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

2. 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

3. 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

4. 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

5. 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

6. 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

7. 2 4 11 Harmful/pathogenic effects Health promoting functions / effects
Ps. aeruginosa
Pathogenic, including production of toxins
Proteus
No/g Faeces (log10)
Staphylococci
Clostridia 4
Inhibition of growth of harmful bacteria
Production of carcinogens
Veillonellae
Enterococci
Stimulation of immune functions
E. coli
Intestinal putrefaction
Lactobacilli
Streptococci
Aid in digestion or absorption of nutrients
Eubacteria
Bifidobacteria
Bacteroides
Synthesis of vitamins 11
(Gibson & Roberfroid, J. Nutr. 125, 1995)

8. 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

9. or, target the indigenous (prebiotics)
“functional”
food
3. selective effect
2. enters the large
gut

10. Reported prebiotics in use
Oligosaccharide
Soybean oligosaccharides
Isomalto-oligosaccharides
Lactosucrose
Gluco-oligosaccharides
Xylo-oligosaccharides
Lactulose
Galacto-oligosaccharides
Inulin
Fructo-oligosaccharides

11. 31 Volunteers fed Prebiotic Biscuits for 21 days
Active biscuits () Control biscuits ()

12. 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

13. Health effects of gut bacteria
Conditions other than inside the gut (e.g. bone health, eczema)
Bowel and stomach cancer
Inflammation of the colon (IBS, IBD)
Gut infections (e.g. food poisoning)

14. 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

15. 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

16. 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.

17. 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.

18. 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.

19. 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.

20. 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)

21. 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).

22. 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

23. Differences in bacterial
groups (cf. Adults):

24. Differences in bacterial groups (cf. Children):

25. 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

26. 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

27. 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.

28. Link between gut function and ASDs ?
Unusual gut microflora components - release of toxic compounds
Systemic effects
Altered gut function
Probiotic/prebiotic intervention
Undefined interaction
Gut Dysbiosis
Overgrowth of bacteria such as clostridia
Gut flora
acquisition
Alterations in gut microflora
GI problems

29. 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)

30. 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.

31. 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)

32. Trial design Double blind placebo-controlled,
randomised crossover study
Group 1
Washout
Probiotic
Placebo
Group 2
Washout
Placebo
Probiotic
-7 days
21 days
21 days
21 days
21 days 1 2 3 4 5 6
Samples 1-6 taken for analysis

33. 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 ”.

34. Changes in faecal bacteria*

35. 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)

36. 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

37. 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