Bacterial Classification and Identification

Bacterial Classification and Identification

Bacterial Classification and Identification

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Microbiological Quality

Microbiological quality of pharmaceuticaland medicinal products is of critical importance

Measured according to 4 categories:

  • Preparations required to be sterile on the dosage form
  • Preparations for topical use and for use on the respiratory tract
  • Preparations for oral and rectal administration
  • Herbal remedies
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Bacterial Classification

  • Classification of bacteria is more difficult than for eukaryotic cells
  • For eukaryotic organisms, a species is defined as a group of closely related organisms, which reproduces sexually to produce fertile offspring
  • Bacteria do not reproduce sexually 
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Classification of Life

  • Based on the nucleotide sequence of rRNA
    • ribosomal RNA
  • Present in all living cells
    • ribosomes are required to build proteins
  • rRNA sequence foms the basis for phylogeny
  • Three domain system proposed by Carl Woese
    • Bacteria (Prokaryotes)
    • Archaea (Prokaryotes)
    • Eukarya (Eukaryotes)
  • Bacteria are often named after a person or a way of describing the bacterium itself
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Bacterial Identification

  • Simpler traditional methods
    • Cultivation - growth requirement
    • Cultivation - selective agar
    • Biochemical profiling
    • Serological testing
  • Complex newer, more rapid methods
    • Nucleic acid techniques
    • MALDI-TOF methods 
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Cultivation

  • Some bacteria may be grown in the laboratory
  • Allows morphologicalc characterisation
  • Spore production, flagella
  • Shape (cocci, bacilli etc.)
  • Staining characteristics
  • Colony morphology
  • Morphology alone can not distinguish between similar looking bacteria
  • Additional techniques are used to narrow down the identification
    • Biochemical profiling
    • Growth requirements and selective media
    • Immunological identification
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Cultivation Considerations

  • Oxygen requirements
    • Aerobic
    • Anaerobic
  • Temperature requirements
  • Salt tolerance
  • Requirements for specific nutrients, e.g. amino acids
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Biochemical Profiling

  • Bacteria can be identified based on their enzymatic activities; widely used to distinguish between bacteria
  • Even closely related microorganisms can be separated on the basis of these tests
  • These tests can be performed in traditional laboratory tests, or in the form of kits
  • Tests for ability to:
    • Ferment various sugars - some are restricted, some vary
    • Produce oxidase - detect activity of cytochrome oxidase if present
    • Hydrogen sulfide production - reacts with iron salts forming a black precipitate
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Selective Media

  • Vogel Johnson Agar
    • Tellurite and lithium chloride (selective)
    • Tellurite - Coagulase reduces to metallic tellurium - black colonies
    • Manitol degradation alters pH - yellow halo
    • Indicates S. aureus
  • MacConkey agar
    • Growth of gram negative (bile salts and crystal violet inhibit most gram positive)
    • Distinguish lactose fermentors 
      • E. coli, Klebsiella: red/pink colonies, lactose positive
      • S. typhimurium and P. aeruginosa: white colonies, lactose negative
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Cultivation/Kit/Selective Media

  • Positives
    • Doesn't require specialised equipment
    • Easy to use
    • Can be automated
  • Negatives
    • Time consuming (18-72 hours of incubation)
    • Not all bacteria can be cultivated under normal laboratory conditions (0.1%)
    • Kits can be expensive
    • Miss-reading of results
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Serological Testing

  • Uses highly specific antibody and antigen interaction
  • Can be used to detect certain pathogens
  • Enzyme Linked Immunosorbent Assay (ELISA)
  • Positives
    • ELISAs are available to test for E.coli, S.aureus, Salmonella and other organisms
    • Highly specific
    • High sensitivity - signal is amplified by the conjugated enzyme
  • Negatives
    • Expensive
    • Can not identify unknown bacteria
    • Complex process (training)
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Nucleic Acid Techniques

  • Polymerase Chain Reaction (PCR) based techniques allow amplification of a known gene of interest for nucleic acid sequencing
  • Sequencing of rRNA provides very accurate identification to genus or species level
  • Can be used to identify unknown/uncultivable organisms
  • Gene sequence is then compared with online database, e.g. NCBI (National Center for Biotechnology Information)
  • BLAST (Basic Local Alignment Search Tool) finds regions of similarity between the sequence and previously identified sequences
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Nucleic Acid Techniques

  • Positives
    • Do not require growth of bacteria for identification
    • Very sensitive
    • High accuracy
  • Negatives
    • Specialised equipment
    • Costly reagents
    • Time consuming
    • Only as good as the reference library
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Nucleic Acid Techniques

  • Genome sequencing
  • Not just bacterial identification, but information about metabolism and growth requirements, pathogenicity (including antibiotic resistance)
  • Becoming more cost effective (around $1000 per genome)
  • Vast amounts of data (time consuming analysis)
  • Requires expensive equipment and reagents
  • Far more expensive than other methods
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Microarrays

  • Thousands of ssDNA (single stranded DNA) probes are fixed to a solid support (chip)
  • Probes could be rRNA which helps identification or antibiotic resistance genes
  • Labelled (fluorescent) target sample is hybridised with probes
  • Positive match shows a bright dot
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MALDI-TOF MS

  • Matrix-assisted laser desorption ionisation time-of-flight mass spectrometry
  • Uknown bacteria mixed with matrix material
  • Bombarded with laser pulses
  • Sample is ionised and passes through electrostatic field (acceleration)
  • Ions pass through flight tube before hitting detector (small ions travel faster than large ones)
  • Generates a unique mass spectrum for different bacteria - check against database
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MALDI-TOF MS

  • Cost effective 
  • Short turnaround times (6-8 minutes vs. 5-48 hours)
  • Precise identification to species level
  • Can't identify species in mixed bacterial populations
  • Reliant on quality and coverage of the database used
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Traditional vs. Newer Methods

  • Traditional methods
    • No specialised equipment
    • Can be done by most laboratories
    • Minimal training
    • 24-72 hours for result
  • Newer methods
    • Specialised equipment required
    • Lower running costs
    • Faster (around 6 hours)
    • High throughput (around 140,000 samples per year)
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