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Journal of Bacteriology, October 2008, p. 6330-6339, Vol. 190, No. 19
0021-9193/08/$08.00+0     doi:10.1128/JB.00707-08
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

Formation of Streptococcus pneumoniae Non-Phase-Variable Colony Variants Is Due to Increased Mutation Frequency Present under Biofilm Growth Conditions{triangledown} ,{dagger}

Magee Allegrucci and Karin Sauer*

Department of Biological Sciences, Binghamton University, Binghamton, New York 13902

Received 19 May 2008/ Accepted 20 July 2008

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-{Delta}spxB, 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-{Delta}spxB 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.

* Corresponding author. Mailing address: Department of Biological Sciences, Binghamton University, State University of New York at Binghamton, 148 Science III, Vestal Parkway East, Binghamton, NY 13902. Phone: (607) 777-3157. Fax: (607) 777-6521. E-mail: ksauer{at}binghamton.edu

{triangledown} Published ahead of print on 25 July 2008.

{dagger} Supplemental material for this article may be found at http://jb.asm.org/.

Journal of Bacteriology, October 2008, p. 6330-6339, Vol. 190, No. 19
0021-9193/08/$08.00+0     doi:10.1128/JB.00707-08
Copyright © 2008, American Society for Microbiology. All Rights Reserved.





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