OBJECTIVETo apply pyrosequencing technique in the detection of the common pathogens in sepsis.

METHODSThe primers for amplification and sequencing in pyrosequencing were designed according to alignment of the bacterial 16S rRNA sequence. Bacterial genomic DNA was extracted for pyrosequencing, and the pathogen species were determined according to the sequencing data obtained.

RESULTSPyrosequencing effectively yielded the sequencing data of the 28 bp sequences of the pathogens and clearly distinguished the pathogen species of Streptococcus pyogenes, Streptococcus pneumonia, Escherichia coli, Pseudomonas aeruginosa, Klebsiella pneumonia, Neisseria meningitides, and Salmonella, but failed to distinguish Staphylococcus epidermidis from Staphylococcus aureus.

CONCLUSIONPyrosequencing technique can effectively distinguish the common pathogens in sepsis at the species level.

Bacteria ; classification ; isolation & purification ; DNA Primers ; DNA, Bacterial ; genetics ; Microbiological Techniques ; Polymerase Chain Reaction ; RNA, Ribosomal, 16S ; Sepsis ; microbiology

A simple, membrane-strip-based lateral-flow (LF) biosensor assay and a high-throughput microtiter plate assay have been combined with a reverse transcriptase polymerase chain reaction (RT-PCR) for the detection of a small number (ten) of viable Mycobacterium (M.) avium subsp. paratuberculosis (MAP) cells in fecal samples. The assays are based on the identification of the RNA of the IS900 element of MAP. For the assay, RNA was extracted from fecal samples spiked with a known quantity of (101 to 106) MAP cells and amplified using RT-PCR and identified by the LF biosensor and the microtiter plate assay. While the LF biosensor assay requires only 30 min of assay time, the overall process took 10 h for the detection of 10 viable cells. The assays are based on an oligonucleotide sandwich hybridization assay format and use either a membrane flow through system with an immobilized DNA probe that hybridizes with the target sequence or a microtiter plate well. Signal amplification is provided when the target sequence hybridizes to a second DNA probe that has been coupled to liposomes encapsulating the dye, sulforhodamine B. The dye in the liposomes provides a signal that can be read visually, quantified with a hand-held reflectometer, or with a fluorescence reader. Specificity analysis of the assays revealed no cross reactivity with other mycobacteria, such as M. avium complex, M. ulcerans, M. marium, M. kansasii, M. abscessus, M. asiaticum, M. phlei, M. fortuitum, M. scrofulaceum, M. intracellulare, M. smegmatis, and M. bovis. The overall assay for the detection of live MAP organisms is comparatively less expensive and quick, especially in comparison to standard MAP detection using a culture method requiring 6-8 weeks of incubation time, and is significantly less expensive than real-time PCR.
Animals ; Bacteriological Techniques ; Biosensing Techniques/*veterinary ; Cattle ; Feces/*microbiology ; Mycobacterium avium subsp. paratuberculosis/*isolation & purification ; RNA, Bacterial/classification/isolation & purification ; Reverse Transcriptase Polymerase Chain Reaction/veterinary ; Sensitivity and Specificity

Objective: High PM concentration is the main feature of increasing haze in developing states, but information on its microbial composition remains very limited. This study aimed to determine the composition of microbiota in PM in Guangzhou, a city located in the tropics in China. Methods: In Guangzhou, from March 5 to 10 , 2016, PM was collected in middle volume air samplers for 23 h daily. The 16S rDNA V4 region of the PM sample extracted DNA was investigated using high-throughput sequence. Results: Among the Guangzhou samples, , , , , and were the dominant microbiota accounting for more than 90% of the total microbiota, and was the dominant gram-negative bacteria, accounting for 21.30%-23.57%. We examined the difference in bacterial distribution of PM between Beijing and Guangzhou at the genus level; was found in both studies, but was only detected in Guangzhou. Conclusion In conclusion, the diversity and specificity of microbial components in Guangzhou PM were studied, which may provide a basis for future pathogenicity research in the tropics.
Air Microbiology ; Air Pollutants ; analysis ; Bacteria ; classification ; isolation & purification ; China ; Cities ; Environmental Monitoring ; Microbiota ; Particle Size ; Particulate Matter ; analysis ; RNA, Bacterial ; analysis ; RNA, Ribosomal, 16S ; analysis

OBJECTIVETo establish multiplex PCR-based assays for detecting H.influenzae and H.parainfluenzae. And the PCR-based assays were applied to detect the carriage rates of H.influenzae and H.parainfluenzae in nasopharyngeal swab specimens which were collected from healthy children.

METHODSMultiplex primers for species-specific PCR were designed by using DNAstar soft based on the sequences of 16S rRNA genes from genus Haemophilus to detect H.influenzae and H.parainfluenzae.

RESULTSThe sensitivity of the 16S rRNA PCR assay for detecting H.influenzae and H.parainfluenzae was 97.53% and 100% respectively, and the specificity was 95.89% and 96.63% respectively. Youden's Index on the ability to detect H.influenzae and H.parainfluenzae was 0.9342 and 0.9663 respectively. 666 nasopharyngeal swab specimens were collected from healthy children. The detection rates of H.influenzae and H.parainfluenzae were 14.11% and 16.07% respectively by using isolation and culture methods. The detection rates of H.influenzae and H.parainfluenzae were 43.54% and 57.96% respectively by 16S rRNA PCR assays. The carriage rates of serotypes a, b, c, d, e, f and non-typeable isolates were 0% (0/666), 0.15% (1/666), 1.20% (8/666), 0.15% (1/666), 1.20% (8/666), 1.80% (12/666), 95.50% (636/666) respectively.

CONCLUSIONThe multiplex PCR assays were very rapid, reliable and feasible methods for detection of H.influenzae and H.parainfluenzae in pharyngeal swab specimens which were compared to conventional isolation and culture methods. 95.5% of H.influenzae strains in healthy children were nontypeable. The encapsulated or typable strains were mainly three serotypes which was c, e, and f serotype.

Haemophilus influenzae ; classification ; genetics ; isolation & purification ; Haemophilus parainfluenzae ; classification ; genetics ; isolation & purification ; Humans ; Multiplex Polymerase Chain Reaction ; methods ; Nasopharynx ; microbiology ; RNA, Bacterial ; genetics ; RNA, Ribosomal, 16S ; genetics ; Sensitivity and Specificity

In the present study, 35 Staphylococcal strain isolated from milk samples of 16 cows from eight farms of three different geographic locations in Central Java, Indonesia, and from milk samples of 19 cows from 19 farms of different geographic locations in Hesse, Germany, were compared pheno- and genotypically. On the basis of cultural and biochemical properties as well as by amplification of the 23S rRNA specific to Staphylococcus aureus, all isolates could be identified as S. aureus. In addition, all S. aureus isolates harboured the genes clfA and coa encoding staphylococcal clumping factor and coagulase, and the gene segments encoding the immunoglobulin G binding region and the X-region of protein A gene spa. By PCR amplification, the genes seb, seg, seh, and sei was observed for the S. aureus cultures isolated in Central Java, Indonesia and the genes sec, sed, seg, seh, sei, sej and tst for the S. aureus cultures isolated in Hesse, Germany. None of the S. aureus of both origins harboured the genes sea, see, eta and etb. All isolates were additionally positive for the genes nuc, fnbA, hla, and set1. The gene hlb was found for 6 cultures from Central Java, Indonesia and 16 cultures from Hesse, Germany. However, the gene fnbB and the gene segments cnaA and cnaB were not present among the strains isolated in Central Java, Indonesia and rare among the strains isolated in Hesse, Germany. It was of interest that most of the S. aureus isolated in Central Java, Indonesia harboured the gene cap5 and most of the strains isolated in Hesse, Germany the gene cap8. The phenotypic and genotypic results of the present study might help to understand the distribution of prevalent S. aureus clones among bovine mastitis isolates of both countries and might help to control S. aureus infections in dairy herds.
Animals ; Bacterial Typing Techniques ; Cattle ; Female ; Genes, Bacterial ; Genotype ; Germany ; Indonesia ; Mastitis, Bovine/*microbiology ; Phenotype ; Polymerase Chain Reaction ; RNA, Bacterial/genetics ; RNA, Ribosomal, 23S/genetics ; Staphylococcal Infections/microbiology/*veterinary ; Staphylococcus aureus/classification/*genetics/isolation&purification

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 make qualitative and quantitative analysis of Archaea in subgingival plaque sample and to investigate the relationship between periodontal disease and Archaea.

METHODSSubgingival plaque was collected from 23 patients with aggressive periodontitis, 29 with chronic periodontitis, 35 with plaque-induced gingivitis and 38 healthy controls. Qualitative and quantitative analysis of methanogenic archaea was performed by amplification of the 16S rRNA genes in the DNA extracted from the plaque samples.

RESULTSArchaea were found in 65% of aggressive periodontitis patients, 72% of chronic periodontitis, 26% of gingivitis and zero of healthy subjects. Quantitative analysis showed the average abundance of archaeal 16S rRNA gene in Archaea-positive patients was different among the three groups. The average 16S rRNA gene copy number from per microg wet plaque was 6.66 x 10(6) in aggressive periodontitis sufferers, 4.47 x 10(6) in chronic periodontitis and 1.78 x 10(6) in gingivitis groups. The prevalence of Archaea and the average Archaea 16S rRNA gene numbers in periodontitis groups were higher than those in gingivitis group (P < 0.05).

CONCLUSIONSThis suggests that Archaea may be implicated as causative agents for periodontitis.

Aggressive Periodontitis ; microbiology ; Archaea ; classification ; genetics ; isolation & purification ; Case-Control Studies ; Chronic Periodontitis ; microbiology ; DNA, Bacterial ; genetics ; Dental Plaque ; microbiology ; Humans ; Periodontal Diseases ; microbiology ; RNA, Ribosomal, 16S ; genetics

OBJECTIVETo analyses the genetic diversity and relationship of Salmonella typhi strains isolated from different years and districts in Guizhou province.

METHODSRibotyping with 16s rDNA probe was used to describe the diversity of the 209 strains which were isolated in 26 counties of Guizhou province, from 1959 to 1999. The antibiotics resistance was also studied.

RESULTSTwenty-six ribotypes were found in all 209 strains, with two dominant types. The strains isolated from local typhoid epidemics belonged to the unique Ribotypes. The major ribotypes of the resistant strains were RT7 and RT1.

CONCLUSIONThe Salmonella typhi isolates from Guizhou diverged obviously. The abundant clones and multi-resistance of the strains might serve the major reasons of the high morbidity of typhoid in Guizhou.

Blotting, Southern ; China ; DNA, Bacterial ; genetics ; DNA, Ribosomal ; genetics ; Phylogeny ; RNA, Ribosomal, 16S ; genetics ; Ribotyping ; Salmonella typhi ; classification ; genetics ; isolation & purification

BACKGROUNDThe bacterial composition of periapical lesions in deciduous teeth has not been well documented. This study was designed to explore the bacterial compositions, especially the dominant bacteria in periapical lesions using 16S rRNA sequencing.

METHODSTissue samples were collected from 11 periapical lesions in deciduous teeth with primary endodontic infections. DNA was extracted from each sample and analyzed using 16S rRNA cloning and sequencing for the identification of bacteria.

RESULTSAll DNA samples were positive for 16S rRNA gene PCR. One hundred and fifty-one phylotypes from 810 clones were identified to eight phyla, and each sample contained an average of 25.9 phylotypes. In addition, 59 phylotypes were detected in more than two samples, and Fusobacterium (F.) nucleatum (8/11), Dialister (D.) invisus (8/11), Campylobacter (C.) gracilis (7/11), Escherichia (E.) coli DH1 (6/11), Aggregatibacter (A.) segnis (6/11), and Streptococcus (S.) mitis (6/11) were the most prevalent species. Furthermore, 45 as-yet-uncultivated phylotypes were also identified.

CONCLUSIONSChronic periapical lesions in deciduous teeth contained polymicrobial infections. F. nucleatum, D. invisus, C. gracilis, E. coli DH1, A. segnis, and S. mitis were the most prevalent species detected by 16S rRNA sequencing.

Bacteria ; classification ; genetics ; isolation & purification ; Bacterial Infections ; microbiology ; Child ; Child, Preschool ; Female ; Humans ; Male ; Periapical Tissue ; microbiology ; RNA, Ribosomal, 16S ; genetics ; Tooth, Deciduous ; microbiology

We investigated the prevalence of Bartonella infections in ticks, mites and small mammals (rodents, insectivores and weasels) collected during 2001 through 2004, from various military installations and training sites in Korea, using PCR and sequence analysis of 16S rRNA, 23S rRNA and groEL heat shock protein genes. The prevalence of Bartonella spp. was 5.2% (n = 1, 305 sample pools) in ticks, 19.1% (n = 21) in mesostigmatid mites and 13.7% (n = 424 individuals) in small mammals. The prevalence within the family Ixodidae was, 4.4% (n = 1, 173) in Haemaphysalis longicornis (scrub tick), 2.7% (n = 74) in H. flava, 5.0% (n = 20) in Ixodes nipponensis, 11.1% (n = 9) in I. turdus, 33.3% (n = 3) in I. persulcatus and 42.3% (n = 26) in Ixodes spp. ticks. In rodents, the prevalence rate was, 6.7% (n = 373) in Apodemus agrarius (striped field mouse) and 11.1% (n = 9) in Eothenomys regulus (Korean red-backed vole) and in an insectivore, Crocidura lasiura, 12.1% (n = 33). Neither of the two weasels were positive for Bartonella spp. Phylogenetic analysis based on amino acid sequence of a portion of the groEL gene amplified from one A. agrarius spleen was identical to B. elizabethae species. We demonstrated the presence of Bartonella DNA in H. longicornis, H. flava and I. nipponensis ticks, indicating that these ticks should be added to the growing list of potential tick vectors and warrants further detailed investigations to disclose their possible roles in Bartonella infection cycles.
Animals ; Bartonella/classification/*isolation&purification ; DNA, Bacterial/isolation&purification ; Disease Vectors ; GroEL Protein/genetics ; Mammals/*microbiology ; Mites/*microbiology ; Polymerase Chain Reaction ; RNA, Ribosomal, 16S/genetics ; RNA, Ribosomal, 23S/genetics ; Ticks/*microbiology