Emergence of the Same Successful Clade among Distinct Populations of emm89 Streptococcus pyogenes in Multiple Geographic Regions

摘要

LETTER The emergence of clades within emm 89 Streptococcus pyogenes isolates that rapidly became the dominant lineages expressing this emm type was recently reported in the United Kingdom ( 1 ) and in a study that included isolates from the United States, Finland, and Iceland (United States/FI/IC) ( 2 ). In the United Kingdom, the emerging clade was associated with the absence of the hasABC locus, responsible for the synthesis of the hyaluronic acid capsule ( 1 ). The study from the United States/FI/IC ( 2 ) highlighted the strict association of the emerging clade with an nga promoter variant, also found in contemporary emm 1 isolates, which results in increased expression of the nga locus. The study from the United Kingdom also examined this region and found that the nga-ifs-slo locus and surrounding sequences of the emerging clade shared 99% DNA identity with that of contemporary emm 1 and emm 12 strains, but the authors do not offer any information on the nga promoter ( 1 ). The acquisition of this region by emm 1 isolates is currently considered the major molecular event triggering the success and enhanced virulence of this clone ( 3 ). Given that the two studies characterized emm 89 strains recovered in overlapping time periods, it was possible that the two were documenting the dissemination of the same clade in different geographic areas, although the information presented did not allow this conclusion since the papers analyze different aspects of the strains. We set out to test this hypothesis by reanalyzing the publicly available data, and we also characterized emm 89 isolates recovered in Portugal to investigate if this clade could be also emerging in southern Europe. While analyzing this data, an additional paper was published focusing on the interplay between expression of the nga-ifs-slo locus and that of capsule in the virulence of the emm 89 strains of the United States/FI/IC study ( 4 ). We analyzed the sequencing reads deposited in public databases from the strains included in the two papers in order to determine the presence of the hasABC locus, the sequence types (STs), the variant of the nga promoter, and possible variations in the nga coding sequence. Briefly, the raw sequencing reads were mapped using Bowtie2 to exemplar sequences of each of the loci together with at least 600?bp of upstream and downstream sequence. A total of 907 strains met the quality standards that we required in our analysis in both the has and nga loci. Multilocus sequence typing (MLST) alleles could be confidently determined for 886 of these strains. A total of 79 isolates presented novel STs due to the presence of new alleles or allelic profiles, which were submitted to the S.?pyogenes MLST database ( http://pubmlst.org/spyogenes/ ) and assigned ST791 to ST804. In addition, we analyzed 125 emm 89 isolates recovered in Portugal between 2000 and 2009, including 26 pharyngitis isolates and 37 invasive isolates which have been previously characterized ( 5 , 6 ), as well as 62 isolates recovered from skin and soft tissue infections (SSTI) (unpublished data). The isolates were screened for the presence of the has locus using previously described primers ( 7 ). The nga gene and its promoter region were amplified and sequenced in a subset of 95 isolates for which MLST data were available. In the three datasets (United Kingdom, United States/FI/IC, and Portugal), all isolates carrying nga promoter variant 3 ( 2 ) lacked the hasABC locus ( Table?1 ). Confirming our analysis, the absence of the hasABC locus in strains carrying nga promoter variant 3 in the United States/FI/IC strains was also independently reported ( 4 ). In contrast, all isolates harboring the capsular locus presented variant 1 or 2 of the nga promoter. Variant 3 isolates were predominantly of ST101 in all datasets (94%), although 4 United Kingdom isolates and 31 United States/FI/IC isolates presented single-locus variants (SLVs) of ST101. These results indicate that the same clade lacking the hyaluronic acid capsule and carrying an altered nga promoter region (designated emm 89-new here) is present in the five countries. In Portugal, emm 89-new was first detected among pharyngitis isolates (in 2004) followed by SSTI isolates (2006) and invasive isolates (2007) ( Fig.?1 ) and also rapidly became dominant among emm 89 strains, being associated with a significant increase in the incidence of this emm type among isolates from SSTI (data not shown). TABLE?1? Characteristics of 1,002 emm 89 strains isolated in the United States/FI/IC ( n = 778), United Kingdom ( n = 129), and Portugal ( n = 125) Promoter variant ( n ) nga allele ( n ) ~(a) NADase allele has locus ST(s) ( n ) ~(b) 1 (195) 3 (1) 2 + 407 (1) 4 (2) 2 + 380 (2) 5 (190) 2 + 407 (135), 803 (44), 795 (2), 799 (1) 6 (1) 3 + 407 (1) 11 (1) 8 + 407 (1) 2 (168) 7 (160) 4 + ~(c) 101 (122), 408 (32), 568 (2), 553 (1), 797 (1) 8 (1) 5 + 101 (1) 9 (2) 6 + 142 (2) 10 (2) 7 + 382 (1) 12 (3) 4 + 101 (3) 3 (639)