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.

FIG. 1.

The emergence of S. pneumoniae serotype 19 variants during biofilm development. Distribution of variants was determined from total CFU, colony size, and mucoidy of colonies on blood agar.

FIG. 2.

Phenotypic and genotypic characteristics of S. pneumoniae serotype 19 wild-type and biofilm-derived variants. (A) Hyperautoaggregation of serotype 19 SCVs in liquid culture. The autoaggregative phenotype was not detected in serotype 19 wild-type (WT) liquid culture. Cultures were inoculated from a blood agar plate and were grown to mid-log phase. (B) Relative hydrophobicity (MATH) results for WT and SCV. Each datum point is based on the results from three replicate measurements. SCVs were significantly (**) more hydrophobic than WT (P < 0.05). (C) Microtiter plate adhesion assay of S. pneumoniae serotype 19 WT and colony variants after 3, 6, and 12 h of initial attachment. The SCV showed significantly (**) greater biofilm adhesion after 12 h than did the WT, MMV, or SMV (P < 0.05). (D) Relative capsule amounts of S. pneumoniae WT and colony variants as determined by using Stains-All reagent. Significantly (**) smaller capsule amounts were detected for SCVs than for wild-type and mucoid variants (P < 0.05). (E) Serotype 19-specific capsule operon and transcript abundance. Open squares indicate transcribed genes; striped squares indicate genes not detected by RT-PCR; filled squares, unknown, as previously described (4). (F) cps19F transcripts (267 bp) determined by RT-PCR were obtained from serotype 19 wild-type and colony variants. Lanes: 1, S. pneumoniae serotype 19 wild type [BS75]; 2, MMV; 3, SMV; 4, SCV; M, 1-kb DNA ladder.

FIG. 3.

Tetracycline treatment and S. pneumoniae SCV biofilm viability. SCV biofilms after 6 days of growth (A) and after treatment with tetracycline (10 μg/ml) for 12 h (B). Flow cell experiments and image acquisition were performed as previously described (2).

FIG. 4.

Role of the suicide gene spxB, catalase, and sodium thiosulfate in the emergence of variants in S. pneumoniae biofilms. (A) Distribution of colony variants in 1-day-old TIGR4 and TIGR4 spxB mutant biofilms. (B and C) Distribution of colony variants in biofilms of S. pneumoniae serotype 3 (BS71) (B) and serotype 19 (BS75) (C) grown for 1 day in the presence of sodium thiosulfate (10 mM) or catalase (4 U/ml; added every 4 h to the growth medium). Distribution of colony variants was determined from total CFU, colony size, and mucoidy of colonies on blood agar. Untreated biofilms were used as controls. Experiments were carried out in triplicate. Results with respect to the emergence of SCVs were significantly (**) different from those seen with untreated S. pneumoniae wild-type biofilms (P < 0.01).

FIG. 5.

Effect of growth conditions and exogenous hydrogen peroxide on the spontaneous mutation rate. The results represent the frequency of the spontaneous rate of mutation to rifampin resistance or sensitivity expressed as a mutation rate (rifampin resistant/rifampin sensitive [Rif^r/Rif^s]) for S. pneumoniae serotype 3, serotype 19, TIGR4, and TIGR4-ΔspxB on the basis of growth conditions (1-day-old biofilm, black bars; planktonic growth conditions, gray bars) (A) and the presence of 0 to 10 mM hydrogen peroxide (S. pneumoniae serotype 3, gray bars; S. pneumoniae serotype 19, striped bars) (B). For planktonic growth conditions, the frequency of the spontaneous rate of mutation to rifampin resistance or sensitivity in the absence or presence of hydrogen peroxide was calculated following growth to the mid-log phase and treatment for 60 min with H₂O₂ added at the concentration indicated. The frequency of spontaneous mutation of biofilms was calculated following 1 day of growth under biofilm growth conditions. Experiments were performed in triplicate for each set of conditions.

FIG. 6.

The emergence of S. pneumoniae serotype 3 (A) and serotype 19 (B) variants upon treatment with hydrogen peroxide under planktonic conditions. Distribution of colony variants was determined from total CFU, colony size, and mucoidy of colonies on blood agar.