Bile Acid 7α-Dehydroxylating Gut Bacteria Secrete Antibiotics that Inhibit Clostridium difficile: Role of Secondary Bile Acids

Abstract

Clostridium scindens biotransforms primary bile acids into secondary bile acids, and is correlated with inhibition of Clostridium difficile growth in vivo. The aim of the current study was to determine how C. scindens regulates C. difficile growth in vitro and if these interactions might relate to the regulation of gut microbiome structure in vivo. The bile acid 7α-dehydroxylating gut bacteria, C. scindens and C. sordellii, were found to secrete the tryptophan-derived antibiotics, 1-acetyl-β-carboline and turbomycin A, respectively. Both antibiotics inhibited growth of C. difficile and other gut bacteria. The secondary bile acids, deoxycholic acid and lithocholic acid, but not cholic acid, enhanced the inhibitory activity of these antibiotics. These antibiotics appear to inhibit cell division of C. difficile. The results help explain how endogenously synthesized antibiotics and secondary bile acids may regulate C. difficile growth and the structure of the gut microbiome in health and disease.

Keywords: 1-acetyl-β-carboline; Clostridium difficile; Clostridium scindens; Clostridium sordellii; cyclic dipeptides; dysbiosis; gut microbiome; turbomycin A.

Figure 1.. C. difficile Growth is Inhibited by C. scindens in the Presence but Not Absence of Cholic Acid

PYF medium containing 50 μM cholic acid (CA) or 50 μM deoxycholic acid (DCA) was inoculated with 106 vegetative cells/ml of C. difficile (Panel A) or C. scindens (Panel B), incubated for a 24 hr. time course, and growth determined at indicated time points by measuring optical density (OD) at 600 nm. PYF medium was inoculated with 106 vegetative cells/ml of C. difficile and C. scindens grown in either mono culture with 50 μM CA (Panel C) or co-culture without CA (Panel D) or with 50 μM CA (Panels E and F), incubated over a 24 hour time course and levels of bacteria estimated at indicated time points by measuring gene dosage of Toxin A gene for C. difficile and baiCD gene for C. scindens (STAR Methods).

Figure 2.. Identification of antibacterial compounds secreted by C. difficile

The purification protocol for antibacterial compounds secreted by C. difficile is described in Figure S1. The mass spectra and ¹H and ¹³C NMR chemical shifts of peaks 1 through 4 were used to identify compounds. Antibacterial compounds were identified as chiral isomers of cyclo(Leu-Pro) for peaks 1 and 2 and chiral isomers of cyclo (Phe-Pro) for peaks 3 and 4.

Figure 3.. Identification of antibacterial compounds secreted by C. sordellii ATCC 9714 and C. scindens ATCC 35704.

The purification protocols for antibacterial compounds secreted by C. sordellii and C. scindens are described in Figure S2. The LC-ESI/MS and ¹H and ¹³C NMR chemical shifts of SDX26–1 and SDX26–2 secreted by C. sordellii were positively identified as turbomycin A and 1,1,1-tris(3-indolyl)-methane, respectively. The LC-ESI/MS and ¹H and ¹³C NMR chemical shift assignments of antibacterial compound (XPSE-27) was positively identified as 1-acetyl-β-carboline.

Figure 4.. Effects of different bile acids on inhibition of growth of C. difficile by turbomycin A and 1-acetyl- β-carboline.

Varying concentrations of Turbomycin A (0, 0.5, 1, 5 and 10 μg/ml) or 1-acetyl-β-carboline (0, 1, 5, 10 and 25 μg/ml) and either cholic acid (CA) (0 to 100 μM), deoxycholic acid (DCA), (0 to 100 μM) or lithocholic acid (LCA), (0 to 25 μM) were added to PYF medium, inoculated with 106 vegetative cells/ml of C. difficile, incubated for 24 hrs. and optical density reading determined at 600 nm. * p< 0.05, ** p< 0.01

Figure 5.. Effect of 1,1,1-tris(3-indolyl)-methane (TIM) and different bile acids (BA) on inhibition of C. difficile ATCC 9689 growth.

A. Varying concentrations of cholic acid (CA) (0 to 100 μM), 12-oxo-lithocholic acid (12-oxo-LCA) (0 to 100 μM), iso-deoxycholic acid (3β-DCA) (0 to 100 μM) or deoxycholic acid (DCA) (0 to 100 μM) were added to PYF medium, inoculated with 106 vegetative cell/ml of C. difficile, incubated for 24 hrs, and O.D. readings at 600 nm determined. B. Experiments were repeated with the addition of TIM (7.5 μg/ml) added to the culture medium. *p<0.05, **p<0.01.

Figure 6.. Changes in C. difficile ATCC 9689 cell morphology induced by antibacterial compounds secreted by C. sordelli ATCC 9714

A) PYF medium control; B) C. difficile grown in 50% C. sordellii spent culture medium; C) C. diffiicle incubated in 100% C. sordellii spent culture medium. Each culture (1ml) was concentrated to 100 μl by centrifugation, 5 μl of cell suspension loaded on to a slide for Gram staining and photo images taken (STAR Methods). D) Cell length was determined using a scale bar generated by Neurolucia 1027 software in several microscopic fields. (Magnification 1000x). ** P<0.01