A new assay for rhamnolipid detection—important virulence factors of Pseudomonas aeruginosa

Abstract

Rhamnolipids (RLs) are heterogeneous glycolipid molecules that are composed of one or two L-rhamnose sugars and one or two β-hydroxy fatty acids, which can vary in their length and branch size. They are biosurfactants, predominantly produced by Pseudomonas aeruginosa and are important virulence factors, playing a major role in P. aeruginosa pathogenesis. Therefore, a fast, accurate and high-throughput method of detecting such molecules is of real importance. Here, we illustrate the ability to detect RL-producing P. aeruginosa strains with high sensitivity, based on an assay involving phospholipid vesicles encapsulated with a fluorescent dye. This vesicle-lysis assay is confirmed to be solely sensitive to RLs. We illustrate a half maximum concentration for vesicle lysis (EC50) of 40 μM (23.2 μg/mL) using pure commercial RLs and highlight the ability to semi-quantify RLs directly from the culture supernatant, requiring no extra extraction or processing steps or technical expertise. We show that this method is consistent with results from thin-layer chromatography detection and dry weight analysis of RLs but find that the widely used orcinol colorimetric test significantly underestimated RL quantity. Finally, we apply this methodology to compare RL production among strains isolated from either chronic or acute infections. We confirm a positive association between RL production and acute infection isolates (p = 0.0008), highlighting the role of RLs in certain infections.

Prototype Development of the Intelligent Hydrogel Wound Dressing and Its Efficacy in the Detection of Model Pathogenic Wound Biofilms

“…Recently, we have demonstrated the potential for development of such diagnostic tests, and the "triggered release" of compounds in response to the onset of clinically relevant infection (CCT and biofilm formation), but not low-level background colonisation. 22,23 This is accomplished through the use of synthetic lipid vesicles loaded with fluorescent self-quenching dye, which are sensitive to the key population-density regulated cytotoxic virulence factors produced by major wound pathogens 19,24,25 . These vesicles were found to be stable under conditions reflective of the un-infected wound environment, 26 but production of cytotoxins by bacterial pathogens results in lysis and dye release, which may be used to provide a clear signal that the CCT has been reached, and clinically relevant infection has been intiated.…”

Rhamnolipids, Microbial Virulence Factors, in Alzheimer’s Disease

“…TLC is usually employed for qualitative or quantitative analysis of RL types [33,46,48,49]. Thus, the presence of RLs in the blood serum isolated sample was further confirmed by TLC, using as positive standards the purified RLs which were isolated from the T. thermophilus HB8 (or E. coli) bacterial cultures grown in sodium gluconate, i.e., the commercial white saponin and rhamnose.…”

Development of a mixed‐species biofilm model and its virulence implications in device related infections

“…S. aureus (MRSA 252) was also used for the initial assessment of mixed‐species biofilm models. All clinical strains used in this study are listed in Table . Bacteria were individually cultured from a single colony in 10 mL of broth in a shaker incubator for 18 h at 37°C, resulting in 10 9 CFU mL −1 in a final culture.…”