This prospective study demonstrates that differences in gut microbiota composition in early infancy are associated with the subsequent development of atopic disease manifestation and sensitisation. The presence of E coli was associated with a higher risk of developing eczema, this risk being increased with increasing numbers of E coli. However, this association was not found when atopic dermatitis was defined according to the UK‐WP criteria. Colonisation with C difficile in early infancy was associated with an increased risk of all atopic outcomes (eczema, atopic dermatitis, recurrent wheeze and atopic sensitisation), independent of the concentration of this bacterium.
This is the first large‐scale prospective study on gut microbiota composition in relation to atopic manifestations. Although previous epidemiological studies were very informative and were the first to suggest a potential role of the gut microbiota in the aetiology of atopic diseases, they were often based on small populations and were not able to adjust for potential confounders. Furthermore, only two previous studies9,11
were prospective and therefore able to determine if differences in the gut microbiota precede the development of atopic symptoms. Another strength of our study is the molecular techniques used, which overcome many of the problems associated with traditional bacteriological culture. Analyses of the gut microbiota using bacteriological culture are biased, since many selective culture media are not absolutely selective. Furthermore, these media do not equally support the growth of the different species comprising a population, and not all bacteria are cultivatable.37,38
The real‐time PCR assays used in the present study are a quantitative culture‐independent approach suitable for high‐throughput analyses of both fresh and frozen samples.
At present, faeces is the only realistic sample available in large non‐invasive epidemiological studies on the gut microbiota. However, a limitation of using faecal samples is that the bacterial composition in the lumen does not reflect the composition of bacteria adhering to the mucosa, and furthermore the composition of bacteria differs throughout the intestinal tract.39
Nevertheless, it can be assumed that although the proportions and activities of the microbiota change with passage through the intestinal tract, most viable and non‐viable intestinal bacteria will still be detectable in the faeces by molecular methods.40
A drawback of this study is the time between collection of the samples by the parents and processing of the samples in the laboratory, which was 1 day for most samples. Ott and colleagues41
demonstrated that the total amount of bacterial DNA as well as the diversity of the microbiota significantly decreased over such a time span. However, they also showed that the similarity (determined by DGGE) of faecal samples processed immediately and processed after 24 h remained high. This means that the dominant microbiota appears to be relatively stable. Furthermore, the aim of the present population‐based study was to examine differences in gut microbiota composition between subjects. It is not likely that the possible change in composition of the samples during transportation was different for infants who developed atopic manifestations later on in life and infants who did not.
In the present study, only one faecal sample per infant was collected at the age of 1 month post partum. There are several reasons why we chose to collect faeces at this age. First, as the gut microbiota is thought to drive the postnatal development of the immune system,12
the first months of life seem to be of major importance. Second, at the age of 1 month colonisation is complete and although the composition may fluctuate, large shifts in the composition do not occur until weaning.42
Finally, as we wanted to exclude the chance of reverse causation, the gut microbiota composition had to be analysed prior to the manifestation of atopic symptoms.
Since manifestation of atopic symptoms and sensitisation to allergens do not always occur together (sensitised infants do not always show symptoms and infants with symptoms are not always sensitised),43
we chose to report the manifestation of atopic symptoms and sensitisation as separate outcome parameters. As a consequence we decided to use the term eczema instead of atopic eczema or atopic dermatitis. However, we did use the term atopic dermatitis for those infants visited at home who fulfilled the UK‐WP criteria, since Williams and colleagues when introducing these criteria originally used the term atopic dermatitis.22
The positive association we found between E coli
and eczema is difficult to compare with previous reports, since several of these studies did not determine E coli9
or measured this bacterium as part of total coliforms,8
or Gram‐negative rods.11
In contrast to the association between E coli
and eczema (based on parents' reports in questionnaires), we did not find an association between E coli
and atopic dermatitis based on the UK‐WP criteria. Selection bias could not explain this difference since non‐response analyses showed that the gut microbiota and the prevalence of eczema and recurrent wheeze was similar for those infants who were visited at home and those who were not. The percentage of infants with atopic dermatitis according to the UK‐WP criteria is much lower than the percentage of infants with eczema reported by parents. This can be explained by the fact that eczema reported by parents is based upon the presence of this condition at any time during the infants' first 2 years of life, whereas many of these infants are probably in remission or have already outgrown this condition at time of the home visit. Therefore, it is possible that E coli
is only associated with a milder eczematous condition that has already disappeared by the time of the home visit. Another explanation for these discrepant findings is that the UK‐WP criteria included specific predilection sites of atopic dermatitis (flexural involvement),22,23,24
whereas the questionnaire data were based on the presence of an itchy rash anywhere (except diaper rash, rash around the eyes and scalp scaling). The increased risk of eczema in infants colonised with E coli
may therefore also be limited to eczema other than at the specific predilection sites for atopic eczema/dermatitis and thus may be non‐atopic. This idea is supported by the fact that we also found no association between E coli
In contrast, C difficile
appears to be associated with a higher risk of atopic eczema since a positive association was also found for atopic dermatitis according to UK‐WP criteria and for sensitisation. Our findings of an association between C difficile
and atopy are in agreement with several previous studies. In a study on microflora‐associated characteristics, allergic infants had higher levels of i‐caproic acid in their stools compared with non‐allergic infants. This short chain fatty acid is suggested to indicate the presence of C difficile
Two studies used IgG serology against C difficile
. Woodcock and colleagues found increased specific IgG against C difficile
in sensitised wheezy infants compared with non‐sensitised non‐wheezy infants.45
Linneberg and colleagues found a positive association between IgG seropositivity against C difficile
and both allergic rhinitis and sensitisation.46
Furthermore, several studies found an association between high numbers of the genus Clostridium and atopic dermatitis and/or sensitisation,9,11,47
although others did not find such an association.10,48
The genus Clostridium is a very heterogeneous group comprising several different clusters.49
It seems therefore unlikely that the members of such a phyologenetically diverse genus like Clostridium all have the same effect on the human host. It is more likely that certain species (such as C difficile
) or a certain cluster of species within this genus is responsible for the increased risk of developing atopic manifestations.
In contrast to several other studies,8,9,10,11,47
we did not find a negative association between allergies and bifidobacteria. A possible explanation is the lack of contrast in our study with respect to bifidobacterial counts, because almost all infants were colonised with high numbers of bifidobacteria. This is probably the consequence of the very young age of our population, an age at which bifidobacteria are known to dominate the gut microbiota.50
Altogether our results support a role of the gut microbiota in the aetiology of atopic diseases. There are several hypotheses by which the associations we found between C difficile
and E coli
and atopic manifestations could be explained. First of all it should be noted that many of the bacteria in the gut are still unknown; differences in E coli
and C difficile
colonisation found in our study could therefore also reflect differences in other unknown bacteria.9
The presence of E coli
and C difficile
could be associated with a decrease in other (unknown) beneficial bacteria. This could result in reduced induction of Treg cells by these beneficial bacteria leading to immune dysregulation. In the absence of optimal levels of immune regulation, an individual may develop a Th1 (such as Crohn's disease or autoimmunity) or Th2 (such as atopic diseases) mediated inflammatory disorder depending on their own Th1/Th2 bias, immunological history and genetic background.7
Secondly, E coli
and C difficile
could have a direct effect on the production of cytokines by antigen‐presenting cells, thereby affecting the differentiation of T cells.51
Another hypothesis is that E coli
and/or C difficile
increase the intestinal permeability (for instance by the production of toxins). This increased permeability of the intestinal barrier could facilitate the penetration of innocuous antigens and subsequent sensitisation.46
Indeed it has been shown that C difficile
toxins A and B compromise the intestinal cell barrier.52,53
Furthermore, increased intestinal permeability has been described in patients with food allergies, eczema and asthma compared with healthy subjects.54,55,56,57
Finally, it has also been suggested that infants susceptible to the development of allergies are also susceptible to aberrant colonisation of the gut. However, this explanation seems less likely since we have controlled for familial history of atopy. Additionally, the fact that differences in the gut microbiota are already present at such a young age preceding atopic symptoms makes this hypothesis less likely.
The consistent findings of a positive association between C difficile
and all atopic symptoms as well as sensitisation strengthen the probability of a causal relationship between the gut microbiota and atopy, and support the potential role of probiotics in the prevention and treatment of these diseases. We have previously examined the external factors influencing the composition of the gut microbiota in early infancy.25
Caesarean section, antibiotic therapy, hospitalisation and formula feeding all caused perturbations in the gut microbiota by increasing colonisation rates and counts of E coli
and C difficile
and/or by decreasing numbers of bifidobacteria. In particular, when the gut microbiota is disrupted by one or more of these external factors, probiotics may be effective in the treatment or prevention of atopic diseases.
Perturbations in the gut microbiota may also be related to other atopic outcomes which manifest at older ages such as asthma, rhinoconjunctivitis and persistent food allergies; long term follow‐up of cohort studies is necessary to examine whether perturbations are also related to these outcomes.
In conclusion, we demonstrated that differences in the gut microbiota composition precede the manifestation of atopic symptoms and atopic sensitisation. In particular, C difficile was associated with all atopic symptoms and sensitisation, whereas E coli appeared to be only associated with (non‐atopic) eczema. Different immunological mechanisms may underlie the effects of E coli and C difficile. This calls for further research on the mechanisms by which intestinal microbes interfere with our (gastrointestinal) immune system.