Preventative Health National Research Flagship

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Preventative Health National Research
Flagship Protective Foods Stream, CRC-3 Project
Gut Bacterial Population Profiles
and Relationships To Diet and Health
Dr. Michael Conlon CSIRO Human Nutrition Adelaide,
Australia
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Broad Focus
Human studies examining the impact of dietary
Resistant Starch (RS) and Fibre on changes in the
population dynamics of colonic bacteria and short
chain fatty acid (SCFA) production
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Diet and Colorectal Cancer

• Based on recent human epidemiological studies red
  meat and processed meat intake is associated with
  colorectal cancer risk whereas fibre intake
  reduces risk
• 500,000 people, followed over 4.8 years
• Positive association between colorectal cancer
  incidents and red and processed meat (but not
  poultry)
• Fibre intake was protective (Norat et al. 2005)
• 150,000 mature adults, followed over 20 years
• Prolonged high intakes of red or processed meat
  were associated with elevated risk of colorectal
  cancer (Chao et al. 2005)
• 60,000 Swedish women, followed over 13.9 years
• Positive association between red meat consumption
  and development of colon cancer (Larsson et al.
  2005)

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Interaction of Complex Systems Influencing Bowel
Health

• Food
• Complex cocktail
• of substances
• -Combinatorial effects
• of components on
• physiology and biochemistry

Gut Microflora -The majority of bacteria in
the colon are poorly characterised due to lack
of culturability, sheer numbers and variety. -The
colonic bacteria carry out a massive range of
reactions relating to metabolism of dietary and
host components.
Bowel Health

• Host
• Individuals have distinct
• genotypes and phenotypes
• and may respond differently
• to diet and harbour different
• bacteria.

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SCFA
SCFA (especially butyrate) - Increased
through colonic fermentation - Reduces colonic
pH (Topping et al. 1996) - Inhibits cell
proliferation (Lupton et al. 1995) - Promotes
cell differentiation and apoptosis
(Godard et al. 1999) Butyrate is the primary
fuel of cells lining the colon and appears
to help maintain a normal colonic phenotype.
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Fibre
Classified broadly as Non-Starch Polysaccharides
(NSP) (eg. Cellulose) and Resistant Starch (RS)
(eg Amylose from grains) All natural
polysaccharides except starch resist digestion by
small intestine enzymic activity, reaching the
large bowel where they provide faecal bulking and
promote laxation. Starch is found primarily as
amylose or amylopectin. Starch is found in food
crops such as cereals, pulses and tubers and
usually found in a compact granular structure. -
approx 30 is amylose and a small amylopectin
RS is starch which passes through the small
intestine undigested and is a highly fermentable
substrate High amylose forms of grain have been
developed by CSIRO and others that increase the
likelihood of starch reaching the large bowel
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CRC-3 Objectives
To understand faecal butyrate concentration
variability within humans with time and across
human populations To understand the influences
of variables such as diet, age and gender (etc)
on this variability To identify individuals who
have high and low faecal butyrate
concentrations To type enteric bacteria against
butyrate producing capacity of human colonic
contents Identify the key bacteria in the colon
and how they respond to diet. Long range
objectives Develop foods which reduce colorectal
cancer risk Develop technology for the analysis
of complex microbial populations (markers of
disease or health risk)
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Colonic Microbial Populations and
SCFA Experimental Program
Human clinical trials

• Pilot trial
• 8 volunteers, regular diet for 12 week study,
  faecal collections
• Established anaerobic protocols, isolated gt21 Bt
  producing species, power analysis for major
  trial, sample storage conditions, in vitro
  fermentation protocols, gt100 bacterial sequences
  added to phylogenetic trees , population
  profiles, FISH analyses
• Dietary intervention
• 46 volunteers
• 14 week crossover dietary intervention.
• 2 phases
• PCR/DGGE analysis of faecal bacteria populations,
  SCFA analyses, in vitro fermentations, FISH
  analyses, potential analyses of bacteria
  populations by functional genes.

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Pilot StudyTotal SCFA output over 48 hours for
12 weeks
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Molecular analysis of microbial populations

Denaturing Gradient Gel Electrophoresis Descriptio
n of the structure of bacterial communities
without the need to culture bacteria.

• DNA extraction
• 16s rDNA PCR amplification
• Polyacrylamide gradient gel
• Silver stain/SYBR Gold
• Density and binary matrix analysis

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Pilot Trial DGGE PopulationAnalysis
-High degree of population stability within each
individual -Populations dominated by 18-23
different bacterial species -Populations were
highly specific to each individual (Plt0.001,
Rgt0.99)
DGGE community fingerprint, demonstrating the
variation in the dominant bacteria present in one
individual over 12 weeks
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nMDS of DGGE
DGGE fingerprints were analysed for similarity
of patterns
(nMDSnon-metric multi-dimensional scaling)
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Bacterial Populations

• Molecular analysis revealed complex populations
  of Clostridia
• Including
• Significant uncultivated clades present in the
  majority of individuals.
• Key clades common to most individuals.
• A number of groups related to different butyrate
  producers in the majority of individuals

Populations are stable Populations are distinct
to individuals Significant novel diversity of
bacteria Potential for butyrate production in all
individuals
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Faecal Butyrate Concentration vs Microbial
Abundance (PCR)

• Total bacteria (excl. Archaea)
• No association

• Bacteroides
• (polysaccharide breakdown nitrogen cycling)
• No association

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Faecal butyrate concentration vs microbial
abundance
Clostridial Cluster XIVa (major
Butyrate-producing bacteria cluster) Positive
association
Methanogens (acetate consumers) Negative
association (CLI)
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Major Trial Diets
Diet 1 Diet 2 High Fibre High Fibre
Resistant starch Bran plus 50g Barleyplus
30g Carrots 50g 3 bean mix 50g Couscous
50g Freekah 50g Bran
2g Hi-Maize 20g Total Dietary Fibre
36g/day
Weeks 0 2 4
6 8
10 12 14
Group1 Normal diet
Diet 1 Normal diet
Diet 2
Group2 Normal diet
Diet 2 Normal diet
Diet 1
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Faecal Butyrate variation
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Preliminary SCFA data

• N 46
• gt1500 SCFA measures
• Phase
• Preliminary SCFA Diet order
• concentration data Significant difference
• Normal Diet v High Fibre x
• Normal diet v Resistant Starch Diet v
• Acetate 12
• Butyrate 23
• Gender
• SCFA concentrations significantly higher in males
  than in females
• pH
• High Fibre diet marginally higher (0.07)
• Resistant Starch diet significantly lower
  (-0.14)

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Ruminococcus bromeii related
F prau related
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UPGMA Analysis ofMajor Trial DGGE
Similarity of DGGE banding profiles (Eub primer)
analysed by UPGMA (unweighted pair group method
with arithmetic mean) Bacteria population
compositions very similar during the normal diet
at the beginning and end of the
study -Resilience of the bacterial
population -Population changes seem rapid given
changes during the intervening period Loss of
bands during high fibre only consumption Biggest
difference in profiles between the high fibre and
high fibre plus high RS diets
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Identification of Bacteria Increased in No. by RS
22 of 29 GDDE bands were found to be upregulated
by high RS Sequencing of bands has revealed -22
of the bands are related to butyrate-producing
Clostridia -2 very close to (appear to be)
Faecalibacterium prausnitzii -2 very close to
Fusobacterium/Roseburia cluster -9 related to
Ruminococcus bromeii (involved in
starch hydrolysis?) -5 are related to
Mollicutes -1 each belong to Bacteroides and
Lactobacilli A large proportion of bacteria
potentially involved in RS fermentation and
butyrate production (and colonic health?) are
poorly characterised
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nMDS plot of Phase I
High Fibre High Fibre plus Resistant Starch
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Study Messages
Human individuals have a distinct gut
microflora Butyrate concentrations vary
considerably between individuals but generally
appear to increase in response to RS The faecal
bacterial population dynamics, but probably not
type, change in response to diet The order of
RS/Fibre consumption may have a significant
effect on some aspects of bacterial responses We
have identified large numbers of
uncharacterised/poorly characterised faecal
bacteria Many of the bacteria closely related to
known butyrate-producers increase as a percentage
of the total population in response to dietary
resistant starch
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Dietary RS appears to be protective in the large
bowel in ways that include - maintenance of the
mucus layer integrity and protection of
underlying tissues - promotion of bacterial
population dynamics that may select against
bacteria causing disease Individuals have a
distinct gut bacteria profile that may result in
each individual having a different risk profile
for disease and a different capacity to utilise
dietary components.
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A greater understanding of the complex
interactions between diet, gut bacteria and host
tissues is likely to lead to strategies to
improve health Technologies that facilitate
this (eg. gene microarrays and chips, as well as
proteomics) by analysing large parcels of
information and then extracting trends are
currently being examined
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Technologies Being Implemented for
Enumeration and Identification of Faecal Bacteria

• Conventional Microbiology (culturing bacteria and
  counting colonies)
• PCR and DGGE
• Fluorescence In Situ Hybridisation (FISH) and an
  automated microscope
• method being developed with Paul Jackway and
  Volker Hilsenstein
• (CMIS)
• Microarrays to detect changes in a large range of
  known bacteria (based
• on 16s rRNA sequences) and functional genes
  (developed by Chris
• McSweeney and others at LI)
• Microbiome chips and proteomics are also being
  discussed

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Enumeration of Faecal Bacteria by FISH
The task is to detect and count particular
species of bacteria found in human faeces A
fluorescent RNA probe is hybridized to bacteria
of interest in the sample Various artefacts
including autofluorescence of the background and
other bacteria, clumping and inhomogeneity of
spatial distribution, non-specificity of probe,
can make this an extremely challenging image
analysis problem A state-of-the-art
segmentation scheme has been developed A
candidate image object must satisfy strict size,
shape, and intensity criteria before being counted
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Dilute faeces stained with a Cy3 (red) labelled
rRNA probe specific for the F. prausnitzii
bacterium.
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Segmentation result. Note the segmenter is
designed to delineate all bright image objects
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Detected F. prausnitzii bacteria after applying
strict size-shape-morphology-brightness criteria.
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Development of a microbial community and
functional gene microarray for the colon
Chris McSweeney Stuart Denman CSIRO Livestock
Industries
Polysaccharides
Fibrolytic flora
Cross-feeding
Fragments
Fibrolytic flora
Saccharolytic flora
Intermediate metabolites of fermentation
H2 tranfer
Acetate
H2
Propionate
Butyrate
CO2
SO4
Hydrogenotrophic flora
Methanogens (CH4)
Acetogens (acetate)
Sulfate - reducers (H2S)
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A functional array to monitor key enzymatic
pathways
Sulfate reduction
Methanogenesis
Butyrate production
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CRC 3 Butyrate and Colonic Health The Team
HSN Adelaide Sandi McOrist Guy Abell Michael
Conlon David Topping Tony Bird Caroline Cooke
Thelma Bridle Kerry Nyland Rhys Bushell Brad
Klingner Robb Muirhead Michelle Vuaran Jennifer
Keogh HSN Clinic
CSIRO P-Health Trevor Lockett Lynne Cobiac
CMIS Ian Saunders Aloke Phatak Harri Kiiveri Paul
Jackway Volker Hilsenstein Richard Beare
CLI Chris McSweeney Andre-Denis Wright