Identifying Bacteria

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Identifying Bacteria
2011-11-08 15:14:47
PMB 112 midterm2

general microbiology midterm 2
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  1. Case 1 - totally unknown organism
    • from an environmental sample
    • from a new disease specimen
    • from an industrial process that is going wrong in an unusual way

    • 1) PCR 16S rRNA from organism and sequence
    • 2) submit sequence to 16S database to find closest relatives
    • - this will identify the genus and sometimes the species of your organism
    • 3) PCR and sequence a set of 7-10 housekeeping genes
    • - this will identify species and possibly strain, if your organism has been identified before (MLST)
    • 4) still not a previously identified species?
    • - characterize and submit description of a new species
  2. case 2 - have an unknown sample from a familiar source, can make an educated guess that organism is one you have seen before
    • clinical samples from patients with various symptoms
    • contaminants from processes

    • bypass 16S rRNA sequencing and go to direct comparisons which can be faster or give information about strains
    • 1) Ribotyping
    • 2) FAME
    • 3) MLST
  3. ribotyping
    • 1) isolate DNA from your unknown organism and known candidates
    • 2) Digest DNA with one or more restriction enzymes
    • 3) Separate fragments by electrophoresis
    • 4) visualize using a probe that hybridizes to rRNA sequences

    • pattern of restriction fragments is based on the rRNA sequence, but no sequencing reactions are performed
    • pattern is unique to species and sometimes to strains
    • technique is useful for determining if your bug is one of a few known species
  4. FAME (fatty acid methyl ester) lipid analysis
    • determine types and proportions of fatty acids present in phospholipids of the bacterial membranes
    • fatty acids differ
    • each species has a fatty acid fingerprint in a database and an algorithm picks the best matches to your organism
    • good for identifying what species you have but not what strain
    • many factors can influence the lipid composition - only useful when growth conditions can be strictly standardized
  5. multilocus sequence typing (MLST)
    • PCR and sequence several housekeeping genes that are present in all of the organisms compared
    • based on sequences, each organism gets a set of allele numbers
    • compare the set of alleles in one organism to sets in the other organisms to determine relatedness
    • best at distinguishing species and strains - has scientific, clinical and epidemiological utility