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

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

We report a case of cat scratch disease in an 8-yr-old girl who presented with fever and enlargement of both axillary lymph nodes. Both aerobic and anaerobic cultures of the lymph node aspirate were negative for microbial growth. Gram staining and Warthin-Starry silver staining did not reveal any organism. Purified DNA from the PCR-amplicon of the 16S-23S rRNA intergenic region was sequenced and showed 99.7% identity with the corresponding sequence of Bartonella henselae strain Houston-1. Our findings suggest that the internal transcribed spacer is a reliable region for PCR identification of Bartonella species. In patients with lymphadenitis, a history of contact with cats or dogs necessitates the use of diagnostic approaches that employ not only the conventional staining and culture but also molecular methods to detect B. henselae.
Animals ; Bartonella henselae/genetics/*isolation & purification ; Cat-Scratch Disease/complications/*diagnosis ; Cats ; Child ; Dogs ; Female ; Humans ; Lymphadenitis/complications ; *Polymerase Chain Reaction ; RNA, Ribosomal, 16S/*genetics ; RNA, Ribosomal, 23S/*genetics ; Republic of Korea ; Sequence Analysis, DNA

The aim of this study was to develop and validate an oligonucleotide suspension array for rapid identification of 15 bacterial species responsible for bacteremia, particularly prevalent in Chinese hospitals. The multiplexed array, based on the QIAGEN LiquiChip Workstation, included 15 oligonucleotide probes which were covalently bound to different bead sets. PCR amplicons of a variable region of the bacterial 23S rRNA genes were hybridized to the bead-bound probes. Thirty-eight strains belonging to 15 species were correctly identified on the basis of their corresponding species-specific hybridization profiles. The results show that the suspension array, in a single assay, can differentiate isolates over a wide range of strains and species, and suggest the potential utility of suspension array system to clinical laboratory diagnosis.
Bacteremia ; diagnosis ; genetics ; Bacterial Typing Techniques ; Bacteriological Techniques ; DNA Probes ; Genetic Techniques ; Listeria monocytogenes ; metabolism ; Nucleic Acid Hybridization ; Oligonucleotide Array Sequence Analysis ; Oligonucleotides ; chemistry ; RNA, Ribosomal ; chemistry ; RNA, Ribosomal, 23S ; genetics ; Stem Cells

We describe 2 cases of pneumonia caused by the same macrolide-resistant Mycoplasma pneumoniae in siblings. M. pneumoniae was identified using real-time PCR. Direct sequence analysis of the 23S rRNA gene revealed a point mutation in V domain (A2063G) of the 23S rRNA gene.
Anti-Bacterial Agents/pharmacology ; Child ; Child, Preschool ; Drug Resistance, Bacterial/drug effects ; Humans ; Macrolides/pharmacology ; Male ; Mutation ; Mycoplasma pneumoniae/*genetics/isolation & purification ; Pneumonia, Mycoplasma/*diagnosis/microbiology/radiography ; RNA, Ribosomal, 23S/*analysis ; Real-Time Polymerase Chain Reaction ; Sequence Analysis, RNA ; Siblings

OBJECTIVETo define the main genotypes in Guizhou agricultural areas by molecular epidemiologic investigation of 21 Borrelia burgdorferi sensu lato of Lyme disease spirochetes and to provide the scientific bases for formulating a preventive policy.

METHODSPolymerase chain reaction (PCR) technique was used to amplify the 23S(rrl)-5S(rrf) intergenic spacer, and amplified products were analyzed by restriction fragment length polymorphism (RFLP) and nucleotide sequencing.

RESULTSThere were two genospecies in the strains: 20 strains belong to Borrelia valaisiana, 1 strain is Borelia sp.

CONCLUSIONBorrelia valaisiana was the main genotype in Guizhou agricultural areas. The harmness of B. valaisiana to human being has been confirmed. In order to efficiently prevent the harmness of agent to the people in Guizhou agriculture areas, we should study the risk further.

Base Sequence ; Borrelia burgdorferi ; classification ; genetics ; China ; DNA, Bacterial ; chemistry ; genetics ; DNA, Ribosomal Spacer ; genetics ; Humans ; Lyme Disease ; microbiology ; Molecular Sequence Data ; Polymerase Chain Reaction ; Polymorphism, Restriction Fragment Length ; RNA, Ribosomal, 23S ; genetics ; RNA, Ribosomal, 5S ; genetics ; Ribotyping ; Sequence Analysis, DNA

OBJECTIVEUsing 16S rDNA and 23S rDNA genes as the target sequences to develop a system based on oligonucleotide microarray and to detect the seven clinical pathogenic bacteria, commonly seen.

METHODSDouble polymerase chain reaction (PCR) was applied to amplify the segments of 16S rDNA and 23S rDNA genes of the target bacteria. An oligonucleotide microarray was constructed to simultaneously detect EHEC O157:H7, Vibrio parahaemolyticus, Salmonella sp., Vibrio cholerae, Listeria monocytogenes, Campylobacter jejuni and Shigella sp. Specificity, sensitivity and reproducibility of the microarray during detection were checked. And then microarray was used to detect the microbes in stool specimens of 81 patients with diarrhea and vomiting.

RESULTSThe double PCR method could simultaneously amplify the target sequences of 16S rDNA and 23S rDNA genes of the seven pathogens. The sensitivity of the developed oligonucleotide microarray could reach 10(3) cfu/ml but no positive results were presented for non-targeted bacteria. The coefficients of differentiation in one lot or among different lots of the microarray slices were 3.89% - 5.81%. The positive detection rate of the stool specimens by oligonucleotide microarray was 39.5% (32/81), with a coincidence of 96.3% (78/81) for the patients and another coincidence of 96.8% (31/32) for bacterial genus or species identification, when comparing to the results by routine bacteriological examinations.

CONCLUSIONThe established assay in this study based on oligonucleotide microarray to detect the seven pathogenic bacteria has many advantages such as convenient, rapid, accurate, stable and high flux, which is suitable for clinical specimen examination and epidemiological field investigation.

Bacteria ; isolation & purification ; Campylobacter jejuni ; isolation & purification ; DNA, Bacterial ; genetics ; DNA, Ribosomal ; genetics ; Escherichia coli O157 ; isolation & purification ; Humans ; Listeria monocytogenes ; isolation & purification ; Oligonucleotide Array Sequence Analysis ; methods ; RNA, Ribosomal, 16S ; genetics ; RNA, Ribosomal, 23S ; genetics ; Reproducibility of Results ; Salmonella ; isolation & purification ; Sensitivity and Specificity ; Shigella ; isolation & purification ; Vibrio cholerae ; isolation & purification ; Vibrio parahaemolyticus ; isolation & purification

OBJECTIVETo establish polymerase chain reaction (PCR) technique for the detection of specific genes related to species of genus Bartonella, and for diagnosing clinically suspected cat-scratch disease (CSD) case complicated with pneumonia on both lungs. The appearance of Bartonella infectious diseases calls for genus and species detection and tools for identification in order to make clinical diagnosis and carry on epidemiological studies.

METHODSOne pair of primer TIle.455p-TAla.885n was designed based on the fact that tRNA(Ile)-tRNA(Ala) intergenic spacer region in 16S-23S rRNA intergenic spacer (ITS) of genus Bartonella were high variable sequences flanked by completely conserved tRNA-encoding genes. 16S-23S rRNA was longer than that which had been described in other bacteria. Two published pairs of primers were used to directly detect the specific gene fragments of Bartonella species DNA extracts from human blood, followed by PCR product Sequencing and nucleotide base sequence analysis.

RESULTSAmplification products of the three pairs of primers had the same predicted size of those in Bartonella spp. According to the different length of electrophoresis bank, the sample was identified as a species of genus Bartonella other than the positive control. Sequence analysis showed that the nuleotide sequence from the PCR product of primer TIle.455p-TAla.885n was identical to the Bartonella isolated from Yunnan in China.

CONCLUSIONSPCR-based assay provided a simple and rapid means to detect pathogenic Bartonella species in humans and mammalian hosts as well as in arthropod vecters. This study suggested that this pathogenic Bartonella species existed in patients in northern and southern parts of China.

Animals ; Bartonella ; genetics ; isolation & purification ; Bartonella Infections ; diagnosis ; microbiology ; Base Sequence ; Cat-Scratch Disease ; diagnosis ; microbiology ; Cats ; China ; Diagnosis, Differential ; Humans ; Male ; Middle Aged ; Molecular Sequence Data ; Polymerase Chain Reaction ; methods ; RNA, Ribosomal, 16S ; genetics ; RNA, Ribosomal, 23S ; genetics ; RNA, Transfer, Ala ; chemistry ; genetics ; RNA, Transfer, Ile ; chemistry ; genetics ; Sequence Analysis, DNA ; Sequence Homology, Nucleic Acid

A slowly growing, non-chromogenic mycobacterial strain was isolated from sputum and bronchial lavage fluid samples of a patient presenting with productive cough, blood-tinged sputum, low-grade fever, and weakness. A positive acid-fast bacilli sputum smear result prompted the initiation of an anti-tuberculosis regimen. Multiplex real-time PCR showed a negative result for Mycobacterium tuberculosis complex and a positive result for nontuberculous mycobacteria. The DNA chip test confirmed this organism as a member of the genus Mycobacterium, but could not specify the species. Interestingly, the mycolic acid patterns obtained by HPLC nearly overlapped with those of M. simulans. The sequences of the Mycobacterium 16S rRNA gene and 16S-23S internal transcribed spacer region were unique and were found to have 100% similarity with those of M. riyadhense. After a review of the literature, we report this case as the first Korean case of M. riyadhense lung infection.
Adult ; Antitubercular Agents/pharmacology ; Chromatography, High Pressure Liquid ; Female ; Humans ; Lung Diseases/*microbiology ; Microbial Sensitivity Tests ; Mycobacterium/classification/drug effects/*isolation & purification ; Mycobacterium Infections/microbiology ; Mycobacterium tuberculosis/genetics/isolation & purification ; Mycolic Acids/analysis ; Oligonucleotide Array Sequence Analysis ; Phylogeny ; RNA, Ribosomal, 16S/chemistry/genetics ; RNA, Ribosomal, 23S/chemistry/genetics ; Republic of Korea ; Sequence Analysis, DNA