Complete Nucleotide Sequence of pGA45, a 140,698-bp IncFIIY Plasmid Encoding blaIMI-3-Mediated Carbapenem Resistance, from River Sediment

Plasmid pGA45 was isolated from the sediments of Haihe River using Escherichia coli CV601 (gfp-tagged) as recipients and indigenous bacteria from sediment as donors. This plasmid confers reduced susceptibility to imipenem which belongs to carbapenem group. Plasmid pGA45 was fully sequenced on an Illumina HiSeq 2000 sequencing system. The complete sequence of plasmid pGA45 was 140,698 bp in length with an average G + C content of 52.03%. Sequence analysis shows that pGA45 belongs to IncFIIY group and harbors a backbone region which shares high homology and gene synteny to several other IncF plasmids including pNDM1_EC14653, pYDC644, pNDM-Ec1GN574, pRJF866, pKOX_NDM1, and pP10164-NDM. In addition to the backbone region, plasmid pGA45 harbors two notable features including one blaIMI-3-containing region and one type VI secretion system region. The blaIMI-3-containing region is responsible for bacteria carbapenem resistance and the type VI secretion system region is probably involved in bacteria virulence, respectively. Plasmid pGA45 represents the first complete nucleotide sequence of the blaIMI-harboring plasmid from environment sample and the sequencing of this plasmid provided insight into the architecture used for the dissemination of blaIMI carbapenemase genes.


INTRODUCTION
The overuse and misuse of antibiotics have contributed to the emergence and spread of antibiotic resistance genes (ARGs) and multidrug resistance pathogens (Zhang and Zhang, 2011;He et al., 2014). Now ARGs have been recognized as a new type of pollutants (Pruden et al., 2006). Among various ARGs, carbapenem resistance genes, especially plasmid mediated carbapenem resistance genes, have raised worldwide concern, leading to the extensive research on some of these genes and related plasmid architecture (McGann et al., 2012;Tiwari et al., 2012;Villa et al., 2012Villa et al., , 2013Lo et al., 2013;Tiwari and Moganty, 2014). Acquired carbapenem resistance can be resulted from carbapenemases of Amber class A (IMI, GES and KPC), Amber class B (metallo β-lactamases including IMP, VIM and NDM) or Amber class D (OXA-48 and OXA-181) . The bla KPC gene of Amber class A and the metallo β-lactamase genes have been the research focus but there is rare reports about the other carbapenem resistance genes especially little is known about the bla IMI genes and the plasmid architecture involved in the dissemination of this type of genes (Aubron et al., 2005;Yu et al., 2006;Rojo-Bezares et al., 2012;Teo et al., 2013;Chen et al., 2015).
The bla IMI−1 gene was first identified in 1996 and found to be located in the chromosome of Enterobacter cloacae isolates whereas bla IMI−2 was first identified in 2005 in Enterobacter asburiae isolates and found to be related to plasmids (Rasmussen et al., 1996;Aubron et al., 2005). In 2009, a new variant of bla IMI−1 , bla IMI−3 , was identified in Hong Kong in Enterobacter cloacae isolates (Chu et al., 2011). The bla IMI−3 was also located in the conjugative plasmids. The bla IMI -mediated carbapenem resistance is an infrequent mechanism but it has been reported both in clinical strains and environmental bacteria from rivers. Horizontal transfer may occur between environmental bacteria and clinical strains. With the horizontal transfer, the bla IMI genes would broaden their hosts and inevitably pose serious risks to the public health. For further research on the dissemination of bla IMI genes, the full sequence of bla IMI -related plasmid is needed. Here we report the first complete nucleotide sequence of bla IMI -carrying conjugative plasmid from the environment sample.

Studying Sites and Sample Collection
Sediment sample was collected under JinGang Bridge of Haihe River. JinGang Bridge was located in densely populated urban areas with frequent human activities. Sediment sample was collected with a grab sampler and then put into sterile containers. The sample was immediately taken to the laboratory and stored in −20 • C for subsequent experiments after sampling was completed.

Isolation of the Conjugative Plasmids Conferring Resistance to Imipenem
In order to obtain conjugative plasmids which confer resistance to imipenem, amipicillin resistant plasmids were first isolated by filter mating assays and these ampicillin resistant plasmids were then subjected to antibiotic susceptibility testing against imipenem and other antibiotics. The filter mating assays were applied using Escherichia coli CV601 (gfp-tagged, kanamycin and rifampicin resistant) as recipients and sediment samples as donors (Heuer et al., 2002). Transconjugants were selected by Mueller-Hinton agar plates supplemented with ampicillin (100 mg L −1 ), kanamycin (50 mg L −1 ), rifampicin (50 mg L −1 ) and cycloheximide (100 mg L −1 ). E. coli CV601 recipient culture was plated on the same selective plates as controls. The procedure used for filter mating assays was described by Heuer et al. (2002) with slight modification. Briefly, the sediment samples from which the indigenous bacteria were used as donors were doubled to 2 g and the Luria-Bertani (LB) broth used for resuspending the sediment samples was accordingly doubled to 18 ml. After incubation for 2 days, transconjugants were determined by green fluorescence which is resulted from green fluorescence protein (GFP) gene. All the ampicillin resistant transconjugants were then streaked on the ampicillin selective plates. Overnight culture of these transconjugants were stored in −80 • C for further study.

Antibiotic Susceptibility Testing of the Ampicillin Resistant Transconjugants
Kirby-Bauer disk diffusion method was applied to determine which ampicillin resistant transconjugants confer resistance to imipenem. According to the criteria of the Clinical and Laboratory Standards Institute (CLSI), the disks used in this study are as follows: imipenem (10 µg), ampicillin (10 µg), gentamicin (10 µg), streptomycin (10 µg), tetracycline (30 µg), ciprofloxacin (5 µg), sulfamethoxazole (300 µg) and erythromycin (15 µg). E. coli ATCC25922 was used as quality control strain. In this study, one transconjugant designated GA45 was found to confer resistance to imipenem and ampicillin. The conjugative plasmid harbored by GA45 was named pGA45 and stored for further analysis.

Conjugative Transfer Experiments and the Role Determination of pGA45 in Recipient Strains
To assess the conjugative frequency of plasmid pGA45, liquid mating assays were employed using E. coli CV601 (pGA45) as donor strains and E. coli J53 (azide and nalidixic acid resistance) as recipient strains. For liquid mating assay, overnight cultures of donor and recipient strains were centrifuged, washed and adjusted to the optical density of 0.6 at the wavelength of 600 nm (OD 600 ) with LB broth. Then 0.5 ml cultures of each donor and recipient strains were mixed and make up to the volume of 5 ml with LB broth. After incubation of 16 h in 37 • C, transconjugants were selected on LB plates containing azide (200 mg L −1 ), nalidixic acid (20 mg L −1 ) and ampicillin (100 mg L −1 ). Conjugative frequency was determined by the following formula: conjugative frequency = transconjugants (CFU/ml)/recipients (CFU/ml). The E. coli J53 transconjugants were then tested against imipenem to confirm the role of pGA45 in recipient strains. The results showed that pGA45 also conferred resistance to imipenem in recipient strains.

Plasmid Sequencing and Bioinformatics
Plasmid DNA from the E. coli J53 transconjugants was extracted using a Qiagen plasmid midikit (Qiagen, Inc). The plasmid DNA was sequenced on an Illumina HiSeq 2000 sequencing system. Sequencing reads were de novo assembled into contigs using the SOAPdenovo 2.04 software (Li et al., 2008(Li et al., , 2010. Gaps between contigs were closed by PCR with standard Sanger sequencing. Glimmer 3.02 was used to predict putative open reading frames (ORFs) (Salzberg et al., 1998;Delcher et al., 1999Delcher et al., , 2007

Nucleotide Sequence Accession Number
The complete nucleotide sequence of pGA45 was deposited in GenBank under accession no. KT780723.

RESULTS
Sequencing of plasmid pGA45 generated 237,937,000 reads in total. Reads either of low quality or representing the host chromosome contamination through comparison with the sequence of reference strain E. coli MG1655 were filtered. In the end, 34 contigs were obtained and then assembled into 10 scaffolds. Through PCR and Sanger sequencing, gaps between contigs and scaffolds were closed. The complete sequence of plasmid pGA45 was 140,698 bp in length with an average G + C content of 52.03% (Figure 1). Analysis of the sequence identified 157 ORFs, 64 of which were transcribed in the opposite direction. The backbone of this plasmid included the replication region, stability region 1, and transfer region (51524 bp), making up 36.6% of the total sequence. This backbone region shared high homology and gene synteny to several other IncF plasmids including pNDM1_EC14653 (Wu et al., 2015), pYDC644 (GenBank accession no.: KR351290), pNDM-Ec1GN574 (GenBank accession no.: KJ812998), pRJF866 (Qu et al., 2015), pKOX_NDM1 (Huang et al., 2013) (86% query coverage and 97% nucleotide identity, NCBI database) and pP10164-NDM (Sun et al., 2015) (86% query coverage and 94% nucleotide identity, NCBI database). Notably, all these plasmids were recently sequenced and four of them were found in China. Thus, to our best of knowledge, pGA45 and other recently sequenced plasmids represent a new IncF subtype and this type of plasmids exhibit a high prevalence in China. By contrast, the rest parts of pGA45 [including the variable region, the stability region 2 and the type VI secretion system (T6SS) region] showed no significant similarities with other sequenced plasmids in GenBank.
The replication region (1,431 bp) of pGA45 (positions 75496-76926), including the replication initiation protein gene repA and replication regulatory protein gene repA2, shared 93% nucleotide similarity with the six IncF plasmids mentioned above with 100% query coverage. Plasmid pGA45 was further assigned to the IncFII Y incompatibility group through sequence queries against the plasmid MLST databases 1 .
The genes on plasmid pGA45 that are responsible for plasmid stability and maintenance included umuC-umuD genes 1 http://pubmlst.org/plasmid which confer resistance to UV light, relE-relB genes encoding a toxin-antitoxin system, ardA gene with antirestriction function, parA-parB genes for partition, psiA-psiB genes involved in the bacterial SOS inhibition and ssb gene involved in recombination and repair.
The variable region of plasmid pGA45 contained two resistance genes including one ARG bla IMI−3 and one copper resistance gene copC (with 54% coverage and 94% amino acid identity to Klebsiella pneumoniae subsp. pneumoniae DSM 30104, NCBI database). Plasmid pGA45 also harbored a T6SS region (position 82987-114739bp) which may be related to bacterial pathogenesis.

DISCUSSION
The bla IMI−3 gene from the variable region was the only ARG harbored by pGA45. The bla IMI−3 -containing region (position 58145-74139 bp) was bracketed by one copy of insertion sequence ISEc36 2 and one copy of insertion sequence highly similar to ISEcl1 2 (with 100% coverage and 94% nucleotide identity to ISEcl1) in the same orientation (Figure 2). ISEc36 was first identified in a bla IMI−2 -bearing E. coli W635 strain (Rojo-Bezares et al., 2012). In this strain, bla IMI−2 was detected upstream the IMI-2R gene. However, in plasmid pGA45, bla IMI−3 and IMI-3R changed positions with each other and IMI-3R was located upstream the bla IMI−3 gene. Another well characterized bla IMI -containing structure was from Enterobacter cloacae plasmid pT103 (GenBank accession no.: NG_036022.1) (Yu et al., 2006). In this partially sequenced plasmid, the bla IMI -containing region comprises two ISEc13 2 (one is partial) elements flanking the bla IMI−3 and bla IMI−3R genes in the opposite directions and one partial ISEc36 located downstream of this region. In all these characterized bla IMI -containing regions, bla IMI genes have close relationships with insertion sequence ISEc36. Therefore, ISEc36 may play an essential role in the dissemination of bla IMI genes between different plasmids. It is also noteworthy to point out that the six plasmids mentioned above similar to pGA45 in backbones mainly have two different bacterial hosts, Klebsiella pneumoniae and Enterobacter cloacae. In addition, the identified bla IMI genes were mostly from Enterobacter species. In view of this, the most probable hosts for plasmid pGA45 were Enterobacter species.
Another notable feature harbored by pGA45 was the T6SS region. Compared to other similar IncFII plasmids, the T6SS region was unique to pGA45. This region was most closely related to the T6SS system of plant pathogen Erwinia amylovora not only in nucleotide identity (71% coverage and 82% nucleotide identity) but also in gene organization. Previous studies showed that bla IMI−2 and bla IMI−3 genes were located on plasmids with sizes ranging from 48.5 to 80 kb (one of these plasmids had been identified to belong to IncF group). In this study, pGA45 was much bigger than these plasmids. This perhaps resulted from the FIGURE 1 | Circular map of plasmid pGA45 (GenBank KT780723). The rings show from outside to inside (i) position of predicted coding sequences in the clockwise direction, (ii) position of predicted coding sequences in the counterclockwise direction, (iii) different regions of plasmid pGA45, (iv) GC plot in a 10,000-bp window, (v) GC skew in a 10,000-bp window. Each predicted coding sequence is color-coded by its function as shown in the figure. integration of this T6SS region (31753 bp). The T6SS region of plasmid pGA45 was flanked by a copy of ISEc25-like element (with 100% coverage and 85% nucleotide identity to ISEc25) downstream and two transposase genes (with weak amino acid identity to known transposase) upstream. The T6SS region of plasmid pGA45 comprised 14 T6SS-related genes including vasD (with 93% coverage and 64.46% amino acid identity to Erwinia amylovora ATCC 49946, KEGG database),  The T6SS was a recently discovered phage-like secretion apparatus. First reported in Vibrio cholerae and Pseudomonas aeruginosa, T6SS was likely to be involved in bacterial pathogenesis through acting like a potential nano-syringe for the translocation of effector proteins into the host cell (Pukatzki et al., 2006;Sarris et al., 2011). The Hcp (haemolysin coregulated protein) and VgrG (valine-glycine repeat G protein) proteins are putative effectors for T6SS (Russell et al., 2014) and the genes encoding these two proteins are also found to be located in the T6SS region of plasmid pGA45. T6SSrelated genes are harbored by many kinds of Gram-negative bacterial pathogens which can result in human or animal diseases. In this study, plasmid pGA45 was isolated from river sediment which was collected from urban section of Haihe River. This area was densely populated and strongly affected by human activities. In previous published literatures, most of the bla IMI isolates were from in clinical settings. Therefore, the occurrence of T6SS and bla IMI−3 -containing plasmid pGA45 in the river environment are a potential risk for human health and the horizontal transfer of this plasmid between Enterobacteriaceae bacteria may aggravate this situation.

CONCLUSION
This report demonstrated the complete nucleotide sequence of the bla IMI -harboring plasmid. The sequencing of this plasmid provided insight into the architecture used for the dissemination of bla IMI carbapenemase genes. In addition to the bla IMI gene, plasmid pGA45 also harbored a T6SS cluster probably involved in bacteria virulence. Notably, this plasmid was isolated from environment sample, which will increase the risks of obtaining infections resulted from various types of pathogens carrying this plasmid.

AUTHOR CONTRIBUTIONS
DM and YL designed experiments; BD carried out experiments and analyzed experimental results; BD wrote the manuscript.

ACKNOWLEDGMENTS
We are greatly thankful to Professor Holger Heuer (Julius Kühn-Institut) for providing E. coli CV601 and E. coli J53. This work was supported by the National Natural Science Foundation of China (Grants 41473085 and 31470440, 31270542), the Ministry of Education, People's Republic of China as an innovative research team project (grant No. IRT13024).