Multiple self-splicing introns in the 16S rRNA genes of giant sulfur bacteria

The gene encoding the small subunit rRNA serves as a prominent tool for the phylogenetic analysis and classification of Bacteria and Archaea owing to its high degree of conservation and its fundamental function in living organisms. Here we show that the 16S rRNA genes of not-yet-cultivated large sulfur bacteria, among them the largest known bacterium Thiomargarita namibiensis, regularly contain numerous self-splicing introns of variable length. The 16S rRNA genes can thus be enlarged to up to 3.5 kb. Remarkably, introns have never been identified in bacterial 16S rRNA genes before, although they are the most frequently sequenced genes today. This may be caused in part by a bias during the PCR amplification step that discriminates against longer homologs, as we show experimentally. Such length heterogeneity of 16S rRNA genes has so far never been considered when constructing 16S rRNA-based clone libraries, even though an elongation of rRNA genes due to intervening sequences has been reported previously. The detection of elongated 16S rRNA genes has profound implications for common methods in molecular ecology and may cause systematic biases in several techniques. In this study, catalyzed reporter deposition-fluorescence in situ hybridization on both ribosomes and rRNA precursor molecules as well as in vitro splicing experiments were performed and confirmed self-splicing of the introns. Accordingly, the introns do not inhibit the formation of functional ribosomes.     

Antibiotic effects on bacterial profile in osteonecrosis of the jaw

Oral Diseases (2011) 18 , 85–95 Objective: Oral infection is considered to play a critical role in the pathogenesis of bisphosphonate‐related osteonecrosis of the jaw (BRONJ), and antibiotic therapy has become a mainstay of BRONJ therapy. This study was aimed to investigate the effect of antibiotics on bacterial diversity in BRONJ tissues. Materials and methods: The bacterial profile from soft tissues associated with the BRONJ lesion was determined using 16S rRNA‐based denaturing gradient gel electrophoresis (DGGE) and sequencing. Twenty BRONJ subjects classified as stage 0–2 were enrolled in this study, and patient groups were divided into an antibiotic cohort (n = 10) treated with systemic antibiotic and a non‐antibiotic cohort (n = 10) with no prior antibiotic therapy. Results: The DGGE fingerprints indicated no significant differences in bacterial diversity of BRONJ tissue samples. Patients on antibiotics had higher relative abundance of phylum Firmicutes with bacterial species, Streptococcus intermedius, Lactobacillus gasseri, Mogibacterium timidum, and Solobacterium moorei, whereas patients without antibiotics had greater amounts of Parvimonas micra and Streptococcus anginosus. Thirty percent of bacterial populations were uncultured (yet‐to be cultured) phylotypes. Conclusion: This study using limited sample size indicated that oral antibiotic therapy may have a limited efficacy on the bacterial population associated with BRONJ lesions.     

Using high throughput sequencing to explore the biodiversity in oral bacterial communities

High throughput sequencing of 16S ribosomal RNA gene amplicons is a cost-effective method for characterization of oral bacterial communities. However, before undertaking large-scale studies, it is necessary to understand the technique-associated limitations and intrinsic variability of the oral ecosystem. In this work we evaluated bias in species representation using an in vitro-assembled mock community of oral bacteria. We then characterized the bacterial communities in saliva and buccal mucosa of five healthy subjects to investigate the power of high throughput sequencing in revealing their diversity and biogeography patterns. Mock community analysis showed primer and DNA isolation biases and an overestimation of diversity that was reduced after eliminating singleton operational taxonomic units (OTUs). Sequencing of salivary and mucosal communities found a total of 455 OTUs (0.3% dissimilarity) with only 78 of these present in all subjects. We demonstrate that this variability was partly the result of incomplete richness coverage even at great sequencing depths, and so comparing communities by their structure was more effective than comparisons based solely on membership. With respect to oral biogeography, we found inter-subject variability in community structure was lower than site differences between salivary and mucosal communities within subjects. These differences were evident at very low sequencing depths and were mostly caused by the abundance of Streptococcus mitis and Gemella haemolysans in mucosa. In summary, we present an experimental and data analysis framework that will facilitate design and interpretation of pyrosequencing-based studies. Despite challenges associated with this technique, we demonstrate its power for evaluation of oral diversity and biogeography patterns.     

A Canadian Working Group report on fecal microbial therapy: microbial ecosystems therapeutics

A working group from across Canada comprised of clinician and basic scientists, epidemiologists, ethicists, Health Canada regulatory authorities and representatives of major funding agencies (Canadian Institutes of Health Research and the Crohn's and Colitis Foundation of Canada) met to review the current experience with fecal microbial therapy and to identify the key areas of study required to move this field forward. The report highlights the promise of fecal microbial therapy and related synthetic stool therapy (together called 'microbial ecosystems therapeutics') for the treatment of Clostridium difficile colitis and, possibly, other disorders. It identifies pressing clinical issues that need to be addressed as well as social, ethical and regulatory barriers to the use of these important therapies.     

Immune activation and increased IL‐21R expression are associated with the loss of memory B cells during HIV‐1 infection

Abstract. Ruffin N, Lantto R, Pensieroso S, Sammicheli S, Hejdeman B, Rethi B, Chiodi F (Karolinska Institutet, Stockholm; and South Hospital, Stockholm, Sweden). Immune activation and increased IL‐21R expression are associated with the loss of memory B cells during HIV‐1 infection. J Intern Med 2012; 272: 492–503. Objectives. Microbial translocation and chronic immune activation were previously shown to be associated with impairment of T cell functions and disease progression during infection with human immunodeficiency virus type‐1 (HIV‐1); however, their impact on B cell function and number remains unknown. By measuring markers of immune activation and molecules involved in apoptosis regulation, we have evaluated the association between microbial translocation and loss of memory B cells in HIV‐1‐infected patients. Methods. Markers of activation [the interleukin‐21 receptor (IL‐21R) and CD38] and apoptosis (Bim, Bcl‐2 and annexin V) were measured in B cell subpopulations by multicolour flow cytometry. Levels of soluble CD14 (sCD14) and lipopolysaccharide (LPS), measures of microbial translocation, were determined in plasma. Purified B cells were also exposed in vitro to Toll‐like receptor (TLR) ligands. Results. IL‐21R expression was higher in cells from HIV‐1‐infected patients, compared with control subjects, with the highest levels in nontreated patients. An inverse correlation was observed between IL‐21R expression and percentages of circulating resting memory (RM) B cells. IL‐21R‐positive memory B cells were also more susceptible to spontaneous apoptosis and displayed lower levels of Bcl‐2. It is interesting that the levels of sCD14, which are increased during HIV‐1 infection, were correlated with decreased percentages of RM B cells and high IL‐21R expression. In the plasma of HIV‐1‐infected individuals, a correlation was found between sCD14 and LPS levels. TLR activation of B cells in vitro resulted in IL‐21R up‐regulation. Conclusions. Microbial translocation and the associated immune activation during HIV‐1 infection may lead to high expression levels of the IL‐21R activation marker in RM B cells, a feature associated with increased apoptosis and a reduced number of these cells in the circulation.     

老年糖尿病患者泌尿道感染的病原谱及药敏分析

目的了解老年糖尿病患者泌尿道感染的常见病原菌及药敏情况。方法采用细菌培养鉴定、药敏试验方法,对老年糖尿病合并泌尿道感染患者的70株中段尿分离菌株进行统计学分析。结果70株病原菌中,革兰氏阴性菌占65．7％;革兰氏阳性菌占21．4％;真菌占12．9％。革兰氏阴性菌中大肠埃希菌最多;革兰氏阳性菌中溶血葡萄球菌最多,未发现对万古霉素耐药的肠球菌。真菌以白假丝酵母菌为主。革兰氏阴性菌敏感性高的抗生素：美洛培南和亚胺培南、头孢吡肟、阿米卡星、哌拉西林／他唑巴坦;革兰氏阳性菌中,溶血葡萄球菌对夫西地酸、利福平、米诺环素、替考拉宁、万古霉素、呋喃妥因、喹奴普汀／达福普汀高度敏感,粪肠球菌及屎肠球菌仅对替考拉宁、万古霉素敏感性较高。白假丝酵母菌对氟康唑、伊曲康唑等药物敏感性较高。结论老年糖尿病患者合并泌尿道感染,常见的致病菌为革兰氏阴性菌。因细菌耐药率高,应及早进行中段尿培养及药敏试验。     

维医异常黑胆质体液质高血压人群和正常黑胆质体液质健康人群肠道菌群多样性分析

目的了解维吾尔医学异常黑胆质体液质高血压人群和正常黑胆质体液质健康人群肠道菌群结构多样性的差异。方法以20例异常黑胆质体液质高血压患者和20例正常黑胆质体液质者粪便中的细菌总DNA为模板,以细菌通用引物进行16SrDNA的V6～V8区域PCR扩增和DGGE分析;切取特异条带,进行克隆、测序和核酸序列比对。结果两组体液质人群检测的共有优势条带为厚壁菌门(Firmicutes)和拟杆菌门(Bacteroidetes),检测的差异条带为肠道产丁酸细菌和疣微菌门(Verrucomicrobia),其在两组中出现的频率差异均有统计学意义(P<0.05)。结论 PCR-DGGE技术检测异常黑胆质体液质高血压人群与正常黑胆质体液质人群肠道菌群的多样性存在差异,其中直肠真杆菌和多形拟杆菌的存在可能会导致异常黑胆质体液质高血压的形成和发展。     

中药外源性污染物检测技术的现代研究

中药外源性污染物残留现已成为引起中药不良反应的重要原因之一,按照其来源不同主要包括农/兽药残留、重金属污染、真菌毒素残留、病原微生物污染及其他有机污染物残留。对中药的主要外源性污染物的检查对象及检测方法的现代研究进行综述,为补充、完善、提升中药质量安全体系提供参考和依据。     

百两金皂苷A的微生物转化及其产物的细胞毒活性研究

目的利用燕麦曲霉Aspergillus avenaceus AA 3.4454对百两金皂苷A进行微生物转化,并对转化产物进行细胞毒活性研究。方法将百两金皂苷A放置燕麦曲霉液体培养基中,28℃、160 r/min条件下共培养3 d后,利用多种柱色谱分离转化产物,采用核磁共振等波谱手段鉴定结构;采用MTT法测定转化产物对肿瘤细胞系的细胞毒活性。结果从百两金皂苷A的燕麦曲霉液体发酵液中分离出3个主要转化产物,其结构分别鉴定为西克拉明皂苷元A-3β-O-{α-D-半乳吡喃糖基-(1→4)-[β-D-木吡喃糖基-(1→2)]-β-D-吡喃葡萄糖基-(1→4)-[β-D-吡喃葡萄糖基-(1→2)]-α-L-阿拉伯吡喃糖苷}(1)、西克拉明皂苷元A-3β-O-{α-D-半乳吡喃糖基-(1→4)-β-D-吡喃葡萄糖基-(1→2)-[β-D-木吡喃糖基-(1→2)-β-D-吡喃葡萄糖基-(1→4)]-α-L-阿拉伯吡喃糖苷}(2)、西克拉明皂苷元A-3β-O-{β-D-木吡喃糖基-(1→2)-β-D-吡喃葡萄糖基-(1→4)-[β-D-吡喃葡萄糖基-(1→2)]-[α-D-半乳吡喃糖基-(1→3)]-α-L-阿拉伯吡喃糖苷}(3)。结论首次利用燕麦曲霉对百两金皂苷A进行微生物转化并分离得到在糖链的不同位置增加了α-D型半乳糖的转化产物,3个转化产物均为新化合物,均具有一定的细胞毒活性,且转化产物1对大细胞肺癌细胞的细胞毒活性略优于底物。