PREreview of Cell arrangement impacts metabolic activity and antibiotic tolerance inPseudomonas aeruginosabiofilms

Reviewed by: Hashmath Fatimah, Liu Xuyu, Clarence Sim, Samantha Quah, Sreelakshmi Cheruvalli, Hana Marican, Nicole Loh and Viduthalai Rasheedkhan Regina

Singapore Centre for Environmental Life Science Engineering, Nanyang Technological University, Singapore

Summary:

Overall, there is generally a good flow to the paper. It shows a step-by-step approach taken to study the cellular arrangement of P. aeruginosa macrocolony biofilm. This was first done by visualising the cellular organisation within the biofilm, and determining if resource availability affects the organisation, followed by elucidating the genetic determinants that gives rise to this cellular arrangement, and then investigating the effects of this cellular arrangement on substrate distribution and transport as well as metabolic activity across the biofilm depth. Lastly, the implications of the cellular arrangement on the susceptibility of the biofilm to antibiotic treatments was touched upon. Results were well-presented in the figures and schematic diagrams were useful in guiding the reader to interpret the results.

Major comments:

Pg 5 – Experiment to study how changes in tryptone concentration alters cellular arrangement in P. aeruginosa macrocolonies:

Comparing the metabolic activity and the cell arrangement, it is clear that actively growing cells are existing as vertical striations. With 0.25% tryptone concentration, vertical striations were observed at the bottom of the biofilm, suggesting that these cells might be using the anaerobic/fermentation pathways. Since this inverse phenotype is observed only under low concentrations of tryptone, this experiment is inconclusive in identifying the effects of nutrient availability on the organisation of cells. Furthermore, if the experiment was run beyond 72 hours with 1% tryptone concentration, would it show the same phenotype or would it show a phenotype that resembles the biofilm with 0.25% tryptone? Moreover, there was no clear explanation for the choice of tryptone as a variable for this assay. Additional experiments are required to test the effect of other nutrients to eliminate confounding variables e.g. switching between metabolic pathways.

Pg 10 – Assessing cell death across biofilm depth using PI staining:

PI staining may not be a good representation of cell death as cells may still be viable with membrane perturbations. Combining a metabolic reporter such as fluorescein diacetate (FDA) would be a better alternative for this experiment.

Minor comments:

mScarlet fluorescence was shown yellow in colour in the fluorescence micrographs in Figures 1 – 4, but in Figure 5, mScarlet fluorescence was coloured red while eGFP expression was coloured yellow. The same colour could have been used to represent the same fluorescent marker in the different experiments performed to avoid confusion in interpreting the results. 

Competing interests

The author declares that they have no competing interests.