Human plasma enhances the expression of Staphylococcal microbial surface components recognizing adhesive matrix molecules promoting biofilm formation and increases antimicrobial tolerance In Vitro

Abstract

Background: Microbial biofilms have been associated with the development of chronic human infections and represent a clinical challenge given their increased antimicrobial tolerance. Staphylococcus aureus is a major human pathogen causing a diverse range of diseases, of which biofilms are often involved. Staphylococcal attachment and the formation of biofilms have been shown to be facilitated by host factors that accumulate on surfaces. To better understand how host factors enhance staphylococcal biofilm formation, we evaluated the effect of whole human plasma on biofilm formation in clinical isolates of S. aureus and the expression of seven microbial surface components recognizing adhesive matrix molecules (MSCRAMMs) known to be involved in biofilm formation by quantitative real-time PCR. We also evaluated whether plasma augmented changes in S. aureus biofilm morphology and antimicrobial resistance.

Results: Exposure of clinical isolates of S. aureus to human plasma (10%) within media, and to a lesser extent when coated onto plates, significantly enhanced biofilm formation in all of the clinical isolates tested. Compared to biofilms grown under non-supplemented conditions, plasma-augmented biofilms displayed significant changes in both the biofilm phenotype and cell morphology as determined by confocal scanning laser microscopy (CLSM) and scanning electron microscopy (SEM), respectively. Exposure of bacteria to plasma resulted in a significant fold-increase in MSCRAMM expression in both a time and isolate-dependent manner. Additionally, plasma-augmented biofilms displayed an increased tolerance to vancomycin compared to biofilms grown in non-supplemented media.

Conclusions: Collectively, these studies support previous findings demonstrating a role for host factors in biofilm formation and provide further insight into how plasma, a preferred growth medium for staphylococcal biofilm formation enhances as well as augments other intrinsic properties of S. aureus biofilms. Consequently, these findings indicate that incorporation of host factors may be necessary to better replicate in vivo conditions and for the best utility of a clinical biofilm assay to evaluate the process of biofilm formation and treatments.

Figure 1

Visualization of staphylococcal biofilms grown in the presence of human plasma by CLSM and SEM. A-B) CLSM images biofilms of a methicillin-resistant (MRSA-3) and methicillin susceptible (UAMS-1) isolates of S. aureus grown overnight on coverslips in media supplemented with or without human plasma (10% Plasma). Biofilms were stained with a bacterial and a biofilm matrix stain to visualize the bacterial cells and extracellular polymeric matrix, respectively. Images were captured at 20X magnification. C-D) SEM analysis of biofilms of the clinical isolates listed above, following exposure to human plasma. Images were captured at 40,000X. Inlayed size bars represent 200 nm.

Figure 2

Plasma augmented biofilms have reduced susceptibility to vancomycin in vitro. A-B) Viability of bacteria (Log₁₀ CFU/mL) within plasma augmented (MHB-10% Plasma, light bars) and non-augmented (MHB, dark bars) biofilms of a methicillin resistant (MRSA 3) and methicillin susceptible (UAMS-1) strain of S. aureus following overnight exposure to vancomycin (0, 2, 16, 128, and 1024 μg/mL). Bar graphs are indicative of the mean bacterial CFU ± SD. P-values generated via One-way ANOVA with a Dunnett’s post hoc test. C-D) SEM analysis of plasma and non-plasma augmented biofilms from the isolates listed above, following exposure to vancomycin. Images were captured at 2,500X. Size bars represent 10 μm.