qEV Isolation combined with Tunable Resistive Pulse Sensing (TRPS) has been used to isolate and characterise extracellular vesicles (EVs) derived from a range of samples and cell types. In an article published by Chiang et al., the qEV10 was used to isolate EVs from large cultures of a nosocomial (originating in a hospital) bacterial pathogen, and the Exoid was used to determine the zeta potential changes in EVs isolated from bacteria growing under different antibiotic stress conditions.
Following its initial isolation from a mosquito, Elizabethkingia anopheles has become recognised for its involvement in a range of healthcare-related infections including bacteraemia, lower respiratory tract infection, pneumonia, and neonatal meningitis. In most instances, these cases have been identified in immunocompromised people or premature babies, who are not only more vulnerable to E. anopheles infection but also the morbidity and mortality caused is significantly higher.
In addition, the presence of multiple antimicrobial resistance in infecting strains has led to increased clinical challenges, as options for successful antibiotic treatment become sparse. Thus, insights of E. anopheles infection mechanisms may help in the future development of effective antimicrobial therapy or progress on vaccines against the pathogen.
Like their mammalian counterparts, bacterial EVs can originate by different biogenesis mechanisms. Outer membrane vesicles (OMVs), however, are EVs derived specifically from the outer membrane of Gram-negative bacterial cells. OMVs carry a diverse molecular cocktail that can have numerous biological roles important for different stages of a bacteria’s life cycle and for communication with other bacteria or its host. Moreover, the fast pace of bacterial growth allows a rapid cellular physiological response to environmental cues, translating into quick release of new OMVs adjusted to the stimulus.
Download the app note to learn more about bacterial EVs: Isolation of Bacterial Extracellular Vesicles: What’s Different and What’s the Same?
Various stress conditions have been reported to affect OMV production, composition and biogenesis mechanisms – including antibiotics. Importantly, to study OMVs and their compositional changes, it is critical to have a reliable OMV isolation method that supports the fast and accurate purification of EVs. For a better understanding of how the bacteria deploys its traits for survival, virulence and antibiotic resistance, the aim of this study was to investigate the role of E. anopheles OMVs under stress conditions induced by antibiotics.
To answer this question, the authors firstly characterised the antimicrobial susceptibility of two E. anopheles strains by disk diffusion and microdilution assays, confirming the multiresistant phenotype of strain C08. OMVs were then isolated from C08 cultures grown with sub-minimum inhibitory (sub-MIC) concentrations of different antibiotics.
Briefly, bacterial cells were removed from culture by centrifugation (6,000 xg, 20 min, 4⁰C) and filtration (0.22 µm). Cell-free supernatant was concentrated by 100 kDa tangential flow filtration (TFF) down to 10 mL. Concentrated supernatant was run through a qEV10 column, with the OMV-containing volumes collected, washed and pelleted by ultracentrifugation (150,000 xg, 3 h, 4⁰C).
Read more: Highlights of Research Conducted Using the qEV10
Resulting purified OMVs were characterised in morphology, particle counts and size, and the zeta potential of OMVs was measured using the Exoid. Finally, in this study, proteins associated with OMVs from bacteria grown with antibiotic imipenem or untreated were identified by liquid chromatography-tandem mass spectrometry and further analysed for protein enrichment.
OMV characterisation showed clear variability in analysed parameters with bacteria grown under different antibiotics, highlighting that OMVs from chloramphenicol-treated bacteria had the highest protein content; OMVs from imipenem-treated bacteria had the highest concentration and size homogeneity; OMVs from amikacin-treated bacteria had the second most negative zeta potential (first was untreated); and minocycline-treated bacteria produced the most OMVs per cell.
Proteomic analysis revealed that OMVs from imipenem-treated cells had 20 unique proteins (out of 126 identified in total) and Gene Ontology analysis showed a significant change in pathway enrichment associated with carbohydrate transport and metabolism. Localisation of identified OMV proteins was similar in imipenem-treated and untreated cells, with 51-52% being from the outer membrane.
Protein-Protein Interaction network analysis confirmed differences in protein enrichment, which were further corroborated with in vitro assays: OMVs purified from imipenem-treated bacteria reduced the viability of lung epithelial cells and reduced macrophages’ bacterial killing rates. Furthermore, OMVs from imipenem-treated bacteria showed no differences in hydrolytic (carbapenemase) activity against imipenem compared to OMVs from untreated cells, suggesting no role in antibiotic resistance
Overall, OMVs from E. anopheles exposed to imipenem were shown to shift into an immunologically relevant phenotype – one that seems to be crucial for bacterial survival rather than a boost to antibiotic resistance. Broad physical characterisation of OMVs released under other antibiotic conditions studied here, suggests that other OMV compositional changes may be worth investigating, seeking for potential biological significance in E. anopheles infection.
Learn more about Tunable Resistive Pulse Sensing (TRPS)