Dissemination of KPC-2-Encoding IncX6 Plasmids Among Multiple Enterobacteriaceae Species in a Single Chinese Hospital

Forty-five KPC-producing Enterobacteriaceae strains were isolated from multiple departments in a Chinese public hospital from 2014 to 2015. Genome sequencing of four representative strains, namely Proteus mirabilis GN2, Serratia marcescens GN26, Morganella morganii GN28, and Klebsiella aerogenes E20, indicated the presence of blaKPC-2-carrying IncX6 plasmids pGN2-KPC, pGN26-KPC, pGN28-KPC, and pE20-KPC in the four strains, respectively. These plasmids were genetically closely related to one another and to the only previously sequenced IncX6 plasmid, pKPC3_SZ. Each of the plasmids carried a single accessory module containing the blaKPC-2/3-carrying ΔTn6296 derivatives. The ΔTn6292 element from pGN26-KPC also contained qnrS, which was absent from all other plasmids. Overall, pKPC3_SZ-like blaKPC-carrying IncX6 plasmids were detected by PCR in 44.4% of the KPC-producing isolates, which included K. aerogenes, P. mirabilis, S. marcescens, M. morganii, Escherichia coli, and Klebsiella pneumoniae, and were obtained from six different departments of the hospital. Data presented herein provided insights into the genomic diversity and evolution of IncX6 plasmids, as well as the dissemination and epidemiology of blaKPC-carrying IncX6 plasmids among Enterobacteriaceae in a hospital setting.


INTRODUCTION
Klebsiella pneumoniae carbapenamase (KPC), a class A β-lactamase, can hydrolyze almost all β-lactams, including carbapenems (Bush and Fisher, 2011). At least 31 variants (KPC-2 to KPC-32; KPC-1 is essentially identical to KPC-2) of the KPC enzyme have been identified to date 1 . Two Tn3-family unit transposons, Tn4401 and Tn6296, which are genetically divergent from each other, represent the two major prototype genetic platforms carrying bla KPC genes (Wang et al., 2015). Tn4401 and its derivatives are frequently identified in KPC-encoding plasmids of different incompatibility groups in bacterial isolates from European and American countries, but are rarely found in isolates from China . The bla KPC genetic environment in isolates from China is predominantly associated with Tn6296 and its derivatives (Wang et al., 2015).
Plasmids belonging to incompatibility group X (IncX) are 30-80 kb in size and were initially discovered in the pre-antibiotic era (Datta and Hughes, 1983). IncX plasmids have a narrow host range and are mainly circulated among Enterobacteriaceae species (Norman et al., 2008). The backbones of all IncX plasmids have a pir-parA-hns-hha-topB-pilX (tivB)-actX-taxC (rlx)-taxA (dtr) organization, but are quite divergent with respect to nucleotide and amino acid sequences similarity (Johnson et al., 2012). Comparative genomic analysis has shown that IncX plasmids can be phylogenetically grouped into seven major IncX subgroups, IncX1 to IncX6 (Du et al., 2016), along with another IncX6 subgroup (Bustamante and Iredell, 2017) that is re-designated herein IncX7.
This study provides evidence for the dissemination of genetically highly similar KPC-2-encoding IncX6 plasmids among at least six Enterobacteriaceae species collected in a Chinese public hospital from 2014 to 2015. The complete nucleotide sequences of plasmids pGN2-KPC, pGN26-KPC, pGN28-KPC, and pE20-KPC, extracted from strains belonging to four representative species, were determined to be genetically closely related to the IncX6 reference plasmid pKPC3_SZ. In addition, all five plasmids carried a single accessory region that harbored the bla KPC-2/3 gene.

Bacterial Identification
Bacterial species identification was performed on the basis of 16S rRNA gene sequencing (Frank et al., 2008). The major plasmidborne carbapenemase and extended-spectrum β-lactamase genes were screened for by PCR (Chen et al., 2014). All PCR amplicons were sequenced using an ABI 3730 Sequencer (Life Technologies, Carlsbad, CA, United States) using the primers used for PCR.

Plasmid Transfer
Plasmid conjugal transfer experiments were carried out using rifampin-resistant Escherichia coli strain EC600 (LacZ-, NalR, RifR) as the recipient and each of Proteus mirabilis GN2, Serratia marcescens GN26, Morganella morganii GN28, and Klebsiella aerogenes E20 as the donor (Feng et al., 2016). Aliquots (3 ml) of overnight cultures of each of the donor and recipient strains were mixed together, harvested, and resuspended in 80 µl of brain heart infusion broth (BD Biosciences, Franklin Lakes, NJ, United States). The mixture was spotted onto a 1-cm 2 hydrophilic nylon membrane filter with a 0.45-µm pore size (Millipore, Billerica, MA, United States) placed onto the surface of a brain heart infusion agar (BD Biosciences, Franklin Lakes, NJ, United States) plate. Plates were incubated for mating at 37 • C for 12-18 h. Bacteria were washed from the filter membrane and spotted on Mueller-Hinton agar (BD Biosciences, Franklin Lakes, NJ, United States) plates containing 1 mg/ml rifampin and 2 µg/ml imipenem to select the transconjugants containing the bla KPC marker.

Phenotypic Assays
Activity of Ambler class A/B/D carbapenemases in bacterial cell extracts was determined by a modified CarbaNP test (Feng et al., 2016). Bacterial antimicrobial susceptibility was examined using the broth dilution method, and interpreted as per the Clinical and Laboratory Standards Institute guidelines (CLSI, 2015).

Genomic DNA Sequencing and Sequence Assembly
Genomic DNA was isolated from Enterobacteriaceae isolates GN2, GN26, GN28, and E20 using a Blood and Cell Culture DNA Maxi Kit (Qiagen, Hilden, Germany). Genome sequencing was performed for isolate GN2 using a sheared DNA library with an average size of 15 kb (ranging from 10 to 20 kb) on a PacBio RSII sequencer (Pacific Biosciences, Menlo Park, CA, United States), as well as with a paired-end library with an average insert size of 400 bp (ranging from 150 to 600 kb) on a HiSeq sequencer (Illumina, San Diego, CA, United States). The paired-end short Illumina reads were used to correct the long PacBio reads using proovread (Hackl et al., 2014), then the corrected PacBio reads were assembled de novo using SMARTdenovo 2 .
Genomic DNA from isolates GN26, GN28, and E20 was sequenced from a mate-pair libraries with an average insert size of 5 kb (ranging from 2 to 10 kb) using a MiSeq sequencer (Illumina, San Diego, CA, United States). DNA contigs that were not matched with the reference chromosome sequences of S. marcescens (GenBank accession number HG738868), M. morganii (GenBank accession number CP023505) or K. aerogenes (GenBank accession number FO203355) were assembled based on their contig coverage values using Newbler 2.6 (Nederbragt, 2014). Gaps between contigs were filled using a combination of PCR and Sanger sequencing using an ABI 3730 Sequencer.
These 45 bla KPC -carrying isolates consisted of K. pneumonia (n = 31), K. aerogenes (n = 6), S. marcescens (n = 5), and one isolate each of M. morganii, E. coli, and P. mirabilis. In total, 36 of the isolates were recovered from sputum specimens, while the remaining isolates were obtained from urine specimens. 3 https://inkscape.org/en/ The 45 isolates came from 10 different hospital departments: 25 from the Intensive Care Unit, 6 from the Department of Gerontology, 5 from the Department of Respiratory Medicine, 2 from the Department of Neurology, 2 from the Department of Urology, and 1 each from the Department of General Surgery, the Department of Neurosurgery, the Emergency Department, the Department of Endocrinology, and the Department of Traditional Chinese Medicine (Supplementary Table S1). Thirty-six (80.0%) of the 45 isolates carried one or more β-lactamase genes [bla TEM , bla SHV , bla CTX-M-1G (Group) , bla CTX-M-9G , bla OXA-1 and bla OXA-2 ] in addition to bla KPC .

pKPC3_SZ-Like IncX6 Plasmids From bla KPC-2 -Carrying Isolates
Four bla KPC -positive isolates, P. mirabilis GN2, S. marcescens GN26, M. morganii GN28, and K. aerogenes E20, were arbitrarily selected for genome sequencing. GN2 was isolated from the urine specimens of an elderly female with urinary tract infection in 2014, while GN26 and GN28 (in 2015) and E20 (in 2014) were isolated from sputum specimens from three different elderly males suffering from pulmonary infections. These four patients were admitted to the hospital because of primary diseases consisting of myocardial infarction, cerebral infarction sequelae, cerebral contusion and pneumonia, respectively, and developed the above hospital-acquired infections during hospitalization.
GN2, GN26, GN28, and E20 each contained an IncX6 plasmid, designated pGN2-KPC, pGN26-KPC, pGN28-KPC, and pE20-KPC, respectively. These plasmids were 45.6-46.3 kb in length, with 62-65 predicted ORFs ( Table 1). The modular structure of each plasmid was divided into the backbone regions along with a single accessory module, which was defined as an acquired DNA region associated with mobile elements, and was inserted into the backbone (Figure 1 and Supplementary Figure S1). A total of three resistance genes were identified: bla KPC-2 was located in all four plasmids, while bla TEM-1 was identified in pGN2-KPC, pGN26-KPC, and pGN28-KPC, and qnrS1 was found in pGN26-KPC. All these resistance genes were located in the accessory modules.
bla KPC -positive E. coli transconjugants GN2-KPC-EC600, GN26-KPC-EC600, GN28-KPC-EC600, and E20-KPC-EC600. Class A carbapenemase activity was detected for all transconjugants, and resulted from the production of the KPC-2 enzyme. Both the wild-type and transconjugant strains were resistant to ampicillin, cefepime, meropenem, and aztreonam. Moreover, GN26 was resistant to ciprofloxacin, but its transconjugant was intermediately resistant to this drug due to the presence of qnrS1 known to mediate the low-level resistance to fluoroquinolones ( Table 2). In conclusion, each of GN2, GN26, GN28, and E20 harbored a conjugative bla KPC -carrying IncX6 plasmid, which accounted for the carbapenem resistance phenotype. Based on the complete sequences of the five IncX6 plasmids (pGN2-KPC, pGN26-KPC, pGN28-KPC, pE20-KPC, and pKPC3_SZ), a total of nine genes were arbitrarily selected to screen for the prevalence of pKPC3_SZ-like IncX6 plasmids among the 45 bla KPC -positive isolates. Of these nine selected genes, eight [replication initiation: repA (replication initiation protein); maintenance: parA (partitioning ATPase), topB (type III topoisomerase), dnaJ (molecular chaperone), and ftsH (cell division protein); conjugal transfer: tivB3-4 (P-type type IV secretion, inner-membrane component of translocation channel and ATPase), tivB6 (P-type type IV secretion, inner-membrane component of translocation channel), and tivB10 (P-type type IV secretion, outer-membrane component of translocation channel)] were from backbone regions, while the remaining one was quinolone-resistance gene qnrS1. PCR analysis and amplicon sequencing showed that all eight backbone genes were present in 24 isolates, including 11 K. pneumoniae isolates, all 6 K. aerogenes isolates, 4 S. marcescens isolates, and 1 isolate  each of M. morganii, E. coli, and P. mirabilis, indicating that these isolates harbored IncX6 plasmids. All five replication and maintenance genes, but none of the three conjugal transfer genes, were detected in another S. marcescens isolate, probably indicating that this isolate contained an IncX6 plasmid that had lost the conjugal transfer genes. None of the eight selected genes were detected in the remaining 20 isolates, signifying that these isolates did not carry IncX6 plasmids. qnrS1 was detected in 26 K. pneumoniae isolates, 6 K. aerogenes isolates, and 4 S. marcescens isolates, denoting coexistence of bla KPC and qnrS1 in these isolates.

Genomic Comparison of IncX6 Plasmids
pGN2-KPC, pGN26-KPC, pGN28-KPC, and pE20-KPC showed the highest sequence identity to the IncX6 reference plasmid pKPC3_SZ (Du et al., 2016), with >92% query coverage and >99% nucleotide identity. The major backbone genes or gene loci included repA and its iterons (replication initiation), parA and topB-hha-hns (maintenance), and rlx, dtr, tivB, cpl, and eex (conjugal transfer). repA coded for the IncX6-specific replication initiation protein and was not identified in any other available sequences. A 253-bp region containing seven imperfect GGTTTTTAAATCCCGata direct repeats was located 73-bp upstream of repA, and may function as iterons that bind the RepA protein. ParA was the partitioning ATPase responsible for plasmid segregation and stability (Schumacher, 2008), however, centromere-binding protein ParB and its binding sites parC could be not located. The gene expression modulation (gem) region, composed of topB, hha (transcriptional regulator), and hns (histone-like DNA-binding protein), was involved in plasmid maintenance (Norman et al., 2008). The conjugal transfer region was composed of a complete set of P-type conjugative DNA transfer genes, including rlx and dtr (DNA transfer; encoding relaxase Rlx and an auxiliary protein, Dtr), tivB1-tivB11 (encoding P-type type IV secretion system elaborating the pilus for mating pair formation), cpl (encoding a coupling protein that links DNA transfer and mating pair formation), and eex (entry exclusion preventing nucleoprotein transport between donors) (De La Cruz et al., 2010;Chen et al., 2013;Thomas et al., 2017). The backbones of these five plasmids displayed only two major modular differences (Figure 1): (i) a 128-bp duplication in cpl of pGN2-KPC resulted in frameshift mutation, turning cpl into a pseudogene but retaining the conjugal transfer ability of pGN2-KPC, and (ii) a 71-bp deletion within orf393 (coding for an XRE-family Helix-turn-helix protein) was identified in pGN28-KPC, again causing the hypothetical gene orf393 to become a pseudogene.
The Tn6296 derivatives from these five plasmids were slightly different from each other, with deletions and insertions relative to the prototype Tn6296 (Figure 2 and Supplementary  Table S2). First, Tn1722-5 was lost from all five Tn6296 elements. Second, a 70-bp deletion within tnpA of Tn6376 and a 70-bp deletion within tnpA of ISKpn6 were found in pE20-KPC and pGN26-KPC, respectively, leading to frameshift mutations of these two coding regions. Third, the insertion of a 624-bp bla TEM-1 -containing region between ISKpn27 and bla KPC-2/3 was identified in pGN26-KPC, pGN2-KPC, pGN28-KPC, and pKPC3_SZ. Two promoters, consisting of the intrinsic P1 promoter and an upstream Tn6376-provided P2 promoter, were found to govern the bla KPC-2 expression of Tn6296 (Wang et al., 2015). The insertion of the bla TEM-1 -containing region resulted in the loss of IRL Tn6376 and P1 (Supplementary Figure  S2), leaving P2 as the only promoter for bla KPC expression. Finally, the 3 -terminal regions of these five Tn6296 derivatives were truncated in different formats: (i) Tn1722-3 was absent in each of pE20-KPC, pGN2-KPC, and pGN28-KPC due to connection of ISKpn19; (ii) in pGN26-KPC, the qnrS1-containing Tn6292 element (6.4 kb in length) was connected with a 1.6-kb Tn6376 remnant with deletion of IRL-tnpA-res and truncation of tnpR, and the introduction of Tn6292 into pGN26-KPC probably resulted from homologous recombination between Tn6292 and a pre-existing ISKpn19 element (as observed in pE20-KPC, pGN2-KPC, and pGN28-KPC), with ISKpn19 acting as the common region necessary for recombination; and (iii) Tn1722-3' was also lost in pKPC3_SZ, although none of the ISKpn19-related elements or any other regions were found to be adjacent to the 3 -end of Tn6296.
In summary, complex transposition and homologous recombination events, particularly those involving the three prototype mobile elements Tn6296, Tn6292, and ISKpn19, occurred to promote the assembly and mobilization of the bla KPC regions in these plasmids.
Previously sequenced IncX plasmids mostly belong to the IncX1-IncX4 subgroups, with very few representatives of IncX5-IncX7 plasmids. Currently, only five IncX6 plasmids have been fully sequenced, including pKPC3_SZ (Du et al., 2016) and the pGN2-KPC, pGN26-KPC, pGN28-KPC, and pE20-KPC plasmids sequenced in the current study. The five plasmids all originate from clinical isolates belonging to different Enterobacteriaceae species, namely E. cloacae, P. mirabilis, S. marcescens, M. morganii, and K. aerogenes, respectively, all of which come from China. Each of these five IncX6 plasmids contains a single accessory module containing two or three resistance genes, with all five carrying bla KPC-2/3 , pKPC3_SZ, pGN2-KPC, and pGN28-KPC harboring bla TEM-1 , and pGN26-KPC containing both bla TEM-1 and qnrS1. None of these IncX6 plasmids encodes multi-drug resistance. IncX6 plasmids appear to be an important vehicle for bla KPC genes in China, and the core genetic environments of bla KPC genes are close derivatives of Tn6296.
Genomic and epidemiological analyses herein show that bla KPC -carrying IncX6 plasmids are present in 44.4% of the analyzed bla KPC -positive isolates from a single hospital and have disseminated among at least six different Enterobacteriaceae species from six distinct departments of this hospital, indicating wide spread of these plasmids in this hospital. Further studies are needed to determine the prevalence of IncX6 plasmids among various geographic areas to understand the contribution of IncX6 plasmids to bla KPC epidemiology among Enterobacteriaceae isolates.

SUPPLEMENTARY MATERIAL
The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fmicb. 2018.00478/full#supplementary-material FIGURE S1 | Schematic maps of IncX6 plasmids. Genes are denoted by arrows, and the backbone and accessory module regions are highlighted in black and gray, 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.
FIGURE S2 | Alignment of promoter-proximal regions of bla KPC-2 . The 898-354 bp upstream sequences together with the start codon of the bla KPC-2 genes from IncX6 plasmids and Tn6296 are aligned by MUSCLE. Shown are core promoter regions, −35 and −10 elements, transcription starts, Shine-Dalgarno (SD) sequences for ribosome recognition and translation starts.