An optimised GAS-pharyngeal cell biofilm model

Abstract

Group A Streptococcus (GAS) causes 700 million infections and accounts for half a million deaths per year. Biofilm formation has been implicated in both pharyngeal and dermal GAS infections. In vitro, plate-based assays have shown that several GAS M-types form biofilms, and multiple GAS virulence factors have been linked to biofilm formation. Although the contributions of these plate-based studies have been valuable, most have failed to mimic the host environment, with many studies utilising abiotic surfaces. GAS is a human specific pathogen, and colonisation and subsequent biofilm formation is likely facilitated by distinct interactions with host tissue surfaces. As such, a host cell-GAS model has been optimised to support and grow GAS biofilms of a variety of GAS M-types. Improvements and adjustments to the crystal violet biofilm biomass assay have also been tailored to reproducibly detect delicate GAS biofilms. We propose 72 h as an optimal growth period for yielding detectable biofilm biomass. GAS biofilms formed are robust and durable, and can be reproducibly assessed via staining/washing intensive assays such as crystal violet with the aid of methanol fixation prior to staining. Lastly, SEM imaging of GAS biofilms formed by this model revealed GAS cocci chains arranged into three-dimensional aggregated structures with EPS matrix material. Taken together, we outline an efficacious GAS biofilm pharyngeal cell model that can support long-term GAS biofilm formation, with biofilms formed closely resembling those seen in vivo.

Figure 1

Schematic outlining the process of Detroit 562 pharyngeal cell monolayer formation. Schematic shows collagen coating, seeding with Detroit 562 pharyngeal cells, and finally an example well containing a 3.7% PFA fixed ~ 95% confluent monolayer of Detroit 562 pharyngeal cells. Example monolayer image taken at ×10 objective in an Incucyte^® S3 Live-Cell Analysis System.

Figure 2

Detroit 562 monolayer development was monitored at both 28 (a,b) and 48 h (c,d). Coating with collagen I (a,c) facilitated Detroit 562 monolayer formation comparatively greater than uncoated wells (b,d). Images were taken at ×10 objective at the Incucyte and representative of 3 biological replicates with 1 technical replicate each. Each well was initially seeded with 2 × 10⁵ Detroit 562 cells/mL.

Figure 3

M1 and M12 GAS were assessed for the ability to form biofilm on plastic and Detroit 562 pharyngeal cell monolayers. 48 h biofilms were formed and biofilm biomass ascertained via crystal violet staining. Monolayers with THY (no GAS biofilm) served as media sterility controls and background staining controls, with absorbance values (supplementary data Table 1) subtracted from those of biofilm samples. Data represents mean ± SEM, ** (P ≤ 0.01) and **** (P ≤ 0.0001); n = 3 biological replicates, with 3 technical replicates each.

Figure 4

Methanol fixation improves M1 and M12 GAS biofilm biomass detection. 48 h GAS biofilms were formed from planktonic GAS that had initially adhered to the Detroit 562 pharyngeal cell monolayer after 2 h incubation. Biofilm biomass was ascertained via crystal violet staining. Monolayers with THY (no GAS biofilm) served as media sterility controls and background staining controls, with absorbance values subtracted from those of biofilm samples. Data represents mean ± SEM, * (P ≤ 0.05) and ** (P ≤ 0.01); n = 3 biological replicates, with 3 technical replicates each.

Figure 5

72 h is an optimal period for GAS biofilm formation. M1 and M12 were assessed for GAS biofilm formation at 72 and 96 h. 72 h yielded significantly more biofilm than 96 h. Biofilm biomass was determined via crystal violet staining. Monolayers with THY (no GAS biofilm) served as media sterility controls and background staining controls, with absorbance values subtracted from those of biofilm samples. Data represents mean ± SEM, ** (P ≤ 0.01) and *** (P ≤ 0.001); n = 3 biological replicates, with 3 technical replicates each.

Figure 6

Assessing the utility of the optimised methodology on additional GAS M types (M3, 98, and 108). Biofilm biomass was determined via crystal violet staining. Monolayers with THY (no GAS biofilm) served as media sterility controls and background staining controls, with absorbance values subtracted from those of biofilm samples. Data represents mean ± SEM, ** (P ≤ 0.01) and *** (P ≤ 0.001); n = 3 biological replicates, with 3 technical replicates each.

Figure 7

Representative 72 h M1 (a,b), M12 (c,d), and M3 (e,f) GAS biofilms visualised by scanning electron microscopy at 500 and ×15,000 magnification. GAS biofilms show chained cocci (white arrows) arranged into three dimensional aggregated structures with EPS (black arrows) upon the Detroit 562 monolayers (smaller white arrows). Detroit 562 monolayers (without biofilm) (g,h) were also imaged at 500 and ×5000 magnification. Images represent 3 biological replicates, with 3 technical each.