Establishment of a universal size standard strain for use with the PulseNet standardized pulsed-field gel electrophoresis protocols: converting the national databases to the new size standard

Abstract

The PulseNet National Database, established by the Centers for Disease Control and Prevention in 1996, consists of pulsed-field gel electrophoresis (PFGE) patterns obtained from isolates of food-borne pathogens (currently Escherichia coli O157:H7, Salmonella, Shigella, and Listeria) and textual information about the isolates. Electronic images and accompanying text are submitted from over 60 U.S. public health and food regulatory agency laboratories. The PFGE patterns are generated according to highly standardized PFGE protocols. Normalization and accurate comparison of gel images require the use of a well-characterized size standard in at least three lanes of each gel. Originally, a well-characterized strain of each organism was chosen as the reference standard for that particular database. The increasing number of databases, difficulty in identifying an organism-specific standard for each database, the increased range of band sizes generated by the use of additional restriction endonucleases, and the maintenance of many different organism-specific strains encouraged us to search for a more versatile and universal DNA size marker. A Salmonella serotype Braenderup strain (H9812) was chosen as the universal size standard. This strain was subjected to rigorous testing in our laboratories to ensure that it met the desired criteria, including coverage of a wide range of DNA fragment sizes, even distribution of bands, and stability of the PFGE pattern. The strategy used to convert and compare data generated by the new and old reference standards is described.

FIG. 1.

Original standards used for the normalization and analysis of E. coli O157:H7, L. monocytogenes, Salmonella, and Shigella. Approximate kilobase values for positions used for normalization are indicated. Lanes are from tiffs of gels run using the PulseNet standardized methods for that organism. Electrophoresis switch times are indicated for each organism.

FIG. 2.

PFGE image showing the E. coli O157:H7 (G5244) size standard (lanes 1, 6, and 10), three test strains restricted with XbaI (lanes 2 to 4) and BlnI (lanes 7 to 9), and the new universal standard (Salmonella serotype Braenderup H9812; lane 5).

FIG. 3.

Approximate band sizes in kilobases of the Salmonella serotype Braenderup reference standard (H9812) restricted with XbaI and run under the PulseNet standardized electrophoresis conditions specific for each organism.

FIG. 4.

Dendrograms of tiff images of gel lanes containing AM01144 (old Salmonella standard), alternating with H9812 (new standard), and normalized with both old and new standards. (The first column to the right of the dendrogram indicates the pattern for the standard, and the second column indicates which reference system was used for normalization.) Shown are results before optimization (A) and after the calibration curve optimizations (B) for alternating lanes of AM01144 and H9812.

FIG. 5.

Dendrogram showing 26 Salmonella isolates run with both the AM01144 and the H9812 standards on same gel; the tiff was renamed and normalized with each standard. Isolates normalized with the original Salmonella standard strain (AM01144) are designated by one or two letters; the same isolate normalized with the universal standard (H9812) is designated by the same combination of letters followed by -H. Isolates designated with the same uppercase letter but a different lowercase letter are epidemiologically related.