Temporal modelling of the biofilm lifecycle (TMBL) establishes kinetic analysis of plate-based bacterial biofilm dynamics

Abstract

Bacterial biofilms are critical to pathogenesis and infection. They are associated with rising rates of antimicrobial resistance. Biofilms are correlated with worse clinical outcomes, making them important to infectious diseases research. There is a gap in knowledge surrounding biofilm kinetics and dynamics which makes biofilm research difficult to translate from bench to bedside. To address this gap, this work employs a well-characterized crystal violet biomass accrual and planktonic cell density assay across a clinically relevant time course and expands statistical analysis to include kinetic information in a protocol termed the TMBL (Temporal Mapping of the Biofilm Lifecycle) assay. TMBL's statistical framework quantitatively compares biofilm communities across time, species, and media conditions in a 96-well format. Measurements from TMBL can reliably be condensed into response features that inform the time-dependent behavior of adherent biomass and planktonic cell populations. Staphylococcus aureus and Pseudomonas aeruginosa biofilms were grown in conditions of metal starvation in nutrient-variable media to demonstrate the rigor and translational potential of this strategy. Significant differences in single-species biofilm formation are seen in metal-deplete conditions as compared to their controls which is consistent with the consensus literature on nutritional immunity that metal availability drives transcriptomic and metabolomic changes in numerous pathogens. Taken together, these results suggest that kinetic analysis of biofilm by TMBL represents a statistically and biologically rigorous approach to studying the biofilm lifecycle as a time-dependent process. In addition to current methods to study the impact of microbe and environmental factors on the biofilm lifecycle, this kinetic assay can inform biological discovery in biofilm formation and maintenance.

Keywords: Biofilm formation; Crystal violet; Nutritional immunity; Pseudomonas aeruginosa; Staphylococcus aureus; Time-dependent.

Figure 1:. Schematic procedure for temporal measurement of biofilm.

Overnight culture of the microbe of interest in nutrient-rich media is grown to early stationary phase (12–16 hours), then diluted into media conditions of interest and aliquoted into 96-well plates. These plates (1/desired timepoint) are then incubated at 37°C, and at 12-hour intervals for 4 days, 1 plate is pulled. Each plate undergoes aspiration of the planktonic bacterial population which is then measured for optical density at 600 nm. The remaining population(s) in wells are then washed with PBS, dried, stained with CV, washed and de-stained with 70% v/v ethanol solution. The de-stained biomass is measured at optical density 595 nm.

Figure 2:. Nutrient-rich media support bacterial biomass accumulation in a species- and kinetics-dependent manner.

(A and B) Measurement of accrued biomass on polystyrene 96-well flat-bottom plate as a function of time (A) and of planktonic cell density (B). (C and E) Area under the curve analyses for growth-time replicates of adhered biomass (C) and planktonic biomass (E). (D and F) These replicates were also analyzed using the slope of the growth-time curve for each replicate as the response variable of adhered biomass (D) and planktonic biomass (F). At least 2 biological and 12 technical replicates were performed for each bacterial strain. Comparisons between the strains were made by Wilcoxon Rank Sum tests. Comparisons between test and control conditions without specific notation were not significant. *** p-value <0.001

Figure 3:. Staphylococcus aureus biomass accrual and planktonic growth are significantly affected by bioavailable zinc concentration in the growth environment.

(A and B) Measurement of accrued biomass on polystyrene 96-well flat-bottom plate as a function of time (A) and of planktonic cell density (B) in nutrient rich media containing increasing concentrations of small molecule chelator TPEN. (C and E) Area under the curve analyses show the area under the curve for growth-time replicates of adhered biomass (C) and planktonic biomass (E). (D and F) These replicates were also analyzed using the slope of the growth-time curve for each replicate as the response variable of adhered biomass (D) and planktonic biomass (F). Two biological and twelve technical replicates were used for each concentration of TPEN. All comparisons made by Kruskal-Wallis Rank Sum test for overall difference among all concentrations with follow-up Wilcoxon Rank Sum tests comparing control replicates to replicates at each TPEN concentration. Comparisons between test and control conditions without specific notation were not significant. * p<0.05 ** p<0.01 *** p<0.001

Figure 4:. Pseudomonas aeruginosa biomass accrual and planktonic growth are significantly affected by bioavailable zinc concentration in the growth environment.

(A and B) Measurement of accrued biomass on polystyrene 96-well flat-bottom plate as a function of time (A) and of planktonic cell density (B) in nutrient rich media containing increasing concentrations of small molecule chelator TPEN. (C and E) Area under the curve analyses show the area under the curve for growth-time replicates of adhered biomass (C) and planktonic biomass (E). (D and F) These replicates were also analyzed using the slope of the growth-time curve for each replicate as the response variable of adhered biomass (D) and planktonic biomass (F). Two biological and twelve technical replicates were used for each concentration of TPEN. All comparisons made by Kruskal-Wallis Rank Sum test for overall difference among all concentrations with follow-up Wilcoxon Rank Sum tests comparing control replicates to replicates at each TPEN concentration. Comparisons between test and control conditions without specific notation were not significant. * p<0.05 ** p<0.01 *** p<0.001

Figure 5:. Staphylococcus aureus biomass and planktonic growth are responsive to nutritional immune factor calprotectin and additional metal-binding S100 host proteins.

(A and B) Measurement of accrued biomass on polystyrene 96-well flat-bottom plate as a function of time (A) and of planktonic cell density (B) in media with applied S100 proteins and zinc-binding defective mutants. (C and E) Area under the curve analyses show the area under the curve for growth-time replicates of adhered biomass (C) and planktonic biomass (E). (D and F) These replicates were also analyzed using the slope of the growth-time curve for each replicate as the response variable of adhered biomass (D) and planktonic biomass (F). Two biological and twelve technical replicates were used for each S100 protein. All comparisons made by Kruskal-Wallis Rank Sum test for overall difference among all concentrations with follow-up Wilcoxon Rank Sum tests comparing CP replicates to replicates of each S100 protein. Comparisons between test and control conditions without specific notation were not significant. * p<0.05 ** p<0.01 *** p<0.001

Figure 6:. Staphylococcus aureus biomass and planktonic growth are significantly affected by individual metal starvation.

(A and B) Measurement of accrued biomass on polystyrene 96-well flat-bottom plate as a function of time (A) and of planktonic cell density (B) in nutrient-poor (relative to TSB and LB) media RPMI which had been depleted of metals by Chelex. Aliquots were then supplemented back with metals at a known concentration sufficient for S. aureus growth (denoted as Full Addback on Fig. 6A–F). (C and E) Area under the curve analyses show the area under the curve for growth-time replicates of adhered biomass (C) and planktonic biomass (E). (D and F) These replicates were also analyzed using the slope of the growth-time curve for each replicate as the response variable of adhered biomass (D) and planktonic biomass (F). Two biological and twelve technical replicates were used for each media condition. All comparisons made by Kruskal-Wallis Rank Sum test for overall difference among all concentrations with follow-up Wilcoxon Rank Sum tests comparing Full Addback replicates to replicates of each metal depleted condition. Comparisons between test and control conditions without specific notation were not significant. * p<0.05 ** p<0.01 *** p<0.001