Circular map of plasmid pTR3 and pTR4.

<p>The open reading frames are marked along the map by arrows and significant ones are labeled. The <i>bla</i>_NDM-1 gene (red) is located in a region with several transposon/IS-related genes (gray). The region corresponding to the IncN2 backbone of pJIE137 is indicated by a black line. Positions of the two resistance regions (a class 1 integron/Tn and a complex IS<i>Ecp1</i>-<i>bla</i>_CTX-M-62 transposition unit) present in pJIE137 but missing in pTR3/4 are marked by the arrowheads. The CUP-related region between <i>repA</i> and <i>stbABC</i> is missing in p271A. G+C% are shown in the inner circle.</p

Antimicrobial susceptibility test among <i>bla</i>_NDM-1 carrying isolates and their transconjugants.

*<p>43320 and 44951, clinical isolates from patient 1 and 2 respectively; TCJ-P1 and TCJ-P2, transconjugants from 43320 and 44951 respectively.</p>†<p>The MIC is presented according to the concentration of trimethoprim.</p

Schematic diagram of the NDM-1 region of pTR3 and pTR4, compared to those from the other known plasmids.

<p>The <i>bla</i>_NDM-1 (red), and nearby IS elements (various colors) are shown. ORFs are depicted with arrows and the IRs were depicted by short vertical bars. The regions corresponding to possible vestiges of unknown IS identified in pTR3/4 and p271A are marked by yellow rectangles. Nucleotide sequences of the two regions are shown in the boxes, of which the 39-bp putative IRs are underlined. Corresponding repeat sequences in the boxes are shown in the same color. Differences are shown in lower case.</p

The <i>pks</i> colibactin gene cluster (GM1) in KPHPI208 and the <i>pks</i> colibactin gene cluster in the <i>E. coli</i> IHE3034 genome.

<p>The regions spanning the genes responsible for colibactin production were depicted as arrows according to the directions of transcription. The <i>attO</i> sites in the left were marked by solid triangles. The VNTR locus between <i>clbR</i> and <i>clbB</i> was marked by empty triangles. The 53-kb regions indicated by dotted lines are ∼100% identical. The locations of the four PCR amplicons in studying the prevalence of the colibactin genes among <i>K. pneumoniae</i> clinical isolates were marked. The 768-bp region spanning the <i>clbA</i> gene, which was deleted in <i>ΔclbA</i> strain, was indicated.</p

<i>K. pneumoniae</i> 1084 induced colibactin-related DSBs <i>in vitro</i>.

<p>BNL cells were left uninfected (A) or were infected with <i>K. pneumoniae</i> 1084 (B), Δ<i>clbA</i> (C), or with Δ<i>clbA</i> complemented with <i>clbA</i> coding plasmid pYC502 (D) at an MOI of 100. After 4 h infection, the cells were washed, co-cultured with gentamycin (100 µg/ml), and were examined by confocal microscopy for DNA (blue, stained with Hoechst 33342), for membrane glycoproteins (red, stained with ConA), and for γH2AX (green, recognized by Alexa488-anti-γH2AX antibodies) (scale bar, 20 µm). (E) Quantification of γH2AX-positive cells. Error bars represent SEs from three experiments. (F) Western blot analyses of γH2AX or H3 in BNL cells recovered at 2, 4, and 6 h after a 4 h transient infection with <i>K. pneumoniae</i> 1084 (lanes 1–3), Δ<i>clbA</i> (lanes 4–6), or with Δ<i>clbA</i> complemented with <i>clbA</i>-coding plasmid pYC502 (lanes 7–9). (G) Western blot analyses of γH2AX and H3 in uninfected BNL cells harvested from serum recovery for 2, 4, 6, 24, 48, and 72 h. (H) Clonogenic assays. BNL cells were uninfected (Control) or transiently infected with <i>K. pneumoniae</i> 1084, Δ<i>clbA</i>, or with Δ<i>clbA</i>-pYC502 for 4 hours. Colonies formed after 14-day incubation stained with 0.5% of crystal violet. A representative image is presented. (I) Quantification of colony formation. Error bars represents SEMs from three experiments. An asterisk (*) represents a significant increase in the <i>K. pneumoniae</i>-infected group in comparison with the uninfected control by the Student's <i>t</i> test (two-tailed; <i>P</i><0.05).</p

Tandem duplication of the <i>bla</i>_NDM-1 gene in pKPX-1 and pECX-1.

<p>(<b>A</b>) Diagrammatic representation of the analysis of <i>bla</i>_NDM-1 copy number by Southern blot. The probe is shown with a red arrow, and the tandem duplication of the 8588-bp repeat is indicated by the bracket. The asterisks indicate the methylated <i>Nru</i>I sites. The sizes of <i>Bam</i>HI or <i>Hind</i>III digested fragments depend on the copy number of the repeat. The pound sign indicates 79.1 kb and 71.8 kb for <i>Bam</i>HI and <i>Hind</i>III restrictions, respectively, when there are 8 copies of the tandem repeat, as in the case of pECX-1. (<b>B</b>) Sequencing read distribution and Southern analysis of the <i>bla</i>_NDM-1 region for pECX-1. The upper panel shows the relative coverage depth of the repeat region and its flanking sequences. The average coverage of <i>bla</i>_NDM-1 is 7–8 fold of those sequences of the immediately adjacent regions, suggesting that there are eight copies of the repeat. As shown in the lower panel, Southern analysis confirms this model of tandem duplication. (<b>C</b>) <i>Bla</i>_NDM-1 copy number variation detected by the Southern analysis. Sequence depth of the region revealed an average of 3–4 copies of the repeat sequence in pKPX. <i>Bam</i>HI and <i>Hind</i>III digestion gave a series of ladder bands, corresponding to different copy numbers of the repeat. By contrast, <i>Avr</i>II and <i>Nru</i>I both deliberated a single major band of 8.6 kb, representing the unit length of the tandem repeats.</p

Restriction analysis of the KPX plasmids by PFGE.

<p>Plasmid DNA isolated from the KPX isolate was analyzed using pulsed field gel electrophoresis (PFGE). The restriction patterns of the plasmid DNA were compared to those of pECX-1 and pECX-2 in <i>Escherichia coli</i>. The size of the DNA markers is shown in kilobases (kb) on the left side.</p

Sequence analysis of KPX plasmids.

<p>Two circular sequences are shown for the organization of pKPX-1 (<b>A</b>) and pKPX-2 (<b>B</b>). Mapping shotgun sequencing reads of pECX-1 to the pKPX-1 is indicated by the red half-circle. A large part of the plasmid, corresponding to the nucleotide positions 23,125 to 145,377 of pKPX-1, was not found in pECX-1. Only the part on the left side, totaling 128,191-bp, is retained. Two genes encoding chloramphenicol and amikacin resistance were identified by functional library screening. Their positions in the deleted region are indicated. Nucleotides are numbered according to the replication origin. Genes are color coded: yellow, β-lactamase; red, antimicrobial resistance associated; blue, plasmid replication and partitioning; black, transposases or IS elements; and white, other coding sequences of miscellaneous features. The arrows on the open reading frames (ORFs) indicate the gene orientation. Gene clusters involved in gene transfer or mobility are marked in green. <i>Xba</i>I and <i>Avr</i>II restriction sites are shown inside the circle.</p

Antimicrobial resistance determinants in pKPX-1 and pKPX-2.

*<p>Nonfunctional; ^† Deleted in pECX-1.</p><p>Abbreviations: AAC, aminoglycoside acetyltransferase; APH, aminoglycoside phosphotransferase; ANT, aminoglycoside nucleotdyltransferase; Rmt, 16S rRNA methyltransferase; Qnr, quinolone resistance protein; TetA, tetracycline efflux protein; Cat, chloramphenicol acetyltransferase; ARR-2, rifampin ADP-ribosyltransferase; DHPS, dihydropteroate synthase; DHFR, dihydrofolate reductase; Mph, macrolide phosphotransferase.</p

Minimal inhibitory concentrations (MICs)^* for different antimicrobial agents of <i>K. pneumoniae</i> KPX, <i>E. coli</i> DH10B, and <i>E. coli</i> transformants of DNA derived from <i>K. pneumoniae</i> KPX.

*<p>MIC interpretations are based on Clinical and Laboratory Standards Institute (CLSI) breakpoints (CLSI M100-S21, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0062774#pone.0062774-Clinical1" target="_blank">[28]</a>), except for polymyxin E and tigecycline, which are based on EUCAST (<a href="http://www.eucast.org/clinical_breakpoints/" target="_blank">http://www.eucast.org/clinical_breakpoints/</a>) breakpoints.</p