Ajg_1547.tex

The Effect of a Multispecies Probiotic on the IntestinalMicrobiota and Bowel Movements in Healthy VolunteersTaking the Antibiotic AmoxycillinCatherina J.M. Koning, M.Sc.,1 Daisy M.A.E. Jonkers, Ph.D.,1,2 Ellen E. Stobberingh, Ph.D.,2Linda Mulder, M.Sc.,3 Frans M. Rombouts, Ph.D.,4 and Reinhold W. Stockbr¨ugger, M.D., Ph.D.11Division of Gastroenterology-Hepatology and 2Department of Medical Microbiology, University HospitalMaastricht, Maastricht, The Netherlands; 3Winclove Bio Industries B.V., Amsterdam, The Netherlands; and4Laboratory of Food Microbiology, Department of Agrotechnology and Food Sciences, Wageningen University,Wageningen, The Netherlands One of the side effects of antimicrobial therapy is a disturbance of the intestinal microbiotapotentially resulting in antibiotic-associated diarrhea (AAD). In this placebo-controlled double-blindstudy, the effect of a multispecies probiotic on the composition and metabolic activity of theintestinal microbiota and bowel habits was studied in healthy volunteers taking amoxycillin.
Forty-one healthy volunteers were given 500 mg amoxycillin twice daily for 7 days and wererandomized to either 5 g of a multispecies probiotic, Ecologic daily for 14 days. Feces and questionnaires were collected on day 0, 7, 14, and 63. Feces wasanalyzed as to the composition of the intestinal microbiota, and β-glucosidase activity, endotoxinconcentration, Clostridium difficile toxin A, short chain fatty acids (SCFAs), and pH were determined.
Bowel movements were scored according to the Bristol stool form scale.
Mean number of enterococci increased significantly from log 4.1 at day 0 to log 5.8 (day 7) and log6.9 (day 14) cfu/g feces (P < 0.05) during probiotic intake. Although no other significant differenceswere observed between both intervention groups, within each group significant changes were foundover time in both microbial composition and metabolic activity. Moreover, bowel movements with afrequency ≥3 per day for at least 2 days and/or a consistency ≥5 for at least 2 days were reportedless frequently in the probiotic compared to the placebo group (48% vs 79%, P < 0.05).
Apart from an increase in enterococci no significant differences in microbial composition andmetabolic activity were observed in the probiotic compared with the placebo group. However,changes over time were present in both groups, which differed significantly between the probioticand the placebo arm, suggesting that the amoxycillin effect was modulated by probiotic intake.
Moreover, the intake of a multispecies probiotic significantly reduced diarrhea-like bowelmovements in healthy volunteers receiving amoxycillin.
INTRODUCTION
the cases of AAD are thought to be due to a disturbance ofthe intestinal microbiota by antibiotics, which is associated One of the collateral effects of antimicrobial therapy is with loss of colonization resistance (leading to overgrowth antibiotic-associated diarrhea (AAD), which can occur of potential pathogens), changes in carbohydrate digestion shortly after antibiotic intake up to 8 wk after cessation (1, 2).
and production of short-chain fatty acids (SCFAs), altered The incidence of AAD ranges from 5-39%, depending on the metabolism of bile acids, and changes in both the mucosal definition of diarrhea, the type of antibiotic used, and host fac- and systemic immune response (7). In addition, antibiotics tors (2). In general, amoxycillin, amoxycillin/clavulanic acid, may have direct allergic and toxic effects on the mucosa, di- clindamycin, and cephalosporines are associated with a high rect effects on immune-cell function, and pharmacological risk of AAD (3). AAD may range from mild disturbances to effects on intestinal motility (7-9). Possible consequences severe pseudomembranous colitis due to Clostridium difficile of AAD in health-care facilities include an increase in the (4). This bacterium is thought to be the causative agent in up incidence of nosocomial infections and an increase in mor- to 20% of AAD patients; however, the mechanisms causing bidity and mortality, longer hospitalization, and higher costs the majority of cases of AAD are not clear (5, 6). Most of of care (2). Although in general practice AAD is often merely Koning et al.
considered a nuisance, it may lead to a lack of compliance of The objective of this placebo-controlled, randomized, antibiotic intake, which is associated with the development double-blind study was to evaluate the effect of a multi- of antibiotic resistance (10). Furthermore, antibiotic use and species probiotic on the composition of the intestinal mi- the subsequent disturbance of the intestinal microbiota is a crobiota in healthy volunteers during and after amoxycillin risk factor for the development of irritable bowel syndrome intake. Moreover, the effect of the probiotic on the metabolic activity of the intestinal microbiota and on bowel habits was Probiotics, which are defined as "mono- or mixed cultures of live microorganisms that, when applied to animal or hu-man, beneficially affect the host by improving the propertiesof the indigenous microbiota," may prevent and restore an imbalance caused by antibiotics and are therefore of increas-ing interest for the prevention and treatment of AAD (13).
Subjects
Several probiotic strains have been used in controlled stud- Healthy volunteers between 18 and 65 yr of age were eligible ies, aiming at the prevention and treatment of AAD, such for the study. Exclusion criteria were: smoking, pregnancy, as Lactobacillus acidophilus, Lactobacillus rhamnosus GG, lactation, hypersensitivity to β-lactam antibiotics or tetracy- Bifidobacterium longum, Enterococcus faecium, and Saccha- cline, pre-existing bowel pathology (including irritable bowel romyces boulardii, and resulted in a significant decrease in syndrome, inflammatory bowel disease, diverticulitis, and the incidence of diarrhea (0-10% in the probiotic versus 14- cancer), treatment with immune-suppressive medication or 27% in the placebo group) (14-22). However, other studies immune-compromised subjects, diarrhea or constipation (in failed to show any benefit from probiotics in the prevention the last 3 days prior to inclusion), allergic and inflammatory of AAD (23-25). Two meta-analyses on the use of probiotics reactions, as well as infections within 2 wk prior to inclu- in the prevention of AAD evaluated nine and seven placebo- sion. Furthermore, the volunteers were not allowed to use: controlled, double-blind trials, and reported an odds ratio of (a) gastric acid inhibitors, laxatives, antidiarrhea medication, 0.37 and a relative risk of 0.40, respectively, in favor of pro- or antibiotics for at least 2 months before the start of and dur- biotic administration (26, 27). A recent meta-analysis, which ing the study; (b) corticosteroids for at least 4 wk before the included 25 randomized controlled trials, confirmed these start of and during the study; (c) other probiotics and prebi- findings (relative risk of 0.43), and showed that the probi- otics for at least 2 wk before the start of and during the study.
otic efficacy could be attributed to three types of probiotics: Finally, participants were asked to continue their ordinary di- S. boulardii, L. rhamnosus GG, and probiotic mixtures (28).
etary habits. All volunteers gave written informed consent.
Most studies on probiotics and AAD have only investigated The study was approved by the medical ethics committee of the development of diarrhea (i.e., clinical outcome); only in the University Hospital Maastricht, The Netherlands.
a few studies the effect on the composition of the fecal mi-crobiota was also examined, albeit to a very limited degree Study Design
The study was executed according to a parallel, randomized, It has recently been demonstrated that multispecies and, to placebo-controlled, double-blind design. The total duration a lesser extent, multistrain probiotics have certain advantages of the intervention and follow-up period was 63 days. Vol- over monostrain preparations. Mixed preparations may com- unteers received 500 mg amoxycillin twice daily from day plement each other's effect through synergism and/or sym- 1-7 and were randomized to receive either 5 g of a multi- biosis (31). Ouwehand et al. reported, for example, that the species probiotic or 5 g placebo twice daily from day 1-14.
in vitro adhesion of B. lactis Bb12 was more than doubled This resulted in three time periods defined as: day 1-7, "the by the presence of L. rhamnosus GG and L. delbrueckii spp.
antibiotic/probiotic period," in which all volunteers received Bulgaricus (32). A progressive increase in B. lactis growth amoxycillin in combination with either probiotic or placebo; and acidification in the presence of L. acidophilus in vitro day 8-14, "the probiotic only period," in which volunteers was demonstrated by Gomes et al. (33).
received either probiotic or placebo; day 15-63, "the post- It is well known that the composition of the fecal mi- treatment follow-up period." Amoxycillin was taken with crobiota and its role in colonization resistance, but also milk before breakfast and dinner, while the placebo or pro- its metabolic activity (producing several bacterial enzymes, biotic was taken before lunch and before bedtime. The time short chain fatty acids [SCFAs], amines, and bacteriocins), between antibiotic and probiotic intake had to be at least 2 h.
will affect the host. However, few studies have assessed the Fresh fecal samples were collected on day 0 (i.e., baseline), 7, influence of probiotics on the metabolic activity of the intesti- 14, and 63. On the same day, a questionnaire was filled out in- nal microbiota during and after antibiotic treatment. Com- cluding questions on bowel movements (stool frequency and monly, studies on AAD concerned the use of monospecies consistency [ranging from 1 = hard lumps to 7 = completely probiotics. The results of these studies are difficult to gener- watery] according to the Bristol stool form scale (34)), use of alize, since there is a lot of variation between species; proper- pre- and probiotics, other medication taken, (drastic) change ties that apply to one strain are not necessarily applicable to of eating habits, and compliance. In addition, a short ques- tionnaire on bowel habits and side effects (nausea, abdominal Effect of a Multispecies Probiotic on the Intestinal Microbiota and Bowel Movements
cramps, bloating, flatulence, or other) had to be completed emulsion (36). Before inoculation of the egg yolk-neomycin daily during probiotic/placebo intake.
agar, fecal dilutions were heated at 80◦C for 10 min.
Blood agar and eosin-methylene blue agar plates were in- Probiotic
cubated aerobically at 37◦C for 24 h. Sabouraud GM+C and The multispecies probiotic (Ecologic AAD) and the placebo KF-streptococcus agar plates were incubated aerobically at were kindly provided by Winclove Bio Industries, Amster- 37◦C and 42◦C, respectively, for 48 h. Fastidious anaerobic, dam, The Netherlands. Ecologic AAD consists of 10 differ- bile-esculine, egg yolk, and LAMVAB agar plates were in- ent bacterial species at each 108 colony forming units (cfu)/g cubated under anaerobic conditions at 37◦C for 48 h.
the total dose being 109 cfu/g (B. bifidum W23, B. lactisW18, B. longum W51, E. faecium W54, L. acidophilus W37 Viability of E. faecium W54
and W55, L. paracasei W72, L. plantarum W62, L. rham- From each KF-streptococcus agar plate two dominant nosus W71, and L. salivarius W24), 5% mineral mix (potas- colonies were isolated on both day 7 and 14 and purified sium chloride [∼67.3%], magnesium sulphate [∼32.6%] and on blood agar plates. Isolates were frozen at −80◦C awaiting manganese sulphate [∼0.1%]) and 15% Raftilose synergy1 further identification. At the end of the study, the enterococci (inulin enriched with oligofructose). Each participant con- of 10 individuals (i.e., 40 colonies), who had received probi- otic treatment, were typed by pulse field gel electrophoreses twice daily for 2 wk. Sachets were dissolved in lukewarm (PFGE), using SmaI according to the method described by water, left for 10 min, stirred, and thereafter ingested. The van den Braak et al. (37, 38).
placebo sachets were indistinguishable in color, smell, andtaste from the probiotic sachets but contained no bacteria.
Bacterial Enzyme Activity
Bacterial β-glucosidase activity was determined as previ-
Sample Processing
ously described (39). Briefly, fecal dilutions were mixed (1:1) Fecal samples were brought to the laboratory within 12 h after with 0.1 M PBS (pH 6.8), sonicated for 1 min, and centrifuged defecation and divided into three portions: (a) ten grams was at 1,700 g for 15 min. The supernatants were lyophilized for centrifuged at 47,000 g for 2 h at 4◦C to obtain fecal water, 75 min by Speed-Vac (Savant DNA 120, GMI, Inc., Ram- which was frozen immediately in 2-fold at −80◦C for anal- sey, MN) and the remaining fractions were used to determine ysis of endotoxin concentrations and determination of pH; β-glucosidase (at 420 nm) activity by using p-nitrophenyl (b) five grams was diluted (1:4) with peptone water (Oxoid β-D-glucopyranoside as a substrate.
CM9, Basingstoke, Hants, U.K.) supplemented with cysteine(2.1 mM) and glycerol (30%). Bacterial cultures of the fe- Endotoxin
cal dilution were performed immediately and the remainder The endotoxin (i.e., lipopolysaccharide) concentration was was frozen at −20◦C for the subsequent analyses of enzyme determined in fecal water using the Limulus amoebocyte activities, Clostridium difficile toxin A, and SCFAs, (c) the lysate endochrome technique (Endosafe, end point chro- remaining fecal sample (1-15 grams) was frozen directly at mogenic analysis endochrome test kit, Charles River, Kent, U.K.). The analysis was performed according to the man-ufacturer's specifications under pyrogen-free conditions.
Bacteriological Culture
Pyrogen-free water was used to dilute the fecal samples, and Tenfold serial dilutions of the fecal dilution were made the test-solutions and as negative control. The detection range in physiological saline (0.85%) with cysteine-HCl (0.05%) of the assay was 0.015 to 0.12 EU/mL (9 EU/ng). Concen- and 40 µL of these dilutions was inoculated using a spiral tration of fecal endotoxin was expressed as nanogram of en- plater (Eddy Jet v1.2, IUL-instruments, Barcelona, Spain) onto the following agar plates: blood agar (Oxoid CM271)for total (facultative) aerobic bacteria, eosin-methylene blue Clostridium difficile Toxin A
(methylthioninium chloride) agar (Oxoid CM69) for enter- Clostridium difficile toxin A was determined using an obacteriaceae, KF-streptococcus agar (Oxoid CM701) for en- enzyme-linked fluorescent immunoassay technique (VI- terococci, fastidious anaerobic agar (Laboratory M LabGo, C. difficile Toxin A II assay, bioMerieux, Lyon, Lancashire, U.K.) for total (facultative) anaerobic bacteria, France). The analysis was performed according to the bile-esculine agar (Becton Dickinson 287920, La Pont de manufacturer's specifications using the VIDAS system Claix, France) for Bacteriodes spp., LAMVAB agar for lac- tobacilli, Sabouraud agar with gentamicin and chlorampheni-col (GM+C) (Becton Dickinson 254041) for yeasts and egg Short-Chain Fatty Acids
yolk-neomycin agar for spore-forming clostridia. LAMVAB SCFAs were measured in the fecal dilutions using gas- agar was prepared according to the method described by liquid chromatography. The gas-liquid chromatography sys- Hartemink et al. (35). Egg yolk-neomycin agar was prepared tem consisted of a CP9002 gas chromatograph equipped by adding a sterile neomycin solution (final concentration, with a flame ionization detector in conjunction with Mae- 100 µg/mL) to egg yolk agar with freshly prepared egg yolk stro software (Chrompack, Middelburg, The Netherlands) for Koning et al.
calculations. The chromatographic column used was WCOT Based on data from previous probiotic studies, it was esti- fused silica (25 m × 0.32 mm id), coated with FFAP-CB df mated that 19 volunteers per treatment group would provide 0.3. This column was used in an isothermal mode at 140◦C 80% power to detect a one log difference in numbers of spe- and both the injector and detector temperature were 270◦C.
cific microorganisms cultured, assuming a variance of 1.1 and The sample size was 1.0 µL, which was split 50:1 to give a a 2-sided significance level of 0.05.
0.02-µL sample on the column. Helium was used as the car-rier gas with a head pressure of 0.8 bar. SCFAs were extractedand analyzed as previously described (39).
Subjects
pH
Forty healthy volunteers completed the study, 19 in the pro- The pH of fecal water was determined using a PHM standard biotic (5 men and 14 women, mean age 25.5, SD 10.2 yr) pH meter with a PHC3006 electrode (Radiometer Nederland and 21 in the placebo group (10 men and 11 women, mean age 28.2, SD 11.5 yr). One subject in the probiotic group wasfound to be allergic to amoxycillin and had to be excluded.
Protein Concentration
Two subjects in the placebo group did not complete the daily Homogenized fecal samples were diluted (1:99) in 0.1 M questionnaire. On day 14, subjects delivered fecal samples to PBS (pH 6.8, 5-7◦C) and added to BioRad Assay Protein the hospital and subsequently handed in the daily question- Dye Reagent (1:1). After 30 min, the absorbance was read at naire. As a consequence information from the questionnaire 595 nm. Concentrations of proteins were calculated from a was available for day 1-13. The compliance for antibiotic standard curve for proteins ranging from 0-120 µg/mL and intake was at least 93%, and for probiotic/placebo intake at expressed as mg total protein per gram feces.
least 97% in both groups. One subject in the placebo groupand three in the probiotic group incidentally (i.e., maximally Defecation Score
twice a week) consumed yogurt containing L. rhamnosus GG In this study a diarrhea-like defecation has been defined as a between day 14 and 7 before starting the study. Moreover, in defecation frequency ≥3 per day and/or a fecal consistency the probiotic group one other subject incidentally consumed ≥5 per day, on the Bristol stool form scale, for at least 2 that probiotic during the first 2 wk of the study. Apart from one subject in the probiotic group taking omeprazole oncedaily on day 45 and 46 of the study, no medication poten- Statistics
tially affecting the intestinal microbiota was taken during the The treatment allocation was concealed to all investigators and volunteers, until the study had been completed and allanalyses had been performed.
Bacteriological Culture
The primary outcome of this study was to compare the During probiotic intake, a significant increase in the mean changes that occurred in the composition of the intesti- number of fecal enterococci was found on day 7 (5.8 vs 4.0 nal microbiota during and after amoxycillin intake between log cfu/g feces, P < 0.02) and on day 14 (6.9 vs 4.3, P < probiotic- and placebo-treated subjects. Secondary outcomes 0.001) in the probiotic group compared to the placebo group were the changes that occurred in the metabolic activity of the (Table 1). Moreover, the mean number of fecal enterococci intestinal microbiota and changes in defecation score during within the probiotic group increased significantly during an- and after amoxycillin between probiotic- and placebo-treated tibiotic/probiotic intake (day 7) and increased further during probiotic therapy alone (day 14). A significant decrease in the Only data from volunteers who completed the study, had mean number of fecal enterococci was observed 7 wk after a probiotic/placebo and antibiotic compliance of ≥90%, and cessation of probiotic intake (P < 0.05) having returned to delivered all four fecal samples were included in the data pretreatment level (Table 1). No further differences in either aerobic or anaerobic bacterial species could be seen between Statistical evaluation of differences between groups and the probiotic and the placebo group.
changes within groups (at all time points during the study However, group-specific differences were observed over period) was carried out using linear mixed model analysis.
time: within the probiotic group a significant decrease was In this analysis the fixed effects were day and treatment and found in total aerobes (day 63 versus day 7) and significant the random effect was subject. For two-group comparisons increases were observed over time in total anaerobes (day 14 of independent ordinal and interval values the nonparamet- versus day 0) and Bacteroides spp. (day 7 and day 14 versus ric Mann-Whitney U-test was used while the nonparametric day 0) (P < 0.05). Within the placebo group a significant Wilcoxon signed-ranked test was used for comparison of re- increase was found over time in enterococci (day 14 versus lated ordinal and interval values. All tests were conducted day 0) and significant decreases were found in lactobacilli using SPSS version 11.0 (SPSS Inc, Chicago, IL) and a P (day 7 versus 0) and spore-forming clostridia (day 7 versus value below 0.05 was considered statistically significant.
day 0 and 63) (P < 0.05) (Table 1).
Effect of a Multispecies Probiotic on the Intestinal Microbiota and Bowel Movements
Table 1. Numbers of Bacteria Cultured Expressed as Log cfu/g Feces
∗Between group difference P < 0.02.
†within group decrease t = 63 versus t = 7, P < 0.05.
‡within group increase t = 7/14 versus t = 0/63 and t = 7 versus t = 14, P < 0.05.
§within group increase t = 14 versus t = 0, P < 0.05.
¶within group increase t = 14 versus t = 0, P < 0.05.
within group increase t = 7/14 versus t = 0, P < 0.05.
∗∗within group decrease t = 7 versus t = 0/63, P < 0.05.
††within group decrease t = 7 versus t = 0, P < 0.05.
For all bacterial species studied in both the probiotic and Metabolic Activity
the placebo group, values on day 63 did not differ significantly β-Glucosidase activity did not differ significantly between the probiotic and the placebo group during the total study period The PFGE profile of 39 out of the 40 enterococci strains, (Table 2). Within both groups, a decrease in β-glucosidase isolated from the feces of the healthy volunteers receiving was observed at day 7 (significant for the placebo group), probiotic, was similar to that of the orally administrated pro- which increased again on day 63 (significant for the probiotic biotic E. faecium W54 strain.
group). In both groups, the β-glucosidase activity returned Table 2. Metabolic Activity: β-Glucosidase Activity (Expressed as mg/60 min/g Feces); SCFA Concentration (Expressed in mmol/g Feces)
and pH
β-glucosidase
Probiotic∗
Placebo†
SCFA
Acetic acid
Probiotic‡
Placebo§
Probiotic¶
Probiotic
Placebo∗∗
Probiotic
∗within group increase t = 63 versus t = 7, P < 0.05 and a tendency to a within group decrease t = 7 versus t = 0, and increase t = 63 versus t = 14, P < 0.06.
†within group decrease t = 7 versus t = 0, P < 0.05.
‡within group decrease t = 7/14 versus t = 0, P < 0.05.
§within group increase t = 14 versus t = 7, P < 0.05 and within group decrease t = 63 versus t = 14, P < 0.05.
¶within group decrease t = 14/63 versus t = 7.
within group decrease t = 7/14 versus t = 0, P < 0.05 and within group increase t = 63 versus t = 7/14, P < 0.05.
∗∗within group decrease t = 7/63 versus t = 0, P < 0.05.
Koning et al.
Figure 1. Linear regression of fecal consistency (scored with the Bristol stool form scale) in relation to the protein concentration per gram
feces (R = 0.61, P < 0.01). Consistency ranging from 1 (hard lumps) to 7 (watery) according to the Bristol stool form scale. Red dots
represent fecal samples from each volunteer collected on day 14.
to baseline values 7 wk after cessation of amoxycillin intake the probiotic group. This effect was also observed for acetic acid. Furthermore, an increase in propionic acid concentra- Endotoxin concentrations (mean ± SEM), expressed as tions was observed on day 7 in the probiotic group.
log ng/mL fecal water, did not differ significantly between No significant changes were found in the pH of the fecal the probiotic and the placebo group on day 0 (2.15 ± 0.07 vs water between and within both groups, during the total study 2.04 ± 0.08), day 7 (2.30 ± 0.06 vs 2.22 ± 0.07), and day 14 (1.92 ± 0.10 vs 1.89 ± 0.11). However, in both groups, a At all time points, a negative correlation (P < 0.05) was small but not significant increase in mean endotoxin concen- observed between the amount of protein per gram feces and tration was observed on day 7, whereas 1 wk after cessation the consistency score (except at t = 63 days). In addition, of antibiotic intake (day 14) a significant decrease (P < 0.05) a positive correlation (P < 0.05) was observed between the in endotoxin concentration was observed compared to day 7.
amount of fecal water per 10 g of feces and the consistency Clostridium toxin A was detected in the feces of two vol- score at all time points (an example is shown in Fig. 1).
unteers in the placebo group 1 wk after cessation of antibioticintake (day 14) and in one volunteer in the probiotic group at Defecation Score
The mean defecation frequency and consistency before an- No significant differences between the groups were ob- tibiotic and probiotic intake (day 0, i.e., baseline), during the served for any of the SCFAs tested (Table 2). However, within antibiotic/probiotic period (day 1-7), and during the probiotic both groups changes were found over time. Butyric acid con- only period (day 8-13) are listed in Table 3. The defecation centrations significantly decreased in both groups during an- frequency during the probiotic only period (day 8-13) was tibiotic intake, but by day 63 had recovered to baseline in significantly lower (P < 0.05) in the probiotic than in the Table 3. Mean Daily Fecal Frequency and Consistency Scores Before Antibiotic/Probiotic Intake (Day 0), During Antibiotic/Probiotic Intake
(Day 1-7), and During Probiotic Intake Alone (Day 8-13)
∗Consistency ranging from 1 (hard lumps) to 7 (watery) according to the Bristol stool form scale.
†Significant difference between probiotic and placebo group (P < 0.05).
Effect of a Multispecies Probiotic on the Intestinal Microbiota and Bowel Movements
Table 4. Defecation Score Between Day 1 and Day 13
versus the placebo group, apart from a significant increase in fecal enterococci. Although no other differences were ob- served between groups, group-specific changes were seenover time. Such changes were also observed for metabolic activity. Finally, a significantly better defecation score (de- crease in diarrhea-like bowel movements) was observed in the probiotic group versus the placebo group.
In AAD, differences in efficacy have been reported for dif- a consistency† ≥ 5 for at least ferent bacterial species, bacterial strains, and probiotic mix- tures (28). The efficacy of multispecies probiotic mixtures is further supported by the successful use of the multispecies probiotic VSL#3 in several gastrointestinal disorders (40- 42). In the present study, we used a multispecies probiotic †Consistency ranging from 1 (hard lumps) to 7 (watery) according to the Bristol stool containing 10 different probiotic strains selected on the basis of their in vitro ability to inhibit growth of Clostridium spp.
and to survive a low pH (2.5) as well as bile and digestive en- placebo group. During the total probiotic period (day 1-14), zymes (pancreatin and pepsin) (data not shown). In addition, diarrhea-like bowel movements were reported less frequently their resistance profile against a wide range of antibiotics was in the probiotic (48%) than in the placebo (79%) group (P < taken into account to prevent possible transfer of resistance from the probiotic bacteria to the indigenous microbiota. Fi-nally, the combination of strains chosen was tested to exclude Side Effects
The placebo and probiotic group were comparable regard- The composition of the fecal microbiota regarding total ing the percentage (Fig. 2) and severity of side effects re- aerobic bacteria, clostridia, and lactobacilli counts before in- ported: 79% mild-moderate symptoms in the probiotic group tervention was comparable with previous findings in healthy versus 90% mild-moderate symptoms in the placebo group.
volunteers using the same culture methods (39, 43). How- Side effects most frequently reported were nausea, abdomi- ever, in this study lower mean fecal bacterial concentrations nal cramps, bloating, and flatulence. Finally, side effects were were found for total anaerobic bacteria and Bacteroides spp., significantly more frequent during the antibiotic/probiotic pe- riod (day 1-7) than during the probiotic only period (day One of the possible mechanisms by which probiotics exert 8-13) (P < 0.05), for both the probiotic and placebo group their effect is by affecting the composition of the intesti- nal microbiota and preventing the overgrowth of possiblepathogens. Only the fecal microbiota was investigated in thisstudy, even though mucosa-associated bacteria may also be DISCUSSION
very relevant. Due to interindividual variation and possible In this placebo-controlled double-blind study, investigating sampling error, various biopsies ought to have been taken at the effect of a multispecies probiotic in healthy volunteers all the different time points of the study. Considering the in- after amoxycillin intake, no differences in the composition of vasiveness and the potential risks this was considered not to the intestinal microbiota were observed in the probiotic group be ethically acceptable in healthy volunteers.
The consumption of the multispecies probiotic, containing E. faecium, was associated with a significant increase in the concentration of fecal enterococci in the probiotic group from log 4.1 cfu/g to log 5.8 cfu/g on day 7 and to log 6.9 cfu/g on day 14. This increase disappeared 7 wk after cessation of probiotic intake, demonstrating that the consumption of this multispecies probiotic, containing E. faecium, can tran- siently alter the number of viable enterococci. Considering a consumption of 1 × 109 cfu E. faecium per day, present in the multispecies product, and a fecal volume of 100 g per day, the recovery of around log 7 enterococci per gram feces after probiotic intake indicates that E. faecium is able to survive passage through the gastrointestinal tract very well. More- over, the PFGE patterns of the enterococci isolated from the Figure 2. Side effects. ∗Significant difference (P < 0.05) in the
fecal samples were similar to the orally administered E. fae- percentage of volunteers with side effects during day 1-7 versus cium. A study in which a monospecies E. faecium probiotic (4.5-7.5 × 109 cfu daily) was given to healthy volunteers also Koning et al.
found a high increase (100-fold) in the total number of ente- 5-20% (52, 53), which can further increase with length of stay rococci (44). Furthermore, the recovery of viable enterococci in the placebo group was not affected during amoxycillin in- A change in the composition of the intestinal micro- take, indicating that in this study amoxycillin had little effect biota might affect its metabolic characteristics, such as β- on the indigenous enterococci population.
glucosidase activity. β-Glucosidase has been implicated in In contrast to the counts of enterococci, twice daily probi- carcinogenesis, since it is able to hydrolyse dietary substrates otic consumption containing 3 × 109 cfu lactobacilli (L. sali- into carcinogenic compounds (55, 56). A decrease of this ac- varius, L. plantarum, and L. rhamnosus) did not significantly tivity is therefore potentially beneficial. It has been demon- increase the number of fecal lactobacilli. However, a previous strated that a change in the composition of the intestinal mi- study by our group, in which 20 healthy volunteers consumed crobiota or the intake of Lactobacillus spp. can influence L. plantarum 299v for 4 wk did show a 1000-fold increase β-glucosidase activity, although the results differ between in the mean number of fecal lactobacilli (39). An increase in strains and populations studied (39, 46, 57-61). In the present the mean fecal number of lactobacilli was also observed by study β-glucosidase activity decreased in both groups during others after a 6-month consumption of L. rhamnosus and a 3- amoxycillin intake and returned to baseline values 7 wk after wk consumption of L. acidophilus (45, 46). However, during cessation of amoxycillin. No effect of probiotic intake was amoxycillin intake a decrease was observed in the total num- ber of fecal lactobacilli in the placebo group but not in the The endotoxin concentrations in both groups increased probiotic group. Comparable results were observed in a study during amoxycillin intake, though not significantly, and de- performed by Plummer et al. in which probiotic supplemen- creased significantly 1 wk after cessation of amoxycillin in- tation was given during H. pylori eradication therapy (30).
take. This is in accordance with evidence from several studies These findings indicate that probiotic intake might prevent a showing that antibiotics increase the bioavailability of endo- decrease of lactobacilli caused by antibiotic intake.
toxin originating from Gram-negative bacteria (62-65). The Apart from lactobacilli and the E. faecium, the multispecies level of intestinal endotoxin, however, does not only correlate probiotic used also contained bifidobacteria. However, no bi- with the number of Gram-negative bacteria, which is in line fidobacteria were cultured, due to insufficient selectivity and with the fact that no changes were seen in total counts of enter- sensitivity of media available. In future studies, quantification obacteriaceae, but can also be associated with the metabolic of bifidobacteria ought to be performed with molecular-based activity associated with proliferation (62). The clinical sig- nificance of antibiotic-induced endotoxin release remains to Looking at both intervention groups, specific changes dur- be clarified. There is evidence that endotoxemia may be of ing and after amoxycillin intake were observed, indicating an importance in patients with increased gut permeability and effect of amoxycillin intake on the gut microbiota. These re- that probiotics show potential in preventing loss of gut bar- sults are in line with the literature (47-50). The various effects rier integrity (66, 67). Some studies suggest that a reduction over time in the probiotic group compared to the placebo in intestinal endotoxin concentration may be associated with group suggest that the intake of the multispecies probiotic decreased endotoxin leakage across the gut wall, and sub- had an impact on the microbiota during amoxycillin intake, sequently with the control of endotoxin-related conditions possibly contributing to the better defecation score. This pro- (68). In our study, probiotic intake had no effect on intestinal biotic effect on the microbiota is partly caused by the bacteria themselves, as is reflected in the increase of enterococci in the The major SCFAs arising from the bacterial fermenta- probiotic group. In addition, the increase in, for example, the tion of nondigestible carbohydrates are acetic acid, propi- total anaerobic microbiota and the absence of a decrease in onic acid, and butyric acid. They serve as important energy the spore-forming clostridia during amoxycillin intake sug- sources (mostly butyric acid) for colonocytes, are associated gests that the probiotic bacteria were able to induce a change with the regulation of water and electrolyte transport, and de- in the intestinal environment favoring the growth of these crease colonic pH, thereby inhibiting overgrowth of potential commensal organisms. The fact that the differences between pathogens (69). In a study with 31 severe AAD patients dis- the groups were not significant is probably due to the high turbances in the intestinal microbiota were observed as was a reduction of the amounts of all major fecal SCFAs (70). SCFA Alteration of the colonic microbiota due to antibiotic treat- concentrations and anaerobic cultural counts also decreased ment can result in overgrowth of C. difficile in the colon. How- after systemic ceftriaxone treatment in 10 healthy volunteers ever, no increase in Clostridium spp. was observed in either (71). Probiotics, by interacting with the intestinal microbiota group during or after antibiotic therapy. Moreover, during and being saccharolytic, can alter SCFA concentrations in the antibiotic therapy Clostridium toxin A was not detected in colon. Studies have demonstrated different effects on SCFA the stool of any of the volunteers. This was to be expected concentrations after probiotic intake, with some showing no as the prevalence of C. difficile colonization among healthy effect (39, 43, 72-74), and others showing either an increase adults is very low, generally less than 2% (51). The spores (75, 76) or a decrease in specific SCFA concentrations (61).
of these bacteria are usually acquired from hospitals and Possible explanations for these inconsistent findings are the long-term-care facilities where the prevalence ranges from techniques applied, the populations studied, and the different Effect of a Multispecies Probiotic on the Intestinal Microbiota and Bowel Movements
probiotic strains used. In the present study, decreased acetic amoxycillin effect, which differed between the probiotic and acid and butyric acid concentrations were observed during the placebo group. Moreover, the intake of a multispecies pro- antibiotic treatment, only returning to baseline 7 wk after ces- biotic significantly reduced diarrhea-like bowel movements sation of antibiotic intake in the probiotic group. Moreover, in healthy volunteers receiving amoxycillin. Although the an increased propionic acid concentration was observed in the changes over time in microbial composition and metabolic probiotic group at day 7. In contrast, the main fermentation activity by themselves were small, the sum of potentially ben- products of the bacteria present in the multispecies probi- eficial changes may have contributed to the improved defe- otic are lactate, acetate, and formate (the latter only formed cation score observed. The present study therefore supports by bifidobacteria) and do not include propionate. In this re- the hypothesis that multispecies probiotics could be used in spect, metabolic cross-feeding is likely to have occurred as the prevention of AAD, as they affect the composition and lactate can be converted into butyrate or propionate. Which function of the intestinal microbiota.
metabolic pathway is utilized depends on the compositionof the microbiota as well as environmental conditions, and ACKNOWLEDGMENTS
shows high interindividual variation (77, 78). In general, theoverall SCFA concentration seemed to be higher in the probi- The authors would like to thank Ing. M. Hazen and L. Hoff- otic group, which could be one of the explanations for the less man for their technical assistance and B. Winkens, Ph.D., diarrhea-like defecation score in this group, due to a better for his help with the statistical analyses. This research was water and electrolyte absorption (79). It should be noted that funded by SenterNovem, an agency of the Dutch Ministry of only 1-5% of the amount of SCFAs produced is excreted in Economic Affairs (grant no. TSGE 1041).
the feces and that changes in SCFA concentration can be dueto both changes in production and/or absorption and altered STUDY HIGHLIGHTS
This study demonstrated that the intake of a multispecies What Is Current Knowledge
probiotic resulted in a significantly better defecation score r The disruption of the intestinal microbiota by an- (decrease in diarrhea-like bowel movements), which is in ac- tibiotics may result in antibiotic-associated diarrhea cordance with previous studies showing that probiotics sig- nificantly reduce the relative risk of developing AAD (26- r Clinical studies show that probiotics seem efficacious 28). Fecal consistency was estimated by the validated Bristol stool form scale. The significant correlation of the consis- r Beneficial probiotic effects differ per probiotic bacte- tency with both the amount of protein and fecal water per gram feces supports the validity of this scale.
During antibiotic intake a significant number of side ef- What Is New Here
fects was reported in both groups, but their numbers did not r Amoxycillin intake affects both the microbial compo- differ between the probiotic and the placebo group. These sition and metabolic aspects of the fecal microbiota.
results suggest that the multispecies probiotic that was able r The multispecies probiotic Ecologic AAD affects to decrease diarrhea-like defecation does not reduce other both the composition as well as the metabolic activ- gastrointestinal side effects, but also does not result in ad- ity of the fecal microbiota in healthy volunteers taking verse events. The clinical relevance of the improved defeca- tion score has to be further studied in specific patient popula- r The multispecies probiotic Ecologic AAD causes a tions who have an increased risk of AAD due to host factors small but significant reduction in diarrhea-like bowel (age, immune status), hospitalization status, and exposure to movements in healthy volunteers taking amoxycillin.
The compliance rates for both antibiotic and probi- otic/placebo intake were high in this study, although we read-ily admit that this was self-reported. We also acknowledge Reprint requests and correspondence: C.J.M. Koning, Division of
that many gastrointestinal bacteria remain uncultured and that Gastroenterology-Hepatology, University Hospital Maastricht, P.O.
Box 5800, 6202 AZ Maastricht, The Netherlands.
molecular-based techniques would allow a more complete Received December 15, 2006; accepted August 5, 2007. assessment of microbial diversity. However, culture providesinformation on quantitative alterations in viable counts of REFERENCES
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CONFLICT OF INTEREST
Potential competing interests: Linda Mulder and Frans M.
Guarantor of the article: R.W. Stockbr¨ugger, M.D., Ph.D.
Rombouts are employees of Winclove Bio industries B.V.
Specific author contributions: Catherina J.M. Koning was
Catherina J.M. Koning is supported, in part, by a grant from the principal investigator. Catherina J.M. Koning and Daisy SenterNovem and, in part, by Winclove Bio Industries B.V.

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