The effect of Lactobacillus acidophilus NCFM on E. coli gastroenteritis

Foodborne infections occur frequently. In European countries 10-25% of the
population suffer from at least one foodborne infection per year. This number
increases sharply in travelers to tropical countries, with incidences up to
80%. After some days of diarrhea, stomach pain, nausea or vomiting, most
infections are self-limited and cured. However, such ordinary infections can be
life-threatening 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.
Lactobacillus acidophilus NCFM* is a probiotic strain originally isolated from
the faeces of a healthy adult. The strain has been marketed without any known
side effects since the 1970*s. Its complete genome sequence indicates the
strain does not carry any known antibiotic resistance genes.6 Human studies
have shown that the strain survives gastrointestinal transit.7,8 Thanks to the
existence of a strain specific primer pair, it can be fully traced.8 The
species L. acidophilus has also been granted Qualified Presumption of Safety
status by the European Food Safety Authority.9 Human intervention studies have
shown that consumption of the strain affects the composition3 and activity4 of
the faecal microbiota. The strain also affects the small intestinal
microbiota.10,11 Consumption of the strain, together with an oral cholera
vaccine has been shown to lead to a more rapid immune response.5 In combination
with another strain (Bifidobacterium lactis Bi-07), the strain has been
observed to reduce diarrhoea.12 The strain also improved general health and
well being in children. Although the diarrhea incidence was low in the study
and no difference could be observed, the respiratory infection rate was
dramatically reduced.13 In addition to these human studies, animal studies have
shown that administration of the strain protects immune-deficient mice from
Candida albicans infection14 and modulates various functions of the immune
system.15
In the present double-blind, placebo-controlled, randomized parallel study, the
effect of oral Lactobacillus acidophilus NCFM* vs placebo will be studied on
the resistance of humans to enterotoxigenic Escherichia coli infection (ETEC).
The main hypothesis is that Lactobacillus acidophilus NCFM* 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 Lactobacillus acidophilus NCFM* 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 Lactobacillus acidophilus NCFM* will
decrease fecal ETEC excretion by 1 log10 units. As a consequence, diarrhea
severity, as measured by total daily faecal 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
Lactobacillus acidophilus NCFM* or placebo. Subjects will be instructed to
maintain their usual pattern of physical activity and their habitual diet,
except for their dairy intake and products containing 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 vaccine, low-calcium milk and low-calcium vanilla custard will be provided
to the subjects for the entire study (also see chapter 9). The low-calcium
dairy products will be produced under GMP conditions and supplied by NIZO food
research. The subjects are not allowed to consume other dairy products during
the study. The probiotic Lactobacillus acidophilus NCFM* will be supplied as a
powder in capsules. The placebo is also a powder of identical appearance and
consists of the carrier material (microcrystalline cellulose). Subjects will be
instructed to open a capsule and mix its contents with a glass of supplied
dairy (150 ml per portion) and drink it immediately after preparation. This
will be done twice daily, preferably at breakfast and dinner, and during the
entire study. Each probiotic capsule contains 10E9 CFU Lactobacillus
acidophilus NCFM*. 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 single oral dose of attenuated ETEC strain E1392-75-2A at
a dose of 10E10 CFU. 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 (50 ml) containing
the ETEC strain at the above-mentioned dose. This is followed by consumption of
150 ml low-calcium milk with either added Lactobacillus acidophilus NCFM* or
placebo, depending on assignment. Subjects go home, but are not allowed to eat
and drink 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 2 time points 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, transported to the lab, weighed,
homogenized, and analyzed for ETEC within 24 hours after defecation.
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).

The PRETEC study is a dietary intervention with oral probiotics. Twice daily,
preferably at breakfast and dinner, subjects consume 10E9 colony-forming units
of Lactobacillus acidophilus NCFM. The probiotic is a dry powder and has to be
mixed with the supplied dairy products (low-calcium milk and vanilla custard,
150 ml each). The control group receives a powder of identical appearance which
consists of carrier material only, to be used with the supplied dairy 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. In a
previous ETEC study of NIZO food research (Bovee-Oudenhoven et al.
Gastroenterol 2003; 125: 469-476) using the same infection dose, almost all
subjects consuming dietary placebo experienced mild diarrhea for 2 days.
Reported infection symptoms were: stomach pain (34%), headache (25%), bloating
(22%) and a slight rise of body temperature (13%), indicating a mild type of
foodborne infection. Other symptoms, e.g. nausea and vomiting, were not
reported and complications are not expected.

Safety information on Lactobacillus acidophilus NCFM*:
The strain has been marketed without any known side effects since the 1970*s.
Its complete genome has been sequenced and published (Alterman E et al. Proc
Natl Acad Sci USA 2005; 102: 3906-3912). The sequence indicates that the strain
does not carry any known antibiotic resistance genes. The species L.
acidophilus has also been granted Qualified Presumption of Safety status by the
European Food Safety Authority (Andreoletti O et al. EFSA J 2008; 923: 1-48).
Thanks to the existence of a strain specific primer pair, it can be fully
traced (Ouwehand AC et al. Br J Nutr 2009; 101: 367-375).

Benefits for subjects:
There are no direct benefits for the subjects from participation to the PRETEC
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 Lactobacillus acidophilus NCFM* 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.