Shifting national surveillance of Shigella infections toward geno‐serotyping by the development of a tailored Luminex assay and NGS workflow

Abstract

The phylogenetically closely related Shigella species and enteroinvasive Escherichia coli (EIEC) are responsible for millions of episodes of bacterial dysenteriae worldwide. Given its distinct epidemiology and public health relevance, only Shigellae are subject to mandatory reporting and follow‐up by public health authorities. However, many clinical laboratories struggle to differentiate non‐EIEC, EIEC, and Shigella in their current workflows, leading to inaccuracies in surveillance and rising numbers of misidentified E. coli samples at the National Reference Centre (NRC). In this paper, we describe two novel tools to enhance Shigella surveillance. First, we developed a low‐cost Luminex‐based multiplex assay combining five genetic markers for species identification with 11 markers for serotype prediction for S. sonnei and S. flexneri isolates. Using a test panel of 254 clinical samples, this assay has a sensitivity of 100% in differentiation of EIEC/Shigella pathotype from non‐EIEC strains, and 68.7% success rate in distinction of Shigella and EIEC. A novel, and particularly successful marker was a Shigella‐specific deletion in the spermidine acetyltransferase gene speG, reflecting its metabolic decay. For Shigella serotype prediction, the multiplex assay scored a sensitivity and specificity of 96.6% and 98.4%, respectively. All discrepancies were analyzed with whole‐genome sequencing and shown to be related to causative mutations (stop codons, indels, and promoter mutations) in glycosyltransferase genes. This observation spurred the development of an in silico workflow which extracts the Shigella serotype from Next‐Generation Sequencing (NGS) data, taking into account gene functionality. Both tools will be implemented in the workflow of the NRC, and will play a major role in the shift from phenotypic to genotyping‐based surveillance of shigellosis in Belgium.

Outbreak of Central American born Shigella sonnei in two youth camps in Belgium in the summer of 2019

“…DNA libraries were prepared using the Nextera XT DNA Sample Preparation Kit (Illumina) and sequenced on a Illumina MiSeq. Fastq files were further processed as described by Ventola et al 2019 [ 3 ].…”

Historical, current, and emerging tools for identification and serotyping of Shigella

“…The average nucleotide identity (ANI) between two genomes has been suggested as a valid alternative to the wet-laboratory DDH methods for species delineation. Genomes can be defined as members of the same species if sharing ≥ 95–96% ANI [ 82 ]. Various ANI-based approaches and software are currently available to compare the genomes in silico [ 45 ].…”

“…Likewise, Ventola et al proposed two novel tools to be implemented in the National Reference of Salmonella and Shigella of Belgium for Shigella surveillance. The first tool consisted of a cost-effective Luminex assay based on a modular multiplex oligonucleotide ligation-PCR procedure targeting five genetic markers for species identification and 11 serotype markers for S. sonnei and S. flexneri, in a single test [82,102]. The second tool is a WGS-based workflow for automated prediction of Shigella serotypes, focusing on gene functionality [82].…”

Phylogenomic Investigation of Increasing Fluoroquinolone Resistance among Belgian Cases of Shigellosis between 2013 and 2018 Indicates Both Travel-Related Imports and Domestic Circulation

“…The predicted genotypic susceptibility was compared to the observed phenotypic resistance to ciprofloxacin. Serotypes for S. flexneri samples were determined in silico using a WGS-based workflow described previously [21]. Serotyping is not performed for S. sonnei and was therefore not evaluated.…”

“…National surveillance in Belgium is performed by the National Reference Centre (NRC) for shigellosis, which receives annually approximately 400 Shigella cultures on a voluntary basis from peripheral laboratories [21]. Traditionally, Shigella spp.…”