DISPATCHES Enhanced Subtyping Scheme for Salmonella Enteritidis Jie Zheng,* Christine E. Keys,† Shaohua Zhao,‡ Jianghong Meng,* and Eric W. Brown† To improve pulsed-field gel electrophoresis–based strain discrimination of 76 Salmonella Enteritidis strains, we evalu- ated 6 macro-restriction endonucleases, separately and in various combinations. One 3-enzyme subset, Sfi I/PacI/NotI, was highly discriminatory. Five different indices, including the Simpson diversity index, supported this 3-enzyme combina- tion for improved differentiation of S. Enteritidis. S ince 1987, Salmonella Enteritidis has been one of the most frequently isolated salmonellae associated with foodborne outbreaks (1). Illness from S. Enteritidis is linked to consumption of chickens, eggs, and foods that contain eggs (2). S. Enteritidis presents an interesting chal- lenge from an epidemiologic perspective. Several reports documented a limited number of genotypes among ecologi- cally diverse S. Enteritidis, reinforcing the notion that most S. Enteritidis strains are derived from a few endemic clones (3,4). Pulsed-field gel electrophoresis (PFGE) is an integral subtyping tool used by several national public health net- works (e.g., PulseNet, FoodNet, and VetNet) to differenti- ate outbreak strain clusters (5). The genetic homogeneity of S. Enteritidis, however, confounds many subtyping ap- proaches, including PFGE (6,7). Conventional PFGE pro- tocols lack discriminatory power to cull the subtle genotyp- ic differences that distinguish S. Enteritidis strains. A more discriminatory scheme that incorporates combinations of potentially more informative enzymes may be attainable. We explored the discriminatory power of 6 enzymes, indi- vidually and in combination, to identify a more informative PFGE-based subtyping scheme for this important food- borne pathogen. The Study We examined 76 strains of S. Enteritidis and 74 strains of S. Typhimurium. Strains were isolated from poultry and poultry-related sources and were obtained from the Center for Veterinary Medicine and Center for Food Safety and Applied Nutrition of the US Food and Drug Administration and from the University of Georgia. After screening nu- merous restriction enzymes, the 6 selected were XbaI, BlnI, and SpeI, all used in PulseNet protocols (5); SfiI and PacI, previously used to improve resolution in PFGE studies in- volving Escherichia coli O157:H7 (8,9); and NotI, found to yield an optimal number of cut sites (10). The standard PulseNet PFGE protocol for non-typhoidal Salmonella was performed as described (11,12). Individual run conditions are listed in Table 1. Five diversity indices were used to assess discrimina- tory potential among enzymes. First, unique PFGE patterns or pattern combinations, when analyzing >2 enzymes, were tallied. Second, the mean number of strains per polytomy (an unresolved strain cluster) was calculated as the number of polytomous strains divided by the number of polytomies in the tree. Third, the percentage of polytomous strains (of 76 S. Enteritidis strains) was calculated. Fourth, the node: strain ratio was calculated as the number of nodes (bifurcat- ing tree forks) divided by 76 S. Enteritidis strains. A node: strain value closer to 1 indicated a more resolved tree. Fi- nally, the Simpson diversity index was calculated as a nu- merical assessment of the relative discriminatory potential of each enzyme and enzyme combination (13). XbaI-BlnI patterns from 76 S. Enteritidis strains and 74 S. Typhimurium strains were analyzed simultaneously for a direct comparison of PFGE diversity. The resultant dendrogram yielded striking topologic differences between the 2 serovars (Figure 1). S. Typhimurium strains were al- most entirely resolved; nearly every strain possessed its own branch on the dendrogram. In contrast, S. Enteritidis strain discrimination was sharply weaker, affirming exten- sive genetic homogeneity among strains. For example, 6 polytomies were evident in the S. Enteritidis portion of the tree, 5 of which comprised 4 or more strains and 1 of which comprised 24 strains. In total, 76% of S. Enteritidis strains occupied unresolved clusters with an average of ≈10 strains per cluster. Moreover, the S. Typhimurium dendrogram retained a nearly 1:1 ratio of nodes to strains, indicating that almost every strain retained a unique XbaI/BlnI pattern combination. S. Enteritidis, however, yielded a node:strain ratio of 1:3, indicating a relatively poorly bifurcated tree. Together, these observations highlighted the difficulty in differentiating S. Enteritidis with conventional PFGE ap- proaches. To develop a more discriminatory scheme for S. En- teritidis, we examined pattern diversity for 4 additional restriction endonucleases (SpeI, SfiI, PacI, and NotI). Di- versity indices associated with each of the 6 enzymes are listed in Table 2. Many of the indices designated NotI as being effective for discriminating S. Enteritidis. Among the 6 enzymes, NotI yielded the highest number of unique patterns (n = 26), the fewest average number of strains per polytomy (5.2), the lowest percentage of strains captured 1932 Emerging Infectious Diseases  www.cdc.gov/eid  Vol. 13, No. 12, December 2007 *University of Maryland, College Park, Maryland, USA; †US Food and Drug Administration, College Park, Maryland, USA; and ‡US Food and Drug Administration, Laurel, Maryland, USA