5. prof dana stanley central qld uni - how to influence and manage the gut microflora

1. How to influence and manage the gut
microflora
Dana Stanley

2. Rise of Molecular Microbiology
Until mid-1990’s we were confident we
knew all about bacteria
Still, there was unexplained difference in
culture and microscope-based cell counts
and diversity???
16S rRNA gene sequence is species
specific i.e. acts like a
fingerprint

3. So, what did we learn in the last
20 years???
Use of culture-free methods provided
massive amount of information:
Manuscriptspublishedonfecalmicrobiota

4. Roles of Intestinal Bacteria
Highly adapted, mutualistic relationship
Bacterial (prokaryotic) cells outnumber our
(eukaryotic) own cells 10 fold
Proportion of culturable bacteria is less than
1% of bacterial diversity for some
environments
Proportion of culturable bacteria in human
gut is approximated to 25%

5. Human host has about 20,000 genes
Microbiota has 100 times more unique
genes
Only 1% of DNA we carry is ours!!!
Microbiota is biggest contributor of
metabolites and protein products in our
body
Microbiota is now regarded as another
organ of human body
Roles of Intestinal Bacteria

6. Digest indigestible nutrients from food
Produce vitamins and hormones
Regulate development of the gut
Growth and differentiation of gut epithelial cells
Prevent growth of pathogens
Assist in establishment of immune system
Play a role in anxiety, stress hormone levels
Control number of brain receptors
Roles of Intestinal Bacteria

7. Microbiota Genocide
Massive loss of diversity and richness
“Western lifestyle”, More than 50% of
today’s diet is made up of refined
carbohydrates
5-fold increase in GI transit TIME
US stats: by the year of 2030, see another
76 m obese adults, Additional 6–8.5 m cases
of diabetes, 6–7 m cases of cardiovascular
disease, 492,000–669,000 cases of cancer,
leading to loss of 26–55 m life years and a
dramatic increase in costs of care (calculated
to be $50–68b/year

8. WHY?
Insulinotrophic food
Leaky guts (modern grain has 20 times
more gluten than very old and less
productive strains)
Volunteers living for 1 month on a
Western-style diet demonstrated, in a
crossover study, a 71% increase in plasma
levels of endotoxin activity (endotoxemia)
Colonic “bioreactor” is running empty
Confused gut

9. Everything is leaking!!!
“Alzheimer’s disease [147], cardiovascular diseases [148,149],
chronic liver diseases [150–152],chronic kidney disease
[153,154], chronic obstructive pulmonarydiseases
(COPD)[155], diabetes [156], inflammatory bowel
diseases(IBD) [157,158], irritable bowel syndrome (IBS) [159],
paradontal diseases such as paradontosis [160,161] and
polycystic ovarysyndrome (PCOS) [161]. Leaky gut is also
seen in a large variety of other conditions, such as alcoholism
[162], autoimmune diseases [163], chronic encephalopathy
[164], chronic fatigue syndrome [35,39], mental depression
[165,166] and other, idiopathic, conditions, which are mainly
observed in the Western world.” (Bengmark 2013)
Atherosclerotic plaques come from leaky oral mucosa, CF
from leaky lungs, leaky skin, brain barrier, placenta…
Medicine!!!!
The disaster of human nutrition!!!!

10. Chicken microbiota problem?
Poor reproducibility betwean flock
In some cases there was extreme (p<1e-20)
difference in community richness and
diversity
Refocused work on understanding trial
variation

11. Microbiota differences across
three replicate trials
Red – Trail 1
Blue – Trial 2
Green – Trial 3

12. Why sudden interest in poo
in agriculture?
Undisputable evidence on role of intestinal
bacteria in food digestion, fat metabolism,
obesity, muscle gain etc potential to
improve productivity!
Evidence of gut bacteria modulating immune
system development and disease resistance
we can improve animal health!
Results are applicable to all
ag animals

13. Human Gut
Microbiota is major player in obesity and
weight gain
Germ-free animals need 30% more calories to
compensate for the lack of presence of bacteria
Control mice have 47% more fat than germ-
free
Fecal transplant from obese mice induced
obesity in recipient mice

14. Research focus
1. Microbiota remodelling
Development
Timing of probiotic delivery
At-hatch administration
Fibre and the gut
2. Alternative to AGPs
Herbs and spices
Natural products (biochar, bentonite, zeolite)
3. Nanoparticles and other food
manipulations
4. Litter quality
NIR

15. How does it develop?
Early, middle and late
colonisers
Our data shows
different story to
current understanding
of microbiota
development in chicken
Has to be manipulated
from the hatch day to
make it work

16. Can it be manipulated?
Microbial
interactions and batch
effect are further
complicating things
Our aim: to develop a
timed and defined
probiotic delivery
system to ensure
comparable
populations in the
guts of chickens
across Australia

17. At-hatch manipulation
Weights of the birds at 4 (A) and 28 days (B).
Control PBS inoculated birds were significantly heavier between the
days 2 and 6 post hatch. Between day 6 and 24 there were no
significant differences, however, probiotic inoculated group became
significantly heavier starting from day 24 to the end of the trail.
At-hatch administration of 3 persistent Lactobacillus strains,
only one colonised

18. Effects are more visible on
mucosa-associated bacteria
Out of the 3 OTUs
detected in inoculum,
only one, identical to L.
ingluviei was detected in
birds using sequencing
methodology and was
present in all birds
across all sampling
points.

19. TSS normalised, square root transformed
abundance of genus Bacteroides and an
OTU most similar (blastn on 16S Microbial
database) to Escherichia fergusonii.
In all experiments we
noticed:
Reduction of pathogens
Increase in beneficial
strains

20. Another attempt
Early, middle and late
colonisers
Should we add
Enterococcus first?

21. Just keep testing…
We inoculated birds with range of pb (L
johnsonii, L. reuteri, L kitasatonis, Lmix,
B. subtillis, different ferments and
SynGenx
Then we introduced DEX leaky gut
challenge during week 6
Protection noted in
L.johnsonii
L.kitastionis
Lacto Mix
SynGenx

22. To clean or not?
The caecum and ileum of PBS control birds
showed higher Richness Index than the
Lactobacillus inoculated birds (A), likely due to
the strong dominance of Lactobacillus shown in
both caeca and ileum of inoculated birds
(P=0.026) (B). Differences in gut morphology
and colour of the intestinal content were
obvious (C).
Using quail for germ –free
model
Inoculate with PB and keep
under very clean conditions for
one week
Then re-introduce to the flock
The birds never recovered
microbial richness, remained
on 20-30 species

23. To clean or not?

24. Biochar Project
We started with biochar, bentonite and
zeolite
OTU level redundancy
analysis (RDA) plot
comparing chicken faecal
samples of birds fed control
diet (CTRL) and groups with
feed supplemented with
biochar (BC), bentonite (BT)
and zeolite (ZT).

25. Overall effect on microbiota was moderate
Additives did not alter weights after 23
weeks
Additives did not change richness
Weight(g)

26. All 3 additives reduce
Proteobacteria
Influence of biochar (BC),
bentonite (BT) and zeolite
(ZT) supplementation on
abundance of genera of the
phylum Proteobacteria
Epsilonproteobacteria and
Gammaproteobacteria
The Epsilonproteobacteria
was comprised of only two
genera - Campylobacter and
Helicobacter
Campylobacter OTU269490
was 100 % identical to
Campylobacter jejuni subsp.
Jejuni
H. pullorum

27. Zeolite Controls Enterobacteria
Gammaproteobacteria were made up of family Enterobacteriaceae (P=0.036, r=-0.3).
CTR, ZT1 ZT2 and ZT4 indicate control, 1%, 2% and 4% zeolite in feed respectively;
letters p, c and f in the graph title indicate phylum, class and family taxonomic level.
Enterobacteriaceae family includes many human and poultry pathogenic genera
(Klebsiella, Salmonella, Escherichia, Enterobacter, Yersinia, Proteus, Shigella,
Serratia, and Citrobacter).
The reduction of
Proteobacteria was mostly
due to the reduction in its
class Gammaproteobacteria
(P=0.018) that reached a
mean of 15% in the control
group. Pearson analysis
indicated significant negative
correlation (P=0.016, r=-0.33)
between abundance of
Proteobacteria and zeolite
concentration as well as in
Gammaproteobacteria
(P=0.036, r=-0.3).

28. Zeolite Controls Enterobacteria

29. Biochar Concentration
A number of genera correlated with
concentration of BC in feed
Bifidobacteria were unaffected, Lactobacillus
promoted and Campylobacter reduced

30. Fraser Coast Trial
We performed an animal trial on n>5000 birds
with 2% biochar supplementation in an organic
farm setting (Fraser Coast poultry farm)
2%BC protected treated birds from Spotty liver
outbreak

31. The downstream effect-soil
We used poultry manure from both control
and biochar amended feed sheds of the
Fraser Coast trial to produce compost and
granulated both raw manures and
composted manures.

32. This manure was then used in fertilisation of a capsicum
crop, growing on a ferrosol
The BC reduced genera (P<5E-5) all contain known human
and plant pathogens (Dietzia, Corynebacterium, Georgenia).
Another five pathogen containing genera were significantly
reduced by biochar (Aerococcus, Gordonia, Facklamia,
Streptococcus and Brevibacterium), with the former three
acknowledged as novel emerging plant or human pathogens
Thus, residual biochar in poultry manure was able to
improve soil quality and consequently improve plant health
Fraser Coast Trial

33. Fraser Coast Trial

34. Farm 2
Comparing Farm 1 and Farm 2
F1 (Blue BC, grey CTR)
F2 (orange BC, red CTR)

35. Farm 2 Trial
CTRLBC
Spotty Liver test: C. hepaticus

36. Farm 2 Trial
CTRLBC
Spotty Liver test: C. hepaticus – remaining
samples

37. Oregano in-vitro
We generated
microbiological
media that supports
2608 species (OTUs)
originating from 50
genera, with 23 of
those being genera
that are not yet
cultured.

38. Oregano in-vitro
Condition AA PA IBA BA VA Total SCFA
Cont 3.55 0.12 0.02 0.11 0.02 3.81
Oreg 7.48 0.22 0.06 1.49 0.01 9.26
Significance 0.0013 ns ns 0.014 ns 0.022
A recent study selected 46 strains of
Lactococcus from about 2,600

39. Oregano in-vivo
In-vivo trial completed, more pathogens
confirmed as reduced, mostly Proteobacteria
Taxa P ▾
Positive
Samples
Proteus 0.000063 140
Klebsiella 0.0014 23
Salinicoccus 0.0023 39
Granulicatella 0.0041 19
Jeotgalicoccus 0.006 60
Unclassified.Bacillales 0.0065 21
Serratia 0.0068 43
Bifidobacterium 0.0082 16
Unclassified.Pseudomonadaceae 0.017 55
Unclassified.Planococcaceae 0.018 103
Unclassified.GMD14H09 0.018 11
Oligella 0.023 15
Unclassified.Enterobacteriaceae 0.025 168
Unclassified.Carnobacteriaceae 0.026 15
Unclassified.Gemellales 0.027 68
Staphylococcus 0.027 107
Unclassified.Weeksellaceae 0.032 21
Lysinibacillus 0.033 21
Aerococcus 0.038 17
Unclassified.Lactobacillales 0.044 146
Unclassified.JG30KFCM45 0.055 20
Unclassified.Leuconostocaceae 0.056 33

40. NEXT-Total Pathogen Control Mix
The in-vitro mix data matrix will be 2–way
clustered and presented as a heat map that will be
used to select exact product combinations for the in
vivo chicken trial stage

41. Prasai, Walsh, Midmore and Bhattarai
(2016) Effect of biochar, zeolite and
bentonite feed supplements on egg
yield and excreta attributes. Journal of
Animal Science
Biochar and N emissions
Compost tumblers

42. Prasai, Walsh, Midmore, and
Bhattarai (2017)
Effect of biochar, zeolite and
bentonite feed supplements on egg
yield and excreta attributes.
CSIRO Animal Production
Science.
http://www.publish.csiro.au/an/pdf/
AN16290

43. Granulation of litter
Prasai, Walsh, Midmore, Jones and
Bhattarai (2017) Manure from biochar,
bentonite, and zeolite feed
supplemented poultry: Moisture
retention and granulation properties.
Journal of Environmental Management

44. Granule use for fertilizer and carbon
sequestration – pot and field trials
Pudasaini, K., Walsh, K., Ashwath, N. and Battarai,
T. (2016) Effects of biochar addition on plant
available water of a loamy sandy soil and
consequences on cowpea growth Acta
Horticulturae 1112, 357-364

45. Machine vision (deep learning)

46. My long-term collaborators…
Rob Moore, RMIT
Hao Van (RMIT)
Bob Hughes, SARDI
CQU’s Professor Kerry Walsh
And Surya Bhattarai

48. THANK YOU!!!