Doi:10.1016/j.vaccine.2006.05.048

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 Abstract
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.
Keywords: Probiotics; Respiratory tract infections; Immune system 1. Introduction
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.
E-mail address: (M. de Vrese).
the administration of Bifidobacterium breve YIT4064 aug- 0264-410X/$ – see front matter 2006 Elsevier Ltd. All rights reserved.
doi: 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- lus gasseri PA 16/8, Bifidobacterium longum SP 07/3, B. ters who consumed milk containing Lactobacillus rhamnosus bifidum MF 20/5 (Tribion harmonisTM 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 preparation 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 caseiDN-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 2. Methods
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 ingested (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 and Chlamydiapneumoniae.
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-test.
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), χ2-tests (incidence of infections) and independent The mean absolute numbers of cells per gram stool Student’s t-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 3. Results
(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) −1␮L blood (mean ± S.E.M.). TH, T helper cells (CD4+). TC + TS cytotoxic T cells + T suppressor cells (CD8+).
* p-Values from independent Student’s t-test.
4. Discussion
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 marginally (p < 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 Bifidobacterium longum 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.
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