Bacteria and gut health Glenn Gibson INYS Workshop. 30 th Nov 2006 Departmet of Food Biosciences The University of Reading.

Bacteria and gut health

Glenn Gibson

INYS Workshop. 30th Nov 2006

Departmet of Food BiosciencesThe University of Reading

What are bacteria?

• Single cell organisms • No nucleus but very clever!• Reproduce by splitting in half• Round, rod, spiral, comma, square• 99.9% are “friendly”• 5,000,000,000,000,000,000,000,000,000,000

bacteria exist on the Planet Earth• They regenerate 1/2 million times quicker than

humans

Where do they come from?

• Birth: initial colonisers

• First week: conditions

favourable for growth

• Breast fed: bifidobacteria predominate

• Formula fed: similar profile to days 4-7

• Key development stages occur

Microflora of the stomach

• Low pH• Transit time ca. 30 minutes• HCl + pepsin• Colonisers need to invade the mucosal layer• Numbers are low• H. pylori is notorious

Microflora of the small intestine

• Transit time is 2-4h• The organ is a long narrow tube• Bile salts and pancreatic secretions affect

colonisation by the indigenous flora• Typical numbers are (only!) around 1 million

per mL contents

Microflora of the Large Intestine

• 150cm in length • Typical transit time of 24-72h• The most heavily colonised organ in the human

body• Antimicrobial intake, stress, poor diet and living

conditions all affect the flora composition• Up to 1000 species• Most of the bacteria in your body

(1,000,000,000,000,000) are here

(Gibson & Roberfroid, J. Nutr. 125, 1995)

Ps. aeruginosa

Proteus

Staphylococci

Clostridia

Veillonellae

Enterococci

E. coli

Lactobacilli

Streptococci

Eubacteria

Bifidobacteria

Bacteroides

Pathogenic, including

production of toxins

Production of carcinogens

Intestinal putrefaction

Inhibition of growth of harmful bacteria

Stimulation of immune functions

Aid in digestion or

absorption of nutrients

Synthesis of vitamins

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Harmful/pathogenic effects Health promoting functions / effects

What are probiotics?• Live microbes in the diet

• Do the products match up?

• Delivery of probiotic bacteria to the lower gut is required

• >80 positive human trials are reported

Gastric Juices

Bile

or, target the indigenous (prebiotics) 1.“functional”

food

2. enters the largegut

3. selective effect

Reported prebiotics in useOligosaccharide

Soybean oligosaccharides

Isomalto-oligosaccharides

Lactosucrose

Gluco-oligosaccharides

Xylo-oligosaccharides

Lactulose

Galacto-oligosaccharides

Inulin

Fructo-oligosaccharides

31 Volunteers fed Prebiotic Biscuits for 21 days

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Total bacteria

Bacteroides spp.

Bifidobacterium spp.

Clostridium spp.

Lactobacillus spp.

Active biscuits () Control biscuits ()

Enhancing functionality

• Distally targeted activities

• Anti-adhesive effects

• Species level changes

• Activities at low dosage without side effects

• Maintenance of effects in real foods

• Health effects

Health effects of gut bacteria

Gut infections (e.g. food poisoning)

Inflammation of the colon (IBS,

IBD)

Conditions other than inside the gut (e.g. bone health,

eczema)

Bowel and stomach cancer

How does it work?

• Prebiotics in infant monkey

feed stopped E. coli infection

• It was seen that the “good”

bacteria had grown up to

cause this result

• Monkeys and humans are

99.6% the same

Prebiotic foods researched and developed at UoR

• Yoghurts and fermented milks

• Health drinks, spreads• Infant and weaning foods• Cereals• Biscuits• Juices• Pet food• Farm animals

ASDs and the human gut microflora

Some clinical reports show that some children with ASDs may suffer from intestinal dysfunction presenting various different symptoms:

Diarrhoea Constipation Excess wind Abdominal discomfort Bloated “stomach”

No direct evidence of human gut microflora imbalance.

Intestinal Bacteria in ASDs

Clostridia and ASDs

• Bolte (1998)Clostridium tetani suggested as pathogenic in ASDs - production of neurotoxins absorbed from gut leading cognitive abnormalities.

• Sandler et al (2000) Reported on the use of oral vancomycin therapy in ASDs. Significant improvements with antibiotic treatment were noted but with a short term benefit.

Intestinal Bacteria in ASDs• Finegold et al (CID 2002) (Bacteriology in Reading)

Improved symptomology with oral vancomycin

Study compared species found in the stools of regressive autistic children and control children in USA.

Higher number in autistic group for clostridial counts.

Autistic children yielded 9 species of clostridia not found in control group in stool samples.

Overall counts of clostridia were higher in the stools of the autistic children – through culture methods.

Theoretical Overview

• Can imbalances in the gut microflora contribute: – towards certain symptoms of autism?– towards gastrointestinal problems in autism?

• Particular gut bacteria may be important.

• Control of these species maybe of relevance.

Aim of the study

● Characterisation of the gut microflora of autistic and Autistic Spectrum disorders (ASDs) Subjects

● Builds upon work with USA autistic children

● Evaluate the need and monitor the effectiveness of probiotic strategies in the treatment and alleviation of GI symptoms (some already used)

Approaches Profile of human gut bacteria in ASDs subjects using

molecular based techniques:• Quantitative study • Fluorescent in situ Hybridisation (FISH)

• Qualitative study DGGE separation of amplified PCR products

Comparison to gut flora of healthy children, adults and siblings.

To identify a probiotic that has anti-bacterial activity against relevant microbes.

Questionnaire – for retrospective correlation of bacterial profile and characteristics (Gastrointestinal function, Diet, Medical history).

Subjects

• Number of children taking part in the study: - 60 with confirmed diagnosis - 11 females and 49 Males

• Patients had gastrointestinal symptoms, such as diarrhoea and constipation, and were on gluten and casein-free diets.

• Control groups: - 10 non ASDs children- 10 siblings of ASD children

- 50 adults

1.E+05

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Non-autistic autistic

Differences in bacterial groups (cf. Adults):

Differences in bacterial groups (cf. Children):

1.E+05

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Bif Bac Clos Lac Clos-Eub DAPI

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Healthy Autistic Siblings

Bacteriology results

• Clostridia counts from autistic subjects were higher in comparison with the control groups

• No major change in other bacterial groups but overall differences were proven (due to clostridia).

• In our hands, such a clostridial predominance has not been hitherto observed in any other study group

Inhibitory activity of different probiotics against Clostridium spp.

• Inhibitory activity tested using co-culture.• Test for anti-pathogenic activity against

clostridia.• Different strains of Lactobacillus and

Bifidobacterium spp. were selected.• Extrapolate to other clostridia as based upon

DGGE data

Results• Filtered broths from L. plantarum showed

inhibitory effects upon the growth of clostridia.

• L. plantarum broth did not lose the inhibitory activity after being boiled for 10 min, and showed stronger inhibition.

• The strain has had its entire genome sequenced

• All the other probiotic strains tested showed no inhibition of the clostridia.

Link between gut function and ASDs ?

Gut flora

acquisition

Alterations in gut microflora

Undefined interaction

Overgrowth of bacteria such as clostridia

Systemic effects

Unusual gut microflora components - release of toxic compounds

Gut Dysbiosis

Probiotic/prebiotic intervention

Altered gut function

GI problems

Experimental objectives

To study the:

• Effect of L. plantarum WCFS1 on autistic

gut microflora. Packaged in Belgium

• Impact on gastrointestinal health.

• Psychological impact on children (behaviour, communication, emotional problems)

Inclusion/exclusion criteria

Inclusion Criteria: - Diagnosis of ASD- Age 4-16 years of age- Signed consent form

Exclusion Criteria: - Intake of other probiotics or prebiotics - Administration of antibiotics- If the child is Involved in other similar study or

involving an experimental drug/medication.

Recruitment

• Step 1 Through databases - 166• Step 2 Through advertising/ media - unknown• Total responses: Yes – 211. No – 72. • Step 3 Exclusion Criteria 1: distance to Reading 116• Step 4 Agreement to experimental protocol: 85 • Step 5 Exclusion criteria 2: 69• Step 6 Final consent – 62• 23 withdrew before commencing the intervention• Final number – 39

(Time 7 months)

2 3 4 5 6121 days 21 days21 days 21 days-7 days

Group 1

Group 2

Trial designDouble blind placebo-controlled,

randomised crossover study

Samples 1-6 taken for analysis

WashoutProbiotic WashoutPlacebo

Washout WashoutPlacebo Probiotic

Analysis

Samples - molecular analysis by FISH

Volunteer Diary - assess tolerance of probiotic, stool frequency and consistency, abdominal pain, intestinal bloating, flatulence.

Psychology study - Assess psychological impact of probiotic administration through a standardised “Development Behaviour Checklist ”.

Changes in faecal bacteria

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Pre-treatment Active Placebo

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• Volunteer diaries:

• Improved in stool consistency (formed stools) (P=0.006) and reduced flatulence (P=0.01).

• Various differences in abdominal pain (P=0.10) and intestinal bloating ((P=0.055)

Total behaviour scorings showed a non-significant difference in the interventions but the probiotic gave reductions. Both placebo and probiotic gave significant reductions cf. pre-treatment (P<0.05)

Comments from the parents shows that the probiotic has helped their general well being and gut symptoms in particular

• 17 volunteers completed the study (although 22 provided data for analysis)

Parent´s observations

Treatment period:• More calm, relaxed, not stressed, no mood change, content• Improved ability to listen & concentrate• Highest grades at school so far• Better formed stools

Washout Period:• Heartbreaking to see the improvements deteriorating• Loose stools & undigested food particles• Abdominal pain• Depressed mood• Poor grades at school

Preliminary conclusions

• From FISH

Lactobacillus/enterococci group higher in active (P<0.05) compared to placebo. Small effect on clostridia

• From Diaries

General bowel function of the children improved

‘Cautious optimism’ but overall the trial failed