Establishment of the gut microbiota in infancy
Ia Adlerberth Clinical bacteriology, Dept. Infectious diseases Sahlgrenska Academy, University of Göteborg The gut microbiota may have profound effects on the host
• Effects on the host´s immune system • Effects on the host´s metabolism • Effects on the host´s central nervous system
The gut microbiota may be involved in the development of a number of conditions and disease states
Irritable bowel syndrome Inflammatory bowel disease allergy Celiac disease metabolic syndrome obesity Autism spectrum disorders atherosclerosis
The gut microbiota may have profound effects on the immune system of the host
Germfree animals: • Few (1/10) IgA-producing plasmacells in the gut mucosa • Few T-lymphocytes in the gut mucosa • Low levels of serum-IgG and serum-IgA • Reduced capacity to develop immunological tolerance to harmless antigens (e.g. ovalbumin)
Does the early gut microbiota composition influence the risk of allergy development? Microbiota of the gastrointestinal tract
Distal ileum 105-8 bacteria/ml Stomach Lactobacilli, streptococci <102 bacteria/ml E. coli, other enterobacteria Helicobacter pylori (laktobacilli) Bacteroides (streptococci) Clostridium
Colon Duodenum, jejunum 1011 bacteria/g <105 bacteria/ml streptococci, lactobacilli, Granulicatella, Neisseria Veillonella The colonic microbiota
1/2-1 kg 1014 bacteria hundreds of species >99% anaerobic bacteria
<50% belong to identified culturable species The colonic microbiota
Phylum Genera
Firmicutes Eubacterium, Ruminicoccus, Clostridium, Coprococcus, Blautia, Anaerostipes, Roseburia, Dorea, Butyrivibrio, Bryantella, Lachnobacterium, Lachnospira, Faecalibacterum, Anaerotruncus, Oscillospira, Papillibacter, Subdoligranulum, Sporobacter, Megasphaera, Veillonella, Dialister, Finegoldia, Peptostreptococcus, Streptococcus, Enterococcus
Bacteroidetes Bacteroides, Parabacteroides, Alistipes, Prevotella, Butyricimonas, Odoribacter, Porphyromonas
Actinobacteria Bifidobacterium, Collinsella, Egghertella
Fusobacteria Fusobacterium, Leptotrichia
Proteobacteria E. coli, Oxalobacter
Verrucomicrobia Akkermansia muciniphila The gut microbiota Adults
• A highly complex ecological system hundreds of species • Dominance of anaerobic bacteria (1000:1)
• The majority of gut anaerobes has not yet been cultured
Newborn infants
• A less compex ecological system • Initially few species, increases over time • Initial proliferation of facultative bacteria • Fewer unculturable bacteria
Acquisition of a complex microflora
11 facultatives anaerobes
10 Ruminococcus Akkermansia Eubacterium x 1000 Clostridium Faecalibacterium 7 Bacteroides 10 Peptostreptococcus Bifidobacterium Fusobacterium non-culturable bacteria Streptococcus Anaerostipes Veillonella Staphylococcus Lachnospira hundreds of species Enterococcus Lactobacillus Coprococcus
E. coli , enterobacteria Roseburia Population Population counts 1 wk 1 mo 1 yr adult Age
A complex ”adult-type” microflora is acquired at some years of age What determines which bacteria will establish in the gut microbiota?
• which bacteria are the infant exposed to?
• which bacteria manage to colonize, expand and persist in the gut microbiota? (selection) Sources of bacterial exposure
bacterial environment close contacts load animals
breastmilk other feeds
mother Enviromental bacterial exposure
The early gut microbiota differ between infants born in developing and industrialized societies Colonization by E. coli in the first days of life
Lahore, Pakistan Göteborg, Sweden
100 100 80 80 60 60 40 40
% koloniserade % 20 20 % koloniserade % 0 0 1 2 3 4 days 1 2 3 4 days
Adlerberth et al. Acta Paediatr 1991 Differences in gut colonization pattern between infants in different countries Sweden - Pakistan • Earlier colonization by E. coli and other enterobacteria, and a more diverse enterobacterial flora in Pakistani neonates Adlerberth et al. 1991 Sweden - Ethiopia • Earlier colonization by lactobacilli, E. coli and other enterobacteria, and enterococci in Ethiopian neonates
• More staphylococci in Swedish neonates Bennet et al. 1991 Sweden - Estonia • Earlier colonization by lactobacilli and Eubacterium in Estonia
• More clostridia, especially Clostridium difficile in Swedish one year old infants. Sepp et al. 1997, 1999 Sweden – UK – Germany – Spain - Italy South-North gradient: South: More Bacteroides, enterobacteria and lactobacilli in 6-week-old infants. North: More bifidobacteria, Atopobium, C. perfringens + C. difficile
Fallani et al. 2010 Changing colonization pattern in Western countries?
Colonization by E. coli
100
80
60
% colonized % 40 Sweden, England, Italy* Adlerberth et al. 2007
20 Studies from Western countries 1975-1989 Pakistan 1983 Adlerberth et al. 1991
3 7 14 28d 2mo 6mo 12mo
*236 vaginally delivered infants born 1998-2002, ALLERGYFLORA
Changing colonization pattern in Western countries?
Less ”turn-over” of E. coli-strains in the gut microbiota today?
Sweden, 1998-2001 2.1 E. coli-strains/12 months Nowrouzian et al. 2003
Sweden, 1980s 4.2 strains/11-18 months Kühn et al. 1986
Pakistan, 1980s 8.5 strains/6 months Adlerberth et al. 1998
0 6 12 18 months age Bacteria stimulate the immune system only when first colonizing the gut Secretory-IgA produced in response to the bacteria will plasma coat the bacteria and prevent cells further translocation over the gut mucosa
S-IgA Shroff et al., Infect. Immun, 1995
Slow turn-over – little immune activation? Changing colonization pattern in Western countries?
Colonization by Bacteroides
100
80
60 % colonized % 40 Sweden, England, Italy* Adlerberth et al. 2007 20 Studies from Western countries 1975-1989 Nigeria Rotimi et al. 1985
7 14 28d 2mo 6mo 12mo
*236 vaginally delivered infants born 1998-2002, ALLERGYFLORA
Changing colonization pattern in Western countries?
Staphylococci, classical skin bacteria, colonize in the absence of competition from ”true” gut bacteria
100
80
60
40 % koloniserade % CoNS 1975-1989 CoNS Sweden, England, Italy* 20 S. aureus Sweden, England, Italy* S. aureus 1975-1989
3 7 14 28d 2mo 6mo 12mo
*236 vaginally delivered infants born 1998-2002, ALLERGYFLORA Adlerberth et al. JACI 2007 S. aureus attain relatively high counts in the early gut microbiota
And may persist for several months…
73% of strains were resident (>3w) Median persistence: 17weeks
Lindberg E et al. Pediatr Res. 2000 Dec;48(6):741-7 Changing colonization pattern in Western countries?
C. difficile in the gut microbiota of Swedish infants - indicating ”immaturity” of the gut microbiota?
Unpublished data removed
The modern Western colonization pattern
100 Late colonization by ”classical” gut 80 microbes like E. coli och Bacteroides 60 Bacteroides little exposure to faecal bacteria
% colonized % 40 E. coli in modern Western societies? 20
7d 2mo 6mo 12mo
100 Staphylococci are the first 80 colonizers and persist for many 60 months in the infantile gut
% colonized % 40 absence of competition from CoNS 20 S. aureus ”classical” gut microbes? 7d 2mo 6mo 12mo
Frequent colonization by C. difficile
sign of an ”immature” gut microbiota? Influence of 87 sectio- and 237 vaginally delivered infants delivery mode on ALLERGYFLORA Sweden, England, Italy gut colonization pattern Vaginal delivery Caesaean section
E. coli Bacteroides bifidobacteria 100 100 100
80 80 80
60 60 60 40 40 40
20 20 20 P< 0.001 P< 0.001 P< 0.001 0 0 0 3d 1w 2w 4w 8w 6m 12m 3d 1w 2w 4w 8w 6m 12m 3d 1w 2w 4w 8w 6m 12m
Klebsiella other Enterobacteriaceae clostridia 100 100 100
80 80 80 60 60 60
40 40 40
Cumulative colonization (%) colonization Cumulative 20 20 20 P=0.003 P< 0.001 P< 0.001 0 0 0 3d 1w 2w 4w 8w 6m 12m 3d 1w 2w 4w 8w 6m 12m 3d 1w 2w 4w 8w 6m 12m
Adlerberth et al. JACI 2007 anaerobes ALLERGYFLORA 10 facultatives 10 infants delivered by x 1000 caesarean section 107 had lower anaerobe/facultative ratio at 12 months age
Population counts (p=0.040)
Adlerberth et al. JACI 2007 1 wk 1 mo 1 yr adult Slower maturation of Age the gut microbiota in sectio-delivered infants
Unpublished data removed Reduced diversity in the gut microbiota of infants delivered by caesaren section
HE Jakobsson et al. Gut. 2014 Apr;63(4):559-66.
Lower diversity in sectio-delivered infants at most time- points from 1 week to 1 year of age
Reduced diversity especially within phylum Bacteroidetes In developing countries: Sectio delivered infants ”catch up” more rapidly Rotimi 1985, Adlerberth 1991, Bennet 1991
Nigerian neonates
E. coli Bacteroides
100 100 80 80 60 60 40 vaginal 40 % colonized % sectio 20 20
1 2 3 6 d 1 2 3 6 days
Rotimi et al. 1985 Close human and animal contacts increase exposure to gut bacteria gut colonization pattern Influence of siblings on Lower anaerobe/facultative ratio by 12m age in first inby 12mage ratio anaerobe/facultative Lower Cumulative (%)
100 20 40 60 80 0
3d
1w
2w Unpublished Unpublished data removed
4w
Klebsiella 8w P=0.01
6m
12m
100 20 40 60 80 0
3d other
1w
Enterobacteriaceae ALLERGYFLORA Sweden, England,Italy 2w
4w siblings (n=149) siblings
8w P=0.02
6m
12m
100
Adlerberth - 20 40 60 80
0 born children (p<0.001).children born
first the gut microbiota in Slower maturation of
3d
1w no no (n=175) siblings - born infants?
2w et al et
4w . JACI 2007 JACI .
8w P< 0.001 clostridia
6m
12m
Having older siblings is associated with increased bacterial diversity and increased bacterial richness in the gut microbiota at 18 months of age Laursen et al. BMC Microbiol. 2015 Aug 1;15:154. Does out of home-day care influence gut colonization pattern?
Thompson et al 2015, Front Cell Infect Microbiol. 2015 Feb 5;5:3.
Exclusively breastfed and non-exclusively breastefed infants, sampled before and after the introduction of solid foods.
4/9 infants attended out of home-daycare.
Samples from infants in out of home-daycare had significantly higher diversity and species richness compared to samples from infants cared for at home (p<0.05). Influence of pets on gut ALLERGYFLORA Sweden, England, Italy colonization pattern Pets in household at 6 mo age:
No significant association with time to colonization by E. coli, other Enterobacteriaceae, staphylococci, enterococci, Bacteroides, Bifidobacterium, Lactobacillus, Clostridium Adlerberth et al. JACI 2007
Unpublished data removed Influence of early feeding pattern on gut colonization pattern
• breastmilk, formula and other feeds may be a source of colonizing bacteria
• breastmilk promotes the growth of some and suppresses the growth of other bacteria Differences in the gut microbiota between breast- and bottle-fed infants
Bifidobacterium more in breastfed (13/36 studies) Staphylococcus more in breastfed (5/12 studies)
Lactobacillus no difference (10/16 studies)
Bacteroides less in breastfed (11/23 studies) Clostridium less in breastfed (16/20 studies) Enterococcus less in breastfed (13/18 studies) Enterobacteriaceae less in breastfed (13/28 studies)
(Veillonella less in breastfed) (Peptostreptococcus less in breastfed) (Akkermansia less in breastfed)
Breastfed infants have a less diverse gut microbiota than bottlefed infants Diversity increases rapidly after weaning Risk factors for allergy development:
• born in a Western society • delivered by caesarean section • first-born • little contact with animals
These factors influence acquisition of gut microbiota
- results in delayed acquisition of a complex gut microbiota?
which, in turn, increases the risk of allergy development? Gut microbiota and allergy development ALLERGYFLORA 15 infants who developed atopic eczema and 20 infants who remained healthy for the first 18 months of life
Faecal samples from 1 week of age were analyzed by T-RFLP and TGGE.
Low bacterial richness in the early samples was associated with atopic eczema at 18 months age.
Wang et al. J Allergy Clin Immunol. 2008 Jan;121(1):129-34. ALLERGYFLORA
Agnes Wold, Ia Adlerberth, David P Strachan, Lia Orfei, Michael R Perkin, Erika Lindberg, Forough Nowrouzian Anthony R Coates, David Coleman Nils Åberg, Robert Saalman, Bill Hesselmar St George´s, University of London Inga-Lisa Strannegård Göteborg university Paolo M Matricardi Charité Medical University, Berlin Siv Ahrné, Göran Molin Salvatore Tripodi, Carmen L Bonanno, Valentina Dept. Of Food Technology Panetta Lund university Sandro Pertini Hospital, Rome FARMFLORA: Annika Ljung, Gunhild Lindhagen, Susanne Johansen, Margareta Ceder