Optimized methods for the targeted surveillance of extended-spectrum beta-lactamase-producing Escherichia coli in human stool

Gallichan, Sarah and Forrest, Sally and Picton-Barlow, Esther and McKeown, Claudia and Moore, Maria and Heinz, Eva and Feasey, Nicholas A. and Lewis, Joseph M. and Graf, Fabrice E. (2024) Optimized methods for the targeted surveillance of extended-spectrum beta-lactamase-producing Escherichia coli in human stool. Microbiology Spectrum. ISSN 2165-0497 (https://doi.org/10.1128/spectrum.01058-24)

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Abstract

Understanding transmission pathways of important opportunistic, drug-resistant pathogens, such as extended-spectrum beta-lactamase (ESBL)-producing Escherichia coli, is essential to implementing targeted prevention strategies to interrupt transmission and reduce the number of infections. To link transmission of ESBL-producing E. coli (ESBL-EC) between two sources, single-nucleotide resolution of E. coli strains, as well as E. coli diversity within and between samples, is required. However, the microbiological methods to best track these pathogens are unclear. Here, we compared different steps in the microbiological workflow to determine the impact different pre-enrichment broths, pre-enrichment incubation times, selection in pre-enrichment, selective plating, and DNA extraction methods had on recovering ESBL-EC from human stool samples, with the aim to acquire high-quality DNA for sequencing and genomic epidemiology. We demonstrate that using a 4-h pre-enrichment in Buffered Peptone Water, plating on cefotaxime-supplemented MacConkey agar and extracting DNA using Lucigen MasterPure DNA Purification kit improves the recovery of ESBL-EC from human stool and produced high-quality DNA for whole-genome sequencing. We conclude that our optimized workflow can be applied for single-nucleotide variant analysis of an ESBL-EC from stool.

ORCID iDs

Gallichan, Sarah, Forrest, Sally, Picton-Barlow, Esther, McKeown, Claudia, Moore, Maria, Heinz, Eva ORCID logoORCID: https://orcid.org/0000-0003-4413-3756, Feasey, Nicholas A., Lewis, Joseph M. and Graf, Fabrice E.; Altman, Deena R.
CORE (COnnecting REpositories)