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. 2004 Oct;70(10):6181-7.
doi: 10.1128/AEM.70.10.6181-6187.2004.

Persistence of Streptococcus mutans in stationary-phase batch cultures and biofilms

John A Renye Jr  1 , Patrick J Piggot, Lolita Daneo-Moore, Bettina A Buttaro
Affiliations

Persistence of Streptococcus mutans in stationary-phase batch cultures and biofilms

John A Renye Jr et al. Appl Environ Microbiol. 2004 Oct.

Abstract

Streptococcus mutans is a member of oral plaque biofilms and is considered the major etiological agent of dental caries. We have characterized the survival of S. mutans strain UA159 in both batch cultures and biofilms. Bacteria grown in batch cultures in a chemically defined medium, FMC, containing an excess of glucose or sucrose caused the pH to decrease to 4.0 at the entry into stationary phase, and they survived for about 3 days. Survival was extended up to 11 days when the medium contained a limiting concentration of glucose or sucrose that was depleted by the time the bacteria reached stationary phase. Sugar-limited cultures maintained a pH of 7.0 throughout stationary phase. Their survival was shortened to 3 days by the addition of exogenous lactic acid at the entry into stationary phase. Sugar starvation did not lead to comparable survival in biofilms. Although the pH remained at 7.0, bacteria could no longer be cultured from biofilms 4 days after the imposition of glucose or sucrose starvation; BacLight staining results did not agree with survival results based on culturability. In both batch cultures and biofilms, survival could be extended by the addition of 0.5% mucin to the medium. Batch survival increased to an average of 26 (+/-8) days, and an average of 2.7 x 10(5) CFU per chamber were still present in biofilms that were starved of sucrose for 12 days.

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Figures

FIG. 1.
FIG. 1.
Survival of S. mutans UA159 in batch cultures containing an excess or limiting concentration of glucose. S. mutans grown in FMC plus 6 mM glucose (filled rectangles) or THB (filled triangles) was in a glucose-limiting medium, and bacteria grown in FMC plus 100 mM glucose (open rectangles) or THB plus 100 mM glucose (open triangles) were in a medium with excess glucose. Samples were taken after entry into stationary phase, serially diluted, and plated on TH agar. The minimum level of detection for this experiment was 10 CFU ml−1. The results are from one experiment and are representative of three independent experiments.
FIG. 2.
FIG. 2.
S. mutans UA159 biofilm development in the presence of glucose or sucrose. Flow chambers were used to grow biofilms as previously described (27). Flow chambers were maintained in parallel, with chambers removed for imaging after 2, 7, 14, and 24 h of growth in FMC containing glucose or sucrose. The biofilms were stained with the BacLight viability stain and were imaged by confocal scanning laser microscopy. Stack images were created from individual slices from both the SYTO 9 and PI channels by use of the Olympus Fluoview system. Images were converted to a gray scale in Adobe Photoshop 7.0. Mature biofilms in the presence of sucrose were observed to contain both isolated (24 h, top) and clumped (24 h, bottom) microcolonies. Bar, 20 μm for the 2-h images and 50 μm for the 7- to 24-h images. The images are representative of two independent experiments.
FIG. 3.
FIG. 3.
BacLight staining of S. mutans UA159 in a biofilm after 5 days of sucrose starvation. The biofilm was established for 16 h in FMC with 3 mM sucrose, after which the medium reservoir was replaced with biofilm starvation medium. The biofilm was maintained in the starvation medium for 5 days and then stained with BacLight and imaged by confocal scanning laser microscopy. Individual sections were collected and compiled to create a maximum projection image by use of the Olympus Fluoview system. The image is representative of three independent experiments.
FIG. 4.
FIG. 4.
Growth of S. mutans UA159 in FMC containing 6 mM glucose and 0.5% mucin in batch cultures. Growth curves are shown for S. mutans UA159 in FMC plus 6 mM glucose (diamonds), FMC plus 6 mM glucose and 0.5% mucin (squares). An overnight culture grown in FMC with 24 mM glucose was diluted 25-fold into fresh medium, and growth was assayed by measuring the CFU on TH agar (A) or OD675 (B). The growth curves measured by optical densities and CFU were from independent experiments. The data shown are the averages of three experiments ± standard deviations.
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