Complete sequences of KPC-2-encoding plasmid p628-KPC and CTX-M-55-encoding p628-CTXM coexisted in Klebsiella pneumoniae

A carbapenem-resistant Klebsiella pneumoniae strain 628 was isolated from a human case of intracranial infection in a Chinese teaching hospital. Strain 628 produces KPC-2 and CTX-M-55 encoded by two different conjugative plasmids, i.e., the IncFIIK plasmid p628-KPC and the IncI1 plasmid p628-CTXM respectively. blaKPC−2 is captured by a Tn1722-based unit transposon with a linear structure. ΔTn3-ISKpn27-blaKPC−2-ΔISKpn6-ΔTn1722 and this transposon together with a mercury resistance (mer) gene locus constitutes a 34 kb acquired drug-resistance region. blaKPC−2 has two transcription starts (nucleotides G and C located at 39 and 250 bp upstream of its coding region respectively) which correspond to two promoters, i.e., the intrinsic P1 and the upstream ISKpn27/Tn3-provided P2 with the core −35/−10 elements TAATCC/TTACAT and TTGACA/AATAAT respectively. blaCTX−M−55 is mobilized in an ISEcp1-blaCTX−M−55-Δorf477 transposition unit and appears to be the sole drug-resistant determinant in p628-CTXM. blaCTX−M−55 possesses a single transcription start (nucleotides G located at 116 bp upstream of its coding region) corresponding to the ISEcp1-provided P1 promoter with the core −35/−10 element TTGAAA/TACAAT. All the above detected promoters display a characteristic of constitutive expression. Coexistence of blaKPC and blaCTX−M in K. pneumoniae has been reported many times but this is the first report to gain deep insights into genetic platforms, promoters, and expression of the two coexisting bla genes with determination of entire nucleotide sequences of the two corresponding plasmids.


Introduction
KPC-producing Klebsiella pneumoniae has spread worldwide and became an emerging pathogen with serious clinical and infection control implications (Tzouvelekis et al., 2012;Munoz-Price et al., 2013). Coexistence of bla KPC and bla CTX−M in K. pneumoniae has been reported in several countries, such as bla KPC−2 /bla CTX−M−1 group, bla KPC−2 /bla CTX−M−2 group, and bla KPC−2 /bla CTX−M−8 group in Brazil (Peirano et al., 2009), bla KPC−2 /bla CTX−M−10 , bla KPC−2 /bla CTX−M−15 , and bla KPC−3 /bla CTX−M−2 in Israel (Leavitt et al., 2007(Leavitt et al., , 2010, bla KPC−2 /bla CTX−M−14 in China (Cai et al., 2008), and bla KPC−2 /bla CTX−M−15 in Greece (Souli et al., 2010). However, all these studies are confined to PCR detection and sequencing of bla genes, lacking deeper characterization of mechanisms of drug resistance. This study describes co-production of KPC-2 and CTX-M-55 in a clinical K. pneumoniae strain 628 from China. The bla KPC−2 and bla CTX−M−55 genes are encoded by two different conjugative plasmids, p628-KPC and p628-CTXM respectively. The complete nucleotide sequences of p628-KPC and p628-CTXM are determined and then compared with other genetically closely related plasmids to gain deep insights into genetic structures of relevant plasmids and resistance gene loci. In addition, the promoters and their expression characteristics of these two plasmid-borne bla genes are dissected experimentally.

Bacterial Strains and Identification
K. pneumoniae strain 628 was isolated from the cerebrospinal fluid specimen of a 64-year-old male with intracranial infection in a Chinese teaching hospital in October 2010. Bacterial species identification was performed using Bruker MALDI Biotyper (Bruker Daltonics, Bremen, Germany) and 16s rRNA gene sequencing (Frank et al., 2008). The major carbapenemase and extended-spectrum beta-lactamase (ESBL) genes were detected by PCR, followed by sequencing on an ABI Sequencer (Applied Biosystems, Foster City, CA, USA) (Chen et al., 2015). Bacterial antimicrobial susceptibility was tested by using VITEK 2 and judged by CLSI standard (CLSI, 2012).

Plasmid Transfer
Plasmid conjugal transfer experiments were carried out with Escherichia coli EC600 (LacZ − , Nal R , Rif R ) being used as recipient and strain 628 as donor. Three milliliter of overnight culture of each of donor and recipient bacteria were mixed together, harvested and resuspended in 80 µl of Brain Heart Infusion broth (BD Biosciences, San Jose, CA, USA). The mixture was spotted on a 1 cm 2 filter membrane that was placed on Brain Heart Infusion agar (BD Biosciences, San Jose, CA, USA) plate, and then incubated for mating at 37 • C for 12 to 18 h. Bacteria were washed from filter membrane and spotted on Muller-Hinton agar (BD Biosciences, San Jose, CA, USA) plate containing 1000 mg/L rifampin (Merck, Darmstadt, Germany) and 200 mg/L ampicillin (Merck, Darmstadt, Germany) for selection of bla CTX−M -or bla KPC -positive E. coli transconjugants.

Determination of Plasmid DNA Sequence
Plasmid DNA was isolated from the cell culture of E. coli transconjugant using Qiagen large construct kit (Qiagen, Hilden, Germany) and then sequenced by using whole-genome shotgun strategy in combination with Illumina HiSeq 2500 (Illumina, San Diego, CA, USA) sequencing technology. The contigs were assembled with Velvet and the gaps were filled through combinatorial PCR and Sanger Sequencing on ABI 3730 Sequencer. The genes were predicted with GeneMarkS ™ and further annotated by BLASTP and BLASTN against UniProt and NR databases.

RNA Isolation and Primer Extension Assay
Bacteria were cultured overnight in Mueller-Hinton broth (BD Biosciences, San Jose, CA, USA). Total RNAs were extracted from harvested bacterial cells using TRIzol Reagent (Life Technologies, Carlsbad, CA, USA). RNA quality was monitored by agarose gel electrophoresis, and RNA quantity was determined by spectrophotometry. Each of the [γ-32 P] ATP end-labeled primers GCTCAGTGGAACGAAAAC, AGCCGCCAAAGTCCTGTTCG, and CATGGGATTCCTTATT CTG, which corresponded to bla KPC−2 promoter P2, bla KPC−2 promoter P1, and bla CTX−M−55 promoter P1 respectively, was annealed with total RNA sample for primer extension assay as described previously (Zhang et al., 2011). For different cell cultures in a single experiment, equal amounts of total RNA were used as starting materials. The corresponding end-labeled primers were also used for sequencing the PCR amplicons generated by the primer pairs TCAGCGACATCGTCAACC/GGTCGTGTTTCCCTTTAGCC, TCAGGTGGCACTTTTCGG/GGTCGTGTTTCCCTTTAGCC, and AGACCTTTCGTTTGAAGTATG/AGCTTATTCATCGCC ACGTT for bla KPC−2 promoter P2, bla KPC−2 promoter P1, and bla CTX−M−55 promoter P1 respectively. DNA sequencing was carried out using AccuPower & Top DNA Sequencing Kit (Bioneer, Daejeon, Korea). Primer extension products and sequencing materials were analyzed on 8 M urea-6% polyacrylamide gel electrophoresis. Radioactive species were detected by autoradiography.

Nucleotide Sequence Accession Numbers
The complete sequences of plasmids p628-KPC and p628-CTXM were submitted to GenBank under accession numbers KP987218 and KP987217 respectively.

Complete Nucleotide Sequence of p628-KPC
The entire nucleotide sequence of p628-KPC is 105,008 bp in length, forming a circular plasmid with an average G+C content of 53.22 and a total of 127 open reading frames (ORFs) annotated  ( Figure 1A). p628-KPC belongs to the IncFII K incompatibility group and harbors IncFII K repA and the second IncFIB-like repA2, both of which encode replication initiation proteins.

Complete Nucleotide Sequence of p628-CTXM
The p628-CTXM genome consists of an 85,338 bp circular DNA molecule with an average G+C content of 49.71 and harbors a total of 92 ORFs annotated ( Figure 1B). p628-KPC belongs to the IncI1 incompatibility group expressing the replication initiation protein RepZ. The p628-CTXM backbone, 82,357 bp in length, contains DNA regions for plasmid replication (repY, repZ, and inc), conjugal transfer (tra, trb, pil, etc) and transfer leading (imp, yfa to yfh, yga to ygg, etc), which show >98% sequence identity to the corresponding regions of the IncI1 plasmids R64 from Salmonella enterica serovar Typhimurium (Sampei et al., 2010), pKHSB1 from Shigella sonnei (Holt et al., 2013) and pEK204 from E. coli O25:H4-ST131 clone (Woodford et al., 2009) (Figure 2B). Another backbone component is the plasmid stability region, composed of three genes yafA, yafB, and yagA, which is highly conserved among p628-CTXM, pKHSB1, and pEK204; by contrast, the corresponding segment of R64 is a 17.8 kb region which harbors at least 18 genes and especially include those encoding site-specific recombination (resD and yefA) and partition (parAB) of replicated DNA into daughter cells during cell division (Sampei et al., 2010) (Figure 2B).
p628-CTXM harbors a single accessory module, a 2980 bp ISEcp1-related element, which interrupts yagA (a member of the plasmid stability region) (Figures 1B, 2B). Two distinct Tn3-related elements, 7935 and 8014 bp in length, are inserted downstream of yagA in pKHSB1 and pEK204 respectively. There are still additional accessory modules including ISCro1 and IS421 for pKHSB1, and IS66 for pEK204.
Genetic Surroundings of bla  As characterized in European and American countries, the bla KPC genes are located in a Tn3-family transposon named Tn4401, which is present on a wide variety of plasmids varying in size, structure and replicon (Naas et al., 2008;Kitchel et al., 2009Kitchel et al., , 2010Chen et al., 2012;Bryant et al., 2013;Chmelnitsky et al., 2014). At least eight isoforms of Tn4401 have been named, i.e., Tn4401a to Tn4401g and a separate Tn4401d (Table S1 in Supplementary Material). Several unnamed Tn4401 isoforms have been also reported recently (Cuzon et al., 2011;Li et al., 2011;Ho et al., 2013b;Naas et al., 2013;Perez-Chaparro et al., 2014). Tn4401b is considered as the prototype one, and the other isoforms result from occurrence of distinct deletion or insertion events at different sites.

Genetic Surroundings of bla CTX−M−55
R64, p628-CTXM, pKHSB1, and pEK204 carry a 17 kb IS2-based mobile element, a 2980 bp ISEcp1-based transposition unit, a 7935 bp Tn3-based element, and an 8014 bp Tn3-based element respectively; each of them is the sole determinant for antibiotics resistance of the corresponding plasmid (Figure 4). For R64, stepwise insertions occur to eventually assemble the IS2-based element: insertion of IS2 into arsA1, that of Tn6082 into IS2, that of IS1133 into Tn6082, and finally that of Tn10 into IS1133; the tet locus carried by Tn10 and the strAB operon carried by Tn6082 account for resistance to tetracycline and streptomycin respectively.
A  2006; Zong et al., 2010). In p628-KPC, the plasmid backbone gene yagA is disrupted by ISEcp1-bla CTX−M−55 -orf477. In pKHSB1 and pEK204, a bla TEM−1 -carrying Tn3 transposon is inserted at the site downstream of yagA and the Tn3 tnpA gene is further disrupted by ISEcp1-bla CTX−M−15 -orf477 and ISEcp1-bla CTX−M−3 -orf477, respectively. In addition, these two inserted ISEcp1-based structures differ from each other with respect to targeting sites and oriented directions (Figure 4).

Expression of bla KPC−2 and bla CTX−M−55
Each of the bla KPC−2 genes in Tn4401a, b, d, f, and g has two transcription starts, i.e., nucleotides G and C located at 39 and 289 bp upstream of bla KPC−2 , which correspond to the two promoters P1 and P2 (re-designated P2 ISKpn7 herein) with core −35/−10 elements TAATCC/TTACAT and TTGACA/TATCTT respectively (Naas et al., 2012). By contrast, bla KPC−2 from Tn4401c or e has only P1, while P2 ISKpn7 is absent due to presence of 215 or 255 bp deletion within bla KPC−2 upstream region respectively (Naas et al., 2012).
In this work, the primer extension assay detected two transcription starts, i.e., nucleotides G and C located at 39 and 250 bp upstream of bla KPC−2 Tn1722−based from p628-KPC respectively; the corresponding two promoters were designated P1 and P2 ISKpn27/Tn3 with the core −35/−10 elements TAATCC/TTACAT and TTGACA/AATAAT respectively (Figures 5, 6). The first 74 bp fragments upstream of bla KPC−2 Tn4401b and bla KPC−2 Tn1722−based are essentially identical; the P1 promoter is located within this 74 bp region and thereby shared by bla KPC−2 Tn4401b and bla KPC−2 Tn1722−based (Figure 6). The next 280 bp region upstream of the above 74 bp fragment for bla KPC−2 Tn4401b is dramatically divergent at nucleotide level from the counterpart for bla KPC−2 Tn1722−based ; these two distinct 280 bp regions contain P2 ISKpn7 and P2 ISKpn27/Tn3 respectively. The −35 element of P2 ISKpn7 is provided by ISKpn7 inserted at 319 bp upstream of bla KPC−2 Tn4401b , while the −35 and −10 elements of P2 ISKpn27/Tn3 are provided by ISKpn27 and Tn3 inserted at 281 and 75 bp upstream of bla KPC−2 Tn1722−based respectively (Figure 6). moreover, the ISEcp1-provided promoter P1 is stronger and more important than the intrinsic P2 promoter in the 127 bp spacer (Ma et al., 2011). The above result is applicable to the bla CTX−M−55 genes with the 127 bp spacer (Figure 7), because their ISEcp1+spacer region is identical to the counterpart of bla CTX−M−3 AF550415 . In the present study, the primer extension assays detected a transcription start, i.e., nucleotides G located at 116 bp upstream of bla CTX−M−55 (Figure 5) (Figure 7). In addition, the primer extension assay showed that addition of increasing amounts of imipenem or ampicillin during cultivation of indicated strains 628, 628-KPC-EC600 and 628-CTXM-EC600 had no effect on activity of all the above promoters detected for bla KPC−2 or bla CTX−M−55 , denoting constitutive expression of the above two resistance genes (Figure 5).

Concluding Remarks
KPC-2 and CTX-M-55 enzymes are produced by two different conjugative plasmids, p628-KPC and p628-CTXM respectively, in K. pneumoniae strain 628, and the sequences of these two plasmids are >98% identical to other relevant plasmids carrying the same resistance determinants previously sequenced. The detected bla KPC−2 gene is captured by a Tn1722-based unit transposon carried by an IncFII K -type multi-drugresistant plasmid p628-KPC, and this gene has two different promoters, the intrinsic P1 and the ISKpn27/Tn3-provided P2, both characteristic of constitutive expression. The detected bla CTX−M−55 gene, being the sole drug-resistant determinant in the plasmid, is mobilized in an ISEcp1-based transposition unit carried by an IncI1 plasmid p628-CTXM, and this gene has a single ISEcp1-provided promoter driving bla CTX−M−55 expression in a constitutive manner. Coexistence of bla KPC and bla CTX−M in K. pneumoniae has been reported many times, but this is the first report to gain deep insights into genetic platforms, promoters, and expression of the two coexisted bla genes. The IncFII K and IncI1 plasmids have been frequently identified to carry horizontally acquired drug-resistant gene modules and could be transmitted across a number of bacterial species (Woodford et al., 2009;Jiang et al., 2010;Sampei et al., 2010;Holt et al., 2013), and increased surveillance of these drug-resistant plasmids is needed.

Supplementary Material
The Supplementary Material for this article can be found online at: http://journal.frontiersin.org/article/10.3389/fmicb. 2015.00838