The intestinal microbiota is a complex ecosystem of great importance for health and is composed of both aerobic and anaerobic bacteria.1 The gastrointestinal tract may be considered sterile during intrauterine life and is colonised after birth. Bacterial colonisation depends on several factors, particularly the type of childbirth, vaginal or caesarean,2 and the method by which the child is fed during the first months of life.

Evidence suggests that possible imbalances in the composition of the intestinal microbiota and its relationship with the host may be implicated in the occurrence of allergic diseases.3 This hypothesis has led to a number of investigations to elucidate the relationship between the composition and function of the intestinal microbiota and the onset of allergic diseases.4

Penders et al.5 reviewed 18 studies on the association between the intestinal microbiota and allergic diseases. This study evaluated papers published between 1999 and 2006, primarily observational studies that compared healthy and allergic individuals. The profile of the intestinal microbiota of individuals with eczema/atopic dermatitis, food allergy, wheezing, allergic rhinitis, asthma and/or sensitisation was evaluated. The composition of the faecal microbiota was analysed using different laboratory techniques, from traditional bacterial culture to novel molecular biology techniques. Most studies indicated an association between microbiota composition and atopic symptoms and/or sensitisation. However, it has not yet been possible to distinguish protective microorganisms from those associated with increased risk of allergic diseases. The differences in the results were due to differences in the types of studies and laboratory techniques used to assess the composition of the intestinal microbiota.

In this context, this systematic study aimed to review the literature published between 2007 and 2013 that addresses the intestinal microbiota in allergic diseases.

In this review, all studies published between 2007 and 2013 that compared the intestinal microbiota of allergic patients (atopic dermatitis, food allergy, wheezing, allergic rhinitis, asthma and/or atopic sensitisation) with those of healthy individuals were evaluated. Clinical trials with interventions were not included except when the composition of the intestinal microbiota on admission was evaluated before the intervention.

The studies were evaluated for sample size, place of study, age range, study design, criteria for the diagnosis of allergic diseases, and composition of the faecal microbiota.

To identify studies addressing this topic, an extensive literature search was conducted using the following databases: 1. PubMed, a database developed by the National Center for Biotechnology Information (NCBI) at the National Library of Medicine (NLM), which integrates information from NCBI databases with the PubMed database and includes MEDLINE, nucleotide sequences, protein sequences, macromolecular structures, and whole genome sequences; 2. IBECS, a database that uses the LILACS methodology and allows the selection of scientific studies on health sciences published in Spain; and 3. LILACS, a database produced by the institutions that form the Latin American and Caribbean System on Health Sciences Information. LILACS has catalogued technical–scientific data in the health sciences produced by Latin American and Caribbean authors published since 1982.6

For the search, the following keywords and limits were used: (“Intestines” [Mesh] OR intestin* OR gut OR gastrointestin* OR enteric) AND (flora OR microbiolog* OR microflora OR bacteria OR bacterial OR colonisation OR colonisation OR microbes OR microbial OR microbiota) AND (hypersensitivity OR atopic dermatitis OR allergic OR allergy OR atopic OR atopy OR eczema OR rhinitis OR asthma), as previously reported.5

Initially, 1134 studies were found in PubMed, IBECS, and LILACS databases. Of these, one study was found in duplicate that was catalogued in both IBECS and LILACS. Therefore, an analysis of titles and/or abstracts of 1133 studies was performed considering the inclusion criteria. After this analysis, 1105 studies were excluded, and 28 studies remained. Of the 28 studies, seven were excluded after analysis of the full article because they did not meet the inclusion criteria; i.e., four were intervention studies that did not provide information on the microbiota at admission,7–10 two involved children born to mothers who received probiotic intervention during gestation,11,12 and one did not compare the microbiota of allergic and non-allergic patients.13 Therefore, 21 articles were included in the analysis (Fig. 1).

Figure 1.

Study flowchart.

(0.11MB).

In Tables 1 and 2, the abstracts are presented according to the period in which stool collections were performed in relation to the assessment of allergic diseases. Table 1 lists 16 studies wherein stool collection was performed before assessment of the presence of allergic diseases; and Table 2 lists five studies wherein stool collection was performed during the assessment.

Among the 16 studies that performed stool collection before the assessment, 14 were prospective, and two were non-prospective studies, totalling 4246 stool samples included for analysis. In five prospective studies, the experimental groups contained more than 300 children14–18 who were monitored for 18 months to seven years. In four studies, the groups comprised between 90 and 160 children19–22 who were monitored for 12 months to three years, and in five studies, the groups comprised fewer than 60 children23–27 who were monitored for six months to five years. Among the non-prospective studies, 35 children aged 18 months28 and 58 children aged five years29 were evaluated (Table 1).

The other five studies in which stool collection was performed close to the period of allergy assessment comprised case–control studies involving 18–61 children aged between three months and 14 years, totalling 170 analyses.30–34 In the study of Suzuki et al.,32 children (both cases and controls) were monitored from six months to two years of age (Table 2).

The criteria used to define allergic diseases involved mostly clinical assessment and the performance of skin tests and/or quantity of IgE antibodies in 11 studies,15–17,23,25,28,29,31–34 in addition to the use of questionnaires in two other studies.22,30 Three studies20,21,26 performed only clinical assessments, two studies14,18 used tests and questionnaires, and three studies19,24,27 only used questionnaires. The studies that used tests include those by Penders et al.,10,14 Gore et al.,31 and Storro et al.,22 wherein IgE antibodies were quantified in foods and/or inhalants, and those by Suzuki et al.,23,32 Sjögren et al.,25 and Johansson et al.,29 wherein skin tests were used. Other studies measured IgE antibodies and performed skin tests. The only criterion for the classification of allergies included the use of the International Study of Asthma and Allergies in Childhood (ISAAC)36 questionnaires in the studies by Songjinda et al.24 and Verhulst et al.19 and the Asthma Predictive Index35 in the studies by Vael et al.20,21

For the analysis of faecal microbiota, culture-independent techniques, such as real-time PCR, which was used in seven studies,14,16,18,22,24,25,29 or conventional PCR,23,27,31,34 predominated. Techniques that allowed the assessment of microbial diversity were used in four studies.17,26,28,30 Four other studies19–21,32 used only culture-dependent techniques, and both culture-dependent and culture-independent techniques were used in two studies15,33 (Tables 1 and 2).

In stool samples analysed during the first year of life, a lower bacterial diversity was observed in three studies,17,26,28 with a predominance of Firmicutes,27 higher counts of Bacteroidaceae,24 and a higher prevalence of anaerobic bacteria,19,20 such as Bacteroides fragilis,20,21Escherichia coli,14 and Clostridium difficile,14,18 in the microbiota of children who developed allergies later in life (Table 1). In three studies, the prevalence of C. difficile,25E. coli,22 and Staphylococcus aureus19 in the microbiota of allergic children was lower than that of the controls (Table 1), and three other studies found no significant differences in bacterial counts31–33 (Table 2).

Bifidobacteria were investigated in ten studies where the stool was collected before the assessment of the clinical outcome (allergy) and in five studies where the stool was collected during the assessment. The most prevalent species in the early microbiota among infants (up to 12 months of age) who developed allergies were Bifidobacterium catenulatum,23,31Bifidobacterium bifidum,23 and Bifidobacterium longum,22 and the least prevalent were Bifidobacterium adolescentis25 and B. bifidum29 (Table 1). In the faecal microbiota of five-year-old children, a higher prevalence of B. adolescentis and a lower prevalence of B. catenulatum30 were found (Table 2).

Allergic children exhibited a lower prevalence of Lactobacillus25,29 and Lactobacillus paracasei16 in the first weeks of life. An increased gram-negative/gram-positive ratio at 12 months of age was associated with IgE>100kU/L at 18 months of age15 (Table 1).

Allergic children at two years of age had higher counts of Bacteroides and a lower prevalence of S. aureus32 in their faecal microbiota, and children between four and 14 years of age had lower counts of Akkermansia muciniphila, Faecalibacterium prausnitzii, and Clostridium.34 Stsepetova et al.30 also found a lower microbial diversity in the faecal microbiota of children younger than five years old (Table 2).

Our study indicated differences in the microbiota of allergic children compared with non-allergic children, as previously reported by Penders et al.5 Twenty studies out of the 21 published between 2007 and 2013 indicated associations between some abnormalities in the intestinal microbiota and atopic symptoms or sensitisation, whereas in the review conducted previously, 14 studies out of the 17 published between 1999 and 2006 indicated such an association. Furthermore, an increased number of studies in the period from 2007 to 2013 used advanced laboratory techniques and a larger sample size.

As reported in three pioneering studies conducted in Estonia and Sweden and reviewed by Penders et al.,5 investigations into the role of the faecal microbiota in the aetiology of allergic diseases began in the 1990s and involved healthy one-year-old children in Estonia, where the prevalence of childhood allergy was low, and in Sweden, where allergy prevalence was high.37 Lactobacilli and eubacteria were the most prevalent bacterial groups, particularly among Estonian children, and Swedish children harboured higher counts of C. difficile. Based on these observations, the first study comparing the composition of the faecal microbiota of allergic and healthy children was conducted.38 The results of this study indicated that allergic children in both countries were colonised less frequently with lactobacilli compared with non-allergic children and harboured higher counts of facultative aerobic microorganisms, particularly coliforms in Estonia and S. aureus in Sweden. A prospective study39 by the same research group found a lower prevalence of colonisation with bifidobacteria and enterococci in the first year of life, higher counts of clostridia at three months of age, and a higher prevalence of S. aureus at six months of age among children who developed allergic diseases.

Of the 21 studies reviewed herein, 16 studies performed stool collection before the assessment of allergies and evaluated 4246 individuals, most of whom were children monitored for up to seven years. In the other studies where stool collection was performed concomitantly with the assessment of allergies, the small number of subjects evaluated (N=170) should be noted, with small experimental groups (≤37 individuals) that included infants to 14-year-olds. We observed differences in both the study design and the techniques used to analyse the faecal microbiota, with a predominance of molecular techniques.

Molecular biology techniques, which were performed in 85.7% (18/21) of the studies analysed, are able to overcome the limitations of traditional bacterial culture techniques, such as low sensitivity and low reproducibility, due to the large number of species able to be identified.40 These techniques, including fluorescence in situ hybridisation (FISH), PCR combined with denaturing- or temperature-gradient gel electrophoresis (PCR-DGGE/PCR-TGGE), and real-time PCR, are molecular techniques with wide applicability for the study of intestinal microbiota.41 The greatest advantages of these methods are speed, specificity, sensitivity, and the possibility of quantification. In addition, samples can be frozen for future analysis.42 These tools have provided detailed molecular characterisation of the composition and concentration of faecal microorganisms and led to the discovery of a large number of bacterial genera and species, thereby increasing by more than 50% the number of organisms known to compose the faecal microbiota.4

Many epidemiological methods have been used to assess the distribution, determinants of disease prevalence and to analyse cause-and-effect associations. The selection of the methods used to evaluate the association between disease and environment depends on several factors because each of these methods has advantages and limitations.5 Observational studies serve a variety of purposes, including providing an initial assessment of the possible causes of a disease and the magnitude of previously observed associations. Therefore, cohort, case–control, and cross-sectional studies represent different approaches that can be employed to investigate the incidence of related events in a population in a given period of time.43

In contrast to studies published before 2007, most of which were cross-sectional or transversal, the studies evaluated in this review were predominantly cohort studies. In 16 birth cohort studies, stool collection was performed in the first weeks of life; other studies involved stool collection from subjects by the age of two years.

Assuming that the chronology of events can be easily determined in cohort studies, i.e., exposure is followed by the clinical outcome,44 it can be hypothesised that in the first weeks of life, the process of microbiota development would determine the level of exposure, which would then culminate in the outcome studied, i.e., allergic disease. In the early years of life, the intestinal microbiota of children is simple but becomes more complex over time, and bacterial diversity increases. Bacteria that can be identified in this initial phase include the genera Bifidobacterium, Ruminococcus, Enterococcus, Clostridium, and Enterobacter.41 In a recent study using molecular techniques, changes were observed in the intestinal microbial population at different ages; e.g., E. coli was more frequent at 10 days of life among children whose mothers were also positive for this bacterium, and this frequency remained stable during the first year of life in 80–85% of the study group. However, at two years of age, this frequency decreased to 45% in children who were previously positive. In contrast, B. longum is considered to be one of the most persistent colonisers; at 10 days of life, B. longum was detected in almost all newborns who harboured the bacteria at three days of life, and even at two years of age, this species persisted in most children who were previously positive. At the age of four months, bifidobacteria represented 57.6% of the total intestinal microbiota, whereas the prevalence of Lactobacillales and Streptococcus organisms had decreased.45

Certain bacteria are associated with a reduced risk of allergic diseases, whereas others were reported to be associated with an increased risk of allergic diseases. The comparison of these results is difficult because of differences in several variables, including the methodology used in these studies, sample size, differences in the protocols, and the selection of the microorganisms present in the colonic microbiota.

Despite these limitations, we observed lower biodiversity in the colonic microbiota of allergic children compared with non-allergic children, with a predominance of Firmicutes, in addition to an increased count of Bacteroidaceae; a higher prevalence of the anaerobic bacteria B. fragilis, E. coli, C. difficile, B. catenulatum, B. bifidum, and B. longum; and a lower prevalence of B. adolescentis, B. bifidum, and Lactobacillus.

The authors have no conflict of interest to declare.