Bacterial Classification and Identification
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- Created by: amazingemilyjones
- Created on: 10-04-19 20:29
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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