A newly constructed primer pair for the PCR amplification, cloning and sequencing of the flagellin (flaA) gene from isolatesof urease-negative Campylobacter lari.

A newly constructed primer pair (lari-Af/lari-Ar) designed to generate a product of the flagellin (flaA) gene for urease-negative Campylobacter lari produced a PCR amplicon of about 1700 bp for 16 isolates from 7 seagulls, 5 humans, 3 food animals and one mussel in Japan and Northern Ireland. Nucleotide sequencing and alignments of the flaA amplicons from these isolates demonstrated that the deduced amino acid sequences of the possible open reading frame were 564-572 amino acid residues in length with calculated molecular weights of 58,804 to 59,463. The deduced amino acid sequence similarity analysis strongly suggested that the ORF of the flaA from the 16 isolates showed 70-75% sequence similarities to those of Campylobacter jejuni isolates.

[Relationship between of carotid ultrasonography in Japanese hypertensive subjects for intima-media thickness and plaque, and candidate gene polymorphism--possibility of early detection of arteriosclerotic disease].

The purpose of this study was to determine the relationship between organic changes in carotid artery walls and candidate gene polymorphism in Japanese sufferers of essential hypertension. Carotid Ultrasonography was used to measure intima-media thickness (IMT) and presence of plaque formation. Patients were divided into two groups; a hypertension (HT) group and a healthy control (C).

Phenotypic characterisation of flagellin and flagella of urease-positive thermophilic campylobacters.

In this study, flagellin is purified biochemically from eight urease-positive thermophilic camplylobacters (UPTC) isolated from river water, sea water and mussels, and purified also from two isolates of Campylobacter jejuni and C. coli and fractionated by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE). Results showed that no flagellin components were detected in the two Japanese UPTC isolates (CF89-12 and CF89-14) and the two UPTC NCTC strains (NCTC12893 and NCTC12894).

Nucleotide sequence analysis of the recA gene and discrimination of the three isolates of urease-positive thermophilic Campylobacter (UPTC) isolated from seagulls (Larus spp.) in Northern Ireland.

Nucleotide sequencing after TA cloning of the amplicon of the almost-full length recA gene from three strains of UPTC (A1, A2, and A3) isolated from seagulls in Northern Ireland, the phenotypical and genotypical characteristics of which have been demonstrated to be indistinguishable, clarified nucleotide differences at three nucleotide positions among the three strains. In conclusion, the nucleotide sequences of the recA gene were found to discriminate among the three strains of UPTC, A1, A2, and A3, which are indistinguishable phenotypically and genotypically. Thus, the present study strongly suggests that nucleotide sequence data of the amplicon of a suitable gene or region could aid in discriminating among isolates of the UPTC group, which are indistinguishable phenotypically and genotypically.

Molecular cloning, nucleotide sequencing and characterization of the flagellin gene from isolates of urease-positive thermophilic Campylobacter.

A primer pair which was expected to generate an amplicon of the estimated size (approximately 1700 base pair (bp)) of the flaA gene for Campylobacter jejuni amplified products of approximately 1450 bp for 33 of the 44 isolates of urease-positive thermophilic Campylobacter (UPTC). The primer pair, however, failed to amplify fragments for 11 isolates of UPTC, for all of the 12 isolates of urease-negative C. lari and for one isolate of C.

Recent advances in molecular epidemiology and detection of Taylorella equigenitalis associated with contagious equine metritis (CEM).

In the present review article, recent molecular advances relating to studies with Taylorella equigenitalis, as well as the recently described second species of the genus Taylorella, namely Taylorella asinigenitalis, have been described. Molecular genotyping of T. equigenitalis strains by pulsed-field gel electrophoresis (PFGE) after digestion with the suitable restriction enzyme(s) enabled the effective discrimination of strains, thus allowing the examination of the scientific mechanism(s) for its occurrence and transmission of contagious equine metritis (CEM).

Structural analysis and genetic variation of the 16S-23S rDNA internal spacer region from Micrococcus luteus strains.

Aims: To clone and sequence the 16S-23S ribosomal DNA (rDNA) internal spacer region (ISR) from Micrococcus luteus.

Methods And Results: The primer pair for 16S-23S rDNA ISR amplified a fragment of about 850 bp in length for two strains, JCM3347 and JCM3348 and a fragment of about 790 bp for a strain, ATCC9341. After sequencing the ISRs were identified by the comparison of the ISRs and the flanking regions of ISR.

Studies on the genomic heterogeneity of Micrococcus luteus strains by macro-restriction analysis using pulsed-field gel electrophoresis.

Macro-restriction analysis by means of double digestion using DraI and VspI demonstrated that they cleaved the genomic DNAs from Micrococcus luteus JCM1464(T), JCM3347, and JCM3348 into four to five fragments in a distinguishable manner by pulsed-field gel electrophoresis (PFGE). Separate digestion with DraI and VspI cleaved the genomic DNA from M. luteus ATCC9341 into a relatively limited number of restriction fragments (six pieces).

Characterization of urease-positive thermophilic Campylobacter subspecies by multilocus enzyme electrophoresis typing.

Thirty-one urease-positive thermophilic Campylobacter (UPTC) isolates, including three reference strains (NCTC12892, NCTC12895 and NCTC12896), and three Campylobacter lari isolates, which were isolated from several countries and sources, were compared genotypically by using multilocus enzyme electrophoresis (MLEE). We examined allelic variation around seven enzyme loci, including the adenylate kinase, alkaline phosphatase, catalase, fumarase, malic enzyme, malate dehydrogenase, and L-phenylalanyl-L-leucine peptidase loci. MLEE typing revealed the presence of 23 different electrophoretic types (ETs) among the 31 UPTC isolates, and 14 isolates shared six electrophoretic profiles.

[Antimicrobial susceptibility tests and resistant strain of Helicobacter pylori].

The antimicrobial susceptibility test was necessary for the eradication therapy of Helicobacter pylori infections. This is because, clarithromycin resistant strains has became an increasing problem. In this study, we used the antimicrobial susceptibility test which was compare with the agar gradient method, Etest, and broth microdilution method (dry plate) with 4 antimicrobial agents.

flaA-like sequences containing internal termination codons (TAG) in urease-positive thermophilic Campylobacter isolated in Japan.

Aims: To demonstrate two flaA-like sequences containing two internal termination codons (TAG) in two Japanese strains of urease-positive thermophilic Campylobacter (UPTC).

Methods And Results: A primer pair of A1 and A2, which ought to generate a product of approx. 1700 bp of the flaA gene for Campylobacter jejuni, was used to amplify products of approx.

First isolation of urease-positive thermophilic Campylobacter (UPTC) from crows (Corvus levaillantii) in Japan.

Two strains of urease-positive thermophilic Campylobacter (UPTC), designated YC98-1 and YC98-2, were identified by biochemical characterization after isolation from the intestinal contents of crows around Yokohama City, Japan, in 1998. The biochemical characteristics of these strains were identical to those of strains described previously. Pulsed-field gel electrophoresis (PFGE) after separate digestion with ApaI, SalI, and SmaI of the genomic DNA from the two strains indicated that respective PFGE profiles were distinctly different and distinguishable from each other.

Molecular genotyping of isolates of Actinobacillus actinomycetemcomitans by pulsed-field gel electrophoresis after separate digestion with Sse8387I and XhoI.

Genomic DNA from 18 Japanese clinical isolates of Actinobacillus actinomycetemcomitans was obtained from six periodontitis patients and analyzed using pulsed-field gel electrophoresis (PFGE) after separate digestion with Sse8387I and with XhoI. Three isolates from an identical patient were found to share an identical PFGE profile, and isolates from distinct patients were found to have PFGE profiles distinctly different from each other. Consequently, the 18 Japanese clinical isolates were discriminated into six distinct genotypes by means of PFGE.

Cloning and sequencing of 16S rDNA and 16S-23S rDNA internal spacer region (ISR) from urease-positive thermophilic Campylobacter (UPTC).

Aims: To clone and sequence the 16S rDNA and 16S-23S rDNA internal spacer region (ISR) from urease-positive thermophilic Campylobacter (UPTC).

Methods And Results: The primer sets for 16S rDNA and 16S-23S rDNA ISR amplified almost the full length of 16S rDNA and 16S-23S rDNA ISR. About 1500 bp for 16S rDNA and about 720 bp for 16S-23S rDNA ISR of the rrn operon of four strains of UPTC were identified after molecular cloning and sequencing.

Effects of selected metal ions on photodegradation of organophosphorus pesticides sensitized by humic acids.

Selected metal ions having paramagnetic property were found to exert inhibition effects on aquatic photodegradation of organophosphorus pesticides sensitized by humic acids, according to the increasing order of Cr(III) < Co(II) < Mn(II) < Cu(II). Basic factors dominating the metal-ion effects were clarified on the basis of the fluorescence quenching as well as radical scavenging abilities of metal ions complexed with humic acids.

Demonstration of heterogeneous genotypes of Taylorella equigenitalis isolated from horses in six European countries by pulsed-field gel electrophoresis.

Forty-six isolates of Taylorella equigenitalis were analysed by pulsed-field gel electrophoresis (PFGE) after separate digestion of the genomic DNA with ApaI and with NotI. The isolates had been obtained from horses in six European countries and were classified into 18 genotypes. In Belgium, 2 genotypes were detected in 2 isolates, in England 9 among 15, in Finland 2 in 2, in France 2 among 10, in Sweden 3 among 5, and in Switzerland 3 among 12.

Demonstration of genome DNA diversity of strains of urease-positive thermophilic Campylobacter isolated from the natural environment by pulsed-field gel electrophoresis analysis.

Pulsed-field gel electrophoresis (PFGE) analysis was carried out after separate digestion with Apa I, Sal I and Sma I of the genomic DNA from sixteen isolates of urease-positive thermophilic Campylobacter (UPTC) obtained from the natural environment, namely from oysters and mussels, in Northern Ireland. Five NCTC strains previously isolated in England were used for the analysis. Although the eight isolates of UPTC in Northern Ireland and a strain of UPTC in England showed that one or no fragments appeared after digestion with Apa I around 1,900 to 1,640 kb region of the gel, Apa I was shown to cut the genomic DNA from all of the other twelve strains of UPTC and Sal I and Sma I from all of the 21 strains in a distinctly different and distinguishable manner.

Comparison of the value of pulsed-field gel electrophoresis, random amplified polymorphic DNA and amplified rDNA restriction analysis for subtyping Taylorella equigenitalis.

Eight strains of Taylorella equigenitalis were identified by a polymerase chain reaction using a primer pair specific to the 16S rDNA of T equigenitalis. These eight strains were chosen because they had previously been shown to represent eight distinct genotypes by pulsed-field gel electrophoresis analysis after separate digestion of the genomic DNA with ApaI or NotI. The eight strains could be classified into six or seven types by random amplified polymorphic DNA analysis using different kinds of primers.

recA genotyping of Salmonella enteritidis phage type 4 isolates by restriction fragment length polymorphism analysis.

Aims: To subtype Salmonella enteritidis phage type 4 isolates by using recA genotyping.

Methods And Results: Random amplified polymorphic DNA analysis using a primer ERIC2 of 76 isolates of Salmonella enteritidis phage type 4 obtained in Northern Ireland in 1998 and in 1999 demonstrated the presence of five genotypes. Restriction fragment length polymorphism analysis, using a degenerate primer pair designed to amplify a segment (about 640 bp in length) of the recA gene from several members of the Enterobacteriaceae with restriction enzymes, HhaI and Sau3AI, showed that the resulting fragments could differentiate the isolates into three groups, respectively.