Nitrogen removal techniques based on Anammox process are developing rapidly these years. The distribution and diversity of Anammox have become important research directions. A variety of Anammox have been detected till now, of which only Kuenenia and Brocadia are often detected in wastewater treatment systems. In addition, in a single niche there is only one type of Anammox bacteria. However, the distribution mechanism and transformation of Anammox bacteria in different niches are still ambiguous. Therefore, the distribution of Anammox in various conditions was summarized and analyzed in this article. And the key factors influencing the distribution of Anammox were concluded, including substrate concentration and the specific growth rate, sludge properties and microbial niche, the joint action and influence of multiple factors. The engineering significance research on the distribution and influencing factors of Anammox bacteria in the sewage system and proposed research prospects were expounded.
Ammonia ; chemistry ; Anaerobiosis ; Bacteria ; Nitrogen ; chemistry ; RNA, Ribosomal, 16S ; Sewage ; microbiology ; Waste Disposal, Fluid ; Waste Water

Ligustrazine is an important active ingredient of the traditional Chinese medicine Chuanxiong Rhizoma, but its content is a controversial topic. The endophytes of medicinal plants have the ability to produce the same active substances as the host, so this report focused on the endophytic Bacillus subtilis, to study the origin of ligustrazine in Chuanxiong Rhizoma preliminarily by inoculating the isolated endophytic B. subtilis to the Chuanxiong Rhizoma medium for solid state fermentation. Tissue grinding method was used to isolate the endogenetic B. subtilis. The morphological features, conventional physiological and biochemical reactions and 16S rRNA molecular techniques were combined to identify the endogenetic strains. Then, the strains that grew well in the medicinal matrix of Chuanxiong Rhizoma were screened out for further fermentation studies. The solid-state fermentation was performed at 37 °C for 30 d using Chuanxiong Rhizoma fermentation medium (40 g Chuanxiong Rhizoma powder, 100 mL sterile water, 121 °C, sterilization for 25 minutes). UPLC was used to detect the contents of ligustrazine, acetoin in the Chuanxiong Rhizoma fermentation medium and Chuanxiong Rhizoma. All the five strains were Gram-positive and had spores. Phylogenetic analysis of the 16S rRNA sequence showed that the endophytes were B. subtilis. The results of UPLC showed that ligustrazine was detected in the Chuanxiong Rhizoma fermentation medium inoculated with endogenetic B. subtilis LB3, LB3-2-1, LB4, LB5 and LB6-2, while not detected neither in blank Chuanxiong Rhizoma fermentation medium nor in Chuanxiong Rhizoma. This study showed that the endogenetic B. subtilis of Ligusticum chuanxiong Hort. can make use of Chuanxiong Rhizoma fermentation medium to produce ligustrazine. Endogenetic B. subtilis has a certain correlation with the accumulation of ligustrazine in Rhizoma Chuanxiong. We speculate that the ligustrazine may be derived from the catabolism of endogenetic B. subtilis in Ligusticum chuanxiong.
Bacillus subtilis ; Endophytes ; Fermentation ; Ligusticum ; chemistry ; microbiology ; Phylogeny ; Pyrazines ; analysis ; RNA, Ribosomal, 16S ; Rhizome ; chemistry

For the purpose of seeking new antibiotics, researchers usually modify the already-existing ones. However, this strategy has been extensively used and is close to its limits, especially in the case of aminoglycosides, and it is difficult to find a proper aminoglycoside antibiotic for novel modification. In this paper, we reported the design, synthesis, and evaluation of a series of 5-epi-neamine derivatives based on the structural information of bacterial 16S RNA A-site binding with aminoglycosides. Bioassay results showed that our design strategy was feasible. Our study offers a new way to search for structurally novel aminoglycosides. Meanwhile, our study provides valuable structure-activity relationship information, which will lead to better understanding and exploitation of the drug target, and improved development of new aminoglycoside antibiotics.
Aminoglycosides/chemistry* ; Anti-Bacterial Agents/chemistry* ; RNA, Ribosomal, 16S/metabolism* ; Structure-Activity Relationship ; Biological Assay

An anaerobic sequencing batch reactor (ASBR) was used to treat thermal denitration tail liquid and microbial community was studied. Activated sludge was taken from the reactor for scanning electron microscope analysis. The images showed that the dominant cells in the flora were oval cocci. Its diameter was about 0.7 μm. Through a series of molecular biology methods such as extracting total DNA from the sludge, PCR amplification, positive clone authentication and sequencing, we obtained the 16S rDNA sequences of the flora. Phylogenetic tree and clone library were established. The universal bacteria primers of 27F-1492R PCR amplification system obtained 85 clones and could be divided into 21 OTUS. The proportions were as follows: Proteobacteria 61.18%; Acidobacteria 17.65%; Chlorobi 8.24%; Chlorofexi 5.88%; Gemmatimonadetes 3.53%; Nitrospirae 2.35% and Planctomycetes 1.18%. The specific anammox bacterial primers of pla46rc-630r and AMX368-AMX820 PCR amplification system obtained 45 clones. They were divided into 3 OTUS. Candidatus brocadia sp. occupied 95.6% and unknown strains occupied 4.4%.
Ammonia ; chemistry ; Bacteria ; Phylogeny ; Polymerase Chain Reaction ; RNA, Ribosomal, 16S ; Sewage ; microbiology

Polyurethane (PUR) plastics is widely used because of its unique physical and chemical properties. However, unreasonable disposal of the vast amount of used PUR plastics has caused serious environmental pollution. The efficient degradation and utilization of used PUR plastics by means of microorganisms has become one of the current research hotspots, and efficient PUR degrading microbes are the key to the biological treatment of PUR plastics. In this study, an Impranil DLN-degrading bacteria G-11 was isolated from used PUR plastic samples collected from landfill, and its PUR-degrading characteristics were studied. Strain G-11 was identified as Amycolatopsis sp. through 16S rRNA gene sequence alignment. PUR degradation experiment showed that the weight loss rate of the commercial PUR plastics upon treatment of strain G-11 was 4.67%. Scanning electron microscope (SEM) showed that the surface structure of G-11-treated PUR plastics was destroyed with an eroded morphology. Contact angle and thermogravimetry analysis (TGA) showed that the hydrophilicity of PUR plastics increased along with decreased thermal stability upon treatment by strain G-11, which were consistent with the weight loss and morphological observation. These results indicated that strain G-11 isolated from landfill has potential application in biodegradation of waste PUR plastics.
Plastics/metabolism* ; Polyurethanes/chemistry* ; RNA, Ribosomal, 16S ; Bacteria/genetics* ; Biodegradation, Environmental

BACKGROUNDAccurate identification of bacterial isolates is an essential task in clinical microbiology. This study compared culturing to analyzing 16S rRNA gene sequences as methods to identify bacteria in clinical samples. We developed a key technique to directly identify bacteria in clinical samples via nucleic acid sequences, thus improving the ability to confirm pathogens.

METHODSWe obtained 225 samples from Beijing Tongren Hospital and examined them by conventional culture and 16S rDNA sequencing to identify pathogens. This study made use of a modified sample pre-treatment technique which came from our laboratory to extract DNA. 16S rDNA was amplified by PCR. The amplified product was sequenced on a CEQ8000 capillary sequencer. Sequences were uploaded to the GenBank BLAST database for comparison.

RESULTSAmong the positively cultivated bacterial strains, seven strains were identified differently by Vitek32 and by 16S rDNA sequencing. Twelve samples that were negative by standard culturing were determined to have pathogens by sequence analysis.

CONCLUSIONThe use of 16S rRNA gene sequencing can improve clinical microbiology by providing better identification of unidentified bacteria or providing reference identification of unusual strains.

Bacteria ; isolation & purification ; DNA, Ribosomal ; chemistry ; Humans ; Polymerase Chain Reaction ; RNA, Ribosomal, 16S ; genetics ; Sequence Analysis, DNA ; methods

An expanded-granular sludge bed (EGSB) reactor was set-up with artificial water by seeding a 60 d stored ANAMMOX sludge. The nitrogen removal efficiency of ANAMMOX enrichment culture in the reactor was determined. In addition, the main microbial populations and the relative abundance of ANAMMOX bacteria were investigated by molecular approaches. Results show that the maximum nitrogen removal rate was 3.0 kg-N·m(-3)·d(-1) after 185 d, and the ammonium and nitrite removal efficiencies were all over 85%. Analysis of 16S rRNA gene-cloning indicates that the main microbial population in the ANAMMOX enrichment culture was changed from Candidatus Brocadiafulgid and Candidatus Brocadia brasiliensis (0 day) to Candidatus Jettenia asiatica (185 day). Fluorescence in situ hybridization analysis shows that the relative abundance of ANAMMOX bacteria was increased from (57.69 ± 4.79)% to (83.32 ± 4.40)%. The results of qPCR further indicate that the gene copies of ANAMMOX bacteria in the granules were increased from 1.14 x 10(11) copies/g wet weight to 3.69 x 10(11) copies/g wet weight.
Ammonia ; chemistry ; Anaerobiosis ; Bacteria ; classification ; Bioreactors ; microbiology ; In Situ Hybridization, Fluorescence ; Nitrites ; chemistry ; Nitrogen ; chemistry ; RNA, Ribosomal, 16S ; Sewage ; microbiology

Marine Actinobacteria are emerging as new resources for bioactive natural products with promise in novel drug discovery. In recent years, the richness and diversity of marine Actinobacteria from the South China Sea and their ability in producing bioactive products have been investigated. The objective of this work is to isolate and identify bioactive secondary metabolites from a marine actinobacterium SCSIO 1934 derived from sediments of South China Sea. The strain was identified as a Streptomyces spieces by analyzing its 16S rDNA sequence. Streptomyces sp. SCSIO 1934 was fermented under optimized conditions and seven bioactive secondary metabolites were isolated and purified by chromatographic methods including colum chromatography over silica gel and Sephadex LH-20. Their structures were elucidated as 17-O-demethylgeldanamycin (1), lebstatin (2), 17-O-demethyllebstatin (3), nigericin (4), nigericin sodium salt (5), abierixin (6), respectively, by detailed NMR spectroscopic data (1H, 13C, COSY, HSQC and HMBC). This work provided a new marine actinobacterium Streptomyces sp. SCSIO 1934, capable of producing diverse bioactive natural products.
Anti-Bacterial Agents ; chemistry ; China ; DNA, Ribosomal ; chemistry ; genetics ; Geologic Sediments ; microbiology ; Oceans and Seas ; RNA, Ribosomal, 16S ; genetics ; Streptomyces ; chemistry ; classification ; genetics ; isolation & purification

OBJECTIVETo establish the specific 16S-23S rRNA gene spacer regions in different bacteria using polymerase chain reaction (PCR), restriction fragment length polymorphism (RFLP), DNA cloning and sequences analysis.

METHODSA pair of primers were selected from highly conserved sequences adjacent to the 16S-23S rRNA spacer region. Bacterial DNA from sixty-one strains of standard bacteria and corresponding clinical isolates representative of 20 genera and 26 species was amplified by PCR, and further analyzed by RFLP, DNA cloning and sequences analysis. Furthermore, all specimens were examined by bacterial culturing and PCR-RFLP analysis. The evaluation of these assays in practical clinic practice was also discussed.

RESULTSRestriction enzyme analysis revealed one, two or three bands or more observed among the 26 different standard strains. The sensitivity of PCR reached 2.5 colony-forming unit (CFU), and there was no cross reaction with human genomic DNA, fungus or virus. Fourteen species could be distinguished immediately by PCR, while another 10 species were further identified by Hinf I or Alu I digestion. The only difference between K.pneumoniae and E. durans was located at the site of the 779th nucleotide according to the sequence analysis and only XmaIII digestion could distinguish one from another. Of 42 specimens from septicemic neonates, 15 were identified as positive by blood culture at a rate of 35.7%. However, 27 specimens identified as positive by PCR, with a rate of 64.2%, a method significantly more effective than blood culture (P < 0.01). Of 6 cerebrospinal fluid (CSF) specimens, one tested positive for S.epidermidis was also positive by PCR, two culture negative were positive by PCR and diagnosed as S.epidermidis according to the DNA pattern. One positive for C.neoformans was negative by PCR. The other two specimens were negative by both PCR and culture.

CONCLUSIONSThe method of detecting bacterial 16S-23S rRNA spacer regions using PCR-RFLP techniques was specific, sensitive, rapid and accurate in providing a new technique for detecting pathogens in clinical bacterial infections.

Bacteria ; genetics ; isolation & purification ; DNA, Bacterial ; analysis ; chemistry ; DNA, Ribosomal ; analysis ; chemistry ; Genes, rRNA ; Humans ; Polymerase Chain Reaction ; Polymorphism, Restriction Fragment Length ; RNA, Ribosomal, 16S ; genetics ; RNA, Ribosomal, 23S ; genetics ; Sensitivity and Specificity ; Sequence Analysis, DNA

OBJECTIVETo establish the specific 16S-23S rRNA gene spacer regions pattern in different bacteria using polymerase chain reaction (PCR), restriction fragment length polymorphism (RFLP), DNA cloning and sequences analysis.

METHODSA pair of primers were selected from highly conserved sequences adjacent to the 16S-23S rRNA spacer region. Bacterial DNA of sixty-one strains of standard bacteria and corresponding clinical isolates representative of 20 genera and 27 species was amplified by PCR, and further studied by RFLP, DNA cloning and sequences analysis. Meanwhile, all specimens were examined by bacterial culturing and PCR-RFLP analysis.

RESULTSThe 27 different standard strains showed one, two, three or more than three bands. The sensitivity of PCR reached 2.5 colony-forming unit (CFU), and there was no cross reaction to the human, fungal or viral genomic DNAs. Fifteen species could be distinguished immediately by PCR, while another 10 species were further identified by Hinf I or Alu I digestion. Klebsiella pneumoniae (Kp) and Enterococcus durans (Ed) could not be differentiated from each other by Alu I or Hinf I digestion. The spacer sequences of the Kp and Ed were 908 bp and 909 bp, respectively, and they differed only at the site of the 779th nucleotide. The former was G, and the latter was A. The 760 - 790 bp sequence of Kp was as follows: CGACTGCACCGCCTCCTAC / GGCCGCGTATTC. The 760 - 790 bp sequence of Ed was as follows: CGACTGCAC CGCCTCCTAC / AGCCGCGTATTC. Only one enzyme XmaIII, could discriminate the two. The cleaving site of XmaIII is C downward arrow GGCCG. Kp DNA was cleaved into 778 bp and 130 bp fragments, while E. durans was not. Of 42 specimens with suspected septicemia, 15 were positive (35.7%) on blood culture, and 27 on PCR (64.29%). The positive rate of PCR was significantly higher than that of blood culture (P < 0.01). Of the six CSF specimens, one was positive for Staphylococcus epidermidis (Se) on culture as well as by PCR, while two specimens which were negative on cultures were positive by PCR and were diagnosed as Se according to its DNA pattern. One specimen was culture-positive for Cryptococcus neoformans (Cn) but was negative by PCR. The other two specimens were negative by both PCR and culture. Fifteen blood samples from healthy children were negative by both blood culture and PCR.

CONCLUSIONSThe method of detecting bacterial 16S-23S rRNA spacer regions using PCR-RFLP techniques was specific, sensitive, rapid and accurate in detecting pathogens in clinical bacterial infections.

Bacterial Infections ; diagnosis ; microbiology ; DNA, Bacterial ; chemistry ; genetics ; DNA, Ribosomal Spacer ; genetics ; Humans ; Polymerase Chain Reaction ; Polymorphism, Restriction Fragment Length ; RNA, Ribosomal, 16S ; genetics ; RNA, Ribosomal, 23S ; genetics ; Sequence Analysis, DNA