Antibacterial properties of bile acid oxazoline compounds

347

3023

ANTIBACTERIAL PROPERTIES OF BILE ACID OXAZOLINE COMPOUNDS Phillip B. Hylemonl, Rebecca J. Frickel and Erwin H. Mosbach2 IDepartment of Microbiology and Immunology, Medical College of Virginia, Virginia Commonwealth University, Richmond, Va. 23298 and 2Lipid Research Laboratory, Department of Surgery, Beth Israel Medical Center, New York, New York 10003 Remceived 8-16-82 ABSTRACT Chenooxazoline3 (50-100 PM) inhibited (>50%) both 7a and 7B-dehydroxylase activities in whole cells and cell extracts of Eubacterium sp. V.lP.1. 12708. Chenooxazoline (250 uM) and methylchenooxazoline (>25 PM) but not lithooxazoline (A.100 PM) inhibited growing cultures of Eubacterium sp. V.P.I. 12708. Chenooxazoline (100 PM) also inhibited the cs of certain members of the genera Eubacterium, Clostridium, Bacteroides and Staphylococcus but not Pseudomonas, Escherichia, Salmone=ii-a-or the eucaryotic microorganism, Saccharomyces cerevisiae ((w INTRODUCTION Chenodeoxycholic acid has been used to induce cholesterol gallstone dissolution in man (1); however, hepatotoxicity has been associated with feeding chenodeoxycholic acid to laboratory animals presumably due to the formation of lithocholic acid by microbial 7a-dehydroxylation (2-3). Ursodeoxycholic acid, the 78-epimer of chenodeoxycholic acid, has been reported to dissolve cholesterol gallstones, but does not appear to induce the hepatotoxicity seen during chenodeoxycholic acid feeding to laboratory animals (4).

The difference has been attributed to the

decreased formation of lithocholic acid from ursodeoxycholic acid as compared to chenodeoxycholic acid.

Recently, White _et al. (5) reported

that Eubacterium sp. V.P.I. 12708 can directly 'I-dehydroxylate ursodeoxycholic acid but at a slower rate than chenodeoxycholic acid.

The

treatment of cholesterol gallstones with either chenodeoxycholic acid or ursodeoxycholic acid might be improved if an inhibitor of 7a and 7Bdehydroxylase activities were discovered.

In this regard, Ayengar et

-al. (6) have recently chemically synthesized a series of bile acid

Vohine

90,

Number 3

S

TBROXD6

Sepptember, 1982

oxazoline compounds (Fig. 1) that when fed to rats markedly altered fecal bile acid patterns (7). chenooxazoline

After two weeks of treatment with methyl-

(0.1% of the diet) there was an almost complete replace-

Fig. 1. Chemical structures of lithooxazoline (A), chenooxazoline (8) and methylchenooxazoline (C).

S

Cl?DROTDI)

349

ment of hyodeoxycholic acid3 with w-muricholic acid3 indicating a suppression of the bacterial 78-dehydroxylase.

However, it was not clear from

these studies if the bile acid oxazoline compound inhibited the 7dehydroxylation activity of certain intestinal bacteria or the growth of the 7-dehydroxylating microorganisms. The results presented in this communication suggest that certain bile acid oxazoline compounds can inhibit both 7a and 7B-dehydroxylase activities in Eubacterium sp. V.P.I. 12708 and have antibacterial activity against a variety of anaerobic intestinal bacteria. MATERIALS AND METHODS Eubacterium sp. V.P.I. 12708 and Clostridium difficile V.P.I. 2642 were cultured anaerobically under Nz in Brain Heart Infusion (Difco) medium essentially as described previously (8). Bacteroides species and Clostridium sordellii V.P.I. 11801 were grown in Peptone-Yeast Extractmucose-(PYG)as described by Holdeman and Moore (9). Pseudomonas aeru inosa PA0 was cultured in glucose basal salts medium as ---.b+ descn e previously (10). Salmonella typhimurium was grown in nutrient broth (Difco) and Escherichia coli K-12 was rown in Luria broth. Sta hylococcus epidermidis (clinical isolate 7 was grown in T-soy broth Tati% glucose. Saccharom ces cerevisiae ATCC 9763 was grown Growth of all microorganisms was in Sabauraud dextrose brothi determined by measuring culture turbidity with a Klett-Summerson colorimeter equipped with a number 66 filter. Cell viability studies of Eubacterium sp. V.P.I. 12708 were determiined by diluting cells in anaerobic salts solution (9) and plating on BHI agar medium. Agar plates were incubated anaerobically in Gas-Pak (BBL) jars for 48 h at 37°C. Colonies were then counted to determine cell viability. To determine the effects of bile acid oxazoline compounds (in MeOH) on growth of microorganisms, varying concentrations (10 uM, 25 PM, 50 IJM,, and 100 uM) were added to 10 ml early log phase cultures. Turbidity and cell viability measurements were determined in culture samples taken before and following the addition of bile acid oxazoline compounds. Controls were run with methanol alone and no addition. To determine whether other bacteria may be inhibited by bile acid oxazoline compounds, selected species of aerobic, facultative and anaerobic bacteria as well as Saccharomyces cerevisiae were used in growth inhibition studies. Experiments were performed essentially as described for Eubacterium sp. V.P.I. 12708 and illustrated in Fig. 3.

350

S

TEIROIDN

Enzyme Assay for 7-Dehydroxylase: Enzymatic 7-dehydroxylation of [24-l'+CJ chenodeoxycholic acid, L24-14C] cholic acid and [II, 12-3H]ursodeoxycholic acid was measured by a radiochromatographic assay procedure described previously (8). Varying concentrations (50 PM, 100 PM, 200 PM, 400 uM) of bile acid oxazoline compounds (in MeOH) were added to the reaction mixtures. Assays were initiated by the addition of whole cells or cell extracts (l-2 mg protein) and were incubated anaerobically (37°C) under an argon gas atmosphere for 2 minutes. Enzyme activity was terminated by acidification. The reaction mixtures were extracted with ethyl acetate and radiolabeled bile acids were quantitated as described previously (5). Bile acid oxazoline derivatives were synthesized as described previously (6). RESULTS The data presented in Table 1 show that growing cultures of certain members of the genera Eubacterium, Clostridium, Bacteroides and Staphylococcus are sensitive to one or more bile acid oxazoline compounds. Aerobic and facultative gram-negative bacteria were resistant under these conditions, except for Salmonella typhimurium TA 100.

Interest-

ingly, resting cell suspensions of j$. fragilis ATCC 25285 also appeared to be resistant to lysis by chenooxazoline.

Moreover, the eucaryotic

microorganism, Saccharomyces cerevisiae was resistant to chenooxazoline at concentrations as high as 0.4 mM. Experiments were then performed to determine if selected bile acid oxazoline compounds could inhibit 7a and 7kdehydroxylase

acitivities in

whole cells and cell extracts of Eubacterium sp. V.P.I. 12708. oxazoline, methylchenooxazoline

Cheno-

and lithooxazoline were tested for their

ability to inhibit 7~ and 7B-dehydroxylase activities.

The results

presented in Fig. 2 (_Aand B) show a concentration dependent inhibition of both 7~ and 7B-dehydroxylase activities in both whole cells and cell extracts of this bacterium when chenooxazoline was added to reaction

S Table 1. -Urganism

-.l?DR.OIDS

351

Effect of Bile Acid Oxazoline Compounds on Growth of Selected Microorganisms oxazoline derivatives Chenomethylchenolithooxazolinea oxazolinea oxazolinea

On (+I Anaerobe Eubacterium so. E.species E. species nostridium sordellii C.diffici4e & (-) Anaerobe Bacteroides distasonis B.vulgatus

SNTd NT S S

RC R Ie R S

:

I I

S R

S S NT

iT R

:

:

S I

R

NT

NT

I: S

NT R S

R R R

z(O.4 mM)

NT

R NT

VP1 12708 22C30.2 22c50.2 VP1 11801 VP1 2642

Sb S s S S

VP1 4243 VP1 4245

S

VP1 2393

;

ATCC 25285 ATCC 25285 -(cell suspension) VP1 5482 B. thetaiotaomicron VP1 2302 B. thetaiotaomicron & (-1 Aerobe Pseudomonas aeruginosa PA0 Kc-) FacuZtative Escherichia coli K-12 SalmoneTitmmurium clinical Salmomtyphimurium TA-100

?3YT~FacuZtative Staphylococcus epidermidis Saclcaromyces cerevisiae ATcc 9763

aFinal concentration (0.1 mM); bS = sensitive; cR = resistant; dNT = not tested; eI = Inhibition of growth but no apparent lysis. mixtures containing either [14C]-chenodeoxycholic acid or [3H]-ursodeoxycholic acid.

In the case of chenodeoyycolic acid 7a-dehydroxyla-

tion, 50 JIM to lOO.uM concentrations were required to inhibit enzyme activity by 50%.

Ursodeoxycholic acid 7B-dehydroxylation was somewhat

more sensitive to chenooxazoline inhibition (Fig. 2).

Cholic acid

7a-dehydroxylation was also inhibited by chenooxazoline (data not shown). Methylchenooxazoline

and lithooxazoline did not produce appre-

ciable inhibition of 7a-dehydroxylase activity.

352

S

“b

TIlROIDB

0.1

0.2

CHENOOXAZOLINE

0.3

0.4

(mM 1

Fig. 2. Effect of chenooxazoline on 7a (0) and 7B(O)-dehydroxylase activities in whole cells (A) and cell extracts (B) of Eubacterium sp. V.P.I. 12708. The initial activities of 7a and 7B-dehydroxylase activities in whole cells and cell extracts were (1399, 330 nmol/hr/mg protein) and (502, 275 nmol/hr/mg protein), respectively.

S

TBROXDBI

353

The next objective of this investigation was to determine if bile acid oxazoline compounds were capable of inhibiting the growth of selected intestinal bacteria.

Therefore, varying concentrations (0.01 mM

to 0.1 mM) of methylchenooxazoline,

chenooxazoline and lithooxazoline

were added to growing cultures of Eubacterium sp. V.P.I. 12708 under analerobic conditions.

The data shown in Fig. 3 indicate a marked de-

crease in turbidity of cultures of this organism approximately 30 minutes following the addition of methylchenooxazoline

(> 25 FM) and

chenooxazoline (2 50 FM) but not lithooxazoline (2 100 PM).

Cell

viability studies were in accord with the turbidity measurements.

When

chenodeoxycholic acid (100 MM) or lithocholic acid (100 uM) were added to growing cultures there was no detectable inhibition of growth. Growth inhibition also occurred when bile acid oxazoline compounds were added to growing of Clostridium sordellii and &. difficile (Fig. 4). DISCUSSION The present investigation reports two major observations.

First,

chenooxazoline is an inhibitor of both 7a and 7s-dehydroxylase activities in whole cells and cell extracts of Eubacterium sp. V.P.I. 12708. Thirs compound, or analogous more active derivatives, could theoretically implrove treatment of cholesterol gallstones by chenodeoxycholic acid or ursodeoxycholic acid by inhibiting both 7a and 7s-dehydroxylase activity.

This may have several important effects including:

(a) allowing

chenodeoxycholic acid or ursodeoxycholic acid to remain within the enterohepatic circulation for longer time periods;

(b) prevent the forma-

tion of potentially toxic lithocholic acid (11); and (c) prevent the formation of deoxycholic acid.

In this regard, Low-Beer and Nutter (12)

354

S

TBEOXDb

Fig. 3. Effect of methylchenooxazoline (top), lithooxazoline (middle) and chenooxazoline (bottom) on growth of Eubacterium sp. V.P.I. 12708. Symbols indicate varying concentrations of each compound: (R) 0.1 mM; (0) 0.05 mM; (A) 0.025 mM; (0) 0.01 mM; (A) methanol control; (e) no addition control.

S

TIlROXDb

355

200 -

F p IOO-J 80ii % d t

60-

f t”

40-

:: Y y

20-

addition

-

addition

IO’

,

I

0

I

I

0

3 2 HOURS

,

1

4

5

Fig. 4. Effect of methylchenooxazoline (m), chenooxazoline (A) and lithooxazoline on growth of Clostridium sordellii (upper panel) and &. difficile (lower panel). All compounds were tested at 100 $+l final Methanol (@) and no addition (0) controls are also coGentration. shown for each organism.

(A)

have reported that the formation of deoxycholic acid increases the cholesterol saturation index of biliary bile in man. The other surprising result of this investigation was the discovery that bile acid oxazoline compounds have antibacterial properties.

The

antibacterial activity of these compounds occurred at low concentrations (25 to 100 PM) and appeared to have a requirement for a specific steroid structure in addition to the oxazoline at C-24.

Methylchenooxazoline

and chenooxazoline were more effective than lithooxazoline in inhibiting the growth of sensitive bacteria (Figs. 3 and 4).

These two observa-

tions give a satisfactory explanation for the virtual absence of secondary bile acids in rats fed methylchenooxazoline

(7).

The mechanism of bile acid oxazoline inhibition of bacterial growth is presently unknown.

However, we do not believe that the inhibition is

strictly due to the detergent properties of these molecules.

This is

based on the observation that inhibition occurred at very low bile acid oxazoline concentrations, required growing cells and appeared to be highly specific for steroid structure.

We have observed that within a

bacterial population resistant cells arise very rapidly.

It is not

clear if this resistance represents a selection of genetically different bacteria or a physiological change in the bacterial cells. ACKNOWLEDGEMENTS This work was supported by USPHS research grants AM 26262 from the National Institute of Arthritis, Metabolism and Digestive Disease and HL-24061 from the National Heart, Lung and Blood Institute.

S

TIlICOIDS

357

REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9.

10. 11. 12.

Thistle, J. L. and A. F. Hofmann. N. Engl. J. Med. 289: 655-659 (1973). Dyrszka, H. G. Salen, G. Zaki, T. Chen and E. H. Mosbach. Gastroenterology 70: 93-104 (1976). Morrissey, K. P., C. K. McSherry, R. L. Swarm, W. H. Nieman and J. E. Deitrick. Surgery 77: 851-860 (1975). Stiehl, A., P. Czygan,B. Kommerell, H. J. Weiss and K. H. Holtmuller. Gastroenterology 75: 1016-1020 (1978). White, 8. A., R. J. Fricke andT. 8. Hylemon. J. Lipid Res. 23: 145-153 (1982). Ayengar, N. K. N., A. K. Singhal, C. K. McSherry and E. H. Mosbach. Steroids 38: 333-345 (1981). Mosbach, r H., A. K. Singhal, N. K. N. Ayengar, P. S. May and C. K. McSherry. In: Bile Acids and Lipids (W. Gerok, W. Paumgartner and A. Stiehl, eds.), MTP Press Ltd., Lancaster, England (1981). White, B. A., R. H. Lipsky, R. J. Fricke and P. B. Hylemon. Steroids 35: 103-109 (1980). Holdeman,T. V. and W. E. C. Moore (eds.) Anaerobe Laboratory Manual, 2nd ed., Virginia Polytechnic Institute and State University, Blacksburg, (1973). Hylemon, P. B. and P. V. Phibbs, Jr. Biochem. Biophys. Res. Commun. 48: 1041-1048 (1972). Palmer, r H. Arch. Intern. Med. 130: 606-617 (1972). Low-Beer, T. S. and S. Nutter, LaGt 2: 1063-1065 (1978).

3The following abbreviations have been used: Chenooxazoline = 2-(3a, 7a-dihydroxy-24-nor-5@-cholan-23-yl-4, 4-dimethyl-2-oxazoline (B, fig. 1); methylchenooxazoline = 2-(3cl, 76-dihydroxy-7c-methyl-24-nor-58cholan-23-yl)-4, 4-dimethyl- oxazoline (c, fig. 1); lithooxazoline = 2-(3a-hydroxy-24-nor-58-cholanyl)-4, 4-dimethyl-2-oxazoline (A, fig. 1); aj-muricholic acid = 3a, 68, 7B-trihydroxy-5@-cholan-24-oic acid; hyodeoxycholic acid = 3a, 6a-dihydroxy-5B-cholan-24-oic acid.