POST-SURGICAL GUT MICROBIOMEUnderstanding intestinal dysfunction

Gut microbiotaMore than 1014 resident bacteria in humanGITThere is a life-long bidirectional and symbiotic relationship with the hostGut microbiota: the total microbial ecosystemGut microbiome: the collective genomic content;150 times the human gene pool

Importance: digestionAnaerobic microbial fermentation of fibers in hind gutThe volatile fatty acids, the main end products, supply

75%-80% of the absorbed energy from poor quality hay20%-30% of the total energy requirements of a horse

Importance: normal development of immune system

Importance: intestinal barrierGerm free mice compared to conventional mice showed

Impaired intestinal morphology, cell renewal propertiesDecreased total intestinal surface areaAberrant intestinal morphology

Shorter ileal villiSmall intestinal crypts

Lower rate of ileal and Peyer's patches turn over8 days after colonization cell renewal reverted to the same degree of conventional mice

Importance: intestinal barrier

Germ free mice showed also:

Reduced level of antimicrobial peptides secreted by gut epitheliumLower number of mucin-secreting goblet cellsThinner less stable and compact mucin layer

Importance: Health and diseaseReduced bacterial biodiversity (dysbiosis)

In obese compared to lean individualsIn infants with colic compared to those without colic

Gut microbial ecosystem differs between children with or without atopic eczemaWith or without autism

probiotic or prebiotic supplementation to liver disease patientsIncreased intestinal stability (reduced permeability)Decreased bacterial translocation to the liverAmeliorated symptoms of hepatic encephalopathy due to decreased ammonia production

Importance: health and diseaseMarked differences of gut microbita between horses with colitis compared with normal horsesHorses fed diets rich in readily fermentable carbohydrate are:

at increased risk of developing laminitis, simple colonic obstruction and distension with concurrent changes in hind gut bacterial population compared with forage fed animals

Methods to study gut microbiota

Microbita study: Culture based techniques

Isolation and culture on selective and non-selective mediaidentification through:

colony morphological characters, growth patterns on different medias, biochemical characters

Culture based techniques: pros and cons

ProsRelatively inexpensiveWidely available, Possible physiological and biochemical studies, Can provide a good indication of ecosystem complexity

ConsMost of the bacterial community is unculturable

only 10% of direct microscopic count from small intestine and 30% from caecum and colon of horse are culturable60-70% of human gut bacteria are not culturable

methods of sampling, transport, cultivation and identification are inconsistent among the different studies; results discrepancyvery laborious and time consuming

Study Method

sample M.Os

Number (log10 CFU/g)*

Studied effect Result

Medina et al., 2002

Culture

Colonic content 

Total anaerobesLactobacillistreptococci

8.86.96.9

Reliability of saccharomyces cerevisiae to limit the negative impact of concentrate overload

The decrease in pH and the increase in lactic acid production were ameliorated

Milinovich et al., 2008

qPCRCecal content

Total bacteriaEHSS

1210

Changes in cecal microbiota in response to oligofructose induced laminitis

Drastic increase in quine hindgut streptococcal species count

Culture vs. molecular based techniques: horse’s gut

* log number of the colony forming units per gram of sample (faeces or gut content)Total bacterial counts obtained by culture-based methods are many logs less than the results of qPCR

Molecular-based techniques

Samples to study: Feces Vs. intestinal content

A non-invasive surrogate for the lower intestinal microbiota Human Microbiome Project, 2012.Reflect the shifts in caecal microbiome in horses with experimentally induced laminitis yet, the relative abundance is not reflected Milinovich et al., 2007.Represent the microbial diversity of the distal colon (dorsal colon) Hastie et al., 2008.

Fecal samples preservationNo difference in microbial composition between samples that processed directly after defecation and those stored for 24 hours at room temperature Carroll et al. (2012.long-term storage of faecal samples at -80 for up to 6 months has no detrimental effects on microbial community structure and diversity Wu et al. (2010.

Microbita study: Molecular-based techniques basis

The 16s rRNA gene constitute the fundamental basis of molecular studies of microbial communities

50S

30S

23S

5S

16s

Plus 32 proteins

Plus 21 proteins70S

RNA

16s rRNA: Reliable phylogenetic marker

homologous function and universal location in all micro-organismsContains conserved regions; did not change over timeUnique nine variable regions (v1-v9). universal primer can be used to amplify nearly all rRNA genes of all microorganismsHas appropriate length (about 1.5 kbp) compared to 5S (short) and 23S (long) rRNA molecules.

Part of 16s rRNA gene

Microbiota studies: Overview of techniques to characterize the gut microbiota

Faecal sample FISH

DNA isolation

Amplification of 16s rRNA gene by PCR

Separation of 16srRNA (DNA Finger

printing)Band

resolution

Microbiome shotgun

sequencing

Direct sequencing of 16S rRNA amplicons

Sequencing of cloned 16s rRNA amplicons

Quantitative PCR

DNA microarrays

Band excision and sequencing

Probe hybridization

Culture

Extraction of DNA and amplification of 16S rRNA genes

Fraher, M. H. et al. (2012)

Molecular techniques: FISH

Fluorescence in situ hybridization

Molecular techniques: DNA microarray

DNA Microarray

DNA finger printing: DGGE, TGGE

Conventional electrophoresis

DNA fingerprinting

454 pyrosequencing

T. Liloglou, 17 Jul2013

Pyrosequencing

39% 36% 37% 32% 39% 36% 38% 40% 43%

EE SS AA TT CC GG TT5

GG AA TT TT CC10

TT GG TT CC GG15

TT GG TT GG CC20

TT TT AA GG TT25

CC TT GG TT CC30

AA GG TT CC TT35

GG TT CC GG TT40

GG TT AA GG TT45

CC TT GG TT CC50

GG GG TT0

10

20

30

40

50

60

A2 : Y GGGT AT T T T YGY GT GGTGTTTTGYGGTYGTYGTYGTTGTGGTYGTTYGGGGTGGGGTGTGAGGAGGGGA

T. Liloglou, 17 Jul2013

3’ – TGTCGAGTCAGGTAAATTTTGAAAGCCA – 5’ 5’ - ACAGCTCAG

Pyrosequencing: Principle

TCCA CTTTAAAA TTTCGGT

4×3×2×1×

T C A CT A T C G T

Dispensation Order Sequence

454 Sequencing vs. other techniquesProvided an open ended view of microbial communitiesNon sequencing techniques: only known bacterial groups can be detected using pre-designated oligonucleotide probes.Sequences can't be directly assigned to speciesSequences are annotated into operational taxonomic units (OTU) (phylotypes)OUT includes cluster of similar sequences with identity threshold of 97%

Pitfalls of molecular-based techniques: Extraction bias

Rigorous processing; excessive fragmentation of bacterial genome; formation of more chimeric DNAThe different extraction methods have resulted in

extraction of different quantities of DNAvariation in phylotype abundance and composition of microbial communities

Pitfalls of molecular-based techniques: Inhibitors

Inhibitory substances in sample; can inhibit the PCR amplification of the extracted DNA:

Bile salts and complex polysaccharides in faecesCollagen in foodHeme, immunoglobulin g and lactoferrin in blood Humic acid in soilMelanin and myoglobin in tissues polysaccharides in plants; proteinases and calcium ions in milkurea in urine

Pitfalls of molecular-based techniques: Amplification bias

Differential or preferential PCR amplificationFormation of PCR artefacts (chimeric molecules, deletion mutant, and point mutant)DNA contamination (false positive) 16s rRNA sequence variation due to variation in number of rRNA gene regions (rrn operon) Hairpin lope, primer dimers formation,

Pitfalls of molecular-based techniques: Universal primers are not universal

No single set of primers can guarantee amplification of all prokaryotic 16s rRNA genesThe coverage rate of twenty nine known primers including 13 Archaea-specific, 9 Eubacteria-specific, and 7 universal primers based on RDP database.

Average coverage rate of these primers are 85%, 77.4% and 83.3% respectively30.6% of known primers have a coverage rate less than 90%.

Molecular studies of horse’s gut: Colitis vs. normalFirmicutes (68.1% control, 30.3 colitis),Bacteroids (14.2% control, 40.0% colitis) Proteobacteria (control 10.2%, 18.7 colitis),Costa et al., 2012454 pyrosequencing

Horse’s gut: forage vs. concentrate fed and SCOD

Daly et al., 2012: Quantitative oligonucleotide hybridization, Colonic content

Lachnospiraceae

Bacteroides

Bacillus-Lactobacillus-Streptococcus group

Horse’s gut: forage vs. concentrate fed and SCOD

Daly et al., 2012: Quantitative oligonucleotide hybridization, Colonic content

Fibrobacter spp.

Ruminococcaceae

Normal horses vs. those with chronic laminitis

Steelman et al., 20122. Pyrosequencing fecal samples

17 29

Normal horses vs. those with chronic laminitis

Steelman et al., 20122. Pyrosequencing fecal samples

108