Commensal modulation of Clostridioides difficile infection in vivoMadeline Graham1, Nicholas DiBenedetto1*, Mary Delaney1,2, Aidan Pavao1, Vladimir Yeliseyev1,

Lynn Bry1,2

1. Massachusetts Host-Microbiome Center, Department of Pathology, Brigham & Women's Hospital, Harvard Medical School. Boston, MA. 2. Clinical Microbiology Laboratory, Department of Pathology, Brigham & Women's Hospital, Harvard Medical School. Boston, MA. * Current address: Department of Molecular Biology and Microbiology, Tufts University School of Medicine. Boston, MA.

Background Results

Methods

Conclusions

Clostridioides difficile infection (CDI) places a substantial burden onhealthcare systems. Disruptions in the commensal microbiota due toantibiotic treatment increase the risk of developing CDI. Treatment forrecurrent CDI that does not respond to antibiotics entails the replacement ofcommensal microbiota via fecal microbiota transplantation (FMT). WhileFMT can be effective against CDI, it poses risks, particularly for susceptiblepatient populations. Specific bacteriotherapeutics with well-definedmechanisms of action against CDI would be more effective and and comewith fewer risks to patients. The present study aims to assess the effects ofa select commensal species, Clostridium scindens (CSCI), that has beenhypothesized to protect against CDI by its production of 2° bile acids, andhas shown promise in animal models as a CDI bacteriotherapeutic.

Using a gnotobiotic mouse model, we previously studied the effects of othercommensal Clostridia with proposed activity to modulate CDI in vivo:Paraclostridium bifermentans (PBI) and Clostridium sardiniense (CSAR).The present work investigates the effects of CSCI on CDI. Cecal contentswere analyzed for C. difficile vegetative and spore biomass, toxin Bcontent, tissue damage, and short-chain fatty acid profiles.

0 1 2 30

50

100

25

75

13 14Days

% S

urvi

val

Cdiff (n=24)CSCI+Cdiff (n=16)PBI+Cdiff (n=18)

CSAR+Cdiff (n=6)

A. B. C. D.

K. CSCI+Cdiff 14dJ. PBI+Cdiff 14dF. Cdiff 24h H. PBI+Cdiff 24hE. GF colon G. CSCI+Cdiff 24h I. CSAR+Cdiff 24h

Figure 2. C. scindens protects germfree mice from acute lethal C. difficile infection but not against chronic disease. A: Survival curves. B: Log10 µg/gextracellular toxin B in cecal contents. Different letters within time points represent significance values of p<0.05 by Kruskal-Wallis and Dunn’s multiple comparisonstest. Significance values of comparisons between 24h and 14d values in CSCI+C difficile and PBI+C. difficile and comparisons between CSCI+C difficile and PBI+C.difficile at 14d by Mann-Whitney test: *0.01≤p≤0.05; ***p<0.001. C: Log10 C. difficile vegetative CFU and D: spore biomass/g cecal contents. E-I: H&E-stained colontissue; E-H: 200X, I: 40X, J-K: 100X. E: Normal germfree mucosa. F: C. difficile-infected mice at 24h showing transmural neutrophilic infiltrates and surface epithelialloss. G: CSCI+C. difficile-infected mice at 24h showing epithelial stranding. H: PBI+C. difficile-infected mice at 24h showing epithelial stranding and neutrophilicinfiltrates. I: CSAR+C. difficile-infected mice at 24h showing epithelial loss and severe submucosal edema. J: PBI+C. difficile-infected mice at 14d showing intactepithelium. K: CSCI+C. difficile-infected mice at 14d showing marked epithelial hyperplasia.

We show that while CSCI demonstrates acute protection against lethal CDI,the commensal is not as effective as PBI, which conferred 100% survival andtissue resolution with long-term survival. While most mice with CSCI survivedacutely, they demonstrated higher toxin B levels and chronic colonic damage.These findings demonstrate the efficacy of select commensals and indicatethat while CSCI has been touted as a key protective against CDI, it lacks theefficacy of other Clostridial species such as PBI.

Figure 3. Cecal SCFA profiles of different colonization states. Color scale indicates mM SCFA/g of cecal contents.

Figure 1. Predicted mechanisms of C. scindens activity against C. difficileinfection in vivo. A: Mechanism of C. scindens modulation of C. difficile infection by suppressing spore germination. B: Gnotobiotic experiment overview.

A.

B.

Survival Toxin B Log10 µg/g Cecal Contents

Cdiff

CSCI+Cdiff

PBI+Cdiff

CSAR+Cdiff

Cdiff

CSCI+Cdiff

PBI+Cdiff

CSAR+Cdiff

CSCI+Cdiff

PBI+Cdiff

0.1

1

10

100

1000

Log1

0 To

xin

B µ

g/g

Cec

al C

onte

nts

Cdiff

CSCI+Cdiff

PBI+Cdiff

CSAR+Cdiff

20hr 24hr 14d

Toxin B GramCecal Contents

aa

a

b

cc

d d

✱✱✱

✱✱✱

Cdiff

CSCI+Cdiff

PBI+Cdiff

CSAR+Cdiff

Cdiff

CSCI+Cdiff

PBI+Cdiff

CSAR+Cdiff

CSCI+Cdiff

PBI+Cdiff

5

6

7

8

9

Log1

0 Ve

geta

tive

CFU

/g C

ecal

Con

tent

s Cdiff

CSCI+Cdiff

PBI+Cdiff

CSAR+Cdiff

20hr 24hr 14d

✱

a,b

bb

b c,dc

d

c

Log10 C. difficile VegetativeCFU/g Cecal Contents

Cdiff

CSCI+Cdiff

PBI+Cdiff

CSAR+Cdiff

Cdiff

CSCI+Cdiff

PBI+Cdiff

CSAR+Cdiff

CSCI+Cdiff

PBI+Cdiff

5

6

7

8

9

Log1

0 S

pore

CFU

/g C

ecal

Con

tent

s

20hr 24hr 14d

a

ba, b

a

d

cc c

✱

✱

Log10 C. difficile SporeCFU/g Cecal Contents