Assessment of resolution and intercenter reproducibility of results of genotyping Staphylococcus aureus by pulsed-field gel electrophoresis of SmaI macrorestriction fragments: a multicenter study

Abstract

Twenty well-characterized isolates of methicillin-resistant Staphylococcus aureus were used to study the optimal resolution and interlaboratory reproducibility of pulsed-field gel electrophoresis (PFGE) of DNA macrorestriction fragments. Five identical isolates (one PFGE type), 5 isolates that produced related PFGE subtypes, and 10 isolates with unique PFGE patterns were analyzed blindly in 12 different laboratories by in-house protocols. In several laboratories a standardized PFGE protocol with a commercial kit was applied successfully as well. Eight of the centers correctly identified the genetic homogeneity of the identical isolates by both the in-house and standard protocols. Four of 12 laboratories failed to produce interpretable data by the standardized protocol, due to technical problems (primarily plug preparation). With the five related isolates, five of eight participants identified the same subtype interrelationships with both in-house and standard protocols. However, two participants identified multiple strain types in this group or classified some of the isolates as unrelated isolates rather than as subtypes. The remaining laboratory failed to distinguish differences between some of the related isolates by utilizing both the in-house and standardized protocols. There were large differences in the relative genome lengths of the isolates as calculated on the basis of the gel pictures. By visual inspection, the numbers of restriction fragments and overall banding pattern similarity in the three groups of isolates showed interlaboratory concordance, but centralized computer analysis of data from four laboratories yielded percent similarity values of only 85% for the group of identical isolates. The differences between the data sets obtained with in-house and standardized protocols could be the experimental parameters which differed with respect to the brand of equipment used, imaging software, running time (20 to 48 h), and pulsing conditions. In conclusion, it appears that the standardization of PFGE depends on controlling a variety of experimental intricacies, as is the case with other bacterial typing procedures.

FIG. 1

Comparative analysis of gel pictures obtained after PFGE of DNA macrorestriction fragments derived from the panel of 20 MRSA isolates. From top to bottom the experimental outputs of participating centers 8, 1, and 7 are shown. White arrowheads in the two top panels highlight potential fragment doublets. Note that these two pictures clearly overlap with respect to resolution and number of DNA fragments. The arrowhead in the lower panel identifies a floating plug. Although the lower panel shows a lesser degree of band resolution, it has to be emphasized that pattern identification obtained from this gel picture was as expected except for patterns belonging to isolates 9 and 10. However, the quality of the PFGE profiles shown in the lower panel is markedly inferior to those in the upper two panels. Numbering above the lanes corresponds with strain numbers, L identifies the lambda concatamers and N indicates the macrorestriction pattern generated for the S. aureus NCTC 8325 reference strain.

FIG. 2

Dendrogram of results of seven data sets, four in-house (IH) and 3 standardized GP kits (KIT) from four centers (Ctr). The numbers after the colons are lane numbers. The percent similarity scale is based on UPGMA clustering of Dice coefficients generated by Bio Image software.