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Bacterial Infections

The Clostridium difficile spo0A Gene Is a Persistence and Transmission Factor

Laura J. Deakin, Simon Clare, Robert P. Fagan, Lisa F. Dawson, Derek J. Pickard, Michael R. West, Brendan W. Wren, Neil F. Fairweather, Gordon Dougan, Trevor D. Lawley
A. J. Bäumler, Editor
Laura J. Deakin
aMicrobial Pathogenesis Laboratory, Wellcome Trust Sanger Institute, Hinxton, United Kingdom
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Simon Clare
aMicrobial Pathogenesis Laboratory, Wellcome Trust Sanger Institute, Hinxton, United Kingdom
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Robert P. Fagan
bCenter for Molecular Microbiology and Infection, Imperial College of London, London, United Kingdom
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Lisa F. Dawson
cDepartment of Pathogen Molecular Biology, London School of Hygiene and Tropical Medicine, University of London, London, United Kingdom
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Derek J. Pickard
aMicrobial Pathogenesis Laboratory, Wellcome Trust Sanger Institute, Hinxton, United Kingdom
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Michael R. West
aMicrobial Pathogenesis Laboratory, Wellcome Trust Sanger Institute, Hinxton, United Kingdom
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Brendan W. Wren
cDepartment of Pathogen Molecular Biology, London School of Hygiene and Tropical Medicine, University of London, London, United Kingdom
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Neil F. Fairweather
bCenter for Molecular Microbiology and Infection, Imperial College of London, London, United Kingdom
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Gordon Dougan
aMicrobial Pathogenesis Laboratory, Wellcome Trust Sanger Institute, Hinxton, United Kingdom
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Trevor D. Lawley
dBacterial Pathogenesis Laboratory, Wellcome Trust Sanger Institute, Hinxton, United Kingdom
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A. J. Bäumler
Roles: Editor
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DOI: 10.1128/IAI.00147-12
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  • Fig 1
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    Fig 1

    Genetic and phenotypic characterization of C. difficile 630 and R20291 spo0A mutants. (A) C. difficile cultures were grown in Wilson's broth for 48 h under anaerobic conditions, and then total cell and spore counts were determined. Genetic complementation of the spo0A mutation restored spore formation to levels statistically comparable to those of the parental strains (P > 0.05), according to the Student t test. The dashed horizontal line indicates the detection limit. The error bars indicate standard deviations. (B) Representative indirect-immunofluorescence images of C. difficile cultures stained with vegetative-cell-specific (green) and spore-specific (red) polyclonal antibodies and visualized with FITC-conjugated and Cy3-conjugated secondary antibodies, respectively. Scale bar = 5 μm.

  • Fig 2
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    Fig 2

    C. difficile spo0A mutants produce elevated levels of TcdA and TcdB and exhibit increased virulence in mice. (A) Kaplan-Meier survival curve of mice infected with C. difficile 630, R20291, 630Δspo0A, or R20291Δspo0A. (B) Representative images demonstrating epithelial cell damage in hematoxylin- and eosin-stained cecal sections of mice infected with C. difficile R20291 or R20291Δspo0A. Magnification, ×20. (C) Sandwich ELISA indicating the relative levels of TcdA and TcdB produced by C. difficile R20291 or R20291Δspo0A after 30 h of growth in Wilson's broth under anaerobic conditions. Data are from 3 independent experiments performed in triplicate. The error bars indicate standard deviations.

  • Fig 3
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    Fig 3

    C. difficile spo0A mediates intestinal persistence in mice. Shown are the CI time courses of C. difficile R20291 and R20291Δspo0A (A) and 630 and 630Δspo0A (B). Individual CI values are illustrated as open circles, and the horizontal bars represent the geometric means. The second through fourth (A) and third and fourth (B) spo0A mutants are attenuated, as determined by the Mann-Whitney test.

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    Fig 4

    The C. difficile spo0A gene is required for relapsing disease in mice. (A and B) Representative fecal shedding profiles from mice (n = 5 per group) infected with C. difficile 630, R20291, 630Δspo0A, or R20291Δspo0A. The mice were pretreated with clindamycin (clin) (represented as a gray line) for 7 days prior to infection via oral gavage. Following infection, the mice received a series of 7- to 10-day courses of vancomycin (van) (represented as green lines) during which fecal shedding of C. difficile decreased to below the detection limit of the assay in all groups of mice. The dashed horizontal lines indicate the detection limit.

  • Fig 5
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    Fig 5

    Host-to-host transmission of C. difficile is mediated by spo0A. (A) Average fecal shedding of C. difficile by mice (n = 5 per group) at 5 days postinfection. The dashed horizontal line indicates the detection limit. The error bars indicate standard deviations. (B) Transmission efficiencies of C. difficile 630, R20291, 630Δspo0A, and R20291Δspo0A, demonstrating the percentages of naïve recipient mice that acquired infection following exposure to infected donor mice via mingling, contact, airborne, or environmental transmission. The efficiency of transmission was determined as described in Materials and Methods.

Tables

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  • Table 1

    Strains and plasmids used in this study

    Strain or plasmidCharacteristicsSource
    Strains
        E. coli CA434Conjugation donor26
        C. difficile
            Wild-type 630Virulent and multidrug-resistant PCR ribotype 012, isolated from a patient with pseudomembranous colitis in Zurich, Switzerland (1985)29
            630Erythromycin-sensitive derivative of wild-type C. difficile 63016
            R20291Hypervirulent and epidemic PCR ribotype 027, isolated from a hospital outbreak in Stoke Mandeville, UK (2004–2005)33
            630Δspo0AC. difficile 630ΔermB spo0A::ermBThis study
            R20291Δspo0AC. difficile R20291 spo0A::ermBThis study
            630Δspo0A+pspo0AComplemented 630Δspo0A mutantThis study
            R20291Δspo0A+pspo0AComplemented R20291Δspo0A mutantThis study
    Plasmids
        pMTL007First-generation ClosTron plasmid with catP marker and intron containing ermB RAM; intron expression is induced using IPTG.14
        pMTL007C-E2Second-generation ClosTron plasmid with catP marker and intron containing ermB RAM; contains a constitutive fdx promoter to direct intron expression13
        pRPF101E. coli-C. difficile shuttle vectorThis study
        pspo0ApRPF101 containing the 825-bp spo0A coding region (and upstream promoter)This study
  • Table 2

    Summary of transmission routes

    Type of transmission (1 h)Characteristics
    MinglingDonor and recipient mice were able to freely touch and interact with no restriction in movement; potential for coprophagy
    ContactDonor and recipient mice were separated by a single porous barrier and were able to touch each other; no potential for coprophagy
    AirborneDonor and recipient mice were separated by a double porous barrier and were unable to touch each other; no potential for coprophagy
    EnvironmentalRecipient mice were housed in a contaminated donor cage, which had been left for 24 h under ambient oxygen conditions. Donor feces were removed to prevent transmission via coprophagy

Additional Files

  • Figures
  • Tables
  • Supplemental material

    Files in this Data Supplement:

    • Supplemental file 1 -

      Table S1. Oligonucleotides used in this study.

      PDF, 137K

    • Supplemental file 2 -

      Fig. S1. Schematic diagram demonstrating the experimental models used to investigate the role of the C. difficile spo0A gene in host transmission.

      PDF, 120K

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The Clostridium difficile spo0A Gene Is a Persistence and Transmission Factor
Laura J. Deakin, Simon Clare, Robert P. Fagan, Lisa F. Dawson, Derek J. Pickard, Michael R. West, Brendan W. Wren, Neil F. Fairweather, Gordon Dougan, Trevor D. Lawley
Infection and Immunity Jul 2012, 80 (8) 2704-2711; DOI: 10.1128/IAI.00147-12

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The Clostridium difficile spo0A Gene Is a Persistence and Transmission Factor
Laura J. Deakin, Simon Clare, Robert P. Fagan, Lisa F. Dawson, Derek J. Pickard, Michael R. West, Brendan W. Wren, Neil F. Fairweather, Gordon Dougan, Trevor D. Lawley
Infection and Immunity Jul 2012, 80 (8) 2704-2711; DOI: 10.1128/IAI.00147-12
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