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. 2005 May;187(10):3319-28.
doi: 10.1128/JB.187.10.3319-3328.2005.

Persistence of Streptococcus pyogenes in stationary-phase cultures

Daniel N Wood  1 , Michelle A Chaussee, Michael S Chaussee, Bettina A Buttaro
Affiliations

Persistence of Streptococcus pyogenes in stationary-phase cultures

Daniel N Wood et al. J Bacteriol. 2005 May.

Abstract

In addition to causing fulminant disease, Streptococcus pyogenes may be asymptomatically carried between recurrent episodes of pharyngitis. To better understand streptococcal carriage, we characterized in vitro long-term stationary-phase survival (>4 weeks) of S. pyogenes. When grown in sugar-limited Todd-Hewitt broth, S. pyogenes cells remained culturable for more than 1 year. Both Todd-Hewitt supplemented with excess glucose and chemically defined medium allowed survival for less than 1 week. After 4 weeks of survival in sugar-limited Todd-Hewitt broth, at least 10(3) CFU per ml remained. When stained with fluorescent live-dead viability stain, there were a number of cells with intact membranes that were nonculturable. Under conditions that did not support persistence, these cells disappeared 2 weeks after loss of culturability. In persistent cultures, these may be cells that are dying during cell turnover. After more than 4 weeks in stationary phase, the culturable cells formed two alternative colony phenotypes: atypical large colonies and microcolonies. Protein expression in two independently isolated microcolony strains, from 14-week cultures, was examined by use of two-dimensional electrophoresis. The proteomes of these two strains exhibited extensive changes compared to the parental strain. While some of these changes were common to the two strains, many of the changes were unique to a single strain. Some of the common changes were in metabolic pathways, suggesting a possible alternate metabolism for the persisters. Overall, these data suggest that under certain in vitro conditions, S. pyogenes cells can persist for greater than 1 year as a dynamic population.

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Figures

FIG. 1.
FIG. 1.
The stationary-phase survival kinetics of four strains of S. pyogenes. Four different strains of S. pyogenes were inoculated into TH broth and monitored for exit from exponential phase (T0). Stationary-phase culture aliquots were then removed, serially diluted in sterile TH broth, and plated on TH agar plates to determine the number of CFU per milliliter. Bars in the graph represent the mean number of CFU per milliliter for each time point. Each data set is the mean of results from at least two independent experiments, quantitated in duplicate. Error bars represent the standard deviation between these experiments.
FIG. 2.
FIG. 2.
The effects of pH decreases on S. pyogenes survival time. S. pyogenes M49-CS101 was inoculated into TH broth, grown to stationary phase, and aged 1 week. Exogenous HCl or lactic acid was then added from 2 M stocks to final concentrations of 25.0, 12.5, or 6.3 mM in these aged cultures. Survival was assayed by spotting culture aliquots on TH agar plates at 24-h time intervals after addition of exogenous acid. The formation of any S. pyogenes colonies within the culture spot was scored positive for survival. Since 20 μl of culture was plated to assay for survival, the lower limit of detection for this assay was 50 CFU ml−1. Bars in the graph represent the mean duration of survival for each culture condition. For this graph, T0 is at the point of addition of exogenous acid to 1-week-old cultures. The terminal pH range, after addition of exogenous acid, is noted next to each culture condition. Each data set is the mean of results from eight independent experiments. Error bars represent the standard deviation between these experiments.
FIG. 3.
FIG. 3.
Phenotypic variants of surviving, stationary-phase S. pyogenes. S. pyogenes M49-CS101 was inoculated into TH broth, grown to stationary phase, and aged approximately 8 weeks. Culture aliquots were then removed, serially diluted, and plated on TH agar plates. S. pyogenes M49-CS101 microcolonies and alternative large colonies from the 8-week culture are pictured in panel B, while the early-stationary-phase (<12 h) precursor colonies are pictured in panel A. On plate medium, early-stationary-phase M49-CS101 cells grow with a dense center, yielding colonies with a “fried egg” appearance. These pictures are scaled relative to one another.
FIG. 4.
FIG. 4.
mRNA expression of typical M49-CS101 colonies and stationary-phase-derived microcolony phenotypes Alt. 1 and Alt. 2. mRNA was isolated from mid-exponential-phase cells (OD600 of 0.50 to 0.65) grown in TH broth. The RNA concentration was determined by spectrophotometric measurement at OD260, appropriate dilutions were made, and RNA from each strain was run on a denaturing agarose gel. Equal loading was confirmed visually by ethidium bromide staining. RNA concentrations are noted above the lanes in each image. These RNA gels were subsequently Northern blotted, and the binding of DNA probes, generated by PCR-mediated digoxigenin incorporation, was determined by CSPD development followed by autoradiography. Specific probes are indicated beside each blot. Fresh RNA was isolated for each experiment, and the results presented here are representative of those from two independently derived M49-CS101, Alt. 1, and Alt. 2 RNA preparations.
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