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Mechanisms of Resistance

Complete Nucleotide Sequence of a 92-Kilobase Plasmid Harboring the CTX-M-15 Extended-Spectrum Beta-Lactamase Involved in an Outbreak in Long-Term-Care Facilities in Toronto, Canada

David A. Boyd, Shaun Tyler, Sara Christianson, Allison McGeer, Matthew P. Muller, Barbara M. Willey, Elizabeth Bryce, Michael Gardam, Patrice Nordmann, Michael R. Mulvey, ; Canadian Nosocomial Infection Surveillance Program, Health Canada
David A. Boyd
1Nosocomial Infections, National Microbiology Laboratory, Health Canada, Winnipeg, Manitoba
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Shaun Tyler
1Nosocomial Infections, National Microbiology Laboratory, Health Canada, Winnipeg, Manitoba
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Sara Christianson
1Nosocomial Infections, National Microbiology Laboratory, Health Canada, Winnipeg, Manitoba
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Allison McGeer
2Mount Sinai Hospital
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Matthew P. Muller
2Mount Sinai Hospital
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Barbara M. Willey
2Mount Sinai Hospital
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Elizabeth Bryce
3The Vancouver General Hospital, Vancouver, British Columbia, Canada
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Michael Gardam
4University Health Network, Toronto, Ontario
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Patrice Nordmann
5Hôpital de Bicêtre, Assistance Publique/Hôpitaux de Paris, Paris, France
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Michael R. Mulvey
1Nosocomial Infections, National Microbiology Laboratory, Health Canada, Winnipeg, Manitoba
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  • For correspondence: michael_mulvey@hc-sc.gc.ca
DOI: 10.1128/AAC.48.10.3758-3764.2004
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  • FIG. 1.
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    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.
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    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.
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    FIG. 3.

    Putative hybrid promoter regions of aac(6′)-Ib/blaOXA-1 (A) and aac(3)-II (B) as found in pC15-1a.

Tables

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  • TABLE 1.

    Characteristics of the E. coli strains harboring pC15-1a used in this studya

    StrainDate of isolationLocationbPFGE profilecPlasmidResistance phenotyped
    N00-06662000LTC outbreakA1pC15-1aAmp Cfz Cro Cip Gen Tob
    ESBL351 Dec. 1999VGHB1pC15-1a variantAmc Amp Cfz Caz Cro Cip Gen Tzp Tob Sxt
    ESBL1233 April 2000VGHC1pC15-2aAmp Cfz Cro Cip Sxt
    ESBL30420 May 2000VGHD1pC15-1aAmp Cfz Cro Cip Gen Tob Sxt
    ESBL36130 Aug. 2000VGHE1pC15-1aAmp Cfz Cro Gen Tob Sxt
    ESBL37014 Sept. 2000VGHF1pC15-1aAmp Cfz Cro Cip Gen Tob Sxt
    ESBL37317 Sept. 2000VGHF1NDeAmc Amp Cfz Cro Cip Gen Tob Sxt
    ESBL37418 Sept. 2000VGHF1NDAmp Cfz Cro Cip Gen Tob Sxt
    ESBL4757 May 2000UHNG1pC15-1aAmc Amp Cfz Cro Cip Gen Tob Sxt
    ESBL48027 Sept. 2000UHNH1pC15-2bAmp 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

    VariationaR100 coordinatesbEffect in pC15-1ac
    Substitution29694G-to-T substitution introduces stop codon and truncates ycjB
    Deletion29885-29902After the early stop introduced by substitution above in pC15-1a, a 6-aa deletion (LLLLLL) in the R100 ycjB region
    Deletion30210C deleted; causes frameshift in ydaA approximately midway in ORF; resulting protein diverges at this point
    Deletion33242-332472-aa deletion (SS; AGTAGC) in repetitive region of yddA
    Deletion36157-45253Deletion of Tn10; inverted repeats from IS10-L and IS10-R are still present
    Insertion47797/47798IS682 variant (not 100% identical); disrupts yehA; last 8 aa originate from IS682 sequence
    Insertion48227/482281-bp insertion in noncoding region downstream of yehA
    Deletion58087-58489AAC deleted; causes fusion of yfhA and yfiA; last 28 aa of ORF are from yfiA
    Substitution65886T-to-G substitution eliminates stop between traN and traZ; also noted in R100-1 (AF005044)
    Deletion78171-781876-aa deletion (PQQPQQ; CCACAACAGCCACAACAG) in repetitive region of traD
    Insertion86627-866281-bp insertion in noncoding region downstream of yigA
    Insertion86648-86649IS1a inserted here; found in R100 but located in region not contained in pC15-1a
    Insertion86640-866488-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.

    • ↵ b Based on GenBank accession number AP000342 .

    • ↵ 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

    LocationFeature(s) and/or open reading frame(s)Gene(s)% HomologyaGenBank accession no.
    1-3664Tn5403; transposase, resolvase tnpA, tnpR99.8 X75779
    3663-9641Central core of Tn1721 (bp 4091-10096); partial transposases, PecM-like transcriptional regulator, tetracycline resistance protein, tetracycline resistance repressor tnpA partial, pecM-like, tetA, tetR99.4 X61367
    9642-10461IS26 tnpA 100 AF550679
    10462-10499Partial chloramphenicol O-acetyltransferase catB3 100 AJ310778
    10718-11548Beta-lactamase bla OXA-1 100 AF326777
    11679-12278Aminoglycoside N6′-acetyltransferase aac(6′)-Ib100 AY259086
    12325-13144IS26 tpnA 100 AF550679
    13145-13475Partial chloramphenicol O-acetyltransferase catB3 100 AJ310778
    13476-14295IS26 tnpA 100 AF550679
    14339-15199Aminoglycoside N3′-acetyltransferase aac(3)-II100 AY138987
    15212-15754Hypothetical; identical to hypothetical protein AAN34369 in Acinetobacter baumanniihypo100 AY138987
    15825-16157IS3 family transposase; Agrobacterium tumefaciens C58 OrfAorfA95.1 (aa) NP356057
    16154-16894Partial; putative transposase protein of Sinorhizobium melilotiorfB89.5 (aa) NP387207
    16896-17715IS26 tnpA 99.6 AF550679
    17716-19960Tn3-like partial transposase; disrupted by ISEcp1/CTX-M transposition element tnpA 100 AB103092
    20353-21228Beta-lactamase bla CTX-M-15 100 AY044436
    21277-22932ISEcp1 tnpA 100 AY044436
    22933-23146Tn3-like partial transposase; disrupted by ISEcp1/CTX-M transposition element tnpA 100 AB103092
    23145-25057Tn3-like resolvase, beta-lactamase tnpR, blaTEM-199.7 AB103092
    25057-27528Partial transposase, identical to Tn21 tnpA from R100 tnpA 100 NC_002134
    27531-27628Partial resolvase, identical to Tn21 tnpR from R100 tnpR 100 NC_002134
    27619-28438IS26 tnpA 100AF550679
    • ↵ 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.

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Complete Nucleotide Sequence of a 92-Kilobase Plasmid Harboring the CTX-M-15 Extended-Spectrum Beta-Lactamase Involved in an Outbreak in Long-Term-Care Facilities in Toronto, Canada
David A. Boyd, Shaun Tyler, Sara Christianson, Allison McGeer, Matthew P. Muller, Barbara M. Willey, Elizabeth Bryce, Michael Gardam, Patrice Nordmann, Michael R. Mulvey Canadian Nosocomial Infection Surveillance Program, Health Canada
Antimicrobial Agents and Chemotherapy Sep 2004, 48 (10) 3758-3764; DOI: 10.1128/AAC.48.10.3758-3764.2004

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Complete Nucleotide Sequence of a 92-Kilobase Plasmid Harboring the CTX-M-15 Extended-Spectrum Beta-Lactamase Involved in an Outbreak in Long-Term-Care Facilities in Toronto, Canada
David A. Boyd, Shaun Tyler, Sara Christianson, Allison McGeer, Matthew P. Muller, Barbara M. Willey, Elizabeth Bryce, Michael Gardam, Patrice Nordmann, Michael R. Mulvey Canadian Nosocomial Infection Surveillance Program, Health Canada
Antimicrobial Agents and Chemotherapy Sep 2004, 48 (10) 3758-3764; DOI: 10.1128/AAC.48.10.3758-3764.2004
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KEYWORDS

Cross Infection
Escherichia coli
Escherichia coli Infections
plasmids
Skilled Nursing Facilities
beta-lactamases

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