Subtyping of Salmonella enterica Serotype Enteritidis Strains by Manual and Automated Pst I- Sph I Ribotyping

Abstract

Salmonella enterica subsp. enterica serotype Enteritidis is not readily subtyped beyond the level of phage type (PT). A recently developed method for ribotyping of this organism, which uses a mixture of PstI and SphI (PS) for restriction of DNA (PS ribotyping), has proved useful for further subtyping of a number of PTs of this organism, including PT 4. However, it has not been extensively tested with PT 8. In the present study the PS ribotyping method was used to investigate outbreaks of both S. enterica serotype Enteritidis PT 4 and PT 8 and provided subtyping data that were consistent with information obtained from epidemiologic investigations. The method proved to be more discriminatory than phage typing and pulsed-field gel electrophoresis (PFGE) combined and was useful for investigating a pseudo-outbreak involving isolates that had identical PTs and PFGE types but that could not be linked epidemiologically. Several PS ribotypes were found within the cluster of isolates indistinguishable by other subtyping methods, confirming the epidemiologic findings. Although the PS ribotyping method proved to have a superior discriminatory ability in resolving clusters, it did not have high enough throughput for use in outbreak investigations. This method has therefore been adapted for use in automated ribotyping with a RiboPrinter, and the results were compared with those obtained by manual ribotyping. Both methods produce equivalent results and are useful for obtaining epidemiologically relevant subtyping data for S. enterica serotype Enteritidis, including PT 8 strains not extensively tested previously.

Comparison of Multiple-Locus Variable-Number Tandem Repeat Analysis, Pulsed-Field Gel Electrophoresis, and Phage Typing for Subtype Analysis of Salmonella enterica Serotype Enteritidis

“…Current Salmonella serotype Enteritidis subtyping methods for the most part do not meet the proposed criteria, with the main problem being suboptimal discriminatory power. The method of choice for the national PulseNet surveillance system has been PFGE, although ribotyping may provide higher discriminatory power than does PFGE with a single enzyme (6). Ribotyping is not widely used for Salmonella serotype Enteritidis subtyping in the United States, however, and suffers from the many of the same data portability issues as PFGE does.…”

“…Plasmid profiling, single-enzyme ribotyping, and random amplified polymorphic DNA (RAPD) analysis also have limited discriminatory power for Salmonella serotype Enteritidis and, in addition, suffer from poor reproducibility (14,20,21,27,38,39,49). Two-enzyme ribotyping (PstI-SphI) may provide higher discriminatory power than other methods (6); however, manual ribotyping is labor-intensive while automated ribotyping is prohibitively expensive. Amplified fragment length polymorphism appears to be one of the more discriminatory methods for subtyping Salmonella serotype Enteritidis, but amplified fragment length polymorphism used with epidemiologically linked outbreak isolates of Salmonella serotype Enteritidis has revealed a lack of subtype stability (8,42).…”

Genomic Diversity Within Phage Types ofSalmonella entericassp.entericaSerotypes Enteritidis and Typhimurium

“…Serotypes Enteritidis and Typhimurium are the most prevalent serotypes in many countries (Herikstad et al, 2002;Fisher, 2004a). Some of the phage types of these serotypes have been reported to predominate in certain geographical areas (Hasenson et al, 1992;Fantasia and Filetici, 1994;Schroeter et al, 1994;Grimont et al, 1999;Tschäpe et al, 1999;Wall and Ward, 1999;van de Giessen et al, 1999;Clark et al, 2003).…”

Pulsed field, ribotype and integron analysis of multidrug resistant isolates of Salmonella enterica serovar Newport

“…Automated ribotyping has been investigated as a method to subtype Salmonella spp. (Clark et al, 2003;Fontana et al, 2003). Although the discriminatory power of ribotyping has been reported to be less than PFGE, when used in conjunction with PFGE, the results can be more useful than if either method was used alone (Fontana et al, 2003).…”