Background: The present study was designed to compare the faecal microbiota and concentrations of faecal short-chain fatty acid and ammonia between healthy and cow’s milk protein allergic (CMPA) infants. Methods: The population comprised 92 infants aged 2–12 months who were nonallergic (n = 46) or diagnosed as having CMPA (n = 46). Faecal samples were analyzed by fluorescent in situ hybridization and flow cytometry, using a panel of 10 rRNA targeted group- and species-specific oligonucleotide probes. Acetic, propionic, butyric, isocaproic and branched-chain short fatty acids (BCSFA) were measured by gas-liquid chromatography, lactate by enzymatic reaction, and pH and ammonia levels were determined. Results: CMPA infant faeces had significantly higher proportions of the Clostridium coccoides group and Atopobium cluster and a higher sum of the proportions of the different bacterial groups in comparison to healthy infant faeces. Faecal pH and ammonia did not significantly differ between CMPA and healthy infants. Faeces concentrations and percentages of butyric acid and BCSFA were higher in CMPA infants than in healthy infants. Conclusions: The findings clearly set a link between a dysbiosis in gut microbiota composition and the pathogenesis of CMPA. No single species or genus appeared to play an essential role, but dysbiosis led to biomarkers of CMPA among bacterial fermentation products.

1.
Høst A: Frecuency of cow’s milk allergy in childhood. Ann Allergy 2002;89(6 suppl 1): 33–37.
2.
Penders J, Stobberingh EE, van den Brandt PA, Thijs C: The role of the intestinal microbiota in the development of atopic disorders. Allergy 2007;62:1223–1236.
3.
Björkstén B, Naaber P, Sepp E, Mikelsaar M: The intestinal microflora in allergy Estonian and Swedish 2-year-old children. Clin Exp Allergy 1999;29:342–346.
4.
Björkstén B, Sepp E, Julge K, Voor T, Mikelsaar M: Allergy development and the intestinal microflora during the first year of life. J Allergy Clin Immunol 2001;108:516–520.
5.
Ouwehand AC, Isolauri E, He F, Hashimoto H, Benno Y, Salminen S: Differences in Bifidobacterium flora composition in allergic and healthy infants. J Allergy Clin Immunol 2001;108:144–145.
6.
Hayashi H, Sakamoto M, Benno Y: Phylogenetic analysis of the gut microbiota using 16S rDNA clone libraries and strictly anaerobic cultural based methods. Microbiol Immunol 2002;46:535–548.
7.
Rigottier-Gois L, Le Bourhis AG, Gramet G, Rochet V, Doré J: Fluorescent hybridisation combined with FC and hybridisation of total RNA to analyse the composition of microbial communities in human faeces using 16S rRNA probes. FEMS Microbiol Ecol 2003;43:237–245.
8.
Kirjavainen PV, Apostolou E, Arvola T, Salminen S, Gibson GR, Isolauri E: Characterizing the composition of intestinal microflora as a prospective treatment targed in infant allergy disease. FEMS Immunol Med Microbiol 2001;32:1–7.
9.
Kalliomäki M, Kirjavainen P, Eerola E, Kero P, Salminen S, Isolauri E: Distinct patterns of neonatal gut microflora in infants in whom atopy was and was not developing. J Allergy Clin Immunol 2001;107:129–134.
10.
Mah KW, Björkstén B, Lee BW, van Bever HP, Shek LP, Tan TN, Lee YK, Chua KY: Distinct pattern of commensal gut microbiota in toddlers with eczema. Int Arch Allergy Immunol 2006;140:157–163.
11.
Parret AM, Edwards CA, Lokerse E: Colonic fermentation capacity in vitro: development during weaning in breast-fed infants is slower for complex carbohydrates than for sugars. Am J Clin Nutr 1997;65:927–923.
12.
Böttcher MF, Norin EK, Sandin A, Midtvedt T, Björkstén B: Microflora-associated characteristics in faeces from allergic and nonallergic infants. Clin Exp Allergy 2000;30:1590–1596.
13.
Sandin A, Brabäck L, Norin E, Björkstén B: Faecal short chain fatty acid pattern and allergy in early childhood. Acta Paediatr 2009;98:823–827.
14.
Rochet V, Rigottier-Gois L, Beguet F, Doré J: Composition of human intestinal flora analysed by fluorescent in situ hybridisation using group-specific 16S rRNA-targeted oligonucleotide probes. Genet Selec Evol 2001;33:S339–S352.
15.
Rigottier-Gois L, Rochet V, Garrec N, Suau A, Doré J: Enumeration of Bacteroides species in human faeces by fluorescent in situ hybridisation combined with flow cytometry using 16S rRNA probes. Syst Appl Microbiol 2003;26:110–118.
16.
Amann RI, Krumholz L, Stahl DA: Fluorescent-oligonucleotide probing of whole cells for determinative, phylogenetic, and environmental studies in microbiology. J Bacteriol 1990;172:762–770.
17.
Walner G, Amann R, Beisker W: Optimizing fluorescent in situ hybridization with ribosomal-RNA-targeted oligonucleotide probes for flow cytometric identification of microorganism. Cytometry 1993;14:136–143.
18.
Fallani M, Rigottier-Gois L, Aguilera M, Bridonneau C, Collignon A, Edwards CA, Corthier G, Doré J: Clostridium difficile and Clostridium perfringens species detected in infant faecal microbiota using 16S rRNA targeted probes. J Microbiol Methods 2006;67:150–161.
19.
Harmsen HJ, Wildebwer-Veloo AC, Grijpstra J, Knol J, Degener JE, Welling GW: Development of 16S rRNA-based probes for the Coriobacterium group and the Atopobium cluster and their application for enumeration of Coriobacteriaceae in human faeces from volunteers of different age groups. Appl Environ Microbiol 2000;66:4523–4527.
20.
Franks AH, Harmsen HJM, Raangs GC, Jansen GJ, Schut F, Welling GW: Variations of bacterial populations in human faeces measured by fluorescent in situ hybridization with group-specific 16S rRNA-targeted oligonucleotide probes. Appl Environ Microbiol 1998;64:3336–3345.
21.
Langendijk PS, Schut F, Jansen GJ, Raangs GC, Kamphuis GR, Wilkinson MHF, Welling GW: Quantitative fluorescence in situ hybridization of Bifidobacterium spp with genius-specific 16S ribosomal-RNA targeted probes and its application in faecal samples. Appl Environ Microbiol 1995;61:3069–3075.
22.
Manz W, Amman R, Ludwig W, Vancanneyt M, Schleifer KH: Application of a suite of 16S rRNA-specific probes designed to investigate bacteria of the phylum cytophaga-flavobacter-bacteroides in the natural environment. Microbiology UK 1996;142:1097–1106.
23.
Sghir A, Gramet G, Suau A, Rochet V, Pochart P, Doré J: Quantification of bacterial groups within human faecal flora by oligonucleotide probe hybridization. Appl Environ Microbiol 2000;66:2263–2266.
24.
Harmsen HJM, Elfferich P, Schut F, Weling GW: A 16S rRNA-targeted probe for detection of Lactobacilli and Enterococci in faecal samples by fluorescent in situ hybridization. Microb Ecol Health Dis 1999;11:3–12.
25.
Lay C, Sutren M, Rochet V, Saunier K, Doré J, Rigottier-Gois L: Desing and validation of 16S rRNA probes to enumerate members of the Clostridium leptum subgroup in human faecal microbiota. Environ Microbiol 2005;7:933–946.
26.
Van Nuenen MH, Venema K, van der Woude JC, Kuipers EJ: The metabolic activity of faecal microbiota from healthy individuals and patients with inflammatory bowel disease. Dig Dis Sci 2004;49:485–491.
27.
Sepp E, Julge K, Milkasaar M, Björksten B: Intestinal microbiota and immunoglobulin E responses in 5-year-old Estonian children. Clin Exp Allergy 2005;35:1141–1146.
28.
Sjögren YM, Jenmalm MC, Bötcher MF, Björkstén B, Sverremark-Ekström E: Altered early infant gut microbiota in children developing allergy up to 5 years of age. Clin Exp Allergy 2009;39:518–526.
29.
Watanabe S, Narisawa Y, Arase S, Okamatsu H, Ikenaga T, Tajiri Y, Kumemura M: Differences in faecal microflora between patients with atopic dermatitis and healthy control subjects. J Allergy Clin Immunol 2003;111:587–591.
30.
Murray CS, Tannock GW, Simon M-A, Harmsen HJM, Welling GW, Custovic A, Woodcokc A: Faecal microbiota in sensitized wheezy and non-sensitized non-wheezy children: a nested case-control study. Clin Exp Allergy 2005;35:741–745.
31.
Høverstad T, Midtvedt T, Bøhmer T: Short-chain fatty acids in intestinal content of germ-free mice monocontaminated with E. coli or Cl. Difficile. Scand J Gastroenterol 1985;20:373–380.
32.
Umesaki Y, Setojama H, Matsumoto S, Imaoka A, Itoh K: Differential roles of segmented filamentous bacteria and clostridia in development of the intestinal immune system. Infect Immun 1999;67:3504–3511.
33.
Wold AE: The hygiene hypothesis revised: is the rising frequency of allergy due to changes in the intestinal flora? Allergy 1998;53(46 suppl):20–25.
34.
Lin J, Peng L, Itzkowitz S, Holzman IR, Babyatsky MW: Short-chain fatty acid induces intestinal mucosal injury in newborn rats and down-regulates intestinal trefoil factor gene expression in vivo and in vitro. J Pediatr Gastroenterol Nutr 2005;41:607–611.
35.
Butel MJ, Roland N, Hibert A, Popot F, Favre A, Tessedre AC, Bensaada M, Rimbault A, Szylit O: Clostridial pathogenicity in experimental necrotising enterocolitis in gnobiotic quails and protective role of bifidobacteria. J Med Microbiol 1998;47:391–399.
36.
Penders J, Stobberingh E, Thijs C, Adams H, Vink C, van Ree R, van den Brandt PA: Molecular firgerprinting of the intestinal microbiota of infants in whom atopic eczema was or was not developing. Clin Exp Allergy 2006;36:1602–1608.
37.
Suau A, Bonnet R, Sutren M, Godon JJ, Gibson GR, Collins MD, Doré J: Direct analysis of genes encoding 16S rRNA from complex communities reveals many novel molecular species within the human gut. Appl Environ Microbiol 1999;65:4799–4807.
38.
Alles MS, Hartemink R, Meyboon S, Harryvan JL, van Laere KM, Nagengst FM, Hautvast JG: Effect of transgalactooligosaccharides on the composition of the human intestinal microflora and on putative risk markers for colon cancer. Am J Clin Nutr 1999;69:980–991.
Copyright / Drug Dosage / Disclaimer
Copyright: All rights reserved. No part of this publication may be translated into other languages, reproduced or utilized in any form or by any means, electronic or mechanical, including photocopying, recording, microcopying, or by any information storage and retrieval system, without permission in writing from the publisher.
Drug Dosage: The authors and the publisher have exerted every effort to ensure that drug selection and dosage set forth in this text are in accord with current recommendations and practice at the time of publication. However, in view of ongoing research, changes in government regulations, and the constant flow of information relating to drug therapy and drug reactions, the reader is urged to check the package insert for each drug for any changes in indications and dosage and for added warnings and precautions. This is particularly important when the recommended agent is a new and/or infrequently employed drug.
Disclaimer: The statements, opinions and data contained in this publication are solely those of the individual authors and contributors and not of the publishers and the editor(s). The appearance of advertisements or/and product references in the publication is not a warranty, endorsement, or approval of the products or services advertised or of their effectiveness, quality or safety. The publisher and the editor(s) disclaim responsibility for any injury to persons or property resulting from any ideas, methods, instructions or products referred to in the content or advertisements.
You do not currently have access to this content.