# Determining genotype and antimicrobial resistance of Salmonella Typhi in environmental samples by amplicon sequencing

## TL;DR

This study shows that wastewater can be used to detect and analyze Salmonella Typhi, including its genetic makeup and antibiotic resistance, using sequencing techniques.

## Contribution

A novel amplicon sequencing protocol was developed to genotype and determine antimicrobial resistance in S. Typhi directly from wastewater.

## Key findings

- A targeted amplicon sequencing protocol was successfully used to identify S. Typhi genotypes and AMR in wastewater samples.
- Out of 110 wastewater samples, 8 yielded genotype and/or AMR data consistent with clinical isolates from India.
- The method could complement clinical surveillance and inform public health interventions for typhoid fever.

## Abstract

Estimates of the burden of typhoid fever due to Salmonella enterica serovar Typhi (S. Typhi) rely on data from clinical surveillance, which is rarely done in low income settings and is also limited by the poor sensitivity of the assays used and the reliance on health seeking by patients. Environmental surveillance for S. Typhi shed by symptomatic and asymptomatic individuals in wastewater offers a sensitive surveillance tool that could help to inform burden estimates. Sequencing S. Typhi direct from wastewater concentrates has the potential to identify circulating genotypes and associated antimicrobial resistance (AMR) genes, supporting public health interventions such as vaccination and antimicrobial usage.

We designed a multiplex targeted amplicon sequencing protocol for genotyping and determining AMR in S. Typhi from wastewater samples, targeting SNPs that identify genotypes of interest and both chromosomal and plasmid-borne AMR. PCR products were sequenced using the Oxford Nanopore Technologies (ONT) MinION, and genotypes and AMR identified using the GenoTyphi program.

We tested this approach on samples from south India from both hospital outflow and wastewater collected from the community. All samples tested were suspected to be positive for S. Typhi following quantitative PCR for ttr, tviB, and staG gene targets. Out of 110 samples tested we were able to determine a genotype and/or AMR for 8. All samples that gave a genotype call suggested a genotype consistent with those found in clinical cases in India during the same time period and produced consensus sequences that clustered with S. Typhi when included in a phylogenetic tree.

In this study, we provide proof of concept data for amplicon sequencing of S. Typhi in wastewater which with further optimisation could be used to complement clinical surveillance data or provide data on S. Typhi presence in the absence of clinical surveillance. This information can inform public health interventions, and the concept could be applied to other pathogens of interest for genotyping from environmental surveillance samples.

This study investigated the application of wastewater surveillance for detection of S. Typhi. Wastewater surveillance is of increasing interest for public health as a potential lower-cost alternative or complement to clinical surveillance. As well as confirming if S. Typhi is present in a sample, obtaining sequence information would allow the genotype and antimicrobial resistance present to be determined. Culturing S. Typhi from wastewater samples is difficult, preventing the application of traditional sequencing approaches using culture isolates. We therefore developed an amplicon sequencing protocol with a primer scheme targeting known genotype-defining single nucleotide polymorphisms and AMR genes that we tested on samples in south India.

This was implemented as a targeted multiplex PCR using four primer panels, with sequencing of the products performed on the Oxford Nanopore Technologies (ONT) MinION. We were able to identify the genotype and AMR profile for 8 of 110 samples previously shown by quantitative PCR to contain S. Typhi gene targets (ttr, staG, and tviB).

The results from this study show promise for the application of targeted next-generation sequencing to characterise S. Typhi in wastewater.