Streptococcal toxic shock syndrome (STSS) is a re-emerging infectious disease in Japan and many other developed countries. Epidemiological studies have revealed that the M1 serotype of Streptococcus pyogenes is the most dominant causative isolate of STSS. Recent characterization of M1 isolates revealed that the mutation of covS, one of the two-component regulatory systems, plays an important role in STSS by altering protein expression. We analyzed the M1 S. pyogenes clinical isolates before or after 1990 in Japan, using two-dimensional gel electrophoresis (2-DE) and pulsed-field gel electrophoresis (PFGE). PFGE profiles were different between the isolates before and after 1990. Markedly different profiles among isolates after 1990 from STSS and pharyngitis patients were detected. Sequence analysis of two-component regulatory systems showed that covS mutations were detected not only in STSS but also in three pharyngitis isolates, in which proteins from the culture supernatant displayed the invasive type. The mutated CovS detected in the pharyngitis isolates had impaired function on the production of streptococcal pyrogenic exotoxin B (SpeB) analyzed by 2-DE. These results suggest that several covS mutations that lead to the malfunction of the CovS protein occurred even in pharyngeal infection.

The administration of high-dose clindamycin plus benzylpenicillin has been recommended for the treatment of streptococcal toxic shock-like syndrome caused by Streptococcus pyogenes, and clindamycin has been found to be more effective than beta-lactams in retrospective analyses of human cases. Although therapeutic doses of clindamycin have also been shown to be effective against experimental infections and clindamycin has great efficacy against the production of bacterial exoproteins, we recently reported that the level of production of some exoproteins was unchanged or even increased by a subinhibitory dose of clindamycin when it is added upon the initiation of bacterial culture and the treated cultures were analyzed by two-dimensional gel electrophoresis. In this study we further examined the effect of clindamycin on the production of exoproteins by adding it to Streptococcus pyogenes cultures during various growth phases. We found that the levels of production of some proteins, NAD ؉ glycohydrolase, streptolysin O, and streptococcal inhibitor of complement, were increased when clindamycin was added at early-log-phase growth, which was the result that was seen when clindamycin was added at the beginning of culture. However, clindamycin inhibited the production of most types of proteins when it was administered to Streptococcus pyogenes cultures at mid-log-phase growth. In csrS-or mga-knockout bacterial strains, the increase in exoproteins seen in parental strains was considerably inhibited. Our study indicates that the in vitro effect of clindamycin on the production of exoproteins greatly depends on the growth phase of bacteria and some regulatory factors of Streptococcus pyogenes that are involved in this phenomenon.Streptococcus pyogenes is a gram-positive bacterium that is one of the most common agents of upper respiratory tract infections, especially the acute pharyngitis that occurs mainly in children. It is also responsible for poststreptococcal diseases such as rheumatic fever and glomerulonephritis, in addition to increasing numbers of invasive infections, such as streptococcal toxic shock-like syndrome (TSLS), necrotizing fasciitis, bacteremia, and multiple-organ failure (15,20,23).Because S. pyogenes is exquisitely susceptible to ␤-lactam antibiotics, benzylpenicillin (PCG) has been recommended for the treatment of most S. pyogenes infections. However, it has been reported that PCG has reduced efficacy against aggressive infections like TSLS, which is a condition in which large numbers of organisms are present. Inocula with large numbers of organisms reach the stationary phase of growth quickly, and PCG is less effective against slowly growing organisms (22, 24). Moreover, certain penicillin-binding proteins have been shown not to be expressed by S. pyogenes during the stationary phase (24). Conversely, regarding the inhibition of protein synthesis, several investigators have demonstrated that clindamycin (CLI) suppresses the production of a variety of toxins from S. pyogenes (3,13,19,21,22). CLI als...

The administration of high-dose clindamycin (CLI) along with penicillin is recommended for the treatment of streptococcal toxic shock syndrome. However, the prevalence of CLI-resistant Streptococcus pyogenes strains is increasing worldwide, and the effect of CLI on CLI-resistant S. pyogenes strains remains unknown. We aimed to evaluate the effect of CLI on the in vitro production of three major virulent exoproteins, namely, streptolysin O (Slo), NAD glycohydrolase (Nga), and streptokinase (Ska), by CLI-resistant S. pyogenes strains. After the incubation of M1 serotype CLI-resistant S. pyogenes D2TY in the presence of 1 g/ml CLI, the amounts of Slo, Nga, and Ska and the levels of slo, nga, and ska mRNA in the supernatant were analyzed by Northern blotting and Western blotting, respectively. The results of both assays showed that the production of Slo, Nga, and Ska was higher with CLI treatment than without CLI treatment. We evaluated the role of the sensor kinase CovS, which is involved in the two-component system of S. pyogenes, in the CLI-induced production of these three exoproteins. Northern blotting analysis revealed that CLI induced the expression of covS mRNA in wild-type strain D2TY. Furthermore, both Northern blotting and Western blotting analyses showed that CLI decreased the levels of expression of Slo, Nga, and Ska in isogenic covS mutant D2TYcovS. These results suggest that CLI increases the production of three virulent exoproteins in CLI-resistant S. pyogenes strains via the action of CovS.Streptococcus pyogenes is a gram-positive bacterium that is responsible for pharyngitis and postinfection diseases such as rheumatic fever and glomerulonephritis. In addition, S. pyogenes causes streptococcal toxic shock syndrome (STSS) (7).The management of STSS requires intensive antibiotic treatment, and the present consensus regarding the antibiotic treatment of STSS is the administration of a high dose of clindamycin (CLI) together with penicillin (16). CLI is a lincosamide that binds to the 50S subunit of the bacterial ribosome and inhibits protein synthesis (25). With regard to the inhibition of protein synthesis, it has been shown that the synthesis of several streptococcal exoproteins, including virulence factors (e.g., SpeA, SpeB, and M protein), is inhibited by CLI at sub-MICs (6,18,21). Another reason for the use of CLI is that it transfers to tissues well (24).In recent years, macrolide-and lincosamide-resistant S. pyogenes strains have gradually spread worldwide (15,20,23). Although the prevalence of macrolide-and lincosamide-resistant S. pyogenes strains has been well investigated, the effects of these drugs on macrolide-and lincosamide-resistant S. pyogenes strains has not been studied. We have previously shown by using two-dimensional gel electrophoresis that the production of streptolysin O (Slo), NAD glycohydrolase (Nga), and streptokinase (Ska) in cultures of S. pyogenes is increased by treatment with CLI at sub-MICs (24). In addition, we examined the role of the two-component sensor kinase, Co...