Summary Advanced Food Microbiology
GI tract composition analysis
Microbial ecology: the interaction of microorganisms with each other and their non-living physical
environment
Microbial populations: assemblages of similar microbes
Microbial communities: mixtures of different microbial populations (GI tract microbial community)
Human GI tract: stomach <103 bacteria, small intestine 105 – 107 colon climax >1011
Molecular versus conventional detection techniques
Culturing disadvantages
Time-consuming & laborious
Suitable selective media not available
Following isolation, further identification is also slow
Drawbacks associated with culture-based techniques are exacerbated when studying anaerobic
habitats. This is especially true of the GI tract environment where a large proportion of the
microbes encountered are strict anaerobes, often non-sporulating
Estimates of cultivability of bacteria from human faeces varies between 10-50%
Molecular technique advantage: overcome the need to cultivate the microorganisms prior to
detection and hence results in a more accurate picture of the microbial diversity and composition.
Need for market molecules suitable for classification
Phylogenetic markers
Ribosomal RNA: universal phylogenetic marker, classification based on evolution rather than
phenotypic characteristics
When >97% similarity, likely to be same species; <97%: species are different
Led to the discovery of Archaea as separate domain
Bacterial rRNA: 16S rRNA, reasonable, manageable size for DNA sequence analysis
- Alternating variable and conserved sequence domains
- Targets for detection & identification of bacteria
Limitations 16s rRNA: useful for distinguishing moderately divergent populations. However, very closely
related populations can be better distinguished by other means. In the future, classification of bacteria
will be based on combinations of two or more unlinked phylogenetic markers or on complete genomes
1. 16S ribosomal RNA (rRNA) gene sequencing and phylogenetic analyses
Aim: identification and phylogenetic analysis
Steps
- DNA isolated directly from feces
– PCR with universal bacterial primer multiple genes by DNA polymerase + primers
– Clone & sequence medium with antibiotic resistance gene
Amplified 16S rRNA gene products are cloned to obtain individual genes, which can be sequenced
– Comparative analysis to 16s rRNA database phylogenetic tree to present phylogenetic relationships
Outcome: % similarity to bacteria; threshold 97%, lower than that: indicates you have something new
that is not in the database (e.g. often termed “uncultured species”), still gives an indication of related
group of microorganism
High Throughput Sequencing Technologies: no cloning of DNA fragments needed
3 arguments in favour: 1. Saving time, 2. Millions of sequence reads at the same time, 3. Cheap
Example: Solaxa or Illumina sequencing, addition of barcodes to known which sample a sequence belong
GI tract composition analysis
Microbial ecology: the interaction of microorganisms with each other and their non-living physical
environment
Microbial populations: assemblages of similar microbes
Microbial communities: mixtures of different microbial populations (GI tract microbial community)
Human GI tract: stomach <103 bacteria, small intestine 105 – 107 colon climax >1011
Molecular versus conventional detection techniques
Culturing disadvantages
Time-consuming & laborious
Suitable selective media not available
Following isolation, further identification is also slow
Drawbacks associated with culture-based techniques are exacerbated when studying anaerobic
habitats. This is especially true of the GI tract environment where a large proportion of the
microbes encountered are strict anaerobes, often non-sporulating
Estimates of cultivability of bacteria from human faeces varies between 10-50%
Molecular technique advantage: overcome the need to cultivate the microorganisms prior to
detection and hence results in a more accurate picture of the microbial diversity and composition.
Need for market molecules suitable for classification
Phylogenetic markers
Ribosomal RNA: universal phylogenetic marker, classification based on evolution rather than
phenotypic characteristics
When >97% similarity, likely to be same species; <97%: species are different
Led to the discovery of Archaea as separate domain
Bacterial rRNA: 16S rRNA, reasonable, manageable size for DNA sequence analysis
- Alternating variable and conserved sequence domains
- Targets for detection & identification of bacteria
Limitations 16s rRNA: useful for distinguishing moderately divergent populations. However, very closely
related populations can be better distinguished by other means. In the future, classification of bacteria
will be based on combinations of two or more unlinked phylogenetic markers or on complete genomes
1. 16S ribosomal RNA (rRNA) gene sequencing and phylogenetic analyses
Aim: identification and phylogenetic analysis
Steps
- DNA isolated directly from feces
– PCR with universal bacterial primer multiple genes by DNA polymerase + primers
– Clone & sequence medium with antibiotic resistance gene
Amplified 16S rRNA gene products are cloned to obtain individual genes, which can be sequenced
– Comparative analysis to 16s rRNA database phylogenetic tree to present phylogenetic relationships
Outcome: % similarity to bacteria; threshold 97%, lower than that: indicates you have something new
that is not in the database (e.g. often termed “uncultured species”), still gives an indication of related
group of microorganism
High Throughput Sequencing Technologies: no cloning of DNA fragments needed
3 arguments in favour: 1. Saving time, 2. Millions of sequence reads at the same time, 3. Cheap
Example: Solaxa or Illumina sequencing, addition of barcodes to known which sample a sequence belong
