Identification and Characterization of Genes Involved in Biofilm Growth and Antibiotic Tolerance in Streptococcus pyogenes

Abstract

Group A Streptococcus (GAS; Streptococcus pyogenes ) causes an array of diseases of varying severity resulting in over 500,000 deaths annually. GAS is invariably susceptible to penicillin in vitro , but treatment failures still occur. This microbial pathogen has been previously shown to form biofilms (defined as complex microbial communities that adhere to a surface, secrete an extracellular matrix, and demonstrate tolerance to antimicrobial agents). However, there is a paucity of data regarding the specific role of this phenotype in GAS pathogenesis. In preliminary studies, we found that the GAS biofilm mode of growth was tolerant to antibiotics when compared to their planktonic counterparts in vitro and in a mouse model of GAS infection. To identify the gene products that are involved in biofilm growth and antibiotic tolerance in GAS, planktonically-grown bacteria were compared to bacteria grown in an in vitro biofilm. An unbiased global transcriptomic, proteomic, and immunoproteomic approach identified differentially-regulated genes and proteins that may contribute to biofilm growth in S. pyogenes. Among the proteins highly up-regulated during biofilm growth were those within the arc operon, which is important for maintaining pH homeostasis in response to acid stress. Further investigation into the function of the arc operon through the use of insertion mutants revealed the operon to have a biofilm-specific role both in growth and susceptibility to antibiotics. Elimination of Arc protein production resulted in a return of penicillin sensitivity of GAS biofilms in a pH-dependent manner in vitro , but did not alter penicillin susceptibility in planktonic culture. This return to penicillin sensitivity was also apparent in a mouse model of nasopharyngeal infection, demonstrating that the biofilm phenotype, and specifically the arc operon, could play a key role in the clinical antibiotic treatment failure observed with GAS. These studies are the first to: (1) show recalcitrance of antimicrobial therapy in a relevant GAS infection model, (2) use the combined global approaches of RNAseq, LC-MS/MS, and immunoproteomics to study microbial biofilms, (3) identify key factors in the antibiotic tolerance in GAS biofilms both in vitro and in vivo , and (4) to readily demonstrate a potential cause of clinical recalcitrance of GAS to clearance by antimicrobial agents.