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

Sylvia H Duncan, Harry J Flint, Colin S Stewart
DOI: http://dx.doi.org/10.1111/j.1574-6968.1998.tb13099.x 283-288 First published online: 1 July 1998


Under both aerobic and anaerobic conditions, the growth of Escherichia coli O157 strain NCTC 12900 was inhibited by the coumarins esculetin, umbelliferone and scopoletin, but not by the coumarin glycoside esculin. Esculin-hydrolysing bacteria from the rumen, the pig gut and the human gut inhibited growth of E. coli in an overlay-plate assay in the presence of esculin. The combined effect of esculetin and volatile fatty acids was greater than the effect of either factor alone suggesting that coumarin glycosides in the diet might reduce the growth or survival of E. coli O157 in the gut. Adding esculin to incubations of mixed rumen contents significantly reduced the survival of E. coli O157.

Key words
  • Escherichia coli O157
  • Rumen
  • Gut
  • Commensal microflora
  • Esculin
  • Esculetin

1 Introduction

The incidence of reported disease caused by enterohaemorrhagic Escherichia coli (EHEC) [1] has recently escalated dramatically; the worst outbreak resulted in 20 deaths in Scotland in 1996 [2]. The rumen and colon provide sources for E. coli O157 shed in ruminant faeces [3, 4] which may contaminate the human food chain, and faecal shedding is also implicated in transmission between humans [5]. Shedding of E. coli by ruminants is influenced by the diet [6]. The growth of E. coli is reduced in the presence of volatile fatty acids (VFA) produced during carbohydrate fermentation in the gut [3, 7] suggesting that when animals are well fed and the VFA levels in the gut are high, the numbers of enterohaemorrhagic and other E. coli may be markedly reduced.

Dietary plant material often contains a great diversity of plant metabolites, some of which demonstrate species-selective effects on the growth of gut bacteria [8]. Coumarins (derivatives of benzo-α-pyrone) occur commonly in plants in the free state and as glycosides [9]. Coumarin (2,3-benzopyrone) has been shown to inhibit the growth of E. coli[10] and cellulolysis by ruminal anaerobic fungi [11] although the effects on commensal anaerobic bacteria remain unknown. We show here that predominant anaerobic species found in the rumen and colonic microbial communities can mediate inhibition of growth of E. coli O157 through conversion of the plant glycoside esculin to the aglycone esculetin. This provides one of the first examples of what may prove to be a more general type of microbial interaction.

2 Materials and methods

2.1 Bacterial strains, media and growth measurements

E. coli O157 strain EC22 was kindly provided by F. Thomson-Carter (University of Aberdeen, UK) and strain NCTC 12900 was from the PHLS, London. The non-O157 serotype strains F34, F36, F38, F310 and F318 were isolated at the Rowett Research Institute [12] from the rumen of sheep. Strain UB281 was supplied by R. Heard (University of Bristol, UK) and Bacteroides ovatus V975 was provided by T.R. Whitehead (USDA, Peoria, IL, USA). Enterococcus faecalis strains 1a, 2a, and 3a were recently isolated from the rumen of an adult sheep rumen at RRI. The Streptococcus bovis strain JB1 was a gift from J.B. Russell, Cornell University and Strep. caprinus strain 3969 was from the Australian Collection of Microorganisms. The other bacterial strains were held in the RRI culture collection. The E. coli strains were grown on LB medium [13] either under aerobic conditions, with shaking at 200 rpm, or under anaerobic conditions, when cysteine HCl (final concentration 0.1%) was added to the medium which was dispensed (5 ml), under O2-free CO2, into 16×125-mm Hungate tubes (Bellco Glass Inc.). Medium SD/E contained 3.0% Difco yeast extract, 1.0% Bacto peptone, 1.5% NaCl and 1.5% agar. Strict anaerobes were cultivated on M2GSC, the M2 medium of Hobson [14] with the omission of lactate and with 0.2% each of glucose, cellobiose, and soluble starch. The coumarins (Sigma Chem. Co., except for esculetin which was from Fluka AG) were prepared in 80% dimethyl sulfoxide (DMSO) stock solutions, filter sterilized (0.2 μm pore size; Gelman Acrodiscs) and added to the autoclaved medium. The volatile fatty acids (VFA) were added as filter-sterilised stock solutions adjusted to pH 7.0, and consisted of acetate, propionate and butyrate in the molar proportions 10:2:1, values within the range encountered in the rumen and the colon. Controls contained DMSO as appropriate. Growth was determined as the absorbance at 650 nm and specific growth rates were determined from the doubling times in the exponential growth phase.

2.2 Overlay plate assay for growth inhibition

Streptococcus and Enterococcus spp. were grown on SD/E medium with 1.5% agar, with or without esculin (5 mM). The plates were spot-inoculated within an anaerobic chamber in an atmosphere of 10% CO2, 10% H2 and 80% N2 (Don Whitley Scientific), then transferred after 1 h into an anaerobic jar under O2-free CO2 and incubated for 24 h at 38°C. The plates were removed, treated with chloroform and overlaid with 5 ml LB medium containing 0.75% agar inoculated with 100 μl of a pre-grown culture of E. coli O157 strain 12900 essentially as described in [15]. The overlaid plates were reincubated aerobically for 24 h at 38°C prior to examination for zones of growth inhibition. For anaerobic bacteria, medium M2GSC was used in place of SD/E, and the spot-inoculated plates were incubated for 48 h prior to testing.

2.3 Enumeration of viable E. coli O157 in mixed rumen contents

Rumen contents were recovered from a cannulated adult sheep fed a mixed hay/barley grain diet and mixed with an equal volume of sterile 0.85% NaCl, with or without 5 mM esculin. The diluted contents were dispensed aseptically under O2-free CO2 in 9.9-ml volumes into sterile Hungate tubes, inoculated with 100 μl of a pre-grown (18 h) culture of E. coli O157 strain 12900 then incubated at 38°C. Aliquots (100 μl) were removed at intervals into 9.9 ml sterile diluent (0.85% NaCl). Serial dilutions were plated on Rainbow O157 agar plates (Biolog), incubated aerobically at 38°C for up to 48 h when the dark grey/black colonies were enumerated.

2.4 Detection of esculin hydrolysis

The E. coli cultures were grown aerobically at 38°C on LB medium (5 ml); the other bacteria were incubated on M2GSC medium (7.5 ml) under O2-free CO2. The media, amended to contain 5 mM esculin, were inoculated with 100 μl of a broth culture pre-grown for 24 h. Esculin hydrolysis was detected using 1% ferric ammonium sulfate 24 h, 48 h, and 72 h after incubation [16].

3 Results

Incubation of E. coli O157 strain 12900 in LB medium under aerobic or anaerobic conditions showed that the presence of free coumarins had a marked effect on the growth of the bacterium. Coumarin was more inhibitory under anaerobic conditions than when tested aerobically (Fig. 1). Although esculetin (6,7-dihydroxy-coumarin) inhibited the growth of Escherichia coli, its glycoside esculin had no effect (Fig. 1). The effects of umbelliferone (7-hydroxycoumarin) and scopoletin (7-hydroxy-6-methoxycoumarin) were similar to those of esculetin (Fig. 1).

Figure 1

Effects of coumarins on the growth of Escherichia coli O157 strain 12900 under (a) aerobic and (b) anaerobic conditions. Cells were grown in LB medium with shaking (aerobic) or with the addition of cysteine HCl and under O2-free CO2 (anaerobic) as described in Section 2. The coumarins were all added at the 5-mM level; control (?), esculin (•), esculetin (▴), coumarin (?), umbelliferone (□), and scopoletin (◯).

A number of bacterial strains selected for their ability to hydrolyse esculin showed clear evidence of inhibition of E. coli only when esculin was added to the basal medium (Table 1). The inhibitory bacteria included rumen strains of Ent. faecalis, Strep. bovis, Mitsuokella multiacidus, Prevotella bryantii, P. brevis, and Selenomonas ruminantium (Table 1). A strain of M. multiacidus isolated from pigs was also inhibitory in the presence of esculin, as were two strains of Bacteroides ovatus isolated from human faeces. Ruminococcus flavefaciens strain 17 which hydrolysed esculin weakly and only tested positive after 48 h incubation in the presence of esculin, did not inhibit the growth of E. coli O157 in its presence (data not shown). Three esculin-negative strains tested, P. ruminicola 23, Bacteroides uniformis 1100, and Fibrobacter succinogenes BL2, did not inhibit growth of E. coli.

View this table:
Table 1

Inhibition of E. coli O157 strain 12900 by commensal bacteria from the gut

Species, strain (origina)Inhibition of E. coli 12900
Control+EsculinEsculin hydrolysis
Enterococcus faecalis 1a (r)++++
Enterococcus faecalis 2a (r)++++
Enterococcus faecalis 3a (r)++++
Streptococcus bovis JB1 (r)++
Streptococcus bovis 26 (r)++
Bacteroides ovatus 1896 (h)+++
Bacteroides ovatus V975 (h)+++
Bacteroides uniformis 1100 (h)
Mitsuokella multiacidus 10935 (p)+++
Mitsuokella multiacidus 46/5 (r)+++
Mitsuokella multiacidus P208-58 (p)++++
Prevotella bryantii B14 (r)+++
Prevotella brevis GA 33 (r)+++
Prevotella ruminicola 23 (r)
Selenomonas ruminantium 2358 (r)++++
Selenomonas ruminantium FB322 (r)++++
Fibrobacter succinogenes BL2 (r)
  • aOrigin of strains: (h) human, (p) pig, (r) rumen. Ent. faecalis and Strep. bovis were grown on SD/E plates, and the remaining anaerobes on M2GSC plates, anaerobically, before overlaying with E. coli 12900 (see Section 2). The zones of growth inhibition were assessed as the areas of no growth of the O157 strain in the plate overlay around the test strain colony and recorded as the diameter of the inhibition zone minus the diameter of the colony; (+) denotes ≤3 mm, (++) denotes >3 mm and ≤9 mm and (+++) denotes >9 mm.

When the growth rate of strain 12900 was measured in the presence or absence of VFA and esculetin, the effects of the two factors were additive. Thus growth could be measured in the presence of 50 mM VFA and 0.5 mM esculetin in anaerobic LB medium, but not in the presence of 100 mM VFA and 0.5 mM esculetin (Table 2).

View this table:
Table 2

Growth rates (h−1) of Escherichia coli O157 strain 12900 in anaerobic LB medium containing combinations of VFA and esculetin

Esculetin (mM)VFA concentration (mM)
  • aDenotes little or no growth; changes in absorbance too small for accurate measurement of growth rates.

Commensal strains of E. coli from the rumen [7, 12] tended to be less sensitive to esculetin than the strains of E. coli O157 (12900 and EC22) when grown in anaerobic M2GSC medium (Table 3). The ability of E. coli strains to grow in the presence of esculetin did not appear to correlate with their ability to hydrolyse esculin. For example, of the strains of E. coli shown in Table 3, only F38 and F310 were able to hydrolyse esculin, although hydrolysis was slow, being detected after 48 h and 72 h incubation, respectively. The O157 strains were slightly less sensitive to the esculetin (0.5 mM) and VFA (100 mM) combination in the rumen fluid M2GSC medium (Table 4) compared with LB medium (Table 2). In M2GSC medium there was little effect of 0.5 mM esculetin and 100 mM VFA on the growth of the rumen strains P. bryantii B14, Bu. fibrisolvens D6/1 and. Sel. ruminantium Z108 but the growth rates of B. ovatus V975 and of R. albus J6 were reduced (Table 4). Growth of E. coli O157 strains was strongly inhibited by 5 mM esculetin in M2GSC medium (not shown). In contrast the Δ OD650 for Bu. fibrosolvens D6/1, P. bryantii B14 and Sel. ruminantium Z108 cultures was decreased only 24%, 12% and 9%, respectively, after 24 h growth in the presence of 5 mM esculetin compared to the untreated control, and by less than 10% in the presence of 2 mM esculetin (data not shown).

View this table:
Table 3

Growth rates (h−1) of E. coli strains on the rumen fluid based M2GSC medium in the presence of esculetin (5 mM) under anaerobic conditions

StrainControl5 mM esculetin
CommensalE. coli
E. coli O157
  • aDenotes little or no growth; changes in absorbance too small for accurate measurement of growth rates.

View this table:
Table 4

Growth rates (h−1) of anaerobic bacteria in the presence of esculetin and esculetin plus 100 mM VFA on M2GSC medium under anaerobic conditions

StrainControl0.5 mM esculetin0.5 mM esculetin+100 mM VFA
B. ovatus V9750.35±0.020.33±0.020.26±0.04
B. ovatus 18960.30±0.050.30±0.00.18±0.02
P. bryantii B140.69±0.040.63±0.00.59±0.01
S. ruminantium Z1080.77±00.70±0.1a0.76±0.02
B. fibrisolvens D6/10.72±0.050.77±0.080.69±0.0
R. albus J60.80±0.050.70±0.0050.21±0.01
E. coli O157 129000.71±0.140.58±0.050.32±0.04
E. coli O157 EC220.63±0.060.57±0.070.37±0.09
  • Average of 3 determations±S.D., except a: 2 replicates±range.

Finally, when E. coli O157 strain 12900 was incubated in mixed rumen contents for 24 h, the numbers of viable E. coli were reduced from around 5×105 ml−1 to below 20 ml−1 when 5 mM esculin was present. In the absence of esculin, the mixed rumen bacteria present had no significant effect on the survival of E. coli (Fig. 2). The numbers of total viable anaerobes in mixed rumen contents were enumerated as 1.2×108 ml−1 initially and dropped to 7.3×106 ml−1 and 6.5×106 ml−1 respectively with and without the addition of esculin over a 24-h incubation period (data not shown). The number of E. coli O157 represents approximately 6% of the total viable anaerobic bacteria in the control incubations, with no added esculin, compared to 0.003% when esculin was added, representing a 2000-fold decrease in the relative proportion of viable E. coli O157 cells in mixed rumen contents containing esculin.

Figure 2

Effect of esculin (5 mM) on the survival of Escherichia coli O157, strain 12900 in rumen contents; (□) control, (•) 5 mM esculetin.

4 Discussion

The mechanism(s) by which coumarins inhibit microbial growth are not understood, but coumarins are known to act as enzyme inhibitors and as antioxidants in biological systems. Esculetin inhibits mammalian xanthine oxidase activity [17] and scavenges superoxide anions [18] and hydroxyl radicals [19]. The free-radical scavenging activity of coumarins in the human gut is seen as a possible health-promoting effect, and the reduction in survival of E. coli O157 reported here offers further evidence of possible beneficial effects of such compounds in the diet.

Coumarins and coumarin glycosides are found in a wide range of forages for farm animals and in fruit and vegetables consumed by humans [9]. Many of the predominant bacterial species found in the gut microflora possess the ability to hydrolyse plant glycosides, as exemplified by the conversion of esculin to the aglycone esculetin, which we show here is a potent inhibitor of the pathogenic E. coli strains. Esculin hydrolysis is presumed to be a property of β-(1-4) glucosidases, and the β-(1-4) glucosidase of P. bryantii B14, for example, has been shown to exhibit a broad specificity, hydrolysing a range of plant glycosides [20]. The finding that esculin-hydrolysing bacteria from the rumen, the pig gut and the human gut can mediate marked inhibition of the growth of E. coli O157 (12900) suggests that the presence of such glycosides in the gut could have a significant effect on the proliferation, survival and transmission of E. coli O157. Furthermore, the combined effects of VFA and esculetin suggest that coumarins may have particularly marked effects in anaerobic regions of the gut.

These findings on the amensalistic interactions, involving the biotransformation of esculin, between the autochthonous rumen and colonic bacteria and pathogenic E. coli provide support for the growing view that appropriate nutrition, including the use of dietary supplements, could provide an important means for combating enteric disease in animals and man. In ruminants, obtaining appropriate conditions in the gut to suppress the numbers of E. coli in the period immediately prior to slaughter may well be an achievable target.


We thank Dr. F. Thomson-Carter for providing Escherichia coli O157 strains. This work is supported by the Scottish Office Agriculture, Environment and Fisheries Department (SOAEFD).


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