 between the chromosome and plasmid in one bacterial strain, and the conjugative plasmid pFM-M19 disseminating Tn6681 and bla OXA-23 between different bacterial species (Fig. 6).

Conclusions
In conclusion, A. johnsonii strain M19, which contains the conjugative plasmid pFM-M19 and Tn6681, is not only a reservoir of bla OXA-23 but also an effective disseminator of bla OXA-23 . To our knowledge, our investigation is the first to provide evidence that bla OXA-23 was transferred into A. johnsonii. The presence of bla OXA-23 on conjugative plasmids of A. johnsonii enhances the risk of carbapenem resistance spread to the environment and needs to be monitored closely.

Nucleotide sequence accession number
Complete sequences of the chromosome of A. johnsonii strain M19 and of plasmid pFM-M19 were deposited in GenBank under accession numbers CP037424 and CP037425, respectively. The 16S rDNA sequence of strain M19 was deposited in GenBank under accession number MT226917.
Authors' contributions GZ conceived and designed and conducted experiments, analyzed and interpreted data, and wrote the manuscript. YZ, JF, PZ and CZ analyzed data and performed the calculation. WZ and YX helped to analyze data and revised the manuscript. RZ and GC led the project and revised the manuscript. All authors read and approved the final manuscript.

Availability of data and materials
All data generated or analyzed during this study are included in this published article [and its supplementary information files] Original data are available from the corresponding author upon reasonable request.

Ethics approval and consent to participate
All activities undertaken did not require Ethics approval. An informed written consent was obtained from the legal guardian of the case.