A randomized, placebo-controlled, double blind human trial into the effect of Protecflor on gastroenteritis caused by attenuated E. Coli

The incidence of gastrointestinal infections is very high. In European
countries 10-25% of the population suffers from at least one foodborne
infection per year. Recent studies suggest that this incidence is
underestimated, and that even 90% of the population may suffer from infectious
diarrhea each year. Usually these infections are self-limiting and only some
days of diarrhea, stomach pain, nausea and vomiting occur. However, such
ordinary infections can be life-theatening in people with reduced resistance
(e.g. young children, elderly, or persons taking immuno-suppressive drugs).
Treatment of foodborne infections with antibiotics is usually non-effective.
Moreover, many bacterial pathogens become resistant to these drugs. Therefore,
it is important to search for alternative means to prevent or treat these
infections. Enhancement of human resistance to foodborne infections is an
attractive option, besides improved hygiene measures. Probiotics can contribute
to enhanced human resistance to infectious disease by excretion of
anti-microbial components of by competing with pathogens for intestinal
nutrients and mucosal adhesion sites.

Protecflor is a blend of probiotic bacteria (Lactobacillus helveticus
Rosell-52, Lactobacillus rhamnosus Rosell-11, Bifidobacterium longum Rosell-175
and probiotic yeast (Saccharomyces boulardii). Each individual strain has been
consumed in nutritional and dietary supplements for more than 20 years without
any reported side effect. Its complete genome sequence indicates the strain
does not carry any known antibiotic resistance genes. Using an in-vitro
digestion system (IViDiS), all strains in Protecflor® have been shown to
survive gastrointestinal conditions (Tompkins et al,. 2011). Survival was best
when Protecflor was given 30 minutes before a meal or together with a meal.
(Tompkins et al., 2011). Thanks to the existence of strain specific primer
pairs, it can be fully traced. All microorganisms in Protecflor have also been
granted Qualified Presumption of Safety status by the European Food Safety
Authority .

S. boulardii has been shown to bind pathogenic bacteria, such as E. coli in
vitro (Gedek , 1998), forming agglutinates that may easy be eliminated through
the gut. S. boulardii reduced the duration of rotaviral diarrhea in children
(Grandy et al., 2010). In addition, human intervention studies with children
have shown that S. boulardii reduced 1) the duration of non-specified diarrhea
(Kurugol et al., 2005, Htwe et al., 2008), 2) the number of children with
prolonged diarrhea (Villarruel et al, 2007) and 3) the number of days of
hospitalization (Kurugol et al, 2005). In addition, S. boulardii reduced the
incidence of diarrhea in travelers (Kollaritsch et al, 1993).

The lactic acid bacteria L. helveticus Rosell-52 and L. rhamnoses Rosell-11 has
been shown to bind to host epithelial cells and to reduce the binding of E.
coli to these epithelial cells (Sherman et al., 2005). The mixture of these
probiotics has been shown to reduce the duration of bacterial and/or viral
diarrhea in children (Tlaskal 1995, Tlaskal 2005)) and diminished the
incidence of Citrobacter freindi and Pseudomonas aeruginosa (Tlaskal, 1995))

The efficacy of Protecflor® in protecting against ETEC diarrhea in rats was
compared with that of solely S. boulardii and with a mixture of the lactic acid
bacteria L. helveticus Rosell-52, L. rhamnoses Rosell-11, and B. longum
Rosell-175. Whereas administration of solely S. boulardiii and the lactic acid
bacteria mixture had minor effects on E. coli-induced diarrhea, Protecflor®
improved stool consistency and shortened the duration of diarrhea (Bisson
2009). The protection against diarrhea was accompanied with a decreased
secretion of the pro-inflammatory cytokines IL1-α, IL-1β, IL-6, IFN-γ and
TNF-α, and an enhanced secretion of anti-inflammatory cytokines IL-4 and IL-10
(Bisson 2009). Protecflor® also showed synergistic effects in preventing weight
loss in E. coli-infected rats (Bisson 2009). Protecflor tended to shorten
duration of rotaviral diarrhea in Bolivian children (Grandy et al., 2010).

In the present double-blind, placebo-controlled, randomized parallel study, the
effect of oral Protecflor vs placebo will be studied on the reistace of humans
to enterotoxigenic Escherichiea coli (ETEC) infection. The main hypothesis is
that Protecflor will improve human resistance to ETEC as measured by decreased
faecal excretion of ETEC with time and less ETEC-induced daily faecal output.

We want to study whether consumption of Protecflor can improve the resistance
of humans to intestinal ETEC infection. An important parameter of intestinal
resistance is the so-called colonization resistance. The colonization
resistance is inversely related to the faecal excretion of a pathogen with
time. The hypothesis is that Protecflor will decrease fecal ETEC excretion by 1
log10 units. As a consequence, diarrhea severity, as measured by total daily
fecal output, will likely be reduced as well

Subjects, recruited from the Wageningen/Ede area, will participate in a
randomized, double-blind, placebo-controlled, parallel intervention study of 4
weeks after receipt of signed informed consent. Subjects consume either
Protecflor® or placebo. Subjects will be instructed to maintain their usual
pattern of physical activity and their habitual diet, except for their dairy
intake and intake of products with high amounts of prebiotic fibers and
probiotics. Dairy has a high calcium content and contributes significantly to
total daily calcium intake. To standardize and decrease dietary calcium intake
of the subjects, in order to increase the efficacy of the vaccine2, low-calcium
soy products will be provided to the subjects for the entire study (also see
chapter 9). The low-calcium soy products will be purchased from Bio Soya Desert
Provamel Alpro Soja Nederland BV. The subjects are not allowed to consume
other dairy products during the study. The probiotic Protecflor® will be
supplied as a powder in capsules. The placebo is also a capsule made of the
same material (hydroxypropyl-methylcellulose and titanium oxide) and contains a
powder of identical appearance and consists of the carrier material (potato
starch). Subjects will be instructed to consume the low calcium soy products at
breakfast and dinner. In addition, subjects are requested to ingest a capsule
twice daily, one at breakfast and one at dinner, and during the entire study.
Each probiotic capsule contains 5 x109 Protectflor®. The capsules will be
provided in a bottle and can be stored at room temperature.

After an adaptation period of 2 weeks to the intervention products, subjects
will be infected with a single oral dose of attenuated ETEC strain E1392-75-2A
at a dose of 1010 CFU.2 Oral infection will occur between 10.00 h and 11.00 h
AM. Before taking ETEC, subjects are not allowed to eat for 4 hrs and not to
drink for 2 hrs . Thereafter, and under supervision of the project team, they
will get a NaHCO3 solution (100 ml 2% NaHCO3) to neutralize the gastric acid.
After 5 minutes, they get a fruit juice (100 ml) containing the ETEC strain at
the above-mentioned dose. Subjects go home, but are not allowed to drink and
eat for 1 hour.

Before and after infection, the subjects are asked to fill in a 2x24 hrs
nutrition diary and report and estimate amounts of all foods and drinks eaten.
Bowel habits (defaecation frequency) and frequency and severity of
gastrointestinal symptoms (flatulence, bloating, abdominal pains and cramps)
are self-recorded daily in a diary, using Visual Analogue Scales (VAS; range
0-5 from none to severe) wherever appropriate. Blood samples (10 ml) will be
taken by qualified staff of a local hospital on 1 time point before on 2 time
points (day 3 and 14) after ETEC infection. Before (on 2 separate days) and
after ETEC infection (on 5 separate days), 24 hrs faecal samples will be
collected. All materials and information needed for proper collection of the
faecal samples (stool collection kit) will be supplied by NIZO food research
and delivered to the subjects. Feces will be refrigerated immediately after
defecation. Subjects will be asked to store feces in mini-freezers, supplied by
NIZO food research. Every 3 days, the frozen feces will be transported to the
lab, weighed, homogenized, and analyzed for ETEC by QPCR Homogenized faecal
sub-samples will be frozen and stored (at -20 oC) for later analyses. Diarrhea
will be quantified by analyses of fecal wet and dry weight. Results will be
compared with self-reported information on stool consistency (Bristol stool
scale). A time schedule of the PROTETEC study is added as appendix A.

The PROTETEC study is a dietary intervention with oral probiotics. Twice daily,
at breakfast and dinner, subjects consume 5 x 10E9 colony forming units
Protecflor. The probiotics are supplied as capsule containing dry powder, and
need to be consumed together with the supplied soy products. The control group
receives a placebo capsule of identical appearance which consists of carrier
material only, to be used with the supplied soy products as mentioned above.

Safety information on ETEC strain:
ETEC strain E1392-75-2A (supplier: Acambis, Cambridge, UK) is a spontaneous
mutant unable to produce toxins. The strain obtained is 100% pure. Because of
its streptomycin-resistance it can be discriminated from other E. coli species
that are part of the endogenous microbiotia and excreted in faeces. ETEC
E1392-75-2A is sensitive to ciproxin, which is a commonly used antibiotic for
treatment of E. coli infections in humans. Vaccination experiments with this
ETEC strain in humans are published by e.g. Tacket et al. (Vaccines against
enterotoxigenic E. coli infections. In: New generation vaccines. Eds. Levine et
al., Marcel Dekker Inc. New York, 1997:875-883). In their study, after oral
administration of 1010 CFU, 15% of the vaccinated persons suffered from
self-limited, mild diarrhea with spontaneous recovery after 1-3 days. The most
recent human intervention study at NIZO food research with this strain
performed in 2010 showed that 79% of infected volunteers suffered from a mild
and transient diarrhea for 1-3 days when orally dosed with 10E10 colony forming
units. Besides this, 74% of the volunteers experienced a mild abdominal pain,
63% reported bloating and 26% reported fever. Other symptoms, e.g. nausea and
vomiting, were not reported and complications are not expected.

Safety information on Protecflor:
The active substance in Protecflor consists of the probiotic yeast
Saccharomyces cervisiae var boulardii (CNCM I-1079) combined with 3 strains of
lactic acid bacteria Lactobacillus helveticus R0052 (CNCM I-1722),
Lactobacillus rhamnosus R0011 (CNCM I-1720) and Bifidobacterium longum R0175
(CNCM I-3470). Each of the individual strains in Protecflor have been consumed
in nutritional and dietary supplements for more than 20 years without any
reported side-effects and have not been identified in infections. All strains
are registered in the National Collection of Microorganism Cultures (CNCM) at
the Intitute Pasteur in France. All four species present in Protecflor haven
been granted Qualified Presumption of Safety status by the European Food Safety
Authority (EFSA 2011). The International Dairy Federation in collaboration with
the European Food and Feed Cultures Association (IDF/EFFCA 2002) assembled a
list of microorganisms with a documented history of safe use in food.
Saccharomyces cervisiae var boulardii, Lactobacillus helveticus, Lactobacillus
rhamnosus and Bifidobacterium longum are on that list. The genomes of the 4
microorganisms in Protecflor have been sequenced and annotated (internal
results Lallemand). The genome for Lactobacillus rhamnosus has been published
(Tompkins et al, 2012). The four strains in Protecflor have been tested for
antibiotic resistance genes using minimal inhibitory concentration breakpoints,
according to the recommendations of the FEEDAP Panel (2008). None of the
strains carried any known antibiotic resistance gene (internal results
Lallemand). Further testing confirmed the lack of antibiotic resistance genes
(internal results Lallemand). Primer sets exists for all four microorganisms in
Protecflor, which allow the detection and enumerations of the strains in
various samples (internal results Lallemand).

Benefits for subjects:
There are no direct benefits for the subjects from participation to the
PROTETEC study. The single oral administration of the ETEC vaccine strain to
the subjects offers no protection against E. coli infections in the future.
Previous studies with this vaccine strain have shown that single oral
administration leads to a rise of specific serum antibody titers, but the
quality and quantity of the effect is considered inadequate for significant
protection against subsequent infections. Only after repeated vaccinations
protection would be induced against a very specific (and thus small) group of
bacterial pathogens. Although, there are not direct benefits for the study
subjects, a positive study outcome can offer advantages for population groups
in the future. When Protecflor® does improve resistance to ETEC infection, it
will be possible to decrease intestinal infection incidence by providing
relatively simple dietary advices and probiotic products, e.g. to travelers to
tropical countries