Extracellular Polymeric Matrix Production and Relaxation under Fluid Shear and Mechanical Pressure in Staphylococcus aureus Biofilms

Abstract

The viscoelasticity of a biofilm's EPS (extracellular polymeric substance) matrix conveys protection against mechanical challenges, but adaptive responses of biofilm inhabitants to produce EPS are not well known. Here, we compare the responses of a biofilm of an EPS-producing (ATCC 12600) and a non-EPS producing (5298) Staphylococcus aureus strain to fluid shear and mechanical challenge. Confocal laser scanning microscopy confirmed absence of calcofluor-white-stainable EPS in biofilms of S. aureus 5298. Attenuated total reflection Fourier transform infrared (ATR-FTIR) spectroscopy combined with tribometry indicated that polysaccharide production per bacterium in the initial adhering layer was higher during growth at high shear than at low shear and that this increased EPS production extended to entire biofilms, as indicated by tribometrically measured coefficients of friction (CoF). CoF of biofilms grown under high fluid shear were higher than those when grown under low shear, likely due to wash-off polysaccharides. Measurement of a biofilm's CoF implies application of mechanical pressure that yielded an immediate increase in the polysaccharide band area of S. aureus ATCC 12600 biofilms due to their compression. Compression decreased after relief of pressure to the level observed prior to mechanical pressure. For biofilms grown under high shear, this coincided with a higher percent whiteness in optical coherence tomography-images indicative of water outflow, returning back into the biofilm during stress relaxation. Biofilms grown under low shear, however, were stimulated during tribometry to produce EPS, also after relief of stress. Knowledge of factors that govern EPS production and water flow in biofilms will allow better control of biofilms under mechanical challenge and better understanding of the barrier properties of biofilms against antimicrobial penetration.IMPORTANCE Adaptive responses of biofilm inhabitants in nature to environmental challenges such as fluid shear and mechanical pressure often involve EPS production with the aim of protecting biofilm inhabitants. EPS can assist biofilm bacteria in remaining attached or can impede antimicrobial penetration. The TriboChemist is a recently introduced instrument, allowing the study of initially adhering bacteria to a germanium crystal using ATR-FTIR spectroscopy, while simultaneously allowing measurement of the coefficient of friction of a biofilm, which serves as an indicator of the EPS content of a biofilm. EPS production can be stimulated by both fluid shear during growth and mechanical pressure, while increased EPS production can continue after pressure relaxation of the biofilm. Since EPS is pivotal in the protection of biofilm inhabitants against mechanical and chemical challenges, knowledge of the factors that make biofilm inhabitants decide to produce EPS, as provided in this study, is important for the development of biofilm control measures.

Keywords: FTIR; biofilm relaxation; biofilms; friction; tribochemistry; viscosity.

FIG 1

Schematic presentation of the TriboChemist and its possibilities for measuring the lubricity and molecular composition of biofilms grown on a Ge crystal using a combination of a sliding wear tester and FTIR spectroscopy.

FIG 2

CLSM images (top views and XZ cross sections) together with biovolumes (table inset) of EPS and of live and dead bacteria in staphylococcal biofilms grown in the parallel plate flow chamber of the TriboChemist under low and high shear and after staining with calcofluor-white and Live/Dead BacLight bacterial viability stain. Live bacteria are green-fluorescent and dead ones are red-fluorescent, while EPS show calcofluor-white blue fluorescence. (a and b) Biofilms of non-EPS producing S. aureus 5298 under low (0.16 s⁻¹) (a) and high (0.79 s⁻¹) (b) shear. (c and d) Biofilms of EPS-producing S. aureus ATCC 12600 under low (c) and high (d) shear. Data in the table inset are averages over two images ± standard deviations. An asterisk (*) refers to significant differences (P < 0.05) between data obtained at low and high shear. Bar, 50 μm.

FIG 3

Influence of fluid shear on the friction between S. aureus biofilms and a PDMS ball. Friction was measured on biofilms of non-EPS-producing S. aureus 5298 (red lines and columns) and EPS-producing S. aureus ATCC 12600 (black lines and columns) grown on Ge crystal surfaces under low (0.16 s⁻¹) shear rates (LSR) and high (0.79 s⁻¹) shear rates (HSR), as well as on a wetted, bare Ge crystal without biofilm (blue line and column). (a) Friction forces as a function of time during a single shear stroke with a PDMS ball (sliding speed 0.5 mm/s; loading force 0.45 N). (b) Root mean square (RMS) values of the CoF as averaged over a single stroke (see panel a). Data represent averages over triplicate experiments with separate bacterial cultures and with error bars indicating standard deviations. An asterisk (*) refers to significant differences at P < 0.05.

FIG 4

Examples of the FTIR absorption bands in biofilms of EPS-producing S. aureus ATCC 12600 grown under high (0.79 s⁻¹) fluid shear on a Ge crystal surface prior to and during mechanical pressure (speed of the sliding PDMS ball, 0.5 mm/s; loading force, 450 mN). (a) Polysaccharide absorption band (950 to 1,200 cm⁻¹), (b) phosphate absorption band (1,150 to 1,310 cm⁻¹), (c) amide absorption band (1,480 to 1,780 cm⁻¹) with the band components at 1,643 and 1,549 cm⁻¹ indicating the amide I and amide II bands, respectively. Note that water bending also occurs at 1,643 cm⁻¹ and interferes with the amide I band, and (d) water stretching band (2,650 to 3,800 cm⁻¹) with the band components at 3,308 and 3,469 cm⁻¹, indicating bound and free water, respectively. A.U., absorbance units.

FIG 5

FTIR absorption band areas and wavenumbers of staphylococcal biofilms as a function of the RMS value of their CoF during mechanical deformation for non-EPS-producing S. aureus 5298 and EPS-producing S. aureus ATCC 12600, grown on Ge crystal surfaces under low (0.16 s⁻¹) and high (0.79 s⁻¹) shear rates. (a) Polysaccharide absorption band (around 1,073 cm⁻¹), (b) phosphate absorption band (around 1,244 cm⁻¹), and (c) amide II absorption band (around 1,549 cm⁻¹). Results of triplicate experiments with separate bacterial cultures are indicated by different symbols of the same type. The solid lines represent the best fit to a linear function with R² values indicated, while the dotted lines show the 95% confidence intervals. P values indicate significant differences of the slope from zero.

FIG 6

FTIR absorption band areas and wavenumbers for water stretching in staphylococcal biofilms as a function of the RMS value of their CoF for non-EPS-producing S. aureus 5298 and EPS-producing S. aureus ATCC 12600 strains, grown on Ge crystal surfaces under low (0.16 s⁻¹) and high (0.79 s⁻¹) shear rates. (a) Bound water absorption band (around 3,308 cm⁻¹) and (b) free water absorption band (around 3,469 cm⁻¹). Results of triplicate experiments with separate bacterial cultures are indicated by different symbols of the same type. The solid lines represent the best fit to a linear function with R² values indicated, while the dotted lines show the 95% confidence intervals. P values indicate significant differences of the slope from zero.

FIG 7

Percentage changes in FTIR absorption band areas and wavenumber shifts as a function of relaxation time after exertion of mechanical pressure on biofilms of non-EPS-producing S. aureus 5298 and EPS-producing S. aureus ATCC 12600 strains, grown on Ge crystal surfaces under low (0.16 s⁻¹) and high (0.79 s⁻¹) shear rates (speed of the sliding PDMS ball, 0.5 mm/s; loading force, 450 mN). (a) Polysaccharide absorption band (around 1,073 cm⁻¹) and (b) phosphate absorption band (around 1,244 cm⁻¹). Percentage changes in absorption band area and wavenumber shifts were expressed relative to the values observed before exertion of mechanical pressure. Data represent averages over triplicate experiments with separate bacterial cultures and with error bars indicating standard deviations.

FIG 8

Examples of OCT images of S. aureus ATCC 12600 biofilms grown on glass surfaces under low (0.16 s⁻¹) and high (0.79 s⁻¹) shear rates prior to growth, immediately during compression, and after relaxation. (a) OCT images of staphylococcal biofilms. Biofilms are contained and compressed between two glass slides, visible as highly reflecting horizontal lines above and below the biofilm. Images represent a part (300 × 150 pixels) of an entire OCT image, as used to calculate the percent whiteness of a biofilm. (b) Percent whiteness of the biofilms (bacterial density) as a function of time after growth, during compression and relaxation. Percent whiteness represents the whiteness of the biofilm on a scale from 0% (all water) to 100% (all bacteria, no matrix) in the images after correction for the autoscaling of the OCT in images to be compared, which is heavily influenced by reflection from the substratum surface. (c) Thickness of the biofilms as a function of time after growth, during compression and relaxation.