Staphylococcus aureus Strain-Dependent Biofilm Formation in Bone-Like Environment

Abstract

Staphylococcus aureus species is an important threat for hospital healthcare because of frequent colonization of indwelling medical devices such as bone and joint prostheses through biofilm formations, leading to therapeutic failure. Furthermore, bacteria within biofilm are less sensitive to the host immune system responses and to potential antibiotic treatments. We suggested that the periprosthetic bone environment is stressful for bacteria, influencing biofilm development. To provide insights into S. aureus biofilm properties of three strains [including one methicillin-resistant S. aureus (MRSA)] under this specific environment, we assessed several parameters related to bone conditions and expected to affect biofilm characteristics. We reported that the three strains harbored different behaviors in response to the lack of oxygen, casamino acids and glucose starvation, and high concentration of magnesium. Each strain presented different biofilm biomass and live adherent cells proportion, or matrix production and composition. However, the three strains shared common responses in a bone-like environment: a similar production of extracellular DNA and engagement of the SOS response. This study is a step toward a better understanding of periprosthetic joint infections and highlights targets, which could be common among S. aureus strains and for future antibiofilm strategies.

Keywords: MRSA; MSSA; biofilms; bone microenvironment; prosthetic joint infection.

FIGURE 1

Different biofilm structures according to S. aureus strains. Biofilms were grown aerobically for 24 h in minimal medium and imaged by light microscopy. Representative images are shown. The scale bars indicate 40 μm.

FIGURE 2

Impact of hypoxia on S. aureus biofilm formation. Biofilms were grown in minimal medium with (white histograms) or without (black histograms) oxygen for 24 h before absorbance measurement, live adherent bacteria numeration, and scanning electron microscopy acquisition. (A) Results represent the ratio of crystal violet absorbance (biofilm biomass) on planktonic growth absorbance. Experiments were performed at least four independent times with three technical replicates for each. (B) Results represent the percentage of live adherent bacteria among all bacteria. Experiments were performed at least four independent times with two technical replicates for each. Error bars represent standard errors for each average value. (C) Representative images of scanning electronic microscopy acquisitions at 15,000 × are shown. Statistical analyses were performed using the exact non-parametric Wilcoxon–Mann–Whitney test for independent samples: ¤¤¤Statistically different from aerobic culture condition (p < 0.001). The scale bars indicate 1 μm.

FIGURE 3

Effect of hypoxia on matrix formation and gene regulation of S. aureus CIP 53.154. Biofilms were grown in minimal medium with or without oxygen for 24 h before staining and confocal laser scanning microscopy acquisitions or RT-qPCR analysis. (A) Results represent the percentage of dead or damaged bacteria (stained by PI) among live bacteria (stained by SYTO^TM 9) acquired by confocal microscopy. Grid histograms represent biofilm growth with oxygen, whereas non-grid histogram represents growth without oxygen. (B) Results represent the volume measurement of SYTO^TM 9 (green histograms, live bacteria), SYPRO^® Ruby (magenta, protein), WGA (blue, PIA), and TOTO^TM-3 (orange, extracellular DNA) acquired by confocal microscopy. Grid histograms represent biofilm growth with oxygen, whereas non-grid histogram represents growth without oxygen. (C) Representative images of confocal microscopy acquisitions are shown. The scale bars indicate 20 μm. Experiments were performed two independent times with three representative acquisitions for each coverslip. (D) Results represented relative mRNA expressions of bacteria within biofilms grown hypoxically vs. those grown aerobically. Experiments were performed eight independent times with two technical replicates for each. Results are presented as box and whiskers: whiskers represent minimum and maximum, the bottom and top of the box are the 15th and 85th percentiles, and the black band inside the box stands for the median. Statistical analyses were performed using the exact non-parametric Wilcoxon–Mann–Whitney test for independent samples: ^∗∗∗Statistically different from aerobic culture condition (p < 0.001).

FIGURE 4

Impact of starvation, magnesium excess, and hypoxia on S. aureus biofilm formation. Biofilms were grown in minimal medium (control) or modified minimal medium (no CAA, without casamino acids; 10 × Mg, 10 × minimal medium concentration; no Gluc, without glucose) without oxygen for 24 h before absorbance measurement and live adherent bacteria numeration. (A) Results represent the ratio of crystal violet absorbance (biofilm biomass) on planktonic growth absorbance. Experiments were performed at least four independent times with three technical replicates for each. (B) Results represent the percentage of live adherent bacteria among all bacteria. Experiments were performed at least four independent times with two technical replicates for each. Error bars represent standard errors for each average value. Red grid line represents results of biofilms grown with oxygen in minimal medium. Statistical analyses were performed using the exact non-parametric Wilcoxon–Mann–Whitney test for independent samples: ¤,¤¤,¤¤¤Statistically different from aerobic control (p < 0.05; p < 0.01; p < 0.001); *, **, ***Statistically different from hypoxic control (p < 0.05; p < 0.01; p < 0.001).

FIGURE 5

Impact of starvation, magnesium excess, and hypoxia on S. aureus biofilm matrix. Biofilms were grown in minimal medium (control) or modified minimal medium (no CAA, without casamino acids; 10 × Mg, 10 × minimal medium concentration; no Gluc, without glucose) without oxygen for 24 h before imaging. Representative images of scanning electronic microscopy acquisitions at 15,000 × are shown. The scale bar indicates 1 μm.

FIGURE 6

Impact of starvation, magnesium excess, and hypoxia on S. aureus biofilm matrix components. Biofilms were grown in minimal medium (MM) or modified minimal medium (BLE: no CAA, without casamino acids; 10 × Mg, 10 × minimal medium concentration; no Gluc, without glucose) without oxygen for 24 h before confocal scanning electron microscopy imaging. (A) Results represent the percentage of dead or damaged bacteria (stained by PI) among live bacteria (stained by SYTO^TM 9). (B) Results represent the volume measurement of SYTO^TM 9 (green histograms; live bacteria), SYPRO^® Ruby (magenta: protein), WGA (blue: PIA) and TOTO^TM-3 (orange: extracellular DNA) of bacteria grown in MM without oxygen. (C) Results represent the volume measurement as previously described in BLE without oxygen. (D) Representative images are shown. Experiments were performed two independent times with three representative acquisitions for each coverslip. Experiments were performed two independent times with three representative acquisitions for each coverslip. Error bars represent standard errors for each average value. Scale bar = 20 μm.

FIGURE 7

Impact of starvation, magnesium excess, and hypoxia on S. aureus gene regulation. Biofilms were grown in minimal medium (MM) or in modified minimal medium (BLE: no CAA, without casamino acids; 10 × Mg, 10 × minimal medium concentration; no Gluc, without glucose) without oxygen for 24 h before RT-qPCR analysis. Results represent the ratio of relative mRNA expressions of bacteria within biofilms grown in BLE vs. those grown in minimal medium. Experiments were performed eight independent times with two technical replicates for each. Results are presented as box and whiskers: whiskers represent minimum and maximum, the bottom and top of the box are the 15th and 85th percentiles, and the black band inside the box stands for the median. Statistical analyses were performed using the exact non-parametric Wilcoxon–Mann–Whitney test for independent samples: *, **, ***Statistically different from minimal medium without oxygen (p < 0.05; p < 0.01; p < 0.001).