Comparative 16S rRNA Analysis Research Articles

Efficacy of Isolated Probiotic Bacteria from Piglet Nostrils in Fattening Pigs

Probiotics applications in animal husbandry can increase feed intake, growth and immune responses. The aim of this study was to isolate the probiotic bacteria from piglet nostrils and test their efficacy in fattening pigs. Nasal swabs collected from 110 healthy piglets were processed on Man, Rogosa and Sharpe (MRS) agar plates for lactic acid bacteria isolation. The biochemical standard tests were used for identification of isolates organisms including: Gram staining, catalase production, tolerance to bile salts, acid and base and the utilization of proteins, starch and fat. The antimicrobial activity and resistance against antibiotics were also determined. Comparative 16S rRNA analysis confirmed the identity of selected strains as Enterococcus italicus . Thirty weaned piglets (mixed sex and 35-day old) were divided into three groups (five replicates with two animals) consisting of a control group, a positive control group fed with commercial feed and antibiotic, and an experimental group fed with commercial feed and probiotics. Porcine reproductive and respiratory syndrome (PRRS) vaccination were performed twice. Administration of Enterococcus italicus had no impact on average daily gain, whereas it affected feed intake and feed conversion ratio (p 0.05).

Comparative 16S rRNA signatures and multilocus sequence analysis for the genus Salinicola and description of Salinicola acroporae sp. nov., isolated from coral Acropora digitifera.

A novel Gram-negative, aerobic, motile marine bacterium, strain S4-41(T), was isolated from mucus of the coral Acropora digitifera from the Andaman Sea. Heterotrophic growth was observed in 0-25 % NaCl, at 15-45 °C and pH 4.5-9. In phylogenetic trees, strain S4-41(T) was grouped within the genus Salinicola but formed a separate branch distant from a cluster composed of Salinicola salarius M27(T) and Salinicola socius SMB35(T). DNA-DNA relatedness between strain S4-41(T) and these reference strains were well below 70 %. Q-9 was the sole respiratory quinone. The DNA G+C content was determined to be 63.6 mol%. Based on a polyphasic analysis, strain S4-41(T) is concluded to represent a novel species in the genus Salinicola for which the name Salinicola acroporae sp. nov. is proposed. The type strain is S4-41(T) (=JCM 30412(T) = LMG 28587(T)). Comparative 16S rRNA analysis of the genera Salinicola, Kushneria, Chromohalobacter and Cobetia revealed the presence of genus specific sequence signatures. Multilocus sequence analysis based on concatenated sequences of rRNAs (16S and 23S) and four protein coding housekeeping genes (atpA, gyrB, secA, rpoD) was found to be unnecessary for phylogenetic studies of the genus Salinicola.

Bacterial symbionts of Bathymodiolus mussels and Escarpia tubeworms from Chapopote, an asphalt seep in the southern Gulf of Mexico

Chemosynthetic life was recently discovered at Chapopote, an asphalt hydrocarbon seep in the southern Gulf of Mexico. Preliminary morphological analyses indicated that one tubeworm and two mussel species colonize Chapopote. Our molecular analyses identified the tubeworm as Escarpia sp., and the mussels as Bathymodiolus heckerae and B. brooksi. Comparative 16S rRNA analysis and FISH showed that all three species harbour intracellular sulfur-oxidizing symbionts highly similar or identical to those found in the same host species from northern Gulf of Mexico (nGoM). The mussels also harbour methane-oxidizing symbionts, and these shared highly similar to identical 16S rRNA sequences to their nGoM conspecifics. We discovered a novel symbiont in B. heckerae, which is closely related to hydrocarbon-degrading bacteria of the genus Cycloclasticus. In B. heckerae, we found key genes for the use of aromatic compounds, and its stable carbon isotope values were consistently higher than B. brooksi, indicating that the novel symbiont might use isotopically heavy aromatic hydrocarbons from the asphalt seep. This discovery is particularly intriguing because until now only methane and reduced sulfur compounds have been shown to power cold-seep chemosynthetic symbioses. The abundant hydrocarbons available at Chapopote would provide these mussel symbioses with a rich source of nutrition.

The Phylogenetic Diversity of Aerobic Organotrophic Bacteria from the Dagang High-Temperature Oil Field

The distribution and species diversity of aerobic organotrophic bacteria in the Dagan high-temperature oil field (China), which is exploited via flooding, have been studied. Twenty-two strains of the most characteristic thermophilic and mesophilic aerobic organotrophic bacteria have been isolated from the oil stratum. It has been found that, in a laboratory, the mesophilic and thermophilic isolates grow in the temperature, pH, and salinity ranges characteristic of the injection well near-bottom zones or of the oil stratum, respectively, and assimilate a wide range of hydrocarbons, fatty acids, lower alcohols, and crude oil, thus exhibiting adaptation to the environment. Using comparative phylogenetic 16S rRNA analysis, the taxonomic affiliation of the isolates has been established. The aerobic microbial community includes gram-positive bacteria with a high and low G+C content of DNA, and gamma and beta subclasses of Proteobacteria. The thermophilic bacteria belong to the genera Geobacillus and Thermoactinomyces, and the mesophilic strains belong to the genera Bacillus, Micrococcus, Cellulomonas, Pseudomonas, and Acinetobacter. The microbial community of the oil stratum is dominated by known species of the genus Geobacillus (G. subterraneus, G. stearothermophilus, and G. thermoglucosidasius) and a novel species "Geobacillus jurassicus." A number of novel thermophilic oil-oxidizing bacilli have been isolated.

Dual Symbiosis in a Bathymodiolus sp. Mussel from a Methane Seep on the Gabon Continental Margin (Southeast Atlantic): 16S rRNA Phylogeny and Distribution of the Symbionts in Gills

Deep-sea mussels of the genus Bathymodiolus (Bivalvia: Mytilidae) harbor symbiotic bacteria in their gills and are among the dominant invertebrate species at cold seeps and hydrothermal vents. An undescribed Bathymodiolus species was collected at a depth of 3,150 m in a newly discovered cold seep area on the southeast Atlantic margin, close to the Zaire channel. Transmission electron microscopy, comparative 16S rRNA analysis, and fluorescence in situ hybridization indicated that this Bathymodiolus sp. lives in a dual symbiosis with sulfide- and methane-oxidizing bacteria. A distinct distribution pattern of the symbiotic bacteria in the gill epithelium was observed, with the thiotrophic symbiont dominating the apical region and the methanotrophic symbiont more abundant in the basal region of the bacteriocytes. No variations in this distribution pattern or in the relative abundances of the two symbionts were observed in mussels collected from three different mussel beds with methane concentrations ranging from 0.7 to 33.7 microM. The 16S rRNA sequence of the methanotrophic symbiont is most closely related to those of known methanotrophic symbionts from other bathymodiolid mussels. Surprisingly, the thiotrophic Bathymodiolus sp. 16S rRNA sequence does not fall into the monophyletic group of sequences from thiotrophic symbionts of all other Bathymodiolus hosts. While these mussel species all come from vents, this study describes the first thiotrophic sequence from a seep mussel and shows that it is most closely related (99% sequence identity) to an environmental clone sequence obtained from a hydrothermal plume near Japan.

Diversity and Distribution of Methanotrophic Archaea at Cold Seeps

In this study we investigated by using 16S rRNA-based methods the distribution and biomass of archaea in samples from (i) sediments above outcropping methane hydrate at Hydrate Ridge (Cascadia margin off Oregon) and (ii) massive microbial mats enclosing carbonate reefs (Crimea area, Black Sea). The archaeal diversity was low in both locations; there were only four (Hydrate Ridge) and five (Black Sea) different phylogenetic clusters of sequences, most of which belonged to the methanotrophic archaea (ANME). ANME group 2 (ANME-2) sequences were the most abundant and diverse sequences at Hydrate Ridge, whereas ANME-1 sequences dominated the Black Sea mats. Other seep-specific sequences belonged to the newly defined group ANME-3 (related to Methanococcoides spp.) and to the Crenarchaeota of marine benthic group B. Quantitative analysis of the samples by fluorescence in situ hybridization (FISH) showed that ANME-1 and ANME-2 co-occurred at the cold seep sites investigated. At Hydrate Ridge the surface sediments were dominated by aggregates consisting of ANME-2 and members of the Desulfosarcina-Desulfococcus branch (DSS) (ANME-2/DSS aggregates), which accounted for >90% of the total cell biomass. The numbers of ANME-1 cells increased strongly with depth; these cells accounted 1% of all single cells at the surface and more than 30% of all single cells (5% of the total cells) in 7- to 10-cm sediment horizons that were directly above layers of gas hydrate. In the Black Sea microbial mats ANME-1 accounted for about 50% of all cells. ANME-2/DSS aggregates occurred in microenvironments within the mat but accounted for only 1% of the total cells. FISH probes for the ANME-2a and ANME-2c subclusters were designed based on a comparative 16S rRNA analysis. In Hydrate Ridge sediments ANME-2a/DSS and ANME-2c/DSS aggregates differed significantly in morphology and abundance. The relative abundance values for these subgroups were remarkably different at Beggiatoa sites (80% ANME-2a, 20% ANME-2c) and Calyptogena sites (20% ANME-2a, 80% ANME-2c), indicating that there was preferential selection of the groups in the two habitats. These variations in the distribution, diversity, and morphology of methanotrophic consortia are discussed with respect to the presence of microbial ecotypes, niche formation, and biogeography.

Isolation and characterization of aggregate-forming sulfate-reducing and purple sulfur bacteria from the chemocline of meromictic Lake Cadagno, Switzerland

In situ hybridization with specific oligonucleotide probes was used to monitor enrichment cultures of yet uncultured populations of sulfate-reducing and small-celled purple sulfur bacteria found to associate into aggregates in the chemocline of meromictic Lake Cadagno, Switzerland, and to select potential isolates. Enrichment and isolation conditions resembled those of their nearest cultured relatives, the sulfate-reducing bacterium Desulfocapsa thiozymogenes and small-celled purple sulfur bacteria belonging to the genus Lamprocystis, respectively. Based on comparative 16S rRNA analysis and physiological characterization, isolate Cad626 was found to resemble D. thiozymogenes although it differed from the type strain by its ability to grow on lactate and pyruvate. Like D. thiozymogenes, isolate Cad626 was able to disproportionate inorganic sulfur compounds (sulfur, thiosulfate, sulfite) and to grow, although growth on sulfur required a sulfide scavenger (FeOOH). Isolate Cad16 represented small-celled purple sulfur bacteria that belonged to a previously detected, but uncultured population designated F and was related to Lamprocystis purpurea as evidenced by comparative 16S rRNA analysis and the presence of bacteriochlorophyll a and the carotenoid okenone. Mixed cultures of isolates Cad626 and Cad16 resulted in their association in aggregates similar to those observed in the chemocline of Lake Cadagno. Concomitant growth enhancement of both isolates in mixed culture suggested synergistic interactions that presumably resemble a source–sink relationship for sulfide between the sulfate-reducing bacterium growing by sulfur disproportionation and the purple sulfur bacteria acting as biotic scavenger.

Polyphasic Classification of 0.2 μm Filterable Bacteria from the Western Mediterranean Sea

The 0.2 microm filtration of sea water samples from the Mediterranean Sea (Bay of Calvi, Corsica), collected from 10 m and 35 m depth led to the isolation of several gram-negative bacterial strains able to grow on full-strength media as well as on diluted media. The analysis of the 16S rRNA gene sequences and estimation of the phylogenetic relationships of these facultative oligotrophic bacteria indicated that they grouped into two phylogenetic branches. The strains RE10F/2, RE10F/5 (10 m depth samples) and RE35/F12 (35 m depth samples) were assigned to the gamma-subclass, while RE35F/1 (35m depth sample) was assigned to the alpha-4-subclass of the Proteobacteria. The strains RE10/F2 and RE10/F5 were most closely related to species and strains of the Pseudoalteromonas group, whereas the strain RE35F/12 placed adjacent to the family Vibrionaceae. The phylogenetic analysis of strain RE35F/1 revealed that this bacterium clusters with marine strains and species of the aerobic anoxygenic phototrophic bacteria Erythrobacter as well as Erythromicrobium and more distantly to Sphingomonas spp. Supplementary to those genotypic classifications the chemotaxonomic signatures including the major respiratory lipoquinone systems, the cellular fatty acid compositions as well as the polyamine contents of the bacteria were investigated. The isolated organisms displayed differences in their physiological and biochemical properties to already described strains belonging to the same genera or families, as revealed by the comparative 16S rRNA analysis. Despite the fact that these bacteria were isolated from a 0.2 microm filtrate, the cultured organisms which were all rod-shaped, displayed width dimensions ranging from 0.4 up to 0.7 microm, indicating that these bacteria were starvation forms at the time of isolation and not ultramicrobacteria as defined by Torella and Morita (1981) or by Schut et al. (1993). Because our isolated strains represent potentially new taxa, this first investigation on 0.2 pm filterable bacteria from the Western Mediterranean Sea supports the hypothesis that this bacterial fraction contributes to the diversity of marine bacteria.

In situ identification of nocardioform actinomycetes in activated sludge using fluorescent rRNA-targeted oligonucleotide probes.

Hitherto, few environmental samples have been investigated by a 'full cycle rRNA analysis'. Here the results of in situ hybridization experiments with specific rRNA-targeted oligonucleotide probes developed on the basis of new sequences derived from a previously described comparative 16S rRNA analysis of nocardioform actinomycetes in activated sludge are reported. Application of the specific probes enabled identification and discrimination of the distinct populations of nocardioform actinomycetes in activated sludge. One of the specific probes (DLP) detected rod-shaped bacteria which were found in 13 of the 16 investigated sludge samples from various wastewater treatment plants, suggesting their importance in the wastewater treatment process. Another probe (GLP2) hybridized with typically branched filaments of nocardioforms mainly found in samples from enhanced biological phosphorus removal plants, suggesting that these bacteria are involved in sludge foaming. The combination of in situ hybridization with fluorescently labelled rRNA-targeted oligonucleotide probes and confocal laser scanning microscopy improved the detection of nocardioform actinomycetes, which often showed only weak signals inside the activated-sludge flocs.

Evidence for the placement of the gram-negative Catonella morbi (Moore and Moore) and Johnsonella ignava (Moore and Moore) within the Clostridium subphylum of the gram-positive bacteria on the basis of 16S rRNA sequences.

Comparative 16S rRNA analysis was used to determine the phylogenetic positions of Catonella morbi and Johnsonella ignava, which are members of two monospecific genera of gram-negative anaerobic bacilli isolated from human gingival crevices. Both of these genera were found to belong to cluster XIVa (M. D. Collins, P. A. Lawson, A. Willems, J. J. Cordoba, J. Fernandez-Garayzabal, P. Garcia, J. Cai, H. Hippe, and J. A. E. Farrow, Int. J. Syst. Bacteriol. 44:812-826, 1994) of the Clostridium subphylum of gram-positive bacteria. Within this cluster, which contains several Clostridium, Coprococcus, Eubacterium, and Ruminococcus species, C. morbi and J. ignava formed two distinct lines that were separate from all other taxa. Our findings support the separate generic status of the genera Catonella and Johnsonella and show that these genera do not belong to the family Bacteroidaceae but instead belong to the gram-positive Clostridium subphylum.

Bacillus oleroniussp.nov., a member of the hindgut flora of the termiteReticulitermes santonensis(Feytaud)

A new rod-shaped endospore-forming bacterium is described, which was isolated from the hindgut of the termite Reticulitermes santonensis (Feytaud). The isolate stains Gram negative and its DNA has a guanine-plus-cytosine content of 35 mol%. Despite the Gram-staining reaction, both biochemical and physiological features place the isolate in the genus Bacillus and indicate a phenotypic resemblance to the Bacillus firmus-lentus group of species. On the basis of comparative 16S rRNA analysis and some phenotypic features the isolate clearly represents a new species for which the name Bacillus oleronius is proposed. The type strain is Bacillus oleronius Rt 10 (DSM 9356).

Classification of Fish-Pathogenic Vibrios Based on Comparative 16S rRNA Analysis

No systematic classification of fish-pathogenic vibrios has been accomplished previously despite the use of serological, physiological, and genetical classification systems. In this study, a comparative 16S rRNA analysis of 34 strains (representing seven species) of fish-pathogenic vibrios was performed. The 16S rRNA sequences were obtained by using reverse transcriptase. Nearly complete sequences were obtained for nine strains. On the basis of the results of this analysis, the remaining strains were investigated by analyzing selected stretches containing a total of 560 nucleotides. With the exception of a few strains, including ATCC 43313 (serovar O9), our comparative 16S rRNA analysis confirmed that strains preliminarily identified as Vibrio anguillarum were phylogenetically closely related. Strains of V. anguillarum could be divided into groups, with the main group containing serotype O1 and O2 strains isolated from Atlantic salmon, rainbow trout, turbot, cod, and saithe. The other distinctive group was represented by type strain NCMB 6. This strain was nearly indistinguishable from the type strains of Vibrio ordalii and Vibrio damsela on the basis of the 16S rRNA stretches compared. The results of a comparative 16S rRNA analysis justified the status of Vibrio salmonicida as a distinct species. Originally, this species was characterized biochemically as a very homogeneous species. However, two strains, which were isolated from diseased halibut and from the intestines of healthy cod, could not be distinguished from V. salmonicida strains phylogenetically, although they differed from the original species description in several phenotypic traits. Our results indicate that V. salmonicida and Vibrio fischeri form a cluster that is clearly separated from the cluster that includes V. anguillarum.

Molecular characterization of nocardioform actinomycetes in activated sludge by 16S rRNA analysis

The analysis of complex microbiota present in activated sludge is important for the understanding and possible control of severe separation problems in sewage treatment such as sludge bulking or sludge foaming. Previous studies have shown that nocardioform actinomycetes are responsible for these conditions, which not only affect the efficiency of sewage treatment but also represent a threat to public health due to spread of pathogens. However, isolation and identification of these filamentous, nocardioform actinomycetes is hampered by their fastidious nature. Most species are still uncultivable and their taxonomy is unresolved. To study the ecology of these micro-organisms at the molecular level, we have established a clone library of 16S rRNA gene fragments amplified from bulk sludge DNA. A rough indication of the predominant flora in the sludge was given by sequencing randomly chosen clones, which revealed a great diversity of bacteria from different taxa. Colony hybridization with oligonucleotide probe MNP1 detected 27 clones with 16S rDNA inserts from nocardioform actinomycetes and mycobacteria. The sequence data from these clones together with those from randomly chosen clones were used for comparative 16S rRNA analysis and construction of dendrograms. All sequences differed from those of previously sequenced species in the databases. Phenotypic characterization of isolates of nocardioform actinomycetes and mycobacteria cultivated in parallel from the same activated-sludge sample revealed a large discrepancy between the two approaches. Only one 16S rDNA sequence of a cultured isolate was represented in the clone library, indicating that culture conditions could select species which represent only a small fraction of the organisms in the activated sludge.

Comparative 16S rRNA analysis for the identification of slow-growing and uncultivable bacteria from activated sludge

Comparative 16S rRNA analysis for the identification of slow-growing and uncultivable bacteria from activated sludge

Characterization of Desulfomicrobium escambium sp. nov. and proposal to assign Desulfovibrio desulfuricans strain Norway 4 to the genus Desulfomicrobium

A sulfate-reducing bacterium, designated strain ESC1, was isolated and found to be a new species. Strain ESC1 is a strictly anaerobic, gram-negative, non-sporeforming, motile, short, round-ended rod often occurring in pairs. Of 31 fermentative substrates tested, only pyruvate was utilized. Sulfate enhanced growth with pyruvate and allowed growth with ethanol, lactate, formate and hydrogen. Both sulfate and thiosulfate were reduced. Lactate was incompletely oxidized to acetate and CO2. The strain was desulfoviridin negative. The G+C content is 59.9%. These data suggested placement of strain ESC1 in the genus Desulfomicrobium. Comparative 16S rRNA analysis showed that strain ESC1 shares 98% rRNA sequence similarity with Desulfomicrobium baculatum and Desulfovibrio desulfuricans strain Norway 4. The latter two strains shared greater than 99% 16S rRNA sequence similarity. Strain ESC1 has been designated as the new species Desulfomicrobium escambium. We also recommend that D. desulfuricans strain Norway 4 be considered for reclassification as a Desulfomicrobium species.

Phylogenetic evidence for the transfer of Eubacterium suis to the genus Actinomyces as Actinomyces suis comb. nov.

The 16S rRNA primary structures of Eubacterium suis DSM 20639T (T = type strain) and Bifidobacterium bifidum DSM 20456T were determined by sequencing in vitro amplified rDNA. Sequence comparisons indicated that B. bifidum is moderately related to representatives of the genera Actinomyces and Mobiluncus. The closest relative of E. suis is Actinomyces pyogenes. E. suis and A. pyogenes are more closely related phylogenetically to one another than to the other Actinomyces species that have been investigated by using comparative 16S rRNA analysis. Therefore, we propose that E. suis should be transferred to the genus Actinomyces as Actinomyces suis comb. nov.