Non-susceptibility (%) of outpatient <i>E</i>. <i>coli</i> from different age groups to cefotaxime (top) and ciprofloxacin (bottom), Taiwan, 2002–2012.

<p>Pediatric, ≤18 y.o.; adult, 19–64 y.o.; elderly, ≥ 65 y.o. The number of isolates for each age group in 2002, 20024, 2006, 2008, 2010, and 2012 was 29, 42, 41, 38, 39, and 45, respectively for pediatric patients; 186, 228, 245, 254, 304, and 346, respectively for adult patients; and 158, 211, 229, 294, 326, and 392, respectively, for elderly patients.</p

Factors associated with carriage of ESBL and AmpC β-lactamases genes in <i>E</i>. <i>coli</i>.

<p>Factors associated with carriage of ESBL and AmpC β-lactamases genes in <i>E</i>. <i>coli</i>.</p

Ciprofloxacin MIC distribution of outpatient <i>E</i>. <i>coli</i> isolates from different age groups.

<p>Data were based on 234 isolates from pediatric, 1563 isolates from adult, and 1610 isolates from elderly patients.</p

Non-susceptibility (%) of outpatient <i>E</i>. <i>coli</i> stratified by specimen types, age groups, and geographic regions (2002–2012 combined).

<p>Non-susceptibility (%) of outpatient <i>E</i>. <i>coli</i> stratified by specimen types, age groups, and geographic regions (2002–2012 combined).</p

Non-susceptibility^a (%) to key agents among <i>E</i>. <i>coli</i> isolates with and without ESBL or AmpC β-lactamases genes.

<p>Non-susceptibility<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0144103#t003fn001" target="_blank">^a</a> (%) to key agents among <i>E</i>. <i>coli</i> isolates with and without ESBL or AmpC β-lactamases genes.</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

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

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