Genetic characterization of two fully sequenced multi-drug resistant plasmids pP10164-2 and pP10164-3 from Leclercia adecarboxylata

We previously reported the complete sequence of the resistance plasmid pP10164-NDM, harboring blaNDM (conferring carbapenem resistance) and bleMBL (conferring bleomycin resistance), which is recovered from a clinical Leclercia adecarboxylata isolate P10164 from China. This follow-up work disclosed that there were still two multidrug-resistant (MDR) plasmids pP10164-2 and pP10164-3 coexisting in this strain. pP10164-2 and pP10164-3 were completely sequenced and shown to carry a wealth of resistance genes, which encoded the resistance to at least 10 classes of antibiotics (β-lactams. macrolides, quinolones, aminoglycosides, tetracyclines, amphenicols, quaternary ammonium compounds, sulphonamides, trimethoprim, and rifampicin) and 7 kinds of heavy mental (mercury, silver, copper, nickel, chromate, arsenic, and tellurium). All of these antibiotic resistance genes are associated with mobile elements such as transposons, integrons, and insertion sequence-based transposable units, constituting a total of three novel MDR regions, two in pP10164-2 and the other one in pP10164-3. Coexistence of three resistance plasmids pP10164-NDM, pP10164-2 and pP10164-3 makes L. adecarboxylata P10164 tend to become extensively drug-resistant.

We recently reported a fully sequenced resistance plasmid pP10164-NDM, harboring a total of two resistance genes bla NDM (conferring carbapenem resistance) and ble MBL (conferring bleomycin resistance), from the clinical L. adecarboxylata isolate P10164 12 . Strain P10164 is resistant to β -lactams including carbapenems, quinolones, aminoglycosides, macrolides, fosfomycin, tetracyclines, amphenicols, and trimethoprim/sulfamethoxazole but remained susceptible to tigecycline and polymyxin E. This follow-up study provides the evidence for the presence of two additional resistance plasmids pP10164-2 and pP10164-3 in L. adecarboxylata P10164. These two multidrug-resistant (MDR) plasmids were fully sequenced and shown to carry a large amount of antibiotic and heavy metal resistance genes.

Results and Discussion
Overview of plasmids pP10164-2 and pP10164-3. The complete sequences of pP10164-2 and pP10164-3 were determined from the genomic DNA of strain P10164 by high-throughput shotgun sequencing (the mean sequencing coverages are 79 × and 93 × respectively) and PCR-based gap closing. These two plasmids have circularly closed DNA sequences, 313,395 bp and 80,460 bp in length with mean G + C contents of 47.3% and 54.1%, respectively, and they contain 356 and 91 predicted open reading frames (ORFs) in total, respectively ( Fig. 1). The modular structure of each plasmid is discriminated as the backbone with insertion of multiple separate accessory modules.
The pP10164-2 backbone, 205 kb in length, is closely related (97% query coverage and maximum 99% nucleotide identity) to the prototype IncHI2 plasmid R478 from Serratia marcescens 13 , and almost identical (100% coverage and 99% identity) to another IncHI2 plasmid pKST313 from Salmonella enterica serotype Typhimurium 14 . Located in the pP10164-2 backbone are genes or gene clusters that encode the core IncHI2 plasmid determinants such as repHIA and repHI2 (replication initiation), the tra1 and tra2 regions (conjugal transfer), parAB and parM-parR (partition) within tra2, ter (tellurium resistance), klaABC (plasmid maintenance), and ars (arsenic resistance). It has been proposed that the repHIA replicon, the essential trh (conjugal transfer), tra, and oriT (origin of transfer) sequences within tra1 and tra2, and the parAB partitioning module might represent the minimal IncHI2 determinants 13 .
The pP10164-2 accessory regions, which are dramatically different from R478 and pKST313, are composed of the group IIB1 intron Kl.pn.I2, ISKpn26, two IS903D elements, a novel insertion sequence (IS) of IS3-family designated ISLad1, a novel IS element of IS1202 group named ISLad2, and two novel MDR regions designated MDR-1 and MDR-2. The MDR-1 and MDR-2 regions, 61.3 kb and 40.3 kb in length respectively, are adjacent and isolated by a 1.9 kb backbone region composing of two ORFs orf381 and ∆orf666. The pP10164-3 backbone encodes the plasmid replication (repA) and maintenance (parFG and umuCD) functions as well as the residual conjugal transfer determinants (traA, mutated nikAB, and mobC), and overall it exhibits no significant sequence similarity to any known DNA sequences. The deduced replication initiator protein RepA belongs to the Rep_3 superfamily and cannot be assigned into any known incompatibility groups, and it matches various plasmid RepA proteins of unknown incompatibility groups from Leclercia, Cronobacter and Enterobacter with above 93% amino acid identity.
pP10164-3 is quite unusual because it has a relatively small (19 kb in length) backbone but carries much larger accessory contents including the 2.3 kb Kl.pn.I2 intron and a 37.8 kb region composed of a MDR region and a carbohydrate utilization region. The carbohydrate utilization region is sequentially organized as a mutated sequence of a novel IS1-family member (MIS1), a novel 14-gene locus probably accounting for galactan utilization, IS1F, a mutated sequence of a novel IS3-family member (MIS3), and a Tn2555 remnant. Both MIS1 and MIS3 cannot be discriminated as intact IS elements because their transposase genes insB and tnpA, respectively, becomes pseudogenes due to frameshift. The sucrose transposon Tn2555 from E. coli is an IS26-based composite transposon that carries the sucrose utilization gene cluster scrKYABR, two direct IS26 copies on its flanks and, sometimes, a third inverted IS26 copy inside the transposon 15 , while the Tn2555 remnant from pP10164-3 containing only scrK and ∆scrY.
Tn6317 is generated from the insertion of Tn5058b into a backbone remnant of Tn6256, a Tn3-family TnPa38-related transposon from clinical Citrobacter freundii from Italy 16 . Each of the two 39 bp terminal inverted repeats (IRL: inverted repeat left; IRR: inverted repeat right) of Tn6317 is disrupted by IS4321R into two separate parts (IR-5′ plus IR-3′ ), which is also observed in Tn6256. It seems that the Tn5058b insertion is accompanied by not only the truncation of IS4321R but also the loss of downstream IRL-3′ and the core transposition module tnpA (transposase) at the 5′ region of Tn6317 relative to Tn6256 (Fig. 2b). Tn5058b is composed of a Tn5053-family core transposition module tniA (transposase)-tniB (ATP-binding protein)-tniQ (transposition auxiliary protein)-res (resolution site)-tniR (serine resolvase) and two mercury resistance gene clusters named mer1 and mer2, which is delimited by terminal 25 bp IRL and IRR.Tn5058b differs from the prototype Tn5058 (accession number Y17897) from Pseudomonas sp. ED23-33 by the insertion of IS5075 into each of the two internal inverted repeats IIR merT and IIR merR2 . The IS1111-family IS4321 and its close derivative IS5075 are known to target the terminal inverted repeats of the Tn21-subgroup transposons of Tn3 family 17 .
The Tn3-IS26-bla SFO-1 unit is likely derived from a precursor Tn3 [IRL-tnpA-res-tnpR (resolvase)-bla TEM-1 -IRR], which has undergone at least two major evolutionary events ( Fig. 2c): i) The disruption of the 38 bp IRL of Tn3 into IRL-5′ and IRL-3′ by IS4321R; and then ii) the insertion of the IS26-bla SFO-1 -IS26 unit (which is known to be transposable among plasmids 18 ) upstream of IS4321R, leading to the truncation of IS4321R, the loss of IRL-3′ -tnpA-res of Tn3, and the truncation of tnpR of Tn3. The connection of Tn3-IS26-bla SFO-1 with Tn6317 orientated in opposite directions likely results in the loss of the IRR of Tn3, making Tn3-IS26-bla SFO-1 cannot to be discriminated as a transposon due to the absence of one of the paired IRL/IRR routinely bracketing at both ends. Both bla TEM-1 and bla SFO-1 encode class A β -lactamases, whose activity can be inhibited by clavulanic acid. TEM-1 is able to hydrolyze penicillins but not extended-spectrum cephalosporins; by contrast, SFO-1 exhibits significant hydrolytic activity against both penicillins and extended-spectrum cephalosporins, but it has no detectable activity against carbapenems and cephamycins 19 . The bla SFO-1 expression is inducible, which is regulated by the transcriptional activator encoded by ampR that is inversely orientated upstream of bla SFO-1 20 .
Tn1548 is an IS26-based composite transposon from the C. freundii plasmid pCTX-M3 and displays a modular structure IS26-In27-ISCR1-∆ ISEc28-armA-ISEc29-msr(E)-mph(E)-orf543-IS26 21,22 . Notably, Tn1548 lacks the paired short direct repeats (DRs), which represent the target site duplication signals routinely bracketing at both ends of a composite transposon. Tn1548 and various Tn1548-asscoiated elements (with insertion of different class 1 integrons or integron-like sequences between IS26 and ISCR1) are thought to promote the dissemination of the aminoglycoside resistance gene armA, the macrolide resistance operon msr(E)-mph(E), and other classes of antibiotic resistance genes within the inserted integrons 23 . The Tn1548-asscoiated region from pP10164-2 differs from Tn1548 by the replacement of In27 by a novel class 1 integron named In1262, and the deletion of orf543-IS26 originally at the 3′ region of Tn1548 (Fig. 2d). The connection of immediately upstream IS26 and immediately downstream ISCR1 with In1262 leads to the loss of two terminal 25 bp inverted repeats (IRi: inverted repeat initial; IRt: inverted repeat terminal) and the truncation of intI1 (integrase) occurred for this integron. In1262 carries two gene cassettes gcu167 and aacA3 (aminoglycoside resistance):attC aacA3 . The novel gene cassette gcu167 of unknown function contains two consecutive ORFs gcu167a (putative nudix hydrolase) and gcu167b (putative nucleotidase), followed by a single attC gcu167 site.
At least 6 copies of IS26 are found in the MDR-1 region and can be arbitrarily assigned into the four structures IS26-In1262-ISCR1-∆ ISEc28-armA-ISEc29-msr(E)-mph(E)-IS26, IS26-bla SFO-1 -IS26, IS26-In27 pP10164-2 -IS26, and IS26-ydiB-tetA(C)-orf378-tetR(C)-orf477 -IS26. Each of them contains two terminally flanking IS26 elements but cannot be annotated as a composite transposon, because the paired DR sequences are not identified. The common component IS26 would act as an adaptor to mediate massive recombination and transposition events 28,29 , facilitating the assembly of the MDR-1 region with a very complex mosaic structure.
Tn2670 is an IS1-based composite transposon, which is composed of a backbone region with Tn21 inserted within it 30 and originally found in the MDR plasmid R100 (accession number AP000342) from Shigella flexneri. The In2670 backbone consists of two IS1 elements flanking a 1.5 kb central region that harbors the amphenicol resistance gene catA1 and the ybjA gene encoding putative acetyl transferase 30 . ∆ Tn2670 from the pP10164-2 MDR-2 region resembles the In2670 backbone but lacks the right terminal IS1 and, notably, similar structures are found in other IncHI2 plasmids such as pRH-R27 31 and in the chromosomally located resistance island AbaR1 and its derivatives from Acinetobacter baumannii 32 .
Tn6322 is composed of the Tn21 core transposition module tnpAR-res 33 together with a novel mercury resistance gene cluster designated mer3, and the mer3 region differs dramatically (92% coverage and maximum 86% nucleotide identity) from the mer locus from Tn21, indicating the capture of mer3 by the Tn21 core transposition module during the genesis of Tn6322. The mer3 region is mostly similar (100% coverage and maximum 96% nucleotide identity) to the counterpart of the Enterobacter cloacae transposon Tn6005 belonging to the Tn5036/ Tn3926 subgroup of Tn3 family 34 . Tn6322 is flanked of 38 bp IRL/IRR resembling those of Tn21: the IRR is intact, while the IRL shows the insertion of IS5075.
Silver and copper compounds are used as antimicrobial agents in hospitals, and the relevant resistance determinants could serve as hygienic fitness factors and thus improve bacterial survival in hospital environments. In R478, the silver and copper resistance gene clusters, called sil and cop respectively, are located adjacently and associated with an upstream Tn7-like core transposition module tnsABCD. Similar tnsABCD-sil-cop structures are widely found in IncHI2 plasmids such as pMRVIM0813 (accession number KP975077), pSTm-A54650 (LK056646), pKST313 14 and pRH-R27 31 , although considerable variations in both genetic content and nucleotide sequence are observed among different plasmids. Similarly, a multi-heavy metal resistance region ISKpn19-∆ISPpu12-sil-IS1R-orf1623-∆cop-rcn is found in the pP10164-2 MDR-2 region: compared with the prototype tnsABCD-sil-cop structure, ISKpn19-∆ ISPpu12 replaces tnsABCD, the insertion of IS1R-orf1623 (putative metal-dependent hydrolase)-IS903D between sil and cop marked the truncation of cop into ∆copS-copE, and a rcn locus (encoding the RcnA efflux pump responsible for nickel/cobalt detoxification and the rcnA repressor RcnR) is added immediately downstream of ∆copS-copE. Notably, the IncHI2 plasmid pRH-R27 31 carries a very similar structure from ∆ ISPpu12 to rcn with further insertion of a fragment composed of three hypothetical ORFs between IS1R and orf1623 31 . The MDR-2 region of pP10164-2 and the corresponding MDR region of pRH-R27 31 are genetically related and might share a much more recent ancestor, although they contains dramatically different sets of resistance genes upstream of the ∆ ISPpu12 to rcn region.
The Tn6308 backbone is a hybrid of the core transposition module tnpAR-res of Tn1696 and the mer region of Tn21, and it is bordered by the intact 39 bp IRL and the IS5075-disrupted IRR at both ends in the absence of DRs. Tn1696 and Tn21 are both the members of the Tn21 subgroup of Tn3 family, but they have independent histories and origins with limited nucleotide sequence similarity (79 to 96%) between corresponding backbone genes 36 . The res site, originally 120 bp in length, is truncated into an 83 bp remnant in Tn6308 due to the insertion of a class 1 integron In37b. Notably, all the three novel Tn3-family transposons Tn6317, Tn6322, and Tn6308 identified in this work have undergone at least two evolutionary events after their initial transposition into pP10164-2 or pP10164-3: i) the disruption of one or both terminal IR sequences by insertion of IS5075 or IS4321R, making them deficient in further mobilization; and ii) the removal of target site duplication signals, making them lack of terminal DR sequences.
Concluding remarks. This is the first report of detection of MDR plasmids and determination of their complete sequences in L. adecarboxylata. Coexistence of three resistance plasmids pP10164-NDM, pP10164-2 and pP10164-3 makes L. adecarboxylata P10164 tend to become extensively drug-resistant. This bacterial species may serve as a potential reservoir of antimicrobial resistance genes in clinical settings. Data presented here would promote us to gain deeper understanding of plasmid-mediated mechanisms of drug resistance in L. adecarboxylata. Prevalence of the resistance plasmids pP10164-NDM, pP10164-2 and pP10164-3 in L. adecarboxylata and other bacterial species from the clinical settings cultures especially those from immunocompromised patients needs to be elucidated.

Methods
Bacterial genomic DNA were isolated by classical phenol/chloroform method followed by diethyl ether removal of polysaccharides that contaminate genomic DNA 44 , and then sequenced with a paired-end library with an average insert size of 500 bp and a mate-pair library with average insert size of 5,000 bp, using HiSeq 2500 sequencer (Illumina, CA, USA). In order to get complete plasmid sequences, the contigs were assembled with Velvet, and the gaps were filled through combinatorial PCR and Sanger sequencing on ABI 3730 Sequencer (LifeTechnologies, CA, USA).