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PubMed2004

Development of a 16S rRNA primer for the detection of Brevibacterium spp.

AIM: To develop a PCR method for the rapid identification of the genus Brevibacterium. METHODS AND RESULTS: Genus-specific primers were designed by aligning and comparing the 16S sequence of all Brevibacterium spp. with closely related genera. The primer set was tested with all validly described Brevibacterium spp. and their closest neighbours. SIGNIFICANCE: Until today brevibacteria could only be identified with laborious and time-consuming phenotypic characterization. The primer from this study offers a rapid alternative to the detection of Brevibacterium spp. Brevibacteria have been isolated from food, blood, ear discharge, from a wound and from an intravascular catheter.

Blood↗
PubMed2006

Atypical Helicobacter canadensis strains associated with swine.

Forty-two Helicobacter isolates were isolated from swine feces in The Netherlands and Denmark. All 12 isolates sequenced (16S rRNA gene) formed a robust clade with Helicobacter canadensis ( approximately 99% similarity). Species-specific PCR indicated that all of the isolates were H. canadensis isolates. Although the appearance of the porcine isolates was similar to the appearance of H. canadensis, only one of these isolates was able to hydrolyze indoxyl acetate, a cardinal characteristic of this taxon. Examination of the 23S rRNA and hsp60 genes revealed high levels of similarity between the porcine isolates and H. canadensis. However, amplified fragment length polymorphism genomic typing showed that isolates recovered from swine feces were genetically distinct from H. canadensis strains obtained from humans and geese.

Animals↗
PubMed2002

Detection and identification of bacterial pathogens of fish in kidney tissue using terminal restriction fragment length polymorphism (T-RFLP) analysis of 16S rRNA genes.

We report the application of a nucleic acid-based assay that enables direct detection and identification of bacterial pathogens in fish kidney tissue without the need for bacterial culture. The technique, known as terminal restriction fragment length polymorphism (T-RFLP), employs the polymerase chain reaction (PCR) using a primer pair that targets 2 highly conserved regions of the gene that encodes for the 16S small subunit of the bacterial ribosome. Each primer is 5' labeled with a different fluorescent dye, which results in each terminus of the resulting amplicon having a distinguishable fluorescent tag. The amplicon is then digested with a series of 6 restriction endonucleases, followed by size determination of the 2 labeled terminal fragments by capillary electrophoresis with laser-induced fluorescence detection. Comparison of the lengths of the full set of 12 terminal fragments with those predicted based on analyses of GenBank submissions of 16S sequences leads to presumptive identification of the pathogen to at least the genus, but more typically the species level. Results of T-RFLP analyses of genomic DNA from multiple strains of a number of fish bacterial pathogens are presented. The assay is further demonstrated on fish kidney tissue spiked with a known number of cells of Flavobacterium psychrophilum where a detection limit of ca. 30 CFU mg(-1) of tissue was estimated. A similar detection limit was observed for several other gram-negative pathogens. This procedure was also used to detect Aeromonas salmonicida and Renibacterium salmoninarum in the kidney tissue of 2 naturally infected salmonids.

Aeromonas↗
PubMed2003

Bacterial diversity of the Inner Mongolian Baer Soda Lake as revealed by 16S rRNA gene sequence analyses.

Bacterial diversity associated with Baer Soda Lake in Inner Mongolia of China was investigated using a culture-independent method. Bacterial 16S rRNA gene libraries were generated using bacterial oligonucleotide primers, and 16S rRNA gene sequences of 58 clones were analyzed phylogenetically. The library was dominated by 16S rDNAs of Gram-negative bacteria (24% alpha-Proteobacteria, 31% beta-Proteobacteria, 33% gamma-Proteobacteria, and 2% delta-Proteobacteria), with a lower percentage of clones corresponding to Gram-positive bacteria. Forty cloned sequences were similar to that of known bacterial isolates (> 97% sequence similarity), represented by the species of the genera Brevundimonas, Comamonas, Alcaligenes, Stenotrophomonas, and Klebsiella. Eighteen cloned sequences showed less affiliation with known taxa (< 97% sequence similarity) and may represent novel taxa.

Bacteria↗
PubMed2004

Identification of Corynebacterium bovis by endonuclease restriction analysis of the 16S rRNA gene sequence.

Despite its high prevalence within the bovine mammary gland, Corynebacterium bovis is considered a minor pathogen and of limited clinical significance. It has been suggested that intramammary infection with C. bovis may protect quarters against subsequent infection with other pathogens. The literature has produced much conflicting data on the subject. A possible explanation for some of the divergence of opinion on the subject is incorrect identification of isolates in previous studies. This paper describes a novel method for differentiating C. bovis from other lipophilic Corynebacterium species based on endonuclease restriction analysis. The 16S rRNA gene sequences for all known lipophilic Corynebacterium species were obtained from published data and analyzed. It was predicted that endonuclease restriction with HindIII and SmaI could be used to differentiate C. bovis from all other known lipophilic Corynebacterium species. The method was successfully employed to identify 741 of 762 (97.2%) lipophilic Corynebacterium species as C. bovis. Twenty one (2.8%) were identified as species other than C. bovis. Using this technique, it was demonstrated that it is not safe to assume that all lipophilic coryneform organisms isolated from bovine milk samples are C. bovis. This method is an alternative to more traditional methods of identification in large scale studies until methods such as 16S rRNA gene sequencing become more widely available.

Animals↗
PubMed2001

Microbial diversity of the brine-seawater interface of the Kebrit Deep, Red Sea, studied via 16S rRNA gene sequences and cultivation methods.

The brine-seawater interface of the Kebrit Deep, northern Red Sea, was investigated for the presence of microorganisms using phylogenetic analysis combined with cultivation methods. Under strictly anaerobic culture conditions, novel halophiles were isolated. The new rod-shaped isolates belong to the halophilic genus Halanaerobium and are the first representatives of the genus obtained from deep-sea, anaerobic brine pools. Within the genus Halanaerobium, they represent new species which grow chemoorganotrophically at NaCl concentrations ranging from 5 to 34%. The cellular fatty acid compositions are consistent with those of other Halanaerobium representatives, showing unusually large amounts of Delta7 and Delta11 16:1 fatty acids. Phylogenetic analysis of the brine-seawater interface sample revealed the presence of various bacterial 16S rRNA gene sequences dominated by cultivated members of the bacterial domain, with the majority affiliated with the genus Halanaerobium. The new Halanaerobium 16S rRNA clone sequences showed the highest similarity (99.9%) to the sequence of isolate KT-8-13 from the Kebrit Deep brine. In this initial survey, our polyphasic approach demonstrates that novel halophiles thrive in the anaerobic, deep-sea brine pool of the Kebrit Deep, Red Sea. They may contribute significantly to the anaerobic degradation of organic matter enriched at the brine-seawater interface.

Bacteria, Anaerobic↗
PubMedDate not supplied

Genomic ancestry of the American puma (Puma concolor).

Puma concolor, a large American cat species, occupies the most extensive range of any New World terrestrial mammal, spanning 110 degrees of latitude from the Canadian Yukon to the Straits of Magellan. Until the recent Holocene, pumas coexisted with a diverse array of carnivores including the American lion (Panthera atrox), the North American cheetah (Miracynonyx trumani), and the saber toothed tiger (Smilodon fatalis). Genomic DNA specimens from 315 pumas of specified geographic origin (261 contemporary and 54 museum specimens) were collected for molecular genetic and phylogenetic analyses of three mitochondrial gene sequences (16S rRNA, ATPase-8, and NADH-5) plus composite microsatellite genotypes (10 feline loci). Six phylogeographic groupings or subspecies were resolved, and the entire North American population (186 individuals from 15 previously named subspecies) was genetically homogeneous in overall variation relative to central and South American populations. The marked uniformity of mtDNA and a reduction in microsatellite allele size expansion indicates that North American pumas derive from a recent (late Pleistocene circa 10,000 years ago) replacement and recolonization by a small number of founders who themselves originated from a centrum of puma genetic diversity in eastern South America 200,000-300,000 years ago. The recolonization of North American pumas was coincident with a massive late Pleistocene extinction event that eliminated 80% of large vertebrates in North America and may have extirpated pumas from that continent as well.

Americas↗
PubMed2000

Application of 16S rRNA gene sequencing to identify Bordetella hinzii as the causative agent of fatal septicemia.

We report on the first case of fatal septicemia caused by Bordetella hinzii. The causative organism exhibited a biochemical profile identical to that of Bordetella avium with three commercial identification systems (API 20E, API 20 NE, and Vitek GNI+ card). However, its cellular fatty acid profile was not typical for either B. avium or previously reported strains of B. hinzii. Presumptive identification of the patient's isolate was accomplished by traditional biochemical testing, and definitive identification was achieved by 16S rRNA gene sequence analysis. Phenotypic features useful in distinguishing B. hinzii from B. avium were production of alkali from malonate and resistance to several antimicrobial agents.

Aged↗
PubMed2005

The copy-number of plasmids and other genetic elements can be determined by SYBR-Green-based quantitative real-time PCR.

In this study, we explored whether SYBR Green-based quantitative real-time PCR (qPCR) could be used to determine the copy number of a plasmid and whether the method was broadly applicable to chromosomally encoded genetic elements often occurring in multiple copies, such as rRNA genes and insertion sequences (IS). Three different template sources (whole cells, total DNA, and restriction-enzyme digested total DNA) derived from the bacterium Comamonas sp. strain JS46 were analyzed by qPCR using primer-pairs targeting plasmid pJS46 and three chromosomally encoded sequences (16S rDNA, ISCsp1, and IS1071). The difference between threshold cycle values, C(T), of amplicons targeting these elements and of an amplicon targeting the single-copy reference element mnbA (chromosomally encoded) was used to establish DeltaC(T). DeltaC(T) values were then used to derive copy number. For pJS46, qPCR analyses of whole cells and total DNA underestimated the copy number of pJS46 approximately 7-fold and approximately 2.5-fold, respectively, whereas copy number values derived from qPCR analyses of digested total DNA were comparable to those derived from Southern blot (SB) analyses. In contrast, for the chromosomally encoded elements, qPCR analyses of all three template sources gave copy number values that were virtually identical to or differed by approximately 2 from copy number values derived by SB analysis. These data indicate that qPCR can be used to estimate the copy number of various genetic elements but that the accuracy of qPCR-derived values is affected by the template source.

Benzothiazoles↗
PubMed1993

The phylogeny of autotrophic ammonia-oxidizing bacteria as determined by analysis of 16S ribosomal RNA gene sequences.

Partial sequences of the 16S ribosomal RNA genes of eleven autotrophic ammonia-oxidizing bacteria were determined by PCR amplification from small amounts of heat-lysed biomass followed by direct sequencing of PCR products. The sequences were aligned with those of representative Proteobacteria and phylogenetic trees inferred using both parsimony and distance matrix methods. This confirmed that the autotrophic ammonia-oxidizers comprise two major lines of descent within the Proteobacteria. Nitrosomonas spp., Nitrosococcus mobilis, and strains of Nitrosovibrio, Nitrosospira and Nitrosolobus were located in the beta-subdivision. The recovery of Nitrosococcus oceanus strains as a deep branch in the gamma-subdivision supported the RNA catalogue data which had indicated that the genus Nitrosococcus is polyphyletic. The autotrophic ammonia-oxidizing bacteria of the beta-Proteobacteria formed a coherent group which is interpreted as representing a single family. Within this clade, the genera Nitrosovibrio, Nitrosospira and Nitrosolobus exhibited very high levels of homology in their 16S ribosomal RNA gene sequences and can be accommodated within a single genus. Separation of these genera is currently based entirely on gross morphological differences and these can now be considered more appropriate for the identification of species within this group. It is therefore proposed that Nitrosolobus, Nitrosovibrio and Nitrosospira strains be reclassified in a single genus for which the name Nitrosospira has priority.

Ammonia↗
PubMed1998

Phylogenetic evidence for the existence of novel thermophilic bacteria in hot spring sulfur-turf microbial mats in Japan.

So-called sulfur-turf microbial mats, which are macroscopic white filaments or bundles consisting of large sausage-shaped bacteria and elemental sulfur particles, occur in sulfide-containing hot springs in Japan. However, no thermophiles from sulfur-turf mats have yet been isolated as cultivable strains. This study was undertaken to determine the phylogenetic positions of the sausage-shaped bacteria in sulfur-turf mats by direct cloning and sequencing of 16S rRNA genes amplified from the bulk DNAs of the mats. Common clones with 16S rDNA sequences with similarity levels of 94.8 to 99% were isolated from sulfur-turf mat samples from two geographically remote hot springs. Phylogenetic analysis showed that the phylotypes of the common clones formed a major cluster with members of the Aquifex-Hydrogenobacter complex, which represents the most deeply branching lineage of the domain bacteria. Furthermore, the bacteria of the sulfur-turf mat phylotypes formed a clade distinguishable from that of other members of the Aquifex-Hydrogenobacter complex at the order or subclass level. In situ hybridization with clone-specific probes for 16S rRNA revealed that the common phylotype of sulfur-turf mat bacteria is that of the predominant sausage-shaped bacteria.

Bacteria↗
PubMed1990

Brucella abortus 16S rRNA and lipid A reveal a phylogenetic relationship with members of the alpha-2 subdivision of the class Proteobacteria.

On the basis of ribosomal 16S sequence comparison, Brucella abortus has been found to be a member of the alpha-2 subdivision of the class Proteobacteria (formerly named purple photosynthetic bacteria and their nonphototrophic relatives). Within the alpha-2 subgroup, brucellae are specifically related to rickettsiae, agrobacteria, and rhizobiae, organisms that also have the faculty or the obligation of living in close association to eucaryotic cells. The composition of Brucella lipid A suggests a close phylogenetical relationship with members of the alpha-2 group. The chemical analysis of the lipid A fraction revealed that Brucella species contain both glucosamine and diaminoglucose, thus suggesting the presence of a so-called mixed lipid A type. The serological analysis with polyclonal and monoclonal antibodies is in agreement with the existence of mixed lipid A type in B. abortus. The amide-linked fatty acid present as acyl-oxyacyl residues were 3-O-C(16:0)12:0, 3-O-C(16:0)13:0, 3-O-C(16:0)14:0, and 3-O-C(18:0)14:0. The only amide-linked unsubstituted fatty acid detected was 3-OH-C16:0. The ester-linked fatty acids are 3-OH-C16:0, 3-OH-C18:0, C16:0, C17:0, and C18:0. Significant amounts of the large-chain 27-OH-C28:0 were detected together with traces of 25-OH-C26:0 and 29-OH-C30:0. Comparison of the Brucella lipid composition with that of the other Proteobacteria also suggests a close phylogenetical relationship with members of the alpha-2 subdivision. The genealogical grouping of Brucella species with pericellular and intracellular plant and animal pathogens as well as with intracellular plant symbionts suggests a possible evolution of Brucella species from plant-arthropod-associated bacteria.

Bacteria↗
PubMedDate not supplied

[Shewanella genus bacteria isolated from the Black sea water and molluscs].

Six (6) strains of bacteria isolated from the Black Sea water and mollusks Mytilaster lineatus have been assigned to Shewanella genus on the basis of phenotypical and molecular-genetic study. Partial sequence of 16S rRNA gene revealed 100% similarity of three of these strains to S. baltica, although they had some phenotypical differences. Three more strains were genetically related to S. colwelliana and S. affinis (99-100% of 16S rRNA sequences similarity), but they differed from these species as well as from each other in some phenotypical properties, so the question about their taxonomic status still remains open. In aerobic conditions the Black Sea strains of S. baltica adsorbed significant quantities of Fe III from the media. They reduced it to Fe (II) in anaerobic conditions. Strains isolated from mollusks were not able to reduce Fe (III).

Animals↗
PubMed2006

Bacteroides barnesiae sp. nov., Bacteroides salanitronis sp. nov. and Bacteroides gallinarum sp. nov., isolated from chicken caecum.

Eight bacterial strains isolated from the caecum of chicken, BL2(T), BL66, EG3, EG6, M27, BL78(T), C35(T) and C43, were characterized by determining their phenotypic characteristics, cellular fatty acid profiles, menaquinone profiles and phylogenetic positions based on 16S rRNA gene sequence analysis. 16S rRNA gene sequence analysis showed that these isolates belonged to the genus Bacteroides. One group of five strains (BL2(T), BL66, EG3, EG6 and M27) was related most closely to Bacteroides coprocola JCM 12979(T), with approximately 93 % 16S rRNA gene sequence similarity, and to Bacteroides plebeius JCM 12973(T), with about 92 % similarity, and shared >or=99.6 % similarity with each other. Strain BL78(T) exhibited 90.5 % similarity to B. plebeius JCM 12973(T) and 89.8 % similarity to B. coprocola JCM 12979(T) and differed from the above group of five strains at >or=10 % sequence divergence. Strains C35(T) and C43 were related most closely to Bacteroides eggerthii JCM 12986(T), with 95.1 % sequence similarity, to Bacteroides stercoris JCM 9496(T), with 94.6 % similarity, and to Bacteroides uniformis JCM 5828(T), with 94.4 % similarity, and shared 100 % similarity with each other. From results of phenotypic examination, cellular fatty acid composition analysis, menaquinone composition analysis and DNA G+C contents, the group of five strains as well as strain BL78(T) were shown to differ from the type strains of B. coprocola and B. plebeius. Strain BL78(T) differed from the others based on its menaquinone composition, which included MK-11 and MK-12. Strains C35(T) and C43 could also be differentiated from the type strains of B. eggerthii, B. stercoris and B. uniformis. The group of five strains, strain BL78(T), B. coprocola JCM 12979(T) and B. plebeius JCM 12973(T) showed low levels of DNA-DNA relatedness (<35 %) with each other. High levels of DNA-DNA relatedness were obtained within the group of five strains (>75 %). Strains C35(T) and C43 exhibited a high level of DNA-DNA relatedness (>88 %) with each other, but low levels with B. eggerthii JCM 12986(T) (<40 %), B. stercoris JCM 9496(T) (<37 %) and B. uniformis JCM 5828(T) (<16 %). On the basis of these data, three novel Bacteroides species are proposed: Bacteroides barnesiae sp. nov. (type strain BL2(T)=JCM 13652(T)=DSM 18169(T)), Bacteroides salanitronis sp. nov. (type strain BL78(T)=JCM 13657(T)=DSM 18170(T)) and Bacteroides gallinarum sp. nov. (type strain C35(T)=JCM 13658(T)=DSM 18171(T)).

Animals↗
PubMed2000

Analysis of bacterial DNA in skin and muscle of the Tyrolean iceman offers new insight into the mummification process.

About 80 sequences (16s ribosomal RNA gene) of bacterial DNA in samples of skin and muscle taken directly from the Tyrolean iceman (3350-3100 years B.C.) or recovered during the 1992 archaeological expedition at the Alpine site were analyzed to obtain clues to the natural mummification process that allowed the corpse of the Neolithic shepherd/hunter to be preserved for more than 5,000 years. The investigation was made more complex by the fact that the surface of the mummy had been swabbed with phenol soon after the discovery (September 19, 1991). Our results show that no trace of microbial DNA is left on the actual surface of the body, while the untreated skin still bears the remains of large numbers of bacteria belonging to the genera Sphingomonas, Afipia, Curtobacterium, Microbacterium, Agromyces, and others. Compared to the untreated skin, the iceman's muscle is also very rich in bacterial DNA. However, this DNA comes, with few exceptions, from the species Clostridium algidicarnis. The sharp difference in the bacterial DNA composition of skin and muscle suggests that the remains of the original cadaveric microflora of the latter have not disappeared during the iceman's taphonomic history. On the other hand, the massive presence of C. algidicarnis, a cold-adapted sporigenous, the DNA of which was previously (Ubaldi et al. [1998] Am. J. Phys. Anthropol. 107:285-295) found in the soft tissue of a naturally desiccated Andean mummy, indicates that the hypothesis that the iceman's corpse underwent rapid dehydration by the effect of a warm wind (föhn) is no longer plausible. The results best fit with the hypothesis (Bereuter et al. [1997] Chem. Eur. J. 7:1032-1038) that the body was first covered by snow and ice, and then underwent thawing and, finally, desiccation.

Adult↗
PubMed2002

Phylogenetic and metabolic diversity of bacteria degrading aromatic compounds under denitrifying conditions, and description of Thauera phenylacetica sp. nov., Thauera aminoaromaticasp. nov., and Azoarcus buckelii sp. nov.

Six strains of denitrifying bacteria isolated from various oxic and anoxic habitats on different monocyclic aromatic substrates were characterized by sequencing 16S rRNA genes, determining physiological and morphological traits, and DNA-DNA hybridization. According to these criteria, strains S100, SP and LG356 were identified as members of Thauera aromatica. Strains B5-1 and B5-2 were tentatively affiliated to the species Azoarcus tolulyticus. Strains B4P and S2 were only distantly related to each other and to other described Thauera species. These two strains are proposed as the type strains of two new species, Thauera phenylacetica sp. nov. and Thauera aminoaromaticasp. nov., respectively. By 16S rRNA gene analysis, strain U120 was highly related to the type strains of Azoarcus evansii and Azoarcus anaerobius, whereas corresponding DNA-DNA reassociation values indicated only a low degree of genomic relatedness. Based upon a low DNA similarity value and the presence of distinguishing physiological properties, strain U120 is proposed as the type strain of a new species, Azoarcus buckelii sp. nov. Almost all of the new isolates were obtained with different substrates. The highly varied substrate spectra of the isolates indicates that an even higher diversity of denitrifying bacteria degrading aromatic compounds would be discovered in the different habitats by using a larger spectrum of aromatic substrates for enrichment and isolation.

Azoarcus↗
PubMed2003

Diversity of Actinoplanes and related genera isolated from an Italian soil.

Actinoplanes and related genera are good producers of bioactive secondary metabolites. However, many strains within these genera present similar morphological characteristics, and this prevents an effective discrimination of replicate strains during industrial isolation and screening programs. Using PCR-RFLP analysis of the 23S rDNA gene and of the 16S-23S intergenic spacer, we have analyzed 182 strains of Actinoplanes and related genera obtained through a selective isolation method from a single Italian soil. Combining the 23S and IGS data, 99 unique profiles were observed, and morphologically undistinguishable strains were discriminated. Further analyses on a restricted number of strains through 16S sequencing and hybridization to a probe for secondary metabolism established a good correlation between strain diversity seen by PCR-RFLP and that seen by the other methods. Overall, the data indicate the presence of a high diversity of Actinoplanes and related genera isolated from a single Italian soil.

DNA, Bacterial↗
PubMed2002

Monitoring of a pyrite-oxidising bacterial population using DNA single-strand conformation polymorphism and microscopic techniques.

The single-strand conformation polymorphism (SSCP) technique was used to study the evolution of a bacterial consortium during the batch oxidation of a cobaltiferous pyrite in two types of bio-reactor: a bubble column and a classical stirred tank. Sequencing 16S rDNA revealed the presence of three organisms affiliated to Leptospirillum ferrooxidans, Acidithiobacillus thiooxidans and Sulfobacillus thermosulfidooxidans, respectively. Attempts were made to determine the proportions of bacteria attached to solid particles or freely suspended in the medium using a combination of PCR-SSCP and a microscopic technique. Ac. thiooxidans-related bacteria were dominant in the liquid during the early phase of the batch, but were later supplanted by L. ferrooxidans-related bacteria. L. ferrooxidans-related organisms were always in the majority on the solids. The growth of S. thermosulfidooxidans-related bacteria seemed to be favoured by the bubble-column reactor.

Bacteria↗