Inhibitory activity of gut bacteria against Escherichia coli O157 mediated by dietary plant metabolites

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1 FEMS Microbiology Letters 164 (1998) 283^288 Inhibitory ctivity of gut bcteri ginst Escherichi coli O157 medited by dietry plnt metbolites Sylvi H. Duncn *, Hrry J. Flint, Colin S. Stewrt Rowett Reserch Institute, Greenburn Rod, Bucksburn, Aberdeen, AB21 9SB, UK Received 14 April 1998; revised 4 My 1998; ccepted 11 My 1998 Abstrct Under both erobic nd nerobic conditions, the growth of Escherichi coli O157 strin NCTC ws inhibited by the coumrins esculetin, umbelliferone nd scopoletin, but not by the coumrin glycoside esculin. Esculin-hydrolysing bcteri from the rumen, the pig gut nd the humn gut inhibited growth of E. coli in n overly-plte ssy in the presence of esculin. The combined effect of esculetin nd voltile ftty cids ws greter thn the effect of either fctor lone suggesting tht coumrin glycosides in the diet might reduce the growth or survivl of E. coli O157 in the gut. Adding esculin to incubtions of mixed rumen contents significntly reduced the survivl of E. coli O157. z 1998 Federtion of Europen Microbiologicl Societies. Published by Elsevier Science B.V. All rights reserved. Keywords: Escherichi coli O157; Rumen; Gut; Commensl micro or; Esculin; Esculetin 1. Introduction * Corresponding uthor. Tel.: +44 (1224) ; Fx: +44 (1224) ; E-mil: shd@rri.sri.c.uk The incidence of reported disese cused by enterohemorrhgic Escherichi coli (EHEC) [1] hs recently esclted drmticlly; the worst outbrek resulted in 20 deths in Scotlnd in 1996 [2]. The rumen nd colon provide sources for E. coli O157 shed in ruminnt feces [3,4] which my contminte the humn food chin, nd fecl shedding is lso implicted in trnsmission between humns [5]. Shedding of E. coli by ruminnts is in uenced by the diet [6]. The growth of E. coli is reduced in the presence of voltile ftty cids (VFA) produced during crbohydrte fermenttion in the gut [3,7] suggesting tht when nimls re well fed nd the VFA levels in the gut re high, the numbers of enterohemorrhgic nd other E. coli my be mrkedly reduced. Dietry plnt mteril often contins gret diversity of plnt metbolites, some of which demonstrte species-selective e ects on the growth of gut bcteri [8]. Coumrins (derivtives of benzo-k-pyrone) occur commonly in plnts in the free stte nd s glycosides [9]. Coumrin (2,3-benzopyrone) hs been shown to inhibit the growth of E. coli [10] nd cellulolysis by ruminl nerobic fungi [11] lthough the e ects on commensl nerobic bcteri remin unknown. We show here tht predominnt nerobic species found in the rumen nd colonic microbil communities cn medite inhibition of growth of E. coli O157 through conversion of the / 98 / $19.00 ß 1998 Federtion of Europen Microbiologicl Societies. Published by Elsevier Science B.V. All rights reserved. PII: S (98) Downloded from

2 284 S.H. Duncn et l. / FEMS Microbiology Letters 164 (1998) 283^288 plnt glycoside esculin to the glycone esculetin. This provides one of the rst exmples of wht my prove to be more generl type of microbil interction. 2. Mterils nd methods 2.1. Bcteril strins, medi nd growth mesurements E. coli O157 strin EC22 ws kindly provided by F. Thomson-Crter (University of Aberdeen, UK) nd strin NCTC ws from the PHLS, London. The non-o157 serotype strins F34, F36, F38, F310 nd F318 were isolted t the Rowett Reserch Institute [12] from the rumen of sheep. Strin UB281 ws supplied by R. Herd (University of Bristol, UK) nd Bcteroides ovtus V975 ws provided by T.R. Whitehed (USDA, Peori, IL, USA). Enterococcus feclis strins 1, 2, nd 3 were recently isolted from the rumen of n dult sheep rumen t RRI. The Streptococcus bovis strin JB1 ws gift from J.B. Russell, Cornell University nd Strep. cprinus strin 3969 ws from the Austrlin Collection of Microorgnisms. The other bcteril strins were held in the RRI culture collection. The E. coli strins were grown on LB medium [13] either under erobic conditions, with shking t 200 rpm, or under nerobic conditions, when cysteine HCl ( nl concentrtion 0.1%) ws dded to the medium which ws dispensed (5 ml), under O 2 -free CO 2, into 16U125-mm Hungte tubes (Bellco Glss Inc.). Medium SD/E contined 3.0% Difco yest extrct, 1.0% Bcto peptone, 1.5% NCl nd 1.5% gr. Strict nerobes were cultivted on M2GSC, the M2 medium of Hobson [14] with the omission of lctte nd with 0.2% ech of glucose, cellobiose, nd soluble strch. The coumrins (Sigm Chem. Co., except for esculetin which ws from Fluk AG) were prepred in 80% dimethyl sulfoxide (DMSO) stock solutions, lter sterilized (0.2 Wm pore size; Gelmn Acrodiscs) nd dded to the utoclved medium. The voltile ftty cids (VFA) were dded s ltersterilised stock solutions djusted to ph 7.0, nd consisted of cette, propionte nd butyrte in the molr proportions 10:2:1, vlues within the rnge encountered in the rumen nd the colon. Controls contined DMSO s pproprite. Growth ws determined s the bsorbnce t 650 nm nd speci c growth rtes were determined from the doubling times in the exponentil growth phse Overly plte ssy for growth inhibition Streptococcus nd Enterococcus spp. were grown on SD/E medium with 1.5% gr, with or without esculin (5 mm). The pltes were spot-inoculted within n nerobic chmber in n tmosphere of 10% CO 2, 10% H 2 nd 80% N 2 (Don Whitley Scienti c), then trnsferred fter 1 h into n nerobic jr under O 2 -free CO 2 nd incubted for 24 h t 38³C. The pltes were removed, treted with chloroform nd overlid with 5 ml LB medium contining 0.75% gr inoculted with 100 Wl of pre-grown culture of E. coli O157 strin essentilly s described in [15]. The overlid pltes were reincubted erobiclly for 24 h t 38³C prior to exmintion for zones of growth inhibition. For nerobic bcteri, medium M2GSC ws used in plce of SD/ E, nd the spot-inoculted pltes were incubted for 48 h prior to testing Enumertion of vible E. coli O157 in mixed rumen contents Rumen contents were recovered from cnnulted dult sheep fed mixed hy/brley grin diet nd mixed with n equl volume of sterile 0.85% NCl, with or without 5 mm esculin. The diluted contents were dispensed septiclly under O 2 -free CO 2 in 9.9- ml volumes into sterile Hungte tubes, inoculted with 100 Wl of pre-grown (18 h) culture of E. coli O157 strin then incubted t 38³C. Aliquots (100 Wl) were removed t intervls into 9.9 ml sterile diluent (0.85% NCl). Seril dilutions were plted on Rinbow O157 gr pltes (Biolog), incubted erobiclly t 38³C for up to 48 h when the drk grey/blck colonies were enumerted Detection of esculin hydrolysis The E. coli cultures were grown erobiclly t 38³C on LB medium (5 ml); the other bcteri were incubted on M2GSC medium (7.5 ml) under O 2 -free CO 2. The medi, mended to contin 5 mm esculin, were inoculted with 100 Wl of broth cul- Downloded from

3 S.H. Duncn et l. / FEMS Microbiology Letters 164 (1998) 283^ ture pre-grown for 24 h. Esculin hydrolysis ws detected using 1% ferric mmonium sulfte 24 h, 48 h, nd 72 h fter incubtion [16]. 3. Results Incubtion of E. coli O157 strin in LB medium under erobic or nerobic conditions showed tht the presence of free coumrins hd mrked e ect on the growth of the bcterium. Coumrin ws more inhibitory under nerobic conditions thn when tested erobiclly (Fig. 1). Although esculetin (6,7-dihydroxy-coumrin) inhibited the growth of Escherichi coli, its glycoside esculin hd no e ect (Fig. 1). The e ects of umbelliferone (7- hydroxycoumrin) nd scopoletin (7-hydroxy-6- methoxycoumrin) were similr to those of esculetin (Fig. 1). A number of bcteril strins selected for their bility to hydrolyse esculin showed cler evidence of inhibition of E. coli only when esculin ws dded to the bsl medium (Tble 1). The inhibitory bcteri included rumen strins of Ent. feclis, Strep. bovis, Mitsuokell multicidus, Prevotell bryntii, P. brevis, nd Selenomons ruminntium (Tble 1). A strin of M. multicidus isolted from pigs ws lso inhibitory in the presence of esculin, s were two strins of Bcteroides ovtus isolted from humn feces. Ruminococcus vefciens strin 17 which hydrolysed esculin wekly nd only tested positive fter 48 h incubtion in the presence of esculin, did not inhibit the growth of E. coli O157 in its presence (dt not shown). Three esculin-negtive strins tested, P. ruminicol 23, Bcteroides uniformis 1100, nd Fibrobcter succinogenes BL2, did not inhibit growth of E. coli. When the growth rte of strin ws mesured in the presence or bsence of VFA nd esculetin, the e ects of the two fctors were dditive. Thus growth could be mesured in the presence of 50 mm VFA nd 0.5 mm esculetin in nerobic LB medium, but not in the presence of 100 mm VFA nd 0.5 mm esculetin (Tble 2). Commensl strins of E. coli from the rumen [7,12] tended to be less sensitive to esculetin thn the strins of E. coli O157 (12900 nd EC22) when grown in nerobic M2GSC medium (Tble 3). The Tble 1 Inhibition of E. coli O157 strin by commensl bcteri from the gut Species, strin (origin ) Inhibition of E. coli Control +Esculin Esculin hydrolysis Enterococcus feclis 1 (r) Enterococcus feclis 2 (r) Enterococcus feclis 3 (r) Streptococcus bovis JB1 (r) Streptococcus bovis 26 (r) Bcteroides ovtus 1896 (h) Bcteroides ovtus V975 (h) Bcteroides uniformis 1100 (h) Mitsuokell multicidus (p) Mitsuokell multicidus 46/5 (r) Mitsuokell multicidus P (p) Prevotell bryntii B 1 4 (r) Prevotell brevis GA 33 (r) Prevotell ruminicol 23 (r) Selenomons ruminntium 2358 (r) Selenomons ruminntium FB322 (r) Fibrobcter succinogenes BL2 (r) Origin of strins: (h) humn, (p) pig, (r) rumen. Ent. feclis nd Strep. bovis were grown on SD/E pltes, nd the remining nerobes on M2GSC pltes, nerobiclly, before overlying with E. coli (see Section 2). The zones of growth inhibition were ssessed s the res of no growth of the O157 strin in the plte overly round the test strin colony nd recorded s the dimeter of the inhibition zone minus the dimeter of the colony; (+) denotes 9 3 mm, (++) denotes s 3 mm nd 99 mm nd (+++) denotes s 9 mm. Downloded from

4 286 S.H. Duncn et l. / FEMS Microbiology Letters 164 (1998) 283^288 Fig. 1. E ects of coumrins on the growth of Escherichi coli O157 strin under () erobic nd (b) nerobic conditions. Cells were grown in LB medium with shking (erobic) or with the ddition of cysteine HCl nd under O 2 -free CO 2 (nerobic) s described in Section 2. The coumrins were ll dded t the 5-mM level; control (F), esculin (b), esculetin (R), coumrin (7), umbelliferone (E), nd scopoletin (). bility of E. coli strins to grow in the presence of esculetin did not pper to correlte with their bility to hydrolyse esculin. For exmple, of the strins of E. coli shown in Tble 3, only F38 nd F310 were ble to hydrolyse esculin, lthough hydrolysis ws slow, being detected fter 48 h nd 72 h incubtion, respectively. The O157 strins were slightly less sensitive to the esculetin (0.5 mm) nd VFA (100 mm) combintion in the rumen uid M2GSC medium (Tble 4) compred with LB medium (Tble 2). In M2GSC medium there ws little e ect of 0.5 mm esculetin nd 100 mm VFA on the growth of the rumen strins P. bryntii B 1 4, Bu. brisolvens D6/1 nd. Sel. ruminntium Z108 but the growth rtes of B. ovtus V975 nd of R. lbus J6 were reduced (Tble 4). Growth of E. coli O157 strins ws strongly inhibited by 5 mm esculetin in M2GSC medium (not shown). In contrst the v OD 650 for Bu. brosolvens D6/1, P. bryntii B 1 4 nd Sel. ruminntium Z108 cultures ws decresed only 24%, 12% nd 9%, respectively, fter 24 h growth in the presence of 5 mm esculetin compred to the untreted control, nd by less thn 10% in the presence of 2 mm esculetin (dt not shown). Finlly, when E. coli O157 strin ws incubted in mixed rumen contents for 24 h, the numbers of vible E. coli were reduced from round 5U10 5 ml 31 to below 20 ml 31 when 5 mm esculin ws present. In the bsence of esculin, the mixed rumen bcteri present hd no signi cnt e ect on the survivl of E. coli (Fig. 2). The numbers of totl vible nerobes in mixed rumen contents were enumerted s 1.2U10 8 ml 31 initilly nd dropped to 7.3U10 6 ml 31 nd 6.5U10 6 ml 31 respectively with nd without the ddition of esculin over 24-h incubtion period (dt not shown). The number of E. coli Tble 2 Growth rtes (h 31 )ofescherichi coli O157 strin in nerobic LB medium contining combintions of VFA nd esculetin Esculetin (mm) VFA concentrtion (mm) þ þ þ þ þ þ þ þ þ Denotes little or no growth; chnges in bsorbnce too smll for ccurte mesurement of growth rtes. Downloded from

5 Tble 3 Growth rtes (h 31 ) of E. coli strins on the rumen uid bsed M2GSC medium in the presence of esculetin (5 mm) under nerobic conditions Strin Control 5 mm esculetin Commensl E. coli F þ þ 0.03 F þ þ 0.05 F þ þ 0.04 F þ þ 0.03 F þ 0.03 E. coli O þ 0.13 EC þ 0.06 Denotes little or no growth; chnges in bsorbnce too smll for ccurte mesurement of growth rtes. S.H. Duncn et l. / FEMS Microbiology Letters 164 (1998) 283^ O157 represents pproximtely 6% of the totl vible nerobic bcteri in the control incubtions, with no dded esculin, compred to 0.003% when esculin ws dded, representing 2000-fold decrese in the reltive proportion of vible E. coli O157 cells in mixed rumen contents contining esculin. 4. Discussion Fig. 2. E ect of esculin (5 mm) on the survivl of Escherichi coli O157, strin in rumen contents; (E) control, (b) 5 mm esculetin. The mechnism(s) by which coumrins inhibit microbil growth re not understood, but coumrins re known to ct s enzyme inhibitors nd s ntioxidnts in biologicl systems. Esculetin inhibits mmmlin xnthine oxidse ctivity [17] nd scvenges superoxide nions [18] nd hydroxyl rdicls [19]. The free-rdicl scvenging ctivity of coumrins in the humn gut is seen s possible helthpromoting e ect, nd the reduction in survivl of E. coli O157 reported here o ers further evidence of possible bene cil e ects of such compounds in the diet. Coumrins nd coumrin glycosides re found in wide rnge of forges for frm nimls nd in fruit nd vegetbles consumed by humns [9]. Mny of the predominnt bcteril species found in the gut micro or possess the bility to hydrolyse plnt glycosides, s exempli ed by the conversion of esculin to the glycone esculetin, which we show here is potent inhibitor of the pthogenic E. coli strins. Esculin hydrolysis is presumed to be property of L-(1-4) glucosidses, nd the L-(1-4) glucosidse of P. bryntii B 1 4, for exmple, hs been shown to exhibit Tble 4 Growth rtes (h 31 ) of nerobic bcteri in the presence of esculetin nd esculetin plus 100 mm VFA on M2GSC medium under nerobic conditions Strin Control 0.5 mm esculetin 0.5 mm esculetin+100 mm VFA B. ovtus V þ þ þ 0.04 B. ovtus þ þ þ 0.02 P. bryntii B þ þ þ 0.01 S. ruminntium Z þ þ þ 0.02 B. brisolvens D6/ þ þ þ 0.0 R. lbus J þ þ þ 0.01 E. coli O þ þ þ 0.04 E. coli O157 EC þ þ þ 0.09 Averge of 3 determtions þ S.D., except : 2 replictes þ rnge. Downloded from

6 288 S.H. Duncn et l. / FEMS Microbiology Letters 164 (1998) 283^288 brod speci city, hydrolysing rnge of plnt glycosides [20]. The nding tht esculin-hydrolysing bcteri from the rumen, the pig gut nd the humn gut cn medite mrked inhibition of the growth of E. coli O157 (12900) suggests tht the presence of such glycosides in the gut could hve signi cnt e ect on the prolifertion, survivl nd trnsmission of E. coli O157. Furthermore, the combined e ects of VFA nd esculetin suggest tht coumrins my hve prticulrly mrked e ects in nerobic regions of the gut. These ndings on the menslistic interctions, involving the biotrnsformtion of esculin, between the utochthonous rumen nd colonic bcteri nd pthogenic E. coli provide support for the growing view tht pproprite nutrition, including the use of dietry supplements, could provide n importnt mens for combting enteric disese in nimls nd mn. In ruminnts, obtining pproprite conditions in the gut to suppress the numbers of E. coli in the period immeditely prior to slughter my well be n chievble trget. Acknowledgments We thnk Dr. F. Thomson-Crter for providing Escherichi coli O157 strins. This work is supported by the Scottish O ce Agriculture, Environment nd Fisheries Deprtment (SOAEFD). References [1] Gri n, P.M. nd Tuxe, R.V. (1991) The epidemiology of infections cused by Escherichi coli O157:H7, other enterohemorrgic E. coli, nd the ssocited hemolytic uremic syndrome. Epidemiol. Rev. 13, 60^98. [2] Pennington, T.H. (1997) The Pennington group report on the circumstnces leding to the 1996 outbrek of infection with E. coli O157 in Centrl Scotlnd, the implictions for food sfety nd the lessons to be lerned. The Sttionry O ce, Edinburgh. [3] Rsmussen, M.A., Cry, W.C. Jr., Csey, T.A. nd Whipp, S.C. (1993) Rumen contents s reservoir of enterohemorrhgic Escherichi coli. FEMS Microbiol. Lett. 114, 79^84. [4] Brown, C.A., Hrmon, B.G., Zho, T. nd Doyle, M.P. (1997) Experimentl Escherichi coli O157 crrige in clves. Appl. Environ. Microbiol. 63, 27^32. [5] Krch, H., Russmnn, H., Schmidt, H., Schwrzkopf, A. nd Heesemnn, J. (1995) Long-term shedding nd clonl turnover of enterohemorrhgic Escherichi coli O157 in dirrhel diseses. J. Clin. Microbiol. 33, 1602^1605. [6] Kudv, I.T., Ht eld, P.G. nd Hodve, C.J. (1996) E ect of diet on the shedding of Escherichi coli O157:H7 in sheep model. Appl. Environ. Microbiol. 61, 1363^1370. [7] Scott, K.P. nd Flint, H.J. (1995) Trnsfer of plsmids between strins of Escherichi coli under rumen conditions. J. Appl. Bcteriol. 78, 189^193. [8] Chesson, A., Stewrt, C.S. nd Wllce, R.J. (1982) In uence of plnt phenolic cids on growth nd cellulolytic ctivity of rumen bcteri. Appl. Environ. Microbiol. 44, 597^603. [9] Murry, R.D.H., Mendez, J. nd Brown, S.A. (1982) The Nturl Coumrins. Wiley, Chichester. [10] Roul, Y. (1947) Etude de l toxicite de l coumrine. Action ntgoniste fvorble de diverses vitmines et notmment de l'mide nicotinique en utilisnt le colibcille comme test. Bull. Ste. Chim. Biol. 4-6, 518^525. [11] Moniello, G., Richrdson, A.J., Duncn, S.H. nd Stewrt, C.S. (1996) E ects of coumrin nd sprteine on ttchment to cellulose nd cellulolysis by Neocllimstix frontlis RE1. Appl. Environ. Microbiol. 62, 4666^4668. [12] Flint, H.J., Duncn, S.H. nd Stewrt, C.S. (1987) Trnsmissible ntibiotic resistnce in strins of Escherichi coli isolted from the ovine rumen. Lett. Appl. Microbiol. 5, 47^49. [13] Smbrook, J., Fritsch, E.F. nd Mnitis, T. (1982) Moleculr Cloning ^ Lbortory Mnul, pp. 68. Cold Spring Hrbor Lbortory, Cold Spring Hrbor, NY. [14] Hobson, P.H. (1969) In: Methods in Microbiology, Vol. 3B (Norris, J.R. nd Ribbons, D.W., Eds.) pp. 133^149, Acdemic Press, London. [15] Pugsley, A.P. (1985) Escherichi coli K12 strins for the identi ction nd chrcteriztion of colicins. J. Gen. Microbiol. 131, 369^376. [16] Cown, S.T. (1974) Mnul for the Identi ction of Medicl Bcteri, pp Cmbridge University Press. [17] Chng, W.-S. nd Ching, H.-C. (1995) Structure-ctivity reltionship of coumrins in xnthine oxidse inhibition. Anticncer Res. 15, 1969^1974. [18] Chng,W.-S., Lin, C.-C., Chung, S.-C. nd Ching, H.-C. (1996) Superoxide nion scvenging e ect of coumrins. Am. J. Chin. Med. 24, 1^17. [19] Hirmoto, K., Ojim, N., Sko, K. nd Kikugw, K. (1996) E ect of plnt phenolics on the formtion of the spin-dduct of hydroxyl rdicl nd the DNA strnd breking by hydroxyl rdicl. Biol. Phrm. Bull. 19, 558^563. [20] Wul -Strobel, C.R. nd Wilson, D.B. (1995) Cloning, sequencing nd chrcteristion of membrne-ssocited Prevotell ruminicol L-(1-4) glucosidse with cellodextrinse nd cynoglycosidse ctivities. J. Bcteriol. 177, 5884^5890. Downloded from