Establishment of the 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 • 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 (e.g. ovalbumin)

Does the early gut microbiota composition influence the risk of allergy development? Microbiota of the

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,

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