vii. Microbial Typing Techniques

Question 1

Microbial typing:

Answer 1

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

Question 2

Strains:

Answer 2

Set of bacterial isolates that when typed are indistinguishable from each other and can be differentiated from other bacterial isolates

Question 3

Phenotype:

Answer 3

Physical characteristic eg. Antigens and proteins expressed, biochem rxns, growth requirements, resistance to antibiotics, resistance to bacteriophages

Question 4

Genotype:

Answer 4

Nucleotide sequence found on chromosome of the bacteria

Question 5

What is Microbial Typing?

Answer 5

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

Question 6

Why Microbial Typing?

Answer 6

Question 7

Application:
Outbreak Investigations:

Answer 7

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

Question 8

Application:

Detect recurrent versus new infections in Pt by same ______.

Answer 8

organism

Question 9

Application:

Surveillance:

Answer 9

Predict, observe, and minimise the harm caused by outbreak, epidemic and pandemic

Question 10

Application:

Examine prevalence of organisms:

Answer 10

Endemic in hospital i.e. MRSA, VRE, CRE (identify clusters), strain evolution (changes in antibiotic resistance), detect new emerging pathogens/clones

Question 11

Typing Techniques:

Characteristics of good typing method:

Answer 11

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)

Question 12

Typing Techniques:

Divided into phenotypic and genotypic: Phenotypic tech:

Answer 12

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

Question 13

Typing Techniques:

Genotypic tech:

Answer 13

Genotypic tech: molecular, more discriminatory as DNA-based, discriminatory power still vary btwn dif techs

Question 14

What are the Phenotypic Typing Techniques? (4)

Answer 14

• Serotyping
• Phage typing
• Antibiotic susceptibility-resistance pattern
• Protein Typing

Question 15

What is serotyping?

Answer 15

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)

Question 16

What is phage typing?

Answer 16

Bacteriophages are viruses that infect bacterial cells characterisation by bacteriophage susceptibility pattern

Question 17

Antibiotic susceptibility-resistance pattern: often 1st test used to identify ________ strain - follow this with molecular (genotypic) techs

Answer 17

outbreak

Question 18

Molecular Typing Techniques:

Genome restriction analysis:

Answer 18

Genome restriction analysis: DNA cut into pieces using specific enzymes - resulting fragments analysed on gel/membrane. Eg. Pulse Field Gel Electrophoresis (PFGE)

Question 19

Molecular Typing Techniques:

Pulse-Field Gel Electrophoresis (PFGE):

Answer 19

Question 20

Pulse-Field Gel Electrophoresis (PFGE):

Pros:

Answer 20

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…)

Question 21

Pulse-Field Gel Electrophoresis (PFGE):

Cons:

Answer 21

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

Question 22

PSE based typing:

Specific DNA regions amplified by ___ and analysed on gel. Eg.: amplified fragment length polymorphism (AFLP)

Answer 22

PCR

Question 23

Sequence based typing:

Answer 23

Determine DNA sequence of microorganisms, Multi Locus Sequence Typing (MLST), Whole Genome Sequencing (WGS)

Question 24

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.

Answer 24

microbial
surveillance

Question 25

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 = ________

Answer 25

emerging lineages
Bioinformatics