Importance of whole genome sequencing for the assessment of outbreaks in diagnostic laboratories: analysis of a case series of invasive Streptococcus pyogenes infections

Abstract

Outbreaks of Streptococcus pyogenes hypervirulent clones are constant public health threats. In western Switzerland, an increase of severe cases of S. pyogenes invasive infections was observed between December 2015 and March 2016. Our aim was (i) to investigate these cases by the use of Whole Genome Sequencing (WGS) and (ii) to determine the specific virulome and resistome of each isolate in order to undertake adequate public health measures. Eleven Streptococcus pyogenes strains isolated from 11 patients with severe invasive infections between December 13, 2015 and March 12, 2016 were included in our study. Practically, emm-typing, MLST and WGS were used to investigate the relatedness between the isolates. The presence of virulence and antibiotic resistance genes as well as mutations in transcriptional regulators of virulence and in genes encoding for antibiotic targets were assessed. Three and two groups of isolates shared the same emm-type and ST type, respectively. Single Nucleotide Polymorphism (SNP) analysis revealed 14 to 32 SNPs between the strains of the same emm-type group, ruling out the possibility of a clonal outbreak. Mutations found in covS and rocA could partially explain an increased virulence. As these reassuring results were obtained in less than 10 days, no specific hospital hygiene and no dedicated public health measures had to be undertaken. WGS is a powerful technique to discriminate between closely related strains, excluding an outbreak in less than 10 days. Moreover, WGS provided extensive data on the virulome and resistome of all these strains.

Keywords: Emm-typing; Group a streptococcus; MLST; Outbreak; Streptococcus pyogenes; Whole genome sequencing.

Fig. 1

Phylogenetic representation of the sequenced strains during our study and the complete GAS genomes publicly available on NCBI. The phylogenetic tree was made using Parsnp and is based on the core genome alignment of our 11 isolates (in red), the currently 51 complete genomes available on NCBI (Table S1) and seven other genomes of S. pyogenes recently sequenced in our institute (from CV1 to CV7, taking part in another study). The tree was rooted at midpoint. Stars indicate bootstraps below 0.9

Fig. 2

a Simplified scheme showing the best-known transcriptional regulators of virulence in Streptococcus pyogenes missense and non-sense mutations in covR and covS can increase Streptococcus pyogenes virulence by relieving CovR downregulation of many virulence genes such as the has-operon involved in hyaluronic acid synthesis [9]. RocA positively regulates covR transcription and possibly directly phosphorylates CovR [10]. Mutations resulting in premature truncation of RocA were shown to increase hyaluronic acid production and virulence. Mutations in ropB, a negative transcriptional regulator of many virulence genes, have been linked to more invasive phenotypes [24]. This regulator is also necessary for the expression of speB, a gene encoding for a cysteine protease degrading many Streptococcus pyogenes virulence factors and whose expression is inversely proportional to virulence, though speB is also known to be a major virulence factor involved in tissue invasion and in the pathogenesis of necrotizing fasciitis [9]. PM plasma membrane, HAC hyaluronic acid capsule, P1 promoter region 1 of the has operon. b Enzyme-linked binding protein assay ISR1 (emm3.1) and ISR4 (emm1.0) produced a higher amount of hyaluronic acid. c, d Blood agar plates showing a mucoid phenotype (ISR1) and a non-mucoid phenotype (ISR6)