The characteristics and correlations of vaginal flora in women with cervical lesions / 中华肿瘤杂志

Objective: To explore the characteristics and correlations of vaginal flora in women with cervical lesions. Methods: A total of 132 women, including 41 women diagnosed with normal cervical (NC), 39 patients with low-grade cervical intraepithelial neoplasia (CIN 1), 37 patients with high-grade cervical intraepithelial neoplasia (CIN 2/3) and 15 patients with cervical squamous cell carcinoma (SCC), who came from the gynecological clinic of Second Hospital of Shanxi Medical University during January 2018 to June 2018, were enrolled in this study according to the inclusive and exclusive criteria strictly. The vaginal flora was detected by 16S rDNA sequencing technology. Co-occurrence network analysis was used to investigate the Spearman correlations between different genera of bacteria. Results: The dominant bacteria in NC, CIN 1 and CIN 2/3 groups were Lactobacillus [constituent ratios 79.4% (1 869 598/2 354 098), 63.6% (1 536 466/2 415 100) and 58.3% (1 342 896/2 301 536), respectively], while Peptophilus [20.4% (246 072/1 205 154) ] was the dominant bacteria in SCC group. With the aggravation of cervical lesions, the diversity of vaginal flora gradually increased (Shannon index: F=6.39, P=0.001; Simpson index: F=3.95, P=0.012). During the cervical lesion progress, the ratio of Lactobacillus gradually decreased, the ratio of other anaerobes such as Peptophilus, Sneathia, Prevotella and etc. gradually increased, and the differential bacteria (LDA score >3.5) gradually evolved from Lactobacillus to other anaerobes. The top 10 relative abundance bacteria, spearman correlation coefficient>0.4 and P<0.05 were selected. Co-occurrence network analysis showed that Prevotella, Peptophilus, Porphyrinomonas, Anaerococcus, Sneathia, Atopobium, Gardnerella and Streptococcus were positively correlated in different stages of cervical lesions, while Lactobacillus was negatively correlated with the above anaerobes. It was found that the relationship between vaginal floras in CIN 1 group was the most complex and only Peptophilus was significantly negatively correlated with Lactobacillus in SCC group. Conclusions: The increased diversity and changed correlations between vaginal floras are closely related to cervical lesions. Peptophilus is of great significance in the diagnosis, prediction and early warning of cervical carcinogenesis.

Analysis of the changes in intestinal microecology in the early stage of sepsis rat based on 16S rDNA sequencing / 中华危重病急救医学

Objective:To investigate the changes of intestinal microecology in the early stage of sepsis rat model by 16S rDNA sequencing.Methods:Sixty male Sprague-Dawley (SD) rats were randomly divided into cecal ligation and puncture (CLP) group and sham operation group (Sham group), with 30 rats in each group. In the CLP group, sepsis rat model was reproduced by CLP method; the rats in the Sham group only underwent laparotomy without CLP. At 24 hours after the operation, the intestinal feces and serum samples of 8 rats in each group were collected. The survival rate of the rest rats was observed until the 7th day. The level of serum tumor necrosis factor-α (TNF-α) was detected by enzyme-linked immunosorbent assay (ELISA). Intestinal feces were sequenced by 16S rDNA sequencing technology. The operational taxonomic unit (OTU) data obtained after sequence comparison and clustering was used for α diversity and β diversity analysis, principal coordinate analysis and linear discriminant analysis effect size analysis (LEfSe) to observe the changes of intestinal microecology in early sepsis rats and excavate the marker flora.Results:At 24 hours after the reproduction of the model, the rats in the CLP group showed shortness of breath, scattered hair and other manifestations, and the level of serum TNF-α increased significantly as compared with that in the Sham group (ng/L: 43.95±9.05 vs. 11.08±3.27, P < 0.01). On the 7th day after modeling, the cumulative survival rate of the Sham group was 100%, while that of the CLP group was 31.82%. Diversity analysis showed that there was no significant difference in α diversity parameter between the Sham group and the CLP group (number of species: 520.00±52.15 vs. 492.25±86.61, Chao1 richness estimator: 707.25±65.69 vs. 668.93±96.50, Shannon index: 5.74±0.42 vs. 5.79±0.91, Simpson index: 0.93±0.03 vs. 0.94±0.05, all P > 0.05). However, the β diversity analysis showed that the difference between groups was greater than that within groups whether weighted according to OTU or not (abundance weighted matrix: R = 0.23, P = 0.04; abundance unweighted matrix: R = 0.32, P = 0.01). At the phylum level, the abundance of Proteobacteria and Candidatus_sacchari in the CLP group increased significantly as compared with the Sham group [18.100% (15.271%, 26.665%) vs. 6.974% (2.854%, 9.764%), 0.125% (0.027%, 0.159%)% vs. 0.018% (0.008%, 0.021%), both P < 0.05]. At the genus level, the abundance of opportunistic pathogen including Helicobacter, Ruthenium, Streptococcus, Clostridium ⅩⅧ in the CLP group was significantly higher than that in the Sham group [5.090% (1.812%, 6.598%) vs. 0.083% (0.034%, 0.198%), 0.244% (0.116%, 0.330%) vs. 0.016% (0.008%, 0.029%), 0.006% (0.003%, 0.010%) vs. 0.001% (0%, 0.003%), 0.094% (0.035%, 0.430%) vs. 0.007% (0.003%, 0.030%), all P < 0.05], and the abundance of probiotics such as Alloprevotella and Romboustia was significantly lower than that in the Sham group [7.345% (3.662%, 11.546%) vs. 22.504% (14.403%, 26.928%), 0.113% (0.047%, 0.196%) vs. 1.229% (0.809%, 2.29%), both P < 0.01]. LEfSe analysis showed that the probiotics belonging to Firmicutes were significantly enriched in the Sham group, and Romboustia was the most significantly enriched species. Opportunistic pathogens such as Helicobacter, Streptococcus and Clostridium ⅩⅧ were significantly enriched in the CLP group, Helicobacter_NGSU_ 2015 was the most significantly enriched species. Conclusion:In the early stage of sepsis, the intestinal microbiota structure of rats is significantly changed, which mainly shows that the abundance of Alloprevotella and other probiotics is significantly reduced, while that of Helicobacter and other opportunistic pathogens is significantly increased.

Integrating 16S sequencing and metabolomics study on anti-rheumatic mechanisms against collagen-induced arthritis of Wantong Jingu Tablet / 中国天然药物

Wantong Jingu Tablet (WJT), a mixture of traditional Chinese medicine, was reported to relieve the symptoms of rheumatoid arthritis (RA), but its pharmacological mechanism was not completely understood. The aim of this study was to investigate the therapeutic mechanisms of WJT for RA in vivo. The effects of WJT on joint pathology, as well as the levels of Bax, Bcl-2, caspase-3, cleaved-caspase-3, ERK1/2, pERK1/2, TNF-α, IL-1β, and IL-6 were measured using collagen-induced arthritis (CIA) rats. The intestinal flora composition and the metabolites alteration were analyzed by 16S rDNA sequencing and metabolomics method, respectively. We found that WJT ameliorated the severity of the CIA rats which might be mediated by inducing apoptosis, inactivating the MEK/ERK signals and reducing the production of pro-inflammatory cytokines. WJT, in part, relieved the gut microbiota dysbiosis, especially bacterial phylum Bacteroidetes, Tenericutes and Deferribacteres, as well as bacterial genus Vibrio, Macrococcus and Vagococcus. 3'-N-debenzoyl-2'-deoxytaxol, tubulysin B, and magnoline were significantly associated with the specific genera. We identified serotonin, glutathione disulfide, N-acetylneuraminic acid, naphthalene and thromboxane B2 as targeted molecules via metabolomics. Our findings contributed to the understanding of RA pathogenesis, and WJT played essential roles in gut microbiota health and metabolite modulation in the CIA rats.

Effects of long-term high-fat diet on bone mineral density and intestinal flora / 中华内分泌代谢杂志

Objective:To investigate the effects of long-term high-fat diet on bone mineral density and intestinal flora in mice.Methods:Sixteen male C57BL/6 mice were randomly divided into control group (NC group) and high-fat group (HF group). After 24 weeks of high-fat feeding, biochemical indicators such as blood glucose and blood lipids were detected, bilateral femurs were taken and bone microstructure was analyzed with micro-computered tomography (micro-CT), and changes of intestinal microbial composition and proportion were revealed using 16S rDNA sequencing technology.Results:Compared with the control group, the serum total cholesterol (TC) and low-density lipoprotein cholesterol (LDL-C) in HF group were significantly increased. Micro-CT uncovered that the bone mineral density (Tb.BMD), bone volume fraction (BV/TV) and the number of trabecular bone (Tb.N) decreased, yet structural model index (SMI) and the trabecular fraction (Tb.Sp) increased in the HF group mice. The gut microbiota 16S rDNA sequence analysis showed that the proportion of Proteobacter was significantly increased and the proportions of pachycete, warty microbacterius, and actinomycete were reduced in HF group at the phyla level. The proportion of Bacteroidetes S24-7_norank in the NC group was significantly higher than that in the HF group, and the multilevel discriminant analysis of species differences (LEfSe) identified that the difference was significant, yet the proportion of Bacteroides, Pseudo-Prevotella, Desulfovibrio, Altobacter, and Helicobacter in the HF group were higher than those in the NC group, which were significant differences in Altobacter and Helicobacter at genus level.Conclusion:Long-term high-fat feeding can cause the destruction of femoral trabecular structure, decrease in the number of trabeculus bones, and bone mineral density in C57BL/6 mice. It also leads to significant changes in the composition and proportion of the intestinal flora.

Caracterização da microbiota de queijos artesanais provenientes da Serra da Canastra - MG e da cultura iniciadora natural utilizada em sua produção / Microbiota characterization of artisanal cheeses from Serra da Canastra - MG and their endogenous starter culture

O queijo Canastra possui grande importância na cultura e economia local, é parte do Patrimônio Imaterial do Brasil (IPHAN, 2014) e recebeu o selo de produto com designação de origem em 2012 (INPI, 2016). Sua produção utiliza leite, sal, coalho e uma cultura iniciadora natural, chamada popularmente de pingo. Esse estudo visou a caracterização da microbiota presente no queijo maturado da Serra da Canastra e no pingo utilizado em sua produção utilizando técnicas avançadas de sequenciamento em larga escala para identificação das bactérias e fungos ali presentes. Nossos dados da microbiota bacteriana foram comparados com dados da microbiota de outros queijos brasileiros e do mundo disponíveis na literatura. As principais bactérias encontradas em amostras de pingo pertencem aos gêneros Lactococcus (45.6%), Streptococcus (30.3%), Staphylococcus (5.1%), e em amostras de queijo aos gêneros Lactococcus (22.5%), Streptococcus (27.2%), Corynebacterium (18.8%), Staphylococcus (13.6%), Leuconostoc (6.3%) e Weissella (6%). Os principais gêneros de fungos encontrados nos queijos foram Debaryomycesa (78.6%), Trichosporona (7.8%). Nosso estudo foi capaz de separar a microbiota dos queijos produzidos na Serra da Canastra de outros queijos na Europa e América do Norte, sendo o pH um possível fator de segregação. Também foi observada uma diferença entre a microbiota do queijo Canastra com outros queijos Brasileiros. Além disso, visualizamos que a distância geográfica entre produtores e a sazonalidade possuem um efeito sobre a microbiota dos pingos e queijos. A partir da análise de todos os microrganismos encontrados na microbiota bacteriana, foram detectados táxons que discriminam produtores por suas aplicações de boas práticas de fabricação e por sua infraestrutura. Observamos proporções menores de um táxon de Kocuria Kristinae nos pingos e um de Streptococcus nos queijos e proporções maiores de um táxon de Staphylococcus nos queijos. Também pudemos observar uma diminuição nas proporções de táxons de Debaryomycesa e aumento na proporção de táxons de Trichosporona na composição fúngica dos queijos, possivelmente devido a transição sazonal do período seco para o chuvoso. Usando técnicas moleculares de sequenciamento em larga escala, demonstramos que há uma diferença na microbiota presente em diferentes áreas da Serra da Canastra, um possível efeito da sazonalidade na composição fúngica e bacteriana. E evidenciamos que táxons de Streptococcus, Staphylococcus e Kocuria estão correlacionados às boas práticas de produção e elucidamos a conexão existente entre a microbiota do pingo e a do queijo. Estes resultados podem influenciar o desenvolvimento de métodos de rastreamento de sub-regiões específicas da Canastra e auxiliar os produtores na produção de queijos de boa qualidade, mantendo as características específicas de sua região

The Canastra cheese has great importance for the local culture and economy, being part of the Intangible Heritage of Brazil (IPHAN, 2014). It has received the protected designation of origin certification in 2012 (INPI, 2016). It's made using milk, salt, rennet and a endogenous starter culture, popularly called as "pingo". This study aimed to characterize the microbiota present in the Serra da Canastra's cheese and the pingo used in its production. In order to conduct this research we used next generation sequencing to identify the bacteria and fungi present there. Our bacterial microbiota dataset was compared with microbiota datasets from other Brazilian and world cheeses available in the literature. The main bacteria found were Lactococcus (45.6%), Streptococcus (30.3%) and Staphylococcus (5.1%) in the endogenous starter samples and Lactococcus (22.5%), Streptococcus (27.2%), Corynebacterium (18.8 %), Staphylococcus (13.6%), Leuconostoc (6.3%) and Weissella (6%) in cheese samples. The main fungi found in the cheeses were Debaryomycesa (78.6%) and Trichosporona (7.8%). We were able to separate the microbiota from Serra da Canastra cheeses and other cheeses in Europe and North America, being the pH a possible segregation factor. Furthermore, a difference was also observed between the microbiota of Canastra and other Brazilian cheeses. In addition, we observed that the geographical distance between producers and the seasonality could be affecting the pingos and cheeses microbiota. We found bacterial taxa that could discriminate producers by their good manufacturing practices and their local infrastructure. Low levels of good manufacturing practices (GMPs) were assigned to bigger proportions of a Kocuria Kristinae taxon in the pingos and a Staphylococcus taxon in the cheeses. Also, higher levels of GMPs were assigned to smaller proportions of Streptococcus taxons in the cheeses. Furthermore We could observe a decrease of Debaryomycesa and an increase of Trichosporona proportions in the fungal composition of cheeses. This could be due to a climate transition: from the dry season to the rainy season. Using large-scale sampling coupled with molecular sequencing techniques, we observe a connection between pingo and cheeses microbiota. We show that the microbiota of different areas in Serra da Canastra is different, also, there is a possible effect of seasonality on fungal and bacterial composition. Furthermore, we could see that Streptococcus, Staphylococcus and Kocuria taxons are correlated with good practices. These results may influence the development of tracking methods for specific Canastra subregions and assist producers to manufacture good quality cheeses while maintaining the specific characteristics of their region

Explore Effect of Jieyu Qutan Huazhuo Prescription on Gut-liver Axis of Rats with High-fat Diet Based on 16S rDNA Sequencing / 中国实验方剂学杂志

Objective:To study the effect of Jieyu Qutan Huazhuo prescription（JQHP） on the gut microbiota of rats with high-fat diet，and to explore the effect of Chinese medicine on the regulation of gut microflora and the restoration of gut-liver axis balance. Method:Seventy male SPF Wistar rats were randomly divided into a normal group of 10 and a model group of 60. Mice in the normal group were fed with normal diet and mice in the model group were fed high-fat diet. After 12 weeks，the model group was randomly divided into 6 groups with 10 animals in each group，namely the model group，Xuezhikang group，Liputuo group，and low，medium and high-dose groups of JQHP. The JQHP low-dose，medium-dose and high-dose rats were intragastrically daministered with 0.4，0.8，1.6 g·kg^-1，respectively， rats in Liputuo group with Liputuo 2 mg·kg^-1，rats in Xuezhikang group with Xuezhikang 0.1 g·kg^-1. The rats in the normal group and the model group were intragastrically administered with the same amount of distilled water. Stool were collected after continuous gavaging for 8 weeks，16S rRNA gene sequencing was performed，blood was collected from the abdominal aorta to detect blood lipids，and the liver tissue and ileum tissue were collected for hematoxylin and eosin （HE） staining for pathomorphological observation. Result:Compared with the normal group，the total cholesterol （TC），triglyceride （TG），and low-density lipoprotein （LDL-C） in the model group were significantly increased，while the high-density lipoprotein （HDL-C） was decreased （<italic>P</italic><0.01）. Compared with the model group，TC and TG values were decreased significantly in Xuezhikang group （<italic>P</italic><0.01），HDL-C value was increased （<italic>P</italic><0.05），and in the Liputuo group TC and TG were decreased significantly （<italic>P</italic><0.01）. Compared with the model group，the middle-dose group of JQHP had a certain alleviating effect on liver steatosis and could reduce the infiltration of inflammatory cells. The JQHP could improve the proliferation of lymphoid tissues in the ileal structure，and the middle-dose group has the most significant effect. The results of Shannon curve showed that compared with the normal group，the middle-dose group of JQHP increased significantly （<italic>P</italic><0.01）. Compared with the model group，the middle and high-dose group of JQHP increased （<italic>P</italic><0.05，<italic>P</italic><0.01）. Compared with the middle-dose group of JQHP，the other drug group decreased （<italic>P</italic><0.05，<italic>P</italic><0.01）. Principal component diversity analysis（PCA） showed that the diversity and abundance in the middle-dose JQHP group were higher than those in other drug groups. In linear discriminant analysis（LDA），compared with the normal group，Bacteroidia，Ruminococcaceae，<italic>Bacteroides </italic>S24-7，and <italic>Rumenococcus </italic>UCG-005 were down-regulated in the model group（<italic>P</italic><0.01），while the orders of Desulfovibrionales，Erysipelotrichales and<italic> </italic>Lachnospiraceae were up-regulated in the model group （<italic>P</italic><0.05，<italic>P</italic><0.01）. Compared with the model group，the Bacteroidia，Ruminococcaceae，<italic>Bacteroides</italic> S24-7，and <italic>Rumencoccus</italic> UCG-005 in the middle-dose JQHP group were increased （<italic>P</italic><0.05，<italic>P</italic><0.01），and the orders of Erysipelotrichales were decreased （<italic>P</italic><0.01）. Compared with the middle-dose JQHP group，Bacteroidia，Ruminococcaceae，<italic>Bacteroides</italic> S24-7，and <italic>Rumencoccus</italic> UCG-005 in other drug groups were reduced（<italic>P</italic><0.05，<italic>P</italic><0.01），and the order of Erysipelotrichales and Lachnospiraceae were increased （<italic>P</italic><0.05，<italic>P</italic><0.01）. Conclusion:JQHP can regulate the abundance and diversity of the gut microbiota，improve the state of liver tissue and ileum mucosa，regulate blood lipid levels，and restore the normal intestinal ecological environment. It may be related to the regulation of inflammation-related gut microbiota in order to restore the balance of the gut-liver axis，and the middle-dose JQHP group has the best effect.

Effects of Bupleurum chinense on the diversity of intestinal flora in depressed mice / 中国中药杂志

Bupleuri Radix, serving as the sovereign medicinal in many antidepressant compound preparations, has been proved effective in treating depression in mice, but its effect on the intestinal flora remains unclear. The present study aimed to investigate the effects of Bupleurum chinense(one of the original materials of Bupleuri Radix) on the behaviors and the diversity of intestinal flora of depressed mice. A depression mouse model was induced by repeated social defeat stress. Specifically, C57 BL/6 J male mice were exposed to the attack from the CD-1 mice. Then, C57 BL/6 J male mice were divided into a depression group and a B. chinense group, with normal saline and B. chinense administered(ig) respectively. Sucrose preference test and tail suspension test were conducted during and after the experiment respectively, to analyze the effects of B. chinense on the behaviors of the depressed mice. The feces were collected after the experiment. The V3-V4 16 S rDNA regions of intestinal flora of mice in each group were sequenced by Ion S5 TMXL for the analysis of the number of operational taxonomic units(OTUs), richness, alpha and beta diversity indexes, and differential phyla and genera. The results indicated that B. chinense could decrease depressive-like behaviors of mice, increase sucrose preference, and shorten the time of immobility in tail suspension test. After B. chinense intervention, the relative abundance of Firmicutes was significantly decreased, while that of Bacteroidetes was increased at the phylum level. At the genus level, the relative abundance of Lactobacillus and Lachnoclostridium decreased(P<0.05), while that of Bacteroides, Alistopes, etc. was elevated(P<0.05). The findings demonstrate that B. chinense can regulate the intestinal flora and improve the depressive-like behaviors of mice with depression.

Screening and identification of potassium-dissolving bacteria from different rhizosphere soil of Paris polyphylla var. yunnanensis / 中国中药杂志

The study aiming at exploring the potassium-dissolving capacity of rhizosphere potassium-dissolving bacteria from diffe-rent sources and screen the strains with high potassium-dissolving ability, so as to lay a theoretical foundation for cultivation and quality improvement of Paris polyphylla var. yunnanensis sources. The rhizosphere soil of 10 wild and transplanted species from Yunnan, Sichuan and Guizhou provinces was used as the research object. Potassium-dissolving bacteria were isolated and purified, and their potassium-dissolving capacity was determined by flame spectrophotometry, and identified by physiological, biochemical and molecular biological methods. Twenty-six potassium-dissolving bacteria were purified and 13 were obtained from wild and transplanted strains respectively. It was found through the determination of potassium-dissolving capacity that the potassium-dissolving capacity of 26 strains was significantly different, and the mass concentration of K~+ in the fermentation broth were 1.04-2.75 mg·L~(-1), the mcentration of potassium were 0.01-1.82 mg·L~(-1). The strains were identified as Bacillus, Agrobacterium rhizome and Staphylococcus by physiological, biochemical and 16 S rDNA molecular methods, among them Bacillus amylolyticus(4 strains) was the dominant bacterium of Bacillus. The physiology and biochemistry of rhizosphere potassium-dissolving bacteria in P. polyphylla var. yunnanensis rhizosphere were diffe-rent, and the living environment were different, so the potassium-dissolving capacity also changed. Strain Y4-1 with the highest potassium decomposability was Bacillus amylolytic with a potassium increase of 1.82 mg·L~(-1). The potassium-dissolving ability and the distribution of potassium-dissolving bacteria were different in various habitats. The screening of potassium-dissolving bacteria provided a new strain for the preparation of microbial fertilizer. It is expected that B. amyloidococcus Y4-1 can be used as an ideal strain to cultivate mycorrhizal seedlings of P. polyphylla var. yunnanensis.

Study on Difference of Intestinal Flora in Patients with Primary Insomnia with Different TCM Syndromes / 中国中医药信息杂志

Objective To explore structural differences of intestinal flora in primary insomnia patients with different TCM syndromes through the high-throughput 16S rDNA sequencing analysis. Methods Totally 65 patients with primary insomnia were divided into 22 patients with syndrome of liver depression transforming into fire, 17 patients with deficiency of both heart and spleen syndrome, 26 patients with syndrome of hyperactivity of fire due to yin deficiency, with 47 cases of healthy people as the control group. The fecal flora structure of the subjects was analyzed by high-throughput 16S rDNA sequencing. QIIME software and R language stats package were used to analyze the diversity of flora. Results Totally 1226 different operational taxonomic units (OUTs) were obtained, and there were 180 significant differences among the 4 groups (P<0.05), indicating that the samples were rich in microbial colonies. The mapped reads in group of liver depression transforming into fire and hyperactivity of fire due to yin deficiency were more than the group of deficiency of both heart and spleen and the control group (P<0.05). Unweighted UniFrac analysis showed that the difference among groups was remarkably greater than the difference within group, and the grouping was statistically significant (R=0.103, P=0.002). It suggested that the diversity of intestinal flora was highly correlated with different TCM syndromes of insomnia. There were a total of 57 genera found significant differences among the different groups at the genus level (P<0.05), and 115 species at all species level. The dominant flora of the control group were prevotella, megamonas, clostridium Ⅺ (clostridium ⅩⅧ), weissella, and alloprevotella; The dominant flora of liver depression transforming into fire syndrome were phascolarctobacterium, flavonifractor, eggerthella, and bilophila; The dominant flora of deficiency of both heart and spleen syndrome were sphingomonas and methylobacterium; The dominant flora in hyperactivity of fire due to yin deficiency syndrome group were bacteroides, parabacteroides, parasutterella, butyricimonas, odoribacter. Conclusion The patients with primary insomnia have abundant intestinal flora diversity and diverse flora structure, which may affect the occurrence, development and outcome of different TCM syndromes.

Massilia varians Isolated from a Clinical Specimen

We report a case of Massilia varians isolated from a deep finger wound following orthopedic surgery on an immunocompetent patient. The bacterium was identified by 16S rDNA sequence analysis. This is the first case of M. varians isolated from a clinical specimen since the first report in 2008.

Morphometric and molecular characterization of fungus Pestalotiopsis using nuclear ribosomal DNA analysis.

Taxonomy of the fungus Pestalotiopsis based on morphological characters has been equivocal. Molecular characterization of ten Pestalotiopsis species was done based on nuclear ribosomal DNA internal transcribed spacer (ITS) amplifications. Results of the analyses showed that species of genus Pestalotiopsis are monophyletic. We report ITS length variations, single nucleotide polymorphisms (SNPs) and insertions/deletions (INDELS) among ten species of Pestalotiopsis that did not cause any phylogenetic error at either genus or species designation levels. New gene sequences have been assigned (Gen Accession numbers from HM 190146 to HM 190155) by the National Centre for Biotechnology Information, USA.

Evaluation of foliar fungal endophyte diversity and colonization of medicinal plant Luehea divaricata (Martius et Zuccarini)

Endophyte microorganisms are organisms that live inside plants without causing any apparent damage to their hosts. Since all plants exhibit endophyte microorganisms, it is believed that mutual association is of great importance in nature. Luehea divaricata (Martius & Zuccarini), known popularly in Brazil as agoita-cavalo, is a big-sized tree with a wide distribution in the country that possesses medicinal qualities for: dysentery, leucorrhea, rheumatism, blennorrhoea, tumors, bronchitis, and depuration. This research aims at isolating and molecularly characterizing fungi isolates from L. divaricata by sequence analysis of ITS1-5.8S-ITS2 rDNA. Further, the colonization of endophyte in the host plant by Light and Scanning Electron Microscopy will also be investigated. Whereas, genera Alternaria, Cochliobolus, Diaporthe, Epicoccum, Guignardia, Phoma, and Phomopsis, were identified; rDNA sequence analysis revealed intra-species variability among endophyte isolates of the genus Phomopsis sp. Light and Scanning Electron Microscopy techniques showed the presence of endophyte fungi inside L. divaricata leaves, inhabiting inter- and intra-cellular spaces. These types of extensive colonization and dissemination were reported throughout all the leaf parts in palisade parenchyma, esclerenchyma, spongy parenchyma, adaxial epidermis, and vascular bundle indicating colonization of endophytes in múltiple structural sub-niches in the host plant.

Virulence Characteristics of Sucrose-fermenting Vibrio vulnificus Strains / 대한진단검사의학회지

We identified 6 sucrose-fermenting Vibrio vulnificus strains and examined their virulence characteristics. They were all encapsulated, motile, capable of producing toxins and utilizing transferrin-bound iron, cytotoxic to cultured cells, and virulent enough to kill mice. They could be definitely identified only by genetic identification methods such as PCR, and not by conventional culture-based identification methods such as API 20E (bioMerieux, France). These results indicate that it is essential to adopt genetic approaches as early as possible in order to avoid misdiagnosis of such strains, especially in clinical situations.

A Case of Streptococcus gallolyticus subsp. gallolyticus Infective Endocarditis with Colon Cancer: Identification by 16S Ribosomal DNA Sequencing / 대한진단검사의학회지

Although the association between Streptococcus bovis endocarditis and colon carcinoma is well known, very few cases of S. bovis infection associated with underlying malignancies have been reported in Korea. The S. bovis group has been recently reclassified and renamed as Streptococcus gallolyticus and Streptococcus infantarius subspecies under a new nomenclature system. We report a case of infective endocarditis with colon cancer caused by S. gallolyticus subsp. gallolyticus (previously named S. bovis biotype I). A 59-yr-old woman presented with a 1-month history of fever. Initial blood cultures were positive for gram-positive cocci, and echocardiography showed vegetation on mitral and aortic valves. Antibiotic treatment for infective endocarditis was started. The infecting strain was a catalase-negative and bile-esculin-positive alpha-hemolytic Streptococcus susceptible to penicillin and vancomycin. The strain was identified as S. gallolyticus subsp. gallolyticus with the use of the Vitek 2 GPI and API 20 Strep systems (bioMerieux, USA). The 16S rDNA sequences of the blood culture isolates showed 100% homology with those of S. gallolyticus subsp. gallolyticus reported in GenBank. The identification of the infecting organism, and the subsequent communication among clinical microbiologists and physicians about the changed nomenclature, led to the detection of colon cancer. The patient recovered after treatment with antibiotics, valve surgery, and operation for colon cancer. This is the first report of biochemical and genetic identification of S. gallolyticus subsp. gallolyticus causing infective endocarditis associated with underlying colon cancer in a Korean patient.

Molecular analysis of endophytic bacteria from the genus Bacillus isolated from tropical maize (Zea mays L. ) / Análise molecular de bactérias endofíticas do gênero Bacillus isoladas de milho tropical (Zea mays L. )

Endophytic bacteria play an important role in agriculture by improving plant performance and adaptation against biotic and abiotic stresses. In the present study molecular methods were used for identifying Bacillus endophytic bacteria isolated from Brazilian sweet corn. SDS-PAGE of wholecell protein extract of fortytwo isolates revealed a high number of scrutinable bands. Twenty-four isolates were identified in nine different groups of duplicated bacteria and eighteen were identified as unique. Some high-accumulated polipeptides with variable length were observed in almost isolates. Partial sequencing of 16S ribosomal gene revealed that all isolates are Bacillus sp. and among thirteen isolates with similar protein profiles, two were different strains. Among the forty-two isolates identified by rDNA sequencing, Bacillus subitilis and B. pumilus were the most frequenty species (15 and 12 isolates, respectively) followed by B. licheniformes (7 isolates), B. cereus (5 isolates) and B. amiloliquefascens (3 isolates). According to present results, SDS-PAGE technique could be used as a fast and cheap first tool for identifying interspecific variation in maize endophytic bacterial collections while rDNA sequencing could be applied for analyzing intraspecific variation among isolates with similar protein profile as well as for taxonomic studies.

Bactérias endofíticas desempenham papel importante na agricultura, melhorando a performance e adaptação de plantas contra estresses bióticos e abióticos. No presente estudo, métodos moleculares foram empregados para identificar bactérias endofíticas do gênero Bacillus isoladas de cultivares de milho doce brasileiro. SDS-PAGE de extratos protéicos totais de quarenta e dois isolados revelaram elevado número de bandas escrutináveis. Vinte e quatro isolados formaram nove grupos diferentes de réplicas bactérianas e dezoito foram considerados como únicos. Entre os isolados, alguns polipeptídios, de tamanhos variados, foram altamente acumulados. Seqüenciamento parcial do gene ribosomal 16S revelou que todos os isolados pertencem ao gênero Bacillus e que, entre treze isolados com padrão protéico similar, dois eram linhagens diferentes. Entre os quarenta e dois isolados identificados por seqüenciamento de rDNA, Bacillus subtilis e B. pumilus foram mais frequentes (15 e 12 isolados, respectivamente), seguido por, B. licheniformes (7 isolados), B. cereus (5 isolados) e B. amiloliquefascens (3 isolados). Baseado nos resultados, conclui-se que a técnica de SDS-PAGE poderá ser usada como primeiro procedimento, rápido e barato, para identificar variação inter-específica em coleções de bactérias endofíticas isoladas do milho, enquanto o método de seqüenciamento de rDNA poderá ser aplicado para analisar variações intra-específica entre isolados com padões similares de proteínas e estudos de taxonomia.

Spectrum of Lactobacillus species present in healthy vagina of Indian women.

Background & objectives: Lactobacilli are depleted in vagina of women suffering from recurring episodes of bacterial vaginosis with vaginal pH >5. With the objective of making available probiotic lactobacilli for replenishment in such women, a study was undertaken to isolate and characterize the Lactobacilli present in women with eco-healthy vagina in Delhi. No information is so far available on the species of Lactobacilli resident in vagina of women in India. Methods: Vaginal swabs were taken from 80 women with informed consent after ethical approval and grown in MRS broth. Gram-positive, catalase-negative bacilli generating about 200 bp amplicon by PCR with Lactobacillus genus specifi c primers were further characterized by employing species specifi c primers followed by sequencing of 16S rDNA. Isolates of the same species were differentiated by random amplifi ed polymorphic DNA (RAPD) profi les. Results: The predominant species isolated were L. reuteri present in 26 (32.5%) women, L. fermentum in 20 (25%), and L. salivarius in 13 (16.25%) women. Sequencing of 16S rDNA of 20 isolates showed that except for two isolates of L. plantarum, sequences of the remaining agreed well with PCR identifi cation. None of the isolates had similar RAPD profi le. Interpretation & conclusions: Our fi ndings showed lactobacilli species present in healthy vagina of women in India differ from those reported from other countries. This information would be useful to development of probiotic tablets seeking to replenish the missing lactobacilli for reproductive health of women in India.

A Mycological and Molecular Biological Study of Malassezia dermatis Isolated from Korean / 대한피부과학회지

BACKGROUND: Malassezia (M.) yeasts are lipophilic fungi which are regarded as normal flora of the skin, and are recovered in 75~98% of healthy adults. Gueho et al reclassified the Malassezia yeasts into 7 species (M. furfur, M. obtusa, M. globosa, M. slooffiae, M. sympodialis, M. pachydermatis, M. restricta) on the basis of molecular biology and by employing an interdisciplinary approach of morphology, microstructurology and physiology. Recently novel species of the genus Malassezia have been discovered as a result of molecular analysis. But there are no additional reports in Korea regarding newly reported Malassezia species because most identification and classification of Malassezia in Korea depend on classical methods and research on molecular biologic application is insufficient. OBJECTIVE: Five clinical isolates of M. dermatis were isolated from the skin of healthy subjects without skin disease or seborrheic dermatitis patients using molecular biology techniques for the first time in Korea. Hence the present study describes mycological and molecular biological characteristics of these five isolates as a novel species of M. dermatis. METHODS: Morphological and biochemical analyses, such as colony morphologies, microscopic morphologies and physiological characteristic were done targeting 5 clinical isolates of M. dermatis. Molecular techniques, namely, 26S rDNA PCR-RFLP, 26S rDNA and internal transcribed spacer region 1 (ITS1) sequencing, were done for identification and phylogenetic systematic analysis. RESULTS: Five clinical isolates of M. dermatis showed positive in the catalase test. No growth is obtained on Sabouraud's dextrose agar (SDA) without lipid supplementation but all grew in 0.5% Tween 60 and 0.1% Tween 80 added 2% glucose/1% peptone culture medium. Round and ellipsoidal yeast cells and budding of the yeast cells were observed under microscope, resembling M. sympodialis, M. furfur, and M. nana. The 26S rDNA PCR-RFLP pattern showed the same pattern as M. dermatis (JCM 11348), the standard strain. 26S rDNA and ITS1 sequencing were performed for exact identification, showing 99% accordance with M. dermatis (AB070361), and M. dermatis (AB070356), confirming the species to be new, the first to be reported in Korea. Phylogenetic trees based on the D1/D2 domains of the 26S rDNA sequences and nucleotide sequences of the ITS 1 region showed that the isolates were conspecific and belonged to the genus Malassezia and crusted with M. sympodialis. CONCLUSION: Taking a molecular biological classification approach, we have successfully isolated 5 cases of M. dermatis-the first in Korea. Although it is not known whether M. dermatis plays a role in Malassezia-related skin disease, this species was part of the microflora in both patients with seborrheic dermatitis and healthy subjects.

A Case of Fungal Keratitis Caused by Colletotrichum Species / 대한임상미생물학회지

Colletotrichum is mainly a fungal pathogen of plants, but sporadic cases of human infection have been reported recently. Most of them are fungal keratitis and only a few cases have been reported worldwide. A 63-year-old female farmer developed foreign body sensation and watering in her left eye following trauma by rice leaves. At presentation, her visual acuity decreased and corneal ulcer and inflammation in anterior chamber were observed on a slit lamp examination. Numerous hyphae were found on Gram stain and a rapidly growing mold with cup-shaped acervuli and falcate and nonseptate conidia was observed on fungal culture. As morphological findings did not lead to definite differentiation of the organism, sequencing of the D1-D2 domain of 28S rDNA was performed. It proved to be Colletotrichum species and the patient was treated with amphotericin and natamycin eye drop, but complicated by acute glaucoma. This is the first report of Colletotrichum keratitis in Korea and suggests that its infection should be considered in patients with fungal keratitis.

Molecular and Morphological Characterization of Green Mold, Trichoderma spp. isolated from Oyster Mushrooms

Isolates of Trichoderma spp. collected from Pleurotus ostreatus and P. eryngii beds, which included loosened substrate compactness and development of green colour, were grouped into three species. The occurrence of different species of Trichoderma was as T. cf. virens (70.8%), T. longibrachiatum (16.7%) and T. harzianum (12.5%). The conidia of Trichoderma spp. were ellipsoidal, obovoid and phialides were bowling pins, lageniform and the length of phialides was 3.5~10.0 x 1.3~3.3 micromm. Phialides of T. cf. virens and T. harzianum were tending clustered, but it was solitary disposition in T. longibrachiatum. T. cf. virens was characterized by predominantly effuse conidiation, sparingly branched, and fertile to the apex and it was penicillate type. RAPD analysis could detect variability amongst three different species of Trichoderma using two newly designed URP-primers. However, intra-specific variation could not be detected in all the isolates except for rDNA sequence data classified Trichoderma isolates into three distinct groups representing three species. The profiles of rDNA sequences of isolates representing a species showed high similarity in T. cf. virens and T. harzianum. However, there was a variation in rDNA sequences of isolates representing T. longibrachiatum. The results of present study reveals that molecular techniques of RAPD and rDNA sequencing can greatly aid in classification based on morphology and precise identification of fast evolving species of Trichoderma.

Differentiation of Mycobacterium avium Complex Isolated in Korea by DT1-BT6 PCR

Recently, selective PCR method using DT1 and DT6 sequences was introduced to identify and differentiate the Mycobacterium avium complex (MAC) into M. intracellulare and M. avium. We applied this method to 49 MAC clinical isolates identified by biochemical tests. They were differentiated into 39 strains of M. intracelluare and 10 strains of M. avium. Compared to those results obtained by 16S rDNA sequencing, DT1-DT6 PCR method showed 100% specificity. While the sensitivity of DT6 PCR for M. avium was 100%, that of DT1 PCR for M. intracellulare was 84.6%. These results show heterogeneity of M intracellulare Korea clinical isolates from Korea. In conclusion, although the in-house DTl-DT6 PCR is an easy and convenient method in differentiating MAC members, other methods such as 16S rDNA sequencing analysis should be performed for the correct identification, especially of M intracellulare.