A Meta-Analysis of Probiotic Efficacy for Gastrointestinal
Marina L. Ritchie*, Tamara N. Romanuk
Department of Biology, Dalhousie University, Halifax, Nova Scotia, Canada
Background: Meta-analyses on the effects of probiotics on specific gastrointestinal diseases have generally shown positive
effects on disease prevention and treatment; however, the relative efficacy of probiotic use for treatment and prevention
across different gastrointestinal diseases, with differing etiology and mechanisms of action, has not been addressed.
Methods/Principal Findings: We included randomized controlled trials in humans that used a specified probiotic in the
treatment or prevention of Pouchitis, Infectious diarrhea, Irritable Bowel Syndrome, Helicobacter pylori, Clostridium difficile
Disease, Antibiotic Associated Diarrhea, Traveler’s Diarrhea, or Necrotizing Enterocolitis. Random effects models were used
to evaluate efficacy as pooled relative risks across the eight diseases as well as across probiotic species, single vs. multiple
species, patient ages, dosages, and length of treatment. Probiotics had a positive significant effect across all eight
gastrointestinal diseases with a relative risk of 0.58 (95% (CI) 0.51–0.65). Six of the eight diseases: Pouchitis, Infectious
diarrhea, Irritable Bowel Syndrome, Helicobacter pylori, Clostridium difficile Disease, and Antibiotic Associated Diarrhea,
showed positive significant effects. Traveler’s Diarrhea and Necrotizing Enterocolitis did not show significant effects of
probiotcs. Of the 11 species and species mixtures, all showed positive significant effects except for Lactobacillus acidophilus,
Lactobacillus plantarum, and Bifidobacterium infantis. Across all diseases and probiotic species, positive significant effects of
probiotics were observed for all age groups, single vs. multiple species, and treatment lengths.
Conclusions/Significance: Probiotics are generally beneficial in treatment and prevention of gastrointestinal diseases.
Efficacy was not observed for Traveler’s Diarrhea or Necrotizing Enterocolitis or for the probiotic species L. acidophilus, L.
plantarum, and B. infantis. When choosing to use probiotics in the treatment or prevention of gastrointestinal disease, the
type of disease and probiotic species (strain) are the most important factors to take into consideration.
Citation: Ritchie ML, Romanuk TN (2012) A Meta-Analysis of Probiotic Efficacy for Gastrointestinal Diseases. PLoS ONE 7(4): e34938. doi:10.1371/
Editor: Markus M. Heimesaat, Charite ´, Campus Benjamin Franklin, Germany
Received November 10, 2011; Accepted March 11, 2012; Published April 18, 2012
Copyright: ? 2012 Ritchie, Romanuk. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This research was supported by a National Sciences and Engineering Research Council of Canada (NSERC) Discovery Grant to Tamara Romanuk. The
funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing Interests: The authors have declared that no competing interests exist.
* E-mail: firstname.lastname@example.org
The efficacy of using probiotics in the prevention and treatment
of gastrointestinal diseases has received considerable attention in
recent years [1–5]. In western civilization, there has been an
increase in gut-related health problems, such as autoimmune and
inflammatory diseases . Changes in the gut flora have emerged
as a leading mechanism for the increased prevalence of certain
gastrointestinal diseases [6–8]. Due to improved hygiene and
nutrition, the western human diet contains several thousand times
less bacteria than pre-industrialized diets [6,9]. This is partially
due to the use of processed and sterile foods which contain
artificial sweeteners and preservatives, rather than fresh fruits and
vegetables , or foods containing important microbes for anti-
inflammatory processes [11,12].
Probiotics, products or preparations containing sufficient
amounts of viable microorganisms to alter a host’s microflora
communities , are thought to exert beneficial effects by
providing protective barriers, enhancing immune responses, and
clearing pathogens in the gastrointestinal tract [14–16]. Meta-
analyses or clinical trials on the efficacy of probiotics have been
conducted for a number of common gastrointestinal diseases
including Irritable Bowel Syndrome (IBS) , Helicobacter pylori
infection (HPP) , Necrotizing Enterocolitis (NEC) ,
Pouchitis (Pouch) , Antibiotic Associated diarrhea (AAD)
, Clostridium difficile Disease (CDD) , Infectious diarrhea
(ID) , and Travellers diarrhea (TD) . These studies have
shown that probiotics have significant effects on the prevention
(e.g. ) and treatment (e.g. ) of gastrointestinal disease. While
numerous meta-analyses have been performed on the use of
probiotics in the prevention and treatment of specific diseases (e.g.
[3,5,17]), to our knowledge, a meta-analysis comparing the
efficacy of probiotics across various diseases has not been
conducted. Probiotics have been used to prevent and treat a wide
range of GIT diseases. The GIT diseases considered here can be
grouped into three groups based on symptomology: 1) production
of diarrhea: AAD, CDD, ID, TD, 2) the destruction or
inflammation of tissues in the stomach, large intestine, ileal
reservoir, or bowel: NEC, Pouch, and HPP, 3) abdominal pain,
flatulence, and irregular bowel movements: IBS. The etiology of
re-occurring and chronic inflammation in the gastrointestinal tract
is not definitive . Nevertheless, evidence suggests that an
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imbalance of intestinal bacteria may commence and perpetuate
the inflammation that characterizes the gastrointestinal diseases
related to chronic and re-occurring inflammation [22–24].
Furthermore, pathogenic bacteria can invade tight junctions
between epithelial cells and disturb the barrier function of the
gut, resulting in translocation of pathogenic bacteria that leads to
an inflammatory immune response .
Previous studies have shown probiotic efficacy in treating
[26,27,28]. The primary mechanisms of action of probiotics are
modification of the gut microflora , stabilization of the
indigenous microflora , reductions in the duration of retrovirus
shedding , and a reduction in increased gut permeability
which is caused by retrovirus infection . In diarrhea-related
diseases, probiotics may induce a general immune response, in
addition to increasing IgA antibodies against rotavirus [32,33]. In
inflammatory-related disease, probiotics are thought to decrease
disease activity and promote remission . Reductions in
inflammation are thought to occur by decreasing pathogenic
bacterial growth through the enhancement of barrier functions
which prevents the invasion of tight junctions, by lowering gut pH,
and by stimulating non-specific and specific immune responses
. IBS has been correlated with a lower amount of Lactobacilli
and Bifidobacterium colonies and an increase in anaerobic Clostridium
spp. which has taken place of anaerobic Bifidobacterium spp. and
Bacteriodes spp. [35,36]. Therefore, there are links between humans
consuming lactose and sucrose with an onset of IBS , which is
thought to be caused by providing the pathogenic microbial
population with a nutritional source . As a result, probiotics
such as L. plantarum , and Enterococcus faecum  have been
used to treat IBS because they compete for the same food source.
Not all these mechanisms of action will apply to all the GIT
diseases considered here, thus by comparing probiotic efficacy
across diseases it may be possible to assess the specific functional
responses by which probiotics are operating.
Here we report on a meta-analysis designed to determine
whether probiotics are more or less effective in the prevention and
treatment of eight different gastrointestinal diseases across 11
species or species mixtures of probiotics. We further assessed
whether patient age, dose, length of treatment, and single vs.
multiple probiotic species affect efficacy as previous studies have
shown differences in probiotic efficacy based on these factors.
The objectives of this meta-analysis were to: (i) determine the
overall effect of probiotics on diseases of the gastrointestinal tract
that have previously been shown to be affected by probiotics, (ii)
determine whether certain diseases respond to probiotics more
than others (iii) determine whether different species and species
combinations differed in their overall effect size, and to (iv)
determine whether efficacy differs based on dosage, length of
treatment, and age group.
Search Strategy and Study Selection
The supporting PRISMA checklist is available as supporting
information; see Checklist S1. We conducted a literature search
for randomized controlled efficacy trials in humans for probiotics
used in the prevention and treatment of gastrointestinal disease.
We searched Pubmed (January 1970 to January 2011), Medline
(January 1970 to January 2011), Google Scholar (January 1970 to
January 2011), Embase (January 1970 to January 2011), Biological
Abstracts (January 1970 to January 2011), and Science Direct
(1970 to January 2011). Search terms included: probiotics,
probiotic meta-analysis, Gastrointestinal disease, Diarrhea, Helico-
bacter pylori, Pouchitis, Antibiotic Associated Diarrhea, Irritable
Bowel Syndrome, Travellers Diarrhea, Clostridium difficle Disease,
Necrotising Enterocolitis, Infectious Diarrhea, yogurt, Lactobacillus,
Bifidobacterium, Saccharomyces, Streptococcus, Enterococcus, randomized
control trials, controlled trials, placebo, and control. Searches were
not restricted by language and secondary searches were done by
reference lists, authors and reviews (e.g. Appendix S1). Excluded
trials included case reports or case series, trials of unspecified
probiotics, trials on prebiotics, trials with inconsistent outcome
measures, trials with no specific disease being studied, and trials on
animals other than humans. Eligibility criteria included: random-
ized controlled trials published in peer-reviewed journals, humans
with gastrointestinal disease (AAD, CDD, HPP, IBS, ID, NE,
Pouch, TD), and studies that compared probiotic therapy with
placebo or no therapy. After excluding trials that did not fit the
criteria, a total of 84 suitable trials were identified for analysis
spanning 10,351 patients, 11 probiotic species or mixtures, and
eight diseases. Of the 84 suitable trials that are analyzed in this
meta-analysis, 79 have been cited in meta-analyses on their
specific disease [1–5,13,17,18, and 20].
The primary outcome assessed was prevention in overall
symptoms or treatment of the gastrointestinal diseases. Here we
use prevention and treatment interchangeably when discussing the
effects of probiotics across all diseases as for some diseases (i.e.
CDD; ) probiotics are effective in both prevention and
treatment. For other diseases, probiotics have only shown to have
efficacy in either prevention or treatment. For example, probiotics
are used in the prevention of diarrhea  and in the treatment of
IBS . The outcomes for the efficacy of the eight gastrointestinal
diseases are shown in Table 1.
Data extraction and risk of bias
From each paper we extracted information related to disease,
probiotic species, the dose amount, treatment length, age group,
number of trials, number of patients receiving the probiotic or the
control, and the number of patients that improved following
probiotic/control. A few studies had multiple probiotic treatments
with a common control group and were analyzed separately.
One author (Ritchie) independently reviewed and assessed
inclusion criteria and quality of trials. Each included study was
assessed using a 5-point Jaded scale  based on randomization,
concealment of allocation, blinding of investigators, including
outcome assessors, and completeness of follow-up. Inconsistencies
were resolved by discussion of the authors. Weights for the meta-
analysis are based on sample sizes.
Data synthesis and statistical analysis
A random effects meta-analysis was conducted with inverse
variance weighting using the software MIX version 2.0 Pro .
For each paper the relative risk ratio (RR), which is the ratio of the
probability of the event occurring in the probiotic treatment versus
the control group , was calculated along with 95% confidence
intervals, and summary statistics. Overall RR, heterogeneity (I2), z-
values, and p-values were computed across all studies and for each
comparison. If significant heterogeneity (I2) occurred (p,0.05) the
studies were analyzed using a random effects model with a pooled
relative risk. If the studies were not significant (p.0.05) they were
analyzed using a fixed effect model with a pooled relative risk.
Effect sizes (RR values) that were ,1 favoured the probiotic while
effect sizes that were .1 favoured the placebo. If the 95%
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confidence intervals of effect sizes do not overlap, the RR is
considered significantly different. Publication bias was assessed by
funnel plot asymmetry . Risk ratios were plotted against the
standard error of the risk ratio of each study to identify asymmetry
in the distribution of trials. Potential publication bias is suggested
when there is a gap in the funnel plot. Begg’s regression test was
also used to assess potential publication bias . The Fail safe N-
Method defined as, ‘‘the number of new, unpublished, or un-
retrieved non-significant or ‘‘null result’’ studies that would be
required to exist to lower the significance of a meta-analysis to
some specified level’’  was also used for bias analysis.
Six different factors were included in the meta-analysis: the
disease treated with probiotics (AAD, CDD, IBS, ID, TD, NEC,
Pouch, and HPP), the type of probiotic used (VSL#3, LGG, S.
boulardii, B. infantis, L. acidophilus, L. casei, C. butyricum, E. faecum, L.
plantarium, B. lactis and L. acidophilus combined with B. infantis, the
dose of the probiotic (1–961011, 1012CFU/day; 1–5.56106, 107,
108CFU/day; 1–96109CFU/day; 1–561010CFU/day), the
amount of time the probiotic was administered for (9–240 weeks,
5–8 weeks, 3–4 weeks, 1–2 weeks), the age group of the subjects
receiving probiotics (infants (0–3 yrs), children (3#18 yrs), adult-
s(.18 yrs)) and single versus multiple species of probiotics
Overview of included studies
The literature search yielded 2,420 citations, of which 220 were
screened and 80 were assessed for eligibility. Of these, 6 were
excluded for various reasons (Figure 1), leaving 74 studies that met
the inclusion criteria. Therefore, 84 peer-reviewed trials were
included in the meta-analysis. All trials included in this meta-
analysis had a Jaded quality score of 3 or more, except for 4 of
them which had a score of 2 due to unavailable information
(Materials S1). The median number of patients per trial was 88.5
ranging from 15–756. In total, 10,351 subjects were included in
the studies. Of the 84 trials, 31 (37%) showed a significant
reduction of GI diseases in the probiotic treated patients compared
with the control patients. 53 trials did not reject the null hypothesis
of no difference in the incidence of GI disease for probiotic verses
controls. The pooled estimate of efficacy of probiotics in
prevention or treatment of disease yielded a relative risk of 0.58
(95% CI 0.51–0.65; p,0.001) and a heterogeneity (I2) of 61.24%
(95% CI 51–69; X2p,0.001) showing that across all diseases and
probiotic species, probiotics were effective in the treatment and
prevention of GI diseases (Figure 2).
Effect by types of disease
RR=0.17; 95% CI 0.10–0.30), AAD (n=27; RR=0.43; 95%
CI 0.32–0.56), ID (n=3; RR=0.35; 95% CI 0.13–0.97), IBS
(n=16; RR=0.77; 95% CI 0.65–0.92), HPP (n=13; RR=0.70;
95% CI 0.54–0.91), and CDD (n=6; RR=0.60; 95% CI 0.41–
0.86) yielded significant effect sizes (Figure 3a). Significant effect
sizes were not observed for probiotics for the diseases TD (n=6;
RR=0.92; 95% CI 0.79–1.05) and NEC (n=9; RR=0.54; 95%
CI 0.23–1.24) (Figure 3a). Efficacy for Pouchitis was significantly
greater than for TD, IBS, HPP, CDD, and AAD. When
comparing the diseases that cause diarrhea to those that cause
tissue damage/inflammation and to IBS, no significant effect was
found (Figure 3a).
eight diseases considered,Pouchitis(n=4;
Effect by probiotic species
Across all diseases, eight species yielded significant effect sizes
including: VSL #3 which contains viable lyophilized bacteria of
four species of Lactobacillus (L. casei, L. plantarum, L. acidophilus, and
L. delbrueckii subsp. bulgaricus), three species of Bifidobacterium (B.
longum, B.breve, and B. infantis), and one species of Streptococcus
salivarius subsp. (n=3; RR=0.17; 95% CI 0.09–0.33), E. faecium
(n=2; RR=0.29; 95% CI 0.13–0.64), C. butyricum (n=2;
RR=0.18; 95% CI 0.09–0.37), L. acidophilus combined with B.
infantis (n=3; RR=0.37; 95% CI 0.17–0.83), B. lactis (n=3;
RR=0.59; 95% CI 0.38–0.92), LGG (n=14; RR=0.54; 95% CI
0.39–0.75), L. casei (n=3; RR=0.42; 95% CI 0.24–0.76) and S.
boulardii (n=11; RR=0.46; 95% CI 0.34–0.60) (Figure 3b). The
other three probiotic species (L. acidophilus, L. plantarum, and B.
infantis), did not show significant efficacy (Figure 3b). S. boulardii
showed significantly higher efficacy than L. plantarum and B.
Infantis. C. butyricum had significantly higher efficacy from the
species L. plantarum, L. acidophilus, LGG, L. plantarum and B. Infantis.
VSL #3 had significantly higher efficacy than the species S.
Table 1. List of primary outcomes for the eight gastrointestinal diseases analyzed in this meta-analysis.
Antibiotic Associated Diarrhea (AAD), Traveller’s Diarrhea (TD), and Infectious
The primary outcome for AAD, TD, and ID is defined as diarrhea (3 loose stools/day
for at least 2 days or 5 loose stools/48 h) within 2 months of antibiotic exposure.
Clostridium difficile Disease (CDD) The primary outcome of CDD is defined as a new episode of diarrhea associated with
a positive culture or toxin (A or B) assay within 1 month exposure to antibiotics. The
outcome of prevention of CDD is a new episode of C. difficle positive diarrhea within
1 month of a previous CDD episode.
Irritable Bowel Syndrome (IBS)The primary outcome measures was the improvement in overall symptoms as
defined by the presence or absence of the following physical symptoms: pain,
flatulence, bloating, anxiety, and quality of life or the change in symptom scores from
Helicobacter pylori (HPP)The primary outcome was the improvement of H. pylori eradication rates reducing
side effects with probiotics.
Necrotizing Enterocolitis (NEC)The primary outcome of efficacy of probiotic supplementation in prevention of stage
2 or greater Necrotizing Enterocolitis, and safety in terms of blood culture-positive
septis and any other adverse events reported by investigators.
Pouchitis (Pouch)The primary outcome of efficacy of probiotic supplementation was for the treatment
of Pouchitis with no relapse.
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boulardii, B. infantis, L. plantarum, LGG, B. lactis, and L. acidophilus
(Figure 3b). As L. acidophilus is one of the most common probiotics
we further considered whether differences in efficacy were
observed based on particular strains. We found that when
analyzed alone, L. acidophilis LB did show significant efficacy
(RR=0.40 95% CI 0.20–0.82) and L. acidophilus with no strain
specified did not have a significant effect (RR=1.17 95% CI 0.85–
Effects of age
Across all diseases and probiotic species, significant efficacy was
observed for all of the age groups studied (infants (n=9;
RR=0.41; 95% CI 0.27–0.62, children (n=14; RR=0.36; 95%
CI 0.24–0.55), and adults (n=53; RR=0.64; 95% CI 0.55–0.74)
(Figure 4a). None of the age groups were significantly different
from each other (Figure 4a).
Effects of Dose
Across all diseases and probiotics species, significant efficacy was
observed for three doses: 1–561010CFU/day (n=20; RR=0.51;
95% CI 0.39–0.65), 1–5.56106, 107, 108CFU/day (n=12;
RR=0.60; 95% CI 0.42–0.85), and 1–96109CFU/day (n=25;
RR=0.61; 95% CI 0.49–0.75) (Figure 4b). One dose (1–961011,
1012CFU/day, n=7; RR=0.73; 95% CI 0.46–1.15) did not have
significant efficacy (Figure 4b). None of the dose groups were
significantly different from each other (Figure 4b)
Effect of treatment length probiotic was administered
Subgroup analysis for length of treatment showed significant
efficacy for all of the four groups; 1–2 weeks (n=30; RR=0.53;
95% CI=0.42–0.68), 3–4 weeks (n=21; RR=0.78; 95% CI
0.68–0.89), 5–8 weeks (n=18; RR=0.64; 95% CI=0.51–0.82),
and 9–240 weeks (n=7; RR=0.27; 95% CI 0.14–0.54). The
longest treatment period (9–240 weeks) had significantly higher
efficacy than the 3–4 week treatment length group (Figure 4c).
Effects of single vs. multiple species
To determine whether number of species included in the
probiotic affected efficacy, single species probiotics were compared
to multiple species probiotics. No significant difference between
single and multiple species was observed (single species n=51;
RR=0.73; 95% CI 0.68–0.79, multiple species n=33; RR=0.63;
95% CI 0.53–0.76) (Figure 4d).
The funnel plot had an asymmetrical distribution (Figure 5).
The Egger regression test (p.0.0001) and the Begg rank
correlation test (p.0.0001) showed significant evidence of
publication bias. However, using the fail-safe N method, we
estimated that a total of 3,657 missing studies that would bring the
p-value greater than alpha, were required to overturn the current
results. The trim and fill method was used to correct for
publication bias and yielded an overall effect size of 0.73 (95%
CI 0.63–0.83), compared to the uncorrected overall effect size of
0.58 (95% CI 0.51–0.65).
Across all 11 probiotic species and the eight different
gastrointestinal diseases we found a significant effect of probiotics
on prevention and treatment of gastrointestinal disease with a
RR=0.58 (95% CI 0.51–0.65). Traveler’s Diarrhea and Necro-
tizing Enterocolitis and the species L. acidophilus, L. plantarum, and
B. infantis showed no efficacy. Previous meta-analyses that focused
on efficacy of probiotics in the prevention or treatment of specific
diseases have reported similar results. For example Johnston et al..
 reported a significant effect size (RR=0.43 95% CI 0.25–
0.75) for AAD disease, McFarland & Dublin  reported a
significant effect size (RR=0.78 95% CI 0.62–0.94) for IBS
disease, and Elahi et al..  reported a significant effect size
(OR=0.04 95% CI 0.01–0.14, p,0.0001) for Pouchitis.
Pouchitis (RR=0.17 95% CI 0.10–0.30) had the greatest effect
size of all the diseases analyzed and efficacy of probiotic treatment
for Pouchitis was significantly different than TD, IBS, HPP, CDD,
and AAD. Pouchitis occurs in 50% of patients with ulcerative
colitis after undergoing ileal pouch anal anastomosis (IPAA) .
Pouchitis is caused by inflammation of the ileal pouch that is
caused directly (toxins or invasions in the anal mucosa) or
indirectly (changes in fatty acids and bile salts) . A previous
meta-analysis on the prevention of Pouchitis in patients that have
undergone IPAA surgery showed that probiotics have a positive
effect on the prevention of Pouchitis . Recent evidence
proposes that bacteria play a primary pathogenic role in causing
inflammation in patients with Pouchitis [48–50]. Ruseler-van
Embden  found that individuals with Pouchitis have fewer
Lactobacilli and Bifidobacterium. Efficacy of probiotic treatment in
Pouchitis was significantly higher than efficacy for TD, IBS, HPP,
Figure 1. PRISMA (Preferred Reporting Items for Systematic
reviews and Meta-Analyses) flow diagram showing an over-
view of the study selection process.
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CDD, and AAD (Figure 3a). The high efficacy of probiotics we
observed in the treatment of Pouchitis may be due to a number of
factors related to trial design. For example, treatment of Pouchitis
was limited to VSL #3 and LGG and the patients in Pouchitis
trials were all adults.
ADD, ID, IBS, HPP, and CDD also had effect sizes that were
significant. AAD is present when an individual has three or more
abnormally loose bowel movements over a twenty-four hour
period following antibiotic use . HPP colonization is a
common health problem, especially in developing countries
[3,53], that causes chronic low-level inflammation in the stomach
lining and duodenum leading to the development of gastric and
duodenal ulcers, as well as stomach cancer . When treating
HPP, patients are prescribed antibiotics which results in some
individuals developing AAD. CDD, which is also associated with
antibiotic use, occurs mostly in older adults, and usually only
occurs in hospitalized patients . Probiotics are thought to
restore equilibrium in the gastrointestinal tract and protect against
C. difficile colonization. AAD, HPP colonization, and CDD are
associated with antibiotic treatment [3,4]. Probiotics are thought
to be a useful treatment in these diseases as they occur in part from
alterations of the intestinal microflora . ID is a type of acute
diarrhea that impairs intestinal absorption of nutrients and can
lead to malnutrition . IBS leads to abdominal pain, bloating,
diarrhoea, constipation, and flatulence due to motor and sensory
dysfunction of the gastrointestinal tract .
Our observation of significant efficacy for ADD, ID, IBS, HPP,
and CDD support other recent meta-analyses on specific GIT
diseases. McFarland  showed that AAD is preventable by
probiotics; McFarland & Dublin  demonstrated that probiotics
have a significant effect on the improvement of IBS, and Tong et
al..  suggested that probiotics could be effective in increasing
eradication rates of anti-H. pylori therapy. Although in the latter
study Tong et al..  showed that H. pylori eradication rates were
83.6% for patients with probiotics and 74.8% for patients without,
and thus suggested that larger trials were needed to confirm a
Figure 3. The effect size (risk ratio) for gastrointenstinal diseases and for probiotic species. (A) The effect size including the 95%
confidence intervals for the total events of Antibiotic associated diarrhea (AAD), Clostridium difficile disease (CDD), Helicobacter pylori positive (HPP),
Irritable bowel syndrome (IBS), Infectious diarrhea (ID), Necrotizing Enterocolitis (NE), Traveller’s diarrhea (TD), and Pouchitis during which probiotics
were taken. (B) The effect size including 95% confidence intervals for the type of probiotic species that were used to treat and prevent
gastrointestinal disease. The species that were used were VSL#3, Lactobacillus rhamnosus GG (LGG), Saccromyces boulardii, Bifidobacterium infantis,
Lactobacillus acidophilus, Lactobacillus casei, Clostridium butyricum, Enterococcus faecum, Lactobacillus plantarium, Bifidobacterium lactis and
Lactobacillus acidophilus combined with Bifidobacterium infantis. Risk ratios below one favor the probiotic while risk ratios above one favor the
Figure 2. The effect size (risk ratio) for the overall effects of probiotics in the prevention and treatment of gastrointestinal (GI)
diseases including the 95% confidence intervals. The diseases: Antibiotic associated diarrhea (AAD), Clostridium difficile disease (CDD),
Helicobacter pylori positive (HPP), Irritable bowel syndrome (IBS), Infectious diarrhea (ID), Necrotizing Enterocolitis (NEC), Traveller’s diarrhea (TD), and
Pouchitis are labelled as well as the mean effect sizes for each disease. The author, date, measure (risk ratio (95% CI), and p value are shown. Risk
ratios below one favor the probiotic while risk ratios above one favor the placebo.
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significant effect. Probiotics have also been shown to have
significant efficacy for CDD . Our result for ID represents
the first meta-analysis of probiotic use in ID treatment as only
single trials (e.g. ) have previously been conducted.
Two of the GIT diseases considered here, Traveller’s diarrhea
(TD) and Necrotizing Enterocolitis (NEC), showed no significant
effect of probiotics. TD is a type of acute diarrhea that impairs
intestinal absorption of nutrients and can lead to malnutrition .
Traveller’s diarrhea is typically caused by an amoeba  and is
treated with antibiotics that also lead to diarrhea. Our results
support previous studies by Pozo-Olano et al..  and Katelaris
et al..  who both found probiotics to have no effect in people
suffering with traveller’s diarrhea. In contrast, Hilton et al.. 
showed that LGG can reduce the risk of developing diarrhea by
3.9% per day.
NEC was the only other gastrointestinal disease that did not
show a significant effect for treatment with probiotics. NEC is a
gastrointestinal disease that is a major issue in preterm (,28
weeks’ gestation) neonates and involves infection and inflamma-
tion that causes destruction of the bowel or part of the bowel .
NEC only affects 1% to 5% of neonatal intensive care unit (NICU)
admissions, but it is common worldwide and is the most serious
disorder among hospitalized preterm infants. A possible explana-
tion is that NEC occurs mostly in infants and infants do not have
their immune system or their microbial communities fully
established . Our results, based on ten studies, differ from
those of Deshpande et al..  who showed that probiotics
significantly reduce the risk of NEC (RR=0.36 95% CI 0.20–
0.65) in preterm neonates, however they suggested that probiotics
needed to be assessed in larger trails in order to determine their
short and long term effects in the treatment of NEC. Our meta-
analysis improves on their meta-analysis by adding three studies.
We initially hypothesized that probiotic use might be more
efficacious in some broad types of GI diseases than in others due to
the mechanisms of action of the disease. Specifically, that there
might be differences in efficacy related to diarrheal production
versus inflammation or destruction of tissue, verses abdominal
pain, flatulence and irregular bowel movements (IBS). We found
no support for this hypothesis. AAD, CDD, ID, and TD are
related to diarrhea and NEC, Pouch and HPP are related to
inflammation/destruction of tissue. IBS is characterized by
abdominal pain, increased flatulence and irregular bowel move-
ments. None of these groups differed significantly in probiotic
efficacy and all disease showed significant effects except for NEC
and TD, which are related to inflammation and diarrhea
Previous studies have focused on the effect of one to two species
of probiotics (e.g. [19,61, and 62]) in the prevention of specific GI
diseases. In this study, we analyzed efficacy across 11 probiotic
species and their effects on GI diseases overall. Of the 11 probiotic
Figure 4. The effect size (risk ratio) for the subgroup analyses on age groups, dose, treatment length and multiple or single
probiotic species. (A) The effect size including the 95% confidence intervals for the age groups that had taken the probiotic vs. the controls. Age
groups included were: adults (.18 yrs), children (3#18 yrs) and infants (0–3 yrs). (B) The effect size including the 95% confidence intervals for dose of
probiotic. The doses that were included were: 1–961011, 1012CFU/day; 1–5.56106, 107, 108CFU/day; 1–96109CFU/day; 1–561010CFU/day. (C) The
effect size including the 95% confidence intervals for treatment length. Treatment lengths that were included were: 1–2 weeks, 3–4 weeks, 5–8 weeks
and 9–240 weeks. (D) The effect size including the 95% confidence intervals for multiple or single species of probiotics. Probiotics that contain more
than one species were considered multiple species, while probiotics only administered as one species were considered single species. Risk ratios
below one favor the probiotic while risk ratios above one favor the placebo.
Probiotics and Gastrointestinal Disease
PLoS ONE | www.plosone.org7 April 2012 | Volume 7 | Issue 4 | e34938
species considered, VSL #3 (RR=0.17 95% CI 0.09–0.33) and C.
butyricum (RR=0.18 95% CI 0.09–0.37) had the most significant
effect sizes (Figure 3b). The high statistical efficacy for these species
could be due to the small number of patients analyzed compared
to the other species. For example, C. butyricum had 207 patients and
VSL #3 had 116 patients which are small compared to LGG with
2782 patients. Higher efficacy for these species could also be
related to their use in diseases that also showed high prevention/
treatability with probiotics (e.g. AAD, HPP, Pouchitis), unlike
species that are widely used across many different GI diseases,
such as LGG, which is used in the prevention or treatment of TD,
Pouchitis, CD, AAD, HPP, NEC, and IBS. LGG is used widely in
clinical trials because of its beneficial effects on intestinal immunity
. Furthermore, LGG has shown to inhibit the growth of
Esherichia coli, Streptococci, C. difficile, Bacteriodes fragilis and Salmonella
by producing an antimicrobial substance . S. boulardii, E.
faecum, B. lactis, LGG, L. casei, and L. acidophilis combined with B.
infantis also showed significant efficacy in the treatment and
prevention of GI disease. Our results support recent findings by
McFarland et al..  who showed that S. boulardii prevented AAD
and by Orrhage et al..  who showed that the combination of L.
acidophilus and Bifidobacterium reduced the faecal counts of clostridia
The efficacy of probiotic treatment has been shown to be highly
dependent on the genus, species, and even the strain of bacteria
used . For example, not all lactic acid bacteria have probiotic
effects . In the case of traveller’s diarrhea, acidophilus strain LB
have been found to be effective , whereas other strains of
Acidophilus spp. have not . Also, different probiotics may confer
different degrees of benefit depending on the condition. For
example, McFarland  found that 3 types of probiotics
(Saccharomyces boulardii, Lactobacillus rhamnosus GG and probiotic
mixtures) significantly reduced the development of AAD while, in
the treatment of CDD only Saccharomyces boulardii was effective .
In our meta-analysis L. acidophilis, L. plantarum, and B. infantis did
not have significant effect sizes, showing that they are not effective
across all the GI diseases considered here. In this meta-analysis, all
species of L. acidophilus were first analyzed together. This included
strain LB, a common probiotic as well as unspecific strains. L.
acidophilus (strain LB) a heat stabilized strain also known as
LacteAˆol Fort . In some previous studies, LacteAˆol Fort (L.
acidophilus LB) has shown to be effective in the efficacy of acute
diarrhea, reducing duration and severity [68,69] and in IBS .
In the treatment of HPP, inactive L. acidophilus showed an in vitro
inhibitory effect on the attachment of H. pylori to gastric epithelial
cell lines . In other studies L. acidophilus has not shown
significant effects. For example, Katelaris et al..  found no
protection of TD with L. acidophilus and Witsell et al..  found
no effect of L. acidophilus on AAD. Our results suggest that when
taken without other species, L. acidophilus is not significantly
effective in preventing/treating GI disease (RR=0.82 95% CI
0.47–1.43). This result may be due to analyzing the strains L.
acidophilus LB and L. acidophilus together and strain dependency
could have an effect on the efficacy of GI disease. When analyzed
alone, L. acidophilis LB did show significant efficacy (RR=0.40
95% CI 0.20–0.82) and L. acidophilus with no strain specified did
not have a significant effect (RR=1.17 95% CI 0.85–1.62). Future
studies should compare and report effects of different strains of L.
acidophilus on GI diseases. Sazawal et al..  found that prevention
did not vary significantly for the probiotic species S. boulardii, LGG,
L. acidophilus, or L. bulgaricus. In our meta-analysis L. plantarum and
B. infantis also showed no overall effect on GI disease. Similar
negative results for L. plantarum have been previously shown in the
treatment of IBS [37,72,73]. In contrast, L. plantarum has been
shown to have efficacy in the prevention of CDD . Additional
studies across GI diseases need to be conducted to assess the
specific diseases that respond to L. plantarum. We also found that B.
infantis had no significant effect. There were very few trials
Figure 5. Funnel plot asymmetry used to determine publication bias. Log of the risk ratios were plotted against the standard error of the risk
ratio of each study to identify asymmetry in the distribution of trials. Gaps in the funnel plot suggest potential publication bias. The synthesis
estimate and the 0.01 limit are shown to distinguish asymmetry.
Probiotics and Gastrointestinal Disease
PLoS ONE | www.plosone.org8April 2012 | Volume 7 | Issue 4 | e34938
available in the literature for this species (n=3)  and additional
studies should be done to test efficacy.
Probiotics may be given to patients as either single or multiple
species. While some studies use one probiotic species e.g. B. infantis
 others used multiple strains e.g. VSL #3 [26,76,77]. We
found no significant difference between the efficacies of single or
multiple species across all diseases (Figure 4d). Instead, as discussed
above, the particular strain used is key to efficacy. Since most
studies only included the species of probiotic (e.g. L. acidophilus)
used, it is critical for future studies to include the exact probiotic
Ontogenic changes in the composition of the gut microflora
might also affect efficacy of probiotics [7,78,79,80]. For example,
in the colon of breast-fed infants prior to weaning, the fecal
microbiota is dominated by Bifidobacterium spp., while in adults,
Bifidobacterium spp. are only minor constituents . Likewise, the
colon of elderly individuals has lower proportions of Veillonella spp.
and Bifidobacteria spp. relative to Clostridia spp., Lactobacilli spp., and
Enterobacteria spp. . Ontogenic differences such as these suggest
that efficacy of probiotic-use and potentially overall outcome may
differ based on age. A number of studies have shown that probiotic
efficacy can differ in infants, children, and adults [81,82,83, and
84]. While the administration of probiotics to both infants and
adults results in changes of the microflora present in the feces and
the metabolic activity of the microflora , a number of studies
have shown greater differences between adults and children in the
composition of their fecal microflora communities than exist
within a cohort [82–84], suggesting strong ontogenic differences.
Our results showed no difference in efficacy by age group with
all age groups (infants, children, and adults) showing significant
effect sizes with the use of probiotics for the prevention of GI
disease (Figure 4a). Similar results have been reported by Tong et
al..  who showed that child and adult age group sub-analyses
were both significant for HPP. Likewise, Sazawal et al..  showed
significant results for both children and adults for the prevention of
acute diarrhea. A potential difference in the efficacy of probiotics
based on patient age is an area where additional studies are
needed. Very few trials have been conducted on infants (n=9) or
children (n=14) relative to adults (n=53). For example, Hoveyda
et al..  concluded that IBS was preventable for adults, but could
not assess efficacy in children due to the lack of studies.
Another factor that has been previously considered in probiotic
efficacy is dosage. Our results showed that three of the four dosage
levels were significant in treating disease. Only the dose 1–961011,
1012CFU/day, which was the largest treatment dose, did not
show a significant effect size. However, this result was likely due to
the smaller sample size (n=7) relative to the sample sizes of the
other doses (n=20, 25, and 12), which contributed to a larger 95%
CI. Whorwell et al..  studied the probiotic B. infantis (strain
35624) at three different dosage strengths 106, 108, and 1010and
found 161010CFU (for four weeks) was most effective. The
dosages tested in the studies analyzed here all use dosages well
above the minimum in commercial preparations, which typically
contain more than 1 billion bacterial units . Correct dosage for
specific diseases has been an area of some debate. For example,
Bezkorovainy  suggested that several billion organisms should
be introduced into an organism as not all of the bacteria will reach
target areas due to pH and salinity levels in the esophagus and
stomach which can reduce colony size . Our results suggest
that dosage has relatively minor effects.
In the past, it has been suggested that the treatment length that
patients received the probiotic could be a factor in the treatment
or prevention of disease and longer studies should be implemented
[4,5]. To our knowledge, this is the only meta-analysis that has
examined efficacy according to the length of treatment. Our
results show no significant effect of treatment length on efficacy
(Figure 4c). Taking probiotics for even a week is sufficient in
preventing and treating GI disease.
Several limitations of this meta-analysis are important to
consider in interpreting the results. An important limitation is
heterogeneity in outcome assessment and study design, particu-
larly for the overall analysis which includes different diseases,
strains, dosages, age groups, treatment lengths, and outcomes.
Although the studies were weighted by the number of patients,
heterogeneity still exists; therefore a random effects model was
performed. As in all meta-analyses, results need to be interpreted
Another limitation is that publication bias was observed using
the Begg and Egger method. Thus, a trim and fill method was
used to correct for publication bias. A positive significant effect of
probiotics was still observed. For future studies it would be helpful
to perform fecal samples before and after treatment to distinguish
the changes microbial communities, as well as specify adverse
effects of the treatment and prevention. This data would help
address the question of what changes probiotics are actually
leading to in the microbial ecology of the GIT.
In conclusion, our meta-analysis containing 74 studies, 84 trials
and 10,351 patients shows that in general, probiotics are beneficial
in treatment and prevention of GI diseases. The only GI diseases
where significant effect sizes were not observed were TD and
NEC. This effect may be due to the low number of studies on these
diseases, or in the TD case, the underlying mechanism of disease,
which is often not bacterial. Of the 11 species or species mixtures
only L. acidophilus, L. plantarum and B. infantis showed no efficacy,
however, for L. acidophilus, it was found that the strain LB was
highly effective. No differences in efficacy were observed for age
group or length of treatment or for single vs. multiple species. The
highest dosage considered (1–961011, 1012CFU/day) did not
show a significant effect size, however, due to the small sample
size, this result may be spurious. When choosing probiotics, the
type of disease (treated/prevented) and probiotic species (strain)
used are the most important factors to take into consideration.
raw data on: ID (reference #), Author, Year, Event and group size
(patients given probiotic), Event and group size (patients given
control), patients disease, probiotic species, comparison of mutiple
vs. single probiotics given, dose (1: 1–561010 cfu/day, 2: 1–
96109 cfu/day, 3: 1–5.56106, 107, 108 cfu/day, 4: 1–961011,
1012 cfu/day), treatment lengths, age group, N (number of
patients), rr (relative risk), 95% CI2, 95% CI+, z value, p value,
weight % and quality score.
The supplementary material provided includes
Complete PRISMA search for Pubmed (1970 to
Conceived and designed the experiments: MLR TNR. Performed the
experiments: MLR. Analyzed the data: MLR TNR. Contributed reagents/
materials/analysis tools: MLR. Wrote the paper: MLR.
Probiotics and Gastrointestinal Disease
PLoS ONE | www.plosone.org9April 2012 | Volume 7 | Issue 4 | e34938
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Probiotics and Gastrointestinal Disease
PLoS ONE | www.plosone.org11 April 2012 | Volume 7 | Issue 4 | e34938