OBJECTIVETo investigated the genetic polymorphism of the isolated strains using ribotyping method.

METHODSOne hundred twenty-two strains of V. cholerae O139 isolated from different areas of China from 1993 to 1999 were selected and characterized with ribotyping, including 16s rDNA and 23s rDNA probes.

RESULTSOne hundred twenty-two strains were differentiated into 10 different ribotypes (RT1-RT10) on the basis of rRNA gene probes hybridization (with Bgl I digestion), which consisted of 7 - 9 bands between 12 and 1.5 kb in size. RT1 and RT3, as two predominant ribotypes, comprised most number of the strains which spread to the extensive range. Nine strains, which are negative to ctxAB, zot and RS individually, belong to 4 special ribotypes. The dendrogram revealing genetic relationship among different clones of V. cholerae O139 showed that the clones belonging to RT1 and RT2 had genetic similarity on high degree, although they were isolated from different regions. The two predominant ribotypes (RT1 and RT3) were distant in genetic relationship.

CONCLUSIONResults showed the clonal diversity and the wide area distribution of V. cholerae O139 strains in China, suggesting the multiple origins of O139 epidemics.

Phylogeny ; Polymorphism, Genetic ; RNA, Ribosomal, 16S ; genetics ; RNA, Ribosomal, 23S ; genetics ; Ribotyping ; Species Specificity ; Vibrio cholerae O139 ; classification ; genetics

Toll-like receptors are required for detection of pathogen-associated molecular patterns and play critical roles in protection of host from infection. Murine TLR13 was recently reported to be involved in recognition of bacterial 23S ribosomal RNA sequence that is the binding site of different antibiotics.
Anti-Bacterial Agents ; Binding Sites ; RNA, Ribosomal, 23S ; Staphylococcus aureus ; Toll-Like Receptors

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

OBJECTIVETo establish the approach to detecting two biovars of Ureaplasma urealyticum (Uu) in human semen and to investigate the relationship between the two biovars of Uu infection and the quality of human semen.

METHODSBased on the 16S-23S rRNA intergenic spacer region, three pairs of primers were designed, the species specific primer and two biovars primers (Parvo primer and T960 primer). The two biovars of Uu were detected in the semen from 949 men by semen culture and PCR assay. Meanwhile, semen routine analyses were performed.

RESULTSIn the 949 subjects, 199 were Uu positive both in Uu liquid culture and PCR assay (199/949, 21.1%), of which 136 (136/199, 68.3%) were Parvo biovar, 54 (54/199, 27.1%) T960 biovar, and 9 (9/199, 4.5%) both Parvo and T960 biovars. Compared with the Parvo and the negative groups, human sperm viability was significantly decreased (P < 0.05 ) in the Uu T960 infection group. The difference of sperm motility and density had no statistic significance.

CONCLUSIONA significant correlation has been found between Uu T960 biovar infection and human sperm viability

Adult ; Humans ; Male ; Polymerase Chain Reaction ; RNA, Ribosomal, 16S ; genetics ; RNA, Ribosomal, 23S ; genetics ; Semen ; microbiology ; Sperm Motility ; Ureaplasma urealyticum ; classification ; genetics

Addressing the increasing antibiotic resistance, including clarithromycin resistance, which affects Helicobacter pylori (H. pylori) eradication therapy, is a challenge for clinicians. Antibiotic resistance is the main reason for H. pylori eradication failure and the resistance rate for clarithromycin may drastically increase, up to 38.5%, due to 23S ribosomal RNA point mutations. Therefore, the standard triple regimen is no longer suitable as the first-line treatment in most regions. However, there is a growing interest in personalized care for patients. Increased eradication rates of tailored therapy based on antibiotic susceptibility have been reported using nucleic acid-based techniques for clarithromycin resistance with a focus on the first-line eradication therapy of H. pylori infection. Herein, we discuss the eradication therapy for H. pylori, with a diagnostic test and appropriate treatment for clarithromycin resistance.
Clarithromycin ; Diagnostic Tests, Routine ; Drug Resistance ; Drug Resistance, Microbial ; Helicobacter pylori ; Helicobacter ; Humans ; Point Mutation ; RNA, Ribosomal, 23S

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

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 set up a rapid and simple method for identificating bacteria by 16S-23SrDNA intergenic spacer regions (ISRs).

METHODSPolymorphic products of PCR from ISRs were selected on agarose gel and sequenced directly using purified fragments by excising the gel without cloning. Nucleotide sequences were compared with GenBank databases and analyzed by DNAMAN program.

RESULTSThere was only a single product in streptococcus genus after PCR amplification of 16S-23SrDNA ISRs. Five streptococcal species were obtained from 7 strains of streptococcus. Two major amplicons were consistently generated for 8 strains of Haemophilus influenzae (H. influenzae). The sequence data showed that they all belonged to H. influenzae type b on GenBank databases.

CONCLUSIONPCR and direct sequencing of 16S-23SrDNA ISRs were very successful methods for bacterial species identification.

Base Sequence ; DNA, Ribosomal Spacer ; chemistry ; Haemophilus influenzae ; genetics ; isolation & purification ; Humans ; Molecular Sequence Data ; Polymerase Chain Reaction ; methods ; RNA, Ribosomal, 16S ; genetics ; RNA, Ribosomal, 23S ; genetics ; Streptococcus ; genetics ; isolation & purification

OBJECTIVETo explore the value of detecting bacterial 16S rRNA with 23S rRNA in the diagnosis of periprosthetic joint infection (PJI).

METHODSA prospective study was conducted among 67 patients with previous total hip arthroplasty (THA) undergoing a reoperation for infection (23 patients) or aseptic loosening (44 patients). Bacterial 16S rRNA and 23S rRNA in the interface membrane were detected by real-time PCR and their value in diagnosis of PJI was assessed.

RESULTSThe 16S rRNA and 23S rRNA showed no significant difference in their power in the diagnosis of PJI. The detection of 16S rRNA/23S rRNA showed a higher sensitivity and a greater negative predictive value in PJI diagnosis than the detection of 16S rRNA+23S rRNA (95.7% vs 52.2%, P<0.01; 97.6% vs 79.6%, P=0.01). The specificity, positive predictive value, and accuracy of the 4 diagnostic strategies were not significantly different.

CONCLUSIONSThe diagnostic power of 16S rRNA and 23S rRNA was similar in detecting PJI. Compared with the diagnostic strategy with 16S rRNA+23S rRNA, 16S rRNA/23S rRNA is more sensitive in detecting PJI.

Arthritis, Infectious ; diagnosis ; microbiology ; Arthroplasty, Replacement, Hip ; Humans ; Prospective Studies ; Prosthesis-Related Infections ; diagnosis ; microbiology ; RNA, Bacterial ; isolation & purification ; RNA, Ribosomal, 16S ; isolation & purification ; RNA, Ribosomal, 23S ; isolation & purification ; Real-Time Polymerase Chain Reaction ; Reoperation ; Sensitivity and Specificity