Clinical Laboratory Response to a Mock Outbreak of Invasive Bacterial Infections: a Preparedness Study

Abstract

Large hospital-based clinical laboratories must be prepared to rapidly investigate potential infectious disease outbreaks. To challenge the ability of our molecular diagnostics laboratory to use whole-genome sequencing in a potential outbreak scenario and identify impediments to these efforts, we studied 84 invasive serotype emm59 group A streptococcus (GAS) strains collected in the United States. We performed a rapid-response exercise to the mock outbreak scenario using whole-genome sequencing, genome-wide transcript analysis, and mouse virulence studies. The protocol changes installed in response to the lessons learned were tested in a second iteration. The initial investigation was completed in 9 days. Whole-genome sequencing showed that the invasive infections were caused by multiple subclones of epidemic emm59 GAS strains likely spread to the United States from Canada. The phylogenetic tree showed a strong temporal-spatial structure with diversity in mobile genetic element content, features that are useful for identifying closely related strains and possible transmission events. The genome data informed the epidemiology, identifying multiple patients who likely acquired the organisms through direct person-to-person transmission. Transcriptome analysis unexpectedly revealed significantly altered expression of genes encoding a two-component regulator and the hyaluronic acid capsule virulence factor. Mouse infection studies confirmed a high-virulence capacity of these emm59 organisms. Whole-genome sequencing, coupled with transcriptome analysis and animal virulence studies, can be rapidly performed in a clinical environment to effectively contribute to patient care decisions and public health maneuvers.

The Majority of 9,729 Group A Streptococcus Strains Causing Disease Secrete SpeB Cysteine Protease: Pathogenesis Implications

“…We next assessed secreted SpeB protease activity among 704 emm59 strains that have been characterized by whole-genome sequencing (50,51,55,56,67). Most of these emm59 organisms were recovered as part of comprehensive population-based epidemiologic studies conducted during a countrywide epidemic of severe invasive infections in Canada (57) or were cultured from patients with invasive infections in the United States (53).…”

“…Synonymous single nucleotide polymorphisms (coding for the same amino acid) and polymorphisms identified in multiple strains with a wild-type SpeB phenotype (not SpeB altering) were excluded from the analysis. For SpeB-deficient strains initially found by this analysis strategy to lack a gene polymorphism that could account for the protease-negative phenotype, the whole-genome sequence data were reanalyzed with a second bioinformatics strategy using Trimmomatic, Musket, SMALT, and FreeBayes as described previously (56). FreeBayes (61) is better able than VAAL to identify minority subpopulations that could contribute to an altered SpeB protease phenotype.…”

“…The 704 GAS emm59 invasive strains were collected as part of comprehensive prospective population-based surveillance studies conducted in the United States (n ϭ 50) and Canada (n ϭ 638) or had historic interest (n ϭ 16) (50,51,53,(55)(56)(57).…”

Genomic Landscape of Intrahost Variation in Group A Streptococcus: Repeated and Abundant Mutational Inactivation of the fabT Gene Encoding a Regulator of Fatty Acid Synthesis

“…The sequence reads were corrected using Musket (16) and mapped to the reference serotype M1 strain MGAS5005 (GenBank accession number CP000017) using SMALT (available at http://www.sanger.ac.uk/resources /software/smalt/). Single nucleotide polymorphisms (SNPs) and insertions and deletions (indels) were identified using FreeBayes (available at http: //www.github.com/ekg/Freebayes/) as described previously (17). The nature of the SNPs (coding/noncoding, synonymous/nonsynonymous, etc.)…”

High Incidence of Invasive Group A Streptococcus Disease Caused by Strains of Uncommon emm Types in Thunder Bay, Ontario, Canada

“…Beginning in 2006, Canada experienced an epidemic of invasive GAS (iGAS) infections caused by strains of the previously rare emm59 type (5). A hypervirulent clone, which later disseminated to several areas of the United States, was shown to be responsible for hundreds of cases countrywide (6)(7)(8)(9)(10). Invasive type emm59 strains were frequently isolated from soft tissue infections and were recovered in higher percentages from patients with distinctive risk factors, including alcohol abuse, homelessness, hepatitis C virus (HCV) infection, and intravenous drug use (IVDU) (5,6).…”