vii. Microbial Typing Techniques Flashcards
Microbial typing:
Identification of a bacterial isolate to a strain level (NB: not diagnostic tool), based on phenotypic or genotyping differences, with purpose of controlling nosocomial infections as well as for epidemiology studies
Strains:
Set of bacterial isolates that when typed are indistinguishable from each other and can be differentiated from other bacterial isolates
Phenotype:
Physical characteristic eg. Antigens and proteins expressed, biochem rxns, growth requirements, resistance to antibiotics, resistance to bacteriophages
Genotype:
Nucleotide sequence found on chromosome of the bacteria
What is Microbial Typing?
Members of same bacterial species can have small differences btwn them. These difference are used to further subdivide species to: subspecies, serotypes, strains The process of distinguishing small differences w/in species is known as Typing. Based on phenotypic and genotyping differences within the species
Why Microbial Typing?
Application:
Outbreak Investigations:
Identify/confirm outbreak, find source of transmission (food source), establish origin and mode of transmission of nosocomial outbreaks as well as food-borne/environ diseases, which can have legal implication, determine extent of outbreak and evaluate preventative measures
Application:
Detect recurrent versus new infections in Pt by same ______.
organism
Application:
Surveillance:
Predict, observe, and minimise the harm caused by outbreak, epidemic and pandemic
Application:
Examine prevalence of organisms:
Endemic in hospital i.e. MRSA, VRE, CRE (identify clusters), strain evolution (changes in antibiotic resistance), detect new emerging pathogens/clones
Typing Techniques:
Characteristics of good typing method:
Should be reproducible/standardised (ability of techniques to yield same result when same strain tested repeatedly), should discriminate clearly
and adequately between epidemiology unrelated strains, acceptable turn-around-time, broadly applicable (be able to type broad range of microorganisms, easily available and effort doable, of proven value)
Typing Techniques:
Divided into phenotypic and genotypic: Phenotypic tech:
Phenotypic/observable characteristics express by organism - morphological features, less discriminatory (do not necessarily
discriminate genetic relatedness, limited to small group of microorganisms, certain phenotypic characteristics expressed in relation to environ conditions and pressures
Typing Techniques:
Genotypic tech:
Genotypic tech: molecular, more discriminatory as DNA-based, discriminatory power still vary btwn dif techs
What are the Phenotypic Typing Techniques? (4)
- Serotyping
- Phage typing
- Antibiotic susceptibility-resistance pattern
- Protein Typing
What is serotyping?
Based on interaction of bacterial antigens w/ panel of anti sera. Serotypes are distinct variations w/in a species of bacteria (ie. Variation w/in Salmonella species, there are +2500 serotypes). Based on antigenic determinants of bacterial cell components (outer membrane, flagella, capsule, useful in certain organisms only eg. Salmonella, Shigella, Streptococcus pneumoniae)
What is phage typing?
Bacteriophages are viruses that infect bacterial cells characterisation by bacteriophage susceptibility pattern
Antibiotic susceptibility-resistance pattern: often 1st test used to identify ________ strain - follow this with molecular (genotypic) techs
outbreak
Molecular Typing Techniques:
Genome restriction analysis:
Genome restriction analysis: DNA cut into pieces using specific enzymes - resulting fragments analysed on gel/membrane. Eg. Pulse Field Gel Electrophoresis (PFGE)
Molecular Typing Techniques:
Pulse-Field Gel Electrophoresis (PFGE):
Pulse-Field Gel Electrophoresis (PFGE):
Pros:
Gold standard for many bacteria as good discrimination btwn strains, standardised protocols for many dif bacteria (Salmonells ssp, Listeria monocytogenes, Shigella sp., Campylobacter, Neisseria meningitis, E. Coli…)
Pulse-Field Gel Electrophoresis (PFGE):
Cons:
Cons: Labour intensive - expensive (equip & costly restriction enzymes for high discriminatory power), ambiguous band scoring (variable signal intensities, background noise, differential band motility, gel distortion), takes several days (low through put method), insufficient discriminative power for some isolates, others not typeable
PSE based typing:
Specific DNA regions amplified by ___ and analysed on gel. Eg.: amplified fragment length polymorphism (AFLP)
PCR
Sequence based typing:
Determine DNA sequence of microorganisms, Multi Locus Sequence Typing (MLST), Whole Genome Sequencing (WGS)
WGS: shifting to Whole Genome Sequencing (WGS) as ultimate ______ tying tool (future of microbial typing). Accuracy of WGS supersedes all other typing methods, incl. the gold
standard PFGE. Some countries (USA) even implemented WGS as real-time _________ tool - Track outbreak in real time.
microbial
surveillance
Process of determining complete DNA sequence of organisms’s genome at single time, performed by Next Generation Sequencing (NGS), allow to differentiate between organisms with precision that other typing methods don’t. Facilitates better tracking of outbreaks or ______ _______ within community/country
Advantage of having WGS dats - perform number of analysis using various computer programs/softwares = ________
emerging lineages
Bioinformatics
