Simultaneous Analysis of Multiple Enzymes Increases Accuracy of Pulsed-Field Gel Electrophoresis in Assigning Genetic Relationships among Homogeneous Salmonella Strains

The salmonellae comprise over 2,500 serovars, many of which are known to be intracellular pathogens of mammals, birds, and reptiles (33). Salmonella serovars remain some of the most common food-borne pathogens of humans, causing an estimated 1.4 million Salmonella cases, with 600 deaths, each year (25). The most notable of these include Salmonella enterica serovar Typhimurium and S. enterica serovar Enteritidis, both of which remain the most common etiologic agents of salmonellosis-induced gastroenteritis in humans (http://www.cdc.gov/mmwr/preview/mmwrhtml/mm5714a2.htm). Outbreaks of nontyphoidal salmonellosis associated with the consumption of raw or undercooked foods are attributed regularly to S. enterica, including recent outbreaks from peanut paste, tree nuts, and fresh-cut produce (e.g., tomatoes and peppers). Strain identification is essential for effective investigation of common-source outbreaks. Phage typing (PT) has been used widely to facilitate epidemiologic traceback of S. enterica serovar Enteritidis isolates. It has become clear, however, that most S. enterica serovar Enteritidis isolates are derived from a few endemic clones and belong to a limited number of PTs (35, 38). Pulsed-field gel electrophoresis (PFGE) remains a subtyping “gold standard” for public health investigation and has been shown to be highly effective for epidemiological investigation of Salmonella serovars (1, 16, 26). However, conventional PFGE has shown limited discriminatory power in subtyping certain highly clonal serotypes (e.g., S. enterica serovar Enteritidis and S. enterica serovar Hadar) (19, 24, 36). In the case of S. enterica serovar Enteritidis, in particular, both phage typing and single-enzyme (i.e., XbaI) PFGE often lack the discriminatory power to partition strains into epidemiologically meaningful clusters (3, 30). Most recently, by combining several restriction enzyme data sets into a single analysis, we reported on a highly discriminatory PFGE-based subtyping scheme for S. enterica serovar Enteritidis to enhance the application of this method for differentiating highly homogeneous Salmonella strains (41). This scheme has also been applied successfully to other highly clonal serovars, including S. enterica serovar Saintpaul and S. enterica serovar Hadar, as well as to homogeneous strains of S. enterica serovar Heidelberg and S. enterica serovar Kentucky (40), reinforcing the potential application of this PFGE-based subtyping scheme for a variety of clonally derived serovars. Although the discriminatory power of concatenated PFGE has compared favorably to that of traditional PFGE protocols in differentiating clonal complexes of S. enterica serovar Enteritidis (41), the genetic and epidemiologic accuracy of this method has not been evaluated stringently for Salmonella. Assignment of accurate genetic relationships among strains associated with food-borne outbreaks is critical for effective source tracking and traceback inference. The purpose of this study was to employ a series of genetic and phylogenetic measures to determine the suitability of the concatenated enzyme PFGE method to cluster closely related isolates of Salmonella into meaningful genetic hierarchies that reflect epidemiologically relevant groupings of strains.