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. 2020 May 11;202(11):e00039-20.
doi: 10.1128/JB.00039-20. Print 2020 May 11.

Strain-Dependent Inhibition of Clostridioides difficile by Commensal Clostridia Carrying the Bile Acid-Inducible (bai) Operon

A D Reed  1 , M A Nethery  2 , A Stewart  3 , R Barrangou  2 , C M Theriot  4
Affiliations

Strain-Dependent Inhibition of Clostridioides difficile by Commensal Clostridia Carrying the Bile Acid-Inducible (bai) Operon

A D Reed (V体育官网入口) et al. J Bacteriol. .

Abstract

Clostridioides difficile is one of the leading causes of antibiotic-associated diarrhea. Gut microbiota-derived secondary bile acids and commensal Clostridia that carry the bile acid-inducible (bai) operon are associated with protection from C. difficile infection (CDI), although the mechanism is not known. In this study, we hypothesized that commensal Clostridia are important for providing colonization resistance against C. difficile due to their ability to produce secondary bile acids, as well as potentially competing against C. difficile for similar nutrients VSports手机版. To test this hypothesis, we examined the abilities of four commensal Clostridia carrying the bai operon (Clostridium scindens VPI 12708, C. scindens ATCC 35704, Clostridium hiranonis, and Clostridium hylemonae) to convert cholate (CA) to deoxycholate (DCA) in vitro, and we determined whether the amount of DCA produced was sufficient to inhibit the growth of a clinically relevant C. difficile strain. We also investigated the competitive relationships between these commensals and C. difficile using an in vitro coculture system. We found that inhibition of C. difficile growth by commensal Clostridia supplemented with CA was strain dependent, correlated with the production of ∼2 mM DCA, and increased the expression of bai operon genes. We also found that C. difficile was able to outcompete all four commensal Clostridia in an in vitro coculture system. These studies are instrumental in understanding the relationship between commensal Clostridia and C. difficile in the gut, which is vital for designing targeted bacterial therapeutics. Future studies dissecting the regulation of the bai operon in vitro and in vivo and how this affects CDI will be important. IMPORTANCE Commensal Clostridia carrying the bai operon, such as C. scindens, have been associated with protection against CDI; however, the mechanism for this protection is unknown. Herein, we show four commensal Clostridia that carry the bai operon and affect C. difficile growth in a strain-dependent manner, with and without the addition of cholate. Inhibition of C. difficile by commensals correlated with the efficient conversion of cholate to deoxycholate, a secondary bile acid that inhibits C. difficile germination, growth, and toxin production. Competition studies also revealed that C. difficile was able to outcompete the commensals in an in vitro coculture system. These studies are instrumental in understanding the relationship between commensal Clostridia and C. difficile in the gut, which is vital for designing targeted bacterial therapeutics. .

Keywords: 7α-dehydroxylation; Clostridia; Clostridioides difficile; bile acids; cholate; deoxycholate. V体育安卓版.

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Figures

FIG 1
FIG 1
Genomic variation in selected bai genes carried by commensal Clostridia. Alignment of the bai operon and baiN across Clostridium strains. Each protein sequence was compared against its counterpart in the reference strain Clostridium scindens ATCC 35704, generating the amino acid percent identity labeled within each gene.
FIG 2
FIG 2
C. difficile and C. hylemonae are more resistant to cholate than are other commensal Clostridia tested. The MICs of CA and DCA were tested on C. difficile R20291, C. scindens VPI 12708, C scindens ATCC 35704, C. hiranonis, and C. hylemonae. The MIC was defined as the lowest concentration of compound that showed no visible growth. Growth was defined at 24 h for C. difficile and 48 h for commensal Clostridia. Four biological replicates were performed.
FIG 3
FIG 3
Inhibition of C. difficile by C. scindens grown in medium supplemented with 2.5 mM cholate is correlated with high levels of deoxycholate production. Shown is inhibition of C. difficile after 24 h of growth with supernatants from C. scindens VPI 12708 (A), C. scindens ATCC 35704 (B), C. hiranonis grown without bile acid supplementation or supplemented with 0.25 or 2.5 mM CA (C), and C. hylemonae grown without bile acid supplementation or supplemented with 2.5 mM or 7.0 mM CA (D). The concentrations of CA and DCA in each supernatant are shown to the right of the inhibition data. Experiments were run in duplicate, and three biological replicates were performed. Inhibition by the supernatants was compared to a no-bile-acid C. difficile control consisting of a 4:1 dilution of PBS to BHI. Statistical significance between treatments and the control was determined using one-way ANOVA, with Tukey used for multiple comparisons (**, P < 0.01; ****, P < 0.0001).
FIG 4
FIG 4
C. scindens and C. hiranonis have increased expression in bai operon genes when medium is supplemented with 2.5 mM CA. Shown is expression of baiG, baiE, and baiN in C. scindens ATCC 35704 (A), C. scindens VPI 12708 (B), C. hiranonis in medium without CA or medium supplemented with 0.25 mM or 2.5 mM CA (C), and C. hylemonae in medium without CA or medium supplemented with 2.5 mM or 7.0 mM CA (D). Experiments were run in quadruplicate, and three biological replicates were performed. The expression in medium supplemented with CA was compared to the expression in medium without CA. Statistical significance was determined by one-way ANOVA, with Tukey used for multiple comparisons (*, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001).
FIG 5
FIG 5
C. difficile outcompetes commensal Clostridia in vitro. Competition index for 1:1 competition between C. difficile and C. scindens ATCC 35704, C. scindens VPI 12708, C. hylemonae, and C. hiranonis. The competition index value was determined by comparing the CFU per milliliter of the competition coculture to that of the monoculture for each strain at 24 h. Statistical significance was determined by using Student's t test (**, P < 0.01; ***, P < 0.001; ****, P < 0.0001).
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