Interplay of antibiotics and bacterial inoculum on suture-associated biofilms

Abstract

Background: Biofilms are often antibiotic resistant, and it is unclear if prophylactic antibiotics can effectively prevent biofilm formation. Experiments were designed to test the ability of high (bactericidal) concentrations of ampicillin (AMP), vancomycin (VAN), and oxacillin (OXA) to prevent formation of suture-associated biofilms initiated with low (10(4)) and high (10(7)) numbers of Staphylococcus aureus.

Materials and methods: S. aureus biofilms were cultivated overnight on silk suture incubated in biofilm growth medium supplemented with bactericidal concentrations of AMP, VAN, or OXA. Standard microbiological methods were used to quantify total numbers of viable suture-associated S. aureus. Crystal violet staining followed by spectroscopy was used to quantify biofilm biomass, which includes bacterial cells plus matrix components. To observe the effects of antibiotics on the microscopic appearance of biofilm formation, biofilms were cultivated on glass slides, then stained with fluorescent dyes, and observed by confocal microscopy.

Results: In the presence of a relatively low inoculum (10(4)) of S. aureus cells, bactericidal concentrations of AMP, VAN, or OXA were effective in preventing development of suture-associated biofilms. However, similar concentrations of these antibiotics were typically ineffective in preventing biofilm development on sutures inoculated with 10(7)S. aureus, a concentration relevant to contaminated skin. Confocal microscopy confirmed that bactericidal concentrations of AMP, VAN, or OXA inhibited, but did not prevent, development of S. aureus biofilms.

Conclusion: Bactericidal concentrations of AMP, VAN, or OXA inhibited formation of suture-associated biofilms initiated with low numbers (10(4)), but not high numbers (10(7)), of S. aureus cells.

Fig. 1

Comparative effect of low (10⁴) and high (10⁷) inocula of S. aureus RN6390 (A, B) and ATCC 25923 (C, D) incubated overnight on silk suture in the presence of varying concentrations of AMP, where the MBC for S. aureus 6390 was 0.25 μg/mL and S. aureus 25923 was 0.5 μg/mL. A and C present the numbers of viable S. aureus recovered from suture-associated biofilms, and B and D present the biofilm biomass measured as absorbance of crystal violet. * and ^†indicate a difference compared with the corresponding lower inoculum at P <0.01 and 0.05, respectively. Each bar represents 10 to12 assays.

Fig. 2

Comparative effect of low (10⁴) and high (10⁷) inocula of S. aureus RN6390 (A, B) and ATCC 25923 (C, D) incubated overnight on silk suture in the presence of varying concentrations of VAN, where the MBC for both S. aureus strains was 2 μg/mL. A and C present the numbers of viable S. aureus recovered from suture-associated biofilms, and B and D present the biofilm biomass measured as absorbance of crystal violet. * and ^†indicate a difference compared with the corresponding lower inoculum at P <0.01 and 0.05, respectively. Each bar represents eight assays.

Fig. 3

Comparative effect of low (10⁴) and high (10⁷) inocula of S. aureus RN6390 (A, B) and ATCC 25923 (C, D) incubated overnight on silk suture in the presence of varying concentrations of OXA, where the MBC for both S. aureus strains was 0.5 μg/mL. A and C present the numbers of viable S. aureus recovered from suture-associated biofilms, and B and D present the biofilm biomass measured as absorbance of crystal violet. * and ^†indicate a difference compared with the corresponding lower inoculum at P <0.01 and 0.05, respectively. Each bar represents 12 assays.

Fig. 4

S. aureus (rows A and D, strain 25923; rows B and C, strain 6390) biofilms cultivated overnight on glass slides in the presence of no drug (A), 0.125 μg/mL AMP (B), 2 μg/mL VAN (C), and 0.5 μg/mL OXA (D). Each row contains a light microscopy image using differential interference contrast microscopy, followed by corresponding best focus images from confocal z-stacks that localized cell wall material (red) and genomic DNA (blue) followed by an overlay of these three images. Note robust biofilm only in row A, relatively large S. aureus cells in the presence of AMP and OXA, and the variability in cell wall and genomic DNA staining in antibiotic-treated samples. All images are at the same magnification; scale bar is 10 μm.