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PubMed2006

Electrochemical detection of E. coli 16S rDNA sequence using air-plasma-activated fullerene-impregnated screen printed electrodes.

A new DNA modified electrode for the electrochemical detection of 16S rDNA extracted from Escherichia coli (JCM1649) is proposed. The electrodes were fabricated by screen printing a fullerene-impregnated carbon ink onto a poly(methylmethacrylate) substrate and immobilizing a probe DNA on the surface after activating the electrode with air plasma. The results indicated a dramatic improvement in the surface coverage of the immobilized probe DNA, and of the reduction peak of the redox indicator (Co(phen)(3)(3+)) due to the incorporation of fullerene. By immobilizing the probe onto the fullerene-impregnated screen-printed electrodes, the PCR product of the 16S rDNA extracted from E. coli was directly detected without any pretreatment. A well defined signal difference was observed between the perfectly matching oligonucleotide and the mismatching one, and it was possible to detect the target at the modified electrode. This method enabled us to clearly detect the two base mismatches in the ca. 1500-bases long 16S rDNA sequence.

Air↗
PubMed2006

First record of Leptospira borgpetersenii isolation in the Amami Islands, Japan.

In 2003, a Leptospira survey was performed on Yoroshima Island of the Amami Islands located in the southwestern part of Japan. Seven Leptospira strains were isolated from the field rat Rattus rattus, which were identified as L. borgpetersenii by flaB sequencing, 16S rDNA sequencing and gyrB sequencing, and serovar Javanica was determined by a microscopic agglutination test. NotI-long restriction fragment analysis indicated that these isolates were genetically indistinguishable from an isolate from the Okinawa Islands. The present results suggest that L. borgpetersenii is migrating into the Amami Islands in Japan.

Animals↗
PubMed2003

Identification of signature and primers specific to genus Pseudomonas using mismatched patterns of 16S rDNA sequences.

BACKGROUND: Pseudomonas, a soil bacterium, has been observed as a dominant genus that survives in different habitats with wide hostile conditions. We had a basic assumption that the species level variation in 16S rDNA sequences of a bacterial genus is mainly due to substitutions rather than insertion or deletion of bases. Keeping this in view, the aim was to identify a region of 16S rDNA sequence and within that focus on substitution prone stretches indicating species level variation and to derive patterns from these stretches that are specific to the genus. RESULTS: Repeating elements that are highly conserved across different species of Pseudomonas were considered as guiding markers to locate a region within the 16S gene. Four repeating patterns showing more than 80% consistency across fifty different species of Pseudomonas were identified. The sub-sequences between the repeating patterns yielded a continuous region of 495 bases. The sub-sequences after alignment and using Shanon's entropy measure yielded a consensus pattern. A stretch of 24 base positions in this region, showing maximum variations across the sampled sequences was focused for possible genus specific patterns. Nine patterns in this stretch showed nearly 70% specificity to the target genus. These patterns were further used to obtain a signature that is highly specific to Pseudomonas. The signature region was used to design PCR primers, which yielded a PCR product of 150 bp whose specificity was validated through a sample experiment. CONCLUSIONS: The developed approach was successfully applied to genus Pseudomonas. It could be tried in other bacterial genera to obtain respective signature patterns and thereby PCR primers, for their rapid tracking in the environmental samples.

Base Pair Mismatch↗
PubMed2001

Evaluation of numerical analysis of PFGE-DNA profiles for differentiating Campylobacter fetus subspecies by comparison with phenotypic, PCR and 16S rDNA sequencing methods.

AIMS: To assess the efficacy of numerical analysis of PFGE-DNA profiles for identification and differentiation of Campylobacter fetus subspecies. METHODS AND RESULTS: 31 Camp. fetus strains were examined by phenotypic, PCR- and PFGE-based methods, and the 16S rDNA sequences of 18 strains compared. Numerical analysis of PFGE-DNA profiles divided strains into two clusters at the 86% similarity level. One cluster contained 19 strains clearly identified as Camp. fetus subsp. venerealis. The other cluster comprised 12 strains, of which 10 were unambiguously identified as Camp. fetus subsp. fetus. The remaining two strains were identified as Camp. fetus subsp. venerealis by either phenotypic or PCR methods, but not both. At higher similarity levels, clusters containing isolates from each of two countries were identified, suggesting that certain clones predominate in certain geographical regions. CONCLUSION: Numerical analysis of PFGE-DNA profiles is an effective method for differentiating Camp. fetus subspecies. SIGNIFICANCE AND IMPACT OF THE STUDY: Critical comparison of PFGE, PCR, 16S rDNA sequencing and phenotypic methods for differentiation of Camp. fetus subspecies was attained. Novel phenotypic markers for distinguishing subspecies were identified. Evidence for dominant clones of each subspecies in certain countries was provided.

Animals↗
PubMed1993

Partial sequence of the gene for a serine protease from a halophilic archaeum Haloferax mediterranei R4, and nucleotide sequences of 16S rRNA encoding genes from several halophilic archaea.

A part of the gene coding for a halophilic serine protease from a halophilic archaeum Haloferax mediterranei R4 was amplified by PCR and its 672 nucleotide sequence was determined. Tentative translation to the amino acid sequence suggested that the enzyme was quite similar to halolysin produced by another halophilic archaeum strain 172P1. Nucleotide sequences of 16S rRNA encoding genes from 9 halophilic archaea were determined. Alignment of 19 sequences known so far showed that there are more than 20 positions carrying bases or deletions specific for each halobacterial genus: Halobacterium, Haloarcula, Haloferax, and Halococcus.

Amino Acid Sequence↗
PubMed1996

Bacterial host specificity of Lucinacea endosymbionts: interspecific variation in 16S rRNA sequences.

Three tropical lucinid clams (Codakia orbiculata, Codakia pectinella and Lucina nassula) from a shallow coastal environment have been studied regarding to their thioautotrophic bacterial endosymbionts. The 16S rRNA genes (rDNA) from these three endosymbionts were amplified using PCR. Phylogenetic analysis by distance matrix and parsimony methods always placed the newly examined symbionts within the monophyletic group composed of symbionts of the bivalve superfamily Lucinacea. A same single 16S rRNA sequence was found in C. orbiculata and C. pectinella and was identical to that found in C. orbicularis and Linga pensylvanica, two other lucinids living in the same type of environment. These data indicate that a same symbiont species may be associated with different host species. Lucina nassula host a symbiont with a distinct 16S rDNA sequence, but very closely related to the former.

Animals↗
PubMed2005

Reduced levofloxacin susceptibility and tetracycline resistance in a clinical isolate of Haemophilus quentini identified by 16S rRNA sequencing.

This paper reports the first case of Haemophilus quentini bacteremia with reduced susceptibility to levofloxacin and resistance to nalidixic acid identified by 16S rRNA sequencing. There was an S 84 L substitution in gyrA and an S 84 I substitution in par C. The isolate had co-resistance to ampicillin (beta-lactamase positive) and tetracycline mediated by the tet(B) gene.

Anti-Bacterial Agents↗
PubMed1998

Mitochondrial 16S rDNA sequences and phylogenetic relationships of species of Rhipicephalus and other tick genera among Metastriata (Acari: Ixodidae).

The mitochondrial 16S rRNA gene sequences of the following eight European Metastriata tick species were obtained by direct polymerase-chain-reaction cycle sequencing and silver-staining methods: Rhipicephalus bursa, R. pusillus, R. sanguineus, R. turanicus, Boophilus annulatus, Dermacentor marginatus, Haemaphysalis punctata, and Hyalomma lusitanicum. This mitochondrial gene seems to be a good marker for the establishment of genetic relationships among closely related tick species, but it does not seem to be useful for comparisons of distantly related taxa. The molecular data provide very strong support for the monophyly of the Rhipicephalinae, including Hyalomma spp. However, the genus Rhipicephalus may not be considered a monophyletic group; in all analyses carried out in this study, R. bursa clustered with Boophilus spp. The high percentage of similarity (98.7%) observed between R. sanguineus and R. turanicus sequences would suggest that these species recently diverged within the Rhipicephalus genus. Phylogenetic analyses showed a monophyletic relationship among Amblyomminae taxa. The relationships between Haemaphysalis species and the true placement of this genus within Metastriata could not be resolved.

Animals↗
PubMed1996

Phylogenetic relationships and diversity within the Pasteurella haemolytica complex based on 16S rRNA sequence comparison and outer membrane protein and lipopolysaccharide analysis.

The outer membrane protein (OMP) and lipopolysaccharide (LPS) profiles of 30 untypeable isolates of Pasteurella haemolytica were examined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and compared with the profiles of typeable isolates. The phylogenetic relationships of 28 isolates representing each of the serotypes of P. haemolytica and Pasteurella trehalosi, as well as untypeable isolates of P. haemolytica, were determined by comparing 16S rRNA sequences. The analysis of the OMP and LPS profiles of the untypeable isolates revealed five groups, which were designated untypeable groups 1 (UG1) through UG5. The UG1 and UG2 isolates had OMP and LPS profiles identical to the profiles of certain serotype A1 and A2 isolates, respectively. Furthermore, UG1 isolates originating from cattle and sheep could be clearly differentiated on the basis of their OMP profiles. The OMP and LPS profiles of UG3 isolates were similar appearance to the profiles of serotype A11 isolates, suggesting that these two groups are closely related. The OMP profiles of UG4 and UG5 isolates were unique and different from the OMP profiles of the UG1 through UG3 isolates. A comparison of 16S rRNA sequences revealed that typeable isolates of P. haemolytica could be divided into the following three groups: (i) serotype A1, A5 through A9, A12 through A14, and A16 isolates, (ii) serotype A2 isolates, and (iii) serotype A11 isolates. the isolates belonging to the first group all had identical sequences, whereas the sequences of isolates belonging to the second and third groups differed from the sequences of the isolates belonging to the first group at two and four base positions, respectively. The sequence data for the untypeable isolates confirmed the conclusions derived from the OMP and LPS analysis. Isolates belonging to UG1 and UG2 were identical to serotype A1 and A2 isolates, respectively; isolates belonging to UG3 were related to serotype A11 isolates, although there was some sequence heterogeneity within this group; and isolates belonging to UG4 and UG5 were more distantly related to P. haemolytica than were isolates belonging to UG1 through UG3 and were clearly members of two different species. As expected, isolates of P. trehalosi were event more distantly related to P. haemolytica than were the untypeable isolates, but there was significantly more sequence variation among the four serotypes of this species than there was among the serotypes of P. haemolytica. The correlation of the OMP and LPS data with the 16S rRNA sequence data suggested that OMP and LPS analyses might be useful for preliminary screening and comparing large numbers of isolates in taxonomic and epidemiological studies of the Pasteurellaceae.

Animals↗
PubMed1991

Phylogenetic characterization and in situ localization of the bacterial symbiont of shipworms (Teredinidae: Bivalvia) by using 16S rRNA sequence analysis and oligodeoxynucleotide probe hybridization.

It has been proposed that a bacterium isolated from the gills of shipworms (teredinid mollusks) is, by virtue of its ability both to degrade cellulose and to fix dinitrogen, the symbiont that enables these mollusks to utilize wood as their principal food source. The phylogenetic affiliation of four of these bacteria isolated from wood-boring bivalve mollusks was determined by 16S rRNA sequence analysis by using the reverse transcriptase method with six oligodeoxynucleotide primers. The four bacterial strains tested had indistinguishable 16S rRNA sequences, supporting the previous conclusion, based on phenotypic characterization, that these isolates represent a single species. Evolutionary distance matrix analysis of the RNA sequence indicated that the bacterial symbiont falls within the gamma-3 subdivision of the Proteobacteria and is distinct from other known bacterial genera. In situ localization of the bacterial symbiont in tissue sections of the shipworm Lyrodus pedicellatus was determined by using a 16S rRNA-directed oligodeoxynucleotide hybridization probe specific for the bacterium isolated from shipworm gill tissue. Fluorescence microscopy showed that the specific probe bound to L. pedicellatus tissue at sites coincident with the location of symbiont cells and that it did not bind to other host tissues. This technique provided direct visual evidence that the cellulolytic, nitrogen-fixing bacterial isolates were the symbionts observed within the gill of L. pedicellatus.

Animals↗
PubMed2001

Isolation and 16S rRNA sequence analysis of the beneficial bacteria from the rhizosphere of rice.

The present study deals with the isolation of plant growth promoting rhizobacteria (PGPR) from rice (variety NIAB IRRI-9) and the beneficial effects of these inoculants on two Basmati rice varieties. Nitrogen-fixing activity (acetylene-reduction activity) was detected in the roots and submerged shoots of field-grown rice variety NIAB IRRI-9. Estimation of the population size of diazotrophic bacteria by ARA-based MPN (acetylene reduction assay-based most probable number) in roots and shoots indicated about 10(5)-10(6) counts/g dry weight at panicle initiation and grain filling stages. Four bacterial isolates from rice roots and shoots were obtained in pure culture which produced phytohormone indoleacetic acid (IAA) in the growth medium. Among these, three isolates S1, S4, and R3 reduced acetylene to ethylene in nitrogen-free semi-solid medium. Morphological and physiological characteristics of the isolates indicated that three nitrogen-fixing isolates S1, S4, and R3 belonged to the genus Enterobacter, while the non-fixing isolate R8 belonged to the genus Aeromonas. 16S rRNA sequence of one isolate from root (R8) and one isolate from shoot (S1) was obtained which confirmed identification of the isolates as Aeromonas veronii and Enterobacter cloacae, respectively. The 1517-nucleotide-long sequence of the isolate R8 showed 99% similarity with Aeromonas veronii (accession No. AF099023) while partial 16S rRNA sequence (two stretches of total 1271 nucleotide length) of S1 showed 97% similarity with the sequence of Enterobacter cloacae (accession No. AJ251469). The seedlings of two rice varieties Basmati 385 and Super Basmati were inoculated with the four bacterial isolates from rice and one Azospirillum brasilense strain Wb3, which was isolated from wheat. In the rice variety Basmati 385, maximum increase in root area and plant biomass was obtained in plants inoculated with Enterobacter S1 and Azospirillum Wb3, whereas in the rice variety Super Basmati, inoculation with Enterobacter R3 resulted in maximum increase of root area and plant biomass. Nitrogen fixation was quantified by using 15N isotopic dilution method. Maximum fixation was observed in Basmati 385 with the inoculants Azospirillum Wb3 and Enterobacter S1 where nearly 46% and 41% of the nitrogen was derived from atmosphere (%Ndfa), respectively. In general, higher nitrogen fixation was observed in variety Basmati 385 than in Super Basmati, and different bacterial strains were found more effective as inoculants for the rice varieties Basmati 385 and Super Basmati.

Aeromonas↗
PubMed1992

Phylogenetic relationships of the endosymbionts of mealybugs (Homoptera: Pseudococcidae) based on 16S rDNA sequences.

A portion of the gene coding for the 16S ribosomal RNA from the endosymbionts of three species of mealybugs [Pseudococcus longispinus (Targioni-Tozzetti), Pseudococcus maritimus (Ehrhorn), and Dysmicoccus neobrevipes (Beardsley)] was cloned, sequenced, and compared to a homologous fragment from bacteria representative of aphid endosymbionts as well as major subdivisions of the Proteobacteria. Parsimony analysis of the sequences indicated that the mealybug endosymbionts are related and belong to the beta-subdivision; in contrast, previous studies showed that aphid endosymbionts are part of the gamma-subdivision. These findings suggest that the endosymbiosis of mealybugs is a consequence of a single bacterial infection and indicate that this ancestor was different from the ancestor involved in aphid endosymbiosis.

Animals↗
PubMed1995

Identification of grass-associated and toluene-degrading diazotrophs, Azoarcus spp., by analyses of partial 16S ribosomal DNA sequences.

The genus Azoarcus includes nitrogen-fixing, grass-associated strains as well as denitrying toluene degraders. In order to identify and group members of the genus Azoarcus, phylogenetic analysis based on partial sequences of 16S rRNA genes (16S rDNAs) is proposed. 16S rRNA-targeted PCR using specific primers to exclude amplification in the majority of other members of the beta subclass of the class Proteobacteria was combined with direct sequencing of the PCR products. Tree inference from comparisons of 446-bp rDNA fragments yielded similar results for the three known Azoarcus spp. sequences and for analysis of the complete 16S rDNA sequence. These three species formed a phylogenetically coherent group with representatives of two other Azoarcus species which were subjected to 16S rRNA sequencing in this study. This group was related to Rhodocyclus purpureus and Thauera selenatis. New isolates and also sequences of so far uncultured bacteria from roots of Kallar grass were assigned to the genus Azoarcus as well. Also, strains degrading monoaromatic hydrocarbons anaerobically in the presence of nitrate clustered within this genus, albeit not with grass-associated isolates. All representative members of the five species harboring rhizospheric bacteria were able to form N2O from nitrate and showed anaerobic growth on malic acid with nitrate but not on toluene. In order to visualize different Azoarcus spp. by whole-cell in situ hybridizations, we generated 16S rRNA-targeted, fluorescent probes by in vitro transcription directly from PCR products which spanned the variable region V2. Hybridization was species specific for Azoarcus communis and Azoarcus indigens.(ABSTRACT TRUNCATED AT 250 WORDS)

Base Sequence↗
PubMed2003

The bacterial flora of fresh and chill-stored pork: analysis by cloning and sequencing of 16S rRNA genes.

The composition of the initial bacterial flora of pork and the development of the flora after storage at +4 degrees C for 4 days were analysed by amplification, cloning and sequencing of 16S rDNA. A total of 122 clones were obtained, with lengths of > or =400 nucleotides and > or =95% similarity to database sequences. Nineteen clones were similar to sequences in database not assigned to any genera. Fourteen different genera were represented in clones from fresh meat, with 36.5% of the clones most resembling Acinetobacter and 17.3% resembling Staphylococcus and Macrococcus. After storage, the clones were composed of six different genera, with 44.3% resembling Pseudomonas, 17.1% resembling Aeromonas and only 14.3% resembling Acinetobacter. This study shows that the overall pattern of the initial and chill-stored pork flora, as shown by a molecular approach, was in agreement with results obtained in previous studies using traditional cultivation methods.

Animals↗
PubMed2001

Phylogeny and evolution of African shrews (Mammalia: Soricidae) inferred from 16s rRNA sequences.

Current phylogenetic hypotheses on the African Crocidurinae (Soricidae) are based upon morpho-anatomical, karyological, and allozyme studies. The present study attempts to resolve the interrelationships among African Crocidurinae and their relationships to Eurasian Crocidurinae and to the subfamily Soricinae, on the basis of partial mitochondrial 16s rRNA sequences (549 bp). This is the first molecular study to include all but one of the nine currently recognized African shrew genera. In agreement with current views, two major lineages emerge. The first lineage includes Myosorex and Congosorex and supports the existence of a myosoricine taxon. The second lineage includes the six remaining genera. The genus Sylvisorex appears to be polyphyletic, whereas species of the controversial genus Crocidura are monophyletic. The genus Suncus presumably originated in Africa. The monospecific genera Ruwenzorisorex and Scutisorex and the two representatives of Paracrocidura cluster with species of other genera. Grouping patterns of species from different continents suggest that there have been multiple exchanges between Africa and Eurasia. The time estimates of these exchanges, inferred from two independent fossil-based calibrations of a molecular clock, coincide with the time estimates for migration events in other mammalian taxa.

Animals↗
PubMed2002

Use of 16S rRNA sequencing for identification of Actinobacillus ureae isolated from a cerebrospinal fluid sample.

Actinobacillus ureae, previously Pasteurella ureae, has on rare occasions been described as a cause of human infection. Owing to its rarity, it may not be easily identified in clinical microbiology laboratories by standard tests. This report describes a patient with acute bacterial meningitis due to A. ureae. The identity of the isolate was determined by means of DNA sequence analysis of a portion of the 16S rRNA gene.

Actinobacillus↗
PubMed2000

Phylogenetic affiliation of the pseudomonads based on 16S rRNA sequence.

The broad and vague phenotypic definition allowed the genus Pseudomonas to become a dumping ground for incompletely characterized polarly flagellated, gram-negative, rod-shaped, aerobic bacteria, and a large number of species have been accommodated in the genus Pseudomonas. The 16S rRNA sequences of 128 valid and invalid Pseudomonas species, which included almost valid species of the genus Pseudomonas listed in the Approved Lists of Bacterial Names, were obtained: sequences of 59 species were determined and those of 69 species were obtained from the GenBank/EMBL/DDBJ databases. These sequences were compared with the sequences of other species of the Proteobacteria. Fifty-seven valid or invalid species including Pseudomonas aeruginosa (type species of the genus Pseudomonas Migula 1894) belonged to the genus Pseudomonas (sensu stricto). Seven subclusters were formed in the cluster of the genus Pseudomonas (sensu stricto), and the resulting clusters conformed well to the rRNA-DNA hybridization study by Palleroni (1984). The other species did not belong to the genus Pseudomonas (sensu stricto) and were related to other genera, which were placed in four subclasses of the Proteobacteria (alpha, beta, gamma and gamma-beta subclasses). Twenty-six examined species, which were not included in the cluster of the Pseudomonas (sensu stricto) and have not been transferred to other genera as yet, are listed alphabetically: 'Pseudomonas abikonensis', Pseudomonas antimicrobica, Pseudomonas beijerinckii, Pseudomonas beteli, Pseudomonas boreopolis, 'Pseudomonas butanovora', Pseudomonas carboxydohydrogena, Pseudomonas cissicola, Pseudomonas doudoroffii, Pseudomonas echinoides, Pseudomonas elongata, Pseudomonas flectens, Pseudomonas geniculata, Pseudomonas halophila, Pseudomonas hibiscicola, Pseudomonas huttiensis, Pseudomonas iners, Pseudomonas lanceolata, Pseudomonas lemoignei, Pseudomonas mephitica, Pseudomonas pictorum, Pseudomonas saccharophila, Pseudomonas spinosa, Pseudomonas stanier, Pseudomonas syzygii and Pseudomonas woodsii. The phylogenetic affiliations of these 26 pseudomonads species are shown.

Cloning, Molecular↗