Authors: Mark Smeltzer1, Karen Beenken1, Paul Dunman2, Fionnuala McAleese2, Ellen Murphy2 and Steve Projan2
Title: Differential Gene Expression in Staphylococcus aureus biofilms
Addresses: University of Arkansas for Medical Sciences, Little Rock, AR1 and Wyeth-Ayerst Pharmaceuticals, Pearl River, NY2
Purpose: Staphylococcus aureus is the preeminent musculoskeletal pathogen. Its preeminence is based in part on its ability to form a biofilm, which consists of multiple layers of bacteria encased within a polysaccharide glycocalyx. This glycocalyx protects the bacterium from host defenses and impedes antibiotic therapy. Understanding how the biofilm forms and the nature of the adaptations required for the staphylococci to persist within a biofilm has the potential to both identify novel targets for the development of new therapeutic agents and to increase the efficacy of existing antimicrobials.
Methods: The strain used in these studies (UAMS-1) was isolated from the bone of a patient suffering from osteomyelitis. Using a murine model of catheter-based biofilm formation, we have confirmed that UAMS-1 forms a biofilm in vivo. We have also confirmed that UAMS-1 is virulent in our animal models of musculoskeletal infection. RNA isolated from UAMS-1 grown in planktonic culture or from cells harvested from flow-cell grown biofilms. This RNA was used for genome-scale transcriptional profiling using a comprehensive Affymetrix GeneChip® that includes oligonucleotides representing 4,380 open-reading frames identified in the genome sequence of six S. aureus strains (COL, 8325, EMRSA-16, MSSA-476, N315 and Mu50). Each comparison was done with two different RNA preparations, and each RNA preparation was used to hybridize two chips. Genes that were induced genes were defined as those that exhibited a three-fold increase in expression by comparison to both exponential and post-exponential phase planktonic cultures. Genes that were repressed in biofilms were defined as those that exhibited no more that 33% of the expression level observed in both exponential and post-exponential phase cultures.
Results and Discussion: We identified 44 genes or operons that were induced in biofilms and 49 genes or operons that were repressed. Included among the induced genes were icaA and icaD, both of which are required for S. aureus to produce the polysaccharide intercellular adhesion (PIA) commonly associated with biofilm formation in both S. aureus and S. epidermidis. Other genes of interest include clpB, adhE, ureB and arcC. Identification of clpB, adhE and ureB is important because they were also among the genes identified as induced in biofilms using other methods and/or other bacterial species. Similarly, arcC is among the genes previously found to be regulated in a cell-density dependent manner. It is also important to note that our experiments also identified other genes within the same operons (e.g. ureA, ureC, ureD, ureE and arcA, arcB and arcD). Taken together, these results support the conclusion that our comparisons accurately reflect the changes in gene expression associated with the growth of S. aureus within a biofilm.
Significance: An understanding of how S. aureus forms a biofilm and the adaptive changes required to support the sessile lifestyle associated with biofilms will facilitate the development of therapeutic strategies capable of limiting biofilm formation and thereby increasing the ability to resolve musculoskeletal infections.