Identification of Vibrio parahaemolyticus based on the concatenated DNA sequencing of 16S rRNA and gyrB gene / 上海预防医学

Objective:To establish the concatenated DNA sequencing of 16S ribosomal RNA （16S rRNA） and DNA gyrase subunit B （gyrB） gene， and provide evidence for the identification and classification of Vibrio parahaemolyticus （V. parahaemolyticus）. Methods:Typical strains in the genus of Vibrio spp. was selected， such as V. parahaemolyticus， V. alginolyticus and other species for examination of 16S rRNA and gyrB gene as target. Primers were separately designed to amplify these two nucleotide fragments. Phylogenetic analysis was performed using the concatenated sequences. Results:The concatenated 16S rRNA+gyrB nucleotide sequence of V. parahaemolyticus formed a single cluster in the phylogenetic analysis， which identified the typical strains of Vibro spp. at the species level. Conclusion:In our study， an identification method of V. parahaemolyticus is established based on concatenated 16S rRNA+gyrB nucleotide sequencing. It can identify the strains of V. parahaemolyticus at the species level， which may be applied in phylogenetic analysis and contamination tracing of V. parahaemolyticus in food and drug control.

Frequency of first and second-line drug resistance-associated mutations among resistant Mycobacterium tuberculosis clinical isolates from São Paulo, Brazil

BACKGROUND Tuberculosis (TB) is an infectious disease caused by the bacterium Mycobacterium tuberculosis, and the number of new cases of multidrug resistant TB (MDR-TB), pre extensively drug-resistant TB (pre-XDR-TB) and extensively drug-resistant TB (XDR-TB) has increased considerably worldwide. OBJECTIVES Herein, using 156 M. tuberculosis isolates from 106 patients previously classified as MDR or pre-XDR or XDR isolates, we investigated the genetic mutation profiles associated with phenotypic resistances in patients with MDR-TB, pre-XDR-TB and XDR-TB, treatment outcomes and resistance evolution. METHODS Molecular analyses were performed by partial sequencing of the rpoB, katG, gyrA, gyrB, rrs genes and analysis of the fabG-inhA promoter region. Clinical, epidemiologic and demographic data were obtained from the TB Notification database system of São Paulo (TB-WEB) and the Information System for Special Tuberculosis Treatments (SITE-TB). FINDINGS Drug resistance was attributed to previously known mutations and a novel Asp449Val mutation in gyrB was observed in four isolates from the same patient. Ten patients had more than one isolate evaluated and eight of these patients displayed resistance progression. MAIN CONCLUSIONS The present study is the first to report the frequency of mutations related to second-line drug resistance in MDR-TB, pre-XDR-TB and XDR-TB isolates. The results could lead to the improvement of available technologies for the rapid detection of drug resistant TB.

Comparison of the Utility of dnaJ and 16S rDNA Sequences for Identification of Clinical Isolates of Vibrio Species

BACKGROUND: Among the many Vibrio species that can cause infections in humans, several species can cause a fatal outcome. Therefore, accurate identification of Vibrio species is very important. Since some species show atypical phenotypic features, selecting an appropriate molecular method is necessary to avoid misdiagnosis. METHODS: Vibrio clinical isolates (N=53) and reference strains (N=8) were used in this study. We analyzed the following sequences for identification: dnaJ gene, 16S rDNA, gyrase B (gyrB) V. vulnificus-specific sequence, gyrB V. navarrensis-specific sequence, and V. vulnificus hemolysin gene PCR (Vvh PCR). We performed phylogenetic analysis of the 16S rDNA, dnaJ, and gyrB sequences. Final identification was based on the combined results of all tests described above. Concordance of the 16S rDNA and dnaJ sequence analysis was measured using the Chi-square test. RESULTS: The 61 Vibrio strains were identified as follows, in descending order: V. vulnificus (78.69%), V. parahaemolyticus (6.56%), V. navarrensis (4.92%), V. mimicus (1.64%), V. cholera (1.64%), V. furnissii (1.64%), V. alginolyticus (1.64%), and Grimontia hollisae (1.64%). The accuracy rates of the dnaJ gene and 16S rDNA sequence for identification were 91.80% and 86.89%, respectively. The 16S rDNA and dnaJ sequences showed a concordance rate of 0.45, which indicates moderate agreement. CONCLUSIONS: Our results suggest that analysis of the dnaJ sequence may be a useful method for the identification of clinical isolates of Vibrio species, especially for distinguishing between closely related Vibrio species.

Molecular Detection of Fluoroquinolone Resistance in Multidrug-Resistant Mycobacterium tuberculosis Isolates / 대한임상미생물학회지

BACKGROUND: Fluoroquinolones (FQs) are important drugs for treating multidrug-resistant tuberculosis (MDR-TB). However, due to widespread use of FQs, the resistance rates to FQs have been increasing among Mycobacterium tuberculosis. Rapid and reliable FQ drug susceptibility testing (DST) is crucial for successful treatment of MDR-TB. In this study, the feasibility of molecular detection of FQ resistance was evaluated. METHODS: A total of 95 MDR-TB isolates were collected from Jan through Oct 2009 at the Korean Institute of Tuberculosis. DST for ofloxacin (OFL), levofloxacin, and moxifloxacin was performed using the Lowenstein-Jensen media absolute concentration method. Minimum inhibitory concentrations (MIC) of these were determined using the broth microdilution method. DNA was extracted from cultured isolates using bead beating method. The quinolone resistance-determining region (QRDR) of gyrA and gyrB were amplified and those sequences were analyzed. RESULTS: Of 95 isolates, 79 were resistant to at least one of FQs. Of these, 71 (89.9%) harbored mutation in the QRDR of gyrA or gyrB. None of FQ susceptible strains possessed any mutation in gyrA or gyrB. Mutations in codon 94 of gyrA were most common; only two isolates had mutation in only the gyrB gene. OFL MICs for isolates with gyrA mutation ranged from 1 to 32 microg/mL, but FQ susceptible isolates showed MICs ranging from < or =0.06 to 0.5 microg/mL. CONCLUSION: Mutation analysis of QRDR of gyrA and gyrB showed 89.9% sensitivity and 100% specificity for detecting FQ resistance in MDR-TB. Therefore, molecular DST can be useful for rapid detection of FQ resistance in MDR-TB.

Evaluating the use of loop-mediated isothermal amplification (LAMP) method for detection of Mycobacterium tuberculosis in Indonesian clinical isolates.

Background: Loop-mediated isothermal amplification (LAMP) is a method already claimed as a simple technique to amplify DNA/ RNA using four to six primers as “a set” from conserved sequence of target gene. In this study we optimize the use of LAMP for detection of Mycobacterium tuberculosis in clinical isolates from Indonesia. Methods: Procedures to perform LAMP were optimized, then the method was applied to 122 archieved samples of DNA’s Mtb from clinical TB patients with Acid Fast Bacilli (AFB) smears positive. The samples were obtained in 2008 from 13 provinces in Indonesia for genotyping study, which then become collections of Center for Biomedical and Basic Technology of Health (CBBTH), NIHRD Indonesia. The optimization tests include sensitivity and specificity tests of several sets primers, which were evaluated using 10-fold serially diluted DNA of Mtb H37Rv and 12 species of Mycobacteria. Three equipments consisted of LAMP turbidimeter, heating block and water bath were compared for its ability in DNA amplification. Detection of M. tuberculosis from clinical isolates used set primers specific for gyrB gene, amplicon was detected with UV fluorescence system. Results: The results showed that the highest sensitivity was obtained using the set primers specific for 16S rRNA and gyrB which could detect 10.0 fg to 1.0 pg genomic DNA of Mtb H37Rv. The set primers specific for gyrB gene was the most specific primers. Application of LAMP using gyrB set primers on Indonesian clinical isolates showed 94.2% (114/121) positivity rate. Conclusion: LAMP method is potentially used in TB diagnosis in Indonesia.

PCR based detection of gyrB2 gene from Pseudomonas sp. affected human clinical isolates.

Detection of virulence gene is a key component in determining the pathogenicity of any isolates because these genes act multi-functionally and multi-factorially. Gyrase specific gene primer in combination of PCR technology allows precise detection of DNA gyrase subunit B2 gene (gyrB2) from different virulent microorganisms. In the present study, forward and reverse primers with a length of 20bp for both were used for detection of gyrB2 genes in clinical isolates of Pseudomonas sp. collected from patients suffering from urinary tract infection (UTI). A total of 12 isolates of Pseudomonas sp. viz., Ps1, Ps2, Ps3, Ps4, Ps5, Ps6, Ps7, Ps8, Ps9, Ps10, Ps11 and Ps12 were used in present study in which gyrB2 gene amplified in all 12 isolates and gave the expected 1130bp PCR product after visualization under gel documentation system in 1.2% agarose gel. This PCR was outstanding in the detection of gyrB2 gene in urinary tract infected patients caused by Pseudomonas sp. species.

PCR based detection of gyrB2 gene from Pseudomonas sp. affected human clinical isolates.

Detection of virulence gene is a key component in determining the pathogenicity of any isolates because these genes act multi-functionally and multi-factorially. Gyrase specific gene primer in combination of PCR technology allows precise detection of DNA gyrase subunit B2 gene (gyrB2) from different virulent microorganisms. In the present study, forward and reverse primers with a length of 20bp for both were used for detection of gyrB2 genes in clinical isolates of Pseudomonas sp. collected from patients suffering from urinary tract infection (UTI). A total of 12 isolates of Pseudomonas sp. viz., Ps1, Ps2, Ps3, Ps4, Ps5, Ps6, Ps7, Ps8, Ps9, Ps10, Ps11 and Ps12 were used in present study in which gyrB2 gene amplified in all 12 isolates and gave the expected 1130bp PCR product after visualization under gel documentation system in 1.2% agarose gel. This PCR was outstanding in the detection of gyrB2 gene in urinary tract infected patients caused by Pseudomonas sp. species.

Differentiation of clinical Mycobacterium tuberculosis complex isolates by their GyrB polymorphism.

Purpose: To evaluate the reliability of the gyrB PCR-RFLP technique in differentiating clinical Mycobacterium tuberculosis complex isolates. Materials and Methods: A primer pair MTUB-f and MTUB-r for M. tuberculosis complex (MTBC) was used to differentiate 79 mycobacterial isolates by specific amplification of the 1,020-bp fragment of the gyrB gene (gyrB-PCR1). The MTBC isolates were further differentiated using a set of specific primers MTUB-756-Gf and MTUB-1450-Cr that allowed selective amplification of the gyrB fragment specific for M. tuberculosis (gyrB-PCR2). The DNA polymorphisms in the 1,020-bp gyrB fragment for 7 M. tuberculosis strains confirmed by PCR as well as 2 reference strains; M. tuberculosis H37Rv and M. bovis BCG were analyzed with the restriction enzyme Rsa1. Results: Seventy-seven (97.5%) isolates were positive for gyrB-PCR1 and thus identified as members of M. tuberculosis complex (MTBC) and two (2.6%) isolates were negative and identified as Mycobacteria other than tuberculosis (MOTT). All the M. tuberculosis isolates showed the typical M. tuberculosis specific Rsa1 RFLP patterns (100, 360, 560-bp) while 360 and 480-bp fragments were generated from M. bovis BCG. Conclusion: The gyrB PCR-RFLP using the endonuclease Rsa1 can be used to differentiate M. tuberculosis from M. bovis in clinical isolates.

Mutaciones en genes gyrA y gyrB en cepas de bacilos Gram negativos aisladas en hospitales chilenos y su relación con la resistencia a fluoroquinolonas / Mutations in gyrA and gyrB genes among strains of Gram-negative bacilli isolated from Chilean hospitals and their relation with resistance to fluoroquinolones

Background: A progressive frequency of resistance to fluorquinolones is observed among Gram-negative bacilli. Aim: To investigate the mechanism of resistance to fluoroquinolones mediated by mutations affecting gyrA and gyrB genes in strains of Gram negative bacüli isolated from CMean hospitals. Material and method: Minimal inhibitory concentration of fluoroquinolones was determined in 91 randomly selected nalidixic acid-resistant strains of Escherichia coli, Klebsiella pneumoniae, Acinetobacter baumannii and Pseudomonas aeruginosa, isolated from hospitals of 12 Chilean cities. Quinolone resistance determining region (QRDR) was amplified by PCR and mutations were determined by restriction fragment length polymorphism (RFLP) and DNA sequencing. Results: Strains with mutation in codon 83 of gyrA showed decreased susceptibility to ciprofloxacin with MICs ranging from 0.25 to 1024 fig/ml. The sequencing of PCR products for gyrA indicated amino acid changes in the QRDR region. One strain ofE. coli presented a double mutation, in codon 83 Ser to Leu as well as in codon 87 Asp to Asn. In strains ofK. pneumoniae, however, the change of codon 83 was Ser to Tyr, in A. baumannii was Ser to Leu and in P. aeruginosa was Thr to He. No strains with mutations affecting gyrB were found. Conclusions: Mutations in codon 83 of gyrA is a frequent genetic event involved in the mechanism leading to decreased susceptibility to fluoroquinolone in strains of Gram-negative bacilli.

Evaluation of gyrB as Chromosomal Marker in Bacillus anthracis

Bacillus anthracis is generally accepted as the most potent biological warfare agent because of its highly pathogenic nature and transmission efficiency. Identification of chromosomal markers for the rapid detection of B. anthracis is difficult since significant chromosomal homology exists among B. anthracis, B. cereus and B. thuringiensis. In this study, we tested whether the gyrB sequence could be used as the target for the PCR detection of B. anthracis. The gyrB sequence, composed of 1,923 bp, was identical in 17 Korean B. anthracis isolates. The comparison of gyrB sequence between B. anthracis and B. cereus type strain showed 8.8% difference (105 bp among 1,194 bp), and the gyrB sequence similarities of B. cereus, B. thuringiensis and B. mycoides with B. anthracis were 92.3%, 86.9% and 86.1%, respectively. When polymerase chain reaction was designed and performed based on the gyrB sequence, a specific amplicon (351 bp) could be amplified. These results indicate that gyrB could be useful as a chromosomal marker for the rapid screening of B. anthracis by PCR or differentiation of B. anthracis from other related species by multiplex PCR with other plasmid markers.

Mutations in folP1, rpoB, and gyrB associated with multiple drug resistance in Mycobacterium leprae isolates from South Korea / 나학회지

The emergence of multiple drug resistant Mycobacterium leprae has emphasized the need for early decision of effective antileprosy drug in the treatment for leprosy patients. Mutations in the genes associated with multiple drug resistance in Mycobacterium leprae isolates from 17 South Korean patients, who were already confirmed to have mutations in folP1 gene, were investigated using a PCR - single strand conformation polymorphism (SSCP) - DNA sequencing assay. Two strains, which has double mutations, were found in the two unrelated patients : one missense mutation in folP1 (Arg 55 for Pro) and in rpoB (Gly 522 for Ser), and in folP1 (Ala 53 for Thr) and in gyrB (Asn 426 for Asp), respectively. The patients were treated with the long monotheraphy of dapsone or with the inappropriate regimen of antileprosy drugs. These results emphasize the importance of multi-drug theraphy in order to prevent mult-idrug resistance and assist in the choice of the appropriate regimens for treating leprosy.

Virulence Factors and Genotyping of Vibrio parahaemolyticus in Sea Water and Clinical Isolates

Six strains of Vibrio parahemolyticus isolated from diarrheal patients and the 12 strains from sea water were serotyped and analyzed for biochemical characteristics, antibiotics sensitivity and detection of toxR, gyrB, tdh, and trh genes. Arbitrarily-primed polymerase chain reaction method were performed on the 6 strains from patients and the following results were obtained. 1. The Vibrio parahemolyticus isolated from patients were belonged to 5 different serotypes: 04:K8, 04:KUT, 06: K18, 010:K71 and 03:K6, but those isolated from sea water were belonged to 5 different serotypes: O1:KUT, 02:KUT, 03:K45, 04:K37 and OUT:KUT. All strains explained have different serotypes depending on the different source, 2. Three serotype (04:K8, 04:KUT, 06:K18) isolated from patients were positive for the urease hydrolysis, whereas only one strain of serotype O1:KUT isolated from sea water was positive to the same. Furthermore, the serotype 06:K18 (1 strain) was positive for the fermentation of dulcito1. Both toxR and gyrB genes were detected from all strains isolated. 3. As for control the 2 strains of serotype 03:K6 and 6 strains isolated from patients, serotype 03:K6 were resistant to oxacillin, penicillin, vancomycin. All strains were sensitive to chloramphenicol and tetracycline yet the antibiogram type showed 6 groups from I to VI. 4. DNA probe hybridization method was used to detect genes. The trh1 was detected both from serotype 04:KUT and 06:K18 isolated from patients and the trh2 was also detected from one strain from each 010:K71 and O1:KUT isolated from patients and sea-water respectively, The tdh gene only was detected from two strains of 03:K6 isolated from patients of 1998. The tdh, trh 1 and trh2 were not detected from 7 strains out of 12 strains isolated from sea water whereas the production titer of TDH isolated from patients showed from 2048 times to 4096 times. 5. Four strains of the serotype 03:K6 isolated from Korea, India and Japan as well as 3 strains from Korean patients were tested by AP-PCR to classify serotypes. As for its result the amplicon showed the same in the 4 strains of the serotype 03:K6 whereas the four strains of different serotype from patients are so difference as to explain no inter- relations at all. The result explains that the serotype 03:K6 is the same genes regardless from where it is isolated.

NEW PHYLOGENETIC MARKERS AND THEIR APPLICATIONS / 微生物学通报

Phylogenetic markers are the gene fragments that demonstrate the gene tic relationships between organisms To differentiate close taxa, many new phyl oge n etic markers were used, with which the polyphasic taxonomy was enriched This a r ticle chiefly described the characters of several phylogenetic markers and their applications in the studies of the phylogenetics of bacteria