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

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

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)

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) “functional” food 3. selective effect 2. enters the large gut

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

31 Volunteers fed Prebiotic Biscuits for 21 days 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 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)

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

Differences in bacterial groups (cf. Adults):

Differences in bacterial groups (cf. Children):

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 ? 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

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)

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

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 *

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