Bacterial infectious diseases have always been a major challenge for clinical management and healthy animal husbandry, and Escherichia coli infections are among the most common of these bacterial diseases [1]. In 2022, Lancet report on the Global Survey of Bacterial Resistance and Mortality indicated that E. coli was ranked first in terms of the number of deaths attributable to bacterial resistance [2]. Meanwhile, it has been reported that the antibiotic resistance rate of E. coli has increased dramatically in the past two decades, which has caused great difficulties in clinical treatment. Extended-spectrum β-lactamase-producing Escherichia coli (ESBL-E. coli) has become a new global health problem due to its global distribution and wide spread [3,4]. ESBL-E. coli usually exhibits multi-drug resistance and is frequently transmitted in humans and animals [5,6]. It has been reported that chickens in the poultry industry may be an important reservoir for ESBL-E. coli [1], and ESBL-E. coli is relatively easy to colonize live chickens, leading to infections in both chickens and humans [7]. At the same time, ESBL-E. coli often leads to failure of clinical treatment [8]. Therefore, in view of the rapid and wide spread of ESBL-E. coli globally, there is an urgent need for a comprehensive and in-depth study on the new mechanism of β-lactam resistance formation and spread in E. coli.

The horizontal transfer of drug resistance genes is one of the most important reasons for the development of drug resistance in bacteria [9]. The traditional methods of horizontal gene transfer include conjugation, transformation and transduction, but these three methods have certain limitations. As a class of cup-shaped nanoparticles surrounded by lipid bilayer, EVs is secreted from cells into the extracellular environment. The diameter is usually about 20–200 nm, and contains nucleic acids, proteins, enzymes and other substances [10]. EVs plays biological functions in gene transfer, promoting bacterial metabolism, and intercellular communication, and is an emerging source of antibiotic resistance transfer [10]. EVs-mediated horizontal gene transfer is mainly carried out by wrapping nucleic acids, which helps to protect the nucleic acids in EVs from degradation by external nucleases, and can also achieve the purpose of direct transport over long distances [11]. The most predominant gene encoding extended-spectrum β-lactamase in ESBL-E. coli is blaCTX-M, and blaCTX-M-55 is the most predominant blaCTX-M subtype in chicken-derived ESBL-E. coli. Most ESBL-E. coli have blaCTX-M-55 located on plasmids, and the most common replicon types include transferable plasmids such as IncF, IncI, IncN, IncHI1, IncHI2, and p0111. The plasmids of EVs donor bacteria carrying the blaCTX-M-55 resistance gene used in this experiment include plasmid types such as IncF II, IncI2, IncI1, p0111, IncHI2, IncF II/IncN, and so on. Although it has been reported that EVs can mediate the horizontal transfer of plasmids, given the ubiquity and diversity of blaCTX-M-55 plasmids, it is still unclear whether the type of plasmids has an effect on the vesicle-mediated transfer mode.

Based on this, we took E. coli producing extended-spectrum β-lactamase as the study object to investigate the influence of EVs and β-lactam resistance formation and spread. This study demonstrated the important role of EVs in the transmission of β-lactam resistance in chicken ESBL-E. coli and assessed the risk of EVs-mediated horizontal gene transmission, providing a theoretical basis for elucidation of a new mechanism of EVs-mediated drug resistance transmission.