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Streptococcal toxic shock syndrome (STSS) is primarily caused by Streptococcus pyogenes, but it may also be caused by Streptococcus dysgalactiae subsp. equisimilis (SDSE). The analyses of S. pyogenes have revealed the important roles of NAD + -glycohydrolase (Nga) and CovR/CovS, a two-component regulatory system. We examined these factors in SDSE by analyzing mainly two isogenic SDSE strains (12-10-1 and 12-10-3) from the blood of a patient with STSS. The Nga activities were measured and the nucleotide sequences of covR and covS genes were determined. We detected one nucleotide difference between the covR gene of 12-10-1 and that of 12-10-3, and the Nga activity of 12-10-1 was approximately 6.8-fold more than that of 12-10-3. The introduction of covR of 12-10-3 into 12-10-1 significantly reduced the Nga activity, but the introduction of 12-10-1 covR into itself had only a little effect. In addition, the knockout of covR or covS of 12-10-3 remarkably increased the Nga activity. We are the first to report that strains with wild-type and mutated covR were isolated simultaneously from an SDSE STSS patient and that the CovR/CovS two-component regulatory system is involved in the Nga activity in SDSE as well as in S. pyogenes., (© 2019 APMIS. Published by John Wiley & Sons Ltd.)

Although mef(A) and its subclass mef(E) genes have long been considered to play a central role in macrolide efflux-based resistance, we have previously demonstrated that the msr(D) gene located immediately downstream of the mef(A) gene plays a predominant role in Streptococcus pyogenes macrolide resistance. The mef(A) and mef(E) genes are carried by different genetic elements and the resistance associated with mef(A) was reported to be higher than that associated with mef(E); therefore, we further investigated the functional relevance of mef(A)/mef(E) and its associated msr(D). We established additional mef(A)-, mef(E)-, and their associated msr(D)-knockout strains and confirmed the predominance of msr(D) over mef(A)/mef(E). In addition, we performed experiments introducing mef(A), mef(E), and their associated msr(D) genes into mef(A)/mef(E)-msr(D) double-knockout and mef(A)/mef(E) negative strains. Neither mef(A) nor mef(E) genes had effects on erythromycin resistance. However, both associated msr(D) showed significant effects, and the mef(A)-associated msr(D) exhibited more effect than the mef(E)-associated one. These results suggest that an overall functional predominance of msr(D) over mef(A)/mef(E) is conceivable in efflux-based macrolide resistance in at least some S. pyogenes strains. Furthermore, the higher resistance of mef(A) system over mef(E) system could be derived at least in part from functional differences of mef(A)- and mef(E)-associated msr(D).

In Japan, the number of patients with streptococcal toxic shock syndrome is reported to be increasing. mef(A) gene-positive macrolide-resistant emm1 strains are thought to possibly contribute to the rise in the frequency of STSS. Although analyses of macrolide-resistant mechanisms, including mef(A) resistance, have been performed mainly in Streptococcus pneumoniae, the role of this gene in Streptococcus pyogenes has not been completely investigated. Therefore, to the best of our knowledge, we established the first mef(A)-knockout strain using an emm1-type S. pyogenes strain, and tested its susceptibility to erythromycin, clarithromycin and azithromycin. We found that the antimicrobial susceptibilities were almost identical to those of the parental strain. Hence, we established a knockout strain for another gene, msr(D), that is located immediately downstream of mef(A). The macrolide resistances of the resulting strain significantly decreased, and were further altered when both mef(A) and msr(D) were knocked out. The introduction of the msr(D) gene into a macrolide-sensitive strain conferred more resistance than the introduction of the mef(A) gene. The erythromycin susceptibilities of knockout strains were further dissected using two additional emm4- and emm75-type S. pyogenes strains. We found almost identical results for both strains except for the mef(A) knockout emm4 type, whose susceptibility was altered, although the change was less than that for the msr(D) knockout. These results suggest that both mef(A) and msr(D) are involved in macrolide resistance in S. pyogenes, and that the msr(D) gene plays a more predominant role in macrolide resistance than mef(A).

Streptococcus dysgalactiae subsp. equisimilis isolates (n = 110) were analyzed by PCR to determine whether the gene encoding SICG, a homolog of Streptococcus pyogenes SIC, was present. Nineteen strains (17%) had this gene of which 11 (55%) were isolated from patients with invasive disease. All 19 strains possessed group G carbohydrate. Molecular characterization of emm type revealed that the majority of emm sequences were stG643 and stG2078. Only the N-terminal sequence of SICG was similar to that of SIC in S. pyogenes. Although we found no significant relationship between pathogenic severity and sicG possession, further investigation into the mechanism of SICG may elucidate the virulence in S. dysgalactiae subsp. equisimilis infection.

The molecular mechanism of high level tetracycline resistance in T serotypes 4 and 11 group A streptococcal (GAS) isolates was examined in 61 tetracycline-resistant isolates in Japan. PCR and sequencing analyses revealed that the T serotype/emm genotype, T4/4 isolates carried tet(O) genes, which were genetically homogenous. The T11/11 and T11/89 isolates carried different subtypes of tet(M) genes, which were present on transposons Tn916 and Tn1545, respectively. In addition, these T11 isolates may have obtained the tet(M) gene after the 1990s, because resistance to tetracycline in T11 isolates was rarely found before then. These results strongly suggested that the T4 and T11 GAS isolates acquired tetracycline-resistance via different molecular mechanisms.

Selection of new variants of the streptococcal inhibitor of complement protein has been implicated in the perpetuation of epidemics caused by serotype M1 strains of group A Streptococcus (GAS). The frequency at which new streptococcal inhibitor of complement (Sic) variants arise in an infected individual is not known. To study this issue, the sic gene was sequenced in 100 isolates cultured from throat swabs of each of 20 patients with acute pharyngitis caused by serotype M1 GAS. Five patients were infected with GAS populations expressing 2 Sic variants characterized by deletion of a region of the protein. In contrast, no intrahost variation was detected in the number of a pentanucleotide repeat (CAAAA) that controls production of a bacterial cell-surface collagen-like protein by slipped-strand mispairing. Sic variation occurs at a sufficient frequency in vivo to result in mixed infections on the mucosal surface of human hosts, potentially contributing to pathogen survival.