With the rapid development of genetic engineering and metabolic regulation, antisense technology displays its fascination to the world as a mild regulation genetic tool. Compared with other loss-of-function research methods (e.g. gene knockout), antisense technologies have advantages such as low cost, short period, and easy operation. It has been increasingly used in bacterial metabolic regulation as a powerful genetic tool. This review briefly summarized the latest progress and problems in antisense technologies that are recently used in metabolic engineering of bacteria, and compares the advantages and disadvantages of these technologies.
Bacteria ; genetics ; metabolism ; Genes, Bacterial ; Genetic Engineering ; Metabolic Networks and Pathways ; genetics ; Oligonucleotides, Antisense ; genetics ; RNA, Antisense ; genetics ; RNA, Catalytic ; genetics

OBJECTIVETo explore a new method of rapid and reliable diagnosis of bacterial infectious diseases such as purulent meningitis and septicemia.

METHODSA pair of universal primers and a set of probes (including universal fluorescence probe, Gram-positive probe and Gram-negative probe) were designed based on the bacterial highly conserved region of 16S rRNA gene. By using the FQ-PCR method, 12 standard strains, 23 clinical cultural isolations and the controls such as HBV, Cryptococcus histolyticus, Blastomyces albicans and human DNA were detected with the three kinds of probes. The correlation among the results of the three kinds of probes detection was analyzed.

RESULTSThe determination of 16S rRNA gene with FQ-PCR was a highly specific and sensitive method and not cross-reactive with human DNA, virus or fungi. The least amount of 10 copies of 16S rRNA gene which corresponded to 2 bacteria could be detected with FQ-PCR. Twelve standard strains and 23 clinical cultural isolations were detected by FQ-PCR with the three kinds of probes mentioned above. All samples presented positive results using the universal probe. The results of 16S rRNA gene detected by the Gramjpositive probe were positive to the 18 G+ strains. The results of 16S rRNA gene detected by the Gram-probe were positive to the 17 G- strains.

CONCLUSIONSThe FQ-PCR technique was established for bacteria quantifying and typing using the universal primer and the double type probes. This method was convenient and rapid in detecting, quantifying and typing bacteria, with a high specificity and sensitivity.

Fluorescence ; Genes, rRNA ; Humans ; Polymerase Chain Reaction ; methods ; RNA, Bacterial ; genetics ; RNA, Ribosomal, 16S ; genetics

The important role of intestinal microorganisms in human health has been widely confirmed. At present, most of the studies on intestinal microorganisms are based on amplification of the V3-V4 region of bacterial 16S rRNA gene, and little attention has been paid to archaea. In this study, a primer set which can amplify 16S rRNA gene of both bacteria and archaea at the same time was used. By comparing the community changes before and after probiotics intake, it showed that this primer set is suitable for analyzing the changes of human intestinal bacteria and archaea communities. The fecal samples of volunteers were collected, and the amplification and high-throughput sequencing were carried out by using bacterial primer set (B primer) and bacterial and archaeal universal primer (AB primer); several commonly used rRNA databases were used to determine the amplification ability of the primer set to bacteria and archaea. The results showed that AB primer could display the bacterial community amplified by B primer, and could obtain the sequence of common methanogenic archaea in intestinal tract. AB primer set can analyze the bacteria and archaea in the intestinal tract at the same time by only one amplification and sequencing, which can show the structure of intestinal microbial community more comprehensively, which is suitable for the research of intestinal microorganisms.
Archaea/genetics* ; Bacteria/genetics* ; DNA Primers ; DNA, Bacterial ; Humans ; Microbiota/genetics* ; Phylogeny ; RNA, Ribosomal, 16S/genetics*

The community structure and diversity of the gut microbiota are associated with human diseases. However, the analysis of different community structure might be influenced by experimental approaches such as the quality of DNA extraction. Therefore, evaluating the efficiency of different DNA extraction methods for specific intestinal species is a guideline for obtaining a comprehensive human gut microbial profile, which may assist the in-depth investigation into the structure of the gut microbial community. The aim of this study was to perform a comparative analysis of five different DNA extraction methods. With the aid of qPCR, the efficiency of five DNA extraction kits was evaluated in terms of the purity of the extracted DNA, the DNA concentration, and the abundance of genomic DNA extracted from specific intestinal species. The results showed that the kit Q gave the best extraction results, especially for Gram-positive bacteria such as Lactobacillus and Bifidobacterium. The average DNA concentration of the N kit was lower than that of the Q kit, but there was no significant difference between the two in terms of the purity. Compared to the other three commercial kits (M, PSP, TG), the efficiency of the N kit in extracting the genomic DNA of the specified microorganisms were the least different from those of the Q kit. In contrast, the DNA extracted by the M kit was of higher quality but of lower concentration, and was not very efficient for Gram-positive bacteria. The DNA extracted by the TG and PSP kits was inferior to the other validated kits in terms of the concentration, quality and bacterial abundance. These results provide a basis for the selection of genomic DNA extraction methods in microecological research experiments.
DNA/genetics* ; DNA, Bacterial/genetics* ; Feces/microbiology* ; Humans ; Microbiota/genetics* ; RNA, Ribosomal, 16S/genetics*

OBJECTIVETo establish a set of procedure for recovery and species identification of Legionella from the surface environmental water.

METHODSForty-four water samples were collected in eight parks of Guangzhou city from August to November in 2006. The bacteriologic examination was performed by cultivation on BCYEalpha plate, and 108 presumptive Legionella colonies were picked and their homogeneous relationship was analyzed by using an amplified fragment length polymorphism (AFLP) method. Species identification was carried out by latex agglutination test, biochemical characterization, analysis of cellular fatty acids composition, 16 S rRNA gene and mip gene sequencing.

RESULTSLegionella was recovered among 27 (61.36%) samples of all eight parks, and 31 different strains were identified from those 108 presumptive Legionella isolates by AFLP method, including 20 strains of L. pneumophila, five strains of L. feeleii, four strains of L. longbeachae, one strain of L. oakridgensis and one strain of L. sainthelensi, and L. pneumophila could be easily differentiated by phenotypic and biochemical characteristics, latex agglutination test or analysis of the cellular fatty acids composition . However, uncertain factors were existing in those phenotypic identification methods as compared to the sequence analysis.

CONCLUSIONThe taxonomic analysis of the Legionellae family should be dependent on the 16 S rRNA gene or mip gene.

Bacteriological Techniques ; DNA, Bacterial ; genetics ; Environmental Monitoring ; methods ; Legionella ; genetics ; isolation & purification ; RNA, Bacterial ; RNA, Ribosomal, 16S ; Water Microbiology

OBJECTIVETo type and group the Yersinia pestis strains isolated in China to clarify the geographical distribution of ribotypes of Yersinia pestis.

METHODSGenomic DNA of Yersinia pestis were digested with EcoR I, then hybridized with 16s-23s-5s rRNA gene probe.

RESULTSThese tested strains were divided into 3 ribotypes, the profiles obtained were relatively homogeneous, with most of them differed only by the presence or the absence of 1 - 2 restriction fragments. Ribotype A and B were the most common types, which distributed in a large area in China while ribotype C was the least, only limited to a small area. There was certain correlation between the ribotypes and the plague foci, usually only one ribotype was found in one plague foci.

CONCLUSIONThe ribotypes were stable in the plague foci. Correlation between the ribotypes of Yersinia pestis strains and their geographical origins was noticed. All 3 ribotypes had different origins, however ribotype A and ribotype C seemed to be closer related.

China ; DNA, Bacterial ; genetics ; Geography ; RNA, Ribosomal, 16S ; genetics ; RNA, Ribosomal, 23S ; genetics ; RNA, Ribosomal, 5S ; genetics ; Ribotyping ; Yersinia pestis ; classification ; genetics

Objective To describe the microbiological characteristics of ()CGMCC 12426 and determine and analyze its complete genome sequences.Methods strain CGMCC 12426 genomic DNA sequencing was performed on a single molecule real-time sequencing(SMRT)platform and the annotation was completed in the NCBI Prokaryotic Genomic Annotation Pipeline(pGAP).Results The complete genomic sequences of the released CGMCC 12426 consisted of a 4 138 265-bp circular chromosome and a 74 165-bp plasmid,which resulted in the prediction of 4581 genes including 4222 coding sequences,87 tRNAs,and 30 rRNAs(which included 5S rRNA,16S rRNA,and 23S rRNA).Conclusion The genome sequencing provided a basis for further investigations on the genetic background of and on the metabolic and regulatory mechanisms.
Bacillus subtilis ; genetics ; Genome, Bacterial ; Plasmids ; RNA, Ribosomal, 16S ; genetics ; RNA, Ribosomal, 23S ; genetics ; RNA, Ribosomal, 5S ; genetics ; Sequence Analysis, DNA

In order to clarify dynamic change of microbial community composition and to identify key functional bacteria in the cellulose degradation consortium, we studied several aspects of the biodegradation of filter papers and rice straws by the microbial consortium, the change of substrate degradation, microbial biomass and pH of fermentation broth. We extracted total DNA of the microbial consortium in different degradation stages for high-throughput sequencing of amplicons of bacterial 16 S rRNA genes. Based on the decomposition characteristics test, we defined the 12th, 72nd and 168th hours after inoculation as the initial stage, peak stage and end stage of degradation, respectively. The microbial consortium was mainly composed of 1 phylum, 2 classes, 2 orders, 7 families and 11 genera. With cellulose degradation, bacteria in the consortium showed different growth trends. The relative abundance of Brevibacillus and Caloramator decreased gradually. The relative abundance of Clostridium, Bacillus, Geobacillus and Cohnella increased gradually. The relative abundance of Ureibacillus, Tissierella, Epulopiscium was the highest in peak stage. The relative abundance of Paenibacillus and Ruminococcus did not change obviously in each stage. Above-mentioned 11 main genera all belonged to Firmicutes, which are thermophilic, broad pH adaptable and cellulose or hemicellulose degradable. During cellulose degradation by the microbial consortium, aerobic bacteria were dominant functional bacteria in the initial stage. However, the relative abundance of anaerobic bacteria increased gradually in middle and end stage, and replaced aerobic bacteria to become main bacteria to degrade cellulose.
Bacteria ; classification ; metabolism ; Biodegradation, Environmental ; Cellulose ; metabolism ; DNA, Bacterial ; genetics ; Microbial Consortia ; RNA, Ribosomal, 16S ; genetics

Objective: To explore the changes in bacterial community structure, antibiotic resistance genome, and pathogen virulence genome in river water before and after the river flowing through Haikou City and their transmission and dispersal patterns and to reveal anthropogenic disturbance's effects on microorganisms and resistance genes in the aquatic environment. Methods: The Nandu River was divided into three study areas: the front, middle and rear sections from the upstream before it flowed through Haikou City to the estuary. Three sampling sites were selected in each area, and six copies of the sample were collected in parallel at each site and mixed for 3 L per sample. Microbial community structure, antibiotic resistance, virulence factors, and mobile genetic elements were analyzed through bioinformatic data obtained by metagenomic sequencing and full-length sequencing of 16S rRNA genes. Variations in the distribution of bacterial communities between samples and correlation of transmission patterns were analyzed by principal co-ordinates analysis, procrustes analysis, and Mantel test. Results: As the river flowed through Haikou City, microbes' alpha diversity gradually decreased. Among them, Proteobacteria dominates in the bacterial community in the front, middle, and rear sections, and the relative abundance of Proteobacteria in the middle and rear sections was higher than that in the front segment. The diversity and abundance of antibiotic resistance genes, virulence factors, and mobile genetic elements were all at low levels in the front section and all increased significantly after flow through Haikou City. At the same time, horizontal transmission mediated by mobile genetic elements played a more significant role in the spread of antibiotic-resistance genes and virulence factors. Conclusions: Urbanization significantly impacts river bacteria and the resistance genes, virulence factors, and mobile genetic elements they carry. The Nandu River in Haikou flows through the city, receiving antibiotic-resistant and pathogen-associated bacteria excreted by the population. In contrast, antibiotic-resistant genes and virulence factors are enriched in bacteria, which indicates a threat to environmental health and public health. Comparison of river microbiomes and antibiotic resistance genomes before and after flow through cities is a valuable early warning indicator for monitoring the spread of antibiotic resistance.
Humans ; Rivers ; Virulence Factors/genetics* ; RNA, Ribosomal, 16S/genetics* ; Microbiota/genetics* ; Anti-Bacterial Agents ; Drug Resistance, Microbial/genetics*

BACKGROUND: Nontuberculous mycobacteria (NTM) should be correctly identified to the species level, because of different treatment plans among NTM species. This study was performed to assess the usefulness of real-time PCR and melting curve analysis in the identification of NTM. METHODS: One hundred fifty-two clinical NTM isolates were identified to the species level by PCR-restriction fragment length polymorphism analysis (PRA). Those strains were then identified by multiplex real-time PCR and melting curve analysis on the 16S rRNA gene and hsp65 gene. RESULTS: In the 16S rRNA gene fragment analysis, M. abscessus-M. chelonae group showed melting point at temperatures above 65 degrees C and M. avium complex (MAC; M. avium and M. intracelluare) below 48 degrees C, which differentiated M. abscessus-M. chelonae group and MAC from other NTM. In the hsp65 gene fragment analysis, M. abscessus-M. chelonae group was clearly divided into M. abscessus type I, M. abscessus type II, and M. chelonae according to the melting points at 61.25 degrees C, 66.06 degrees C, and 57.58 degrees C, respectively. CONCLUSIONS: With the multiplex real-time PCR and melting curve analysis of 16S rRNA and hsp65 genes, M. abscessus and M. chelonae were readily identified and MAC were differentiated from other NTM. Especially, M. abscessus and M. chelonae, which were not differentiated from each other with the 16S rRNA gene fragment analysis, were identified with hsp65 gene fragment analysis.
Bacterial Proteins/genetics ; Chaperonins/genetics ; Computer Systems ; DNA, Bacterial/chemistry ; Mycobacteria, Atypical/genetics/*isolation & purification ; Nucleic Acid Denaturation ; Polymerase Chain Reaction/*methods ; RNA, Ribosomal, 16S/genetics