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

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-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. ### Competing Interest Statement The authors have declared no competing interest. ### Funding Statement This work was supported by iiCON (infection innovation consortium) via UK Research and Innovation (107136) and Unilever (MA-2021-00523N). ### Author Declarations I confirm all relevant ethical guidelines have been followed, and any necessary IRB and/or ethics committee approvals have been obtained. Yes The details of the IRB/oversight body that provided approval or exemption for the research described are given below: Research Tissue Bank of Liverpool School of Tropical Medicine gave ethical approval for the use and storage of stool samples (LSTM Research Tissue Bank RTB/2022/007) The National Research Ethics Service Greater Manchester South ethics committee gave ethical approval for the observational cohort study of hospital patients in Liverpool(ref: 22/NW/0343). I confirm that all necessary patient/participant consent has been obtained and the appropriate institutional forms have been archived, and that any patient/participant/sample identifiers included were not known to anyone (e.g., hospital staff, patients or participants themselves) outside the research group so cannot be used to identify individuals. Yes I understand that all clinical trials and any other prospective interventional studies must be registered with an ICMJE-approved registry, such as ClinicalTrials.gov. I confirm that any such study reported in the manuscript has been registered and the trial registration ID is provided (note: if posting a prospective study registered retrospectively, please provide a statement in the trial ID field explaining why the study was not registered in advance). Yes I have followed all appropriate research reporting guidelines, such as any relevant EQUATOR Network research reporting checklist(s) and other pertinent material, if applicable. Yes The R scripts used to generate the figures, supplementary figures and data analyses in this manuscript are available from the GitHub repository. Protocols are available on Protocols.io (dx.doi.org/10.17504/protocols.io.kxygxyk3dl8j/v). Reads from sequenced isolates in this study are accessible in the Sequence Read Archive (SRA) using BioProject ID: PRJNA1095376.