Article Figures & Data
Figures
- FIG. 1.
HpaI digests of pC15-1a (lane A) and pCTX15 (lane B). Fragments hybridizing with blaCTX-M-15, blaTEM-1, and blaOXA-1 probes are indicated. The molecular weight marker was the One Kilobase Extension Ladder (Invitrogen).
- FIG. 2.
(A) Schematic diagram comparing the region between pemK and ycdA from R100 with that from pC15-1a. Tn21, which is inserted between IS1b and ybjA in R100, is not drawn to scale. Junction regions are shown at the bottom; arrows below these sequences indicate the 9-bp region that is repeated in reverse order. (B) Schematic diagram showing the open reading frames of the multidrug resistance region of pC15-1a. Rectangles above the line, open reading frames transcribed from left to right; rectangles below the line, open reading frames transcribed from right to left. The putative origin of the sequence, when known, is shown below the open reading frames. A designation with a prime, e.g., tnpA′, indicates a partial open reading frame. An alignment of the ISEcp1 left inverted repeat (IR-L; reverse complement of the sequence), the ISEcp1 right inverted repeat (IR-R), and the right inverted repeat of the transposition unit (IR-RII) is shown at the bottom.
- FIG. 3.
Putative hybrid promoter regions of aac(6′)-Ib/blaOXA-1 (A) and aac(3)-II (B) as found in pC15-1a.
Tables
- TABLE 1.
Characteristics of the E. coli strains harboring pC15-1a used in this studya
Strain Date of isolation Locationb PFGE profilec Plasmid Resistance phenotyped N00-0666 2000 LTC outbreak A1 pC15-1a Amp Cfz Cro Cip Gen Tob ESBL35 1 Dec. 1999 VGH B1 pC15-1a variant Amc Amp Cfz Caz Cro Cip Gen Tzp Tob Sxt ESBL123 3 April 2000 VGH C1 pC15-2a Amp Cfz Cro Cip Sxt ESBL304 20 May 2000 VGH D1 pC15-1a Amp Cfz Cro Cip Gen Tob Sxt ESBL361 30 Aug. 2000 VGH E1 pC15-1a Amp Cfz Cro Gen Tob Sxt ESBL370 14 Sept. 2000 VGH F1 pC15-1a Amp Cfz Cro Cip Gen Tob Sxt ESBL373 17 Sept. 2000 VGH F1 NDe Amc Amp Cfz Cro Cip Gen Tob Sxt ESBL374 18 Sept. 2000 VGH F1 ND Amp Cfz Cro Cip Gen Tob Sxt ESBL475 7 May 2000 UHN G1 pC15-1a Amc Amp Cfz Cro Cip Gen Tob Sxt ESBL480 27 Sept. 2000 UHN H1 pC15-2b Amp Cfz Cro Cip Gen Tob ↵a All strains were positive by PCR analysis for CTX-M-, TEM-, and OXA-1-type genes and contained beta-lactamases with pls of 5.4, 7.4, and 8.6 as determined by isoelectric focusing.
↵b LTC outbreak; long-term-care outbreak, Toronto region. VGH, Vancouver General Hospital; UHN, University Health Network, Toronto, Ontario, Canada.
↵c The same letter-number combination means that fingerprints were indistinguishable, A different letter-number combination means that fingerprints had >7 band differences.
↵d Abbreviations indicate drug resistance as follows: Amc, amoxicillin-clavulanic acid; Amp, ampicillin; Cfz, cefazolin; Caz, ceftazidime; Cro, ceftriaxone; Cip, ciprofloxacin; Gen, gentamicin; Tzp, piperacillin-tazobactam; Tob, tobramycin; Sxt, trimethoprim-sulfamethoxazole.
↵e ND, not done.
- TABLE 2.
Significant sequence divergences of pC15-1a from R100
Variationa R100 coordinatesb Effect in pC15-1ac Substitution 29694 G-to-T substitution introduces stop codon and truncates ycjB Deletion 29885-29902 After the early stop introduced by substitution above in pC15-1a, a 6-aa deletion (LLLLLL) in the R100 ycjB region Deletion 30210 C deleted; causes frameshift in ydaA approximately midway in ORF; resulting protein diverges at this point Deletion 33242-33247 2-aa deletion (SS; AGTAGC) in repetitive region of yddA Deletion 36157-45253 Deletion of Tn10; inverted repeats from IS10-L and IS10-R are still present Insertion 47797/47798 IS682 variant (not 100% identical); disrupts yehA; last 8 aa originate from IS682 sequence Insertion 48227/48228 1-bp insertion in noncoding region downstream of yehA Deletion 58087-58489 AAC deleted; causes fusion of yfhA and yfiA; last 28 aa of ORF are from yfiA Substitution 65886 T-to-G substitution eliminates stop between traN and traZ; also noted in R100-1 (AF005044) Deletion 78171-78187 6-aa deletion (PQQPQQ; CCACAACAGCCACAACAG) in repetitive region of traD Insertion 86627-86628 1-bp insertion in noncoding region downstream of yigA Insertion 86648-86649 IS1a inserted here; found in R100 but located in region not contained in pC15-1a Insertion 86640-86648 8-bp insertion site of IS1a (GGTGCTATT); duplicated in pC15-1a as direct repeats flanking IS1a ↵a Nucleotide substitutions were included only if they affected the length or arrangement of the R100 coding regions. Locations of deletions in repetitive regions are arbitrary.
↵c Effect of the change at the R100 coordinates on the equivalent region in the pC15-1a R100-derived region. aa, amino acids; ORF, open reading frame.
- TABLE 3.
Features and/or open reading frames of the multidrug resistance region of pC15-1a
Location Feature(s) and/or open reading frame(s) Gene(s) % Homologya GenBank accession no. 1-3664 Tn5403; transposase, resolvase tnpA, tnpR 99.8 X75779 3663-9641 Central core of Tn1721 (bp 4091-10096); partial transposases, PecM-like transcriptional regulator, tetracycline resistance protein, tetracycline resistance repressor tnpA partial, pecM-like, tetA, tetR 99.4 X61367 9642-10461 IS26 tnpA 100 AF550679 10462-10499 Partial chloramphenicol O-acetyltransferase catB3 100 AJ310778 10718-11548 Beta-lactamase blaOXA-1 100 AF326777 11679-12278 Aminoglycoside N6′-acetyltransferase aac(6′)-Ib 100 AY259086 12325-13144 IS26 tpnA 100 AF550679 13145-13475 Partial chloramphenicol O-acetyltransferase catB3 100 AJ310778 13476-14295 IS26 tnpA 100 AF550679 14339-15199 Aminoglycoside N3′-acetyltransferase aac(3)-II 100 AY138987 15212-15754 Hypothetical; identical to hypothetical protein AAN34369 in Acinetobacter baumannii hypo 100 AY138987 15825-16157 IS3 family transposase; Agrobacterium tumefaciens C58 OrfA orfA 95.1 (aa) NP356057 16154-16894 Partial; putative transposase protein of Sinorhizobium meliloti orfB 89.5 (aa) NP387207 16896-17715 IS26 tnpA 99.6 AF550679 17716-19960 Tn3-like partial transposase; disrupted by ISEcp1/CTX-M transposition element tnpA 100 AB103092 20353-21228 Beta-lactamase blaCTX-M-15 100 AY044436 21277-22932 ISEcp1 tnpA 100 AY044436 22933-23146 Tn3-like partial transposase; disrupted by ISEcp1/CTX-M transposition element tnpA 100 AB103092 23145-25057 Tn3-like resolvase, beta-lactamase tnpR, blaTEM-1 99.7 AB103092 25057-27528 Partial transposase, identical to Tn21 tnpA from R100 tnpA 100 NC_002134 27531-27628 Partial resolvase, identical to Tn21 tnpR from R100 tnpR 100 NC_002134 27619-28438 IS26 tnpA 100 AF550679 ↵a Unless otherwise indicated, percent homologies are bases on nucleic acid comparisons. Amino acid (aa) homologies were used if they proved to be more informative of gene function. In many cases, numerous BLAST hits were obtained, all with similar degrees of homology. In such cases, only one was selected for reference purposes.
