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
2
4
11
No/
g F
aece
s (l
og10
)
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
-0.2
-0.1
0
0.1
0.2
0.3
0.4
0.5
0.6
Lo
g10
ce
lls/g
fa
ec
es
.
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
1.E+06
1.E+07
1.E+08
1.E+09
1.E+10
1.E+11
1.E+12
Bif Bac Clos Lac Totalbacteria
Lo
g10
cfu
/g f
aece
s
Non-autistic autistic
Differences in bacterial groups (cf. Adults):
Differences in bacterial groups (cf. Children):
1.E+05
1.E+06
1.E+07
1.E+08
1.E+09
1.E+10
1.E+11
Bif Bac Clos Lac Clos-Eub DAPI
Lo
g10
cfu
/g f
aece
s
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
8
8.5
9
9.5
10
10.5
11
Bif Bac Lac Clos Clos-Eub ATO DAPI
Lo
g1
0 c
fu/g
fa
ec
es
Pre-treatment Active Placebo
*
• 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
Top Related