Deriving group A Streptococcus typing information from short-read whole-genome sequencing data

Abstract

Typing of group A Streptococcus (GAS) is crucial for infection control and epidemiology. While whole-genome sequencing (WGS) is revolutionizing the way that bacterial organisms are typed, it is necessary to provide backward compatibility with currently used typing schemas to facilitate comparisons and understanding of epidemiological trends. Here, we sequenced the genomes of 191 GAS isolates representing 42 different emm types and used bioinformatics tools to derive commonly used GAS typing information directly from the short-read WGS data. We show that emm typing and multilocus sequence typing can be achieved rapidly and efficiently using this approach, which also permits the determination of the presence or absence of genes associated with GAS tissue tropism. We also report on how the WGS data analysis was instrumental in identifying ambiguities present in the commonly used emm type database hosted by the U.S. Centers for Disease Control and Prevention.

FIG 1

Schematics showing emm chromosomal arrangements in different emm GAS types. (A) Many emm types have an emm chromosomal arrangement in which gene emm is found downstream of mga and upstream of scpA. These emm types do not possess emm-like genes. However, some emm types may have other genes between emm and scpA (generically represented by the triangle). All GAS strains in our collection belonging to emm types with this type of emm chromosomal arrangement (listed to the right of scpA) were successfully assigned an emm type using the SRST2 WGS data mapping-based approach. (B) Several GAS emm types contain emm-like genes (mrp and/or enn) in addition to gene emm. We were able to correctly derive emm type information using the same WGS mapping-based approach for several GAS emm types (listed to the right of enn). However, the mapping-based strategy failed in cases where the CDC emm database contained sequences found also in emm-like genes. These emm types (listed below the red box in the gene schematics) were confounded by SRST2 with “emm” types whose sequences actually match either mrp or enn (listed below the green and blue boxes, respectively, in the schematics). The correct emm type could, however, be determined after de novo assembly of WGS data and BLAST and positional analysis. Dotted lines link emm sequences found in the CDC database that were identified in the same strain. Red arrows indicate annealing positions of primers 1 and 2 used in Sanger-based emm typing.

FIG 2

Schematics showing emm chromosomal arrangements in different strains. (A) Strain NGAS320, found to be nontypeable by Sanger sequencing (top) and strain NGAS300, representative of other emm83 GAS strains in our collection (bottom). Although nontypeable by Sanger-based emm typing, NGAS320 was identified as an emm83 by WGS-based emm typing. Further sequence analysis discovered a deletion of 1,390 bp in this strain that knocked out the annealing site of primer 1 used in traditional Sanger-based emm typing. The deletion was confirmed by PCR amplification using primers annealing to the 3′ end of mga and the 5′ end of scpA (indicated by the blue arrows). Red arrows indicate annealing positions of primers 1 and 2 used in Sanger-based emm typing. (B) Strain NGAS128. This GAS has an emm14 gene and was correctly typed by both Sanger-based and WGS-based emm typing. However, the enn gene of this strain possesses sequences identical to those found in the CDC emm database for emm51 (indicated by the blue box).