Presentations

Abstract

Introduction:

Staphylococcus aureus, an invasive bacterial pathogen, is responsible for a plethora of disease states and a major contributor to both healthcare- and community-associated infections worldwide. S. aureus persists as a significant health threat, attributable to antibiotic resistance and immune-evasive strategies. Bacteria grown in biofilms generally exhibit increased antibiotic resistance and greater resistance to the host-immune response. Thus, the aim of the present study was to characterize the expression of select virulence-factor genes in biofilm and identify differences in the expression of these genes in biofilm vs planktonic bacteria.

Methods:

Community-associated S. aureus isolates from patients of the Children's Hospital of the King's Daughters were grown overnight for 16 hours. To develop the biofilm, cultures were incubated with 2% normal human plasma and Columbia broth + 2% NaCl in a 96-well plate. Biofilm was washed, then incubated with either ± 10% or ± 20% normal human serum (NHS) in conditions favoring immune activation. Planktonic bacteria were incubated with ± 20% NHS in borosilicate tubes. For targeted transcriptomic analysis, RNA was extracted and virulence- factor expression was measured via RT-qPCR, normalizing to a housekeeping gene (tpi). The expression fold change was assessed via the ΔΔCT method. The virulence-factor genes assessed (sdrE/bbp, clfA, scn, chp, spA) were based on gene carriage previously determined by the lab. Capsule type, phase of growth, and infection type (blood or skin and soft tissue infection), were used as group discriminators.

Results:

In biofilm, 10% NHS promoted a significant increase in the expression of sdrE alone and in combination with its allelic variant bbp, in isolates with capsule type 5 (CP5) vs capsule type 8 (CP8). Infection type and sensitivity to methicillin did not significantly affect gene expression. In biofilm vs planktonic bacteria, 20% NHS promoted a significant increase in the expression of genes encoding for members of the MSCRAMM adhesin family (sdrE, sdrE + bbp, clfA), as well as scn, a secreted complement inhibitor. Expression of each of the MSCRAMM- associated genes was increased in biofilm and decreased in planktonic bacteria; scn showed increased expression in both phases of growth.

Conclusion:

Increased expression of MSCRAMM-associated genes in biofilm and an associated decreased expression in planktonic bacteria in response to NHS highlights the importance of MSCRAMMs within biofilm and their protective role against host immunity. Overall, these data demonstrate that S. aureus exhibits a differential transcriptional dynamic based on growth phase; the characterization of such supports the development of anti- staphylococcal targeted therapies.