Complete Nucleotide Sequence of IncP-1β Plasmid pDTC28 Reveals a Non-Functional Variant of the blaGES-Type Gene

Plasmid pDTC28 was isolated from the sediments of Haihe River using E. coli CV601 (gfp-tagged) as recipient and indigenous bacteria from the sediment as donors. This plasmid confers reduced susceptibility to tetracycline and sulfamethoxazole. The complete sequence of plasmid pDTC28 was 61,503 bp in length with an average G+C content of 64.09%. Plasmid pDTC28 belongs to the IncP-1β group by phylogenetic analysis. The backbones of plasmid pDTC28 and other IncP-1β plasmids are very classical and conserved, whereas the accessory regions of these plasmids are diverse. A blaGES-5-like gene was found on the accessory region, and this blaGES-5-like gene contained 18 silent mutations and 7 missense mutations compared with the blaGES-5 gene. The mutations resulted in 7 amino acid substitutions in GES-5 carbapenemase, causing the loss of function of the blaGES-5-like gene on plasmid pDTC28 against carbapenems and even β-lactams. The enzyme produced by the blaGES-5-like gene cassette may be a new variant of GES-type enzymes. Thus, the plasmid sequenced in this study will expand our understanding of GES-type β-lactamases and provide insights into the genetic platforms used for the dissemination of GES-type genes.


Introduction
The dissemination of antibiotic resistance genes (ARGs) has been regarded as a threat to public health. The vehicles used for the dissemination of ARGs are mainly conjugative plasmids, among which the IncP-1 group of plasmids may be the most efficient vehicles because of their broad-host-range properties and high transfer frequencies among Gram-negative bacteria. In view of this, it is important to analyze the ARGs contained by the IncP-1 plasmids. To date, many types of ARGs have been found to be located on IncP-1 plasmids, including the clinically important carbapenem resistance gene.
The bla GES-5 gene is always found to be located on plasmids. bla GES-5 -harboring plasmids have various sizes and belong to different incompatible groups, mostly IncQ or IncQ-related groups [15][16][17]. Plasmid pCHE-A, which was found in Canada, belongs to the IncQ group [18]. The plasmids pG5A4Y427, pG5A4Y201, pG5A4Y413 and pG5A4Y217, which were also found in Canada, belong to the MOB Q1 group [15]. The MOB Q1 group was derived from the IncQ group and has close relationships with the IncQ1 plasmid RSF1010. The bla GES-5 gene was also found to be contained by ColE1-like plasmids, such as plasmid pKP-M1144 [19]. Girlich et al. studied two bla GES-5 -harboring plasmids (pRSB113 and pRSB115) that were isolated from the activated sludge of a wastewater treatment plant and found that these two plasmids did not belong to any of the IncP, IncQ, IncN, IncW, or IncA/C groups [20]. To date, there are no reports of the bla GES -5 gene or other bla GES variants carried by the IncP-1 group plasmids. Here, we report the complete sequence of the IncP-1β plasmid pDTC28, which was isolated from river sediments and carries a new non-functional variant of the bla GES -type gene.

Sample Collection
Sediment samples were collected from the Sewage River of Tianjin with a grab sampler and then placed into sterile containers. The sample was immediately taken to the laboratory and stored at -20°C for subsequent experiments after sampling was completed. In this study no specific permissions were required for the sampling activities and we confirm that our study did not involve endangered or protected species.

Source of Plasmid pDTC28
The tetracycline-resistant conjugative plasmid pDTC28 was isolated from the sediments of the Sewage River by filter mating assays using Escherichia coli CV601 (gfp-tagged, kanamycin and rifampicin resistant) as recipient and the sediment sample as donor [21]. The procedure for the filter mating experiments used in this study was as described in Heuer et al. with slight modifications [21]. An overnight culture of the Escherichia coli CV601 recipient strain was centrifuged at 4880 g for 5 min and washed twice with 0.85% sterile sodium chloride. Then, the pellet was resuspended in 2.5 ml Luria-Bertani (LB) broth. We resuspended 2 g of sediment sample in 18 ml LB broth in a 50 ml Erlenmeyer flask with five sterile glass balls (4 mm diameter). Then, the flask was placed on a rotary shaker (200 rpm) for 2 h. After shaking, 4 ml sediment solution was transferred into a 10 ml Eppendorf tube that contained 1 ml Escherichia coli CV601 recipient. The mixture of sediment solution and Escherichia coli CV601 recipient was centrifuged at 4880 g for 5 min. After centrifugation, the pellet was resuspended with 100 μl LB broth and then spotted onto a 0.22 μm sterile filter disk, which was then placed on an LB agar plate supplemented with 100 mg L -1 cycloheximide. The plate was placed upside down in an incubator at 37°C for 2 days. After incubation, the bacteria on the filter disk were resuspended with 2 ml 0.85% sterile sodium chloride solution. Then, the suspension was plated on selective agar plates containing tetracycline (10 mg L -1 ), kanamycin (50 mg L -1 ) and rifampicin (50 mg L -1 ). The cycloheximide (100 mg L -1 ) was also included in the selective agar plates to inhibit fungal growth. The transconjugants were further determined by green fluorescence resulting from the green fluorescence protein (GFP) gene. The Escherichia coli CV601 recipient culture was plated on the same selective plates as the controls. The sediment sample was also plated on the selective plates to rule out the possibility of green fluorescence bacteria from the environment. The isolated tetracycline-resistant transconjugants were subjected to susceptibility testing by the Kirby-Bauer disk diffusion method on Muller-Hinton Agar plates. According to the criteria of the Clinical and Laboratory Standards Institute (CLSI), the antimicrobial agents on the disks used in this study are as follows: ampicillin (10 μg), gentamicin (10 μg), streptomycin (10 μg), tetracycline (30 μg), ciprofloxacin (5 μg), sulfamethoxazole (300 μg), erythromycin (15 μg) and imipenem (10 μg). Escherichia coli ATCC25922 was used as a quality control strain. One transconjugant that conferred resistance to sulfamethoxazole and tetracycline, namely, DTC28, was selected for further analysis. The conjugative plasmid harbored by DTC28 was named pDTC28 and stored for complete genome sequencing.

Conjugative Frequency of Plasmid pDTC28
To assess the conjugative frequency of plasmid pDTC28, liquid mating experiments were performed using Escherichia coli CV601 (pDTC28) as the donor strain and Escherichia coli J53 (azide and nalidixic acid resistance) as the recipient strain. For the liquid mating assay, overnight cultures of donor and recipient strains were centrifuged, washed and adjusted to an optical density of 0.6 at the wavelength of 600 nm (OD 600 ) with LB broth. Then, 0.5 ml cultures of donor and recipient strains were mixed and brought up to a volume of 5 ml with LB broth. After incubation for 16 h at 37°C, transconjugants were selected on LB plates containing azide, nalidixic acid and tetracycline. The concentrations of antibiotics used for selecting transconjugants were described above. The conjugative frequency was determined by the following formula: Conjugative frequency = transconjugants (CFU/ml)/recipients (CFU/ml).

Plasmid Sequencing and Bioinformatics
Plasmid DNA was extracted from the E. coli J53 transconjugants of plasmid pDTC28 using the Qiagen Plasmid Midi Kit (Qiagen Inc., Germany). A shotgun library was generated and then sequenced on an Illumina HiSeq 4000 sequencing system. Sequencing was performed by the Beijing Genomics Institute (BGI, Beijing, China). Sequencing reads were de novo assembled into contigs using the SOAPdenovo 2.04 software [22,23], followed by gap closure through a PCR and Sanger sequencing approach. The putative open reading frames (ORFs) were predicted using Glimmer 3.02 [24][25][26]. All ORFs were translated and aligned with different pro-

Nucleotide Sequence Accession Number
The complete nucleotide sequence of pDTC28 was deposited in GenBank under accession no. KU238092.

Results and Discussion
General Structure of bla GES-5 -harboring IncP-1β Plasmid pDTC28 The complete sequence of plasmid pDTC28 was 61,503 bp in length with an average G+C content of 64.09% (Fig 1). The overall sequence of plasmid pDTC28 can be divided into two main parts: the backbone region (positions 18291 bp-41377 bp, 46514 bp-61134 bp) and the accessory region (positions 157 bp-17566 bp, 42362 bp-45882 bp). Plasmid pDTC28 was found to belong to the IncP-1 group by sequence queries against the GenBank database. The IncP-1 plasmids were originally divided into two subgroups based on the sequence similarities with plasmid RP4 and R751 [29]. RP4 is the prototype of the IncP-1α group, whereas R751 is the prototype of the IncP-1β group. Currently, there are seven subgroups of IncP-1 plasmids including α, β, γ, δ, ε, z and η [30]. A phylogenetic tree was constructed using the trfA proteins of 54 IncP-1 plasmids including plasmid pDTC28 (Fig 2). The tree shows that plasmid pDTC28 belongs to the IncP-1β group and clusters with the IncP-1β prototype R751. The tree also shows that plasmid pDTC28 is slightly different from a smaller IncP-1β clade formed by plasmids pB1, pB12, pNB8c, pLME1, pB4, pTB30, pBAM1 and pRSB223.
The stable inheritance and central control region of plasmid pDTC28 consists of the incC1 gene, which is involved in plasmid active partitioning; the kle genes (kleA, kleB, kleC, kleD, kleE, kleF and kleG), which are involved in plasmid stable inheritance and can enhance the frequency of plasmid active partitioning; the kor genes (korA, korB and korC) and the kfrA gene, which are involved in transcriptional regulation; the klc genes (klcA and klcB), which are also involved in plasmid stable inheritance; and the klu genes (kluA and kluB), which may be involved in plasmid stability (post-segregational killing).

Accessory Region of Plasmid pDTC28
The accessory region of plasmid pDTC28 contained a putative mercury resistance locus (positions 42362 bp-45882 bp), a putative class 1 integron (positions 12198 bp-15593 bp) harboring the bla GES-5 -like gene, tetracycline resistance gene cassettes (positions 5580 bp-7535 bp) and aminoglycoside resistance gene cassettes (positions 8473 bp-9800 bp). This region was 21,031 bp in length and inserted into two loci, one between the tra region and trb region and the other between the oriV region and the trfA gene, altogether accounting for 34.2% of the total sequence.

Mercury Resistance Locus
Plasmid pDTC28 possessed a putative mercury resistance locus (positions 42362 bp-45882 bp) for transporting mercury-derived compounds out of the bacterial cell. This mercury resistance locus contained the merE gene (inner membrane spanning proteins for transporting Hg 2+ to the cytoplasm, where it is reduced by merA), the merD gene, the merA gene (mercuric reductase, enzymatic reduction of Hg 2+ to Hg 0 ), the merP gene (periplasmic Hg 2+ scavenging protein), the merT gene (inner membrane spanning proteins for transporting Hg 2+ to the cytoplasm, where it is reduced by merA) and the merR gene (regulatory protein acting as a transcriptional repressor or activator for regulating the overall expression of the mer operon) [37,38].

Class 1 Integron Harboring the bla GES-5 -like Gene in the Accessory Region
The accessory region of plasmid pDTC28 contains a putative class 1 integron. This integron contains a single gene cassette that shares 97% identity with the bla GES-5 gene with 100% query coverage. The bla GES-5 gene is believed to encode resistance to carbapenems. The GES-type enzymes possess the ability to hydrolyze broad-spectrum cephalosporins. Some variants of GES-type enzymes such as GES-2, -4, -5, -6, -11, and -14 have an enlarged spectrum of activity against carbapenems through amino acid substitutions [10]. Although the bla GES-5 -like gene differs only in a few nucleotides with the canonical bla GES-5 gene, the gene did not confer resistance to carbapenems or even ampicillin in antibiotic susceptibility testing. Compared with the bla GES-5 gene from E. coli (GenBank accession number AY494717), the bla GES-5 -like gene on pDTC28 contains 18 silent mutations and 7 missense mutations. As shown in Fig 4, the mutations resulted in 7 amino acid substitutions in GES-5 carbapenemase. Previous studies have shown that the Ambler positions 104 and 170 of GES-type β-lactamases are hot spots for amino acid substitutions [43]. Amino acids in these two sites play an important role in the interaction with β-lactams [43]. However, the substitutions in this study are not located in these two sites but rather mostly cluster at the two ends of GES-5 carbapenemase. Nonetheless, these substitutions have caused a loss of function of the bla GES-5 -like gene cassette on plasmid pDTC28 against carbapenems and even β-lactams. The enzyme produced by the bla GES-5 -like gene cassette may be a new variant of GES-type enzymes, and further study is needed to determine whether it has any biological role.

Other Antibiotic Resistance Genes in the Accessory Region
Except for the bla GES-5 -like gene, the accessory region of pDTC28 also contains other antibiotic resistance genes. The ΔstrA-strB gene cluster (positions 8473 bp-9800 bp) was found to be located in the accessory region of plasmid pDTC28. The products of the strA-strB gene cluster could catalyze the transfer of a phosphate group to the aminoglycoside molecule [44]. However, like the bla GES-5 -like gene, the strA gene on plasmid pDTC28 also has some differences in nucleotides with the normal strA gene. The strA gene on plasmid pDTC28 showed three mismatches and one insertion in comparison to the strA gene from plasmid RSF1010 (GenBank accession no.: M28829). The mismatches and insertion of the strA gene on plasmid pDTC28 have resulted in the disruption of the strA gene and the loss of function of the strA-strB gene cluster against streptomycin. According to the results of antibiotic susceptibility testing, plasmid pDTC28 did not confer resistance to streptomycin. This result is in correspondence with the sequence analysis. In addition to the ΔstrA-strB gene cluster, plasmid pDTC28 also harbors the tetA-tetR gene cluster. The existence of the tetA-tetR gene cluster is consistent with the susceptibility results of plasmid pDTC28 to tetracycline. In conclusion, we report the first complete sequenced IncP-1β plasmid that harbors the bla GES-5 -like gene and the truncated strA-strB gene cluster. The bla GES-5 -like gene is likely to encode a new variant of GES-type β-lactamase although it does not confer any known resistance phenotype. Therefore, we believe the plasmid sequenced in this study will expand our understanding of GES-type β-lactamases and provide insights into the genetic platforms used for the dissemination of GES-type genes.