Poster BB3 - Antibiotic-induced release of E. coli extracellular vesicles leads to varying levels of endothelial cell activation

Introduction: As the body detects infection, it can initiate an exaggerated immune response resulting in widespread inflammation and tissue damage, which is referred to as sepsis. Common treatments include anti-inflammatories and a broad spectrum of antibiotics. Interestingly, these antibiotics may paradoxically increase release of bacterial extracellular vesicles (bEVs). These bacterial vesicles originate from the outer and inner membranes of gram-negative bacteria and the cytoplasmic membranes of gram-positive bacteria and are produced utilizing processes such as lytic biogenesis pathways and blebbing. Upon interacting with immune cells, bEVs trigger the release of inflammatory cytokines leading to an amplified immune response, contributing to the characteristic hyperinflammatory response of sepsis. To investigate the relationship between antibiotic treatment and bEV production, we assessed eight clinically relevant antibiotics and their impact on Escherichia coli (E. coli) EV release. Subsequently, we stimulated human endothelial cells with the isolated bEVs to evaluate the effect of each antibiotic on ICAM-1 upregulation. Our aim was to validate that the different antibiotic treatments increased bEV release and to uncover how each influences cellular inflammation.

Materials and

Methods: Bacterial extracellular vesicles (bEVs) were generated and isolated following exposure of E. coli to various antibiotics for 3-4 hours, including Amikacin, Cefepime, Ceftriaxone, Ciprofloxacin, Meropenem, Tobramycin, Ertapenem, and Imipinem. The treatment concentrations were standardized at twice the minimum inhibitory concentration for each antibiotic. We utilized Nanoparticle Tracking Analysis (NTA) to measure the concentration and size distribution of particles in liquid samples. Human umbilical vein endothelial cells (HUVECs) were seeded onto a 96-well plate at a density of 40,000 cells/cm2. Upon reaching confluence, the cells were treated with each condition of bEVs and were incubated for 16 hours. The cells were then stained for ICAM-1 and imaged at 10x magnification on a fluorescence microscope.

Results, Conclusions, and Discussions: Our analysis using NTA demonstrated that the diverse antibiotic bEV samples all fell within the range of 1E11-1E12 particles/ml. There was a notable increase in the amount of bEVs released from the E. coli in the presence of the various antibiotics compared to the untreated control. This provided evidence supporting the notion that antibiotic exposure results in enhanced bEV release. Particularly, Ceftriaxone, a beta-lactam antibiotic, exhibited a greater than 5-fold increase in bEV production compared to the control condition, resulting in the highest concentration of bEVs overall. Additionally, staining for ICAM-1 activation in HUVECs following bEV stimulation revealed an increase in ICAM-1 expression across all antibiotic-treated conditions compared to the unstimulated condition. Notably, Ciprofloxacin, Meropenem, Cefepime, and Ceftriaxone consistently showed similar levels of ICAM-1 activation, which put them in the mid-range of the antibiotics in terms of their effect on bEV-induced inflammation. Contrastingly, Tobramycin and Amikacin, both belonging to the aminoglycoside class, demonstrated the highest ICAM-1 activations across all of the antibiotics despite the fact that they did not yield the highest bEV production. This suggests that the inflammatory properties of the bEVs can be modulated by different antibiotic treatments.

In conclusion, our findings underscore the diverse impacts of antibiotics on bEV production, shedding light on the subsequent cellular inflammatory responses. We observed a definite escalation in bEV release in the presence of antibiotics, each with varying properties in terms of ICAM-1 activation. These results highlight the complexity of antibiotic-bEV interactions, emphasizing the need for further investigation. Moving forward, it is crucial to replicate these experiments as well as extend our inquiry to include additional strains of E. coli, which will allow us to deepen our understanding of these antibiotic-bEV interactions. Recognizing these interactions is imperative for developing more effective therapeutic approaches for sepsis, emphasizing the importance of this research in addressing an important clinical challenge.

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