Jakobsen, T. H. | Search bioRxiv

bioRxiv2024

A method for separating bacterial cultures into single cells and defined size fractions of biofilm aggregates

In vitro microbiological experiments that aim to describe differences between planktonic and biofilm aggregate populations are challenging since liquid batch cultures contain a mix of both. Here, we present a simple method for fractioning a bacterial liquid batch culture into aggregates and single cells. Stackable cell strainers with mesh sizes of 30 m and 10 m were used to filtrate 6 day old batch cultures of Pseudomonas aeruginosa to produce size fractions of 0-10m and >30m. By confocal laser scanning microscopy measurements, we show that 95.5% of the total biomass was <10 m in the "0-10m size fraction" and that 92.5% of the total biomass was >30m in the ">30m size fraction". Furthermore, the adjustment of bacterial concentration using CFU/ml was validated by quantifying the total DNA of viable bacteria in the two size fractions after DNase treatment to deplete eDNA and DNA from dead bacteria. Surprisingly, this showed that adjusting the bacterial concentration using CFU/ml was a valid method with no significant differences in total DNA from viable bacteria.

microbiology↗

bioRxiv2023

Inhibition of Pseudomonas aeruginosa quorum sensing by chemical induction of the MexEF-oprN efflux pump

The cell-to-cell communication system quorum sensing (QS), used by various pathogenic bacteria to synchronize gene expression and increase host invasion potentials, is studied as a potential target for persistent infection control. To search for novel molecules targeting the QS system in the Gram-negative opportunistic pathogen Pseudomonas aeruginosa a chemical library consisting of 3280 small compounds from LifeArc was screened. A series of ten conjugated phenones that have not previously been reported to target bacteria were identified as inhibitors of QS in P. aeruginosa. Two lead compounds (ethylthio enynone and propylthio enynone) were resynthesized for verification of activity and further elucidation of the mode of action. The isomeric pure Z-ethylthio enynone was used for RNA sequencing revealing a strong inhibitor of QS-regulated genes and the QS-regulated virulence factors rhamnolipid and pyocyanin were significantly decreased by treatment with the compounds. A transposon mutagenesis screen performed in a newly constructed lasB-gfp monitor strain identified the target of Z-ethylthio enynone in P. aeruginosa to be the MexEF-OprN efflux pump, which was further established using defined mex knockout mutants. Our data indicate that the QS inhibitory capabilities of Z-ethylthio enynone were caused by the drainage of intracellular signal molecules as a response to chemical-induced stimulation of the MexEF-oprN efflux pump thereby inhibiting the auto-generated positive feedback and its enhanced signal-molecule synthesis.

microbiology↗