Understanding Bacterial Differentiation Flashcards
Describe how bacterial biochemistry can differ and how this can be used for selective culture.
How do you distinguish between bacteria?
Morphology.
Motility.
Cell wall composition – i.e. Gram type.
Respiration.
(Aerobic/ Anaerobic/ facultative anaerobe/ microaerophilic)
Colony morphology on specific plates.
Growth on specific media.
Haemolysis.
Substrate utilisation.
(i.e. what can the bacteria metabolise a big list)
Presence and absence of specific enzymes (i.e. catalase, oxidase).
Tolerance to compounds (Enterobacteriaceae tolerate bile salts).
What is biochemical separation?
Once a pure culture has been established, various test can be carried out to asses biochemical differences which could be checked on media alone
The use of multiple tests can categorise bacteria into genera and in some cases species
What are API test strips?
Combine a number of biochemical test & extensive data base for interpretations
Established reference technique
List common tests and how they can support diagnosis.
- Haemolysis
- Catalase Test
- Oxidase Test
- Coagulase Test
- Urease Test
-Fermentation Assays
Describe the Haemolysis test
Tests the ability to lyse erythrocytes
(when on blood agar some lyse or damage blood cells to access iron
Blood Agar used
Three common descriptions for haemolysis:
Alpha - incomplete produces green zone of lysis.
Beta - complete destruction clear zone of lysis.
Gamma - absence of any haemolysis.
Describe the Catalase and Oxidative tests
Illustrate how components relating to respiration can be present or absent in different bacteria and used to help discrimination
Catalase test
-Catalase catalyses the decomposition of hydrogen peroxide to water and oxygen.
-It helps protect cells from oxidative damage by reactive oxygen species (ROS).
-Catalase trigger decomposition of hydrogen peroxide so it fizzes as it produces oxygen.
Oxidase
-Oxidase test identifies bacteria that produce cytochrome c oxidase, Cytochrome c is an enzyme of the bacterial electron transport chain used by bacteria growing aerobically.
-The colour change to blue comes from reduction of test compound.
Describe the Coagulase Test
Shows how properties can impact on the patient
Identify the presence of bound coagulase or clumping factor, which is attached to the cell walls of the bacteria.
Bound coagulase reacts with the fibrinogen in plasma, causing the fibrinogen to precipitate.
Main clinical use:
To separate coagulase positive /negative Staphylococcus aureus and also separate other groups of non-coagulase positive staphylococci S. epidermidis.
Coagulase is often associated with virulence but some coagulase negative strains can cause disease.
Describe the Urease test
pH based indicator test.
In this case urease breaks down urea to CO2 and ammonia.
The indicator is more red the more alkaline it is. A more yellow the more acidic.
Rapid diagnostics.
Describe the fermentation tests
Ability to ferment different sugars
Liquid fermentation
Vial is to collect gases produced within the tube
Assays can be changed for different sugars. For example the sugars lactose, fructose, galactose etc.
All assays are based on a pH change more acid if the sugar can be fermented
There is a pH indicator phenol red and is red colour at neutral pH.
At low pH the phenol red turns yellow
Another pH indicator used is neutral red which appears as pink to red when pH is low
The inverted Durham tube in the assays can collect CO2 which is produced by some bacteria when fermenting the sugars.
MacConkey agar does a fermentation test for lactose in a solid format but as you will see multiple tests can be combined and different bacteria have a almost a bar code of which sugars they can or can not ferment.
Describe how susceptibility to antimicrobials compounds can be used for selective media.
Selective media contain antimicrobial compounds which inhibit groups of bacteria you do not want allowing those you want to isolate to grow.
Antimicrobials are compounds which inhibits bacterial growth.
Some are selective only acting on certain groups of bacteria.
Example: Certain enteric bacteria (intestinal bacteria) tend to be resistant to bile
salts. If you add bile salts to media it will inhibit n a lot of non-enteric bacteria. This
means the media can be used to select for intestinal bacterial.
Antibiotics can also be used to selected for certain groups of bacteria as some bacteria have intrinsic resistances certain antibiotic groups lacking targets for the drugs.
A whole range of supplements is available to add to media to allow you select out bacteria you do not want.
What is Typing?
Separation of bacteria within the same species
Allows linkage between bacteria derived from the same lineage or source
What are methods of Typing?
Recognition of variable surface structures
–Serotyping
–Phage typing
DNA variation
–Ribosome sequencing
–Multi locus Sequence Typing (MLST)
–Probe based tests
–PCR
Other
–Mass spectometry profiles
Explain how phage typing can be used to differentiate between bacteria
Uses lytic bacteriophage – virus that infects bacteria.
Pattern of susceptibility of a bacterial isolate establishes it phage type (PT).
Phage attaches to surface proteins
These structures can subtly vary between different isolates of the same species or
simply be present or absent
Multiple phage are assessed for their ability to infect the bacteria
The pattern of susceptibility leads to a phage type
Explain how use of antibodies can be used to differentiate between bacteria
Serotyping
Use of antibodies that recognize variable structures. Antibody-antigen interaction is very specific!
Example antigen:
O-antigen is the lipopolysaccharide (LPS) of Gram-negative bacteria
Very subtle structural changes between the O-antigen can lead to changes in binding.
Some O-antigen core structures are more conserved so define genera/species of bacteria.
Some more variable used to define different lineages within the same species.
Antibody targets are specific to groups of bacteria.
How do serotyping kits work?
Use of latex beads linked with antibodies
Bacteria is lysed and antibodies with blue latex in suspension on card are added.
The lysate (proteins, sugars & lipids etc are mixed with the antibodies
Postive result
Antibodies and antigens bind leading to agglutination of latex beads
Negative result
antibody doesnt bind to antigen
Explain the difference between serology and serotyping.
Serotyping uses antibodies
defines closely related bacteria using antibodies which bind known antigens of those bacteria
Serology measures antibodies
uses sera from patients to measure if antibodies have been produced by a patient to a specific pathogen
can be used as a diagnostic tool
E.g. ELISA
Describe Molecular typing, DNA & pathogen identification in their role to differentiate bacteria in epidemiology.
When DNA replicates every so often mistakes occur (mutations)
Over time mutations can accumulate and can distinguish populations that have grown up independently.
This can be used to separate very closely related bacteria.
Genes may also be lost / gained they directly relate to properties of the bacteria.
Molecular diagnostics is a rapidly developing field, current methodologies include:
PCR/qPCR – Polymerase chain reaction Sequencing MLST – Multi locus sequence typing Mass spectrophotometry (MALDI-TOF)
Describe PCR/qPCR and its role to differentiate bacteria in epidemiology.
cyclic amplification of specific piece of DNA
Use of primers –
small pieces of DNA that are specific and complementary to the target gene to amplify
Three Steps
Denaturation of DNA strands
Annealing of primers Amplification of PCR product
Identification of specific pathogens
Quantification (qPCR)
PCR product can be sequenced variation
What are the advantages of PCR?
Rapid (hours)
Cheap
Specific
High sensitivity
small amounts can be detected
Minimal equipment and consumables
no need for lots of different media, incubators, biochemical tests
Sample can be frozen
What are the disadvantages of PCR?
Can not distinguish between viable or dead bacteria as bacterial DNA is stable within the environment
Antibiotic sensitivity testing cannot be performed
(specific AMR genes could be amplified by PCR but you do not know then by which bacteria produced if it is a mix)
Describe MLST – Multi locus sequence typing and its role to differentiate bacteria in epidemiology.
A method to classify bacterial/fungal isolates to allow epidemiological studies/outbreak tracing etc
Typically 7 housekeeping genes are studied, these genes are essential so each strain will have them!
Each strain is characterized by a profile of 7 allele numbers; the allelic profile
If the sequence is different (even by just 1 nucleotide) to all others, it is considered to be a new allele
and is assigned a unique allele number
Each unique allelic profile is given a one digit sequence type (ST)
Describe Mass spectrophotometry (MALDI-TOF) and its role to differentiate bacteria in epidemiology.
Newly developed method
Requires a single colony -species identification
Pprofile using graph produced
