Systematic screening of 42 vancomycin-resistant Enterococcus faecium strains for resistance, biofilm, and desiccation in simulated microgravity

Abstract

Vancomycin-resistant Enterococcus faecium (VRE) presents significant challenges in healthcare, particularly for hospitalized and immunocompromised patients, including astronauts with dysregulated immune function. We investigated 42 clinical E. faecium isolates in simulated microgravity (sim. µg) using a 2-D Clinostat, with standard gravity conditions (1 g) as a control. Isolates were tested against 22 antibiotics and characterized for biofilm formation and desiccation tolerance. Results showed varied responses in minimum inhibitory concentration (MIC) values for seven antibiotics after sim. µg exposure. Additionally, 55% of isolates showed a trend of increased biofilm production, and 59% improved desiccation tolerance. This investigation provides initial insights into E. faecium's changes in response to simulated spaceflight, revealing shifts in antibiotic resistance, biofilm formation, and desiccation tolerance. The observed adaptability emphasizes the need to further understand VRE's resilience to microgravity, which is crucial for preventing infections and ensuring crew health on future long-duration space missions.

Fig. 1. Adherence of VRE (vancomycin-resistant E. faecium) isolates tested with crystal violet (0.5%) biofilm assay (n = 15).

Initial: adherent cell formation was initially evaluated with the crystal violet assay before any treatment. 1 g: isolates were incubated in normal gravity as a control for 7 days. Sim. µg: isolates were incubated under sim. µg for 7 days by clinorotation. Isolates were then tested in their ability to adhere and potentially form biofilms after 24 h at 37 °C in measuring the optical density (OD_600nm) in a microplate reader. Measurements were done in triplicates and calculated error bars show the standard deviation of each sample (n = 3). Significance was determined by two-sample t-test; the conditions corresponding to the significance levels are shown in the parentheses: *p < 0.05; **p < 0.01; ***p < 0.001.

Fig. 2. Adherence of VVE-B (vancomycin-variable E. faecium) isolates tested with crystal violet (0.5%) biofilm assay (n = 7).

Initial: adherent cell formation was initially evaluated with the crystal violet assay before any treatment. 1 g: isolates were incubated in normal gravity as a control for 7 days. Sim. µg: isolates were incubated under sim. µg for 7 days by clinorotation. Isolates were then tested in their ability to adhere and potentially form biofilms after 24 h at 37 °C in measuring the optical density (OD_600nm) in a microplate reader. Measurements were done in triplicates and calculated error bars show the standard deviation of each sample (n = 3). Significance was determined by two-sample t-test; the conditions corresponding to the significance levels are shown in the parentheses: **p < 0.01; ***p < 0.001.

Fig. 3. Adherence of VSE (vancomycin susceptible E. faecium) isolates tested with crystal violet (0.5%) biofilm assay (n = 20).

Initial: adherent cell formation was initially evaluated with the crystal violet assay before any treatment. 1 g: isolates were incubated in normal gravity as a control for 7 days. Sim. µg: isolates were incubated under sim. µg for 7 days by clinorotation. Isolates were then tested in their ability to adhere and potentially form biofilms after 24 h at 37 °C in measuring the optical density (OD_600nm) in a microplate reader. Measurements were done in triplicates and calculated error bars show the standard deviation of each sample (n = 3). Significance was determined by two-sample t-test. The conditions corresponding to the significance levels are shown in the parentheses: *p < 0.05; ***p < 0.001.

Fig. 4. Reduction (%) of alamarBlue Cell Viability Reagent after 3 h incubation with VRE (vancomycin-resistant E. faecium) isolates after 24 h of desiccation.

Isolates were incubated in normal gravity (1 g) and under sim. µg for 7 days (n = 15). These isolates were then dried for 24 h and tested in their viability after desiccation by the Cell Viability Reagent alamarBlue. Measurements were done in triplicates and calculated error bars show the standard deviation of each sample (n = 3). Due to technical constraints, the assay was designed without the initial measurement to ensure that all samples fit within a single run of the measurement, maintaining consistency and accuracy across all data points. Significance was determined by two-sample t-test; The conditions corresponding to the significance levels are shown in the parentheses: *p < 0.05.

Fig. 5. Reduction (%) of alamarBlue Cell Viability Reagent after 3 h incubation with VVE-B (vancomycin-variable E. faecium) isolates after 24 h of desiccation.

Isolates were incubated in normal gravity (1 g) and under sim. µg for 7 days (n = 15). These isolates were then dried for 24 h and tested in their viability after desiccation by the Cell Viability Reagent alamarBlue. Measurements were done in triplicates and calculated error bars show the standard deviation of each sample (n = 3). Due to technical constraints, the assay was designed without the initial measurement to ensure that all samples fit within a single run of the measurement, maintaining consistency and accuracy across all data points. Significance was determined by two-sample t-test. Significance was determined by two-sample t-test; The conditions corresponding to the significance levels are shown in the parentheses: *p < 0.05.

Fig. 6. Reduction (%) of alamarBlue Cell Viability Reagent after 3 h incubation with VSE (vancomycin susceptible E. faecium) isolates after 24 h of desiccation.

Isolates were incubated in normal gravity (1 g) and under sim. µg for 7 days (n = 20). These isolates were then dried for 24 h and tested in their viability after desiccation by the Cell Viability Reagent alamarBlue. Measurements were done in triplicates and calculated error bars show the standard deviation of each sample (n = 3). Due to technical constraints, the assay was designed without the initial measurement to ensure that all samples fit within a single run of the measurement, maintaining consistency and accuracy across all data points. Significance was determined by two-sample t-test. Significance was determined by two-sample t-test; the conditions corresponding to the significance levels are shown in the parentheses: *p < 0.05.

Fig. 7. Workflow overview from E. faecium sample preparation to final analysis (antibiotic susceptibility testing, biofilm formation, desiccation tolerance) after simulation of microgravity for 7 days by 2D-Clinorotation.

MFU McFarland Unit, MIC minimal inhibitory concentration, sim. µg simulated microgravity, NaCl Sodium Chloride 0.85%. Created with BioRender.com.