Comparison of multiple-locus variable-number tandem repeat analysis, pulsed-field gel electrophoresis, and phage typing for subtype analysis of Salmonella enterica serotype Enteritidis

Abstract

Strain subtyping is an important tool for detection of outbreaks caused by Salmonella enterica serotype Enteritidis. Current subtyping methods, however, yield less than optimal subtype discrimination. In this study, we describe the development and evaluation of a multiple-locus variable-number tandem repeat analysis (MLVA) method for subtyping Salmonella serotype Enteritidis. The discrimination ability and epidemiological concordance of MLVA were compared with those of pulsed-field gel electrophoresis (PFGE) and phage typing. MLVA provided greater discrimination among non-epidemiologically linked isolates than did PFGE or phage typing. Epidemiologic concordance was evaluated by typing 40 isolates from four food-borne disease outbreaks. MLVA, PFGE, and, to a lesser extent, phage typing exhibited consistent subtypes within an outbreak. MLVA was better able to differentiate isolates between the individual outbreaks than either PFGE or phage typing. The reproducibility of MLVA was evaluated by subtyping sequential isolates from an infected individual and by testing isolates following multiple passages and freeze-thaw cycles. PFGE and MLVA patterns were reproducible for isolates that were frozen and passaged multiple times. However, 2 of 12 sequential isolates obtained from an individual over the course of 36 days had an MLVA type that differed at one locus and one isolate had a different phage type. Overall, MLVA typing of Salmonella serotype Enteritidis had enhanced resolution, good reproducibility, and good epidemiological concordance. These results indicate that MLVA may be a useful tool for detection and investigation of outbreaks caused by Salmonella serotype Enteritidis.

FIG. 1.

Minimum spanning tree of MLVA of sporadic Salmonella serotype Enteritidis isolates. A categorical coefficient and the priority rule using the highest number of single-locus changes were used for generation of the minimum spanning tree. Each circle in the tree represents a different MLVA type, with the MLVA type number indicated by the number in the circle. Heavy short lines connecting two MLVA types denote types differing by a single MLVA locus while thin longer lines connect double-locus variants, and dotted lines indicate the most likely connection between two types differing by more than two MLVA loci. The number of loci that differ between two MLVA types is indicated on the lines connecting the MLVA types. The colors of the circles indicate the numbers of isolates with a particular MLVA type as indicated on the figure. Clusters were defined as MLVA types having a maximum distance of changes at two loci and a minimum cluster size of two types. Cluster A is shaded in green, cluster B in blue, and cluster C in pink.