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Mechanisms of Resistance

Homologous Recombination within Large Chromosomal Regions Facilitates Acquisition of β-Lactam and Vancomycin Resistance in Enterococcus faecium

Mónica García-Solache, Francois Lebreton, Robert E. McLaughlin, James D. Whiteaker, Michael S. Gilmore, Louis B. Rice
Mónica García-Solache
aDepartment of Medicine, Rhode Island Hospital, Warren Alpert Medical School of Brown University, Providence, Rhode Island, USA
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  • ORCID record for Mónica García-Solache
Francois Lebreton
bDepartments of Ophthalmology, Microbiology and Immunology, Massachusetts Eye and Ear Infirmary, Harvard Medical School, Boston, Massachusetts, USA
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Robert E. McLaughlin
cInfection Bioscience, AstraZeneca R&D Boston, Waltham, Massachusetts, USA
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James D. Whiteaker
cInfection Bioscience, AstraZeneca R&D Boston, Waltham, Massachusetts, USA
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Michael S. Gilmore
bDepartments of Ophthalmology, Microbiology and Immunology, Massachusetts Eye and Ear Infirmary, Harvard Medical School, Boston, Massachusetts, USA
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Louis B. Rice
aDepartment of Medicine, Rhode Island Hospital, Warren Alpert Medical School of Brown University, Providence, Rhode Island, USA
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DOI: 10.1128/AAC.00488-16
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  • FIG 1
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    FIG 1

    Relative expression levels of pbp5 and vanB ligase in the naive TC-A transconjugant (TCA P0) or in the resulting selected ones (TCA-A P9, which was continuously passaged in the presence of ampicillin for ∼400 generations, and TCA-V P13, which was continuously passaged in the presence of vancomycin for ∼400 generations). The parental strains (D344RRF and C68) were included for comparison. The parents and the three versions of TC-A were grown with either ampicillin or vancomycin, and relative expression was calculated using the levels of expression for the corresponding group without antibiotic treatment. Expression levels of 16S rRNA were used to normalize the data. Error bars indicate the standard errors of the means for biological triplicates. (A) pbp5 expression is not modified with respect to that for the control (untreated cells) in the presence of half the MIC of ampicillin. pbp5 expression was induced in C68 and the three different TC-A groups in the presence of vancomycin. (B) vanB ligase expression was very low in the absence of vancomycin. In the presence of the antibiotic, there was a significant induction of vanB ligase expression. Unpassaged TC-A (P0) had lower vanB ligase expression levels than C68 and the passaged groups, which correlates with a lower vancomycin MIC. White bars, BHI-grown cells (calibrator); gray bars, ampicillin-grown cells; black bars, vancomycin-grown cells. ****, P < 0.0001; ***, P < 0.0002; *, P < 0.05.

  • FIG 2
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    FIG 2

    pbp5 and TC-D insertion site. (A) pbp5 operon cartoon showing the differences between strains D344RRF and C68. In C68, an insertion (C) 153 bp downstream of the ribosomal binding site causes a frameshift introducing a premature stop codon 309 bp downstream of the start of the gene, possibly generating a nonfunctional gene. (B) The sequence of the pbp5 gene in C68 codes for an extra serine and has two amino acid substitutions compared with the sequence of D344RRF (red). (C) Alignment of D344RRF, TC-D, and C68 in the region of integration of Tn5382 carrying vanB. Green, the vancomycin resistance-carrying transposon; light blue, the transposon (Tn) ends flanking the vancomycin resistance-carrying transposon; light gray, the segments in which the C68 genome replaced that of D344RRF in the transconjugant; purple, the crossover regions (COR) flanked by the SNV used to identify the region. Note the additional C68 integration in TC-D upstream of the pbp5 operon. pbp5 from D344RRF was not replaced by that of C68 in this transconjugant. SNV, single nucleotide variant.

  • FIG 3
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    FIG 3

    Crossover regions. (A) Cartoon representation of crossover regions in all individual transconjugants. The area of the crossover is measured from the last SNV corresponding to the donor strain, C68, to the first SNV corresponding to the recipient strain, D344RRF. Transconjugants are organized by groups with shared left crossover regions. Notice the additional crossover regions in TC-D and TC-M (light gray). The amount of chromosomal DNA integrated in addition to Tn5382 is measured from the left or right end of the element up to the last SNV between D344RRF and each transconjugant. The amount of integrated DNA is shown in kilobases. (B) Plot of GC and AT contents of C68 (donor) and D344RRF (recipient) in the chromosomal region where crossovers occurred. Blue, GC content; green, AT content. The high-GC-content area in C68 corresponding to Tn5382 carrying vanB is highlighted between red lines.

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  • TABLE 1

    Antibiotic resistance profile of the parental strains and selected transconjugants

    StrainResistance traitsaOrigin (reference)
    C68Ampr, Vanr, Eryr, Tetr, Strr, GenrClinical isolate (13)
    D344RRFAmpr,b Rifr, Fusr, Eryr, Tetr, Strr, KanrDerived from clinical isolate D344R (22)
    TransconjugantsAmpr, Vanr, Rifr, Fusr, Eryr, Tetr, Strr, KanrTransconjugants obtained from mating C68 and D344RRF during this work
    • ↵a Amp, ampicillin; Van, vancomycin; Ery, erythromycin; Tet, tetracycline; Str, streptomycin; Gen, gentamicin; Rif, rifampin; Fus, fusidic acid; Kan, kanamycin. Boldface indicates the parental antibiotic resistance genotype that was acquired by the transconjugants.

    • ↵b Intermediate resistance levels.

  • TABLE 2

    MICs for parental strains and transconjugants

    StrainaMICb (μg/ml)
    VanAmpFusRif
    C68>400>4001.6<0.2
    D344RRF1.612.5>400>400
    TC-A P010050>400>400
    TC-A BHI P13400200>400>400
    TC-A Van P13400>400>400>400
    TC-A Amp P9400400>400>400
    TC-B P020050>400>400
    TC-B BHI P13400>400>400>400
    TC-B Van P13400200>400>400
    TC-B Amp P9400>400>400>400
    TC-C2525200>400
    TC-D5025400>400
    TC-E5050200>400
    TC-F50100400>400
    TC-G2512.5200400
    TC-H5025200>400
    TC-I20025>400>400
    TC-J20025>400>400
    TC-K100100>400>400
    TC-L506.3>400>400
    TC-M10025>400>400
    TC-N12.56.3200200
    • ↵a BHI, Van, and Amp, the strains were passaged on BHI, vancomycin, and ampicillin, respectively, for the indicated number of passages.

    • ↵b Van, vancomycin; Amp, ampicillin; Fus, fusidic acid; Rif, rifampin.

  • TABLE 3

    Integration site and size of integrated DNA in the studied transconjugants

    TCPosition of integration siteaSize (kb) of integrated DNAAdditional integration site
    First SNV of CORb left of vanBFirst SNV of COR right of vanB (integrase site)Minimum amt transferredcCOR between the two delimiting SNVs
    LeftRight
    TC-A−49.87+100.8184.6574307
    TC-B−10.6+49.994.53,1124,315
    TC-C−16.7+27.778.46,2291,159
    TC-D0+2.936.96542,4669,098d
    TC-E−14.9+15.1642,0052,171
    TC-F−10.9+47.992.83,1122,049
    TC-G−61.8+27.6123.42,8781,157
    TC-H−60.2+81.3175.5593126
    TC-I−16.9+124174.96,18211
    TC-J−16.9+79.9130.86,1821,240
    TC-K−10.9+78.7123.63,112185
    TC-L−16.9+48.499.36,182947
    TC-M−10.9+3680.93,1137,37311,719e
    TC-N−14.9+5.954.82,005371
    • ↵a Considering the first position of Tn5382 to the left side (negative values) and the last position of Tn5382 to the right side (positive values).

    • ↵b COR, crossover region.

    • ↵c Including the 34-kb Tn5382 element harboring vanB.

    • ↵d Position −14 left of vanB.

    • ↵e Position −16.9 of vanB.

Additional Files

  • Figures
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  • Supplemental material

    • Supplemental file 1 -

      Supplemental Tables S1 to S4

      PDF, 159K

    • Supplemental file 2 -

      Supplemental Figures S1 and S2

      PDF, 3.6M

    • Supplemental file 3 -

      Supplemental Table S5: (A) pLRM23 gene content, (B) putative pRIH77 gene content, and (C) pLRM23 coverage and pRIH77 coverage in parents and transconjugants.

      XLSX, 52K

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Homologous Recombination within Large Chromosomal Regions Facilitates Acquisition of β-Lactam and Vancomycin Resistance in Enterococcus faecium
Mónica García-Solache, Francois Lebreton, Robert E. McLaughlin, James D. Whiteaker, Michael S. Gilmore, Louis B. Rice
Antimicrobial Agents and Chemotherapy Sep 2016, 60 (10) 5777-5786; DOI: 10.1128/AAC.00488-16

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Homologous Recombination within Large Chromosomal Regions Facilitates Acquisition of β-Lactam and Vancomycin Resistance in Enterococcus faecium
Mónica García-Solache, Francois Lebreton, Robert E. McLaughlin, James D. Whiteaker, Michael S. Gilmore, Louis B. Rice
Antimicrobial Agents and Chemotherapy Sep 2016, 60 (10) 5777-5786; DOI: 10.1128/AAC.00488-16
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