Bacteria and gut health

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

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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

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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

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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

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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

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

• Live microbes in the diet
• Do the products match up?
• Delivery of probiotic bacteria to the lower gut
  is required
• gt80 positive human trials are reported

Gastric Juices Bile
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or, target the indigenous (prebiotics)

• functional
• food

3. selective effect
2. enters the large gut
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Reported prebiotics in use
Oligosaccharide
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31 Volunteers fed Prebiotic Biscuits for 21 days
Active biscuits (?) Control biscuits (?)
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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

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

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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

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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.

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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.

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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.

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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.

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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)

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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).

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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

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Differences in bacterial groups (cf. Adults)
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Differences in bacterial groups (cf. Children)
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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

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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

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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.

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

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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.

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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)

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

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Changes in faecal bacteria

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• Volunteer diaries
• Improved in stool consistency (formed stools)
  (P0.006) and reduced flatulence (P0.01).
• Various differences in abdominal pain (P0.10)
  and intestinal bloating ((P0.055)
• 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 (Plt0.05)
  
• Comments from the parents shows that the
  probiotic has helped their general well being and
  gut symptoms in particular

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Parents 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

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Preliminary conclusions

• From FISH
• Lactobacillus/enterococci group higher in active
  (Plt0.05) compared to placebo. Small effect on
  clostridia
• From Diaries
• General bowel function of the children improved
• Cautious optimism but overall the trial failed