The distribution of adipose tissue and the metabolic profile in hypertensive and normotensive adults

Fifty isolates belonging to the six validly described species of the genus Thermus (T. aquaticus, T. filiformis, T. thermophilus, T. scotoductus, T. brockianus, and T. oshimai) isolated from hot springs of different geographical areas were compared using macrorestriction analysis of genomic DNA and ribotyping. With the exception of presumed clones, the macrorestriction patterns of isolates obtained with EcoRI or NdeI were distinct. However, isolates belonging to the same species exhibited similar profiles particularly when they were isolated from the same hot spring. The estimated genomic size of strains of the Thermus spp. varied between approximately 1.8 and 2.5 Mbp. Ribotyping with BamHI and HindIII produced 30 and 35 distinct ribotypes, respectively. In spite of the variability of the hybridization patterns produced, the ribotypes obtained for isolates belonging to the same species also shared, in general, several fragments of identical size, and these fragments were similar when isolates originated from the same spring.     

Molecular evolution of the internal transcribed spacers (ITS1 and ITS2) and phylogenetic relationships among species of the family Cucurbitaceae

Phylogenetic relationships of different members of the family Cucurbitaceae were estimated from sequences of the internal transcribed spacer (ITS1 and ITS2) regions of the nuclear ribosomal RNA genes. Twenty-six species of different genera belonging to different tribes and several subtribes were analyzed. The whole ITS regions were amplified by PCR technique and cloned, and three to five different clones of each species were sequenced; for some species PCR products were sequenced directly. ITS1 and ITS2 regions are slightly variable in length, with each length appearing genus-specific. A substitution rate of 3.62 x 10(-9) substitutions per site per year was calculated assuming 40 MYA separation time. Phylogenetic relationships inferred from ITS sequences of some species is in agreement with morphological data, but deviations to the taxonomic classification were also observed. A polyphyletic origin of the New World species must be considered. In the genus Cucurbita different "types" of ITS sequences within one species exist, possibly due to the high frequency of introgression during domestication or due to polyploidization events; in contrast, low intraspecific variability was detectable in the genus Cucumis, indicating different stages of speciation.     

Rearrangements in the genomes of Vibrio cholerae strains belonging to different serovars and biovars

The intron-encoded enzyme I-CeuI provides an excellent tool for rapidly examining the organization of genomes of related species of bacteria. Vibrio cholerae strains belonging to serovars O1 and O139 have 9 I-ceuI sites in their genomes, and V. cholerae strains belonging to serovars non-O1 and non-O139 have 10 I-ceuI sites in their genomes. This information can be used as a criterion to differentiate O1 strains from non-O1 and non-O139 strains. To our knowledge, intraspecies variation in the number of rrn operons has not been reported in any other organism. Our data revealed extensive restriction fragment length polymorphism based on a comparison of the I-ceuI digestion profiles of strains belonging to different serovars and biovars. From the analysis of partial digestion products, I-CeuI macrorestriction maps of several classical, El Tor, and O139 strains were constructed. While the linkage maps are conserved within biovars, linkage maps vary substantially between biovars.     

Bacterial whole cell protein profiles of the rRNA group II pseudomonads

Studies on bacterial whole cell protein profiles showed that members of the rRNA group II pseudomonads were distinct from other non-fluorescent and fluorescent pseudomonads, including Pseudomonas aeruginosa, the type species of the genus Pseudomonas. Strains of Ps. andropogonis, Ps. caryophylli, Ps. gladioli pv. gladioli, Ps. pickettii, Ps. pseudomallei and Ps. rubrisubalbicans showed uniform and distinct protein patterns, while strains of Ps. solanacearum and Ps. cepacia displayed differences within species. Numerical analysis of their protein profiles with GelManager and Taxan programs generated dendrograms comprising 16 clusters at 89% similarity. Each cluster included strains belonging to the same species with the exception of Ps. solanacearum, which fragmented into three clusters. Pseudomonas solanacearum showed different protein patterns correlating with different biovars and the two divisions of Cook et al. (1989), as well as the results of 16S rRNA gene sequencing. The whole cell protein profiles of a total of 83 strains belonging to 14 bacterial species were numerically analysed.     

Role of the multifunctional Trio protein in the control of the Rac1 and RhoA gtpase signaling pathways

Two rapid spectroscopic approaches for whole-organism fingerprinting of pyrolysis-mass spectrometry (PyMS) and Fourier transform-infrared spectroscopy (FT-IR) were used to analyze a group of 29 clinical and reference Candida isolates. These strains had been identified by conventional means as belonging to one of the three species Candida albicans, C. dubliniensis (previously reported as atypical C. albicans), and C. stellatoidea (which is also closely related to C. albicans). To observe the relationships of the 29 isolates as judged by PyMS and FT-IR, the spectral data were clustered by discriminant analysis. On visual inspection of the cluster analyses from both methods, three distinct clusters, which were discrete for each of the Candida species, could be seen. Moreover, these phenetic classifications were found to be very similar to those obtained by genotypic studies which examined the HinfI restriction enzyme digestion patterns of genomic DNA and by use of the 27A C. albicans-specific probe. Both spectroscopic techniques are rapid (typically, 2 min for PyMS and 10 s for FT-IR) and were shown to be capable of successfully discriminating between closely related isolates of C. albicans, C. dubliniensis, and C. stellatoidea. We believe that these whole-organism fingerprinting methods could provide opportunities for automation in clinical microbial laboratories, improving turnaround times and the use of resources.     

Assessment of apparent vena caval penetration by the Greenfield filter

Bacteria belonging to the family Vibrionaceae were suspended using saline and a solution prepared from a marine-cations supplement. The effect of this on the profile of oxidized substrates obtained when using Biolog GN MicroPlates was investigated. Thirty-nine species belonging to the genera Aeromonas, Listonella, Photobacterium, and Vibrio were studied. Of the strains studied, species of Listonella, Photobacterium, and Vibrio could be expected to benefit from a marine-cations supplement that contained Na+, K+, and Mg2+. Bacteria that are not of marine origin are usually suspended in normal saline. Of the 39 species examined, 9 were not included in the Biolog data base and were not identified. Of the 30 remaining species, 50% were identified correctly using either of the suspending solutions. A further 20% were correctly identified only when suspended in saline. Three species, or 10%, were correctly identified only after suspension in the marine-cations supplemented solution. The remaining 20% of species were not correctly identified by either method. Generally, more substrates were oxidized when the bacteria had been suspended in the more complex salts solution. Usually, when identifications were incorrect, the use of the marine-cations supplemented suspending solution had resulted in many more substrates being oxidized. Based on these results, it would be preferable to use saline to suspend the cells when using Biolog for identification of species of Vibrionaceae. A salts solution containing a marine-cations supplement would be preferable for environmental studies where the objective is to determine profiles of substrates that the bacteria have the potential to oxidize. If identifications are done using marine-cations supplemented suspending solution, it would be advisable to include reference cultures to determine the effect of the supplement. Of the Vibrio and Listonella species associated with human clinical specimens, 8 out of the 11 studied were identified correctly when either of the suspending solutions was used.     

Composition and enzymatic activity of the extracellular matrix secreted by germlings of botrytis cinerea

A total of 122 Streptococcus suis serotype 2 strains were characterized thoroughly by comparing clinical and pathological observations, ribotype profiles, and antimicrobial resistance. Twenty-one different ribotype profiles were found and compared by cluster analysis, resulting in the identification of three ribotype clusters. A total of 58% of all strains investigated were of two ribotypes belonging to different ribotype clusters. A remarkable relationship existed between the observed ribotype profiles and the clinical-pathological observations because strains of one of the two dominant ribotypes were almost exclusively isolated from pigs with meningitis, while strains of the other dominant ribotype were never associated with meningitis. This second ribotype was isolated only from pigs with pneumonia, endocarditis, pericarditis, or septicemia. Cluster analysis revealed that strains belonging to the same ribotype cluster as one of the dominant ribotypes came from pigs that showed clinical signs similar to those of pigs infected with strains with the respective dominant ribotype profiles. Furthermore, strains belonging to different ribotype clusters had totally different patterns of resistance to antibiotics because strains isolated from pigs with meningitis were resistant to sulfamethazoxazole and strains isolated from pigs with pneumonia, endocarditis, pericarditis, or septicemia were resistant to tetracycline.     

Neisseria weaveri sp. nov. (formerly CDC group M-5), from dog bite wounds of humans

The taxonomic relationships of strains belonging to Centers for Disease Control group M-5 were examined. Previous studies of rRNA cistron similarities placed this organism on the Neisseriaceae rRNA branch of rRNA superfamily III; the closest neighbors included the genus Neisseria and groups EF-4a and EF-4b. The group M-5 strains were characterized by a range of phenotypic tests, and their G + C contents and DNA-DNA relatedness levels were determined. In addition, a numerical taxonomic analysis of the whole-cell protein patterns (obtained by sodium dodecyl sulfate-polyacrylamide gel electrophoresis) of group M-5 and related taxa was performed. The strains studied included 45 group M-5 strains, the type strains of six Neisseria species or subspecies, three group EF-4a reference strains, and three group EF-4b reference strains plus the type strain of the phenotypically similar organism Oligella urethralis. Our results showed that the group M-5 strains were members of a homogeneous taxon distinct from phylogenetically closely related taxa. The genomic divergence as revealed by levels of rRNA cistron similarity and phenotypic characteristics indicate that group M-5 can be considered a new species of the genus Neisseria. We therefore propose the new species Neisseria weaveri, with NCTC 12742 (= CCUG 4007 = ISL775/91 = LMG 5135) as the type strain. N. weaveri strains are strictly aerobic, gram-negative, nonmotile, rod-shaped organisms which are catalase and oxidase positive, nonsaccharolytic, and able to grow on MacConkey agar and do not reduce nitrate but generally reduce nitrite.(ABSTRACT TRUNCATED AT 250 WORDS)     

Taxonomic relationships between distinct potato virus Y isolates based on detailed comparisons of the viral coat proteins and 3'-nontranslated regions

Detailed comparisons were made of the sequences of the coat protein (CP) cistrons and 3'-nontranslated regions (3'-NTR) of 21 (geographically) distinct isolates of potato virus Y (PVY) and a virus isolate initially described as pepper mottle virus (PepMoV). Multiple sequence alignments and phylogenetic relationships based on these alignments resulted into a subgrouping of virus isolates which largely corresponded with the historical strain differentiation based on biological criteria as host range, symptomatology and serology. Virus isolates belonging to the same subgroup shared a number of characteristic CP amino acid and 3'-NTR nucleotide residues indicating that, by using sequences from the 3'-terminal region of the potyvirus genome, a distinction could be made between different isolates of one virus species as well as between different virus species. RNA secondary structure analysis of the 3'-NTR of twelve PVY isolates revealed four major stem-loop structures of which, surprisingly, the loop sequences gave a similar clustering of isolates as resulting from the overall comparisons of CP and 3'-NTR sequences. This implies a biological significance of these structural elements.     

Comparative analysis of antigens from different Brucella species using immunoblotting with antisera from immunized rabbits

Brucella antigens recognized by IgG antibodies in cell lysates from various Brucella species differing by the origin, biological, and virulent properties (including the reference, vaccine, and newly isolated strains) were compared by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Proteins in SDS-cell lysates were separated by 12% SDS-PAGE and protein gels were stained with Coomassie brilliant blue R-250 and Silver reagent. SDS-PAGE showed differences in the protein profiles of 15 strains of different species. Immunoblotting revealed that rabbit S-antisera contained IgG reacting with S-LPS and identical proteins of 90 to 16 kDa belonging to B, melitensis, B. suis, B. abortus, and B. neotomae strains. B. canis strains had 4 antigens reacting with these antisera, whereas B. ovis had none. No agglutinating antibody were detected by the standard tube agglutination test with smooth Brucella strains in rabbit R-antisera. By contrast, immunoblotting analysis with these sera demonstrated common 90-16 kDa antigens in the strains of B. melitensis, B. suis, B. abortus, B. neotomae, and B. canis. B. ovis possessed none of these antigens. These results confirm that all Brucella species except B. ovis possess common protein antigens reacting with IgG.     

Evolution of histone H4 and H3 genes in different ciliate lineages

The histones H4 are known as highly conserved proteins. However, in ciliates a high degree of variation was found compared both to other eukaryotes and between the ciliate species. To date, only H4 histones of species belonging to two distantly related classes have been investigated. In order to obtain more detailed information on histone H4 variation in ciliates we undertook a comprehensive sequence analysis of PCR-amplified internal H4 fragments from 12 species belonging to seven out of the nine currently recognized ciliate classes. In addition, we used PCR primers to amplify longer fragments of H3 and H4 genes including the intergenic region. The encoded amino acid sequences reveal a high number of differences when compared with those of other eukaryotes and the ciliate species investigated. Furthermore, in some species H4 gene variants were detected, which result in amino acid differences. The greatest number of substitutions and insertions found was in the amino terminal region of the H4 histones. However, all sequences possess a conserved region corresponding to those of all other eukaryotic H4 histones. The histone gene variations were used to reconstruct phylogenetic relationships. The tree from our data matches perfectly with the ribosomal RNA data: The heterotrichs, which were considered as a late branching lineage, diverge at the base of the ciliate tree and groups formerly thought to represent ancestral lineages now appear as highly derived ciliates.     

Differentiation of bifidobacteria by use of pulsed-field gel electrophoresis and polymerase chain reaction

Several different genomic fingerprints can be obtained from various commercially-important species of Bifidobacterium using pulsed-field gel electrophoresis (PFGE) following digestion of DNA with XbaI and SpeI. Four different genomic finger printings were discernible for reference strains of Bifidobacterium animalis, five for B. bifidum, three for B. breve, five for B. infantis and three for B. longum. Standard commercially-available industrial strains of B. animalis are identical to the reference strain ATCC 27536, previously isolated from chicken feces. There was more genomic heterogeneity among industrial strains of B. longum, in that only one gave profiles similar to the type strain of this species (ATCC 15707). The other 14 commercially-available strains of B. longum (mainly isolated from Japanese commercial preparations) were divided into four new molecular types based on their PFGE patterns. The PFGE method indicated that only five distinct strains of B. longum and one strain of B. animalis are used in commercial preparations. Additionally, the use of polymerase chain reaction amplification of portions of 16S rDNA provides a highly specific technique to discriminate between the species B. breve, B. infantis and B. longum.     

Changes in the rabbit temporomandibular joint associated with posterior displacement of the mandible

We report a detailed evolutionary study of the RNase P- and RNase MRP- associated RNAs. The analyses were performed on all the available complete sequences of RNase MRP (vertebrates, yeast, plant), nuclear RNase P (vertebrates, yeast), and mitochondrial RNase P (yeast) RNAs. For the first time the phylogenetic distance between these sequences and the nucleotide substitution rates have been quantitatively measured.The analyses were performed by considering the optimal multiple alignments obtained mostly by maximizing similarity between primary sequences. RNase P RNA and MRP RNA display evolutionary dynamics following the molecular clock. Both have similar rates and evolve about one order of magnitude faster than the corresponding small rRNA sequences which have been, so far, the most common gene markers used for phylogeny. However, small rRNAs evolve too slowly to solve close phylogenetic relationships such as those between mammals. The quicker rate of RNase P and MRP RNA allowed us to assess phylogenetic relationships between mammals and other vertebrate species and yeast strains. The phylogenetic data obtained with yeasts perfectly agree with those obtained by functional assays, thus demonstrating the potential offered by this approach for laboratory experiments.     

Ribotyping: a tool for taxonomy and identification of the Nocardia asteroides complex species

Ribotyping has been evaluated as a taxonomic tool for studying bacteria belonging to the Nocardia asteroides complex. The probe used was obtained by PCR from DNA extracted from the type strain Nocardia asteroides ATCC 19247, a sequence that codes partly for 16S rRNA. Interpretation of hybridization after EcoRI restriction of total DNA of 21 strains of the N. asteroides complex allowed for the identification of only four different ribotypes: two related to N. asteroides sensu stricto, one related to N. farcinica and one related to N. nova. These results confirm the classification established by Tsukamura in 1982 (M. Tsukamura, Microbiol. Immunol. 26:1101-1119, 1982) as well as the heterogeneity of N. asteroides sensu stricto species. Ribotyping may also provide a major identification technique for the species belonging to the N. asteroides complex.     

Validation of random amplified polymorphic DNA assays by ribotyping as tools for epidemiological surveys of Pasteurella from animals

Two collections of strains of Pasteurella were studied for epidemiological purposes by ribotyping and random amplified polymorphic DNA (RAPD) assays. These strains were isolated through two different structures of animal productions: cattle and rabbit. Forty strains of P. haemolytica from cattle reared in independent breeding-herds belonged to only 3 ribotypes after digestion with HindIII and PvuII. No further discrimination of these strains was obtained by RAPD assays. All these 40 strains showed more than 90% of similarity. This result was consistent with the hypothesis of a clonal dissemination of these strains in bovine herds, possible favoured by the large use of antibiotics. Forty-one strains of P. multocida were isolated in rabbits flocks belonging to 16 breeders. Six of these were linked by commercial relationships. Twenty-eight out of the 29 strains isolated through this commercial network belonged to only three ribotypes whereas the 12 strains from independant breeders belonged to 9 ribotypes. Results of RAPD assays were in accordance with those of ribotyping and validate the use of RAPD assays for epidemiological studies of Pasteurella strains.     

The expanding small heat-shock protein family, and structure predictions of the conserved "alpha-crystallin domain"

The ever-increasing number of proteins identified as belonging to the family of small heat-shock proteins (shsps) and alpha-crystallins enables us to reassess the phylogeny of this ubiquitous protein family. While the prokaryotic and fungal representatives are not properly resolved, most of the plant and animal shsps and related proteins are clearly grouped in distinct clades, reflecting a history of repeated gene duplications. The members of the shsp family are characterized by the presence of a conserved homologous "alpha-crystallin domain," which sometimes is present in duplicate. Predictions are made of secondary structure and solvent accessibility of this domain, which together with hydropathy profiles and intron positions support the presence of two similar hydrophobic beta-sheet-rich motifs, connected by a hydrophilic alpha-helical region. Together with an overview of the newly characterized members of the shsp family, these data help to define this family as being involved as stable structural proteins and as molecular chaperones during normal development and induced under pathological and stressful conditions.     

Isolation of Arthrobacter spp. from clinical specimens and description of Arthrobacter cumminsii sp. nov. and Arthrobacter woluwensis sp. nov

Arthrobacter spp. are very widely distributed in the environment (e.g., soil) but have not been described as causing disease in humans. Over a 6-year period, two reference laboratories isolated or received 11 strains which were eventually identified as belonging to the genus Arthrobacter. These strains had been initially identified as Centers for Disease Control and Prevention coryneform group B-1 and B-3 bacteria (whitishgrayish colonies of 2 mm or greater in diameter after 24 h of incubation, respiratory metabolism, absent or weak acid production from sugars, and hydrolysis of gelatin). However, chemotaxonomic investigations revealed lysine as the diamino acid of the cell wall and the presence of branched cellular fatty acids (with anteiso-pentadecanoic acid predominating) which was compatible with an assignment of the 11 isolates to the genus Arthrobacter only. Peptidoglycan and 16S rRNA gene sequence analyses demonstrated that three of the strains studied were representatives of a new Arthrobacter species for which the name Arthrobacter cumminsii sp. nov. is proposed and that one other strain represented a second new Arthrobacter species for which the name Arthrobacter woluwensis sp. nov. is proposed. This report is the first on the isolation of Arthrobacter spp. from clinical specimens.     

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.     

Q-methodology: definition and application in health care informatics

Three different approaches (whole-cell protein profiles, DNA fingerprinting combined with pulsed-field gel electrophoresis and analysis of rDNA genes) were used to characterize thirty-one strains of Lactobacillus delbrueckii subsp. bulgaricus from different dairy products, and three type strains belonging to L. delbrueckii subsp. delbrueckii, L. delbrueckii subsp. lactis and L. delbrueckii subsp. bulgaricus. Moreover, the number and different forms of rrn operons in L. delbrueckii and its subspecies were defined. At the strain typing level, Notl macrorestriction analysis permitted grouping of the 32 strains of L. delbrueckii subsp. bulgaricus into 23 restriction patterns, providing a high degree of discriminatory power. Among whole-cell protein profiles, PCR analysis of rDNA genes and ribotyping, the latter method seemed to be the most reliable approach to characterizing the subspecies belonging to L. delbrueckii. Ribotyping combined with enzymes such as HindIII and EcoRI showed that at least six rrn operons were present in L. delbrueckii and its subspecies; two forms of rrn operons were present in the subspecies lactis and bulgaricus and four forms were present in the subspecies delbrueckii.     

PCR-amplified 16S and 23S rDNA restriction analysis for the identification of Acinetobacter strains at the DNA group level

The genus Acinetobacter is phenotypically rather homogeneous, but genotypically heterogeneous. In this study, a simple method based on restriction analysis of a PCR-amplified large fragment (4.5 kb) of most of the ribosomal operon (16S and 23S ribosomal genes and the spacer in-between) was investigated. Sixty-seven collection strains belonging to the 20 DNA groups proposed until 1993 were studied. Using the enzyme Sau3AI, 25 DNA profiles were obtained. Strains belonging to DNA groups 1, 3, 6, TU13 and TU15 showed two profiles each, and DNA groups 4, 5 and 7 showed profiles with variants showing less intensive additional bands. The remaining 12 groups showed 12 different profiles. The profiles obtained were DNA-group-specific except for one profile which was shared between the unnamed DNA group 3 and a rarely encountered genotypically related DNA group. These two DNA groups could be separated by using the enzyme Hinf1. Twenty-five additional clinical isolates previously characterized by standard DNA-DNA hybridization were selected in a double-blind fashion for identification at the DNA group level to check the reliability of the assay. All strains were correctly identified at the DNA group level. PCR-amplified 16S and 23S rDNA restriction analysis is both an accurate and rapid method for the identification of Acinetobacter at the DNA group level.