Illustration of a common framework for relating multiple typing methods by application to macrolide-resistant Streptococcus pyogenes

Abstract

The studies that correlate the results obtained by different typing methodologies rely solely on qualitative comparisons of the groups defined by each methodology. We propose a framework of measures for the quantitative assessment of correspondences between different typing methods as a first step to the global mapping of type equivalences. A collection of 325 macrolide-resistant Streptococcus pyogenes isolates associated with pharyngitis cases in Portugal was used to benchmark the proposed measures. All isolates were characterized by macrolide resistance phenotyping, T serotyping, emm sequence typing, and pulsed-field gel electrophoresis (PFGE), using SmaI or Cfr9I and SfiI. A subset of 41 isolates, representing each PFGE cluster, was also characterized by multilocus sequence typing (MLST). The application of Adjusted Rand and Wallace indices allowed the evaluation of the strength and the directionality of the correspondences between the various typing methods and showed that if PFGE or MLST data are available one can confidently predict the emm type (Wallace coefficients of 0.952 for both methods). In contrast, emm typing was a poor predictor of PFGE cluster or MLST sequence type (Wallace coefficients of 0.803 and 0.655, respectively). This was confirmed by the analysis of the larger data set available from http://spyogenes.mlst.net and underscores the necessity of performing PFGE or MLST to unambiguously define clones in S. pyogenes.

FIG. 1.

PFGE profiles of three pairs of macrolide-resistant isolates generated after digestion with either SmaI, Cfr9I, SfiI, or ApaI. The isolates in pair 1 were refractory to cleavage with SmaI similarly to what was previously reported in the literature for isolates presenting the M phenotype (21) and showed identical profiles upon digestion with Cfr9I, SfiI, and ApaI. The isolates in pair 2 were digested by both SmaI and Cfr9I and, as expected, the profile of each isolate was identical with either endonuclease. The isolates in pair 2 showed different SmaI/Cfr9I and ApaI profiles but were indistinguishable by SfiI. The isolates in pair 3 were refractory to cleavage with SmaI and presented different profiles upon digestion with Cfr9I (four-band difference). These isolates presented identical SfiI profiles but exhibited different profiles with ApaI. m, lambda ladder PFG marker (New England Biolabs, Beverly, Ma.).

FIG. 2.

Panel A. Adjusted Rand values for all possible cutoff values in each of the SmaI/Cfr9I and SfiI dendrograms (in panels B and C). Panel B. Dice/UPGMA dendrogram for SfiI band patterns of the 325 isolates. Panel C. Dice/UPGMA dendrogram for SmaI/Cfr9I band patterns of the 325 isolates. Panel D. Visual representation of cluster congruence between SmaI/Cfr9I and SfiI clusters at the cutoff levels indicated in panels B and C.

FIG. 3.

(A) Representation of correspondences between the typing methods used in the 41-strain subset, calculated by Wallace coefficients. The arrows represent Wallace coefficients of >0.60. (B) Representation of correspondences between the typing methods used in the 325-strain data set and on the 795 strains from www.mlst.net, calculated by Wallace coefficients. The arrows represent Wallace coefficients of >0.60.

FIG. 4.

Relationship between MLST and emm sequence typing for the 41 strains. In this example, the figure illustrates the fact that the majority of strains with emm type 22, the most frequent emm type in this subset, presented ST 46, and two isolates presented ST 52. However, the majority of the strains with ST 52 have emm sequence type 28.