title: Formation of Streptococcus pneumoniae non-phase-variable colony variants is due to increased mutation frequency present under biofilm growth conditions.
abstract: In this report, we show that biofilm formation by Streptococcus pneumoniae serotype 19 gives rise to variants (the small mucoid variant [SMV] and the acapsular small-colony variant [SCV]) differing in capsule production, attachment, and biofilm formation compared to wild-type strains. All biofilm-derived variants harbored SNPs in cps19F. SCVs reverted to SMV, but no reversion to the wild-type phenotype was noted, indicating that these variants were distinct from opaque- and transparent-phase variants. The SCV-SMV reversion frequency was dependent on growth conditions and treatment with tetracycline. Increased reversion rates were coincident with antibiotic treatment, implicating oxidative stress as a trigger for the SCV-SMV switch. We, therefore, evaluated the role played by hydrogen peroxide, the oxidizing chemical, in the reversion and emergence of variants. Biofilms of S. pneumoniae TIGR4-DeltaspxB, defective in hydrogen peroxide production, showed a significant reduction in variant formation. Similarly, supplementing the medium with catalase or sodium thiosulfate yielded a significant reduction in variants formed by wild-type biofilms. Resistance to rifampin, an indicator for mutation frequency, was found to increase approximately 55-fold in biofilms compared to planktonic cells for each of the three wild-type strains examined. In contrast, TIGR4-DeltaspxB grown as a biofilm showed no increase in rifampin resistance compared to the same cells grown planktonically. Furthermore, addition of 2.5 and 10 mM hydrogen peroxide to planktonic cells resulted in a 12- and 160-fold increase in mutation frequency, respectively, and gave rise to variants similar in appearance, biofilm-related phenotypes, and distribution of biofilm-derived variants. The results suggest that hydrogen peroxide and environmental conditions specific to biofilms are responsible for the development of non-phase-variable colony variants.
authors.affiliation: Department of Biological Sciences, Binghamton University, Binghamton, NY 13902, USA.
authors.forename: Magee
authors.initials: M
authors.lastname: Allegrucci
authors.forename: Karin
authors.initials: K
authors.lastname: Sauer
mesh_terms: D018441:Biofilms / Q000187:drug effects / Q000254:growth & development*; D002374:Catalase / Q000494:pharmacology; D005838:Genotype; D006861:Hydrogen Peroxide / Q000378:metabolism / Q000494:pharmacology; D018613:Microscopy, Confocal; D009154:Mutation; D010641:Phenotype; D020641:Polymorphism, Single Nucleotide; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013296:Streptococcus pneumoniae / Q000235:genetics / Q000378:metabolism / Q000502:physiology*; D013752:Tetracycline / Q000494:pharmacology; D013885:Thiosulfates / Q000494:pharmacology; D014031:Tobramycin / Q000494:pharmacology; D014158:Transcription, Genetic / Q000187:drug effects
chemical_list: D013885:Thiosulfates; D006861:Hydrogen Peroxide; D002374:Catalase; D013752:Tetracycline; C017717:sodium thiosulfate; D014031:Tobramycin