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. 2009 Sep;77(9):3661-9.
doi: 10.1128/IAI.00558-09. Epub 2009 Jun 29.

V体育ios版 - Antibiotic treatment of clostridium difficile carrier mice triggers a supershedder state, spore-mediated transmission, and severe disease in immunocompromised hosts

Trevor D Lawley  1 , Simon Clare, Alan W Walker, David Goulding, Richard A Stabler, Nicholas Croucher, Piero Mastroeni, Paul Scott, Claire Raisen, Lynda Mottram, Neil F Fairweather, Brendan W Wren, Julian Parkhill, Gordon Dougan
Affiliations

Antibiotic treatment of clostridium difficile carrier mice triggers a supershedder state, spore-mediated transmission, and severe disease in immunocompromised hosts

Trevor D Lawley et al. Infect Immun. 2009 Sep.

VSports手机版 - Abstract

Clostridium difficile persists in hospitals by exploiting an infection cycle that is dependent on humans shedding highly resistant and infectious spores. Here we show that human virulent C. difficile can asymptomatically colonize the intestines of immunocompetent mice, establishing a carrier state that persists for many months. C VSports手机版. difficile carrier mice consistently shed low levels of spores but, surprisingly, do not transmit infection to cohabiting mice. However, antibiotic treatment of carriers triggers a highly contagious supershedder state, characterized by a dramatic reduction in the intestinal microbiota species diversity, C. difficile overgrowth, and excretion of high levels of spores. Stopping antibiotic treatment normally leads to recovery of the intestinal microbiota species diversity and suppresses C. difficile levels, although some mice persist in the supershedding state for extended periods. Spore-mediated transmission to immunocompetent mice treated with antibiotics results in self-limiting mucosal inflammation of the large intestine. In contrast, transmission to mice whose innate immune responses are compromised (Myd88(-/-)) leads to a severe intestinal disease that is often fatal. Thus, mice can be used to investigate distinct stages of the C. difficile infection cycle and can serve as a valuable surrogate for studying the spore-mediated transmission and interactions between C. difficile and the host and its microbiota, and the results obtained should guide infection control measures. .

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VSports在线直播 - Figures

FIG. 1.
FIG. 1.
Induction of high-level fecal shedding of C. difficile spores by clindamycin treatment of carrier mice shedding low levels of spores. (a) Long-term fecal shedding from individual mice (five mice per cage) infected with C. difficile M68. The horizontal dashed line indicates the detection limit, 101 CFU/g. (b) Clindamycin treatment (1 mg/day via oral gavage) (arrows) of carriers (five mice per cage) reproducibly resulted in rapid induction of high-level shedding of C. difficile that gradually returned to low-level shedding 12 to 16 days after treatment was discontinued. In contrast, PBS treatment of carrier mice (five mice per cage) (arrows) did not affect the levels of C. difficile cultured from feces, although these mice were prone to high-level shedding of C. difficile after clindamycin treatment (see panel a). In this experiment mice were pretreated with neomycin, although prior antibiotic treatment is not necessary to establish the carrier state, as shown in Fig. 2. (c) Levels of C. difficile vegetative and spore forms (supershedder total) and spores (supershedder spores) excreted by supershedders and of spores excreted by carrier mice. Five mice were used for each sample.
FIG. 2.
FIG. 2.
Antibiotic-induced C. difficile supershedding state is associated with reduced intestinal microbiota diversity. (a) Average fecal shedding of C. difficile by carrier mice (five mice per cage) treated with clindamycin to induce a transient supershedder state. The error bars indicate standard deviations. DNA was extracted from fresh feces from each mouse at the indicated time points (green arrowheads) to create 16S rRNA gene clone libraries. The detection limit was 103 CFU C. difficile/g feces. (b) Temporal shifts in the intestinal bacterial community after clindamycin treatment of carrier mice as determined by 16S rRNA gene analysis. The levels of predominant bacterial groups are expressed as percentages of the clone libraries using pie charts. On day 23 1,400 clones were included (average, 280 clones/mouse), on day 25 1,202 clones were included (average, 241 clones/mouse), on day 27 1,204 clones were included (average, 241 clones/mouse), on day 29 1,081 clones were included (average, 216 clones/mouse), and on day 33 1,167 clones were included (average, 233 clones/mouse). (c) SDI for each phase of the microbiota community structure determined by 16S rRNA gene phylotypes.
FIG. 3.
FIG. 3.
Host transmission of C. difficile via supershedding mice and environmental spore contamination. (a) Transmission efficiency, indicating the percentage of naïve mice that acquired C. difficile infection from donor mice. The number of naïve mice housed with no donor was 10, the number of naïve mice housed with carrier mice was 15, and the number of naïve mice housed with supershedding mice was 20. (b) Percentage of naïve mice that acquired C. difficile infection from a spore-contaminated environment. The number of naïve mice housed in a contaminated environment with no sporicide was 15, the number of naïve mice housed in a contaminated environment treated for 1 min with sporicide was 10, the number of naïve mice housed in a contaminated environment treated for 5 min with sporicide was 10, and the number of naïve mice housed in a contaminated environment treated for 20 min with sporicide was 10. Groups of 5 or 10 naïve mice were housed in cages previously contaminated by supershedders. The transmission efficiency was determined as described in Materials and Methods.
FIG. 4.
FIG. 4.
Supershedders efficiently transmit C. difficile disease to naïve recipients. (a to f) Representative images that illustrate epithelial damage and the inflammatory response in ceca of recipient mice (100 mice) after clindamycin treatment (see Fig. S3 in the supplemental material). (a) Toluidine blue staining of the cecum from a clindamycin-treated control mouse. (b) Toluidine blue staining demonstrating edema and immune cell infiltrate (arrows) within the cecal submucosa of a C. difficile-infected mouse. (c) Transmission electron micrograph of the intact epithelial brush border (black arrow) and tight junctions (white arrows) of the cecum from a clindamycin-treated control mouse. (d) Disrupted tight junction and microvillus effacement within the cecal submucosa of a C. difficile-infected mouse (arrow) (compare with panel c). (e) C. difficile vegetative cells and spores associated with epithelial effacement and cellular invasion and immunogold labeling of C. difficile within the lumen (inset). (f) Matt of C. difficile cells (arrows) overlaying damaged and necrotic microvilli.
FIG. 5.
FIG. 5.
Myd88−/− mice are susceptible to severe C. difficile disease. (a) Average weights of wild-type, Igh6−/−, and Myd88−/− mice that were infected with C. difficile via transmission (green arrow) and subsequently treated with clindamycin (red bar). Wt, wild type. (b) Hemotoxylin- and eosin-stained sections of cecal mucosa from wild-type, Igh6−/−, and Myd88−/− mice that were treated with clindamycin but not infected and from mice treated with clindamycin and infected with C. difficile. Infection with C. difficile results in moderate intestinal inflammation (cecum) in wild-type and Igh6−/− mice and more severe intestinal inflammation in Myd88−/− mice. Magnification, ×20.
FIG. 6.
FIG. 6.
Relationship between C. difficile carrier state and supershedding state in response to antibiotic treatment and role of the host immune system in severe disease. C. difficile carrier mice possess a stable and diverse intestinal microbiota community (small rods) which includes low levels of C. difficile (red rods with spores). As a result, carrier mice excrete low levels of spores and are poor donors for C. difficile infection. Antibiotic treatment of carrier mice reduces the diversity of the intestinal microbiota community, allowing C. difficile to proliferate and sporulate (red circles). As a result, a supershedding state occurs with high levels of spore excretion and efficient host-to-host transmission. Withdrawing the antibiotic allows the intestinal microbiota to rediversify and suppresses C. difficile levels, thereby reestablishing the carrier state and reducing the contagiousness of the host. Transmission of C. difficile to Myd88-deficient mice, but not transmission of C. difficile to Igh6−/− mice, results in severe disease, implicating Toll-like receptor signaling in protection against virulent C. difficile overgrowth. Wt, wild type.
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