Cell arrangement impacts metabolic activity and antibiotic tolerance in Pseudomonas aeruginosa biofilms
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Abstract
Cells must access resources to survive, and the anatomy of multicellular structures influences this access. In diverse multicellular eukaryotes, resources are provided by internal conduits that allow substances to travel more readily through tissue than they would via diffusion. Microbes growing in multicellular structures, called biofilms, are also affected by differential access to resources and we hypothesized that this is influenced by the physical arrangement of the cells. In this study, we examined the microanatomy of biofilms formed by the pathogenic bacterium Pseudomonas aeruginosa and discovered that clonal cells form striations that are packed lengthwise across most of a mature biofilm’s depth. We identified mutants, including those defective in pilus function and in O-antigen attachment, that show alterations to this lengthwise packing phenotype. Consistent with the notion that cellular arrangement affects access to resources within the biofilm, we found that while the wild type shows even distribution of tested substrates across depth, the mutants show accumulation of substrates at the biofilm boundaries. Furthermore, we found that altered cellular arrangement within biofilms affects the localization of metabolic activity, the survival of resident cells, and the susceptibility of subpopulations to antibiotic treatment. Our observations provide insight into cellular features that determine biofilm microanatomy, with consequences for physiological differentiation and drug sensitivity.
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This Zenodo record is a permanently preserved version of a PREreview. You can view the complete PREreview at https://prereview.org/reviews/8410727.
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 …
This Zenodo record is a permanently preserved version of a PREreview. You can view the complete PREreview at https://prereview.org/reviews/8410727.
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.
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