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1.19.5.10 - Understanding Bacterial Differentiation 1 - Coggle Diagram
1.19.5.10 - Understanding Bacterial Differentiation 1
epidemiology
what is the cause
is there a common casue between cases
is there a common pathogens linking it to the same source
genus and species
bacterial biochemistry
biochemical separation
pure cultures can be used to assess biochemical differences which could not be checked on media alone
superseded by molecular techniques
DNA sequencing
using multiple tests to categorise bacteria into genera and species
diagnostic approach
API test strips
combine a number of biochemical tests and extensive data base for interpretation
established reference technique
specific assays/reactions
distinguishing different bacteria
morphology
motility
cell wall comparison
respiration
aerobic
anaerobic
facultative anaerobe
microaerophilic
colony morphology on specific plates
growth on specific media
haemolysis
substrate utilisation
presence and absence of sepcific enzymes
tolerance to compounds
lineann taxonomy
tests
haemolysis
catalase tests
oxidase tests
coagulase tests
urease tests
fermentation assays
haemolysis
assess the ability of the bacteria to lyse erythrocytes
when grown on blood agar some lyse (brake open) or damage blood cells to access iron
blood agar - enriched medium
common descriptions
alpha
incomplete produces green zone of lysis
beta
complete destruction clear zone of lysis
gamma
absence of any haemolysis
In some bacterial species the ability to haemolyse is indicative of how virulent the bacteria are as iron is a limiting factor in the mammalian host
catalase & oxidase test
how components relating to respiration can be present or absent in different bacteria and used to help discrimination
catalase test
fizzes as it produces oxygen
protect cells from oxidative damage by reactive oxygen species (ROS)
catalyses the decomposition of hydrogen peroxide to water and oxygen
reflect common parts of their respiratory biochemistry
oxidase tests
Cytochrome c is an enzyme of the bacterial electron transport chain used by bacteria growing aerobically
colour change to blue comes from reduction of test compound
identifies bacteria that produce cytochrome c oxidase
coagulase tests
identify the presence of bound coagulase or clumping factor
shows how properties can impact on the patient
coagulase reacts with the fibrinogen in plasma, causing the fibrinogen to precipitate
clinical use
Coagulase is often associated with virulence but some coagulase negative strains can cause disease
(To separate coagulase positive /negative Staphylococcus aureus and also separate other groups of non-coagulase positive staphylococci S. epidermidis)
urease test
pH based indicator test
breaks urea down to CO2 and ammonia
the indicator is more red the more alkaline it is
yellow = more acidic
rapid diagnostics
ability to ferment different sugars
The assays can be changed for different sugars
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
MacConkey agar does a fermentation test for lactose in a solid format
The inverted Durham tube in the assays can collect CO2 which is produced by some bacteria when fermenting the sugars
multiple tests can be combined and different bacteria have a almost a bar code of which sugars they can or can not ferment
selective media
Antimicrobials are compounds which inhibits bacterial growth. Some are selective only acting on certain groups of bact
Selective media contain antimicrobial compounds which inhibit groups of bacteria you do not want allowing those you want to isolate to grow
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
MacConkey Agar
bacteria which ferment lactose
decrease the pH of the medium
the decrease is detected by neutral red
when pH drops the neutral red is absorbed and the bacterial appear as bright pink/red colonies
typing
separation of bacteria within the same species is typing
typing allows linkage of bacteria derived from the same lineage or source
DNA Variation
ribosome sequencing
MLST
probes
PCR
mass spectrometry
recognition of variable surface structures
phage typing
serotyping
phage typing
phage is a virus that can infect bacteria
pattern of susceptibility is the phage type
phage attaches to the surface structures
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
serotyping
Use of antibodies that recognize variable structures. Antibody-antigen interaction is very specific!
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
O-antigen is the lipopolysaccharide (LPS) of Gram-negative bacteria
Some more variable used to define different lineages within the same species
Antibody targets are specific to groups of bacteria
often uses latex beads with antibodies attached to them
if there is antibody binding, there are precipitations where the latex beads fall out of suspension
serology vs serotyping
Serotyping uses antibodies
defines closely related bacteria using antibodies which bind known antigens of those bacteria.
Serology measures antibodies
Common method is ELISA
If an animal is infected, it ‘may’ generate an antibody response to the pathogen.
Can infer infection based on antibody level.
Does not confirm current infection
Cross reaction between related species
May indicate exposure
Positive result from vaccinated animals.
uses sera from patients to measure if antibodies have been produced by a patient to a specific pathogen
diagnostic tool
Molecular typing, DNA & pathogen identification
allow for identifying species
When DNA replicates every so often mistakes occur (mutations)
Over time mutations can accumulate and can distinguish populations that have grown up independently
Genes may also be lost / gained they directly relate to properties of the bacteria
This can be used to separate very closely related bacteria
Molecular diagnostics is a rapidly developing field, current methodologies include
PCR
Sequencing
MLST (multi locus sequence typing)
Mass Spec
PCR
PCR – 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
Amplification of PCR product
Annealing of primers
Denaturation of DNA strands
Quantification (qPCR)
PCR product can be sequenced
variation
Identification of specific pathogens
PCR/qPCR: Advantages & Disadvantages compared to culture based methods
Disadvantages
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
cannot distinguish between viable or dead bacteria
Advantages
rapid
cheap
specific
high sensitivity
minimal equipment and consumables
sample can be frozen
MLST
A method to classify bacterial/fungal isolates to allow epidemiological studies/outbreak tracing etc
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
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
Each unique allelic profile is given a one digit sequence type (ST)
mass spec
Suspended
In relevant buffer
shot with laser
Putative isolate from media picked.
produces +ve charged ion
Ion accelerator send to detector
Result mass spectra (MS)
protein matched against known bacteria
AMR profile in future?
summary
Bacterial phage (phage typing) and antibodies (serotyping) can be used to differentiate bacteria.
In contrast to serotyping that uses antibodies to identify bacteria, serology measures antibodies to determine the host immune response to specific microbes.
Selective media contain antimicrobial compounds which inhibit groups of bacteria you do not want allowing those you want to isolate to grow (ie MacConkey, XLD)
Molecular methods for diagnostics & typing include PCR, MLST and Mass spectrophotometry.
Biochemical differences can be used to differentiate between bacterial groups and species using specific tests (ie catalase, oxidase) or API test strips