Ultra-/Small Angle X-ray Scattering (USAXS/SAXS) and Static Light Scattering (SLS) Modeling as a Tool to Determine Structural Changes and Effect on Growth in S. epidermidis

Abstract

Usually, to characterize bacterial cells' susceptibility to antimicrobials, basic microbiology techniques such as serial dilutions or disk assays are used. In this work, we present an approach focused on combining static light scattering (SLS) and ultra-/small angle X-ray scattering (USAXS/SAXS). This approach was used to support microbiology techniques, with the aim of understanding the structural changes caused to bacteria when they are exposed to different stresses like pH, oxidation, and surfactants. Using USAXS/SAXS and SLS data, we developed a detailed multiscale model for a Gram-positive bacterium, S. epidermidis, and we extracted information regarding changes in the overall size and cell thickness induced by different stresses (i.e., pH and hydrogen peroxide). Increasing the concentration of hydrogen peroxide leads to a progressive reduction in cell wall thickness. Moreover, the concomitant use of pH and hydrogen peroxide provides evidence for a synergy in inhibiting the S. epidermidis growth. These promising results will be used as a starting base to further investigate more complex formulations and improve/refine the data modeling of bacteria in the small angle scattering regime.

Keywords: SAXS; USAXS; antimicrobial; bacteria; modeling; small angle scattering; static light scattering.

Figure 1

(A) Schematic representation of the cross section of a bacterial cell in the case of a Gram-positive cell wall. Similarly, to the Gram-negative scheme: the cell cytoplasm is separated from the periplasm (PP) by the inner membrane (IM) composed by lipopolysaccharides and proteins where lipoteichoic acids (LPA) are bound, protruding through the peptidoglycan (PG) layer. On Gram-positive cells, the wall is mainly composed by a thick PG layer with attached surface proteins (SP) and teichoic acids (TA). (B) SEM micrograph of a S. epidermidis cell (during division). (C) Scattering length density profile for a Gram-positive membrane (dashed gray line) and simplified version of the same profile (continuous red line) used in the core–shell model for X-rays (see eq 1).

Figure 2

USAXS scattering profile for a dispersion of S. epidermidis at pH 7. Markers represent the data, and the red line represents the best fit according to the model proposed in Section 2.5.

Figure 3

(A) Core–shell model fittings (lines) applied to static light scattering profiles (dots) obtained for a range of concentrations of cells dispersed in water. Fluorescence microscopy micrograph of a cell dispersion containing (B) 10⁸ CFU/mL and (C) 10⁷ CFU/mL.

Figure 4

Light scattering profiles for S. epidermidis cells dispersed in water after exposure to 0.01, 0.05, and 0.1 wt % of Tween 20 and correspondent spherical core–shell model fitting. For the sake of clarity, data and respective fittings curves were shifted along the y-axis.

Figure 5

(A) Example of Gompertz model fitting a set of growth data for S. epidermidis cells previously exposed for 1 h to PBS at different pH values from 2 to 12. (B) USAXS/SAXS scattering profile of S. epidermidis cells at pH 2 (red markers), 7 (green markers), and 12 (blue markers) and fits (continuous black line) obtained for pH 7 and 12.

Figure 6

(A) Initial lag phase time after treatment with PBS at different pH values with and without hydrogen peroxide. (B) Polarized light micrograph of S. epidermidis cells dispersed in PBS (left to right): untreated, pH = 7, pH = 2 and 0.45 wt % of hydrogen peroxide and, pH = 2 and 9 wt % of hydrogen peroxide. Scale bar corresponds to 10 μm.

Figure 7

Dimensionless Kratky representation for S. epidermidis cells at pH 7 exposed to different hydrogen peroxide concentrations, zoom at low q (A) and high q (B). Legend of panel A applies also to panel B.

Figure 8

(A) USAXS/SAXS scattering profiles of S. epidermidis cells exposed to different concentrations of hydrogen peroxide at pH 7 and respective fits (dashed purple lines). In the presence of 4.5 wt % of hydrogen peroxide, it is worth noting the deviation from the fit in the region characteristic of the cell wall (3 × 10^–2 < q < 1.5 × 10^–1 nm^–1). (B) Spherical core–shell model applied to light scattering profile obtained for S. epidermidis cells dispersed in water after exposure to PBS at pH 7 (black) and hydrogen peroxide (gray).