The gtfB gene coding for a glucosyltransferase (GTF) activity of Streptococcus mutans GS-5 was isolated on a 15.4-kilobase DNA fragment by using a lambda L47.1 gene library. The activity was catalyzed by gene products of 150 and 145 kilodaltons which reacted with antibodies directed against both soluble and insoluble glucan-synthesizing GTFs. The enzyme present in crude Escherichia coli extracts synthesized both soluble and insoluble glucans. The enzyme was partially purified from lysates of the lambda DS-76 clone and synthesized both types of glucans in a primer-independent fashion. In addition, the purified enzyme exhibited a pI of approximately 5.0. Southern blot analysis indicated that the cloned GTF gene represented a contiguous nucleotide sequence on the strain GS-5 chromosome. Furthermore, evidence for the existence of a distinct gene sharing partial homology with gtfB was also obtained. The gtfB gene was subcloned into plasmid pACYC184 into E. coli and exhibited GTF activity when carried on GS-5 inserts as small as 5 kilobases. The approximate location of the GTF promoter and the direction of gene transcription were also determined. The cloned enzyme was not secreted through the cytoplasmic membrane of E. coli, since most of the activity was found in the cytoplasm and, in lesser amounts, associated with the cytoplasmic membrane. The gtfB gene was insertionally inactivated by introducing a gene fragment coding for erythromycin resistance into the GTF coding region. After transformation of strain GS-5 with the altered gene, transformants defective in insoluble glucan synthesis were identified. These results indicate that the gtfB gene codes for a GTF involved in insoluble glucan synthesis in strain GS-5.

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It is difficult to separate Streptococcus pneumoniae from the genotypically similar species Streptococcus mitis and Streptococcus oralis, which are commensals of the human oral cavity. A novel nucleic acid amplification technique, loop-mediated isothermal amplification (LAMP), which amplifies DNA under isothermal conditions (63°C) with high specificity, efficiency, and rapidity, was examined regarding its applicability for detecting S. pneumoniae. An S. pneumoniae-specific LAMP primer targeting the lytA gene was designed. The primer specificity was validated using 10 Streptococcus and 7 non-Streptococcus species. Within 60 min, the assay could detect 10 or more copies of purified S. pneumoniae DNA with a sensitivity 1,000 times that of conventional PCR. Clinical isolates of 21 other strains (3 S. oralis, 17 S. mitis, and 1 Streptococcus species) that harbor virulencefactor-encoding genes (lytA or ply) were tried to differentiate S. pneumoniae. The detection of S. pneumoniae in clinical isolates was more selective using the LAMP method than using conventional PCR. Therefore, LAMP appears to be a sensitive and reliable means of diagnosing S. pneumoniae infection.Streptococcus pneumoniae is a common major human pathogen associated with community-acquired pneumonia, septicemia, meningitis, and otitis media (2). While traditional antimicrobial therapy has proven to be an effective treatment, the emergence of antimicrobial-resistant strains has resulted in an increasing number of cases (10). The isolation and identification of pneumococcus is complicated by contamination with alpha-hemolytic streptococci belonging to the normal flora. Pneumococcal detection using classical techniques, including growth-based assays, colonial morphology, optochin susceptibility, bile solubility, and serology, can be time-consuming and can produce equivocal results. Sensitive and specific assays that can be completed promptly in the clinical laboratory are essential for early diagnosis and effective antibiotic therapy. Molecular assays are inherently valuable because detection can be achieved with enhanced sensitivity and specificity and detection is not diminished with nonviable organisms.The molecular methods that have been used to date (3, 6, 17) are expensive and complicated to perform. Varying degrees of success have been realized using PCR-based assays for detecting S. pneumoniae with primers specific to repetitive regions or to genes encoding rRNA (7, 9, 12), pneumolysin (18,19,23), or autolysin (5, 14, 18). Autolysin and pneumolysin, which are encoded by the lytA and ply genes, respectively, represent potential targets for the specific detection of S. pneumoniae. Both genes are essential for pathogenesis and are well-characterized virulence markers (1). The restricted allelic variation of lytA (4, 21) makes this gene an ideal target for specific identification in clinical and epidemiological studies. For example, the gene may allow the differentiation of S. pneumoniae from the genotypically similar species Streptococcus mitis and...

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Abbreviations & Acronyms ICI = immune checkpoint inhibitor IFN-c = interferon-gamma irAE = immune-related adverse event PD-L1 = programmed death-ligand 1 SCC = squamous cell carcinoma