Molecular Strain Typing Methods for Epidemiological Studies

In community or clinical settings, when the same organism is isolated multiple times, whether in the same patient or from different patients, the physician wants to know if the isolates were independently acquired, i.e., came from different sources, or if they came from the same source. With this knowledge, the physician can act to control the transmission of the organism, especially if it is being transmitted from a common source and that source has been identified. Most of the time, these analyses are performed on organisms that have been transmitted noso-comially, but sometimes procedures to determine related-ness are performed on isolates from community outbreak situations.92

There are many laboratory methods that can be used to determine the relatedness of multiple isolates, both phe-

notypic (e.g., by serology and antimicrobial susceptibility patterns) and genotypic (e.g., pulsed field gel electrophoresis and ribotyping).93 The phenotypic methods suffer from a lack of reproducibility, and the ability to discriminate between isolates is not very good. Genotypic methods are used almost exclusively to type bacterial strains to determine the relatedness of multiple isolates and will be summarized in this section.

Plasmid Analysis

Plasmid analysis, or as it was known in the past, plasmid fingerprinting,94 involves isolation and restriction mapping of bacterial plasmids. The same bacterial strain can have different plasmids carrying different phenotypes or resistance patterns. For this analysis, plasmid DNA is isolated from the specimen or culture and then digested with restriction enzymes. Plasmids are distinguished by the pattern of fragments generated when cut with the appropriate enzymes. Restriction analysis can also be performed on chromosomal DNA for organisms with small genomes. For organisms with larger genomes, whole genome restriction enzyme analysis often yields complex patterns that are more difficult to interpret.

Pulsed Field Gel Electrophoresis

Most molecular epidemiological tests are performed using pulsed field gel electrophoresis (PFGE), which can identify organisms with larger genomes or multiple chromosomes. For PFGE analysis, the DNA is digested with restriction enzymes that cut infrequently within the genomic sequences. The resulting large (hundreds of thousands of base pairs) fragments are resolved by PFGE (see Chapter 5 for a more detailed description of this system). Patterns of organisms will differ depending on the chromosomal DNA sequence of the organisms (Fig. 12-5). Tenover and colleagues devised a system to interpret the patterns of a test organism in comparison with the strain of organism known to be involved in the outbreak.95 The interpretation of PFGE results is fairly straightforward and follows the "rule of three" (Table 12.9). This method has been used for typing numerous species, including strains of Pseudomonas aeruginosa, Mycobacterium avium, Escherichia coli, N. gonorrhoeae, VRE, and MRSA. Intralaboratory and interlaboratory computerized databases of band patterns can be stored for reference. A national PFGE database is stored at the Centers for Disease Control and Prevention (CDC) (www.cdc.gov/pulsenet/index.htm). One disadvantage to

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