APPLIED AND ENVIRONMENTAL MICROBIOLOGY, JUlY 1987, p. 1655-16600099-2240/87/071655-06$02.00/0Copyright C) 1987, American Society for Microbiology

Sulfatase Activity in a Peptococcus niger Strain from theHuman Intestinal Microflora

JOHAN R. VAN ELDERE,* GUIDO DE PAUW, AND HENDRICK J. EYSSEN

Rega Institute, University of Leuven, B-3000 Leuven, Belgium

Received 9 February 1987/Accepted 21 April 1987

A strictly anaerobic gram-positive coccus, identffied as Peptococcus niger, that developed sulfatase activitytowards steroid-3-sulfate esters was isolated from human fecal material. This strain desulfated the arylsulfateesters estrone-3-sulfate (100%) and ,1-estradiol-3-sulfate (50%); only trace amounts of desulfated estriol-3-sulfate were found. In addition, alkylsulfatase activity was found towards the 3a-sulfates of 5a-androstane-17-one and 5j1-androstane-17-one and towards the 3p-sulfates of 5ot-androstane-17-one, A5-androstene-17-one,5a-pregnane-20-one, and A5-pregnene-20-one, all of which were 100% desulfated. No sulfatase activity was

found towards the 17-sulfate esters of O-estradiol or A4-androstene-3-one-17ci-ol. The nonsteroid arylsulfateesters paranitrophenyl sulfate, paranitrocatechol sulfate, and phenolphthalein disulfate were desulfated 70, 40,and 40%, respectively. In addition to its sulfatase activity, this strain also developed C-17 oxidoreductaseactivity towards the estrogens and androsta(e)nes and C-3 oxidoreductase activity towards androsta(e)nes andpregna(e)nes.

Gut microorganisms are involved in many complex bio-chemical activities that affect the availability of both naturaland synthetic steroids to the host. Most if not all steroidhormones are taken up by the liver, conjugated with glucu-ronic or sulfuric acid, and excreted via the bile into the gut.Sulfo and glucurono conjugation decrease lipid solubility andreduce reabsorption from the gastrointestinal tract. In thegut, however, steroid glucuronidases of both mucosal andmicrobial origin (9) and steroid sulfatases of bacterial origin(1, 9) deconjugate these esters and thus promote theirenterohepatic circulation. It has already been demonstratedthat mixed cultures of the intestinal microflora of rats andhumans hydrolyze many of these sulfated compounds (9,17). Comparison of the steroids in germfree and conven-tional rats demonstrated larger amounts of free steroids inconventional rats (10). Treatment of humans and rats withantibiotics that suppress the indigenous intestinal microfloraproduced an increase in the excretion of sulfated steroids (1,14). From rat cecal contents, two bile salt-desulfating strainstermed Clostridium sp. strain S, and Clostridium sp. strainS2 have been isolated (12, 22). lodothyronine sulfate-desulfating activity was demonstrated in strains isolatedfrom human and rat intestinal flora (5, 21). To our knowl-edge, no steroid- or bile salt-desulfating bacteria have yetbeen isolated from the indigenous human intestinalmicroflora. In this paper, we report the isolation of a

steroid-desulfating bacterium from the human intestinalmicroflora.

MATERIALS AND METHODS

Media and products used. All experiments were carried outat 37°C in an anaerobic glove box of the type described byAranki et al. (3). The atmosphere consisted of a mixture of80% N2, 10% H2, and 10% CO2.Most experiments were carried out on tryptic soy broth

(TSB) (Bio-Merieux, France) supplemented with 0.1 or 0.2%taurine or on Columbia broth (CB) (Gibco, Bio-Cult Ltd.,Paisley, U.K.) supplemented with 0.025% MgSO4, 0.001%

* Corresponding author.

hemin, 0.0001% vitamin K1 (Konakion; Roche, Basel, Swit-zerland), and 0.1 or 0.2% taurine. The following media were

also used: peptone yeast medium (PY) of Holdeman et al.(11) supplemented with 0.1% taurine; medium PY supple-mented with 0.2% glucose (PYG) and 0.1% taurine; andbrain-heart infusion medium (BHI) (BBL Microbiology Sys-tems, Cockeysville, Md.) supplemented with 0.001% hemin,0.001% vitamin K1 and 0.5% yeast extract. During isolation,a low-sulfur medium (LS medium) was used to avoid inhibi-tion of sulfatase activity by sulfur. The LS medium consistedof 0.004% CaC12, 0.008% MgCl2 6H20, 0.2% NaCl, 0.2%NaHCO3, 0.06% K2HPO4. 3H20, 0.01% KH2PO4, 0.004%ferric ammonium citrate, 0.085% L-arginine hydrochloride,0.085% DL-aspartate, 0.085% glycine, 0.207% L-glutamate,0.0437% L-histidine hydrochloride, 0.0989% L-isoleucine,0.149% L-leucine, 0.131% L-lysine hydrochloride, 0.0805%L-phenylalanine, 0.145% L-proline, 0.078% L-threonine,0.0207% L-tryptophan, 0.0640% L-tyrosine, 0.115% L-valine,0.1% glucose, 0.05% cellobiose, 0.05% arabinose, 0.001%Wensinck vitamin solution (24), 0.0001% vitamin K1,0.0005% hemin, 0. 174% tyramine hydrochloride, and 0.075%methionine; the pH was adjusted to 7.5.Agar plates, prepared by adding 2% agar to the liquid

culture media, and commercial blood plates (tryptic soy agarwith 5% horse blood; Bio-Merieux, France) were used forplating the cultures. Steroid sulfate esters were dissolved inwater or methanol and sterilized by filtration on Millex-HAfilters (0.45-,um pore size; Millipore, Molsheim, France), and50 ,ug/ml of medium was added aseptically to the autoclavedculture media to prevent hydrolysis during sterilization.The autoclaved media were prereduced in the anaerobic

glove box for at least 48 h before inoculation. Inoculatedmedia were incubated for 3 days in the glove box in a closedjar to prevent evaporation. The optical density (OD) of thecultures was measured with a Bausch and Lomb Spectronic20 photometer at 520 nm. A direct microscopic count ofbacterial cells was performed as described by Koch (16). Thenumber of viable cells was determined by making serial10-fold dilutions on TSB with 0.1% taurine and subsequentlyinoculating 0.01 ml on TSB agar plates supplemented with

0.1% taurine.

1655

Steroid

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1656 VAN ELDERE ET AL.

Estrone, ,B-estradiol, dehydroisoandrosterone, 5a-choles-tane, estrone-3-sulfate, estrone-3-glucuronide, dehydroisoan-drosterone sulfate (DHIAS), Sa-androsterone-3a-glucuronide,and 5p-pregnane-3ox-glucuronide were obtained from SigmaChemical Co., St. Louis, Mo. All other steroids and steroidsulfates were purchased from Steraloids, Inc., Wilton, N.H.para-Nitrophenyl sulfate and para-nitrocatechol sulfate werefrom Aldrich, Milwaukee, Wis. Phenolphthalein disulfate wassupplied by EGA-Chemie (Steinheim, Federal Republic ofGermany [FRG]); phenolphthalein glucuronide was obtainedfrom Sigma. para-Nitrophenyl phosphate, para-nitrocatechol,phenol-phthalein, and para-nitrophenol were from JanssenChimica (Beerse, Belgium). Trivial names for steroids are asfollows: estrone, 1,3,5(10)-estratriene-3-ol-17-one, P-estradiol,1,3,5(10)-estraiso-triene-3,171-diol; a-estradiol, 1,3,5(10)-estratriene-3,17a-diol; estriol, 1,3,5(10)-estratriene-3,16a,17,-triol; dehydroepiandrosterone, A5-androstene-3p-ol-17-one;and testosterone, zV'-androstene-17P-ol-3-one.

Isolation and identificaton procedures. Approximately 0.5 gof freshly voided human fecal material was transferred intothe anaerobic glove box, suspended in tubes with 5 ml ofdifferent culture media supplemented with 50 ptg of DHIASper ml, and incubated for 3 days.

Simplification of these mixed fecal cultures was performedby sequential plating of DHIAS-sulfatase-positive cultureson solid media and isolating limited numbers of differentcolonies. This procedure was repeated until pure cultures ofa DHIAS-sulfatase-positive strain were obtained.

Identification was done by the methods of Holdeman et al.(11). However, to obtain sufficient growth, medium PY wassupplemented with 0.1% taurine. The guanine-plus-cytosine(G+C) content of the bacterial DNA was estimated bydetermination of the thermal denaturation temperature (Tm)in a solution containing 15 mM NaCl and 1.5 mM sodiumcitrate, using a double-beam Beckman UV5230 spectropho-tometer at 260 nm with a thermocouple in the sample cuvettefor temperature reading. The molar percent base composi-tion was calculated by the equation G+C = (Tm - 53.9) x2.44 (18).

Study of in vitro transformation. To study transformationof free steroids and desulfation of steroid sulfate esters, 3 mlof liquid culture medium was mixed with 3 ml of a 20% NaClsolution and 50 ,ug of Sa-cholestane as an internal standard.This mixture was extracted three times with 5 ml of diethylether. After evaporating the ether extract to dryness, 0.6 mlof dry pyridine-acetic anhydride (1:1, vol/vol) was added.After 3 h at room temperature the resulting acetate esterswere analyzed by gas-liquid chromatography on packedcolumns of 3% OV-1, 3% OV-17, and 1% QF-1 at 240°C. Theretention times of the free and acetylated reaction productswere compared with those of reference compounds, andmass spectrometry was performed to confirm identification.To determine the desulfation of para-nitrophenyl sulfate,

para-nitrocatechol sulfate, phenolphthalein disulfate,phenolphthalein glucuronide, and para-nitrophenyl phos-phate, 5 ml of grown culture medium was centrifuged for 15min at 6,000 rpm; 3 ml of supernatant was taken andcombined with 3 ml of 1 N NaOH. Absorption was measuredspectrophotometrically at 420 nm for para-nitrophenol, 515nm for para-nitrochatechol, and 560 nm for phenolphthalein.

RESULTS

Isolation. Desulfation (20 to 60% of the total amount ofsubstrate added) in unsimplified fecal cultures was obtained

in PY, PYG, TSB, CB, and LS media after 3 to 4 days ofincubation. In the unsimplified fecal cultures, the highestpercentage of desulfation (60%) was found in LS medium.Growth of more simplified cultures could not, however, besustained in LS medium, and on average, these cultureswere lost after the third subculture. Therefore, the last partof the isolation procedure was performed in PY medium.Concomitantly with simplification of the cultures, desulfa-tion gradually increased towards an average of 90% of thetotal amount of substrate added.

Isolation of a pure DHIAS-desulfating culture was unsuc-cessful until 0.1% taurine was added to the culture medium.Addition of 0.1% taurine led to the appearance of smallcolonies (0.5 to 1 mm diameter) on solid TBS medium thatwere not seen until then and permitted good growth of theseisolates on liquid culture media.Growth conditions and identification. Strictly anaerobic

conditions were required, and 10% CO2 stimulated growth.Pure cultures of the DHIAS-desulfating strain could begrown on the following unsupplemented media: PY and PYG(OD = 0.2 after 3 days), TSB (OD = 0.25), and BHI (OD =0.3). On all media tested, addition of 0.05% or more taurinestimulated growth. Cultures on TSB medium with 0.05%taurine had an OD of 0.63; the cell count increased from 2 x108 cells per ml on TSB medium to 13 x 108 cells per ml onTSB medium with 0.05% taurine. CB and BHI were notoften used because a fine granular black deposit from theproduction of ferrous sulfide formed very quickly afterinoculation in these media.Growth enhancement was also observed when pyruvate,

sodium sulfite, or estrone sulfate was added. Addition ofsodium sulfite (5 mM) to TSB medium increased the OD of3-day-old cultures from 0.25 to 0.5, and the cell count per mlfrom 2 x 108 to 11 x 108. Addition of 5 mM pyruvate to TSBresulted in an OD of 0.45 and cell count of 5 x 108 per ml,while 0.05 mM estrone sulfate led to an OD of 0.43 and cellcount of 4.5 x 108 per ml. The growth-stimulating effect oftaurine and pyruvate increased with higher doses, andgrowth reached OD values of >1 in PY supplemented with0.4% taurine or pyruvate. Taurine (0.05%) plus pyruvate(0.05%) added to TSB medium led to better growth (OD =0.75) than each supplement separately. No improvement ofgrowth was obtained by adding cysteine, cysteic acid,serine, arginine, or methionine at 5 mM. Adding 0.5 or 1% ofdifferent carbohydrates or 0.0001% vitamin K1 or vitamin-rich preparations such as yeast extract and Wensinck vita-min solution (23) produced no improvement either.On liquid CB medium the strain presented as a small (0.6

to 1 ,Lm), gram-positive, nonmotile, somewhat elongatedcoccus. The cells occurred singly, in pairs, and occasionallyin small irregular groups. Surface colonies on PYG agarplates after 3 days of incubation were smooth, small (diam-eter, 0.2 mm), convex, circular, entire, colorless, shiny, andtransparent. Addition of 0.05% taurine increased the diam-eter of these colonies to 1 mm. After 4 days of incubation onCB agar with 0.05% taurine, colonies grew up to 1.5 mm indiameter and developed a black discoloration of the center.

Identification was performed on PY medium supple-mented with 0.05% taurine. The isolated strain did notferment carbohydrates and did not produce gelatinase,indole, esculine hydrolase, hippurate hydrolase, or starchhydrolase. It did produce ammonia, hydrogen sulfide, andcatalase. On PYG with 0.05% taurine, it produced aceticacid (+++), isobutyric acid (+), butyric acid (+ +),isovaleric acid (+ +), phenylacetic acid (+), and caproicacid (+). Addition of 0.5% pyruvate to the PYG plus 0.05%

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TABLE 1. Sulfatase activity of P. niger H4 and DSM 20475

ActivityaCompound

Strain H4 Strain DSM 20475

Steroid sulfatesArylsulfates

Estrone sulfate + (100) + (100)1-Estradiol-3-sulfate + (50) + (50)3-Estradiol-17-sulfate - -

Estriol-3-sulfate trb tr

AlkylsulfatesDHIAS + (100) + (100)5ot-Androstane-17-one-31-sulfate + (100)C + (100)CS5o-Androstane-17-one-3cx-sulfate + (100)C + (0)5P-Androstane-17-one-3a-sulfate + (100)C + (0)5a-Pregnane-20-one-3p-sulfate + (60) + (40)A5-Pregnene-20-one-3p-sulfate + (100) + (100)Testosterone sulfate

Synthetic sulfate esterspara-Nitrocatechol sulfate + (40) + (30)para-Nitrophenyl sulfate + (70) + (70)Phenolphthalein disulfate + (40) + (40)a Numbers in parentheses are the percentage of desulfated product after 3

days of incubation on TSB plus 0.1% taurine and 50 ,ug of steroid sulfates orsynthetic sulfate esters per ml.

b tr = <5%.c Value is for the third subculture.

taurine medium enhanced fatty acid production, whereasaddition of lactate or threonine had no influence. The G+Ccontent of the DNA was 50 mol%.On the basis of these data the strain was tentatively

identified as Peptococcus niger. The API 20A system(Analytab Products, Montalieu, Vercieu, France) identifiedour strain as a Peptococcus sp. The An-Ident System(Analytab Products, Farmingdale, N.Y.) did not reveal anyenzymatic activity except catalase. The newly isolated strainwill be designated P. niger H4.

Sulfatase activity of P. niger H4. P. niger H4 desulfated3-sulfate esters of steroids with an aromatic A ring (arylsul-fates) and with a saturated A ring (alkylsulfates). Thearylsulfates estrone-3-sulfate and 13-estradiol-3-sulfate were100 and 50% desulfated, respectively. Only trace amounts(<5%) of free estriol could be found after incubation withestriol-3-sulfate. The nonsteroid arylsulfates phenolphtha-lein disulfate, para-nitrocatechol sulfate, and para-nitro-phenyl sulfate were 40 to 70% desulfated, but the ,-estra-diol-17-sulfate ester was not desulfated (Table 1). We alsofound sulfatase activity towards the alkylsulfates 3a-sulfate-5a-androstane (androsterone sulfate), 3,-sulfate-5a-andro-stane, 3a-sulfate-5p-androstane (etiocholanolol sulfate), 3p-sulfate-A5-androstene (DHIAS), 3p,-sulfate-5ca-pregnane,and 3p-sulfate-A5-pregnene (pregnenolone sulfate). No other3-sulfate esters of steroid alkylsulfates were tested. DHIASwas 100% desulfated in TSB plus 0.1% taurine from the firstincubation; for the 3I-sulfates of Sa-androstane, Sa-pregnane, and A5-pregnane, we usually found partial desulfa-tion (20 to 70%) in the first culture and complete desulfationin the first subculture. The 3a-sulfates of Sa-androstane and5p-androstane needed three to four sequential incubationsbefore complete desulfation was obtained in TSB with 0.1%taurine. The 17-sulfate ester of A4-androstene-3-one(testosterone) was not desulfated. The type strain P. nigerDSM 20475 (Deutsche Sammlung von Mikroorganismen,Gottingen, FRG) possessed identical sulfatase activity ex-

cept for the 3ca-sulfate ester of 5,- and Sa-androstane-17-one, which were not desulfated.

Deglucuronidation of estrone glucuronide or phenol-phthalein glucuronide was not observed with either P. nigerH4 or DSM 20475.Other enzymatic activities of P. niger 114. P. niger H4

developed 17-oxido reductase activity towards the estrogensestroneand ,-estradiol andtowardstheSa-and 5,-androstanesand the A4- and A5-androstenes (Table 2).

After 3 days of incubation in TSB medium plus 0.1%taurine, we found that 10 to 30% of the desulfated estrone-3-sulfate was reduced to P-estradiol. When free estrone wasadded, only 10% was transformed into P-estradiol. Desul-fated ,-estradiol-3-sulfate was 20 to 60% dehydrogenated toestrone, whereas nonsulfated P-estradiol was only 10%dehydrogenated. Free a-estradiol was not dehydrogenated.

Oxidation or reduction of the oxygen in position 17 in theSa-androstanes was only observed when an oxo or hydroxylgroup was present at C-3. After 3 days in TSB plus 0.1%taurine, the 17 oxo group of sulfated or free androstanes andandrostenes was 20 to 50% hydrogenated to a 17,B-hydroxylgroup; the 17p-hydroxyl group of free androstanes was 10 to40% oxidized to a 17-oxo group; however, the 17a-hydroxylgroup in A4-androstene-3-one was not susceptible todehydrogenation.

P. niger H4 also possessed 3-oxido reductase activity. Inthe nonsulfated androstanes we found the 3p-hydroxyl groupto be relatively resistant to enzymatic activity (less than 1%oxidation). The 3oa-hydroxyl group, after 3 days in TSB plus0.1% taurine, was 5 to 20% converted into a 3p-hydroxylgroup and 5 to 20% oxidized to an oxo group. The 3-oxogroup of the nonsulfated androstanes tested was 60% hydro-genated to a 3a-hydroxyl group and 20 to 30% to a 3,-hydroxyl group. Only 10 to 20% of the substrate remainedunchanged. In incubations with the sulfated androstanes, wefound approximately 10% oxidoreduction products of the3-hydroxyl group. The presence of an oxygen at C-17 wasnot necessary for the oxidoreduction reactions at C-3. Wedid not find any measurable oxidoreduction activity of P.niger H4 towards the C-20 oxygen of the pregnanes tested,and we found only very little activity (<2%) towards the C-3oxygen of these pregnanes.

Incubation on TSB plus 0.2% taurine supplemented with50 ,ug of DHIAS per ml led to the formation of two moretransformation products that were identified by gas-liquidchromatography and mass spectrometry as A4-androstene-3,17-dione (androstenedione) and A4-androstene-3-one-17p-ol (testosterone). The formation of these products entailedoxidation of the desulfated C-3 hydroxyl group with aconcomitant translocation of the double bond from the 5 to 4position. After 3 days of incubation, they each representedapproximately 15% of the total amount of dehydro-isoandrosterone-3-sulfate added. Also, 20% of A5-pregnene-20-one-3,B-sulfate was transformed into /&4-pregnene-3,20-dione (progesterone) after 3 days. The nonsulfateddehydroisoandrosterone and A5-pregnene-3,3-ol-20-one werenot transformed into their 3-one-4-ene derivatives by P.niger H4 on TSB plus 0.2% taurine.A study of the time course of the sulfatase and oxidore-

ductase activity of strain H4 (Fig. 1) showed that DHIAS wasvery quickly desulfated during the exponential growth phaseof the culture (between 4 and 8 h after inoculation of themedium). The hydrogenation of dehydroisoandrosteroneinto A5-androstene-3p,1713-diol proceeded in two phases,with a first maximum of 20% reduction product formed after14 h and a second maximum of 44% after 60 h. Formation of

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TABLE 2. Transformation of steroids by P. niger H4

Substrate Transformation products'

Sa-Androstan-3-oneSa-Androstan-3a-ol5a-Androstan-3,-ol5a-Androstan-17-one5a-Androstan-17p-ol

3-one (21); 3a-ol (72); 3,B-ol (7)3-one (29); 3a-ol (64); 3,B-ol (7)

3,-ol (100)17-one (100)

17p-ol (100)

Sa-Androstan-3,17-dioneSa-Androstan-3a-ol-17-one5a-Androstan-3p-ol-17-one5oS-Androstan-3-one-17,-olSa-Androstan-3a,17p-diol5ac-Androstan-3p,17p-diol5,-Androstan-3-one-17p-ol5 -Androstan-3ot,17p-diol5,B-Androstan-33-ol-17-one5P-Androstan-3p,17p-diolA5-Androsten-3p-ol-17-oneAM-Androsten-3,17-dioneA4-Androsten-3-one-17ot-olA4-Androsten-3-one-17p-ol

3,17-dione (32);3,17-dione (28);3,17-dione (4);3,17-dione (14);3,17-dione (18);3,17-dione (5);

3,17-dione (53);

3,17-dione (26);

3-one-17p-ol (4); 3a-ol-17-one (20); 3a,17,B-diol (25);3-one-17,-ol (4); 3a-ol-17-one (24); 3a,17,-diol (26);

3-one-17p-ol (8); 3a-ol-17-one (19); 3oa,17,-diol (40);3-one-17p-ol (1); 3a-ol-17-one (4); 3a,173-diol (60);

3-one-17j3-ol (2);3a-ol-17-one (4);

3ot,173-diol (63);3oa,171-diol (89);

3p-ol-17-one (15);3p-ol-17-one (14);3,B-ol-17-one (46);3p-ol-17-one (12);3p-ol-17-one (15);3p-ol-17-one (3);3p-ol-17-one (27);

3p-ol-17-one (76);3,B-ol-17-one (30);

3P,1713-diol (4)31,171-diol (4)3P,17,p-diol (S0)3P,17p-diol (7)3P,171-diol (2)3P,17p-diol (92)unidentified (8)unidentified (7)33,1713-diol (24)3P,1713-diol (70)

3,-ol-17-one (53); 3P,17p-diol (47)3-one-17p-ol (47)3-one-17a-ol (100)3-one-17p-ol (74)

Sa-Androstan-3oa-sulfate-17-one5a-Androstan-3,-sulfate-17-oneA5-Androsten-313-sulfate-17-one

Sa-Pregnan-3a-ol-20-one5a-Pregnan-3j3-ol-20-one5ox-Pregnan-3,3-sulfate-20-oneA5-Pregnen-3p-sulfate-20-one

Estrone-3-sulfatej3-Estradiol-3-sulfateEstrone

3-one-17,B-ol (2); 3oa-ol-17-one (50); 3a,17p-diol (41); 3p-ol-17-one (3); 3P,17p-diol (4)3,17-dione (2); 3a,17p-diol (1); 3p-ol-17-one (38); 3P,17,B-diol (59)

3p-ol-17-one (40); 313,17p-diol (60)

3a-ol-20-one (100)3[3-ol-20-one (100)3p-ol-20-one (100)3p-ol-20-one (100)

Estrone (85); ,-estradiol (15)Estrone (69); ,-estradiol (31)Estrone (87); 13-estradiol (13)

a Transformation products after 3 days of incubation on TSB plus 0.1% taurine and 50 pLg of substrate per ml. The numbers in parentheses indicate thepercentage of transformation product formed (mean of four determinations). For the transformation products, only the redox state of the carbons in position 3and 17 is given, since the rest of the molecule was not changed.

80n° 80° w 'v' \I, * o. 08,

S aD/.aeos5Mo /-I

60 \dehydroisoandrosterone

0,6

-40

androste/e.30 ja7-diol o

20 2 60 8 0

TZi02mdehydrosoandroterone/, /v5-ad androstene 3,17.-dioneandrosene-3,7-dion /nd 1A'-androste -androstene -3-one-17-I3-ol

0 20 40 60 s0 100

T m e (hours)

FIG. 1. Time course of the formation of reaction products due to

sulfatase and oxidoreductase activity of P. niger H4 on TSB medium

plus 0.2% taurine and DHIAS (50 p.g/ml). Symbols: U, OD; F-,dehydroisoandrosterone; 0, A5-androstene-33, 17P3-diol; *, A4-

androstene-3 ,17-dione and A4-androstene-3-one-1713-ol.

A4-androstene-3,17-dione plus A4-androstene-3-one-17p-olstarted after 24 h, and these products represented 30% ofthe total amount of substrate added after 3 days. Dehy-droisoandrosterone and A5-androstene-3p,17P-diol each rep-resented approximately 35% after 3 days. P. niger DSM20475 did not perform any of these oxidation-reductionreactions at position 3 or 17.

DISCUSSION

The extent of microbial metabolism of steroid hormones inthe gut during enterohepatic circulation is an importantfactor in fecal hormone excretion and in serum and urinehormone profiles. Antibiotic treatment is known to modifythe steroid composition in feces and urine and in particularto reduce excretion of estrogenic hormones in urine (2, 14,19). This last effect is attributed to the fecal elimination ofunhydrolyzed hormone conjugates such as glucuronides andsulfates following repression of the hydrolyzing intestinalflora. The failure of contraconceptive treatment in womentaking antibiotics, e.g. ampicillin (8), was also ascribed tothis effect (20). The importance of intestinal microbialdesulfation to the enterohepatic circulation of estrone-3-sulfate was also clearly demonstrated in gnotobiotic rats,selectively associated with estrone-desulfating bacteria, thatexcreted estrone clearly more rapidly than germfree ratswithout desulfating bacteria (23).

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STEROID DESULFATION BY P. NIGER 1659

P. niger H4 is, to our knowledge, the first steroid hormone-desulfating strain isolated from the human gastrointestinaltract. It showed a wide range of steroid-3-sulfatase activity;arylsulfates (estrogen sulfates) as well as alkylsulfates[androsta(e)n sulfates and pregna(e)n sulfates] were suitablesubstrates. Two alkylsulfatase-positive strains, Clostridiumsp. strains S, and S2, isolated from rat intestinal flora,desulfate bile salt 3-sulfates (12, 22) but no androsta(e)n orpregna(e)n-3-sulfates (unpublished results). Regarding thePseudomonas aeruginosa isolated from the human intestinalflora (13), no report has yet been published of any sulfataseactivity except for lithocholate-3-sulfate. Steroid sulfataseactivity has also been described for mollusks and mamma-lian tissues. This type of sulfatase appears to be specific forthe 31-sulfates of the A' series of sterols (6). It still has to bedetermined whether one or more enzymes are responsiblefor the alkyldesulfating activity of P. niger H4 and DSM20475.

In contrast to the alkylsulfatase activity, arylsulfataseactivity has been described for several bacteria, perhaps dueto its relatively easy detection by means of chromogenicsubstrates (7). These bacteria were, however, not directlyisolated from the intestinal tract, although some of thembelong to species that are found in the intestinal microflora.Moreover, there is almost no information on the estrogen-desulfating activity of these strains. This is rather surprisingin view of the important role of microbial desulfation in theenterohepatic circulation of estrogens and the frequent useof estrone sulfate in clinical situations. The arylsulfatase ofP. niger H4 and DSM 20475 showed a clear substratespecificity; estrone-3-sulfate was 100% desulfated, and ,B-estradiol-3-sulfate was 50% desulfated, but only traceamounts of estriol-3-sulfate were desulfated.The sulfatases of P. niger H4 and DSM 20475 were specific

for 3-sulfate esters. No 17-sulfatase activity was observedfor either arylsulfates or alkylsulfates. Mixed fecal flora isalso capable of hydrolyzing C17 and C21 sulfate esters (9). Upto this moment no microorganisms with C17 or C21 sulfataseactivity have been described.The growth stimulation by sulfite, taurine, and sulfate

esters but not by sulfate, the absence of growth stimulationby cysteine, and the extensive H2S production seem toindicate that the sulfatase activity of this strain serves mainlyto liberate electron acceptors. Why the sulfon group oftaurine is such a good substrate, not only for this strain butalso for two other desulfating strains (12, 22), remains to bestudied.

Desulfation and deglucuronidation of steroids, apart fromtheir direct effect on steroid fecal excretion, also play acrucial role in the bacterial metabolism of steroids, whichrequires unconjugated molecules (4). In P. niger H4 weobserved that oxidoreduction reactions of the carbon atomin position 3 or 17 took place after desulfation. Oxidoreduc-tion reactions, among other reactions, can transform ste-roids excreted via the bile into biologically less active ormore active substances and may also be implicated in theformation of mutagenic or carcinogenic substances. P. nigerH4 was able to transform estrone into the biologically moreactive 13-estradiol. Oxidation-reduction activity in otherstrains isolated from the intestinal flora was described byJarvenpaa et al. (15) and Winter et al. (25). Intestinaldesulfation and transformation of estrone-3-sulfate to 1-estradiol constitute important variables of the enterohepaticcirculation of estrone-3-sulfate. Factors such as antibiotics(4) or perhaps diet that may influence these reactions canlead to changes in serum hormone profiles.

P. niger H4 also possessed C-17 oxidoreductase activitytowards the androstanes, as was described for Bacteroidesfragilis (25), and also showed C-3 oxidoreductase activitytowards estrogens, androstanes, and pregnanes. In the case

of dehydroisoandrosterone and 3,B-hydroxy-A5-pregnene-20-one, this activity led to introduction of the 4-ene-3-onestructure and thus to the formation of testosterone andprogesterone. Surprisingly, P. niger DSM 20475 developednone of these oxido reductase activities. Our results under-line the potential influence of the intestinal microflora on theoverall metabolism of steroids undergoing enterohepaticcirculation.

ACKNOWLEDGMENT

J. Van Eldere is an aspirant (fellow) of the Belgian NationalFoundation for Scientific Research (N.F.W.O.).

LITERATURE CITED

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