Denaturing gradient gel electrophoresis of neonatal intestinal microbiota in relation to the development of asthma

BackgroundThe extended 'hygiene hypothesis' suggests that the initial composition of the infant gut microbiota is a key determinant in the development of atopic disease. Several studies have demonstrated that the microbiota of allergic and non-allergic infants are different even before the development of symptoms, with a critical time window during the first 6 months of life. The aim of the study was to investigate the association between early intestinal colonisation and the development of asthma in the first 3 years of life using DGGE (denaturing gradient gel electrophoresis).MethodsIn a prospective birth cohort, 110 children were classified according to the API (Asthma Predictive Index). A positive index included wheezing during the first three years of life combined with eczema in the child in the first years of life or with a parental history of asthma. A fecal sample was taken at the age of 3 weeks and analysed with DGGE using universal and genus specific primers.ResultsThe Asthma Predictive Index was positive in 24/110 (22%) of the children. Using universal V3 primers a band corresponding to a Clostridum coccoides XIVa species was significantly associated with a positive API. A Bacteroides fragilis subgroup band was also significantly associated with a positive API. A final DGGE model, including both bands, allowed correct classification of 73% (80/110) of the cases.ConclusionFecal colonisation at age 3 weeks with either a Bacteroides fragilis subgroup or a Clostridium coccoides subcluster XIVa species is an early indicator of possible asthma later in life. These findings need to be confirmed in a new longitudinal follow-up study.

[1]  A. Woodcock,et al.  Fecal microbiota in sensitized wheezy and non‐sensitized non‐wheezy children: a nested case–control study , 2005, Clinical and experimental allergy : journal of the British Society for Allergy and Clinical Immunology.

[2]  B. Franke,et al.  NOD2 mediates anti‐inflammatory signals induced by TLR2 ligands: implications for Crohn's disease , 2004, European journal of immunology.

[3]  N. Saunders,et al.  Rapid extraction of bacterial genomic DNA with guanidium thiocyanate , 1989 .

[4]  S. Salminen,et al.  Aberrant composition of gut microbiota of allergic infants: a target of bifidobacterial therapy at weaning? , 2002, Gut.

[5]  D. Strachan,et al.  Self-reported prevalence of asthma symptoms in children in Australia, England, Germany and New Zealand: an international comparison using the ISAAC protocol. , 1993, The European respiratory journal.

[6]  A. Woodcock,et al.  Clostridium difficile, atopy and wheeze during the first year of life , 2002, Pediatric allergy and immunology : official publication of the European Society of Pediatric Allergy and Immunology.

[7]  J. Swings,et al.  Temporal stability analysis of the microbiota in human feces by denaturing gradient gel electrophoresis using universal and group-specific 16S rRNA gene primers. , 2004, FEMS microbiology ecology.

[8]  T. Shirakawa,et al.  Allergic symptoms and microflora in schoolchildren. , 2004, The Journal of adolescent health : official publication of the Society for Adolescent Medicine.

[9]  J. Doré,et al.  The Firmicutes/Bacteroidetes ratio of the human microbiota changes with age , 2009, BMC Microbiology.

[10]  J. Celedón,et al.  Diversity of the gut microbiota and eczema in early life , 2008, Clinical and molecular allergy : CMA.

[11]  D. P. Strachan,et al.  Hay fever, hygiene, and household size. , 1989, BMJ.

[12]  K. Julge,et al.  Intestinal microbiota and immunoglobulin E responses in 5‐year‐old Estonian children , 2005, Clinical and experimental allergy : journal of the British Society for Allergy and Clinical Immunology.

[13]  J. Castro‐Rodriguez,et al.  A clinical index to define risk of asthma in young children with recurrent wheezing. , 2000, American journal of respiratory and critical care medicine.

[14]  W. D. de Vos,et al.  Molecular Monitoring of Succession of Bacterial Communities in Human Neonates , 2002, Applied and Environmental Microbiology.

[15]  J. Swings,et al.  Culture-Independent Analysis of Probiotic Products by Denaturing Gradient Gel Electrophoresis , 2003, Applied and Environmental Microbiology.

[16]  R. Van Ree,et al.  Molecular fingerprinting of the intestinal microbiota of infants in whom atopic eczema was or was not developing , 2006, Clinical and experimental allergy : journal of the British Society for Allergy and Clinical Immunology.

[17]  H. Gaskins,et al.  Impact of the intestinal microbiota on the development of mucosal defense. , 2008, Clinical infectious diseases : an official publication of the Infectious Diseases Society of America.

[18]  J. Penders,et al.  The role of the intestinal microbiota in the development of atopic disorders , 2007, Allergy.

[19]  G. Roberts,et al.  Statistical Analyses of Complex Denaturing Gradient Gel Electrophoresis Profiles , 2005, Journal of Clinical Microbiology.

[20]  W. D. de Vos,et al.  Molecularly assessed shifts of Bifidobacterium ssp. and less diverse microbial communities are characteristic of 5-year-old allergic children. , 2007, FEMS immunology and medical microbiology.

[21]  Herman Goossens,et al.  Early intestinal Bacteroides fragilis colonisation and development of asthma , 2008, BMC pulmonary medicine.

[22]  O. Ruuskanen,et al.  The hygiene hypothesis of atopic disease--an extended version. , 2004, Journal of pediatric gastroenterology and nutrition.

[23]  J. Doré,et al.  Direct Analysis of Genes Encoding 16S rRNA from Complex Communities Reveals Many Novel Molecular Species within the Human Gut , 1999, Applied and Environmental Microbiology.

[24]  W. D. de Vos,et al.  Development of bacterial and bifidobacterial communities in feces of newborn babies. , 2003, Anaerobe.

[25]  K. McDowell A Clinical Index to Define Risk of Asthma in Young Children with Recurrent Wheezing , 2001 .

[26]  P. Lawson,et al.  The phylogeny of the genus Clostridium: proposal of five new genera and eleven new species combinations. , 1994, International journal of systematic bacteriology.

[27]  Piet A van den Brandt,et al.  Gut microbiota composition and development of atopic manifestations in infancy: the KOALA Birth Cohort Study , 2006, Gut.

[28]  B. Pulendran,et al.  Cutting Edge: Different Toll-Like Receptor Agonists Instruct Dendritic Cells to Induce Distinct Th Responses via Differential Modulation of Extracellular Signal-Regulated Kinase-Mitogen-Activated Protein Kinase and c-Fos 1 , 2003, The Journal of Immunology.

[29]  K. Desager,et al.  The importance of the development of the intestinal microbiota in infancy , 2009, Current opinion in pediatrics.

[30]  M. Sakamoto,et al.  Fluctuation of fecal microbiota in individuals with Japanese cedar pollinosis during the pollen season and influence of probiotic intake. , 2007, Journal of investigational allergology & clinical immunology.