Food and health

The effect of a multi-strain probiotic on the resistance toward Escherichia coli challenge in a randomized, placebo-controlled, double-blind intervention study

Abstract

Background:

Several probiotic strains have been shown to enhance human resistance to infectious disease. It is speculated that these strains may impose this effect by excretion of anti-microbial components, by competing with pathogens for intestinal nutrients and/or mucosal adhesion sites or modulating the immune system.

Objective:

A parallel, double-blind, placebo-controlled 4-week intervention was performed in healthy males, to study the effect of a blend of probiotic bacteria (Lactobacillus helveticus Rosell-52, Lactobacillus rhamnosus Rosell-11, Bifidobacterium longum ssp. longum Rosell-175) and a probiotic yeast (Saccharomyces cerevisiae var boulardii CNCM I-1079) on enterotoxigenic Escherichia coli (ETEC) challenge. Primary outcomes studied were fecal ETEC excretion and total fecal output per day.

Subjects/Methods:

Subjects were randomized to the probiotic (5 × 109 colony-forming units (CFUs); twice daily; n=30) or placebo group (twice daily; n=30). After 2 weeks, subjects were orally challenged with a live attenuated ETEC (3 × 109 CFU), previously demonstrated to induce mild, short-lived symptoms of a foodborne infection. Before and after ETEC challenge, subjects collected 24 h fecal samples. Compliance to study guidelines, stool consistency (Bristol Stool Score), stool frequency, and frequency and severity of gastrointestinal (GI) complaints were recorded by the subjects on a Daily Record Questionnaire.

Results:

ETEC challenge induced a significant increase in fecal ETEC excretion in both groups. However, a statistically significant increase in fecal output was only observed in the probiotic group. ETEC challenge resulted in a decrease in the percentage of fecal dry weight, and an increase in reported Bristol Stool Score, stool frequency and GI complaints. Dietary probiotics significantly decreased the percentage of fecal dry weight. In addition, ETEC increased C-reactive protein, total secretory Immunoglobulin A (IgA) and Immunoglobulin G Colonization Factor Antigen II.

Conclusion:

Dietary probiotics did not increase resistance to oral attenuated ETEC challenge in human subjects.

Introduction

In Europe, 10–25% of the population suffers from at least one foodborne infection per year. This number increases in travelers to tropical countries, with gastrointestinal (GI) diseases accounting for 33% of all illnesses.1 Most infections are self-limiting; however, these infections can be life-threatening in populations with reduced resistance (for example, young children, elderly or persons taking immunosuppressive drugs). Treatment of foodborne infections with antibiotics is usually ineffective, can result in antibiotic-associated diarrhea2 and may lead to antibiotic-resistant bacterial pathogens.3 For these reasons, it is important to search for alternative means to prevent these types of acute infections. Several probiotic strains have been shown to enhance human resistance to infectious disease.4, 5, 6 It is speculated that these strains may impose this effect by excretion of anti-microbial components, by competing with pathogens for intestinal nutrients and/or mucosal adhesion sites or modulating the immune system.4, 5, 6

Previous studies have shown that a probiotic yeast Saccharomyces cerevisiae var. boulardii can increase resistance to acute bacterial diarrheal disease.7, 8 It reduced the duration of non-specified diarrhea,9, 10 reduced the number of children with prolonged diarrhea11 and the number of days of hospitalization,10 and reduced the incidence of diarrhea in travelers.12 In an animal model of traveler’s diarrhea, a multi-strain probiotic product containing Lactobacillus helveticus Rosell-52, L. rhamnosus Rosell-11, Bifidobacterium longum ssp. longum Rosell-175 and the yeast S. cerevisiae var boulardii CNCM I-1079 was able to reduce the severity and duration of symptoms associated with enterotoxigenic Escherichia coli (ETEC). Furthermore, the investigators demonstrated that the combination of bacteria and yeast was more effective than just bacteria or yeast alone.13 As the efficacy of this multi-strain combination has not been evaluated in enterotoxigenic E. coli-induced travelers’ diarrhea in humans, the aim of the present double-blind, placebo-controlled, randomized parallel group study is to determine the effect of the multi-strain probiotic on the resistance to ETEC infection.

Materials and Methods

Subjects

The trial was approved by the Medical Ethics Committee of Wageningen University, the Netherlands (Protocol NL40301.081.12). It was conducted according to the Declaration of Helsinki and registered on clinicaltrial.gov as NCT01709266. Healthy men, age 18–55 years, were recruited by posters mounted in public buildings. The 144 men who responded received a comprehensive brochure of the study and an invitation to attend the study’s information meeting. Eighty-two subjects gave their written informed consent and were screened for eligibility by completing a general medical questionnaire (Figure 1). Subjects were excluded if they reported use of antibiotics, immunosuppressive drugs, antacids, laxatives or anti-diarrheal drugs in the last 3 months before the study, and pre- and probiotics in the last month before the study. Moreover, subjects currently or previously diagnosed with underlying diseases of the GI tract, or those who were lactose intolerant were excluded.

Figure 1
figure1

Flow diagram of study subjects.

Screening

A non-fasting blood sample and a fecal sample were obtained from eligible subjects to establish whether they had either a high antibody titer against IgG Colonization Factor Antigen II (CFAII) or detectable levels of ETEC in their feces. Sera were isolated from whole blood and analyzed for specific IgG against CFAII, a specific immunogenic epitope of ETEC, as described previously.14 In addition, a fresh fecal sample was obtained and analyzed for ETEC and total Lactobacilli, as described earlier.15

Men with either antibody titers against CFAII or detectable levels of ETEC in feces, were excluded from the present study.

Randomization and stratification

The study had two primary outcomes: fecal ETEC excretion and total daily fecal wet weight. Based on two-sided statistical testing for unpaired data, α=0.05 (chance on type-I error) and β=0.20 (chance on type-II error) and to compensate for dropouts, 30 subjects per group were included.

Subjects were stratified according to age, fecal Lactobacilli number and ETEC serum antigen titer (determined at screening), and randomly assigned to the probiotic or placebo group. Stratification and randomization was performed by a non-blinded person not involved in the study. The randomization code of each subject was kept in sealed envelopes and the code was broken after finishing laboratory and statistical analyses.

Study design and dietary intervention

A 4-week parallel, double-blind, placebo-controlled ETEC-challenge study was performed. Subjects were instructed to maintain their usual physical activity level and their habitual diet and to abstain from dairy products and products with high amounts of prebiotic fibers and probiotics. As it has been previously shown that dietary calcium can decrease the efficacy of the ETEC challenge2 and because dairy products are rich in calcium, low-calcium soy custards were provided to subjects as a dairy replacement. During the study, subjects were asked to consume 125 g of soy milk custard twice daily at breakfast and dinner (Alpro Soya, Bio Nature; Gent; Belgium).

The multi-strain probiotic product, Protecflor, was supplied as capsules containing a powder with 5 × 109 CFUs of each of the following strains: L. helveticus Rosell-52, L. rhamnosus Rosell-11, B. longum ssp. longum Rosell-175 and the yeast S. cerevisiae var boulardii CNCM I-1079. The placebo consisted of a capsule of identical appearance composed of the same material (hydroxypropyl-methylcellulose and titanium oxide) and excipients (potato starch, magnesium stearate and ascorbic acid) as the probiotic capsule without the probiotic. Subjects were requested to ingest one capsule twice daily, at breakfast and dinner for the entire 4-week supplementation period of the study. Probiotic dosage and intervention duration was based upon the meta-analysis of McFarland et al.5

Subject compliance to study guidelines

Through the use of online daily diaries, subjects were requested to record capsule intake, soy product intake and indicate whether they kept to the dietary guidelines. Additional compliance was determined by counting the remaining probiotic capsules at study end.

To verify whether subjects kept to the low-calcium dietary restrictions, fecal calcium was analyzed in freeze-dried feces before ETEC challenge (on study days −1 or −2 depending on availability of fecal samples), as described previously.14

Probiotic strains in feces

To verify probiotic persistence in the GI tract and subject compliance to capsule intake, fecal L. helveticus Rosell-52, L. rhamnosus Rosell-11 and B. longum Rosell-175 were quantified by quantitative PCR amplification using an Bio-Rad CFX384 Real-Time System (C1000-Thermal Cycles, Bio-Rad, Veenendaal, The Netherlands) and SYBR Green methodology (IQ SYBR Green Supermix; Bio-Rad; cat no 170-8886). Each sample was analyzed in three dilutions, 10-, 100- and 1000-fold. No template controls and DNA standards from bacterial strains were included on each plate. Primers are presented in Supplementary Table 1.

Quantitation of S. boulardii from stool samples was performed as described by Klein et al.16 In brief, fecal samples (on study days −1 or −2 before ETEC challenge depending on availability of fecal samples) were homogenized, diluted, applied in triplicate to Sabouraud Dextrose Agar containing gentamicin (1 mg/l) and ampicillin (1 mg/l):sulbactam (1 mg/l). The yeast was quantitated in duplicate by counting the CFU and correcting for the serial dilution after 72 h incubation at 37 °C. Colony morphology was checked using Candida albicans (NIZO1670) and S. cerevisiae (NIZO3928) as controls.

Oral ETEC challenge

Following 2 weeks of dietary supplementation (study day 0), 4-h fasted subjects underwent a supervised challenge with a live but attenuated oral ETEC strain at NIZO Food Research (The Netherlands) as described previously.14 The ETEC strain used (Strain E1392/75-2A) is an LT-/ST- CFA/II-positive variant of a previously enterotoxigenic O6:H16 LT’/ST’ strain.17 CFA/II-expressing strains are common in all regions of the world.18 The ETEC strain (3 × 109 CFU) was suspended in 100 ml diluted fruit juice (Roosvice; Koninklijke de Ruijter, Zeist, The Netherlands) set at physiologic pH and osmolarity. Five minutes preceding ingestion of ETEC, subjects drank 100 ml demineralized water containing 2 g sodium bicarbonate (Merck, Darmstadt, Germany) to neutralize gastric acid. Eating and drinking were permitted 1 h after the ETEC challenge.

Primary outcomes

Daily fecal wet weight

Before (on study days −1 and −2) and after ETEC challenge (on study days 1, 2, 3, 4 and 15), 24 h fecal samples were collected. Fecal samples were immediately frozen after defecation in mobile freezers at the subjects’ home. Samples were then transported to the laboratory under frozen conditions, weighed, homogenized by using a stomacher, and aliquots were stored at −20 °C for later analyses. Daily fecal wet weight was measured by weighing all collected stool samples between 0700 hours in the morning until 0700 hours the next day.

Fecal ETEC excretion

Before (on study days −1 and −2) and after ETEC challenge (on study days 1, 2, 3, 4 and 15), DNA was isolated from homogenized wet fecal samples using the QIAamp DNA stool mini kit (QIAgen Benelux, Venlo, The Netherlands). This was done according to the instructions of the manufacturer, with slight modifications; after addition of the lysis buffer, the microbes within the samples were disrupted by bead-beating, using 3 × 1.5 min at 5000 r.p.m. with zirconia/silica beads. Subsequently, ETEC were quantified by quantitative PCR amplification using an Bio-Rad CFX384 Real-Time System (C1000-Thermal Cycles) with Bio-Rad methodology (iTaq Universal Probes Supermix; Bio-Rad; cat no 172-5134). Each sample was analyzed in three dilutions, 10-, 100- and 1000-fold. No template controls and DNA standards from bacterial strains were included on each plate. Primers and probe are given in Supplementary Table 1.

Secondary outcomes

Bowel habits, GI symptoms and diarrhea severity

During the entire study subjects reported information on stool consistency by using the Bristol Stool Scale19 in an online subject diary (LimeSurvey, An Open Source survey tool, LimeSurvey Project, Hamburg, Germany; http://www.limesurvey.org). The Bristol Stool Scale allows subjects to classify stool form into seven types ranging from ‘separate hard lumps like nuts’ (type 1) to ‘watery, no solid pieces, entirely liquid’ (type 7). Moreover, stool frequency (number of bowel movements per day) and frequency and severity of symptoms (flatulence, abdominal bloating, abdominal pains, abdominal cramps, nausea and headache) were self-recorded daily in an online subject diary, using Visual Analogue Scales (VAS) that ranged from absent (VAS 0) to maximum severity (VAS 100).15

Diarrhea was defined by daily total fecal wet weight excretion (primary outcome), the percent fecal dry weight determined after freeze-drying and the Bristol Stool Score (secondary outcomes).

Adverse events

Adverse events (AEs) were established by the medical investigator on basis of: (i) Answers to the daily open question: ‘Daily Health remarks’, and (ii) Questionnaires (VAS>50 on abdominal cramps, abdominal pain, bloating, flatulence, headache and nausea and reporting of vomiting and fever).

Tertiary outcomes

Immune response

Fecal sIgA was measured from supernatants of the fecal homogenates on study day −2 (before ETEC challenge), study day 3 and study day 4 after ETEC challenge. Briefly, homogenates were centrifuged (16 000 g, 5 min) and the supernatants were collected. Concentrations of sIgA were determined by ELISA with Human IgA ELISA Quantitation Kit E80-102 (Bethyl Laboratories Inc., Montgomery, TX, USA) according to the manufacturer’s instructions.

Blood samples (10 ml) were taken by qualified staff of a local hospital at one time point before (on study day −4) and on two time points after (on study days 3 and 15) ETEC challenge. Sera were isolated from whole blood by low-speed centrifugation (20 min at 3000 g and at 10 °C) and stored at −80 °C. Serum C-reactive protein was measured by kinetic determination by photometric measurement at 546 nm of the antigen–antibody reaction between antibodies to human C-reactive protein bound to polystyrene particles and C-reactive protein present in the sample (Hospital Gelderse vallei, Ede, the Netherlands). Specific IgG against CFAII in sera was quantified by direct ELISA as described elsewhere.14

Data and statistical analysis

Intention-to-treat analysis was performed for all outcomes and for all eligible subjects who were orally challenged with ETEC. Continuous variables are presented as means with standard errors of the mean (s.e.m.). The parameters were analyzed using general linear mixed models, linear mixed models or linear models depending on the distribution and amount of available data. In some cases, data were transformed before modeling using, for example, logarithmic or square root transformation to obtain a good model fit to the data. The models had terms for time point, dietary treatment and their interaction. In most cases, also a covariate for the baseline value was used. The mixed models had a random subject-wise intercept term. Hypotheses were tested using contrasts and in the case of multiple comparisons the P-values were adjusted to avoid false-positive findings. The analyses were conducted with R: A Language and Environment for Statistical Computing (version 3.0.1; R Development Core Team, Vienna, Austria). The linear mixed models were computed using R package name: Linear and Nonlinear Mixed Effects Models (version 3.1–109; J. Pinheiro, D. Bates, S. DebRoy, D. Sarkar and R Development Core Team, Vienna, Austria). The general mixed models were computed using R package MASS (Modern Applied Statistics with S. W. N. Venable and B. D. Ripley, Fourth Edition, Springer, New York). The differences between means before versus after ETEC challenge and between means of the probiotic group versus the placebo group were tested two-sided for all study outcomes. For both, P-values <0.05 are considered statistically significant.

Results

Baseline subject characteristics and subject compliance

Baseline subject characteristics are presented in Table 1. Subjects were stratified by age, fecal Lactobacilli counts and serum CFAII titer. No ETEC were detected in fecal samples in any of the subjects, neither at screening nor at day −2 and −1 (before ETEC challenge). The subjects in the dietary groups did not differ in body mass index.

Table 1 Baseline characteristics of the subjectsa

Compliance with the dietary instructions was checked by analysis of calcium excretion in feces at day −2 0r −1 before ETEC challenge. Daily fecal calcium excretion was 523±57 mg/day in the placebo group and 551±40 mg/day in the probiotic group. No difference in total daily fecal calcium excretion was found between the probiotic and placebo group.

Before ETEC challenge (at day −2 or −1), following 2 weeks of supplementation, all subjects in the probiotic group had detectable levels of one or more of the probiotic strains L. helveticus Rosell-52, L. rhamnosus Rosell-11, B. longum ssp. longum Rosell-175 and S. cerevisiae I-1079. In the placebo group, four subjects had detectable levels of S. cerevisiae and eight other subjects had detectable levels of B. longum (Supplementary Table 2).

Daily fecal wet weight

When comparing fecal wet weight before ETEC challenge, a significant increase was observed the first day following the challenge in the probiotic group (pre-challenge: 214±19 g feces/day; day 1: 339±33 g feces/day), and a non-significant increase was observed in the placebo group (pre-challenge: 198±16 g feces/day; day 1: 264±30 g feces/day; effect ETEC at day 1: P=0.0015 for probiotic and P=0.07 for placebo; Figure 2a). However, when comparing total daily fecal wet weight excretion on day 1, there were no significant differences between the placebo and probiotic group. Daily fecal wet weight returned to baseline values after day 2 post-ETEC challenge in both groups.

Figure 2
figure2

Effect of dietary probiotics on the ETEC-induced increase in total fecal wet weight (a) and percentage of fecal dry weight (b). Subjects were orally challenged with 3 × 109 colony-forming units (CFUs) of ETEC on day 0. Total daily fecal wet weight was determined by collecting 24-h stool samples and % fecal dry weight was determined by freeze-drying. Boxplots are depicted using mean (middle horizontal line), s.e.m. (box) and 95% confidence interval of mean (vertical whisker lines). *Significantly different from placebo. #Significantly different from pre-ETEC challenge value (n=22–28 samples per group).

Daily fecal dry weight

Oral ETEC challenge also resulted in a significant decrease in relative fecal dry weight, an objective measure of stool consistency, directly after the oral ETEC challenge compared with the relative fecal dry weight before the oral ETEC challenge. The percentage of fecal dry weight in the placebo group (pre-challenge: 25.1%±0.6; day 2: 24.2%±1.0) returned to normal values on day 2, whereas the values were still decreased in the probiotic group (pre-challenge: 25.0%±0.7; day 2: 20.9%±1.3; effect probiotic at day 2: P=0.029; effect ETEC at day 1: P<0.0002 for probiotic and P<0.0001 for placebo; Figure 2b).

Fecal ETEC excretion

As anticipated, fecal ETEC was detected in all subjects on the first day after ETEC challenge in both the placebo (day 1: 11.3±0.11 log copy10/day ) as well as the probiotic group (day 1: 11.5±0.10 log copy10/day). ETEC gradually decreased the first few days after challenge. At day 15, ETEC numbers were either near or below detection limit in all fecal samples. No differences between the placebo and probiotic group were detected in ETEC excretion (Figure 3).

Figure 3
figure3

Effect of dietary probiotics on fecal ETEC excretion with time (log10 copies/day). Subjects were orally challenged with 3 × 109 colony-forming units (CFUs) of ETEC on day 0. Fecal ETEC excretion was determined by quantitative PCR. Boxplots are depicted using mean (middle horizontal line), s.e.m. (box) and 95% confidence interval of mean (vertical whisker lines).

Reported bowel habits

Oral ETEC challenge resulted in a significant increase in reported Bristol Stool Score the first day after oral ETEC challenge (effect ETEC day 1: P<0.001 for both probiotic and placebo). Bristol Stool Scores one day before ETEC challenge were 4.0±0.1 for the probiotic and 4.2±0.2 for the placebo group. Average Bristol Stool Scores at day 1 after ETEC challenge were 5.2±0.2 for the probiotic and 5.4±0.2 for the placebo group (Figure 4a). ETEC also induced an increase in stool frequency in the probiotic group and a non-significant increase was observed in the placebo group (effect ETEC day 1: P<0.001 for probiotic and P=0.075 for placebo). Average stool frequency at day 1 before ETEC challenge was 1.2±0.1 stools/day for the probiotic group and 1.1±0.1 stools/day for the placebo group. Average stool frequency at day 1 after ETEC challenge was 2.0±0.2 stools/day for the probiotic group and 1.7±0.2 stools/day for the placebo group (Figure 4b). No differences between the placebo and probiotic group were detected in Bristol Stool Score or stool frequency.

Figure 4
figure4

Effect of dietary probiotics on stool consistency (a) and stool frequency (b). Subjects reported stool consistency and stool frequency by using the Bristol Stool Score in a daily online subject diary. The Bristol Stool Score allows subjects to classify stool form into seven types ranging from ‘separate hard lumps like nuts’ (type 1) to ‘watery, no solid pieces, entirely liquid’ (type 7). Subjects were orally challenged with 3 × 109 colony-forming units (CFUs) of ETEC on day 0. Boxplots are depicted using mean (middle horizontal line), s.e.m. (box) and 95% confidence interval of mean (vertical whisker lines). #Significantly different from pre-ETEC challenge value (n=30 per group).

GI symptoms and AEs

For GI symptoms, when comparing the severity of the symptoms before the ETEC challenge to post-challenge (day 1), the oral ETEC challenge induced a significant increase in abdominal bloating in both groups (effect ETEC day 1: P<0.00001 for both probiotic and placebo), abdominal pain (effect ETEC day 1: P<0.001 for both probiotic and placebo) and abdominal cramps (effect ETEC day 1: P<0.001 for both probiotic and placebo). All these ETEC challenge-induced effects peaked at day 1 and 2 after the oral ETEC challenge. No differences between the placebo and probiotic group were detected in GI symptoms (Table 2).

Table 2 Effect of dietary probiotics on reported symptoms, sIgA, C-reactive protein and IgG CFAII with timea

Expected AEs reported the first 2 days after ETEC challenge included abdominal cramps (67% of subjects), abdominal pain (62% of subjects), abdominal bloating (72% of subjects), flatulence (87% of subjects), headache (43% of subjects), nausea (38% of subjects) and vomiting (7% of subjects).20 No serious AEs were reported during the study. AEs (other than reported through the GI symptom questionnaire) were common cold (7% of subjects), flu and migraine (2% of subjects).

Immune response

Fecal secretory IgA and serum C-reactive protein were both shown to increase after the challenge with highest concentrations 3–4 days after the ETEC challenge (effect ETEC on sIgA day 3: P=0.0041 for probiotic and P=0.30 for placebo; effect ETEC on CRP day 3: P<0.00001 for both probiotic and placebo). No differences between the placebo and probiotic group were detected in secretory IgA and C-reactive protein. Serum CFA II-specific IgG also increased significantly after ETEC challenge, at day 15 (effect ETEC on IgG day 15: P<0.00001 for both probiotic and placebo). No differences between the placebo and probiotic group were detected in CFAII-specific IgG (Table 2).

Discussion

Travelers’ diarrhea is the most common health impairment in persons visiting developing countries affecting up to 50–90% of travelers in high-risk areas.21 ETEC is the leading bacterial cause of diarrhea in the developing world, as well as the most common cause of travelers' diarrhea.22 ETEC infection is characterized by profuse and watery diarrhea lasting several days with abdominal cramp, malaise, vomiting and low-grade fever.23 Although it is rare in developed countries and usually benign, travelers' diarrhea represents a considerable socioeconomic burden for both the traveler and the host country.21 For these reasons, much effort has been dedicated to finding a way of preventing such ailment.22

As several probiotic strains have been shown to enhance human resistance to infectious disease4, 5, 6 by excretion of anti-microbial components, by competing with pathogens for intestinal nutrients and mucosal adhesion sites, by strengthening gut barrier integrity and/or by modulating the immune system, the aim of the present study was to investigate whether a probiotic could increase resistance to ETEC. The oral ETEC challenge has been used frequently to elucidate the pathogenesis and immune responses involved with this infection and assess efficacy of various interventions.20 The ETEC-challenge model has been successfully used to study the efficacy of dietary components such as calcium and probiotics on ETEC diarrhea in healthy adults.14, 24

All clinical trials must weigh the ethical dilemmas involved with putting human subjects at risk against the potential benefit of a product designed to treat or prevent disease. The basic concept of the current ETEC challenge study is that we have selected a well-characterized, antibiotic susceptible organism that has been associated with very mild diarrhea and GI symptoms (severity and duration). Moreover, the ETEC strain used (Strain E1392/75-2A) is an LT-/ST- CFA/II-positive variant of a previously enterotoxigenic O6:H16 LT'/ST' strain. Under close supervision, the strain is given to subjects at a dose that induces mild and short-lived symptoms, with complete recovery of reported clinical symptoms within 2 days. Subsequently, subjects are daily monitored throughout the entire study. All recorded disease episodes were self-limiting and did not require early antibiotic treatment.

As shown in previous studies, the model transiently induces symptoms of a foodborne infection increasing fecal pathogen excretion, stool frequency, Bristol Stool Score, reported symptoms, secretory IgA, C-reactive protein, calprotectin and antibody response.14, 20, 24 The attenuated ETEC strain used in the present study induces a small increase in average stool frequency from 1 stool/day to 2 stools/day, and an increase in average daily Bristol Stool Score from 4 to 5. Types 1–2 on the Bristol Stool Scale indicate constipation, with 3 and 4 being the ideal stools (as they are easy to defecate while not containing any excess liquid), and 5–7 tending toward diarrhea.

In addition, the infective dosage of ETEC in the present study was lower (3 × 109 CFU instead of 1 × 1010 CFU) than previously used.14, 24 Analogous, to previous studies, ETEC infection also showed a significant increase in the fecal ETEC excretion. However, the present study failed to reveal a significantly increased daily fecal output in the placebo group, which was a primary study outcome. The increase in fecal output measured in the present study was on average 1.6- and 1.3-fold in the probiotic and placebo group, respectively. This is in clear contrast with previous ETEC challenge studies14, 24 where the fecal output increased significantly by 1.7- to 2-fold on average during the first day after ETEC infection. This difference may be explained by the lower inoculation dose of ETEC used in the present challenge study.

No protection was provided by the probiotic group regarding bowel habits and GI symptoms post-ETEC challenge. A significant increase in fecal output on the first day after ETEC challenge was only observed in the probiotic group. Moreover, dietary probiotics significantly decreased relative fecal dry weight the second day after ETEC challenge. However, these differences did not coincide with self-reporting parameters such as Bristol Stool Score and GI symptoms. The effects of dietary probiotics observed in the present study are in contrast to previous reported studies on traveler’s diarrhea.12, 13

This discrepancy in observed physiological effects might be explained by the mechanism by which probiotics interact with bacterial gut pathogens. It is commonly assumed that competing with pathogens for niches and nutrients ‘competitive exclusion’ is one of the mechanisms by which probiotics may exert their protective effect against gut pathogens.25 However, the results of the present study show that intestinal colonization of the ETEC strain used in the present study was not affected by dietary probiotics, which was illustrated by the unaltered fecal ETEC excretion in the probiotic group compared with the placebo group. It is probable that the positive probiotic effect observed in the past may have been due to other mechanisms than competitive exclusion. Toxin breakdown by proteases,26 binding of toxins27 or inhibition of toxin production28 by probiotics might be an alternative mechanistic explanation. However, as the ETEC strain used in this study does not produce heat-labile (LT) and/or heat-stable (ST) enterotoxins,17 this potential mechanism could not be evaluated in the present challenge model.

In addition, the ETEC strain employed in the study may require another immune-modulatory capacity, such as toxins, for dietary probiotics to suppress disease symptoms. For example, tolerogenic immunomodulation by dietary probiotics may be beneficial to suppress excessive immunological responses to toxin-induced mucosal damage. However, when the disease mechanism is dependent on ETEC colonization and toxin independent (as in the present study), the tolerogenic potential of a probiotic may be ineffective rather than beneficial.

The probiotic combination was well tolerated as no AEs were associated with probiotic intake. The microbes in the combination survived intestinal transit and could be found in high numbers in the fecal samples of the participants during intervention.

In conclusion, the primary objectives of this study were to compare the effects of a blend of probiotic bacteria on total fecal output and fecal ETEC excretion with respect to placebo. Administration of the live but attenuated oral ETEC vaccine induced a significant increase in fecal output only in the probiotic group. However, this diet-induced difference did not coincide with differences in self-reporting parameters such as Bristol Stool Score and GI symptoms. A significant increase in fecal ETEC excretion was observed in both the probiotic and the placebo group. Supplementation of probiotics did not seem to provide benefits in this model in reducing ETEC challenge symptoms in otherwise healthy men. Additional studies should examine the effect of dietary probiotics on ETEC strains that produce heat-labile (LT) and/or heat-stable (ST) enterotoxins.

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Acknowledgements

E Lucas, R Holleman, J Hoolwerf and S van Schalkwijk (NIZO Food Research, Ede, the Netherlands) are thanked for their support of study logistics, and assistance with laboratory analyses. M Kleerebezem (NIZO Food Research, Ede, the Netherlands) is thanked for critically reviewing the manuscript and E Alhoniemi (Pharmatest, Finland) is thanked for performing the statistical analysis. The study was funded by Lallemand Health Solutions Inc., Montreal, Canada.

Author Contributions

All authors have read and approved the final manuscript. SB, RB and TT designed the study. SB and EF conducted the study and analyzed the data. SB, SG, TT and RB wrote the paper. SB and TT had primary responsibility for final content.

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Correspondence to S J M Ten Bruggencate.

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Competing interests

SB and EF declare no conflict of interest. SG and TT are employed by Lallemand Health Solutions Inc., Montreal, Canada, producer and marketer of Protecflor. RB is a consultant to Lallemand Health Solutions Inc.

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Disclosure

The sponsor was involved in the design of the study, interpretation of the data and writing of the manuscript. No external funding, apart from the authors’ institutions, was available for this study.

Registration

The trial was registered on ClinicalTrials.gov, NCT01709266.

Supplementary Information accompanies this paper on European Journal of Clinical Nutrition website

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Ten Bruggencate, S., Girard, S., Floris-Vollenbroek, E. et al. The effect of a multi-strain probiotic on the resistance toward Escherichia coli challenge in a randomized, placebo-controlled, double-blind intervention study. Eur J Clin Nutr 69, 385–391 (2015). https://doi.org/10.1038/ejcn.2014.238

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