Introduction . Streptococcus dysgalactiae subsp. equisimilis (SDSE) is a β-hemolytic streptococcus that causes severe invasive streptococcal infections, especially in the elderly and people with underlying diseases. SDSE strains are primarily characterized by Lancefield group G or C antigens. Hypothesis/Gap Statement. We have previously reported the prevalence of Lancefield group A SDSE (GA-SDSE) strains in Japan and have analysed the draft genome sequences of these strains. As GA-SDSE is a rare type of SDSE, only one complete genome has been sequenced to date. Aim. The present study is focused on genetic characteristics of GA-SDSE strains. In order to examine molecular characteristics, we also tested growth inhibition of other streptococci by GA-SDSE. Methodology. We determined the complete genome sequences of three GA-SDSE strains by two new generation sequencing systems (short-read and long-read sequencing data). Using the sequences, we also conducted a comparative analysis of GA-SDSE and group C/G SDSE strains. In addition, we tested multiplex and quantitative PCRs targeting the GA-SDSE, group G SDSE, and S. pyogenes . Results. We found a group-specific conserved region in GA-SDSE strains that is composed of genes encoding predicted anti-bacteriocin and streptococcal lantibiotic (Sal) proteins. Multiplex and quantitative PCRs targeting the GA-SDSE-specific region were able to distinguish between GA-SDSE, other SDSE, and S. pyogenes strains. The growth of GA-SDSE was suppressed in the presence of group G SDSE, indicating a possible explanation for the low frequency of isolation of GA-SDSE. Conclusion. The comparative genome analysis shows that the genome of GA-SDSE has a distinct arrangement, enabling the differentiation between S. pyogenes , GA-SDSE, and other SDSE strains using our PCR methods.

Streptococcus dysgalactiae subsp. equisimilis (SDSE) causes cellulitis, bacteremia, and invasive diseases, such as streptococcal toxic shock syndrome. Although SDSE infection is more prevalent among elderly individuals and those with diabetes mellitus than infections with Streptococcus pyogenes (Group A streptococci; GAS) and Streptococcus agalactiae (Group B streptococci; GBS), the mechanisms underlying the pathogenicity of SDSE remain unknown. SDSE possesses a gene hylD encoding a hyaluronate lyase (HylD), whose homologue (HylB) is involved in pathogenicity of GBS, while the role of HylD has not been characterized. In this study, we focused on the enzyme HylD produced by SDSE; HylD cleaves hyaluronate (HA) and generates unsaturated disaccharides via a β-elimination reaction. Hyaluronate-agar plate assays revealed that SDSE promoted dramatic HA degradation. SDSE expresses both HylD and an unsaturated glucuronyl hydrolase (UGL) that catalyzes the degradation of HA-derived oligosaccharides; as such, SDSE was more effective at HA degradation than other β-hemolytic streptococci, including GAS and GBS. Although HylD shows some homology to HylB, a similar enzyme produced by GBS, HylD exhibited significantly higher enzymatic activity than HylB at pH 6.0, conditions that are detected in the skin of both elderly individuals and those with diabetes mellitus. We also detected upregulation of transcripts from hylD and ugl genes from SDSE wild-type collected from the mouse peritoneal cavity; upregulated expression of ugl was not observed in ΔhylD SDSE mutants. These results suggested that disaccharides produced by the actions of HylD are capable of triggering downstream pathways that catalyze their destruction. Furthermore, we determined that infection with SDSEΔhylD was significantly less lethal than infection with the parent strain. When mouse skin wounds were infected for 2 days, intensive infiltration of neutrophils was observed around the wound areas Nguyen et al.

Ebola virus is a deadly causative agent with a high mortality rate of up to 90%, therefore it has been classified by the Center for Disease Control and Prevention (CDC) as a category A biological agent. The World Health Organization (WHO) recommended using RT-PCR based assays to rapidly detect the virus. In the present study, we established an in-house assay for detection of Zaire ebolavirus via real-time RT-PCR. The nucleotide sequence of the Zaire ebolavirus nucleoprotein (NP) gene was retrieved from the Genbank for designing primer pairs and probes using Primer Express 3.0 software. The RNA positive control was generated by in vitro RNA transcript synthesis. The optimal components in the 20 μl final volume of the real-time RT-PCR assay were 10 μl 2X QuantiTectProbeRT-PCR master mix, 0,6 μM of each primer, 0,1 μM of the probe, 0,2 μl RT mix and 5 μl of RNA template. The thermal cycle conditions were as follows: 50oC for 30 min, 95°C for 15 min, then 45 cycles of 15 s at 94°C, 60s at 60°C. The limit of detection of the assay was 100 copies/reaction and 1414 FFU/ml with the positive RNA panel and sample panel of RNA extracted from cell culture supernatants of cells infected with Zaire ebolavirus 2014/Gueckedou-C05, respectively. The specificity of this assay was 100% when tested with the positive RNA panel of Ebola virus and other haemorrhagic fever viruses. In conclusion, we successfully established an in-house real-time RT-PCR assay for detection of Zaire ebolavirus in Vietnam with a limit of detection of 1414 FFU/ml and specificity of 100%.

In the published version of the article there was a spelling error in an author's name. Tohru Miyoshi-Akiyama's surname was incorrectly spelled as 'Miyohi-Akiyama' .