Survival of Group A Streptococcus (GAS) is Enhanced Under Desiccated Culture Conditions

Abstract

Streptococcus pyogenes or Group A Streptococcus (GAS) infections are the leading cause of bacterial tonsillopharyngitis. The bacterium can survive and persist within the human host for a long time as it is observed in up to 40% of the population who are considered as carriers. Recurrent tonsillopharyngitis is a particular problem in children which is caused either by relapses due to failed bacterial clearance or by reinfection. A prolonged survival in tonsillar crypts or on inanimate surfaces might be sources for reinfection. We therefore examined 64 clinical GAS isolates from children with tonsillopharyngitis for their long-term survival under either liquid or desiccated culture conditions. After 6 weeks, the overall GAS survival rate was 400-fold increased under desiccated culture conditions compared to liquid culture conditions, but varied depending on the emm-type between 20-fold (emm4) and 14000-fold (emm3). The survival rates of isolates from emm75 were significantly lower which is probably due to their production of hydrogen peroxide up to fatal doses. No hydrogen peroxide production could be detected for other emm-types. Furthermore, 11 isolates from patients with recurrent tonsillopharyngitis were compared to isolates of the same emm-type from patients with single episodes of tonsillopharyngitis. A significant elevated pH value and an increased survival rate for isolates from patients with recurrent infections were observed. In conclusion, significant differences in long-term survival of different GAS isolates as well as survival under desiccated culture conditions might contribute to both failed bacterial clearance and reinfection in patients with recurrent tonsillopharyngitis.

Fig. 1

Stationary-phase survival kinetics of different clinical GAS isolates in BHI. Overall, 64 clinical GAS isolates of 8 different emm-types were grown in BHI until stationary phase (day 0). Further cultivation was performed for another 6 weeks: CFU were determined on Columbia blood agar plates at time points day 0, day 1, day 7 and day 42. Dots in the graph represent the mean of three experiments per isolate, whereas the bars represent the mean per emm-type: a significant effect over time (p < 0.0001) was proven by two-way analysis of variance. A significantly decline was observed for emm75-isolates (Multiple comparisons by Kruskal–Wallis test)

Fig. 2

Effect of desiccation on the stationary-phase survival kinetics of GAS isolates. Overall, 64 clinical GAS isolates were grown in BHI. Culture aliquots of each isolate were transferred to wells and desiccated (black bar) or further cultivated in BHI (gray bar). The mean and standard deviation of CFU is depicted from three experiments per isolate. Statistical analysis was performed by Wilcoxon signed rank test: ***P < 0.001

Fig. 3

Stationary-phase survival kinetics of different clinical GAS isolates after desiccation. Overall, 64 clinical GAS isolates of 8 different emm-types were grown in BHI until stationary phase (day 0). The cultures were air dried under sterile conditions and the CFU were determined on Columbia blood agar plates over a period of 6 weeks at time points day 0, day 1, day 7 and day 42. Dots in the graph represent the mean of three experiments per isolate, whereas the bars represent the mean per emm-type: a significant effect over time (P < 0.0001) was proven by two-way analysis of variance. A significantly decline was observed for emm75-isolates (Multiple comparisons by Kruskal–Wallis test)

Fig. 4

Increased pH values and long-term survival rates of GAS isolates from patients with recurrent tonsillopharyngitis. Long-term survival rates in BHI of 11 GAS isolates of patients with recurrent tonsillopharyngitis (gray bars) compared to GAS isolates of the same emm-type from patients with single episodes of tonsillopharyngitis (white bars). Values for CFU and culture pH represent the mean and standard deviation of three experiments per isolate at time points day 0, day 1, day 7 and day 42. The pH values in the culture of GAS isolated from patients with recurrent tonsillopharyngitis (black circle) and those from patients with single episodes of tonsillopharyngitis (white circle) differ significantly at all time points: ***P < 0.0001. Statistical analysis was performed by Mann—Whitney test