Stability of Repetitive-Sequence PCR Patterns with Respect to Culture Age and Subculture Frequency

Methods for molecular typing of bacteria are powerful tools that can be used to determine whether isolates recovered from different patients or the environment are related and, in so doing, provide evidence for a common source of transmission of infection. Molecular typing can also be used to establish colonization of an individual by the same organism over time or to determine the relatedness of strains of bacteria possessing different antimicrobial resistance profiles. Epidemiological analysis of bacterial isolates has gained popularity over the past three decades, and many distinct approaches have been used for this purpose, including phenotypic and genotypic typing schemes. The limitations of phenotypic analysis have led to the progressive development of genotypic strategies, including analysis of plasmid content and plasmid restriction patterns (6), random amplified polymorphic DNA analysis (8), repetitive-sequence (rep) PCR (2, 13, 14), restriction fragment length polymorphism (RFLP) and pulsed-field gel electrophoresis (PFGE) (4), and multilocus sequence analysis of bacterial DNA (9). While RFLP/PFGE is considered the current “gold standard” for typing bacteria, the process is somewhat time consuming and technically demanding, limiting the laboratory’s ability to process large numbers of organisms simultaneously. Repetitive DNA sequences of approximately 30 to 400 bp have been found interspersed throughout a variety of prokaryotic (and eukaryotic) genomes (10). In bacteria, the most common repeat sequences are highly conserved across species. Some of these sequences, such as the repetitive extragenic palindrome sequence, are thought to play a role in gene regulation (7). Unlike the repetitive sequence analysis used in forensic medicine, the method developed for assessment of bacterial relatedness (termed rep PCR) examines the distance between repeat sequences by using primers directed outwardly from these sequences. If repetitive sequences are within a distance that can be spanned by Taq polymerase extension, products of various molecular sizes (depending upon the distance between repeats) are obtained. The amplified products are separated by gel electrophoresis; the gel image is then scanned and the banding patterns are analyzed visually or by using gel comparison software. Because repetitive sequences are interspersed throughout the genome, rep PCR is potentially capable of surveying the entire genome and has been shown to have similar or better strain differentiation power compared to other methods (12). rep PCR is rapid by comparison to RFLP/PFGE, has been shown to be reproducible, and requires few additional resources other than those already available in most molecular laboratories. Although molecular typing using rep PCR has become relatively popular, some assumptions regarding the performance of this method have been made that have not yet been substantiated. For example, molecular typing analyses have generally relied on DNA extracted from overnight or 24-h subcultures of the strains being examined. Presumably, this practice was adopted to exclude confounding factors that might alter rep PCR banding patterns due to increasing culture age and/or clonal expansion during growth of multiple subcultures. However, the practice of using overnight cultures for analysis might be unnecessary. To challenge this ritual, we evaluated six representative organisms for changes in rep PCR patterns with