ESBL displace: a protocol for an observational study to identify displacing Escherichia coli strain candidates from ESBL-colonized travel returners using phenotypic, genomic sequencing and metagenome analysis

Introduction: Invading extended-spectrum beta-lactamase-producing Escherichia coli (ESBL-PE), non-ESBL E. coli, and other bacteria form a complex environment in the gut. The duration and dynamics of ESBL-PE colonization varies among individuals. Understanding the factors associated with colonization may lead to decolonization strategies. In this study, we aim to identify (i) single E. coli strains and (ii) microbiome networks that correlate with retention or decline of colonization, and (iii) pan-sensitive E. coli strains that potentially could be used to displace ESBL-PE during colonization. Methods and analysis: We recruit healthy travellers to Southeast Asia for a one-year prospective observational follow-up study. We collect and biobank stool, serum, and peripheral blood mononuclear cells (PBMCs) at predefined timepoints. Additional information is collected with questionnaires. We determine the colonization status with ESBL-PE and non-ESBL E. coli and quantify cell densities in stools and ratios over time. We characterize multiple single bacterial isolates per patient and timepoint using whole genome sequencing (WGS) and 16S/ITS amplicon-based and shotgun metagenomics. We determine phylogenetic relationships between isolates, antimicrobial resistance (AMR; phenotypic and genotypic), and virulence genes. We describe the bacterial and fungal stool microbiome alpha and beta diversity on 16S/ITS metagenomic data. We describe patterns in microbiome dynamics to identify features associated with protection or risk of ESBL-PE colonization. Ethics and dissemination: The study is registered (clinicaltrials.gov; NCT04764500 on 09/02/2019) and approved by the Ethics Committee (EKNZ project ID 2019-00044). We will present anonymized results at conferences and in scientific journals. Bacterial sequencing data will be shared via publicly accessible databases according to FAIR principles

Autoinducer 2-Dependent Escherichia coli Biofilm Formation Is Enhanced in a Dual-Species Coculture

Biofilms in nature typically consist of multiple species, and microbial interactions are likely to have crucial effects on biofilm development, structure, and functions. The best-understood form of communication within bacterial communities involves the production, release, and detection of signal molecules (autoinducers), known as quorum sensing. Although autoinducers mainly promote intraspecies communication, autoinducer 2 (AI-2) is produced and detected by a variety of bacteria, thus principally allowing interspecies communication. Here we show the importance of AI-2-mediated signaling in the formation of mixed biofilms by Enterococcus faecalis and Escherichia coli Our results demonstrate that AI-2 produced by E. faecalis promotes collective behaviors of E. coli at lower cell densities, enhancing autoaggregation of E. coli but also leading to chemotaxis-dependent coaggregation between the two species. Finally, we show that formation of such mixed dual-species biofilms increases the stress resistance of both E. coli and E. faecalis IMPORTANCE The role of interspecies communication in the development of mixed microbial communities is becoming increasingly apparent, but specific examples of such communication remain limited. The universal signal molecule AI-2 is well known to regulate cell-density-dependent phenotypes of many bacterial species but, despite its potential for interspecies communication, the role of AI-2 in the establishment of multispecies communities is not well understood. In this study, we explore AI-2 signaling in a dual-species community containing two bacterial species that naturally cooccur in their mammalian hosts, i.e., Escherichia coli and Enterococcus faecalis We show that active production of AI-2 by E. faecalis allows E. coli to perform collective behaviors at low cell densities. Additionally, AI-2- and chemotaxis-dependent coaggregation with E. faecalis creates nucleation zones for rapid growth of E. coli microcolonies in mixed biofilms and enhances the stress resistance of both species

Chemotaxis towards autoinducer 2 mediates autoaggregation in Escherichia coli

Bacteria communicate by producing and sensing extracellular signal molecules called autoinducers. Such intercellular signalling, known as quorum sensing, allows bacteria to coordinate and synchronize behavioural responses at high cell densities. Autoinducer 2 (AI-2) is the only known quorum-sensing molecule produced by Escherichia coli but its physiological role remains elusive, although it is known to regulate biofilm formation and virulence in other bacterial species. Here we show that chemotaxis towards self-produced AI-2 can mediate collective behaviour-autoaggregation-of E. coli. Autoaggregation requires motility and is strongly enhanced by chemotaxis to AI-2 at physiological cell densities. These effects are observed regardless whether cell-cell interactions under particular growth conditions are mediated by the major E. coli adhesin (antigen 43) or by curli fibres. Furthermore, AI-2-dependent autoaggregation enhances bacterial stress resistance and promotes biofilm formation

Chemotaxis and autoinducer-2 signalling mediate colonization and contribute to co-existence of Escherichia coli strains in the murine gut.

Bacteria communicate and coordinate their behaviour at the intra- and interspecies levels by producing and sensing diverse extracellular small molecules called autoinducers. Autoinducer 2 (AI-2) is produced and detected by a variety of bacteria and thus plays an important role in interspecies communication and chemotaxis. Although AI-2 is a major autoinducer molecule present in the mammalian gut and can influence the composition of the murine gut microbiota, its role in bacteria-bacteria and bacteria-host interactions during gut colonization remains unclear. Combining competitive infections in C57BL/6 mice with microscopy and bioinformatic approaches, we show that chemotaxis (cheY) and AI-2 signalling (via lsrB) promote gut colonization by Escherichia coli, which is in turn connected to the ability of the bacteria to utilize fructoselysine (frl operon). We further show that the genomic diversity of E. coli strains with respect to AI-2 signalling allows ecological niche segregation and stable co-existence of different E. coli strains in the mammalian gut. © 2023. The Author(s), under exclusive licence to Springer Nature Limited