Figure 8-19 Procedural steps for pulsed-field gel electrophoresis (PFGE).
these reasons, PFGE has been widely accepted among microbiologists, infection control personnel, and infectious disease specialists as a primary laboratory tool for epidemiology.
The principle of PFGE is to use a specialized electrophoresis device to separate chromosomal fragments produced by enzymatic digestion of intact bacterial chromosomal DNA. Bacterial suspensions are first embedded in agarose plugs, where they are carefully lysed to release intact chromosomal DNA; the DNA is then digested using restriction endonudease enzymes. Enzymes that have relatively few restriction sites on the genomic DNA are selected so that 10 to 20 DNA fragments ranging in size from 10 to 1000 kb are produced (Figure 8-19). Because of the large DNA fragment sizes produced, resolution of the banding patterns requires the use of a pulsed electrical field across the agarose gel that subjects the DNA fragments to different voltages from varying angles at different time intervals.
Although comparison and interpretation of RFLP profiles produced by PFGE can be complex, the basic premise is that strains with the same or highly similar digestion profiles share substantial similarities in their nucleotide sequences and therefore are likely to be most closely related. For example, in Figure 8-18 isolates 1 and 2 have identical RFLP patterns, whereas isolate 3 has only 7 of its 15 bands in common with either isolates 1 or 2. Therefore, isolates 1 and 2 would be considered dosely related, if not identical, whereas isolate 3 would not be considered related to the other two isolates.
One example of PFGE application for the investigation of an outbreak is shown in Figure 8-16.
Following Smal endonuclease enzymatic digestion of DNA from seven vancomycin-resistant Enterococcus faecalis- isolates, RFLP profiles show that the resistant isolates are probably the same strain. Such a finding strongly supports the probability of clonal dissemination of the same vancomycin-resistant strain among the patients from which the organisms were isolated
The discriminatory advantage that PFGE profiles have over phenotype-based typing methods is demonstrated in Figure 8-17. Because all six methicillin-resistant Staphylococcus aureus isolates exhibited identical antimicrobial susceptibility profiles, they were initially thought to be the same strain. However, PFGE profiling established that only isolates B and C were the same.
PFGE can also be used to determine whether a recurring infection in the same patient is due to insufficient original therapy, possibly as a result of developing antimicrobial resistance during therapy, or due to acquisition of a second, more resistant, strain of the same species. Figure 8-18 shows restriction patterns obtained by PFGE, with S. pneumoniae isolated from a patient with an unresolved middle ear infection. The PFGE profile of isolate B, which was My susceptible to penicillin, differs substantially from the profile of isolate G, which was resistant to penicillin. The dear difference in PFGE profiles between the two strains indicates that the patient was most likely reinfected with a second, more resistant, strain. Alternatively^ the patient's original infection may have been a mixture of both strains, with the more resistant one being lost during the original culture workup. In any case, this application of PFGE demonstrates that the method is not only useful for investigating outbreaks or strain dissemination involving several patients but also gives us the ability to investigate questions regarding reinfections, treatment failures, and mixed infections involving more than one strain of the same spedes.
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