Molecular Epidemiology
Introduction
ability to track an organism is important in elucidating pathogenesis, epidemiology and decreasing incidence
- i.e. has resistance been transferred or is it newly acquired?; are several cases of the same strain, or are they different?; where is the origin of an infection that is causing problems in a hospital ward?
the root of all these questions is: how do we tell bacteria apart?
- need to distinguish both between species (easier) and within species (harder)
Phenotypic Techniques
– discriminate based on a wide range of biochemical or serologic characteristics
- Biotyping
: testing for activity of aprox. 20 different enzymes (i.e. lac+ vs. lac-)
- advantages: easy, standardized, inexpensive; good discrimination between species
- disadvantages: poor discrimination within a species
- since better techniques are available, biotyping should no longer be acceptable as a epidemiologic tool
- Antibiotic Resistance Profiling
: discriminate based on resistance to different antibiotics
- advantages: well standardized, accessible, cheap, automated; provides useful clinical data
- disadvantages: resistance may be inconsistantly expressed; resistance determinants are often transferrable; similar resistances can be encoded by different genes
- a good initial screening tool
- Serotyping
: detection of surface antigenic differences using immunologic techniques
- advantages: simple, quick, cheap, readily available
- disadvantages: not well standardized; many strains are untypable; different strains may have the same serotype; sometimes serotype characteristics are transferable
- useful for genera which are well-standardized: salmonellae, shigellae, E.Coli (serotype 0157:H7)
- Bacteriophage Typing
: test susceptibility to lysis by a well-defined set of phages
- advantages: system worked out well for salmonella and s. aureus (for a limited number of genera)
- disadvantages: most genera are not typable; different strains may share the same phage group; technically demanding, requires a phage library
- viable technique for species that have been well standardized
- Protein-Typing/Immunoblotting
: examine mobility of cellular proteins via electrophoresis
- advantages: virtually all strains are typable; has correlated well with other techniques
- disadvantages: requires technical expertise to read complex banding patterns
- reliable but seldom used
- Multilocus Enzyme Electrophoresis
: analyze the electrophoretic mobility of soluble cellular metabolic enzymes
- ssentially detects changes in charge which reflect amino acid substitutions from mutations in chromosomal genes
- advantages: high level of discrimination
- disadvantages: could be too discriminatory; technically difficult
- limited usefulness in small outbreaks and in tracking global movement of certain clones (penicillin resistant pneumococci)
Genotypic Techniques
– discriminate based on DNA sequencing
- Plasmid Profile Analysis
: discriminate based on how many and what size plasmids are carried
- advantages: easy, uses standard equipment
- disadvantages: only useful on stains with plasmids; plasmids are mobile; isolation can be difficult
- restriction enzyme digestion can be helpful with bugs with few plasmids; useful in local outbreaks;
- required when outbreak is through the spread of a plasmid rather than of an individual clone
- Restriction Endonuclease Digestion of Chromosomal DNA
: agarose separations of total genomic DNA
- advantages: simple, standard equipment; patterns more stable than plasmids
- disadvantages: large number of bands Þ smear - difficult to read; instability of plasmids can influence the stability of the genomic DNA (insertion)
- limited use due to better techniques
- Southern Transfer Analysis of Genomic DNA
: transfer smear to nylon membrane and hybridize with probe to give a limited number of bands (probes are generally for ribosomal RNA genes or bacterial insertion sequences)
- advantages: easy, simplifies the complicated digestions of entire genome
- disadvantages: some genera have little variation with riboprobes; insertion sequences may either be unstable or not present at all; plasmids may complicate results
- Agarose Separatoin of Large Genomic Restriction Fragments
: use restriction enzymes that only cut in a few places to yield large fragments then use Pulsed Field Gel Electrophoresis (PFGE) or Field Inversion Gel Electrophoresis (FIGE) to separate them (large fragments normally yield a smear, but variable direction current allows separation
- advantages: all strains typable; limited number of bands easy to interpret; plasmid contam. irrelevent
- disadvantages: high startup costs; technically difficult; small genome changes Þ large changes in restriction pattern
- despite disadvantages, PFGE is considered the gold standard