MHD 2 - Methods to Characterise the Microbiota PDF

Preview

Summary

Dr. Jia Li, Dr. Andreia Vargas-Seymour, Prof Julian Marchesi...

Description

Each region of the body has its own microbiota - Composition is defined by nutrient, pH, temperature and atmospheric conditions in local environment Microbial communities across the body can be studied / characterised collectively using - Cultivation-based methods - Molecular-based methods

16S rRNA gene sequencing is used Some major phyla of bacteria represented within human-associated microbiota - Bacteroidetes, Firmicutes, Actinobacteria, Fusobacteria and Proteobacteria

Characterising the microbiota Individual prokaryotes, viruses, fungi and parasites contributing to the microbiota can be isolated and characterised using - Phenotypic methods - Deal with one or more observable characteristics - Genotypic methods - Dela with one or more genetic characteristics

Terms Taxanomy - The branch of science dedicated to classifying microorganisms and viruses - Provide a point of reference for these entities based on phenotypic and genotypic characteristics Phenotype - Covers morphological, metabolic, physiological and chemical characteristics of a species Genotype - Describes the characteristics of the species’ genome - One or more genes / whole genome can be used to genotypically characterise a species Phylogeny - The study of the evolutionary history of organisms using molecular sequence data - Allow us to group organisms evolved from a common ancestor according to their genetic properties Systematics - The study of the diversity of organisms and their relationships - Underpinned by taxonomy and phylogeny

The Taxonomic Hierarchy Classification allows us to assess the degree of similarity among organisms, with ranking going from broad to narrow Example: E. coli

Species belonging to the same genus are far more similar to one another than in higher groups - Are separated by only 1 or 2 genotypic / phenotypic traits

Broader taxonomic hierarchy

Binomial nomenclature - naming of organisms First term: genus Second term: species - Both in italics (e.g. Escherichia coli)

Cultivation-based Characterisation Species of bacteria are isolated from clinical samples using ____ method - Anaerobic (e.g. Bacteroides) - Microaerophilic (reduced O2 levels compared with atmosphere) (e.g. Helicobacter) - Capnophilic (elevated CO2 levels compared with atmosphere) (e.g. Campylobacter) - Aerobic (e.g. Escherichia coli)

Growth capabilities of microbes under different atmospheric conditions

Obligate aerobes - only when O2 concentration are same as atmosphere Obligate anaerobes - only when complete absence of O2 Facultative anaerobes - better in presence of O2, but can grow in both Microaerophiles - only at low levels of O2 Capnophiles - require increased levels of CO2 Aerotolerant microbes - equally well in both presence and absence of O2

Characterisation of isolated bacteria relies on a combination of phenotypic tests -

-

Gram staining Ability to grow in different conditions Cell shape Simple biochemical tests to see - Which sugars bacteria fermented / produced gas from - What short-chain fatty acids they produced from glucose fermentation Susceptibility to different antimicrobials Characterizing cell-wall fatty acids

Now combine these methods with molecular-based ones → Give more accurate taxonomic assignments (polyphasic approach) Many biochemical tests can be done using kits - API strips (left) - Biolog system (right)

Different types of agar to cultivate different types of bacteria Can culture bacteria using agar-based / broth-based methods Species more likely to be isolated are dictated by - Method chosen - Nutrient composition of the medium - Atmospheric conditions - Incubation temperature (usually human core body 37) Most bacteria can be isolated on agar plates where each colony is assumed to derive from a single bacterium

Mixed microbiota Each different colony potentially represents a different species of bacteria

Initially, mixed cultures are recovered from microbiota samples Individual colonies have to be propagated to purity before identified using phenotypic / genotypic tests

Colonies of a pure culture of Staphylococcus aureus (golden appearance) on nutrient agar

Selective media - used to increase the chances of isolating specific groups of bacteria

MacConkey agar -

Selective and differential medium Allows enrichment of Gram-negative bacteria - Contains bile salts and crystal violet → inhibit Gram-positive bacteria Allows Enterobacteriaceae to be differentiated based on their ability to ferment lactose Contains lactose and pH indicator neutral red

Pink colonies - Enterobacteriaceae capable of fermenting lactose (lactose-positive) - Escherichia, Enterobacter, Klebsiella - Acid produced lowers the pH White colonies - Not capable of fermenting lactose - Salmonella, Proteus, Shigella Identities of isolated colonies can be confirmed quickly using API 20E strips

Chromogenic agar -

Used to identify closely related bacteria Generally only used in clinical / commercial laboratories

Spectrum Chromogenic System -

Allows differentiation of different clinically relevant Gram-negative (Escherichia, Pseudomonas, Proteus, Klebsiella) and Gram-positive (Staphylococcus, Enterococcus) bacteria based on species-specific enzyme activities

Columbia blood agar -

Rich, non-selective medium Used to isolate a range of fastidious aerobic and anaerobic bacteria

Fastidious bacteria have complex nutritional requirements Non-selective medium allow all microbes to grow Nutrient agar is a non-selective medium used for isolation of non-fastidious aerobes and anaerobes

Chocolate agar -

Made by heating blood agar → blood cells burst → release NAD and haemin required for growth by very fastidious bacteria e.g. Neisseria meningitidis (it lacks enzymes necessary for lysing red blood cells)

Anaerobic bacteria are more difficult to work with than aerobic bacteria -

Specialist equipment is required for the isolation and growth

Hungate tubes -

-

Used to prepare anaerobic broth medium - Inoculated with obligately anaerobic bacteria using a needle inserted into the butyl rubber stopper Rubber stopper is sealed by heating → maintain anaerobic environment in tube

Sealed cabinet -

Where anaerobic / microaerophilic environment is established Used to isolate and grow obligately anaerobic / microaerophilic bacteria respectively

Gas pack -

Used in gas jars to isolate and grow facultative / oxygen-tolerant anaerobes or capnophiles Takes ~30 min for required atmospheric conditions to be established after gas packs are activated and jars sealed

Important factors to consider when working with cultured members of the microbiota Difficult to obtain samples from different regions of intestine → Faecal samples are used to study the ecology and activity of bacteria within gastrointestinal tract However Faecal microbiota is not representative of that of the gastrointestinal tract as a whole E.g. strains of the same species isolated from different regions of the gastrointestinal tract metabolise dietary substrates in different ways ** Always keep this in mind when making inferences from in vitro and in vivo studies in relation to specific gastrointestinal diseases, particularly those involving the more proximal regions of the intestine

Faecal and caecal isolates of E. coli metabolise the dietary substance trimethylamine-N-oxide (TMAO) differently (left)

Caecal isolates (blue) produce more TMA from TMAO - TMAO is metabolised by bacteria in the upper gut (small intestine and proximal large intestine)

Molecular-based Characterisation The sequences of some genes change much more slowly over time than others - Housekeeping genes: essential to the proper functioning of a cell The one to characterise individual species of bacteria and archaea and all prokaryotes of the microbiota - 16S ribosomal ribonucleic acid (rRNA) gene Several rRNAs make up the machinery involved in the translation of messenger RNA to protein - rRNAs make up the ribosome (primary site of protein synthesis in cells) - Each rRNA that contributes to ribosome structure is encoded by a different rRNA gene Sequence of rRNA genes change at a relatively constant rate over time irrespective of the taxonomic lineages within which they are present for → Considered as “molecular clocks”

Early studies look at the use of rRNA genes to characterise bacteria relied upon 5S rRNA gene - Cheaper than 16S Now, full length 16S rRNA gene is used in studies of pure cultures of bacteria - Encodes sufficient information to give species identification - Has 2 notable features - Conserved regions: same across all bacteria - Variable regions: differ between species

Uniqueness within V regions enables - Taxonomic positioning - Identification of bacteria

Most studies looking at human microbiota use PCR primers that flank the V3 and V4 regions of 16S rRNA gene - Can amplify up to 450 nucleotides of the gene - Contain sufficient genetic information to describe communities to the genus level Different PCR primer sets can be used → They have different amplification and target efficiencies, giving non-comparable results Collect microbiota sample → extract DNA Amplify V3-V4 regions of the 16S rRNA genes PCR products are tagged with unique identifiers (adapters), one for each of the different microbiota samples PCR products are pooled and run on a sequencer On a Illumina MiSeq machine, it is possible to sequence 96 samples at once, generating up to 100000 sequences per sample (4000 pounds) Process and analyse data using QIIME2 / mothur / DADA2 / phyloseq - Data can be analysed at the phylum to genus level - Data can be reported in terms of abundance (contribution to the total prokaryotic community) - Data can be used various phylogenetic analyses and more complex analyses

American Gut Project To sequence the gut microbiota of as many people as possible Focus on prokaryotes present in faecal samples https://www.youtube.com/watch?v=1uZtCMY-yEw&feature=emb_imp_woyt Can also see faecal samples of pets to determine whether - The person and pet have common bacteria present - Pet-owners can be separated from those without pets on the basis of their mouth / skin / faecal microbiota

Advantage -

-

Cheap Can use tiny quantities of DNA (picograms) as starting material - Previously uncharacterised habitats (skin, respiratory tract, reproductive tract) can now have their microbiotas profiled Large public databases of 16S rRNA gene sequences to inform analyses

Disadvantage Every steps involved can lead to the introduction of bias in data

Sample handling Moving a natural sample to lab without appropriate atmospheric and cold storage will change the relative ratio of taxa → Faecal samples are transported to lab in anaerobic gas jars to minimise sample degradation

Sample storing Freezing samples for a couple of weeks → loss of Bacteroidetes-associated sequences Freeze-thaw cycles can affect populations within samples → Precise details of sample storage and handling are recorded

DNA extraction Release of DNA after cell lysis depends greatly on the structure of the peptidoglycan and outer membranes of the bacteria present in samples Occurence of Gram-positive bacteria is generally underestimated unless DNA extraction incorporates bead-beating to break open these tough bacteria However if too vigorous DNA extraction method is used → damage genes → Increased formation of erroneous PCR products / non-amplification of 16S rRNA genes

PCR -

Differences in the specificity of polymerases Inhibition of the reaction by interfering substances Differential PCR amplification PCR artefacts and contaminated reagents

DNA extraction kits are not sterile = source of contamination → Differences in datasets when there should be none → All microbiota profiling studies should contain a negative control - Sequences that get associated with this sample can be removed from all other samples prior to data analysis so won’t get confound results - Particularly important in studies involving low-biomass samples (e.g. nose, skin, lungs, vagina)...