Optimised methods for the targeted surveillance of extended-spectrum beta-lactamase producingEscherichia coliin human stool

Abstract

AbstractUnderstanding transmission pathways of important opportunistic, drug resistant pathogens, such as extended-spectrum beta-lactamase (ESBL) producingEscherichia coli,is essential to implementing targeted prevention strategies to interrupt transmission and reduce the number of infections. To link transmission of ESBL-producingE. coli(ESBL-EC) between two sources, single nucleotide resolution ofE. colistrains as well asE. colidiversity 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-hour 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 optimised workflow can be applied for single nucleotide variant analysis of an ESBL-EC from stool.