Genetically diverse group A streptococci from children in far-western Nepal share high genetic relatedness with isolates from other countries

Abstract

The genetic diversity of group A streptococci (GAS) throughout much of the world has not been adequately explored. To assess genetic variation among GAS in western Nepal, 120 noninvasive GAS, collected from eight different villages, were genetically characterized using emm typing, sof sequencing, and multilocus sequence typing (MLST). A high level of genetic diversity was observed among these isolates, with 51 genotypes based upon 51 multilocus sequence types (STs), 45 emm sequence types, and 28 sof sequence types. On the basis of shared ST-emm and sof-emm associations, 40 of the 51 genotypes were identical or highly related to genotypes characterized from locations outside of Nepal, even though most of the emm sequence and clonal types are rare among GAS within the United States. When analyzing all known STs highly related to Nepal STs, only one example of similar STs shared between a sof PCR-positive strain and a sof PCR-negative strain was observed. Since previous data indicate free exchange of MLST loci between sof-positive and sof-negative strains, there is possibly selection against the expansion of subclones resulting from horizontal transfers of sof or emm genes between sof-positive and sof-negative strains. All 45 emm types encountered in Nepal have also been documented from other countries. These data, together with data encompassing the past decade of emm type surveillance, support the possibility that most existing GAS emm types have been discovered. Similarly, since most (40/51) strain types were highly related to strains found elsewhere, it is likely that a major fraction of the existing GAS clonal complexes have been discovered.

FIG. 1.

Sequence relationships between deduced N-terminal M protein sequences from study set isolates. M protein sequences consisting of 22 signal sequence residues and 66 N-terminal residues were aligned using the Wisconsin Package 10.3 PileUp program. A phylogram was generated as described in Materials and Methods, using the Wisconsin Package version 10.3 Distances and GrowTree programs followed by entering the data into the TreeView program, selecting the unrooted tree option. The asterisks displayed for M65 and M67 indicate the sole exceptions in this study of M sequences from sof-negative and sof-positive strains clustering on the same half of the dendrogram. It is also interesting that one CDC emm65 reference strain apparently contains an aberrant (deletion) sof gene derivative (www.cdc.gov/ncidod/biotech/strep/emmdata.htm#emm65).

FIG. 2.

Nepal GAS STs in the context of clonal sets defined by eBURST of all known STs and inclusion of double-locus variants of Nepal STs. All STs found during this study are underlined. Clonal sets were determined by eBURST for all STs listed at www.mlst.net and by inclusion of all known double-locus variants of STs found during this study. eBURST forms groups in which each member shares six of seven MLST loci with at least one other member of the set. The solid black line indicates members of each eBURST group. Added double-locus variants of STs obtained in the study are indicated by dashed lines or dashed extensions of the eBURST groups. emm types associated with each ST at the global database (21) are indicated in superscript. Instances where there are multiple emm types for a given ST documented at www.mlst.net are indicated by brackets. Bold STs indicate predicted founder STs as defined by eBURST. These STs are defined as the ST within an eBURST group that has the greatest number of single-locus variants. The superscript A indicates that the ST-emm association is shared by another GAS strain(s) recovered in other geographic locations. ST999 is a six-allele profile that shares four of six identical loci with the other indicated STs. We were unable to amplify the yqiL allele from the emm11 isolates in this study. The single clonal set within a solid square (ST120 founder) includes a single clonal type determined to be sof gene positive (ST331) and several clonal types previously determined to be sof gene negative. This is the only example of a clonal set in this study associated with both sof-positive and sof-negative strains (ST331 was the sole sof-positive strain in the set). The two clonal sets within the dashed square each include the same four study STs depicted in relation to two different eBURST sets.