Introduction {#S1}
============

*Leclercia adecarboxylata*, a Gram-negative rod of the *Enterobacteriaceae* family, exists widely in nature and shares many biochemical features with *Escherichia coli* ([@B2]; [@B32]). As an extremely rare human pathogen, *L. adecarboxylata* causes monomicrobial infection in immune suppressed patients and ones with underlying medical conditions, and it is also found as part of a causative agent of polymicrobial infections in immunocompetent subjects, requiring other coinfecting bacteria to establish infection ([@B2]; [@B32]).

*L. adecarboxylata* is generally susceptible to cephalosporins, carbapenems, tetracyclines, aminoglycosides, quinolones, and chloramphenicol ([@B33]). Nevertheless, *L. adecarboxylata* isolates have evolved to acquire foreign antibiotic resistance genes, which encode extended-spectrum β-lactamases (ESBLs) SHV-12 ([@B20]) and CTX-M-3 ([@B30]), and carbapenemases IMP-1 (GenBank accession number [KJ531212](KJ531212)), IMP-4 ([@B3]; [@B18]), KPC-2 ([@B12]; [@B36]), NDM-1 ([@B34]; [@B15]; [@B28]) and VIM-1 ([@B24]; [@B25]); from these strains, one NDM-encoding plasmid pP10164-NDM (accession number [KP900016](KP900016)) ([@B34]), one VIM-encoding pLec-476cz (accession number [KY320277](KY320277)) ([@B25]), and several KPC-encoding plasmids ([@B36]) have been fully sequenced.

Only a small amount of MDR *L. adecarboxylata* strains, each harboring multiple acquired resistance genes, have been reported ([@B30]; [@B11]; [@B35]). Notably, complete genome sequencing has been applied to only one of those MDR strains designated P10164, which harbors pP10164-NDM that is not a MDR plasmid ([@B34]), and two MDR plasmids pP10164-2 and pP10164-3 (accession numbers [KX710093](KX710093) and [KX710094](KX710094), respectively), as shown in our previous study ([@B35]). This follow-up study disclosed co-occurrence three different multi-drug resistant (MDR) plasmids, p16005813A (unknown incompatibility group), p16005813B (IncFII), and p16005813C (IncR), containing a total of 18 non-redundant resistance genes or gene loci, in a single clinical *L. adecarboxylata* isolate.

Materials and Methods {#S2}
=====================

Bacterial Isolates {#S2.SS1}
------------------

*L. adecarboxylata* 16005813 was isolated in 2016 from a sputum specimen of an infant with pneumonia in a public hospital in Ningbo, China.

Genomic DNA Sequencing and Annotation {#S2.SS2}
-------------------------------------

Genomic DNA was isolated from the 16005813 isolate using an DNeasy UltraClean Microbial Kit or a Large Construct Kit (Qiagen, NW, Germany), and then sequenced from a sheared DNA library with average size of 15 kb (ranged from 10 to 20 kb) on a PacBio RSII sequencer (Pacific Biosciences, CA, United States), as well as a paired-end library with an average insert size of 400 bp (ranged from 150 to 600 kb) on a HiSeq sequencer (Illumina, CA, United States). The paired-end short Illumina reads were used to correct the long PacBio reads utilizing *proovread* ([@B14]), and then the corrected PacBio reads were assembled *de novo* utilizing *SMARTdenovo*^[1](#footnote1){ref-type="fn"}^. Open reading frames (ORFs) and pseudogenes were predicted using *RAST* 2.0 ([@B6]) with default parameters, combined with *BLASTP/BLASTN* ([@B4]) searches against the *UniProtKB/Swiss-Prot* database ([@B5]) and the *RefSeq* database ([@B22]). Annotation of resistance genes, mobile elements, and other features was carried out using the online databases including *CARD* ([@B16]), *ResFinder* ([@B37]), *ISfinder* ([@B31]), *INTEGRALL* ([@B21]), and *Tn Number Registry* ([@B29]). Multiple and pairwise sequence comparisons were performed using *MUSCLE* 3.8.31 ([@B9]) and *BLASTN*, respectively. Gene organization diagrams were drawn in *Inkscape* 0.48.1^[2](#footnote2){ref-type="fn"}^.

Plasmid Transfer {#S2.SS3}
----------------

Plasmid conjugal transfer experiments were carried out with the rifampin-resistant *E. coli* EC600 being used as a recipient and the 16005813 isolate as a donor, as described previously ([@B17]). Bacteria were spotted on Muller-Hinton agar (BD Biosciences, NJ, United States) plates containing 1000 μg/ml rifampin together with 10 μg/ml azithromycin, 2 μg/ml meropenem or 20 μg/ml tetracycline, for selecting an *E. coli* transconjugant that carried *mph* (A) (p16005813A), *bla*~IMP~ (B) (p16005813B), or *tet* (C) (p16005813C), respectively. Plasmid electroporation experiments were carried out as described previously ([@B23]). Bacteria were spotted on Super Optimal Broth agar plates containing 10 μg/ml azithromycin, 2 μg/ml meropenem or 20 μg/ml tetracycline, for selecting an *E. coli* electroporant that carried p16005813A, p16005813B, or p16005813C, respectively. Each transconjugant or electroporant was further confirmed by PCR detection of *rep* (replication initiation) and/or *bla*~IMP--8~ genes carried by relevant plasmids, followed by sequencing of PCR amplicons using the primers for PCR amplification ([Supplementary Table S1](#SM1){ref-type="supplementary-material"}).

Biochemical Assays and Antimicrobial Susceptibility Testing {#S2.SS4}
-----------------------------------------------------------

Activity of carbapenemases in bacterial cell extracts was determined by a modified Carba NP test ([@B10]). Bacterial antimicrobial susceptibility was tested by bioMérieux VITEK 2 and interpreted according to the Clinical and Laboratory Standards Institute (CLSI) guidelines.

Nucleotide Sequence Accession Numbers {#S2.SS5}
-------------------------------------

The complete sequence of plasmids p16005813A, p16005813B, and p16005813C, and that of the 16005813 chromosome, were submitted to GenBank under accession numbers [MK036891](MK036891), [MK036884](MK036884), [MK036885](MK036885), and [CP036199](CP036199), respectively.

Results and Discussion {#S3}
======================

Overview of Sequenced Plasmids {#S3.SS1}
------------------------------

The 16005813 isolate carried three plasmids p16005813A, p16005813B, and p16005813C, which exhibited circularly closed sequences 138,399 bp, 45,490 bp, and 61,463 bp in length and contained 149, 56, and 76 ORFs, respectively ([Table 1](#T1){ref-type="table"}). Each plasmid contained the backbone (responsible for plasmid replication and maintenance, and/or conjugal transfer), with insertion of a single accessory module (acquired DNA region associated and bordered with mobile elements) ([Table 1](#T1){ref-type="table"} and [Figure 1](#F1){ref-type="fig"}). A total of 18 non-redundant genes or gene loci, which were involved in resistance to β-lactams including carbapenems (*bla*~IMP--8~, *bla*~CTX--M--9~ and *bla*~TEM--1~), aminoglycosides (*aacC2, aacA4*, and *aadA2*), tetracyclines \[*tet* (C)\], chloramphenicol (*catA1* and *catB8*), macrolides \[*mph* (A)\], trimethoprim (*dfrA12*), sulfonamide (*sul1*), quaternary ammonium compounds (*qacED1*), chromate (*chrA*), tunicamycin (*tmrB*), copper (*cop*), mercury (*mer*) and arsenic ([@B37]), were found in these three plasmids ([Table 2](#T2){ref-type="table"}). The 16005813 chromosome carried several putative intrinsic resistance genes (including a fosfomycin resistance gene, and several gene loci encoding putative MDR proteins), but harbored none of accessory resistance regions ([Supplementary Table S2](#SM2){ref-type="supplementary-material"}).

###### 

Major features of plasmids analyzed.

  **Category**                  **Plasmids**              
  ----------------------------- -------------- ---------- ------------
  Incomparability group         Unknown        IncFII     IncR
  Total length (bp)             138,399        45,490     61,463
  Total number of ORFs          149            56         76
  Mean G + C content,%          52.5           49.3       53.2
  Length of the backbone (bp)   55,270         28,546     9,191
  Accessory module              MDR region     Tn*6505*   MDR region

![Plasmid schematic maps. Genes are denoted by arrows, and the backbone and accessory module regions are highlighted in black and color, respectively. The innermost circle presents GC-skew \[(G--C)/(G + C)\], with a window size of 500 bp and a step size of 20 bp. The next-to-innermost circle presents GC content.](fmicb-10-02468-g001){#F1}

###### 

Drug resistance genes in plasmids analyzed.

  **Plasmid**   **Resistance gene**   **Resistance phenotype**                  **Nucleotide position**                 **Region located**
  ------------- --------------------- ----------------------------------------- --------------------------------------- --------------------
  p16005813A    *bla*~TEM--1~         β-lactam resistance                       47687.48547 75823.76683 103910.104770   
                *mer* locus           Mercuric resistance                       50156.54118 78292.82254                 
                *mph(A)*              Macrolide resistance                      57038.57943 85174.86079                 
                *sul1*                Sulfonamide resistance                    64581.65420 92668.93507                 
                *qacED1*              Quaternary ammonium compound resistance   65414.65761 93501.93848                 MDR region
                *aadA2*               Aminoglycoside resistance                 65925.66704 94012.94791                 
                *chrA*                Chromate resistance                       61562.62767 89698.90902                 
                *dfrA12*              Trimethoprim resistance                   67124.67621 95211.95708                 
                *aacC2*               Aminoglycoside resistance                 69991.70851 98078.98938                 
                *tmrB*                Tunicamycin resistance                    70864.71406 98951.99493                 
                *sil* locus           Silver resistance                         33066.43739 104918.106694               Plasmid backbone
                *cop* locus           Copper resistance                         107992.115527                           
                *ars* locus           Arsenic resistance                        133081.135957                           
  p16005813B    *mer* locus           Mercuric resistance                       27907.31883                             Tn*6505*
                *bla*~IMP--8~         β-lactam resistance                       33644.34384                             
                *aacA4*               Aminoglycoside resistance                 34483.35037                             
  p16005813C    *mer* locus           Mercuric resistance                       16802.20808                             MDR region
                *catA1*               Chloramphenicol resistance                26717.27376                             
                *bla*~CTX--M--9~      β-lactam resistance                       33236.34087                             
                *sul1*                Sulfonamide resistance                    36384.37223                             
                *catB8*               Chloramphenicol resistance                37721.38353                             
                *aacA4*               Aminoglycoside resistance                 38765.39342                             
                *tet*(C)              Tetracycline resistance                   42678.43868                             
                                                                                                                        

p16005813A, p16005813B, and p16005813C could be transferred from the 16005813 isolate into TOP10 through electroporation, generating the electroporations p16005813A-TOP10, p16005813B-TOP10, and p16005813C-TOP10 ([Table 3](#T3){ref-type="table"}), respectively. Successful conjugation transfer of p16005813B, but not p16005813A and p16005813C, into EC600 generated the transconjugant p16005813B-EC600, which was consistent with the fact that only p16005813B carried a complete set of conjugal transfer genes. Strains 16005813, p16005813B-TOP10, and p16005813B-EC600 had metallo-β-lactamase activity and were resistant to ceftazidime and imipenem ([Table 3](#T3){ref-type="table"}), which was resulted from encoding of IMP enzyme by p16005813B.

###### 

Antimicrobial drug susceptibility profiles.

  **Antibiotics**                 **Minimum inhibitory concentration (mg/L)/antimicrobial susceptibility**                                              
  ------------------------------- -------------------------------------------------------------------------- -------- ------- ------- -------- -------- --------
  Cefazolin                       ≥64R                                                                       8R       ≥64R    ≥64R    ≥64R     ≤4S      ≤4S
  Cefuroxime                      ≥64R                                                                       4S       ≥64R    ≥64R    ≥64R     ≤4S      16I
  Ceftriaxone                     ≥64R                                                                       ≤1S      ≥64R    ≥64R    ≥64R     ≤1S      ≤1S
  Ceftazidime                     ≥64R                                                                       ≤1S      ≥64R    ≥64R    16R      ≤1S      ≤1S
  Imipenem                        8R                                                                         ≤1S      ≥16R    ≥16R    ≤1S      ≤1S      ≤0.25S
  Levofloxacin                    1S                                                                         ≤0.25S   1I      ≤0.5S   ≤0.25S   0.5S     ≤16S
  Gentamicin                      ≥16R                                                                       ≥16R     ≥16R    ≥16R    ≥16R     ≤1S      ≤1S
  Trimethoprim/sulfamethoxazole   ≥320R                                                                      ≥320R    ≥320R   ≥320R   ≤20S     ≤0.25S   ≤20S
  Tetracycline                    ≥16R                                                                       ≤1S      ≤1S     ≤1S     ≥16R     ≤1S      ≤1S

S, sensitive; R, resistant; I, intermediate.

Plasmid Backbone Regions {#S3.SS2}
------------------------

The p16005813A backbone showed very low levels of identity to all DNA sequences available in public databases. The replication initiation gene *repA* of p16005813A showed 100% BLAST coverage and 94% nucleotide identity to the counterparts in pLEC-5e18 (accession number [CP026390](CP026390)) and pLEC-000f (accession number [CP026170](CP026170)) from *L. adecarboxylata*; all these RepA proteins could not assigned into any of known incompatibility groups. A 121-bp region containing six imperfect direct repeats of GTGtGTcataacATG was located 89-bp upstream of *repA* of p16005813A, and might function as RepA-binding iterons. Key determinants for plasmid maintenance include a type Ia partitioning system ParABC, two distinct type II toxin-antitoxin systems VapBC and ParDE involved in post-segregational killing. Residual conjugal transfer determinants TrbA, MobC and mutated NikAB were found, which was consistent with the p16005813A's nature of not self-transmissible.

p16005813B and pECAZ161_KPC (accession number [CP019010](CP019010)) had essentially identical backbones and each of them carried a single accessory module, but the two accessory modules were different with respective to resistance genes and mobile elements harbored, and inserted at two different sites of the backbone. Key backbone genes of p16005813B include a novel replication initiation gene *repA* belonging to the IncFII family, *ccdBA* encoding a type II toxin-antitoxin system, and a set of P-type type IV secretion system genes (*tviF3/12/13/15/17*) involved in conjugal transfer.

The p16005813C backbone was composed of *repB* (replication initiation) and its iterons, *parAB* (type Ia partitioning), *umuCD* (SOS mutagenesis), and *resD* (multimer resolvase), but lacked *retA* (group IIB intron-encoding reverse transcriptase), and *vagCD* (toxin-antitoxin) compared to the IncR single-replicon reference plasmid pHN84KPC (accession number [KY296104](KY296104)) with the most complete IncR backbone. IncR single-replicon plasmids including p16005813 lacked conjugal transfer genes and thus were not self-transmissible ([@B7]; [@B8]).

The MDR Regions {#S3.SS3}
---------------

Each of p16005813A, p16005813B and p16005813C harbored a MDR region as the sole accessory module. The MDR region (83.1 kb in length, [Figure 2](#F2){ref-type="fig"}) of p16005813A was generated from insertion of a 61-kb region into a truncated (losing of *tnsABCD*) version of the *sil--cop* region, which was originally found in the IncHI2 reference plasmid R478 (accession number [BX664015](BX664015)) ([@B13]) and composed of a Tn*7*-like core transposition module *tnsABCD* together with the silver (*sil*) and copper (*cop*) resistance loci. Various derivatives of the *sil--cop* region were presented in IncHI2 plasmids ([@B35]; [@B19]). The 61-kb region comprised Tn*2*, and two 28-kb repeat regions, each of which harbored a *mer*-containing Tn*21* remnant + IS*26--mph*(A)--*mrx--mphR*(A)--IS*6100* unit + *chrA* region as found in pP10159-5 ([@B23]), In27, and *aacC2--tmrB* region as found in pA708-IMP (accession number [MF344567](MF344567)).

![Organization of the MDR region of p16005813A and comparison with related regions. Genes are denoted by arrows. Genes, mobile elements and other features are colored based on function classification. Shading denotes regions of homology (\>95% nucleotide identity). Numbers in brackets indicate e nucleotide positions within corresponding plasmids. The accession numbers of the *sil--cop* region from R478, Tn*21*, and Tn*2* for reference are [BX664015](BX664015), [AF071413](AF071413), and [HM749967](HM749967), respectively.](fmicb-10-02468-g002){#F2}

Tn*6505* (16.9 kb in length, [Figure 3](#F3){ref-type="fig"}) from p16005813B was a novel derivative of Tn*1696* ([@B26]) belonged to the Tn*21* subgroup of Tn*3* transposon family, and flanked by 5 bp direct repeats (DRs; target site duplication signals for transposition). Tn*6505* differed from Tn*1696* by (i) insertion of *bla*~IMP--8~-carrying In655 instead of In4 at the same position within the resolution (*res*) site, and (ii) interruption of IRL~Tn*6505*~ (inverted repeat right of Tn*6505*) by IS*5075* that was a hunter of terminal IRs of Tn*21* subgroup transposons ([@B27]).

![Organization of the MDR region (Tn*6505*) of p16005813B and comparison with related regions. Genes are denoted by arrows. Genes, mobile elements and other features are colored based on function classification. Shading denotes regions of homology (\>95% nucleotide identity). Numbers in brackets indicate e nucleotide positions within corresponding plasmids. The accession number of Tn*1696* for reference is [U12338](U12338).](fmicb-10-02468-g003){#F3}

The MDR region (52.2 kb in length, [Figure 4](#F4){ref-type="fig"}) from p16005813C carried at least four resistance modules, namely Tn*6322* ([@B35]), ΔTn*9* carrying *catA1* ([@B1]), In609, IS*26--tetA*(C)--*tetR*(C)--IS*26* unit ([@B35]). Tn6*322* was composed of a Tn*21* core transposition module *tnpAR--res* together with a *mer* locus, and its IRL was interrupted by IS*4321R* (a homolog of IS*5075*). In609 was a complex class 1 integron carried variable region 1 (VR1: gene cassette *gcuD2*--*aacA4′-17*--*gcuE14*--*catB8*) and VR2 (*bla*~CTX--M--9~).

![Organization of the MDR region of p16005813C and comparison with related regions. Genes are denoted by arrows. Genes, mobile elements and other features are colored based on function classification. Shading denotes regions of homology (\>95% nucleotide identity). Numbers in brackets indicate e nucleotide positions within corresponding plasmids. The accession numbers of Tn*9* and IS*Pa38* for reference are [LN830952](LN830952), and [MH909331](MH909331), respectively.](fmicb-10-02468-g004){#F4}

Conclusion {#S4}
==========

This is the first report of coexistence of three different MDR plasmids, and that of occurrence of IMP-encoding plasmid and *bla*~IMP--8~ gene in *L. adecarboxylata*. *bla*~IMP--8~ was located in In655, representing an ancestral Tn*402*-associated integron (containing a complete *tni*~*Tn*~*~402~* core transposition module) at stage I of evolution as defined previously ([@B17]). The three co-existent MDR plasmids carried a large amount of resistance genes, making the relevant *L. adecarboxylata* isolate tend to be extensively drug resistant. Epidemiological investigation of multi-drug resistant *L. adecarboxylata* needed to be carried out in China.

Data Availability Statement {#S5}
===========================

The datasets generated for this study can be found in the complete sequence of plasmids p16005813A, p16005813B, and p16005813C, and that of the 16005813 chromosome, were submitted to GenBank under accession numbers [MK036891](MK036891), [MK036884](MK036884), [MK036885](MK036885), and [CP036199](CP036199), respectively.

Ethics Statement {#S6}
================

This study uses the clinical bacterial isolate obtained from a public hospital in Ningbo, China. The study needs not to be reviewed or approved by the ethics committee of the hospital, because the bacterial isolate involved in this study was part of the routine hospital laboratory procedure. The research involving biohazards and all related procedures were approved by the Biosafety Committee of the Beijing Institute of Microbiology and Epidemiology.

Author Contributions {#S7}
====================

DZ and ED conceived the study and designed experimental procedures. ZY, LH, QC, XJ, and YX performed the experiments. ZY, LH, DZ, and ED analyzed the data. WY, HY, YZ, BG, and JW contributed to reagents and materials. DZ, ZY, LH, and ED wrote the manuscript.

Conflict of Interest {#conf1}
====================

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

**Funding.** This work was supported by the National Science and Technology Major Project (2018ZX10733402) of China and the Foundation of State Key Laboratory of Pathogen and Biosecurity (SKLPBS1810) of China.

<https://github.com/ruanjue/smartdenovo>

<https://inkscape.org/en/>

Supplementary Material {#S9}
======================

The Supplementary Material for this article can be found online at: <https://www.frontiersin.org/articles/10.3389/fmicb.2019.02468/full#supplementary-material>

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Click here for additional data file.

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Click here for additional data file.

[^1]: Edited by: Costas C. Papagiannitsis, University of Thessaly, Greece

[^2]: Reviewed by: Ibrahim Bitar, Charles University, Czechia; Jaroslav Hrabak, Charles University, Czechia

[^3]: ^†^These authors have contributed equally to this work

[^4]: This article was submitted to Antimicrobials, Resistance and Chemotherapy, a section of the journal Frontiers in Microbiology
