Anti-Inflammatory and Anti-Superbacterial Properties of Sulforaphane from Shepherd's Purse.

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 1ß [IL-1ß], 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.

Virulence factors of uropathogenic Escherichia coli of urinary tract infections and asymptomatic bacteriuria in children.

BACKGROUND/PURPOSE: The clinical aspects of virulence genes of uropathogenic Escherichia coli (UPEC) are not fully understood. This study compared the presence of virulence genes in UPEC isolated from urinary tract infections (UTIs) and asymptomatic bacteriuria (ABU) in children. METHODS: The study included children with UTI (n = 15) or ABU (n = 49) treated at Chung-Ang University Yongsan Hospital between 2010 and 2011. The strains were acquired from each urine sample collected, and 18 major virulence genes were detected by polymerase chain reaction. Antimicrobial susceptibility of all UPEC isolates was determined. RESULTS: Sixty-four E. coli strains were isolated from the urine samples. The most commonly identified virulence gene in both groups was fimH (100.0% in the UTI group and 95.9% in the ABU group). The UTI isolates showed a higher prevalence of papEF and fyuA, and a lower prevalence of feoB than ABU isolates (p < 0.01 for all). The profile of virulence gene, fimH(+)kpsMTII(+)feoB(+) also showed a significant difference between the two groups (p < 0.01). Isolates from ABU were more resistant to most antimicrobials tested. The presence of papEF, feoB, and fyuA also correlated with the antimicrobial susceptibility of UPEC. CONCLUSION: The virulence gene repertoire was different in the UPEC of UTI and ABU. The papEF, feoB, and fyuA genes showed meaningful differences between the two groups and may have an important role in the pathogenesis of overt UTI.

Anti-inflammatory and anti-superbacterial activity of polyphenols isolated from black raspberry.

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-1ß , 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.

Simultaneous detection of Streptococcus pneumoniae, S. mitis, and S. oralis by a novel multiplex PCR assay targeting the gyrB gene.

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.

Development of a 16S-23S rRNA intergenic spacer-based quantitative PCR assay for improved detection and enumeration of Lactococcus garvieae.

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.

Comparative transcriptomic analysis of streptococcus pseudopneumoniae with viridans group streptococci.

BACKGROUND: Streptococcus pseudopneumoniae, is a novel member of the genus Streptococcus, falling close to related members like S. pneumoniae, S. mitis, and S. oralis. Its recent appearance has shed light on streptococcal infections, which has been unclear till recently. In this study, the transcriptome of S. pseudopneumoniae CCUG 49455T was analyzed using the S. pneumoniae R6 microarray platform and compared with those of S. pneumoniae KCTC 5080T, S. mitis KCTC 3556T, and S. oralis KCTC 13048T strains. RESULTS: Comparative transcriptome analysis revealed the extent of genetic relatedness among the species, and implies that S. pseudopneumoniae is the most closely related to S. pneumoniae. A total of 489, 444 and 470 genes were upregulated while 347, 484 and 443 were downregulated relative to S. pneumoniae in S. pseudopneumoniae, S. oralis and S. mitis respectively. Important findings were the up-regulation of TCS (two component systems) and transposase which were found to be specific to S. pseudopneumoniae. CONCLUSIONS: This study provides insight to the current understanding of the genomic content of S. pseudopneumoniae. The comparative transcriptome analysis showed hierarchical clustering of expression data of S. pseudopneumoniae with S. pneumoniae and S. mitis with S. oralis. This proves that transcriptional profiling can facilitate in elucidating the genetic distance between closely related strains.

Characterization of the fungal microbiota (mycobiome) in healthy and dandruff-afflicted human scalps.

The human scalp harbors a vast community of microbial mutualists, the composition of which is difficult to elucidate as many of the microorganisms are not culturable using current culture techniques. Dandruff, a common scalp disorder, is known as a causative factor of a mild seborrheic dermatitis as well as pityriasis versicolor, seborrheic dermatitis, and atopic dermatitis. Lipophilic yeast Malassezia is widely accepted to play a role in dandruff, but relatively few comprehensive studies have been reported. In order to investigate fungal biota and genetic resources of dandruff, we amplified the 26S rRNA gene from samples of healthy scalps and dandruff-afflicted scalps. The sequences were analyzed by a high throughput method using a GS-FLX 454 pyrosequencer. Of the 74,811 total sequence reads, Basidiomycota (Filobasidium spp.) was the most common phylum associated with dandruff. In contrast, Ascomycota (Acremonium spp.) was common in the healthy scalps. Our results elucidate the distribution of fungal communities associated with dandruff and provide new avenues for the potential prevention and treatment of dandruff.

Identification of a pheA gene associated with Streptococcus mitis by using suppression subtractive hybridization.

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.

Comparative genome analysis of Lactococcus garvieae using a suppression subtractive hybridization library: discovery of novel DNA signatures.

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.

Real-time PCR assays for the detection and quantification of Streptococcus pneumoniae.

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.

Identification of the cpsA gene as a specific marker for the discrimination of Streptococcus pneumoniae from viridans group streptococci.

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, particularly Streptococcus mitis and Streptococcus oralis. Although their clinical and pathological manifestations are different, discrimination between S. pneumoniae and its close viridans cocci relatives is still quite difficult. Suppression subtractive hybridization was performed to identify the genomic differences between S. pneumoniae and S. mitis. Thirty-four resulting S. pneumoniae-specific clones were examined by sequence determination and comparative DNA sequence analysis using blast. S. pneumoniae-specific primers were subsequently designed from one of the clonal DNA sequences containing the cps gene (coding for capsular polysaccharide biosynthesis). The primer specificities were evaluated using 49 viridans streptococci including 26 S. pneumoniae, 54 other streptococci, 14 Lactococcus species, 14 Enterococcus species and three Vagococcus species, and compared with the specificities of previously described autolysin (lytA), pneumolysin (ply), Spn9802 and Spn9828 primers. The newly designed cpsA-specific primer set was highly specific to S. pneumoniae and was even better than the existing primers. These findings may help improve the rapid identification and differentiation of S. pneumoniae from closely related members of the viridans group streptococci.

rpoA is a useful gene for identification and classification of Streptococcus pneumoniae from the closely related viridans group streptococci.

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.

Corynebacterium doosanense sp. nov., isolated from activated sludge.

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)).