Article Figures & Data
Figures
- Fig. 1.
Formation of stable atypical small colonies by GAS serotype M2 and M49. GAS serotypes M2 and M49 were incubated at 37°C in 5% CO2 for a number of days in THY broth without addition of fresh medium. At various days after exponential growth, aliquots were withdrawn, plated on THY agar, and incubated at 37°C in 5% CO2. The column indicated by “strain on THY” is representative of the colony dimorphism that was obtained 1 day after exponential growth and continued throughout stationary-phase incubation. To check for formation of stable atypical small isolates, representative colonies from the resulting THY plate were transferred to MH-SBA and incubated under identical conditions. The passage number is the number of times the small-colony isolates from the THY plates were sequentially passaged on MH-SBA. The results are representative of those obtained with at least three independently derived stabilized small colonies.
- Fig. 2.
Growth characteristics of atypical and typical serotype M49 GAS isolates. (A) The survival of passage 3 typical and stabilized small-colony isolates was determined by inoculation of THY with a stabilized small or typical colony, followed by prolonged incubation at 37°C in 5% CO2. At approximately the same time (±3 h) on the indicated days, an aliquot was removed and dilution plated on THY agar. The number of CFU per milliliter was scored after overnight incubation at 37°C in 5% CO2. (B) Growth curves of stabilized small and typical colony isolates. THY was inoculated with passage 3 stabilized small or typical colony isolates and incubated at 37°C in 5% CO2 for 13 to 15 h. The overnight culture was then diluted 1:10, and growth was followed by measuring absorbance at 600 nm.
- Fig. 3.
mRNA expression of passage 3 serotype M49-derived stabilized small and typical colony isolates. mRNA was isolated from passage 3 typical and stabilized small isolates after growth in fresh medium to late logarithmic growth phase. Total RNA concentration recovered was determined by spectrophotometric measurement at OD260, appropriate dilutions were made (indicated above each lane), and even loading of the lanes was confirmed by visual inspection of eithidium bromide-stained gels prior to transfer to membranes. The RNA gels were subsequently Northern blotted, and the binding of specific DIG-labeled PCR probes (indicated below each blot) was determined by CSPD development followed by autoradiography. Fresh RNA was prepared for each blot, and the results obtained were consistent for at least two independently derived typical and stabilized small-colony isolates. Minor bands (especially predominant with the speB probe) represent mRNAs captured by the mass of 23S rRNA. The bands detected by the individual probes were consistent with reported mRNA sizes (45).
- Fig. 4.
Production of capsule by typical, stabilized small-colony isolates and has mutants derived from serotype M49. (A) Hyaluronic acid production of passage 3 stabilized small, typical, and M49-derived capsule synthetase mutants (has−) after overnight growth in CDM was measured by the method of Blumenkrantz and Asboe-Hansen (5). Results are reported as picograms of hyaluronic acid (pgHA) per CFU. (B) Colony morphologies of the capsule synthetase mutants and the typical and stabilized small colonies after overnight growth on MH-SBA.
- Fig. 5.
mRNA expression in passage 5 serotype M49-derived stabilized small and typical colony isolates. mRNA was obtained from late-logarithmic-phase cultures inoculated with passage 5 typical and stabilized small isolates when at least 50% the plated colonies had reverted to a typical large-colony phenotype. The lanes labelled “small” denote mRNA isolated from reverted stabilized small colonies. Total RNA concentration recovered was determined by spectrophotometric measurement at OD260, appropriate dilutions were made (indicated above each lane), and even loading of the lanes was confirmed by visual inspection of ethidium bromide-stained gels prior to transfer to membranes. The RNA gels were subsequently Northern blotted, and the binding of specific DIG-labeled PCR probes (indicated at the bottom of each blot) was determined by CSPD development followed by autoradiography. The bands detected by the individual probes were consistent with reported mRNA sizes (45). Fresh RNA was prepared for each blot. The autoradiograms shown above represent the consistent results obtained for the individual typical and atypical isolates at passage 5. Similar results were obtained with a second independently derived atypical small and typical isolate at passage 9.
Tables
- Table 1.
Antibiotic sensitivity of typical and atypical small colonies
Antibiotic Sensitivity (μg/ml)a Typical Small Cefotaxime 0.12 0.08 Chloramphenicol 3.0 3.0 Clindamycin 0.19 0.094 Cotrimoxazole 0.19 0.25 Erythromycin 0.032 0.064 Gentamicin 48 32 Ofloxacin 4.0 3.0 Penicillin 0.04 0.04 Vancomycin 1.0 0.75 ↵a Determined by the E-test according to the manufacturer’s instructions.
- Table 2.
Phagocytosis sensitivitya of typical and atypical small M49-derived colonies
M49 colony type Passage no. Multiplication factor (mean ± SD) Typical 3 23.6 ± 5.1 Small 3 0.04 ± 0.035 Typical 5 20.3 ± 1.45 Small 5b 8.1 ± 2.5 ↵a Determined by the direct bactericidal assay.
↵b Approximately 50% of the colonies obtained upon plating of the passage 5 small isolate had reverted to typical large colonies. Greater than >95% of the colonies recovered after the phagocytosis resistance assay formed typical-size colonies on THY plates.
