Comparisons of the different bacterial groups detected with FISH-FC across the three feeding methods (fully breast-feeding, formula feeding, and mixed feeding) were determined using a linear model while correcting for the country effect. The adjusted mean values for each feeding type are shown in Figure 2. Breast-fed infants presented significantly greater proportions of bifidobacteria (44.8% vs 29.9%, P < 0.001) and significantly lower proportions of Bacteroides (8.8% vs 15.9%, P < 0.001), C coccoides (3.7% vs 6.9%, P = 0.014), and Lactobacillus groups (0.9% vs 1.9%, P = 0.046) compared with formula-fed babies. Breast-fed infants also presented significantly lower proportions of Bacteroides compared with mixed-fed babies (8.8% vs 13.8%, P = 0.034), whereas proportions of bifidobacteria were still significantly higher in mixed-fed infants than in formula-fed (40.9% vs 29.9%, P = 0.007) infants. Finally, formula-fed babies also presented lower proportions of C perfringens species compared with breast-fed infants (4.6% vs 24.3%, P = 0.006), while no significant difference was observed for C difficile species alone and C perfringens and C difficile detected together.
The mode of delivery had an impact on some bacterial groups. The adjusted mean values of the different bacterial groups detected for each delivery method are shown in Figure 3. Compared with cesarean section, vaginal delivery (67% of the total) was associated with higher average proportions of Bacteroides (16.1% vs 6.9%, P < 0.001) and members of the Atopobium cluster (2.9% vs 0.8%, P < 0.001) and lower proportions of members of the C coccoides group (4.5% vs 8.2%, P < 0.001) and the Streptococcus group (1.4% vs 1.9%, P = 0.048). Vaginally delivered infants also presented a greater proportion for the sum of detected groups compared with the other babies (75.4 vs 67.6, P < 0.001). There was no effect of the mode of delivery on the relative proportions of bifidobacteria.
Newborns who received antibiotics (only 7% of the 606 children investigated) presented significantly higher proportions of enterobacteria (16.6%) compared with those without treatment (6.8%) (P < 0.001). On the contrary, when mothers received antibiotic treatment perinatally and/or during breast-feeding, infants presented significantly lower average proportions of Bacteroides (11.4% vs 15.0%, P = 0.029) and members of the Atopobium cluster (1.5% vs 2.6%, P = 0.044), as well as for the total sum of detected groups (69.6% vs 76.1%, P = 0.005), compared with those whose mother received no treatment during pregnancy. There was a wide range of antibiotics received by individual infants and mothers and differences between countries. Many mothers could not identify which antibiotic was used. It was, therefore, not possible to carry out any detailed analysis.
The aim of this study was to assess the gut microbiota composition of the young European infant by analyzing 606 fecal samples obtained from babies at approximately 6 weeks from 5 countries with different lifestyle characteristics. The present study also investigated the impact of some important variables such as geographic origin, feeding method, mode of delivery, and antibiotic treatment on the early development of the intestinal microbiota of children. There are many differences in diet and lifestyle characteristics across Europe. For example, in Scotland, breast-feeding rates are lower (32) and many infants are weaned before 3 months (33). In contrast, in Scandinavian countries, breast-feeding rates are high and infants are weaned later (34,35). One of the main ways that diet and environment influence the infant is through their effects on the gut microbiota and its metabolism (36), which may have important effects on the health of the infant and later on the longer-term health of the child and adult.
A strong impact of the geographic origin was observed in the present study. This effect was particularly observed for Bacteroides, bifidobacteria, and enterobacteria. Our observations suggest a possible “geographic gradient” in the composition of the gut microbiota in Europe, where the extremes, north (Glasgow and Stockholm) and south (Granada and Reggio Emilia), would present the highest number of differences. It should be noted, however, that the centers from which the infants were recruited in the present study may not be representative of the country in which they were based because only 1 center was used in each case.
The north–south gradient was characterized by higher proportions of bifidobacteria, Atopobium, C perfringens + C difficile, and sum of total detectable bacteria in north European countries, and by higher proportions of Bacteroides, enterobacteria, and lactobacilli in south European countries, whereas C coccoides, C leptum, and streptococci remained unaffected by the country of birth at the age considered. To our knowledge, this is the first cross-sectional study comparing the impact of country of origin on the development of the gut microbiota of babies born in different European countries. A few previous studies have compared the microbiota composition for 2 countries and the majority considered infants born in developing countries as well (42,43). Sepp et al (44) reported high counts of lactobacilli and eubacteria in Estonian infants and increased numbers of clostridia in Swedish babies, with bifidobacteria and anaerobic cocci equally prevailing in both groups, which they related to risk of allergy. It is well established that in Western industrialized countries, routine hygienic procedures aimed at reducing the spread of bacteria in maternity and neonatal wards have strongly influenced the colonization pattern of newborn infants, whereas infants born in developing countries are exposed to a heavier bacterial load from birth and this condition influences the colonization pattern of the gut. The low colonization rate of enterobacteria in infants born in Swedish hospitals, reported by Lundequist et al (38), is probably related to these practices. Meanwhile, 2 recent studies investigated cross-sectional differences (19,20) in microbiota in young and older adults across Europe. In young adults (19), no significant differences with respect to geographic origin were found, but the research included only volunteers from central to northern European countries (Denmark, United Kingdom, the Netherlands, France, Germany). In the other study (20), which compared young adults and elderly adult volunteers from Sweden, France, Germany, and Italy, a significant difference was observed for the Bifidobacterium group, with the population in Italy having 2- to 3-fold higher proportions of bifidobacteria than that in any other country. However, the factors that determine the complex and relatively stable microbiota of adult populations, in which diet and other lifestyle factors may vary considerably, may be different from those that influence the early colonization of the infant gut at 6 weeks of age.
The geographic origin was far more important for the composition of the preweaned infant microbiota than any other parameter. Yet, in accordance with previous culture-based studies (37,38,45–47) and more recent molecular studies (13,16,41), we observed an important impact of the feeding method on the early development of the infant gut microbiota. Breast milk, even in mixed feeding, clearly favored bifidobacteria, whereas in its absence, a more diversified microbiota was established, with higher proportions of Bacteroides and members of the C coccoides and Lactobacillus groups. Comparing several individual studies, Tannock (47) found that numbers of clostridia were always lower in breast-fed babies and that clostridia was the only group predictive of formula feeding. Our observations agreed with this concept, focusing on C coccoides as a potential indicator group.
The mode of delivery also significantly influenced the microbiota composition of the newborns' intestine. Upon vaginal delivery, the infant is predominantly exposed to vagina and fecal bacteria of maternal origin. Conversely, infants born by cesarean delivery have an initial exposure to environmental bacteria from equipment, air, other infants, and nursing staff. Vaginally delivered babies presented higher proportions of Bacteroides and members of the Atopobium, as well as added proportions of detectable bacteria, and lower proportions of members of the C coccoides and the Streptococcus groups compared with those born by cesarean section. Interestingly, the latter showed the same trend as infants born to mothers who received antibiotics during late pregnancy and/or while breast-feeding. A previous study (48) reported a considerable delay in the establishment of a stable microbiota in infants born by cesarean section, characterized by a low incidence of Bacteroides spp and a low isolation rate of other bacteria. Gronlund et al (49) also reported a delay in fecal colonization and a low number of Bacteroides fragilis in cesarean-delivered infants. In a recent study, Penders et al (16), investigating the fecal microbiota of 1032 Dutch infants by quantitative real-time polymerase chain reaction, observed that infants born by cesarean section had lower numbers of Bacteroides and bifidobacteria and were more often colonized with C difficile than were vaginally born infants. Hence, antibiotic treatment and cesarean delivery may promote the same suboptimal development of the microbiota in early infancy.
In conclusion, in this large-scale study, we highlighted the impact of geographic origin, feeding method, delivery mode, and antibiotic treatment on the composition of the fecal microbiota of European infants at 6 weeks of age. Above all, the colonic microbiota of the young healthy infant appeared different across Europe. The potential of a south-to-north gradient among the European countries investigated needs to be researched further to establish the gradient using more centers and to determine the factors responsible.
1. Guarner F, Malagelada JR. Gut flora in health and disease. Lancet 2003; 361:512–519.
2. Kirjavainen PV, Arvola T, Salminen SJ, et al
. Aberrant composition of gut microbiota of allergic infants: a target of bifidobacterial therapy at weaning? Gut 2002; 51:51–55.
3. Penders J, Thijs C, van de Brandt PA, et al
. Gut microflora composition and development of atopic manifestations in infancy: the KOALA birth cohort study. Gut 2007; 56:661–667.
4. Kalliomaki M, Collado MC, Salminen S, et al
. Early differences in fecal microbiota composition in children may predict overweight. Am J Clin Nutr 2008; 87:534–538.
5. Hooper LV. Bacterial contributions to mammalian gut development. Trends Microbiol 2004; 12:129–134.
6. Hooper LV, Midtvedt T, Gordon JI. How host–microbial interactions shape the nutrient environment of the mammalian intestine. Annu Rev Nutr 2002; 22:283–307.
7. Harmsen HJM, Gibson GR, Elfferich P, et al
. Comparison of viable cell counts and fluorescence in situ hybridization using specific rRNA-based probes for the quantification of human fecal bacteria. FEMS Microbiol Lett 2000; 183:125–129.
8. Suau A, Bonnet R, Sutren M, et al
. 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.
9. Amann RI, Ludwig W, Schleifer KH. Phylogenetic identification and in situ
detection of individual microbial cells without cultivation. Microbiol Rev 1995; 59:143–169.
10. Rigottier-Gois L, Le Bourhis AG, Gramet G, et al
. Fluorescent hybridisation combined with flow cytometry
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.
11. Rigottier-Gois L, Rochet V, Garrec N, et al
. 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.
12. Zoetendal EG, Ben-Amor K, Harmsen HJM, et al
. Quantification of uncultured Ruminococcus obeum
-like bacteria in human fecal samples by fluorescent in situ hybridization
and flow cytometry
using 16S rRNA-targeted probes. Appl Environ Microbiol 2002; 68:4225–4232.
13. Harmsen HJM, Wildeboer-Veloo ACM, Raangs GC, et al
. Analysis of intestinal flora development in breast-fed and formula-fed infants by using molecular identification and detection methods. J Pediatr Gastroenterol Nutr 2000; 30:61–67.
14. Favier CF, Vaughan EE, De Vos WM, et al
. Molecular monitoring of succession of bacterial communities in human neonates. Appl Environ Microbiol 2002; 68:219–226.
15. Hopkins MJ, Macfarlane GT, Furrie E, et al
. Characterisation of intestinal bacteria in infant stools using real-time PCR and northern hybridisation analyses. FEMS Microbiol Ecol 2005; 54:77–85.
16. Penders J, Thijs C, Vink C, et al
. Factors influencing the composition of the intestinal microbiota in early infancy. Pediatrics 2006; 118:511–521.
17. Penders J, Vink C, Driessen C, et al
. Quantification of Bifidobacterium
spp., Escherichia coli
and Clostridium difficile
in faecal samples of breast-fed and formula-fed infants by real-time PCR. FEMS Microbiol Lett 2005; 243:141–147.
18. Sakata S, Tonooka T, Ishizeki S, et al
. Culture-independent analysis of fecal microbiota in infants, with special reference to Bifidobacterium
species. FEMS Microbiol Lett 2005; 243:417–423.
19. Lay C, Rigottier-Gois L, Holmstrom K, et al
. Colonic microbiota signatures across five northern European countries. Appl Environ Microbiol 2005; 71:4153–4155.
20. Mueller S, Saunier K, Hanisch C, et al
. Differences in fecal microbiota in different European study populations in relation to age, gender, and country: a cross-sectional study. Appl Environ Microbiol 2006; 72:1027–1033.
21. Rochet V, Rigottier-Gois L, Beguet F, et al
. Composition of human intestinal flora analysed by fluorescent in situ hybridisation using group-specific 16S rRNA-targeted oligonucleotide probes. Genet Select Evol 2001; 33:S339–S352.
22. Lay C, Sutren M, Rochet V, et al
. Design and validation of 16S rRNA probes
to enumerate members of the Clostridium leptum
subgroup in human faecal microbiota. Environ Microbiol 2005; 7:933–946.
23. 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.
24. Wallner G, Amann R, Beisker W. Optimizing fluorescent in situ
hybridization with ribosomal-RNA-targeted oligonucleotide probes for flow cytometric identification of microorganisms. Cytometry 1993; 14:136–143.
25. Fallani M, Rigottier-Gois L, Aguilera M, et al
. Clostridium difficile
and Clostridium perfringens
species detected in infant faecal microbiota using 16S rRNA targeted probes. J Microbiol Methods 2006; 67:150–161.
26. Langendijk PS, Schut F, Jansen GJ, et al
. Quantitative fluorescence in situ hybridization of Bifidobacterium
spp with genis specific 16S ribosomal-RNA targeted probes and its application in fecal samples. Appl Environ Microbiol 1995; 61:3069–3075.
27. Manz W, Amman R, Ludwig W, et al
. 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.
28. Sghir A, Gramet G, Suau A, et al
. Quantification of bacterial groups within human fecal flora by oligonucleotide probe hybridization. Appl Environ Microbiol 2000; 66:2263–2266.
29. Franks AH, Harmsen HJM, Raangs GC, et al
. Variations in bacterial populations in human feces measured by fluorescent in situ hybridization
with group specific 16S rRNA-targeted oligonucleotide probes. Appl Environ Microbiol 1998; 64:3336–3345.
30. Harmsen HJM, Elfferich P, Schut F, et al
. A 16S rRNA-targeted probe for detection of Lactobacilli
in faecal samples by fluorescent in situ hybridization
. Microb Ecol Health Dis 1999; 11:3–12.
31. Harmsen HJM, Wildeboer-Veloo ACM, Grijpstra J, et al
. Development of 16S rRNA-based probes doe the Coriobacterium
group and the Atopobium
cluster and their application for enumeration of Coriobaceteriaceae
in human feces from volunteers of different age groups. Appl Environ Microbiol 2000; 66:4523–4527.
32. Tappin DM, Mackenzie JM, Brown AJ, et al
. Comparison of breastfeeding rates in Scotland in 1990–1 and 197–8. BMJ 2001; 322:1335–1336.
33. Alder EM, Williams FLR, Anderson AS, et al
. What influences the timing of introduction of solid foods to infants. Br J Nutr 2004; 92:527–531.
34. Mikkelsen A, Rinneljungquist L, Borres MP, et al
. Do parents follow breast feeding and weaning recommendations given by pediatric nurses? A study with emphasis on introduction of cows milk protein in allergy risk families. J Pediatr Health Care 2007; 21:238–244.
35. Grjibovski AM, Ehrenblad B, Yngve A. Infant feeding
in Sweden: sociodemographic determinants and associations with adiposity in childhood and adolescence. Int Breastfeed J 2008; 3:23.
36. Alm JS, Swartz J, Bjorksten B, et al
. An anthroposophic lifestyle and intestinal microflora in infancy. Pediatr Allergy Immunol 2002; 13:402–411.
37. Benno Y, Sawada K, Mitsuoka T. The intestinal microflora of infants: composition of fecal flora in breast-fed and bottle-fed infants. Microbiol Immunol 1984; 28:975–986.
38. Lundequist B, Nord CE, Winberg J. The composition of the faecal microflora in breastfed and bottle fed infants from birth to eight weeks. Acta Paediatr Scand 1985; 74:45–51.
39. Stark PL, Lee A. The microbial ecology of the large bowel of breast-fed and formula-fed infants during the first year of life. J Med Microbiol 1982; 15:189–203.
40. Dore J, Sghir A, Hannequart-Gramet G, et al
. Design and evaluation of a 16S rRNA-targeted oligonucleotide probe for specific detection and quantitation of human faecal Bacteroides
populations. Syst Appl Microbiol 1998; 21:65–71.
41. Martin F, Savage SAH, Parrett AM, et al
. Investigation of bacterial colonization of the colon in breast-fed infants using novel techniques. Proc Nutr Soc 2000; 59:64A.
42. Adlerberth I, Carlsson B, de Man P, et al
. Intestinal colonization with Enterobacteriaceae in Pakistani and Swedish hospital-delivered infants. Acta Paediatr Scand 1991; 80:602–610.
43. Bennet R, Eriksson M, Tafari N, et al
. Intestinal bacteria of newborn Ethiopian infants in relation to antibiotic treatment and colonisation by potentially pathogenic gram-negative bacteria. Scand J Infect Dis 1991; 23:63–69.
44. Sepp E, Julge K, Vasar M, et al
. Intestinal microflora of Estonian and Swedish infants. Acta Paediatr 1997; 86:956–961.
45. Adlerberth I, Hanson LA, Wold AE. The ontogeny of the intestinal flora. In: Sanderson IR, Walker WA, editors. Development of the Gastrointestinal Tract. Hamilton, Canada: B.C. Decker; 1999.
46. Conway P. Development of intestinal microbiota. In: Mackie RI, White BA, Isaacson RE, editors. Gastrointestinal Microbiology. New York: Chapman & Hall; 1997. pp. 3–38.
47. Tannock GW. The acquisition of the normal microflora of the gastrointestinal tract. In: Gibson SAW, editor. Human health: the contribution of microorganisms. London: Springer-Verlag; 1994. p. 1–16.
48. Orrhage K, Nord CE. Factors controlling the bacterial colonization of the intestine in breastfed infants. Acta Paediatr
49. Gronlund MM, Lehtonen OP, Eerola E, et al
. Fecal microflora in healthy infants born by different methods of delivery: permanent changes in intestinal flora after cesarean delivery. J Pediatr Gastroenterol Nutr 1999; 28:19–25.