The Clostridium difficile spo0A gene is a persistence and transmission factor

Abstract

Clostridium difficile is a major cause of chronic antibiotic-associated diarrhea and a significant health care-associated pathogen that forms highly resistant and infectious spores. Spo0A is a highly conserved transcriptional regulator that plays a key role in initiating sporulation in Bacillus and Clostridium species. Here, we use a murine model to study the role of the C. difficile spo0A gene during infection and transmission. We demonstrate that C. difficile spo0A mutant derivatives can cause intestinal disease but are unable to persist within and effectively transmit between mice. Thus, the C. difficile Spo0A protein plays a key role in persistent infection, including recurrence and host-to-host transmission in mice.

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

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

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.

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

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.