An important component of recognizing and controlling disease outbreaks inside or outside of a hospital is identification of the reservoir and mode of transmission of the infectious agents involved. Strain typing provides a mechanism for monitoring the spread of drug-resistant pathogens, the evaluation of multiple isolates from a single patient, differentiation of relapse from a new infection, and applications in epidemiology and infection control. Infection control measures often require establishing relatedness among the pathogens isolated during the outbreak. For example, if all the microbial isolates thought to be associated with a nosocomial infection outbreak are shown to be identical or at least very closely related, then a common source or reservoir for those isolates must be identified. If the etiologic agents are not the same, other explanations for the outbreak must be investigated. Because each species of a microorganism comprises an almost limitless number of strains, identification of an organism to the species level is not sufficient for establishing relatedness. Strain typing, the process used to establish the relatedness among organisms belonging to the same species, is required.

Although phenotypic characteristics (e.g., biotyping, serotyping, antimicrobial susceptibility profiles) historically have been used to type strains, these methods often are limited by their inability to consistently discriminate between different strains, their labor intensity, or their lack of reproducibility. In contrast, certain molecular methods do not have these limitations and have enhanced strain-typing capabilities. The molecular typing methods either directly compare nucleotide sequences between strains or produce results that indirectly reflect similarities in nucleotide sequences among “outbreak” organisms. Indirect methods usually involve enzymatic digestion and electrophoresis of microbial DNA to produce RFLPs for comparison and analysis.

Several molecular methods have been investigated for establishing strain relatedness (Table 1). The method chosen primarily depends on the extent to which the following four criteria proposed by Maslow and col leagues are met:

Table1. Examples of Methods to Determine  Strain Relatedness

• Typeability: The method’s capacity to produce clearly interpretable results with most strains of the bacterial species to be tested

• Reproducibility: The method’s capacity to repeatedly obtain the same typing profile result with the same bacterial strain

 • Discriminatory power: The method’s ability to produce results that clearly allow differentiation between unrelated strains of the same bacterial species

• Practicality: The method should be versatile, relatively rapid, inexpensive, technically simple, and provide readily interpretable results

The last criterion, practicality, is especially important for busy clinical microbiology laboratories that provide support for infection control and hospital epidemiology.

Among the molecular methods used for strain typing, pulsed-field gel electrophoresis (PFGE) meets most of Maslow’s criteria for a good typing system and is frequently referred to as the microbial typing “gold standard.” This method is applicable to most of the commonly encountered bacterial pathogens, particularly those frequently associated with nosocomial infections and out breaks such as staphylococci (MRSA), enterococci (vancomycin-resistant enterococci), and gram-negative pathogens, including Escherichia coli, and Klebsiella, Enterobacter, and Acinetobacter spp. For these reasons, PFGE has been widely accepted among microbiologists, infection control personnel, and infectious disease specialists as a primary laboratory tool for epidemiology.

PFGE uses 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 (lysozyme) to release intact chromosomal DNA; the interfering contaminating proteins are then removed by treating the sample with Proteinase K; the DNA is then digested using restriction endonuclease 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 1). 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.

Fig1. Procedural steps for pulsed-field gel electrophoresis (PFGE).

Although comparison and interpretation of RFLP pro files 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 2, 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 closely related, if not identical, whereas isolate 3 would not be considered related to the other two isolates.

Fig2. Restriction patterns generated by pulsed-field gel electrophoresis for two Streptococcus pneumoniae isolates, one that  was susceptible to penicillin (Lane B) and one that was resistant  (Lane C), from the same patient. Restriction fragment length polymorphism analysis indicates that the patient was infected with different strains. Molecular-size markers are shown in Lane A. 

One example of PFGE application for the investigation of an outbreak is shown in Figure 3. After SmaI endonuclease enzymatic digestion of DNA from seven vancomycin-resistant E. 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.

Fig3. Restriction fragment length polymorphisms of  vancomycin-resistant Enterococcus faecalis isolates in Lanes A  through G as determined by pulsed-field gel electrophoresis. All  isolates appear to be the same strain.

The discriminatory advantage that PFGE profiles have over phenotype-based typing methods is demonstrated in Figure 4. Because all six methicillin-resistant S. aureus isolates exhibited identical antimicrobial susceptibility profiles, they were initially thought to be the same strain. However, PFGE profiling established that only iso lates B and C were the same.

Fig4. Although antimicrobial susceptibility profiles indicated  that several methicillin-resistant S. aureus isolates were the same  strain, restriction fragment length polymorphism analysis using  pulsed-field gel electrophoresis (Lanes A through F) demonstrates  that only isolates B and C were the same. 

PFGE can also be used to determine whether a recur ring infection in the same patient is due to insufficient original therapy, possibly as a result of developing antimicrobial resistance during therapy, or to acquisition of a second, more resistant, strain of the same species. Figure 2 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 fully susceptible to penicillin, differs substantially from the profile of isolate C, which was resistant to penicillin. The clear 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 not only is useful for investigating outbreaks or strain dissemination involving several patients, it also gives us the ability to investigate questions regarding reinfections, treatment failures, and mixed infections involving more than one strain of the same species.

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