Abstract
Non-surface attached bacterial aggregates are frequently found in clinical settings associated with chronic infections. Current methods quantifying the extent to which a suspended bacterial population is aggregated mainly rely on: (1) cell size distribution curves that are difficult to be compared numerically among large-scale samples; (2) the average size/proportion of aggregates in a population that do not specify the aggregation patterns. Here we introduce a novel application of Gini coefficient, herein named Aggregation Coefficient (AC), to quantify the aggregation levels of cystic fibrosis Pseudomonas aeruginosa (CF-PA) isolates in vitro using 3D micrographs, Fiji and MATLAB. Different aggregation patterns of five strains were compared statistically using the numerical AC indexes, which correlated well with the size distribution curves plotted by different biovolumes of aggregates. To test the sensitivity of AC, aggregates of the same strains were treated with nitric oxide (NO), a dispersal agent that reduces the biomass of surface attached biofilms. Strains unresponsive to NO were reflected by comparable AC indexes, while those undergoing dispersal showed a significant reduction in AC index, mirroring the changes in average aggregate sizes and proportions. Therefore, AC provides simpler and more descriptive numerical outputs for measuring different aggregation patterns compared to current approaches.
Highlights
Non-surface attached bacterial aggregates are frequently found in clinical settings associated with chronic infections
It is important to quantify and describe the properties of aggregates for treatment and analysis. Whilst software such as COMSTAT11 analysing micrographs have been extensively applied for the quantification of biofilms, so far, the description of bacterial aggregate properties mainly relies on the visual description of microscopic images[12]; average size/volume of aggregates measured in micrographs[13,14,15]; or individual particle measurement using specific equipment such as laser-diffraction particle-size scanning analysis (LDA)[16] or
Results showed that the biofilm formation ability of different strains showed a similar trend in M9, lysogeny broth (LB) and BHI, but the biomass of PAO1, PA10, PA21, PA30, PA58 and PA68 was much higher in M9 than in LB or BHI after 72 hrs
Summary
Non-surface attached bacterial aggregates are frequently found in clinical settings associated with chronic infections. Current methods quantifying the extent to which a suspended bacterial population is aggregated mainly rely on: (1) cell size distribution curves that are difficult to be compared numerically among large-scale samples; (2) the average size/proportion of aggregates in a population that do not specify the aggregation patterns. As for bacterial communities, the most frequent usage of Gini coefficient is for bacteria diversity and species distribution in different environmental settings and biofilm communities[25,26]. This principle has not been reported to describe the unequal size distribution of aggregates within planktonic or biofilm cultures. The inequality of the cell distribution in a population is quantifiable, describing, in a simple way, how compact or scattered are the aggregates, the method being suitable for high throughput comparison among different samples
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