A Global Transcriptomic Analysis of Staphylococcus aureus Biofilm Formation Across Diverse Clonal Lineages

Abstract

A key characteristic of S. aureus infections, and one that also varies phenotypically between clones, is that of biofilm formation, which aids in bacterial persistence through increased adherence and immune evasion. Though there is a general understanding of the process of biofilm formation - adhesion, proliferation, maturation, and dispersal - the tightly orchestrated molecular events behind each stage, and what drives variation between S. aureus strains, has yet to be unraveled. Herein we measure biofilm progression and dispersal in real-time across the 5 major S. aureus CDC-types (USA100-USA500) revealing adherence patterns that differ markedly amongst strains. To gain insight into this, we performed transcriptomic profiling on these isolates at multiple time points, compared to planktonically growing counterparts. Our findings support a model in which eDNA release, followed by increased positive surface charge, perhaps drives initial abiotic attachment. This is seemingly followed by cooperative repression of autolysis and activation of PNAG production, which may indicate a developmental shift in structuring the biofilm matrix. As biofilms mature, diminished translational capacity was apparent, with 53% of all ribosomal proteins downregulated, followed by upregulation of anaerobic respiration enzymes. These findings are noteworthy because reduced cellular activity and an altered metabolic state have been previously shown to contribute to higher antibiotic tolerance and bacterial persistence. In sum, this work is, to our knowledge, the first study to investigate transcriptional regulation during the early, establishing phase of biofilm formation, and to compare global transcriptional regulation both temporally and across multiple clonal lineages. ImportanceStaphylococcus aureus is a highly virulent, opportunistic pathogen and a leading cause of both nosocomial and community acquired infections. Biofilms are associated with persistent, chronic infections and the capacity to form a biofilm varies phenotypically amongst S. aureus clonal lineages. The molecular regulation of biofilm formation has been a popular area of study for this pathogen, however, a comprehensive mapping of the regulatory processes and factors driving strain-specific variation has yet to be elucidated. This study presents transcriptomic analyses of five diverse methicillin-resistant S. aureus isolates during various stages of biofilm formation, tracked in real time. The transcriptomic profiles of all five isolates were compared to identify both core and unique networks of regulation. Importantly, much of what we currently know about biofilms is based on mature, preformed biofilm populations, with little known of the transient regulation driving attachment, proliferation, maturation, and dissemination. We address this issue by investigating transcriptional regulation during the early, establishing phase of biofilm formation, and compare global transcriptional regulation both temporally and across multiple clonal lineages. This study provides a launching point towards understanding the highly orchestrated regulation driving each phase of biofilm development and may inform on future strategies to combat biofilm-mediated infections. Data SummaryO_LIRNA sequencing results have been deposited to the NCBI Gene Expression Omnibus; GEO submission GSE163153 (url - https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE163153) C_LIO_LIOverview and comparisons of gene expression within biofilm and planktonic cell populations for the various Staphylococcus aureus strains are shown in Table S1, S2, S3. Validation and visual depiction of these data can be found as Figure S2 and S3 respectively (available in supplementary material). C_LI