Definitions of the Main Terms Describing Genotypic and Epidemiological Relationships Among Isolates
Adapted from Tenover, F.C., Arbeit, R.D., Goering, R.V., Mickelsen, P.A., Murray, B.E., Persing, D.H., and Swaminathan,B.J. Clin. Microbiol., 33(9), 2233–2239, 1995 [3] and Arbeit, R.D.IDCP,5(4), 260–264, 1996 4].
Isolate-Pure culture of bacteria obtained by subculture of a single colony from a primary isolation plate and presumed to be derived from a single organism.
Strain-Isolate or set of isolates that can be differentiated from other isolates of the same genus and species by phenotypic and/or genotypic characteristics. A strain is a relative concept, a descriptive subdivision of a species based on one or more analyses, and therefore should not be used as a synonym for isolate.
Epidemiologically related isolates-Isolates cultured from specimens taken from patients or the environment during a specific time frame or from a particular area as part of an epidemiological investigation that suggests that the isolates may be derived from a common source.
Genetically related isolates (clones)-Isolates that are indistinguishable from each other by a variety of genetic tests or that are so similar that they are presumed to originate from a common precursor.Such isolates are often referred to as having a particular “genotype”, representing a specific “genetic lineage” or to be “clonal.”
Outbreak-Increased incidence of an infectious disease in a specific place during a given period that is above the baseline rate for that place and time frame.
Outbreak strains- Isolates of the same species that are both genetically and epidemiologically related (e.g., by time, place, and common source); by implication, the outbreak represents a set of isolates that are derived from a common parent.
Endemic strains- Isolates that are recovered frequently from infected or colonized patients in a particular place and that are indistinguishable or closely related to each other by typing methods, but for which no direct or epidemiological relationship can be identified.
From "Guidelines for the validation and application of typing methods for use in bacterial epidemiology"
Common Genotyping Methods-Plasmid Analysis
-Restriction Endonuclease Analysis
-PCR Assays
-Multilocus Enzyme Electrophoresis (MLEE)
-Multilocus Sequence Typing (MLST)
-Pulsed-Field Gel Electrophoresis (PFGE)
-DNA sequencing
-Ribotyping
-PCR Ribotyping
-Restriction Fragment Length Polymorphism (RFLP) studies
-Randomly Amplified Polymorphic DNA (RAPD)
-Amplified Fragment Length Polymorphism (AFLP)
-Repetitive Sequence–Based PCR (rep-PCR)
Pulsed-Field Gel ElectrophoresisSteps:
-Cell lysis and release of intact chromosomal DNA
-Restriction endonuclease digestion of chromosomal DNA
-Separation of the DNA fragments
-Analysis of DNA fragment length polymorphism
PFGE: pulsed-field gel electrophoresis protocol
(M.E.Kaufmann)
From"Pulsed-Field Gel Electrophoresis (PFGE) Technique and its use in Molecular Biology"
PFGE Performance Characteristics
The performance of strain typing technology, including PFGE, is measured by the following criteria:
-Discriminatory power describes the probability that indistinguishable or closely related strains are truly clonal and part of the same chain of transmission. This parameter can be calculated based on Simpson’s index of diversity, using an index greater than 0.95 as acceptable.
-Reproducibility is the ability to obtain the same results upon repeat testing of the same strain.
- Stability is measured by the ability of clonal isolates to consistently express particular markers over time.
-Typeability measures the proportion of isolates within a bacterial species that can be designated a genotype by a molecular typing system.
Restriction Endonuclease Digestion of Chromosomal DNA
SmaI, recognizes the CCC/GGG sequence that cleaves the DNA of most Gram-positive bacteria. SmaI is able to cleave DNA at rarely occurring sites due to the low GC content and AT-rich sequences in Gram-positive bacteria.
XbaI recognizes the T/CTAGA sequence that cleaves that of many Gram-negative bacteria.
From: J Clin Microbiol. 2003 November; 41(11): 4961–4965.
doi: 10.1128/JCM.41.11.4961-4965.2003.
PFGE profiles of 12 isolates of P. mirabilis from mid-stream urine samples, blood samples, and wound swabs from patients in a district general hospital. The isolates in lanes 7 and 8 were from
the urine and blood, respectively, of the same patient. Lanes M, molecular size marker. Relevant sizes of bands are indicated in kilobases.
PFGE is widely used for genotypic characterization of microorganisms. It has been considered the method of choice for the analysis of most bacterial pathogens because of its high discriminatory power and reproducibility. Although PFGE is an important tool for outbreak investigations, it is not indicated for population
analysis of microorganisms. In addition, PFGE is marginally valuable in epidemiological settings of long-term duration encompassing years or decades or at a country to continent level. Its limitations also include substantial measurements of time and resources, and the non-typeability of some strains in some situations. Therefore, a combination of PFGE and PCR-based techniques may be necessary for genotyping a wide range of microorganisms under these circumstances.
PCR-Based Strain Typing Techniques
PCR-based strain typing techniques are designed to generate multiple bands that provide a unique fingerprint for a particular species or strain of microorganism. Unlike diagnostic tests that determine presence or absence of a microorganism (or its nucleic acid) in a specimen,these procedures are used to differentiate epidemiologically unrelated organisms at the species or subspecies level. They must generally produce multiple DNA bands to provide sufficient discrimination power, and these banding patterns must be reproducible run-to-run and among isolates of the same predefined group while clearly distinguishing isolates that epidemiologically or phenotypically fall outside of that group.
