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A multiplex PCR (mPCR) protocol was developed for simultaneous detection of the gyrB gene in Streptococcus pneumoniae , Streptococcus mitis , and Streptococcus oralis , and the specificity was evaluated using 141 coccus strains. Genomic DNAs purified from S. pneumoniae , S. mitis , and S. oralis strains were efficiently detected with size differences, whereas no PCR products were amplified from any of the reference strains tested. A pilot study of 47 human oral swab specimens was conducted in parallel, and the mPCR assay identified S. pneumoniae in 1 sample, S. mitis in 8 samples, and S. oralis in 2 samples, providing a powerful means for characterization at the level of species compared with traditional culture analysis. Our results suggest that the mPCR protocol presented here is a sensitive and promising tool for the rapid detection and discrimination of S. pneumoniae , S. mitis , and S. oralis from clinical specimens.

Lactococcus garvieae is an important foodborne pathogen causing lactococcosis associated with hemorrhagic septicemia in fish worldwide. A real-time quantitative polymerase chain reaction (qPCR) protocol targeting the 16S-23S rRNA intergenic spacer (ITS) region was developed for the detection and enum-eration of L. garvieae. The specificity was evaluated using genomic DNAs extracted from 66 cocci strains. Fourteen L. garvieae strains tested were positive, whereas 52 other strains including Lactococcus lactis ssp. lactis, Lactococcus lactis ssp. hordniae and Lactococcus lactis ssp. cremoris did not show a specific signal. The minimal limit of detection was 2.63 fg of purified genomic DNA, equivalent to 1 genome of L. garvieae. The optimized protocol was applied for the survey of L. garvieae in naturally contaminated fish samples. Our results suggest that the qPCR protocol using ITS is a sensitive and efficient tool for the rapid detection and enumeration of L. garvieae in fish and fish-containing foods.

Lactococcus garvieae is recognized as an emerging pathogen in fish. Several PCR-based methods have been developed for the detection and identification of L. garvieae; however, the sensitivity of these methods is still in question regarding the discrimination of this organism from other closely related species. Two primers, ITSLg30F and ITSLg319R, were designed from the sequence in the 16S-23S internal transcribed spacer (ITS) region and used for the specific detection of L. garvieae. L. garvieae strains including fish isolates were positive by this method. In contrast, previously developed PCR methods showed false-positive results with non-L. garvieae species. Our results indicate that a PCR method using the newly designed ITS primer set provides a sensitive and efficient tool for the detection of L. garvieae in fish and aquaculture environments.

We performed suppression subtractive hybridization to identify genomic differences between Streptococcus mitis and Streptococcus pneumoniae . Based on the pheA gene, a primer set specific to S. mitis detection was found in 18 out of 103 S. mitis -specific clones. Our findings would be useful for discrimination of S. mitis from other closely related cocci in the oral environment.

Streptococcus pneumoniae, the aetiological agent of pneumonia and non-gonococcal urethritis, shares a high degree of DNA sequence identity with the viridans group of streptococci, particularlyStreptococcus mitisandStreptococcus oralis. Although their clinical and pathological manifestations are different, discrimination betweenS. pneumoniaeand its close viridans cocci relatives is still quite difficult. Suppression subtractive hybridization was performed to identify the genomic differences betweenS. pneumoniaeandS. mitis. Thirty-four resultingS. pneumoniae-specific clones were examined by sequence determination and comparative DNA sequence analysis usingblast.S. pneumoniae-specific primers were subsequently designed from one of the clonal DNA sequences containing thecpsgene (coding for capsular polysaccharide biosynthesis). The primer specificities were evaluated using 49 viridans streptococci including 26S. pneumoniae, 54 other streptococci, 14Lactococcusspecies, 14Enterococcusspecies and threeVagococcusspecies, and compared with the specificities of previously described autolysin (lytA), pneumolysin (ply), Spn9802 and Spn9828 primers. The newly designedcpsA-specific primer set was highly specific toS. pneumoniaeand was even better than the existing primers. These findings may help improve the rapid identification and differentiation ofS. pneumoniaefrom closely related members of the viridans group streptococci.

Although the pathogenesis of Streptococcus oralis may be different from that of other viridans group streptococci, S. oralis shares a high degree of DNA sequence similarity with these streptococci. As a result, discrimination of S. oralis from its close relatives has long been considered difficult. This study was conducted to find specific genes that allow for the in vitro identification of S. oralis, but not other oral commensals. Four hundred ninety S. oralis clones obtained by suppressive subtractive hybridization were used for Southern hybridization, and positive clones were sequenced. Of 5 S. oralis-specific clones, newly designed primer sets based on the glucosyltransferase regulatory gene amplified genomic DNA only from S. oralis strains, but not from any of the other 125 strains tested. Our findings may be useful for the future development of efficient diagnostic tools for the rapid identification and differentiation of S. oralis from other oral streptococci strains.

Streptococcus pneumoniae is the main etiologic agent of pneumonia worldwide. Because the members of the viridans group streptococci share a high degree of DNA sequence homologies, phenotypic and genotypic discriminations of S. pneumoniae from the viridans group are difficult. A quantitative real-time PCR assay targeting the capsular polysaccharide biosynthesis gene (cpsA) was developed as a species-specific detection tool for S. pneumoniae. The specificity was evaluated using genomic DNAs extracted from 135 oral cocci strains. Twenty-seven S. pneumoniae strains tested positive, whereas 108 other strains including Streptococcus pseudopneumoniae, Streptococcus mitis, and Streptococcus oralis did not show a specific signal. The linear regression of standard curves indicated high correlations between the log numbers of S. pneumoniae cells and the C(T) values (R(2)=0.99). The minimal limit of detection was 32 fg of purified genomic DNA, equivalent to 14 genomes of S. pneumoniae. This new real-time PCR method may be very useful as a rapid and specific tool for detecting and quantifying S. pneumoniae.

Streptococcus pneumoniae, the leading etiological agent of pneumonia, shares a high degree of DNA sequence homology with the viridans group of streptococci. The clinical and pathological manifestations may present with different features, and discrimination between S. pneumoniae and its close viridans cocci relatives, such as Streptococcus mitis and Streptococcus oralis, is still quite difficult. The 445-bp sequences of the N-terminal region of rpoA from nine S. pneumoniae, seven S. mitis, ten S. oralis, and two related strains were determined and compared with their respective 16S rRNA gene sequences to establish their usefulness in phylogenetic analysis. Pairwise comparisons of rpoA sequences among the species showed higher rates of evolution with lower similarities (92.3-100%) than those of 16S rRNA genes (96.8-100%). The rpoA-based phylogeny generated deeper branches and presented improved discriminatory resolution than the 16S rRNA gene-based phylogeny. These results show that rpoA sequences represent a consistent, but more discriminating, genomic marker than 16S rRNA gene sequences for investigating the evolutionary relationships of Streptococcus at the species level. rpoA could be a useful marker for identifying and classifying S. pneumoniae, S. mitis, and S. oralis from closely related taxa.

The taxonomic position of a Gram-positive, non-motile, non-spore-forming coryneform, isolated from activated sludge and designated strain CAU 212(T), was investigated using a polyphasic approach. Cellular morphology, biochemical tests and chemotaxonomic investigations revealed that strain CAU 212(T) had the characteristics of the genus Corynebacterium. Comparative 16S rRNA gene sequence analysis showed that the organism formed a hitherto-unknown subline within the genus Corynebacterium. Sequence divergence values of more than 4.3 % from recognized Corynebacterium species, together with phenotypic differences, showed that the bacterium represents a previously unrecognized member of the genus Corynebacterium, for which the name Corynebacterium doosanense sp. nov. is proposed. The type strain is CAU 212(T) (=KCTC 19568(T)=CCUG 57284(T)).

Notes on Three Species of the Genus Euphoriomyces (Laboulbeniales) from KoreaTaxonomic Studies on Myelochroa from KoreaMolecular Analysis of Korean Anzia opuntiella (Lichenized Ascomycota) Based on...

The fruit of the black raspberry (Rubus coreanus Miquel) has been employed in traditional medicine, and recent studies have demonstrated its measureable biological activities. However, the root of the black raspberry has not been studied. Therefore, in this study, we evaluated the anti-inflammatory and antibacterial properties of the root and unripe fruit polyphenols of the black raspberry. Both polyphenols proved to have anti-inflammatory activity as evidenced by the decreased nitric oxide (NO), cytokines (IL-1β , IL-6, and IL-10) and prostaglandin E2 (PGE(2)) levels in lipopolysaccharide (LPS)-stimulated RAW 264.7 murine macrophages. However, root polyphenols showed stronger anti-inflammatory activity than fruit polyphenols. LPS-induced mRNA and protein expressions of inducible NO synthase (iNOS) and cyclooxygenase (COX)-2 levels were also decreased, confirming the anti-inflammatory activity. Root polyphenols showed lethal activity against methicillin-resistant Staphylococcus aureus (MRSA), carbapenem-resistant Acinetobacter baumannii (CRAB), and Bacillus anthracis. In contrast, the black raspberry fruit did not demonstrate these properties. These data provide the first demonstration that black raspberry root has potential anti-inflammatory and anti-superbacterial properties that can be exploited as alternatives for use in the food and cosmetic industries and/or as pharmaceuticals.

Lactococcus garvieae, the pathogenic species in the genus Lactococcus, is recognized as an emerging pathogen in fish, animals, and humans. Despite the widespread distribution and emerging clinical significance of L. garvieae, little is known about the genomic content of this microorganism. Suppression subtractive hybridization was performed to identify the genomic differences between L. garvieae and Lactococcus lactis ssp. lactis, its closest phylogenetic neighbor, and the type species of the genus Lactococcus. Twenty-seven clones were specific to L. garvieae and were highly different from Lactococcus lactis in their nucleotide and protein sequences. Lactococcus garvieae primer sets were subsequently designed for two of these clones corresponding to a pyrH gene and a novel DNA signature for application in the specific detection of L. garvieae. The primer specificities were evaluated relative to three previously described 16S rRNA gene–targeted methods using 32 Lactococcus and closely related strains. Both newly designed primer sets were highly specific to L. garvieae and performed better than did the existing primers. Our findings may be useful for developing more stable and accurate tools for the discrimination of L. garvieae from other closely related species.

Streptococcus pneumoniae is the main etiologic agent of pneumonia worldwide. Because the members of the viridans group streptococci share a high degree of DNA sequence homologies, phenotypic and genotypic discriminations of S. pneumoniae from the viridans group are difficult. A quantitative real-time PCR assay targeting the capsular polysaccharide biosynthesis gene (cpsA) was developed as a species-specific detection tool for S. pneumoniae. The specificity was evaluated using genomic DNAs extracted from 135 oral cocci strains. Twenty-seven S. pneumoniae strains tested positive, whereas 108 other strains including Streptococcus pseudopneumoniae, Streptococcus mitis, and Streptococcus oralis did not show a specific signal. The linear regression of standard curves indicated high correlations between the log numbers of S. pneumoniae cells and the C(T) values (R(2)=0.99). The minimal limit of detection was 32 fg of purified genomic DNA, equivalent to 14 genomes of S. pneumoniae. This new real-time PCR method may be very useful as a rapid and specific tool for detecting and quantifying S. pneumoniae.

Streptococcus pneumoniae, the leading etiological agent of pneumonia, shares a high degree of DNA sequence homology with the viridans group of streptococci. The clinical and pathological manifestations may present with different features, and discrimination between S. pneumoniae and its close viridans cocci relatives, such as Streptococcus mitis and Streptococcus oralis, is still quite difficult. The 445-bp sequences of the N-terminal region of rpoA from nine S. pneumoniae, seven S. mitis, ten S. oralis, and two related strains were determined and compared with their respective 16S rRNA gene sequences to establish their usefulness in phylogenetic analysis. Pairwise comparisons of rpoA sequences among the species showed higher rates of evolution with lower similarities (92.3-100%) than those of 16S rRNA genes (96.8-100%). The rpoA-based phylogeny generated deeper branches and presented improved discriminatory resolution than the 16S rRNA gene-based phylogeny. These results show that rpoA sequences represent a consistent, but more discriminating, genomic marker than 16S rRNA gene sequences for investigating the evolutionary relationships of Streptococcus at the species level. rpoA could be a useful marker for identifying and classifying S. pneumoniae, S. mitis, and S. oralis from closely related taxa.

Shepherd's purse, Capsella bursa-pastoris (L.) Medik., has been considered a health food for centuries in Asia and is known to contain the isothiocyanate compound sulforaphane. In this study, we evaluated the anti-inflammatory and antibacterial properties of a sulforaphane-containing solution (SCS) isolated from shepherd's purse. SCS had significant anti-inflammatory activity indicated by the decreased levels of nitric oxide (NO), cytokines (interleukin 1β [IL-1β], IL-6, and IL-10), and prostaglandin E2 (PGE2) in lipopolysaccharide-stimulated RAW 264.7 murine macrophages. In addition, SCS decreased the inducible NO synthase (iNOS) and cyclooxygenase 2 (COX-2) levels, which confirmed the anti-inflammatory activity of SCS. Further, SCS inhibited vancomycin-resistant enterococci (VRE) and Bacillus anthracis. The minimal inhibitory concentration was 250 µg/ml for VRE and 1,000 µg/ml for B. anthracis. Taken together, these data indicate that SCS has potential anti-inflammatory and anti-superbacterial properties, and thus it can be used as a functional food or pharmaceutical.

Although viruses are the most common cause of acute gastroenteritis (AGE) in humans, details about the causative viruses in AGE are largely unknown because many causative viruses are unable to be cultured by current culture techniques. In our study, fecal samples from 10 children under five years of age with unexplained AGE and 10 healthy children were investigated for RNA viruses using random priming (RP)-mediated sequence-independent single primer amplification (SISPA). The causative viruses in cases of cryptogenic diarrhea were then assessed for their potential diagnostic value. Of the 1,129 viral clones identified, rotavirus was most commonly associated with AGE (125 sequences, 22.4%). In contrast, bacteriophage was most common (43 sequences, 13.6%) in healthy children. The remaining 515 viral clones were unidentifiable. These findings suggest that investigation of cases or outbreaks of unexplained diarrhea using a metaviromic strategy is a new avenue for diagnosis.

Bronchial asthma can be triggered by microbial agents in the oropharynx. This study was designed to identify the differences in microbiota of oropharynx of bronchial asthmatic patients in contrast to normal controls. In order to resolve the qualitative and quantitative diversity of the 16S rRNA gene present in the oropharynx microbiota of 4 patients and 4 controls, we compared microbial communities using Sanger sequencing and 376 sequences of 16S rRNA gene were analyzed. Of the total microbial diversity detected in the oropharynx in asthmatic patients 45.6% comprised members of the Firmicutes. In contrast, Proteobacteria (44.0%) dominated the oropharyngeal microbiota in the normal control group. Members of the Bacteroidetes, Fusobacteria, Actinobacteria, TM7, Cyanobacteria and unclassified bacteria were present in both groups. In conclusion, the difference in the microbiota of the oropharynx between patients and normal individuals could trigger symptomatic attacks in bronchial asthma.