nutrients

Article
PHAGE-2 Study: Supplemental Bacteriophages
Extend Bifidobacterium animalis subsp. lactis BL04
Benefits on Gut Health and Microbiota in
Healthy Adults
Diana S. Grubb 1 , Scott D. Wrigley 1 , Kimberley E. Freedman 1 , Yuren Wei 1 ,
Allegra R. Vazquez 1,2 , Roxanne E. Trotter 1 , Taylor C. Wallace 3,4 , Sarah A. Johnson 2 and
Tiffany L. Weir 1, *
1 Intestinal Health Laboratory, Department of Food Science and Human Nutrition, Colorado State University,
Fort Collins, CO 80523, USA; [email protected] (D.S.G.); [email protected] (S.D.W.);
[email protected] (K.E.F.); [email protected] (Y.W.);
[email protected] (A.R.V.); [email protected] (R.E.T.)
2 Functional Foods & Human Health Laboratory, Department of Food Science and Human Nutrition,
Colorado State University, Fort Collins, CO 80523, USA; [email protected]
3 Think Healthy Group, 1301 20th Street, NW, #413, Washington, DC 20036, USA; [email protected]
4 Department of Nutrition and Food Studies, George Mason University, 4400 University Drive, MS:1F7,
Fairfax, VA 22030, USA
* Correspondence: [email protected]; Tel.: +1-970-215-7571




Received: 15 July 2020; Accepted: 13 August 2020; Published: 17 August 2020


Abstract: Probiotics are increasingly used by consumers and practitioners to reduce gastrointestinal
(GI) distress and improve gut function. Here, we sought to determine whether the addition
of supplemental bacteriophages (PreforPro) could enhance the effects of a common probiotic,
Bifidobacterium animalis subsp. lactis (B. lactis) on GI health. A total of 68 participants were enrolled in
a 4-week, randomized, parallel-arm, double-blind, placebo-controlled trial where primary outcomes
included self-assessments of GI health, a daily stool log, and 16s rRNA analysis of gut microbial
populations. We observed within-group improvements in GI inflammation (p = 0.01) and a trending
improvement in colon pain (p = 0.08) in individuals consuming B. lactis with PreforPro, but not
in the group consuming only the probiotic. There was also a larger increase in Lactobacillus and
short-chain fatty acid-producing microbial taxa detected in the stool of participants taking PreforPro
with B. lactis compared to the probiotic alone. Overall, these results suggest the addition of PreforPro
as a combination therapy may alter gut ecology to extend the GI benefits of consuming B. lactis or
other probiotics.

Keywords: bacteriophage; Bifidobacterium; gut microbiota; intestinal health; microbiome; probiotic

1. Introduction
Over the past two decades, research on the gut microbiota has revealed that it plays an important
role in maintaining health and preventing disease [1]. Although novel clinical applications of
microbiota modulation are mainly restricted to fecal microbiota transplantation for treatment of
Clostridium difficile [2], gut-targeted dietary supplements are a large and growing segment of the
public market [3] Among these dietary supplements are probiotics, live beneficial microorganisms that
confer benefits to the host through a variety of mechanisms [4]. In addition to commercial availability
as over-the-counter dietary supplements, they can also be found as natural components in many
fermented foods or supplemented additives in non-fermented food products. Probiotics are being

Nutrients 2020, 12, 2474; doi:10.3390/nu12082474 www.mdpi.com/journal/nutrients

Nutrients 2020, 12, 2474 2 of 15

explored as an accessible, convenient, and relatively low-cost strategy to modify the gut microbiota
for a variety of human health outcomes ranging from improved gastrointestinal (GI) function to the
management of lipid and glucose metabolism [5]. While there is still minimal scientific evidence to
support many of these uses, there are data supporting the efficacy of probiotics for improving GI
function. Efficacy appears to be somewhat dependent on the specific species/strains, or the combination,
used for a given condition; however, a 2012 meta-analysis of randomized controlled trials showed that
8 of the 11 species/mixtures tested were efficacious for a range of GI conditions, including pouchitis,
infectious and C. difficile associated-diarrhea, Irritable Bowel Syndrome (IBS), and antibiotic-associated
diarrhea [6]. A more recent meta-analysis suggests that probiotics may also be beneficial in alleviating
functional constipation in adults [7]. Even in cases where there were insufficient data to demonstrate
probiotic efficacy, adverse effects were minimal [8], suggesting probiotics are a low-risk intervention to
improve GI health in humans.
Bacteriophages, or phages, are also being explored for their potential to selectively modify the gut
microbiota. Phages attach to a specific host bacterium, insert their genetic material into the cell, and
take over the machinery of the host cell to replicate phage components. Once this process is complete,
the cell is destroyed, or lysed, releasing new phage particles. Unlike antibiotics, phages display a
narrow host range, limiting global perturbations to the gut microbiota that can promote dysbiosis,
reduce intestinal homeostasis, and promote disease. In addition to their narrow host range, phages
are ubiquitous in the environment, and many are Generally Regarded as Safe (GRAS) for human
consumption. We recently showed that supplemental consumption of a cocktail of E. coli-targeting
bacteriophages, marketed as PreforPro, was considered safe and tolerable [9]. We also demonstrated
that these phages showed bifidogenic effects after 4 weeks of oral supplementation, with no global
disruption of the gut microbiota [10]. Based on these data, we hypothesized that, in addition to
reducing target host species, phages may also reduce competition among commensal bacteria for
limited resources and produce a potential fuel source in the form of contents released from the dead
bacteria. Therefore, phages taken in combination with a probiotic may extend benefits on GI health
and intestinal environment.
In the current study, we tested whether a combined probiotic and phage-based intervention
extends probiotic impacts on gastrointestinal discomfort and stool consistency in a healthy adult
population. Our randomized, double-blind, placebo-controlled intervention included three parallel
arms in which participants were assigned to consume either Bifidobacterium animalis subsp. lactis
BL04 (B. lactis), B. lactis with a commercial phage cocktail targeting E. coli, or a maltodextrin-based
placebo. We selected this combination because our previous data showed that PreforPro increased
commensal Bifidobacterium [10]. The primary outcome measures included subjective digestive health
questionnaires and stool consistency measurements based on the Bristol Stool Scale. We hypothesized
that probiotic-consumption would improve one or more aspects of digestive health and stool consistency
in our participant population and that addition of the phage cocktail would potentiate these effects.
Specifically, our primary outcome measures included a digestive health questionnaire, stool consistency
measurements, and molecular analysis of the gut microbiota. We hypothesized that consumption
(B. lactis BL04 + PreforPro) would improve one or more aspects of digestive health and stool consistency
in our participant population compared to the probiotic alone.

2. Materials and Methods

2.1. Study Design

The BacterioPHAGE for Gastrointestinal Health 2 Study (PHAGE-2 Study; #NCT04511221)
is a 4-week, randomized, parallel-arm, double-blind, placebo-controlled clinical intervention trial.
The PHAGE 2 Study was designed to test whether combining supplemental bacteriophages with a
probiotic would provide additional benefits to GI health and gut microbiota compared to consuming
a probiotic alone. Data from our previous study [10] were used to determine sample size, based on
Nutrients 2020, 12, 2474 3 of 15

80% power to detect a significant change (alpha = 0.05) in Bifidobacterium levels between the placebo
and treatment groups. The Colorado State University Institutional Review Board approved the study
protocol (CSU #19-9145H) and all 68 participants enrolled in one of the three study arms provided
written informed consent. This trial is registered at ClinicalTrials.gov under #NCT04511221.
Participants were pre-qualified by phone screening and attended an in-person study visit
at Colorado State University’s Food and Nutrition Clinical Research Laboratory (FNCRL) in the
Department of Food Science and Human Nutrition. Participants were fasted at least 8 h and asked to
abstain from exercise for at least 12 h prior to clinic visits. They were also instructed to refrain from
taking any medications or dietary supplements for 24 h prior to their visits. During their initial visit
(baseline), eligibility was confirmed by taking anthropometric measurements of height (cm) and weight
(kg) to calculate BMI and completing a written medical health questionnaire to determine medical
history and current medication use. At both baseline and 4-week (final) visits, participants were asked
to complete a digestive health questionnaire, undergo several measures of cardiovascular function
(reported in Trotter et al. 2020 in press), and provide a venous blood and stool sample. They were also
provided with a stool log and asked to keep a daily record of their bowel movements.
After undergoing sample collections and analysis procedures at the baseline visit, participants
were randomly assigned to 1 of 3 treatments groups: (A) 15 mg rice maltodextrin (placebo); (B) 1 × 109
Colony Forming Units (CFU) Bifidobacterium animalis subspecies lactis strain BL04 (B. lactis BL04); or (C)
1 × 109 CFU B. lactis BL04 + 1 × 106 Plaque Forming Units (PFU) LH01-Myoviridae, LL5-Siphoviridae,
T4D-Myoviridae, and LL12-Myoviridae bacteriophages, marketed as PreforPro (B. lactis BL04 +
PreforPro). Participants were asked to consume one 15-mg capsule per day during the 4-week
intervention period. All treatment capsules contained maltodextrin from rice and medium chain
triglycerides from palm and coconut oil as a filler material. Participants were randomized to treatment
groups using the second generator at www.randomization.com. To assess treatment compliance,
participants returned all unused treatment capsules at their final visit.
Participants were asked to maintain their normal eating and exercise habits throughout the course
of the study. To aid in determining dietary compliance, participants were completed a 2-day diet record
at the beginning and end of the treatment period, including 1 weekday and 1 weekend day, using the
National Institutes of Health, National Cancer Institute Automated Self-Administered 24-h dietary
assessment tool (ASA24; https://asa24.nci.nih.gov). For each of the 2-day diet records, participants
recorded all foods, drinks, and supplements they consumed in a 24-h period and the time of day they
were eating and drinking.

2.2. Participant Characteristics

Healthy, adult participants were recruited from Fort Collins, Colorado and surrounding areas
by referral from local healthcare practitioners, flyers, email, and through word of mouth. Initial
eligibility was determined by a phone screening questionnaire and confirmed onsite at the FNCRL
via interview and BMI assessment by the clinical coordinator. Inclusion and exclusion criteria are
presented in Table 1. Participants were asked to maintain their regular diet and exercise habits as well
as to limit alcohol consumption to 1–2 drinks per day or no more than 8 drinks per week during the
study. They were also asked to abstain from taking any supplemental pre- or probiotics. A summary
of baseline participant characteristics can be found in Table 2.
Nutrients 2020, 12, 2474 4 of 15

Table 1. Participant inclusion and exclusion criteria.

Inclusion Criteria Exclusion Criteria

Men and women Pregnant and breastfeeding women
Taking medication that would influence the endpoints of the study
(statins, metformin, nonsteroidal anti-inflammatory drugs, monoamine
Aged 18–65 years
oxidase inhibitors, blood pressure medications) and taking probiotics
and/or botanical supplements that target the GI tract or gut microbiota.
Normal, overweight, or class 1 Current diagnosis of cancer, liver or kidney disease, gastrointestinal
obese (BMI 20–34.9 kg/m2 ) diseases, and metabolic disorders.
Antibiotic use within the 2 months prior to enrollment.

Table 2. Participant baseline characteristics.

Treatment Male (n) Female (n) Height (cm) Weight (kg) BMI Age
Placebo 8 13 169.9 ± 8.2 71.3 ± 10.6 24.67 ± 2.8 36.5 ± 13.0
B. lactis BL04 10 13 169.9± 8.2 71.5 ± 10.7 24.7 ± 2.8 36.5 ± 13.2
B. lactis BL04 + PreforPro 7 15 170.1 ± 8.22 71.6 ± 10.4 24.7 ± 2.71 36.1 ± 13.3
Data represent mean ± SD.

2.3. Comprehensive Metabolic Panels

Blood samples (100 uL) were collected from the antecubital vein in a lithium heparin tube and
immediately analyzed using a Piccolo Metlyte Plus CRP Reagent Disc, which included glucose, blood
urea nitrogen (BUN), creatinine (CRE), creatinine kinase (CK), NA+, K+, Cl- and C-reactive protein
(CRP), on the Piccolo Xpress Chemistry Blood Analyzer (Abaxis, Union City, CA, USA).

2.4. Digestive Health and Bowel Movement Assessment

Participants completed a digestive health questionnaire at baseline and after 4 weeks on treatment
to assess perceived effects on GI symptoms, as previously described [9]. Briefly, the questionnaire
had 4 sections corresponding to gastric function, GI inflammation, small intestine (SI) and pancreas
pain, and colon function (Supplemental Methods File S1). Participants ranked questions within each
section, choosing no/rarely, occasionally, often, or frequently. Symptom severity was then ranked
according to their priority status outlined in Table 3. The percentage of participants that experienced
low, moderate, and high priority gastrointestinal symptoms at the initial and follow-up visit were
calculated. Initial test scores (baseline) and retest scores (week 4) were compared within treatment
groups across sections of the questionnaire to assess the percentage of participants that experienced an
improvement, worsening, or no change to the severity of their gastrointestinal symptoms after the
4-week intervention.

Table 3. Priority status based on scores from the digestive health questionnaire.

Questionnaire Section
Gastric Function GI Inflammation SI & Pancreas Colon
Low 1–4 1–4 2–8 2–8
Symptom
Moderate 5–8 5–8 9–16 9–16
Severity
High 9–56 9–72 17–80 17–72
Abbreviations: GI, gastrointestinal; SI, small intestine.

Participants were also asked to record all bowel movements during the study and rank them
according to the Bristol Stool chart. Participants were given a copy of the Bristol Stool Chart as a
reference to help determine stool type. Types included Type 1 (separate hard lumps), Type 2 (lumpy
and sausage like), Type 3 (a sausage shape with cracks in the surface), Type 4 (like a smooth, soft
sausage or snake), Type 5 (soft blobs with clear-cut edges), Type 6 (mushy consistency with ragged
Nutrients 2020, 12, 2474 5 of 15

edges) and Type 7 (liquid consistency with no solid pieces). For data analysis, each stool type was
coded as either hard (Types 1 or 2), soft (Types 3, 4, or 5), or diarrhea (Type 6 or 7). Stools categorized
as soft were considered normal, while hard and diarrhea stools were considered abnormal.

2.5. Stool Sample Processing

Stool samples were collected at home, stored refrigerated, and brought into the clinic within 24 h.
In the clinic, they were stored at 4 ◦ C and processed within 24 h in the following manner: two sterile
cotton swabs were inserted into the stool at three separate locations and then stored at −80 ◦ C for DNA
extraction. Approximately 1 g of stool was mixed with PBS buffer containing 10% glycerol and stored
at −80 ◦ C prior to use for phage counts. Remaining stool was transferred to a 100 mL sterile plastic
container and stored at −80 ◦ C for possible future analyses.

2.6. Phage Enumeration

Fecal samples stored in glycerol were thawed at room temperature. Samples were vortexed for
5 min with ceramic beads in 1 mL 2× sodium chloride magnesium sulfide (SM) buffer to homogenize [11].
A 1.5 mL aliquot was removed and centrifuged at 2000× g for 2 min, and supernatant was removed
and centrifuged again at 140,000 rpm for 10 min. An amount of 500 uL of prepared sample was
mixed with 100 uL of E. coli K12 culture (grown overnight in Luria Bertani (LB) broth on a 37 ◦ C
shaker to OD600 = 0.7 − 1.0) and incubated at 37 ◦ C for 5 min. An amount of 3 mL of freshly prepared
LB soft agar was then added to the mixture and immediately poured on LB agar plates which were
pre-warmed to 37 ◦ C for 30 min. Each sample was plated in duplicate and quantified using plated
phage standard dilution plates.

2.7. Microbiota Assessment

Fecal DNA was extracted using the FastDNA® Kit (MP Biomedicals, #116540400) following
manufacturer’s protocol. The V4 region of the 16S rRNA gene was amplified following the Earth
Microbiome Project protocol using the 515F-806R primer set [12] containing a unique 12 bp error
correcting barcode included on the forward primer. Cycling and sequencing conditions were as
previously described [13]. DNA extraction controls, no template PCR controls, and the Zymo mock
community were included on each sequencing plate. Sequence reads were imported into QIIME2
version 2020.2 for analysis [14]. Briefly, the sequence reads were demultiplexed, and concatenated.
Utilizing a Phred score cutoff of 30, upon examining the demultiplexed data, the reverse reads did not
meet the quality filtering parameter. As such analysis proceeded with single-end sequences which
were trimmed to 209 base pairs based on the same quality score. Reads were binned into ASVs
using the DADA2 pipeline [15]. Taxonomic assignments were made using GreenGenes version 13.8.
Samples with low reads or suspected contamination were removed and mitochondrial and chloroplast
sequences were filtered from remaining samples. The resulting feature tables and taxonomy files
were imported into MyPhyloDB version v.1.2.0 [16]. Alpha-diversity was calculated using Faith’s and
Shannon metrics through the QIIME2 diversity plugin. Beta diversity was determined by Bray Curtis
distance measurements and visualized by Principle Coordinates Analysis (PCoA) in MyPhyloDB.
Differences in taxa among treatments were assessed in MyPhyloDB using univariate Analysis of
Covariance (ANCoVA) and multivariate DiffAbund.

2.8. Statistical Analysis

Statistical analysis of clinical parameters (GI questionnaires, stool logs, metabolic panel analytes,
dietary intakes, anthropometrics) and qPCR data was completed using GraphPad Prism, version
8.3.0. All data were analyzed using a 2-way mixed-model ANOVA with Sidak’s post hoc for multiple
comparisons. In addition, baseline adjusted final values were analyzed using a 1-way ANOVA with
Tukey’s post hoc for multiple comparisons. Statistical significance was assigned as p < 0.05 and a
statistical trend was defined as between p < 0.10–0.051. Microbiota alpha-diversity parameters were
Nutrients 2020, 12, 2474 6 of 15
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analyzed
with usingpost-hoc
Tukey’s a one-wayforANOVA
multiplewith a non-parametric
comparisons Kruskal–Wallis
and a negative post
binomial hoc test,
general and model
linear β-diversity
[17]
was analyzed by PERMANOVA with 1000 iterations. Analysis of Covariance (ANCoVA)
were used to determine statistically different taxa between time points and treatment groups. with Tukey’s
post-hoc for multiple comparisons and a negative binomial general linear model [17] were used to
3. Results statistically different taxa between time points and treatment groups.
determine

3. Results
3.1. Participant Characteristics
A total of 153
3.1. Participant individuals were screened for eligibility. From this sample, 93 were eligible and
Characteristics
enrolled in the study. Among these, 25 participants were enrolled in a different RCT with the same
A total of 153 individuals were screened for eligibility. From this sample, 93 were eligible and
inclusion and exclusion protocol (Trotter et al., 2020, in press). Of the 68 enrolled in this branch of the
enrolled in the study. Among these, 25 participants were enrolled in a different RCT with the same
study, two dropped after visit 1. A total of 66 individuals who met all the inclusion and exclusion
inclusion and exclusion protocol (Trotter et al., 2020, in press). Of the 68 enrolled in this branch of the
criteria completed this 4-week, randomized, parallel-arm, double-blind, placebo-controlled clinical
study, two dropped after visit 1. A total of 66 individuals who met all the inclusion and exclusion
trial (Figure 1).
criteria completed this 4-week, randomized, parallel-arm, double-blind, placebo-controlled clinical
trial (Figure 1).

Figure 1. Consort flow diagram of participants through study enrollment to completion.

Figure 1. Consort
Approximately 12% offlow diagram ofinvolved
participants participants through
in the study
study enrollmentadverse
experienced to completion.
events, with the
severity of reported adverse events either mild or moderate. No adverse events were reported as
Approximately
severe 12% ofsuggesting
or life-threatening, participants involved
that there wasin athe study
low risk experienced adverse
associated with both events, withand
treatments, the
severity of reported
no participants adverse
dropped events
out of eitherdue
the study mild or moderate.
to adverse NoThe
events. adverse events
B. lactis BL04were reported
treatment as
group
severe or life-threatening, suggesting that there was a low risk associated with both treatments,
experienced the highest incidence of adverse events, accounting for about 7.5% of the total reported and
no participantsevents.
study-related dropped out of
Major the study due to adverse
treatment-associated adverseevents. The
events B. lactis constipation,
included BL04 treatment group
bloating,
experienced the highestofincidence
flatulence, fatigue/lack energy, orofother.
adverse events, accounting for about 7.5% of the total reported
study-related events. Major treatment-associated adverse events included constipation, bloating,
flatulence, fatigue/lack of energy, or other.
3.2. Study Compliance
Overall compliance with the treatment protocol was about 95% with individual compliance
3.2. Study Compliance
ranging from 73–100%. Under the intention-to-treat analysis principle, all individual data were
Overall
analyzed, compliance
regardless with the treatment
of compliance. protocol was
Within treatment about
groups 95% withwas
compliance individual compliance
as follows: Placebo:
average compliance = 97%, compliance range = 77–100%; B. lactis BL04: average compliance =were
ranging from 73–100%. Under the intention-to-treat analysis principle, all individual data 95%,
analyzed, regardless of compliance. Within treatment groups compliance was as follows: Placebo:
average compliance = 97%, compliance range = 77–100%; B. lactis BL04: average compliance = 95%,
Nutrients 2020, 12, 2474 7 of 15

compliance range = 80–100%; B. lactis BL04 + PreforPro: average compliance = 95%, compliance range
= 73–100%.
The overall number of participants that completed the self-reported 24-h food recalls was low,
with only about 53% of participants completing two dietary records at both baseline and during the
4-week intervention. Within each of the treatment groups, dietary compliance for completing the
food recall was as follows: Placebo: average compliance = 48%, B. lactis BL04: average compliance =
58%, B. lactis BL04 + PreforPro: average compliance = 57%. From the dietary information collected,
there were no significant differences in total calories, macronutrients, fiber, cholesterol, or saturated fat
consumed over the course of the study within or between any of the treatment groups (Table 4). Finally,
variability among and within treatment groups had no influence on body weight during the study as
the average BMI was about 24 kg/m2 (see Table 1) for all groups both pre- and post-treatment and
average weights fluctuated by <1 kg in each treatment group. Likewise, average values for analytes
measured by the Metlyte plus CRP Reagent Disk did not vary significantly across groups or pre- to
post-treatment within groups and all values remained within clinically normal ranges throughout the
study (Supplemental Table S1).

Table 4. Self-reported dietary intake.

Placebo B. lactis BL04 B. lactis BL04 + PreforPro

(n = 10) (n = 14) (n = 12)
Baseline Final Baseline Final Baseline Final
Energy (KCAL) 2269 ± 999 2089 ± 982 1775 ± 735 1853 ± 675 1739 ± 474 1905 ± 719
PRO (g) 99 ± 50 96 ± 50 85 ± 49 74 ± 29 70 ± 18 68 ± 27
TFAT (g) 103 ± 56 86 ± 42 73 ± 27 84 ± 44 66 ± 30 73 ± 29
SFAT(g) 33 ± 18 27 ± 14 21 ± 9 29 ± 21 19 ± 6 23 ± 12
CHOL (mg) 358 ± 259 324 ± 271 282 ± 299 277 ± 281 215 ± 101 180 ± 129
CARB (g) 240 ± 104 225 ± 118 194 ± 103 199 ± 91 209 ± 54 222 ± 71
FIBER (g) 25 ± 11 22 ± 12 27 ± 19 23 ± 16 26 ± 21 24 ± 14
Data represent mean ± SD. No values were statistically significant with a p-value < 0.05. Abbreviations: PRO
(protein), TFAT (total fat), SFAT (saturated fat), CHOL (cholesterol), CARB (carbohydrates).

3.3. GI Health and Digestion

Total point scores for each section of the digestive health questionnaire represent symptom
severity related to gastric function, GI inflammation, small intestine and pancreas pain, and colon
pain. Changes in individual scores, for each section, pre- to post-treatment are shown in Supplemental
Figure S1. There was a significant main effect for Time (p = 0.004) and average scores from the
gastric function section of the questionnaire were significantly reduced (improved) with B. lactis BL04
treatment (Figure 2a; p = 0.046; [CI: −2.851 to −0.019]); although there were no significant changes in
gastric function noted within the placebo or the PreforPro + B. lactis BL04 group or among any of the
treatments. There was also a significant main effect for time (p = 0.005) in perceived improvement in
GI inflammation and a significant reduction in the average symptom severity with the B. lactis BL04 +
PreforPro treatment (Figure 2b; p = 0.005; [CI: −3.509 to −0.307]). While there were no other statistically
significant differences observed, there was also a trend for improved colon pain in individuals on
the B. lactis BL04 + PreforPro treatment (Figure 2d; p = 0.082; [CI: −5.802 to 0.2568]). Notably, while
there were no other significant improvements, there were also no significant worsening of symptoms,
indicating that both treatments, at the levels administered, were tolerable.
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Figure 2. Average symptom severity before and after treatment as determined by gastrointestinal (GI)
questionnaire. Average scores at baseline and after 4-weeks are shown for functional assessments
Figure
of (a) 2. Average symptom
Gastrointestinal severity
function; (b)before and after treatment
gastrointestinal as determined
inflammation; by gastrointestinal
(c) small intestine pain; (GI)
and
questionnaire.
(d) colon pain. Average scores at baseline and after 4-weeks are shown for functional assessments of
(a) Gastrointestinal function; (b) gastrointestinal inflammation; (c) small intestine pain; and (d) colon
When comparing baseline adjusted (final-baseline) means among groups, there were no significant
pain.
differences (Supplemental Figure S2). However, there was a small positive effect, determined using
When
Cohen’s comparing
d, in baseline for
symptom severity adjusted (final-baseline)
the GI Inflammation andmeans among
colon pain groups,
sections withthere were
B. lactis BL04no+
significant
PreforPro whendifferences (Supplemental
compared to the placebo,Figure S2).effect
while the However, there
of B. lactis alone was
wasa negligible
small positive
(Table 5,effect,
[18]).
determined using Cohen’s d, in symptom severity for the GI Inflammation and colon pain sections
with B. lactis BL04 + Table 5. Effect
PreforPro when (Cohen’s d) to
size compared of treatments
the placebo,relative
while to the
the placebo.
effect of B. lactis alone was
negligible (Table 5, [18]). Gastric Function GI Inflammation SI and Pancreas Pain Colon Pain
B. lactis BL04 0.05 −0.13 −0.17 −0.14
Table 5. Effect size
B. lactis BL04 + PreforPro −0.38(Cohen’s d) of treatments
0.30 relative to the0.11
placebo. 0.45

Gastric Function GI Inflammation SI and Pancreas Pain Colon Pain

TheB.GI lactis BL04questionnaire 0.05
health scores corresponded −0.13to priority levels −0.17
of mild (0–24 total −0.14points),
B. lactis BL04 + PreforPro −0.38 0.30 0.11
moderate (25–48 total points), or severe (49–280 total points) symptoms in each of the target 0.45 areas.
Using these priority scores, we were able to calculate whether participants’ symptoms qualitatively
The GIimproved,
worsened, health questionnaire
or stayed thescores corresponded
same during the studyto(Figure
priority 3).levels of mild
Regardless (0–24
of the total points),
treatment group,
moderate (25–48 total points), or severe (49–280 total points) symptoms in each of the
most participants experienced no change in the overall priority level of their perceived GI distress. Only target areas.
Using
a smallthese priorityofscores,
percentage we were
participants able to calculate
experienced whether
an overall participants’
worsening symptoms
of priority qualitatively
status from baseline
worsened, improved, or stayed the same during the study (Figure 3). Regardless
to follow-up, with the lowest percentage of participants in the placebo group (2%) and the highest of the treatment
group, mostinparticipants
percentage the B. lactis experienced no change
group (8%) moving frominathe overall
lower prioritypriority
to a higher level ofscore.
their The
perceived GI
PreforPro
distress. Only a small percentage of participants experienced an overall worsening
+ B. lactis group saw the greatest percentage (24%) of participants with overall improvements in the of priority status
from baseline
priority statusto of follow-up, with the
their symptoms (i.e.,lowest
movedpercentage
from a higherof participants in the placebo
to a lower priority ranking).group (2%) and
In comparison,
the highest percentage in the B. lactis group (8%) moving from a lower to
only 15% of participants on B. lactis alone moved to a lower priority score after treatment.a higher priority score. The
PreforPro + B. lactis group saw the greatest percentage (24%) of participants with overall
improvements in the priority status of their symptoms (i.e., moved from a higher to a lower priority
ranking). In comparison, only 15% of participants on B. lactis alone moved to a lower priority score
after treatment.
The percentage of persons whose ratio of normal (Bristol Type 3, 4 or soft stool) to abnormal
stool (Bristol Type 1, 2, 5, 6 or hard or watery stool) that improved, declined, or stayed the same is
movements that were considered abnormal (hard or diarrhea) to stool that was normal (soft). The
majority of participants experienced no change in stool consistency from week 1 to week 4. However,
a substantial percentage of participants in the B. lactis BL04 (22%) and B. lactis BL04 + PreforPro (32%)
groups experienced a decline in stool consistency from week 1 to week 4. Almost half of those in the
B. lactis 2020,
Nutrients BL0412,+ 2474
PreforPro group saw no change in stool consistency from baseline to follow-up (41%)
9 of 15
while the B. lactis BL04 group saw the greatest improvement in stool consistency over time (39%).

Figure
Figure 3.
3. Priority
Priority changes
changes in
in overall
overall digestive
digestive symptoms
symptoms from
from baseline to follow-up.
baseline to follow-up.

The percentage
Because of persons
participants beganwhose
takingratio ofrespective
their normal (Bristol Type 3,concurrent
treatments 4 or soft stool)
withto abnormalbowel
recording stool
(Bristol
movements,Type 1, 2, 5,may
there 6 or hard
have or watery
been rapidstool) that improved,
changes declined, or
in stool consistency stayed
that the same
occurred withinis shown in
the first
Table
week 6.ofAtreatment,
decline in stool consistency
obscuring was defined
treatment effects. asTherefore,
having a higher
we also proportion
calculatedof bowel
changesmovements
in stool
that were considered
consistency using onlyabnormal
the first (hard or diarrhea)
two recorded daystocompared
stool thatto wasthenormal
reported (soft).
stoolThe majority of
consistency at
participants experienced no change in stool consistency from week 1 to week 4. However,
week 4. We saw higher percentages of participants reporting improved stool consistency in both the a substantial
percentage
Placebo andof B.participants
lactis BL04 groups B. lactis
in the (33% andBL04
52%,(22%) and B. lactis
respectively). MostBL04 + PreforPro
participants in the(32%) groups
B. lactis BL04
+experienced a decline
PreforPro group hadin stool consistency
a decline from week(59%)
in stool consistency 1 to week 4. Almost
from baseline tohalf of thoseThere
follow-up. B. lactis
in thewas also
aBL04 + PreforPro
noticeable group
negative saw noeffect
placebo changewithin 48%
stoolofconsistency
participants from baselineato
reporting follow-up
decline (41%)
in stool while the
consistency
B.
in lactis BL04 group
the placebo group. saw the greatest improvement in stool consistency over time (39%).

Table 6. Stool consistency changes from baseline to follow-up visit.

Stool Changes betweenbetween

Stool Changes Week 1Week
and 1 Stool Changes
Stool Changesbetween
betweenDays and22 and
Days 1 and
and4 Week 4
Week andWeek
Week44
Improved Declined
Improved Declined Same
Same Improved
Improved Declined
Declined Same Same
PlaceboPlacebo 29% 29% 33%33% 38%38% 33%
33% 48% 48% 19% 19%
B. lactis BL04 39% 22% 39% 52% 39% 9%
B.B.
lactis BL04
lactis BL04 + PreforPro
39% 27% 22%32%
39%41%
52%
27% 59%
39% 14%
9%
B. lactis BL04 +
27% No values were
32%found to be41% 27%
statistically different. 59% 14%
PreforPro
No values were found to be statistically different.
Because participants began taking their respective treatments concurrent with recording bowel
movements, there
3.4. Microbiota may have been rapid changes in stool consistency that occurred within the first week
Changes
of treatment, obscuring treatment effects. Therefore, we also calculated changes in stool consistency
usingPhage plating
only the first was conducted
two recorded to confirm
days compared thetopresence of viable
the reported stoolE.consistency
coli-targeting phages
at week in stool
4. We saw
samples.
higher percentages of participants reporting improved stool consistency in both the Placebo and 4-week
No phages were detected in baseline samples from any treatment group or from the B. lactis
samples collected
BL04 groups (33% from individuals
and 52%, in the Most
respectively). Placebo or the B. lactis
participants in theBL04 groups.
B. lactis BL04 About 44% of
+ PreforPro the final
group had
samples collected from individuals assigned to the B. lactis BL04 + PreforPro group had
a decline in stool consistency (59%) from baseline to follow-up. There was also a noticeable negative viable phages
detected,effect
placebo although
with the
48%total number recovered
of participants varied
reporting from in
a decline 7–30,531 pfu/gram with
stool consistency the
in the average
placebo being
group.
5103 pfu/gram.
3.4. Microbiota
Using 16s Changes
rRNA amplicon sequencing, we assessed whether the three treatments impacted the
gut microbial community. The microbiota in all samples was primarily represented by the phyla
Phage plating was conducted to confirm the presence of viable E. coli-targeting phages in stool
Actinobacteria, Bacteroidetes, Firmicutes, Proteobacteria, and Verrucomicrobiota and the proportion
samples. No phages were detected in baseline samples from any treatment group or from the 4-week
of these phyla did not significantly differ between groups or over time. The alpha-diversity metrics
samples collected from individuals in the Placebo or the B. lactis BL04 groups. About 44% of the final
for Faith’s Phylogenetic Diversity (Faith’s PD), Shannon’s diversity, and Pielou’s Evenness did not
samples collected from individuals assigned to the B. lactis BL04 + PreforPro group had viable phages
significantly differ between treatment groups ar within any treatment group over time.
detected, although the total number recovered varied from 7–30,531 pfu/gram with the average being
5103 pfu/gram.
Using 16s rRNA amplicon sequencing, we assessed whether the three treatments impacted the
gut microbial community. The microbiota in all samples was primarily represented by the phyla
Actinobacteria, Bacteroidetes, Firmicutes, Proteobacteria, and Verrucomicrobiota and the proportion
of these phyla did not significantly differ between groups or over time. The alpha-diversity metrics
for Faith’s Phylogenetic Diversity (Faith’s PD), Shannon’s diversity, and Pielou’s Evenness did not
Nutrients 2020, 12, 2474 10 of 15

Nutrients 2020, 12, x FOR PEER REVIEW 10 of 15

significantly differ between treatment groups ar within any treatment group over time. (Supplemental
(Supplemental
Table Table examining
S2). Likewise, S2). Likewise, examining
β-diversity β-diversity
using usingdistances
Bray–Curtis Bray–Curtis distancesbyvisualized
visualized PCoA withby
PCoA with Non-Metric
Non-Metric DimensionalDimensional Scaling
Scaling (NMDS) (NMDS)
revealed revealed no
no significant significant
clustering clusteringas
by treatment, bydetermined
treatment,
as determined
by PERMANOVA by PERMANOVA
(Figure 4). (Figure 4).

Figure4.4.Principle
Figure PrincipleCoordinates
CoordinatesAnalysis
Analysis(PCoA) ofof
(PCoA) Bray–Curtis distances
Bray–Curtis at at
distances baseline and
baseline final
and visit
final for
visit
each treatment group. for each treatment group.

Although
Although no no changes
changes were
were observed
observed in in the
the overall
overall composition
composition and and diversity
diversity of the microbiota,
of the microbiota,
some
some select
select taxa
taxa were
were significantly
significantly increased
increased or or decreased
decreased in in the
the two
two probiotic
probiotic treatment
treatment groups,
groups, but but
not
not in the Placebo. Using the DiffAbund function in MyPhyloDB, we saw some changes in
in the Placebo. Using the DiffAbund function in MyPhyloDB, we saw some changes in specific
specific
taxa. Figure 55 shows
taxa. Figure shows thethe baseline
baseline to to final
final visit
visit changes
changes within
within eacheach treatment
treatment group group with
with the
the blue
blue
bars representing log fold-change (logFC) and the orange bars representing
bars representing log fold-change (logFC) and the orange bars representing the log count per million the log count per million
(logCPM).
(logCPM). Within
Within thethe placebo
placebo group,
group, there there were
were decreases
decreases in in Pseudomonas
Pseudomonas and and Coprobacillus
Coprobacillus and and
increases in Aggregatibacter and Ruminococcus. This increase in Ruminococcus
increases in Aggregatibacter and Ruminococcus. This increase in Ruminococcus (gnavus) was also (gnavus) was also observed
using
observed a less sensitive
using a lessparametric
sensitive ANCoVAparametricanalysisANCoVA (p =analysis
0.04). Lachnobacterium and Lactobacillus
(p = 0.04). Lachnobacterium and
increases were noted with both B. lactis treatment groups; however, the
Lactobacillus increases were noted with both B. lactis treatment groups; however, the increase increase in Lachnobacterium wasin
approximately equal between these groups while there was a ~6-fold (3-logFC)
Lachnobacterium was approximately equal between these groups while there was a ~6-fold (3-logFC) increase in Lactobacillus
observed B. lactis BL04
increase ininLactobacillus + PreforPro
observed compared
in B. lactis BL04 to the probiotic
+ PreforPro alone. to
compared Wethe also saw increases
probiotic alone. We in
Atopobium, Gardnerella/Bifidobacterium, and Clostridium in the group consuming
also saw increases in Atopobium, Gardnerella/Bifidobacterium, and Clostridium in the group consuming phages. The increase
in Clostridium
phages. (citroniae)inwas
The increase also observed
Clostridium in this
(citroniae) wastreatment group when
also observed in this analyzing
treatment with ANCoVA
group when
(p = 0.03). Decreases were noted in Citrobacter and Desulfovibrio, which
analyzing with ANCoVA (p = 0.03). Decreases were noted in Citrobacter and Desulfovibrio, which are taxa often associated with
are
increased
taxa oftengut inflammation
associated withand GI disorders
increased [19,20]. Prevotella
gut inflammation andwas GI concurrently
disorders [19,20]. decreased with both
Prevotella was
groups receiving
concurrently probiotic
decreased treatments,
with both groups although the B.probiotic
receiving lactis BL04 + PreforPro
treatments, showed the
although a slightly greater
B. lactis BL04
decrease. changes in Catenibacterium and Desulfovibrio
+ PreforPro showed a slightly greater decrease. Finally, opposing changes in Catenibacteriumthese
Finally, opposing were observed between and
treatment groups.
Desulfovibrio were observed between these treatment groups.
Additionally,
Additionally,we wequeried
queried ourour sequence
sequencedatadata
for reads that matched
for reads E. coli inE.
that matched BLASTn
coli intoBLASTn
determine to
whether phage consumption altered levels of this taxa. We found
determine whether phage consumption altered levels of this taxa. We found several ASV’s several ASV’s that matched E. coli, that
but
which
matched could
E. not
coli,be confirmed
but which couldbecause notofbe a high degreebecause
confirmed of homology of a in thedegree
high sequenced region, resulting
of homology in the
in
sequenced region, resulting in equal matches with several closely related taxa.putative
equal matches with several closely related taxa. However, an assessment of these However, E. coli
an
reads revealed
assessment a trending
of these decrease
putative E. coli(ANCoVA;
reads revealed p = 0.094) from baseline
a trending decreaseto(ANCoVA;
the final visit p =with B. lactis
0.094) from
BL04 + PreforPro,
baseline to the finalwhich was
visit notB.
with observed in the+ other
lactis BL04 treatment
PreforPro, which groups
was (Supplemental
not observed in Figure S3).
the other
treatment groups (Supplemental Figure S3).
 Nutrients
Nutrients 2020,
2020, 12, x FOR PEER REVIEW
12, 2474 1115
11 of of 15

Figure 5. Changes
Figure in specific
5. Changes taxa
in specific within
taxa treatment
within groups,
treatment identified
groups, using
identified usinga gene-wise negative
a gene-wise negative
binomial generalized linear model (EdgeR). (a) Placebo (b) lactis BL04 (c) lactis BL04+PreforPro,
binomial generalized linear model (EdgeR). (a) Placebo (b) lactis BL04 (c) lactis BL04+PreforPro,LogLog
Fold Change = logFC; log Count Per Million = logCPM.
Fold Change = logFC; log Count Per Million = logCPM.
4. Discussion
4. Discussion
There is growing interest in the incorporation of phages with probiotic dietary supplements.
Phages canThere is growing
target interest in the incorporation
specific pro-inflammatory or pathogenicoforganisms
phages within theprobiotic dietary supplements.
gut and potentially enhance
thePhages can of
GI benefits target specific pro-inflammatory
the probiotics, although evidenceor forpathogenic
these effectsorganisms in theisgut
in human trials and potentially
currently lacking.
enhance
In this the GI benefits
randomized, of theplacebo-controlled
double-blind, probiotics, although evidence
clinical study, for these effects
we observed in human
the effects of B. trials
lactis is
currently lacking. In this randomized, double-blind, placebo-controlled clinical
BL04, with or without the E.coli-targeting phage mixture marketed as PreforPro, on gastrointestinal study, we observed
the effects
well-being andof B. lactis BL04,of
modulation with
theorgut
without the E.coli-targeting
microbiota. The beneficial phage mixture
effect marketed as PreforPro,
of supplementation with
on gastrointestinal well-being and modulation of the gut microbiota.
B. lactis BL04 appeared restricted to improvements in symptoms related to gastric function The beneficialandeffect
stool of
supplementation
consistency in 52% of with B. lactis BL04
participants. appearedwho
Participants restricted
consumed to improvements
B. lactis BL04 in symptomsshowed
+ PreforPro related to
gastric function and stool consistency in 52% of participants. Participants
improvements in digestive symptoms related to GI inflammation and colon pain and had the highest who consumed B. lactis
BL04 + PreforPro showed improvements in digestive symptoms related to
percentage of individuals reporting overall reduced symptom severity. This is largely in agreementGI inflammation and colon
pain
with and had study,
a previous the highest
wherepercentage
Ginden etof al.individuals
reported that reporting overall
PreforPro alonereduced
resultedsymptom severity. This
in an improvement
is largely in agreement with a previous study, where Ginden et al. reported
in gastric function and colon pain, but likewise reported a placebo effect [9]. Additionally, in the that PreforPro alone
population of phage-only consuming participants, a higher percentage of participants reported effect
resulted in an improvement in gastric function and colon pain, but likewise reported a placebo an
[9]. Additionally,
overall worsening ininsymptoms
the population of phage-only
than was reported inconsuming
the currentparticipants,
study. This aindicates
higher percentage
that there of
participants
might be some reported
additive an overall
benefit worsening in
to consuming symptoms
these phages thanwithwas reportedHowever,
a probiotic. in the current study. This
the probiotic
indicates
used that there
in this study, might
B. lactis BL04be alone,
some elicited
additiveminimal
benefit effects
to consuming these population,
on our study phages withwhich a probiotic.
was
However, the probiotic used in this study, B. lactis BL04 alone, elicited minimal
largely healthy adults with no diagnosed GI diseases. Bifidobacterium lactis BL04 is often studied effects on our study
population, which was largely healthy adults with no diagnosed GI diseases. Bifidobacterium lactis
in conjunction with other probiotic strains, and a recent study suggested that a five-strain mixture,
BL04 is often studied in conjunction with other probiotic strains, and a recent study suggested that a
containing B. lactis BL04, improved symptoms of antibiotic-associated diarrhea when given at a high
five-strain mixture, containing B. lactis BL04, improved symptoms of antibiotic-associated diarrhea
dose (>1010 CFU) [21]. Our study used a single strain, lower dose (109 CFU), and tested on a healthy
when given at a high dose (>1010 CFU) [21]. Our study used a single strain, lower dose (109 CFU), and
population. While most people occasionally experience GI distress or irregular bowel movements, it is
tested on a healthy population. While most people occasionally experience GI distress or irregular
likely the effects of this probiotic are more subtle in healthy individuals. It would be interesting to
bowel movements, it is likely the effects of this probiotic are more subtle in healthy individuals. It
would be interesting to determine whether the benefits we observed are more pronounced with a
combination of probiotic and/or in patients with GI disorders.
Nutrients 2020, 12, 2474 12 of 15

determine whether the benefits we observed are more pronounced with a combination of probiotic
and/or in patients with GI disorders.
Consistent with other published phage literature, the combination of B. lactis BL04 + PreforPro
did not significantly impact gut ecology, with no significant shifts in either alpha- or beta-diversity
parameters or large phyla-level changes in taxa. This concurs with our previous study, which looked
at the impacts of the phages alone on gut microbiota [10]. Similarly, E. coli-targeting phages added to a
simulated gut environment with a defined microbial consortium only showed effects on the target
species and did not disrupt commensal organisms [22]. However, various lytic phages administered to
gnotobiotic mice transplanted with a defined human commensal microbiota did demonstrate cascading
effects on many of the non-target species, likely mediated by complex ecological interactions between
bacteria [23]. This consortium was made up of only 10 bacterial species, suggesting that effects on
these species may have been more profound than if they were observed within the context of an intact
human gut containing hundreds of bacterial species.
Despite a lack of global alterations in the microbiota, the treatments were associated with changes
in a small number of taxa. Most notably, we saw an increase in taxa identified as Lactobacillus after
4-weeks of both B. lactis BL04 and B. lactis BL04 + PreforPro. However, the increase was 10-fold
greater than baseline levels when phages were included in the B. lactis treatment compared to a
~4-fold increase seen with the probiotic alone. Oral administration of a lytic phage induced similar
increases in Lactobacillus in a mouse model, as well as increasing Bifidobacterium [24]. It is interesting
to note that we did not observe increased Bifidobacterium in either treatment group, despite daily
consumption of 109 CFU of B. lactis BL04. A similar finding was reported in a study of healthy
volunteers taking Amoxicillin and a multi-species probiotic, which included B. lactis, E. faecalis and
several other Bifidobacteria and Lactobacilli. Using culture-based methods, the investigators only
reported an increase in stool Enterococci [25]. We used a sequencing approach, which is not sufficiently
sensitive to distinguish bacteria at the species level and it is possible that the baseline levels of
Bifidobacterium across our treatment groups were sufficiently high to obscure any treatment effects.
Finally, there were several taxa that have demonstrated inflammatory effects on the gut that were
reduced after treatment of B. lactis BL04 + PreforPro. Specifically, there were decreases in Citrobacter
and Desulfovibrio. Citrobacter overgrowth is associated with bloating, and both Faber et al. [26] and
Gangi et al. [19] reported increased levels of Citrobacter in IBS patients. In the Faber study, symptoms
of bloating and abdominal pain were alleviated after reducing Citrobacter, and other pro-inflammatory
bacteria and yeasts, by antibiotic treatment and supplementation with probiotics [26]. Desulfovibrio
is a sulfate-reducing bacteria that is also often associated with IBS [20] and induces alterations in
colonic architecture, cellular infiltration into the lamina propria, and increased inflammatory immune
responses in mouse models of colitis [27]. Therefore, the reduction of these bacteria may have been
responsible for the symptom reduction reported in our participant population and suggest that future
trials in a population with IBS or colitis may be warranted.
The study has some strengths and limitations worth considering. One major strength is that
this study is a randomized, double-blind, placebo-controlled clinical trial. Randomized controlled
trials are the gold standard for study design since they eliminate much inherent bias that other study
designs introduce. Similarly, compliance across treatments was high at about 95% with no significant
dietary differences in total calories, macronutrients, fiber, cholesterol, or saturated fat within any of the
treatment groups. However, we recognize that there are several limitations to this study as well. First,
although we saw pre- to post-treatment improvements with treatment in several of the GI parameters,
there were no significant differences in score change across groups and the effect size, while significant,
was small. This may be due to the fact that the study was powered from previously collected data [10] to
detect differences in the detection of Bifidobacterium populations among groups but was not specifically
powered for the GI outcomes. Therefore, the study may not have been sufficiently powered to detect
these differences between treatment groups. Another limitation was that true baseline data are lacking
for stool consistency measurements, as these were recorded concurrently with the start of the study
Nutrients 2020, 12, 2474 13 of 15

protocol. To minimize the impact of treatment effects on stool consistency measures, we analyzed
Day 1 and 2 as a baseline in comparison to Week 4 in addition to comparing data from Week 1 to Week
4. Another limitation is that participants had the option of numerically assessing their stool using the
Bristol Stool Chart or provide a designation of hard (H), soft (S), or diarrhea (D). This mixture in data
capture methods prevented the quantitative assessment of stool changes, although it was still useful
for functional assessment. Finally, the high participant burden of recording every bowel movement for
a month could have led to participants not recording all of their stools or recording only their first
bowel movement of the day.
Another limitation of the study was our ability to detect the phages in stool samples
post-consumption. Due to blinding during the study, all collected samples were plated for phage
enumeration. While no baseline samples, nor any in the probiotic alone and placebo group samples
resulted in plaque detection, only about 40% of the samples from the group receiving the bacteriophage
cocktail resulted in plaque formation after incubation with E. coli. There are several possibilities for
the low detection of phages in these samples, including both technical challenges and individual
participant factors. First, in order to reduce variability due to plating and enumeration techniques,
samples were frozen and then batch processed at the end of the study, which may have resulted in
a loss of viability, particularly in samples stored for longer periods. In addition, stool samples are
heterogenous and few studies have looked at the distribution of phages within stool, so it is possible
that our subsample-based approach was insufficient to detect low populations of phages in the stool.
Finally, a study looking at the survival of phages in a simulated digestive tract found that meal
composition, pH and other factors affected phage survival and detection [28]. Therefore, it is likely that
the timing of capsule consumption, the pH of the individual’s GI tract, and the meal composition prior
to sample collection all may have played a role in the survival of phages in the stool. This raises some
interesting possibilities for future research in terms of looking at the timing and delivery of phages,
as well as interindividual variability in efficacy and stool pH to determine the optimal therapeutic
conditions for phage supplementation.
In conclusion, the present study demonstrates that oral supplementation of B. lactis BL04 over the
course of 4 weeks has the potential to improve stool consistency, and B. lactis BL04 with PreforPro
can significantly improve some GI symptoms. In addition, phage consumption did not significantly
disrupt the gut microbial community but was associated with an increased relative abundance of some
beneficial species, like Lactobacillus, and decreases in certain pro-inflammatory taxa. While we did not
observe significant improvements in all aspects of GI well-being or stool consistency, the findings of
this study indicate a low risk for oral supplementation with phages and suggest that probiotic taken
with a phage cocktail may offer a safe solution for the management of occasional GI symptoms in
healthy adults.

Supplementary Materials: The following are available online at http://www.mdpi.com/2072-6643/12/8/2474/s1,

Figure S1: Individual scores at baseline and post-treatment for each of the 4 sections of the gastrointestinal health
questionnaire. Figure S2: Baseline adjusted scores for each section of the gastrointestinal health questionnaire
across treatments. Figure S3: Abundance of unclassified Enterobacteriaceae that putatively belong to Escherichia
coli. Table S1: Metabolic parameters in blood before and after treatment for each group. Table S2: Alpha Diversity
Metrics. File S1: Gastrointestinal (GI) Health Assessment.
Author Contributions: Conceptualization, T.L.W., T.C.W. and S.A.J.; methodology, T.L.W.; data curation, D.S.G.,
K.E.F., S.D.W., R.E.T., A.R.V., S.A.J.; formal analysis, D.S.G., S.D.W., Y.W., K.E.F., and T.L.W.; investigation, D.S.G.,
K.E.F., A.R.V., and R.E.T.; writing—original draft preparation, D.S.G. and T.L.W.; writing—review and editing,
all authors; funding acquisition, T.L.W. and S.A.J. All authors have read and agreed to the published version of
the manuscript.
Funding: This research was funded by Deerland Enzymes (Kennesaw, GA).
Acknowledgments: We would like to acknowledge the students and staff who worked in the FNCRL for always
stepping in to help with blood collection and assisted with REDCap, especially Nicole Litwin, Kiri Michell, and
Lauren Grabos. We’d also like to thank John Deaton and Anamaria Cuentas for training on phage isolation
procedures and Michael Lelko for providing research strains and for technical information on the B. lactis and
PreforPro capsule preparations.
Nutrients 2020, 12, 2474 14 of 15

Conflicts of Interest: The authors declare no conflict of interest. The funders (Deerland Enzymes; Kennesaw, GA)
provided type organisms (B. lactis, E. coli, phage mixture), and protocols for phage counts. They also approved the
design of the study and agreed to publication of the data but had no role in the initial design of the study; in the
collection, analyses, or interpretation of data; or in the writing of the manuscript.

References
1. Fava, F.; Rizzetto, L.; Tuohy, K.M. Gut microbiota and health: Connecting actors across the metabolic system.
Proc. Nutr. Soc. 2018, 78, 177–188. [CrossRef] [PubMed]
2. Ianiro, G.; Murri, R.; Sciumè, G.D.; Impagnatiello, M.; Masucci, L.; Ford, A.C.; Law, G.R.; Tilg, H.;
Sanguinetti, M.; Cauda, R.; et al. Incidence of bloodstream infections, length of hospital stay, and survival
in patients with recurrent clostridioides difficile infection treated with fecal microbiota transplantation or
antibiotics a prospective cohort study. Ann. Intern. Med. 2019, 171, 695–702. [CrossRef] [PubMed]
3. Digestive Health Supplements Market Size|Industry Report, 2019–2025. Available online: https://www.
grandviewresearch.com/industry-analysis/digestive-health-supplements-market (accessed on 5 August 2020).
4. Sanders, M.E.; Benson, A.; Lebeer, S.; Merenstein, D.; Klaenhammer, T.R. Shared mechanisms among
probiotic taxa: Implications for general probiotic claims. Curr. Opin. Biotechnol. 2018, 49, 207–216. [CrossRef]
[PubMed]
5. Goldin, B.; Gorbach, S.L. Clinical Indications for Probiotics: An Overview. Clin. Infect. Dis. 2008, 46,
S96–S100. [CrossRef]
6. Ritchie, M.L.; Romanuk, T.N. A Meta-Analysis of Probiotic Efficacy for Gastrointestinal Diseases. PLoS ONE
2012, 7, e34938. [CrossRef]
7. Zhang, C.; Jiang, J.; Tian, F.; Zhao, J.; Zhang, H.; Zhai, Q.; Chen, W. Meta-analysis of randomized controlled
trials of the effects of probiotics on functional constipation in adults. Clin. Nutr. 2020. [CrossRef]
8. Hungin, A.P.S.; Mulligan, C.; Pot, B.; Whorwell, P.; Agréus, L.; Fracasso, P.; Lionis, C.; Mendive, J.;
De Foy, J.-M.P.; Rubin, G.; et al. Systematic review: Probiotics in the management of lower gastrointestinal
symptoms in clinical practice—An evidence-based international guide. Aliment. Pharmacol. Ther. 2013, 38,
864–886. [CrossRef]
9. Gindin, M.; Febvre, H.P.; Rao, S.; Wallace, T.C.; Weir, T.L. Bacteriophage for Gastrointestinal Health (PHAGE)
Study: Evaluating the Safety and Tolerability of Supplemental Bacteriophage Consumption. J. Am. Coll. Nutr.
2019, 38, 68–75. [CrossRef]
10. Febvre, H.P.; Rao, S.; Gindin, M.; Goodwin, N.D.M.; Finer, E.; Vivanco, J.S.; Lu, S.; Manter, D.K.; Wallace, T.C.;
Weir, T.L. PHAGE Study: Effects of Supplemental Bacteriophage Intake on Inflammation and Gut Microbiota
in Healthy Adults. Nutrients 2019, 11, 666. [CrossRef]
11. Castro-Mejía, J.; Muhammed, M.K.; Kot, W.; Neve, H.; Franz, C.M.A.P.; Hansen, L.H.; Vogensen, F.K.;
Nielsen, D.S. Optimizing protocols for extraction of bacteriophages prior to metagenomic analyses of phage
communities in the human gut. Microbiome 2015, 3, 64. [CrossRef]
12. Caporaso, J.G.; Lauber, C.L.; Walters, W.A.; Berg-Lyons, D.; Huntley, J.; Fierer, N.; Owens, S.; Betley, J.;
Fraser, L.; Bauer, M.; et al. Ultra-high-throughput microbial community analysis on the Illumina HiSeq and
MiSeq platforms. ISME J. 2012, 6, 1621–1624. [CrossRef] [PubMed]
13. Lee, D.M.; E Ecton, K.; Trikha, S.R.J.; Wrigley, S.D.; Thomas, K.N.; Battson, M.L.; Wei, Y.; Johnson, S.A.;
Weir, T.L.; Gentile, C.L. Microbial Metabolite Indole-3-Propionic Acid Supplementation Does Not Protect
Mice from the Cardiometabolic Consequences of a Western Diet. Am. J. Physiol. Gastroint. Liver Physiol. 2020.
[CrossRef] [PubMed]
14. Bolyen, E.; Rideout, J.R.; Dillon, M.R.; Bokulich, N.A.; Abnet, C.C.; Al-Ghalith, G.A.; Alexander, H.; Alm, E.J.;
Arumugam, M.; Asnicar, F.; et al. Reproducible, interactive, scalable and extensible microbiome data science
using QIIME 2. Nat. Biotechnol. 2019, 37, 852–857. [CrossRef] [PubMed]
15. Callahan, B.J.; McMurdie, P.J.; Rosen, M.J.; Han, A.W.; Johnson, A.J.A.; Holmes, S.P. DADA2: High-resolution
sample inference from Illumina amplicon data. Nat. Methods 2016, 13, 581–583. [CrossRef] [PubMed]
16. Manter, D.K.; Korsa, M.; Tebbe, C.; Delgado, J.A. myPhyloDB: A local web server for the storage and analysis
of metagenomic data. Database 2016, 2016, 39. [CrossRef]
17. Chen, Y.; McCarthy, D.; Ritchie, D.; Robinson, M.; Smyth, G.; Hall, E. Edger: Differential Analysis of
Sequence Read Count Data User’s Guide. Available online: https://bioconductor.riken.jp/packages/release/
bioc/vignettes/edgeR/inst/doc/edgeRUsersGuide.pdf (accessed on 8 July 2020).
Nutrients 2020, 12, 2474 15 of 15

18. Cohen, J. Statistical Power Analysis for the Behavioral Sciences Second Edition. Available online: http:
//www.utstat.toronto.edu/~{}brunner/oldclass/378f16/readings/CohenPower.pdf (accessed on 8 July 2020).
19. Ganji, L.; Alebouyeh, M.; Shirazi, M.H.; Eshraghi, S.S.; Mirshafiey, A.; Daryani, N.E.; Zali, M.R. Dysbiosis
of fecal microbiota and high frequency of Citrobacter, Klebsiella spp., and Actinomycetes in patients with
irritable bowel syndrome and gastroenteritis. Gastroenterol. Hepatol. Bed Bench 2016, 9, 325–330. [CrossRef]
20. Loubinoux, J.; Bronowicki, J.P.; Pereira, I.A.; Mougenel, J.L.; Le Faou, A.E. Sulfate-reducing bacteria in human
feces and their association with inflammatory bowel diseases. FEMS Microbiol. Ecol. 2002, 40, 107–112.
[CrossRef]
21. Ouwehand, A.C.; Donglian, C.; Weijian, X.; Stewart, M.; Ni, J.; Stewart, T.; Miller, L.E. Probiotics reduce
symptoms of antibiotic use in a hospital setting: A randomized dose response study. Vaccine 2014, 32,
458–463. [CrossRef]
22. Cieplak, T.; Soffer, N.; Sulakvelidze, A.; Nielsen, D.S. A bacteriophage cocktail targeting Escherichia coli
reduces E. coli in simulated gut conditions, while preserving a non-targeted representative commensal
normal microbiota. Gut Microbes 2018, 9, 391–399. [CrossRef]
23. Hsu, B.B.; Gibson, T.E.; Yeliseyev, V.; Liu, Q.; Lyon, L.; Bry, L.; A Silver, P.; Gerber, G.K. Dynamic Modulation
of the Gut Microbiota and Metabolome by Bacteriophages in a Mouse Model. Cell Host Microbe 2019, 25,
803–814. [CrossRef]
24. Bao, H.-D.; Pang, M.-D.; Olaniran, A.; Zhang, X.-H.; Zhang, H.; Zhou, Y.; Sun, L.-C.; Schmidt, S.; Wang, R.
Alterations in the diversity and composition of mice gut microbiota by lytic or temperate gut phage treatment.
Appl. Microbiol. Biotechnol. 2018, 102, 10219–10230. [CrossRef]
25. Koning, C.J.M.; A E Jonkers, D.M.; E Stobberingh, E.; Mulder, L.; Rombouts, F.M.; Stockbrügger, R.W.
The Effect of a Multispecies Probiotic on the Intestinal Microbiota and Bowel Movements in Healthy
Volunteers Taking the Antibiotic Amoxycillin. Am. J. Gastroenterol. 2008, 103, 178–189. [CrossRef]
26. Faber, S.M. Treatment of abnormal gut flora improves symptoms in patients with irritable bowel syndrome.
Am. J. Gastroenterol. 2000, 95, 2533. [CrossRef]
27. Figliuolo, V.R.; Dos Santos, L.M.; Abalo, A.; Nanini, H.F.; Santos, A.; Brittes, N.M.; Bernardazzi, C.; Souza, H.;
Vieira, L.Q.; Coutinho-Silva, R.; et al. Sulfate-reducing bacteria stimulate gut immune responses and
contribute to inflammation in experimental colitis. Life Sci. 2017, 189, 29–38. [CrossRef] [PubMed]
28. Samtlebe, M.; Denis, S.; Chalancon, S.; Atamer, Z.; Wagner, N.; Neve, H.; Franz, C.; Schmidt, H.;
Blanquet-Diot, S.; Hinrichs, J. Bacteriophages as modulator for the human gut microbiota: Release from dairy
food systems and survival in a dynamic human gastrointestinal model. LWT 2018, 91, 235–241. [CrossRef]

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