Fecal Bacterial Microbiota Study in a Mouse Model of

Niemann Pick Type C1 Disease

Antony Cougnoux, Section of Molecular Dysmorphology, NICHD

Microbiota

• or microbial flora or flora

• the microscopic living organisms of a particular region (soil, cloud, gut, ...)

• include:

• bacteria (Escherichia, Mycobacterium, )

• fungus (saccharomyces, candida ....)

• archaebacteria or archaea

• protist (algae, plasmodium, ...)

• virus (phage, enterovirus,...)

Microbiota analysis

• Studied in human since the late 50s

• Before the molecular biology era

• only cultivated bacteria ~25% of all class

• 16S PCR cloning and sanger sequencing of all the 16S gene cloned

• PCR-DGGE-fingerprinting

• 16S qPCR using specific primers

• Newest approach to microbiota analysis: High throughput sequencing

Why looking at the gut microbiota ?

• Not only quiescent microorganism here

• Wide spectrum of useful function

• Metabolism: vitamin production, polysaccharide processing, lipid absorption

• Immune function: maturate and educate the host immune system

• Hematopoietic function

• Neurotransmitter production

• Barrier for toxic compound ingested

• Full range of biological significance has not been elucidated

• Little is know about the function of the non bacterial organism (viral and fungal) of the gut microbiota, (immunomodulation for some fungi)

• Variation (disbiosis) in microbiota composition associated to several disease (Crohn`disease, diabetes, obesity, “autism”,…)

Normal gut microbiota and disbiosis

• Disbiosis: inbalance in the microbiota composition

Shift in obesity

Fecal bacterial microbiota analysis

• Sample collection

• DNA extraction

• 16S amplification

• Purification

• Sequencing

• Depending on which part of the microbiota you are interested in the sample collection and DNA extraction is different

• Protocol dedicated to fungus, virus, archea

Mouse fecal sample collection

• Handle the mouse and directly collect in a 1.5mL vial (only if you have no space or no single use boxes and few mice)

Wait 5-10 minutes

Remove the mice

Collect fecal pellet and store at -80 C⁰

Or

Do not re-use the same cup for a different mouseThrow after use if: paper cup

Clean and autoclave if: pipet tip or other plastic box

DNA extraction

• Standard extraction time is 1 and a half hours to extract the DNA from a frozen pellet

• Follow the manufacturer instruction

• Qiagen is the most commonly used DNA extraction kit for fecal samples.

• Useful for comparison with other studies

• Depending on your population of interest several different kits could be used, DNA extracted from swabs require a different type of extraction (disrupting beads, DNA free reagent)

Other kit

• Mobio Ultra Clean® Fecal DNA Isolation Kit (M)

• QIAamp® DNA Stool Mini Kit (Q)(QA),

• FastDNA® SPIN Kit (FSp), and

• FastDNA® SPIN Kit for Soil (FSo).

• ZR Fecal DNA Isolation Kit™ (Zymo Research)

• QIAsymphony® Virus/Bacteria Mini Kit (Qiagen) QS

Kit comparison references

• Claassen et al., A comparison of the efficiency of five different commercial DNA extraction kits for extraction of DNA from faecal samples. Journal of Microbiological methods, August 2013, Pages 103–110

• Kennedy et al., The Impact of Different DNA Extraction Kits and Laboratories upon the Assessment of Human Gut Microbiota Composition by 16S rRNA Gene Sequencing. PlosONE, February 24, 2014.

• Ariefdjohan et al., Comparison of DNA extraction kits for PCR-DGGE analysis of human intestinal microbial communities from fecal specimens. Nutrition Journal. 2010, 9:23. 

• Henderson et al., Effect of DNA Extraction Methods and Sampling Techniques on the Apparent Structure of Cow and Sheep Rumen Microbial Communities. Plos ONE September 11, 2013.

• DNA Extraction and Purification, Labome, Anandika Dhaliwal, updated in 2013

Microbiota analysis from other sites

• Much less DNA extracted from the sample

• External contamination

• Use only sterile reagent and consumable

• Clean the bench (Bleach or acetone and ethanol)

• Kit using beads are the most commonly used

• Online protocol for each type of sample from tree roots to clouds

16S PCR amplification

• 8F-1525r: historically used for 16S amplification and cloning

• 8F (5’-AGAGTTTGATCCTGGCTCAG-3’) is the most commonly used forward primer for 16S sequencing

• 1525r (5’-AAGGAGGTGWTCCARCC-3’)is commonly used

• This PCR amplicon (1500 base pairs) will amplify the variable region of all phyla of bacteria used in taxonomic analysis

• PCR program

• 94 C 5 minutes⁰

• 94 C 45 seconds⁰

• 55 C 45 seconds⁰

• 72 C 1 minute 30 seconds 20 cycles⁰

• 72 C 10 minutes⁰

• 4 C ⁰

• PCR purification from large volume 0.8-1mL

• Single band at 1500bp

• MiniElute PCR purification Kit (QIAgen) BUT replace binding buffer by the DNA Gel extraction binding buffer

• OrangeG appear as a bright spot at the same size as primers

• Two or more band

• Isopropanol DNA precipitation (to reduce the volume to purify on gel)

• Gel extraction using MiniElute gel extraction kit (QIAgen)

• Purified product concentration and purity are analyzed using

• Nanodrop spectrophotometer (Thermo Scientific)

• Qubit Fluorometric quantitation (Invitrogen)

• 1% agarose gel separation

PCR purification

Final Sample preparation

• Each sample should be store in an individual vial (Eppendorff 1.5mL) labeled

• Complete the MGL form for the microbiota sample

• Label use for the vial

• Description of the sample

• Sample concentration

• Picture of the purified 16S PCR product separated on 1% agarose gel as an additional file

Tube label

Sample description

Amplicon size

SampleVolume

SampleConcentration

Purification Type

Final Sample preparation