Probiotic bacteria reduced duration and severity but not
the incidence of common cold episodes in a
double blind, randomized, controlled trial
Michael de Vrese , Petra Winkler , Peter Rautenberg , Timm Harder ,
Christian Noah , Christiane Laue , Stephan Ott , Jochen Hampe ,
Stefan Schreiber , Knut Heller , J¨urgen Schrezenmeir
a Institute of Physiology and Biochemistry of Nutrition, Federal Research Centre of Nutrition and Food,
Hermann-Weigmann-Straße 1, D-24103 Kiel, Germany
b Institute for Medical Microbiology and Virology, Christian-Albrechts-University Kiel, D-24105 Kiel, Germany
c Central Veterinary Laboratory, D-24537 Neum¨unster, Germany
d Centre for Biotechnology and Nutrition, D-24229 Kiel, Germany
e Institute for Clinical Molecular Biology, Christian-Albrechts-University Kiel, D-24105 Kiel, Germany
f Institute of Microbiology, Federal Research Centre of Nutrition and Food, D-24103 Kiel, Germany
To investigate the effect of long-term consumption of probiotic bacteria on viral respiratory tract infections (common cold, influenza), a
randomized, double blind, controlled intervention study was performed during two winter/spring periods (3 and 5 month). Four hundred andseventy-nine healthy adults were supplemented daily with vitamins plus minerals with or without probiotic lactobacilli and bifidobacteria.
The intake of the probiotic had no effect on the incidence of common cold infections (verum = 158, control = 153 episodes, influenza was notobserved), but significantly shortened duration of episodes by almost 2 days (7.0 ± 0.5 versus 8.9 ± 1.0 days, p
= 0.045), reduced the severityof symptoms and led to larger increases in cytotoxic T plus T suppressor cell counts and in T helper cell counts.
2006 Elsevier Ltd. All rights reserved.
Probiotics; Respiratory tract infections; Immune system
the colon stimulation of humoral and cellular immu-nity and positive effects on allergy and atopic diseases in
Probiotics are defined live microorganisms which reach
children There is further some evidence for reduction
the intestine in sufficient number and in an active state
of Helicobacter pylori
affection, relief from constipation and
and which thus exert positive health Therefore probi-
options for use in inflammatory diseases of the gut
otic effects mainly result from modulations of the intestinal
The action of probiotics is not exclusively developed in the
microflora or are mediated by the gut associated immune sys-
intestine. By modulating immunological parameters, influ-
tem (GALT) Well proven by investigations in humans
encing absorption and secretion in the intestinal mucosa, after
are the following effects: lower frequency and duration
bacterial translocation, or mediated by products from carbo-
of diarrhea associated with rotavirus infections, antibiotic
hydrate fermentation and other microbial metabolic perfor-
treatment, chemotherapy or lactose intolerance, decrease in
mances probiotic effects also affect other organic systems
unfavourable metabolites and procancerogenic enzymes in
The first evidence that probiotic strains could be effec-
tive in respiratory tract infections came from mice, where
Corresponding author. Tel.: +49 431 609 2471; fax: +49 431 609 2472.
(M. de Vrese).
the administration of Bifidobacterium breve YIT4064 aug-
0264-410X/$ – see front matter 2006 Elsevier Ltd. All rights reserved.
M. de Vrese et al. / Vaccine 24 (2006) 6670–6674
mented anti-influenza IgG production and protected against
The probiotic strains used in this study were Lactobacil-
influenza infection Finnish children in day care cen-
PA 16/8, Biﬁdobacterium longum
SP 07/3, B.
ters who consumed milk containing Lactobacillus rhamnosus
MF 20/5 (Tribion harmonis
TM The number
GG (ATCC 53103) during 7 months in winter had 17%
of viable probiotic bacteria declared for the product dur-
fewer respiratory tract infections than the control group
ing the study period was guaranteed by long term stability
The administration of an Enterococcus faecalis
tests carried out by the supplier. The vitamins and miner-
(Symbioflor®) resulted in fewer relapses of recurrent res-
als in this preparation were according the EC recommended
piratory tract infections, especially bronchitis and sinusitis
daily allowance (RDA). All test preparations were prepared,
In a 3 weeks observation period in elderly people,
packaged, randomized and labeled by the supplier (Merck,
the duration of gastrointestinal plus respiratory illnesses was
significantly lower in the group receiving Lactobacillus casei
DN-114001 than in the control group These studies,
however, neither analyzed the respective pathogen nor dis-tinguished between bacteria- and virus-induced infections at
Two hundred and forty-two and 237 participants were
observed between January and May 2001 or between Decem-
The aim of the present study was to investigate whether the
ber 2001 and June 2002, respectively. The volunteers were
long-term consumption of certain probiotic bacteria affects
asked to take one tablet of the test preparation per day, in
viral respiratory tracts infections, especially influenza or the
addition to their usual diet. They collected the tablets from
common cold, in otherwise healthy adults. As published
the Federal Research Centre for Food and Nutrition every 4
data suggest that probiotics influence duration and severity
weeks. This ensured close contact to the volunteers in order
of infectious diseases rather than the incidence, the mean
duration of episodes was chosen as the primary outcomeparameter.
2.5. Assessment of respiratory tract infections
Respiratory tract infection episodes were defined as the
occurence of at least one specific symptom. To exclude bac-
terial infections, episodes in which antibiotics were admin-istered were not included.
The study was carried out double blind, randomized, and
During the episodes the participants used validated
placebo-controlled, with two parallel arms. It was approved
questionnaires to daily record nasal (running nose, stuffed
by the ethics committee of the Medical Faculty of Christian-
nose, blowing the nose, yellow secretion, bloody secre-
Albrechts-University Kiel, Germany. All participants had
tion, sneezing), pharyngeal (scratchy throat, sore throat,
given their written informed consent before inclusion in the
hoarseness) and bronchial symptoms (cough, secretion, yel-
low secretion), as well as headache, myalgia and reddisheyes (conjunctivitis) using a scale from 0 (no symptoms)
to 6 (severe symptoms). Fever (oral temperature >37.7 ◦C)was recorded daily. These symptom scores were com-
Volunteers were recruited by advertisements. A total of
bined and added up to produce daily and total symptom
479 healthy adults (184 men and 284 women, 18–67 years of
age), not vaccinated against influenza within the last 12 monthand without known congenital or aquired immune defectsor allergies, were enclosed after physical examination. Four
hundred and fifty-four of them completed the study. Duringthe study, neither immune stimulating medication nor abnor-
Cellular immune response was assessed in a randomly
mal physical exercise was allowed, and the volunteers were
drawn subgroup of 122 participants (61 per study group)
asked to maintain their usual eating habits but to refrain from
before and after 14 days of supplementation in the first
eating products containing prebiotics and/or probiotics. The
study period. Blood cells were analyzed by flow cytometry
participants were briefed in an informative meeting and in
using the following surface markers: CD45+ (lymphocytes),
written form before the study started.
CD45+, CD19+ (B-lymphocytes), CD45+, CD3+ (T lym-phocytes), CD45+, CD3+, CD4+ (TH cells), and CD45+,
2.3. Test preparations, blinding, and randomization
CD3+, CD8+ (TS plus TC cells), and CD 45+, CD 56+(natural killer cells). T lymphocyte activation was deter-
Due to logistic reasons test and control preparations were
mined measuring CD69 expression in phytohemagglutinin
given in tablet form. They contained either 5 × 107 cfu of
(PHA) incubated whole blood, and phagocytosis was quan-
the spray dried probiotic bacteria plus vitamins and minerals
tified counting fluorescein-labeled Escherichia coli
(verum) or just the vitamin mineral preparation (control).
M. de Vrese et al. / Vaccine 24 (2006) 6670–6674
On day 2 of each episode, nasal secretions were col-
lected for virus identification. Entero- (Coxsackie-, ECHO-),Rhino-, RS- and parainfluenza 3-viruses were characterizedby RT-PCR and cycle sequencing. Sequence data were ana-lyzed using the DNA Star software package, and were identi-fied by BLAST search. PCR products were used as templatesin a second (nested) PCR round to identify parainfluenza- 1,2 and 4, metapneumo- A and B, influenza- A and B and aden-oviruses as well as Mycoplasma pneumoniae
2.8. Quantiﬁcation of fecal lactobacilli andbiﬁdobacteria
Fig. 1. Effect of three strains of probiotic bacteria (5 × 107 cfu/day) onduration of respiratory tract infections (common cold) episodes. Values are
As both living and killed bacteria may have immunos-
mean ± S.E.M. *p
< 0.05, independent Student’s t
timulatory properties, increase in fecal counts of the bacteriaadministered was assessed in a separate trial by quantitative
real-time PCR. Primers and probes of the assays are basedon the 16S rRNA gene and hybridize to variable regions of
Ninety-five samples of nasal secretion were obtained.
the 16S rRNA gene specific for the bifidobacteria and lacto-
Viruses were identified in 24 of these samples: rhinoviruses
bacillus group. Results were averaged from two independent
in 19, RS viruses in five and enterovirus (not specified) in one.
experiments and related to 1 g of stool.
In one sample, a dual viral infection was found. No influenzavirus infection was detected.
Data are presented as mean ± standard error of the mean
3.4. Fecal increase in probiotic lactobacilli and
(S.E.M.). Differences between verum and control group were
analyzed by Mann–Whitney tests (symptom scores, bacterialcounts), χ
2-tests (incidence of infections) and independent
The mean absolute numbers of cells per gram stool
-tests (other parameters) using Statgraphics Plus®
assessed in a separate trial were significantly increased
4.1 software (Manugistics, Rockville, USA).
from day 0 to day 14 days of probiotic supplementa-tion for both the bifidobacteria (3.14 × 107 ± 5.57 × 106versus
(1.04 × 105 ± 3.69 × 104 versus 1.45 × 105 ± 2.48 × 104).
3.1. Incidence, duration and severity of episodes
Although there was no difference in the total number
of episodes between the test groups (verum = 158, con-trol = 153), the mean duration of an episode was signifi-cantly shorter in the verum than in the control group (relativereduction of 21.5%, p
< 0.05), and the severity of symptoms,especially the total symptoms score (p
< 0.05), was reduced
After the consumption of probiotic bacteria for 14 days,
a significantly larger increase in cytotoxic T cells plus Tsuppressor cells (CD8+) and a smaller increase in the otherimmune cells investigated was observed compared to con-
Fig. 2. Effect of three strains of probiotic bacteria (5 × 107 cfu/day) on
trol There was no change in T cell activation and
severity of respiratory tract infections (common cold) episodes. Values are
phagocytic activity during the observation period (data not
mean ± S.E.M. *p
< 0.05, Mann–Whitney test,
M. de Vrese et al. / Vaccine 24 (2006) 6670–6674
Table 1Effect of probiotic bacteria (5 × 107 cfu/day) on cellular immune parameters
On day 0 and day 14 of the first intervention period immune cell counts were assessed by flow cytometry in blood samples from 122 participants (61 per studygroup). Volunteers with abnormal leucocyte counts before intervention were rejected from the analysis. Results are expressed as
0) −1L blood (mean ± S.E.M.). TH, T helper cells (CD4+). TC + TS cytotoxic T cells + T suppressor cells (CD8+).
-Values from independent Student’s t
Different cell lines such as human PBMCs, monocytes,
dendritic cells, and human intestinal mucosa cells have been
In the present randomized, placebo-controlled, double
shown to release pro- and anti-inflammatory cytokines and
blind intervention study in 479 healthy adults, the long term
to express co-stimulatory molecules in response to certain
effects of the administration of three strains of probiotic
(probiotic) bacteria immunomodulation seems
bacteria on common respiratory tract infections were inves-
to be based on bacterial patterns, such as bacterial cell wall
tigated during two winter/spring periods. The total number
components (e.g. lipoteichoic acid) and CpG motifs of DNA
of episodes registered in this study was 311 (extrapolated to
(cytosine–phosphate–guanosine DNA, frequently found in
1.96 episodes per participant per year), which corresponds to
bacterial and viral genome). They are recognized by toll-like
the rule of thumb that adults catch two to four cold episodes
receptors, which are expressed in intestinal and immune cells
per year. The type and the relative weakness of the symptoms
recorded as well as the fact, that no influenza viruses were
Cytotoxic T cell plus T suppressor cell (CD8+) counts
detected indicate, that mainly, if not exclusively, common
showed a significantly higher increase in the probiotic-treated
cold but no influenza episodes were observed.
group during the first 14 days of supplementation compared
In this investigation, symptoms were recorded using self
to the control group. Cell counts of the other immune cells
assessment based questionnaires. This was justified by stud-
were increased too in the verum group, however, not or only
ies, which demonstrate a good concordance between the
< 0.1) significant. This may be due in part to
individual’s own estimation of symptoms and an evaluation
the fact that both the probiotics and the control preparation
contained, for technical reasons, supplemental vitamins and
The use of probiotic bacteria significantly shortened the
minerals, some of which have immunostimulatory properties;
mean duration of the episodes by about 2 days and reduced
this might have blurred differences between the groups
the severity of symptoms. There, however, was no influ-
In conclusion, the present study presents evidence for the
ence on the over all incidence of respiratory tract infections.
positive effects of long-term consumption of probiotic bac-
Although the endpoints were weaker in the present study,
teria, especially the tested mixture of Lactobacillus gasseri
the effects were in the same order of magnitude as those of
and B. biﬁdum
strains, on the sever-
neuraminidase inhibitors in the case of acute influenza which
ity of viral respiratory tract infections in otherwise healthy
reduced the severity of symptoms and shortened influenza by
adults. This might offer options for use of probiotic bacteria
1 or 2.5 days, respectively, when administered within the first
for prevention or treatment of viral infections of the respira-
48 h of the infection, or prophylactically In contrast
tory tract in addition to the already extensively studied options
to neuraminidase inhibitors, probiotics may be ingested as
part of the daily diet and then do not induce extra costs to thehealth system.
The reduction in the severity and duration of common cold
episodes may be due to immune stimulatory effects of living
 Arbeitsgruppe “Probiotische Mikroorganismenkulturen in Lebens-
or dead probiotic bacteria during gastrointestinal passage.
mitteln” am BgVV. Abschlußbericht. Ern¨ahrungs-Umschau 2000;47:191–5.
Indeed a substantial increase of bacterial DNA of the adminis-
 Cummings JH, Antoine JM, Azpiroz F, Bourdet-Sicard R, Brandtzaeg
tered lactobacilli and bifidobacteria strains in the feces could
P, Calder PC, et al. PASSCLAIM—gut health and immunity. Eur J Nutr
be demonstrated by quantitative RT-PCR.
M. de Vrese et al. / Vaccine 24 (2006) 6670–6674
 Marteau PR, de Vrese M, Cellier CJ, Schrezenmeir J. Protection from
 Turchet P, Laurenzano M, Auboiron S, Antoine JM. Effect of
gastrointestinal diseases with the use of probiotic bacteria. Am J Clin
fermented milk containing the probiotic Lactobacillus casei
114001 on winter infections in free-living elderly subjects: a ran-
 Marteau P, Seksik P, Lepage P, Dore J. Cellular and physiological
domised, controlled pilot study. J Nutr Health Aging 2003;7(2):
effects of probiotics and prebiotics. Mini Rev Med Chem 2004;4(8):
 Honma M. On effect of lactic acid bacteria: biological significance.
 Sullivan A, Nord CE. Probiotics and gastrointestinal diseases. J Intern
 Stansfield SK, Pierre-Louis M, Lerebours G, Augustin A. Vitamin A
 O’Sullivan GC, Kelly P, O’Halloran S, Collins C, Collins JK,
supplementation and increased prevalence of childhood diarrhoea and
Dunne C, et al. Probiotics: an emerging therapy. Curr Pharm Des
acute respiratory infections. Lancet 1993;342:578–82.
 Mossad S, Macknin M, Medendorp S, Mason P. Zinc gluconate
 Blum S, Haller D, Pfeifer A, Schiffrin EJ. Probiotics and immune
lozenges for treating the common cold: a randomised, double-blind,
response. Clin Rev Allergy Immunol 2002;22:287–309.
control-controlled study. Ann Intern Med 1996;125:81–8.
 Rautava S, Kalliomaki M, Isolauri E. New therapeutic strategy for
 MacIntyre S, Pritchard C. Comparisons between the self-assessed
combating the increasing burden of allergic disease: probiotics—a
and observer-assessed presence and severity of colds. Soc Sci Med
Nutrition, Allergy, Mucosal Immunology and Intestinal Microbiota
(NAMI) Research Group report. J Allergy Clin Immunol 2005;116:31–
 Treanor JJ, Hayden FG, Vrooman PS, Barbarash R, Bettis R, Riff
D, et al. Efficacy and safety of the oral neuraminidase inhibitor
 Shanahan F. Physiological basis for novel drug therapies used to
oseltamivir in treating acute influenza: a randomized controlled
treat the inflammatory bowel diseases I. Pathophysiological basis and
trial. US Oral Neuraminidase Study Group. JAMA 2000;283:1016–
prospects for probiotic therapy in inflammatory bowel disease. Am J
Physiol Gastrointest Liver Physiol 2005;288(3):G417–21.
 Hayden FG, Gubareva LA, Monto AS, Klein TC, Elliott MJ, Hammond
 de Vrese M, Schrezenmeir J. Probiotics and non-intestinal infectious
JM, et al. Inhaled Zanamivir for the prevention of influenza in families.
conditions. Br J Nutr 2002;88:59–66.
 Yasui H, Kiyosima J, Hori T, Shida K. Protection against influenza
 Borruel N, Casellas F, Antol´ın M, Llopis M, Carol M, Esp´ıin E, et
infection of mice fed Biﬁdobacterium breve
YIT4064. Clin Diagn Lab
al. Effects of nonpathogenic bacteria on cytokine secretion by human
intestinal mucosa. Am J Gastroenterol 2003;98:865–70.
 Hatakka K, Savilahti E, Ponka A, Meurman JH, Poussa T, Nase L,
 Christensen HR, Frokiaer H, Pestka JJ. Lactobacilli differentially mod-
et al. Effect of long term consumption of probiotic milk on infections
ulate expression of cytokines and maturation surface markers in murine
in children attending day care centres: double blind, randomised trial.
dendritic cells. J Immunol 2002;168(1):171–8.
 Cario E, Podolsky DK. Differential alteration in intestinal epithelial cell
 Habermann W, Zimmermann K, Skarabis H, Kunze R, Rusch V. Influ-
expression of toll-like receptor 3 (TLR3) and TLR4 in inflammatory
ence of a bacterial immunostimulant (human Enterococcus faecalis
bowel disease. Infect Immun 2000;68(12):7010–7.
bacteria) on the recurrence of relapses in patients with chronic bron-
 Muzio M, Bosisio D, Polentarutti N, D’amico G, Stoppacciaro A,
chitis. Arzneimittelforschung 2001;51:931–7.
Mancinelli R, et al. Differential expression and regulation of toll-like
 Habermann W, Zimmermann K, Skarabis H, Kunze R, Rusch V. Reduc-
receptors (TLR) in human leukocytes: selective expression of TLR3 in
tion of acute relapses in patients with chronic recurrent hypertrophic
dendritic cells. J Immunol 2000;164:5998–6004.
sinusitis during treatment with a bacterial immunostimulant (Ente-
 Chandra RK. Effect of vitamin and trace-element supplementation
bacteria of human origin - a medical probiotic).
on immune responses and infections in elderly subjects. Lancet
GUIDELINES FOR APPROVAL OF CONVENTION CENTRES Meetings, Incentives, Conference and Exhibitions (MICE) are today becoming an important segment of the tourism industry. With the opening up of India’s economy, MICE tourism is likely to grow further in the future. Our country therefore needs more Convention and Exhibition Centers to meet the requirement of this lucrative segment of the touri
Inquiry Based Learning using iWorx Systems Case Study #3 – The Stressed Out College Professor Instructions: Week # 1: Design Day After a group discussion of this case, each lab group will develop… a. A hypothesis to be tested. This may be big or small, but must be related in some way to the case. Remember that you are limited in what you can test. b. You will then develop a set