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PubMed1996

Diversity and distribution of subterranean bacteria in groundwater at Oklo in Gabon, Africa, as determined by 16S rRNA gene sequencing.

This paper describes how ground water was sampled, DNA extracted, amplified and cloned and how information available in the ribosomal 16S rRNA gene was used for mapping diversity and distribution of subterranean bacteria in groundwater at the Bangombé site in the Oklo region. The results showed that this site was inhabited by a diversified population of bacteria. Each borehole was dominated by species that did not dominate in any of the other boreholes; a result that probably reflects documented differences in the geochemical environment. Two of the sequences obtained were identified at genus level to represent Acinetobacter and Zoogloea, but most of the 44 sequences found were only distantly related to species in the DNA database. The deepest borehole, BAX01 (105 m), had the highest number of bacteria and also total organic carbon (TOC). This borehole harboured only Proteobacteria beta group sequences while sequences related to Proteobacteria beta, gamma and delta groups and Gram-positive bacteria were found in the other four boreholes. Two of the boreholes, BAX02 (34 m) and BAX04 (10 m) had many 16S rRNA gene sequences in common and also had similar counts of bacteria, content of TOC, pH and equal conductivity, suggesting a hydraulic connection between them.

Acinetobacter↗
PubMed2002

Identification by 16S rRNA gene sequencing of Lactobacillus salivarius bacteremic cholecystitis.

An anaerobic, nonsporulating, gram-positive bacterium was isolated from blood and bile pus cultures of a 70-year-old man with bacteremic acute cholecystitis. The API 20A system showed that it was 70% Actinomyces naeslundii and 30% Bifidobacterium species, whereas the Vitek ANI system and the ATB ID32A Expression system showed that it was "unidentified." The 16S rRNA gene of the strain was amplified and sequenced. There were 3 base differences between the nucleotide sequence of the isolate and that of Lactobacillus salivarius subsp. salivarius or L. salivarius subsp. salicinius, indicating that the isolate was a strain of L. salivarius. The patient responded to cholecystectomy and a 2-week course of antibiotic treatment. Identification of the organism in the present study was important because the duration of antibiotic therapy would have been entirely different depending on the organism. If the bacterium had been identified as Actinomyces, penicillin for 6 months would have been the regimen of choice. However, it was Lactobacillus, and a 2-week course of antibiotic was sufficient.

Aged↗
PubMed1993

Polymerase chain reaction using 16S rRNA gene sequences distinguishes the two biovars of Ureaplasma urealyticum.

Several fundamental phenotypic and genotypic differences have separated strains of the genital mycoplasma Ureaplasma urealyticum into two clusters or biovars. However, the lack of an easily performed and unambiguous test to discriminate between them has hampered investigation of the relationship between these biovars and disease. We determined the 16S rRNA nucleotide sequence of U. urealyticum 27, the serovar 3 standard and representative of the parvo biovar (serovars 1, 3, 6, and 14). This sequence was compared with the published sequence of U. urealyticum T960, which is the type strain and the serovar 8 standard and is representative of the T960 biovar which is composed of the 10 intervening serovars. Homology between the two sequences was 98.8%; differences were exploited to provide primers for biovar-specific polymerase chain reactions (PCRs). The results of these reactions placed all 14 serovar standard strains into the correct biovar. The PCRs were also applied to 10 cloned and 8 noncloned isolates that had been serotyped earlier. For 16 of them, we deduced their biovars from the serotyping data and then confirmed them by PCR. One unpredictable isolate and one nonserotypeable isolate were also classified as to biovar. Thus, we have developed a method for biotyping U. urealyticum that is applicable to both laboratory-adapted strains and wild-type isolates and that is appropriate for testing large numbers of clinical isolates. The amplification by the T960 biovar PCR protocol of DNAs from ureaplasmas of animals and certain Mycoplasma species suggested that the parvo biovar has diverged from the mainstream of the evolution of this clade.

Bacterial Typing Techniques↗
PubMed1999

Variations in the 16S rRNA gene sequence of Yersinia enterocolitica isolates influence the specificity of molecular identification systems.

Four identification systems were used to type Yersinia enterocolitica strain Y11 and Yersinia enterocolitica sensu strictoT. Two systems based on biochemical reaction patterns identified both strains as Yersinia enterocolitica. Two molecular assays targeting the 16S rRNA gene failed to identify either strain Y11 or the type strain. Therefore, both strains were typed by the classical taxonomical approach requiring a determination of the overall base composition and the base sequence similarity using hybridization. Again both strains were classified as Yersinia enterocolitica isolates. Consequently, the sequences of the 16S rRNA gene of both strains were determined and compared. The strains differed in a region where nucleotide changes between species of the genus Yersinia had been described earlier. These differences may explain the failure of the molecular assays to identify the strains. They also demonstrate an independent evolution of the 16S rRNA genes in the species Yersinia enterocolitica sensu stricto suggesting an amendment to the nomenclature to be used in the future.

DNA, Ribosomal↗
PubMed1998

Differentiation of Mycobacterium ulcerans, M. marinum, and M. haemophilum: mapping of their relationships to M. tuberculosis by fatty acid profile analysis, DNA-DNA hybridization, and 16S rRNA gene sequence analysis.

Although Mycobacterium ulcerans, M. marinum, and M. haemophilum are closely related, their exact taxonomic placements have not been determined. We performed gas chromatography of fatty acids and alcohols, as well as DNA-DNA hybridization and 16S rRNA gene sequence analysis, to clarify their relationships to each other and to M. tuberculosis. M. ulcerans and M. marinum were most closely related to one another, and each displayed very strong genetic affinities to M. tuberculosis; they are actually the two mycobacterial species outside the M. tuberculosis complex most closely related to M. tuberculosis. M. haemophilum was more distinct from M. ulcerans and M. marinum, and it appeared to be as related to these two species as to M. tuberculosis. These results are important with regard to the development of diagnostic and epidemiological tools such as species-specific DNA probes and PCR assays for M. ulcerans, M. marinum, and M. haemophilum. In addition, the finding that M. ulcerans and M. marinum are more closely related to M. tuberculosis than are other pathogenic mycobacterial species suggests that they may be evaluated as useful models for studying the pathogenesis of M. tuberculosis. M. marinum may be particularly useful in this regard since strains of this species grow much more rapidly than M. tuberculosis and yet can cause systemic disease in immunocompromised hosts.

Chromatography, Gas↗
PubMed2002

Study of the intra- and interlaboratory reproducibility of partial single base C-sequencing of the 16S rRNA gene and its applicability for the identification of members of the genus Streptococcus.

The use of Single Base C-Sequencing of the first 500 bases of the 16S rRNA-gene (SBCS) combined with capillary electrophoresis was evaluated for the identification of reference strains of 30 different species within the genus Streptococcus. For SBCS, only dd-CTP's are used in the sequencing reactions instead of the four dideoxy bases and the primer is fluorescently labeled. The reproducibility, interlaboratory exchangeability and discriminative power of this method were studied by comparing the patterns obtained in three laboratories under highly standardized conditions. The interlaboratory reproducibility proved to be high, enabling the construction of a common database for the identification of strains belonging to the streptococcal species studied. Most of the examined species generated distinguishable profiles. SBCS did not differentiate between the closely related species S. constellatus and S. intermedius. Also S. thermophilus and S. vestibularis as well as S. mitis and S. pneumoniae showed highly resembling profiles. The previously reported heterogeneity within the species S. equinus was reflected by SBCS. For all other species, strains belonging to the same species generated indistinguishable patterns. In conclusion, Single Base C-sequencing of the first 500 bases of the 16S rRNA-gene could be a useful and widely applicable method for the identification of bacteria at the species level, with the added advantage of being more rapid and easier to automatize than full sequence determination.

Base Sequence↗
PubMed2002

Molecular characterization of DNA encoding 16S-23S rRNA intergenic spacer regions and 16S rRNA of pectolytic Erwinia species.

Sequences of 16S rDNAs and the intergenic spacer (IGS) regions between the 16S and 23S rDNA of bacterial strains from genus Erwinia were determined. Comparison of 16S rDNA sequences from different species and subspecies clearly revealed intraspecies-subspecies homology and interspecies heterogeneity. Phylogenetic analyses of 16S rDNA sequence data revealed that Erwinia spp. formed a discrete monophyletic clade with moderate to high bootstrap values. PCR amplification of the 16S-23S rDNA regions using primers complementary to the 3' end of 16S and 5' end of 23S rRNA genes generated two DNA fragments. The small 16S-23S rDNA IGS regions of Erwinia spp. examined in this study varied considerably in size and nucleotide sequence. Multiple sequence alignment and phylogenetic analysis of small IGS sequence data showed a consistent relationship among the test strains that was roughly in agreement with the 16S rDNA data that reflected the accepted species and subspecies structure of the taxon. Sequence data derived from the large IGS resolved the strains into coherent groups; however, the sequence information would not allow any phylogenetic conclusion, because it failed to reflect the accepted species structure of the test strains.

Base Sequence↗
PubMed1996

The phylogenetic relationship among Ectothiorhodospiraceae: a reevaluation of their taxonomy on the basis of 16S rDNA analyses.

Sequences of the 16S rRNA gene were determined from all type strains of the recognized Ectothiorhodospira species and from a number of additional strains. For the first time, these data resolve the phylogenetic relationships of the Ectothiorhodospiraceae in detail, confirm the established species, and improve the classification of strains of uncertain affiliation. Two major groups that are recognized as separate genera were clearly established. The extremely halophilic species were removed from the genus Ectothiorhodospira and reassigned to the new genus Halorhodospira gen. nov., to recognize that the most halophilic eubacteria are species of this genus. These species are Halorhodospira halophila comb. nov., Halorhodospira halochloris comb. nov., and Halorhodospira abdelmalekii comb. nov. Among the slightly halophilic Ectothiorhodospira species, the classification of strains belonging to Ectothiorhodospira mobilis and Ectothiorhodospira shaposhnikovii was improved. Several strains that were tentatively identified as Ectothiorhodospira mobilis form a separate cluster on the basis of their 16S rDNA sequences and are recognized as two new species: Ectothiorhodospira haloalkaliphila sp. nov., which includes the most alkaliphilic strains originating from strongly alkaline soda lakes, and Ectothiorhodospira marina, describing isolates from the marine environment.

Bacteria↗
PubMed2003

Comparative analysis of Borrelia isolates from southeastern USA based on randomly amplified polymorphic DNA fingerprint and 16S ribosomal gene sequence analyses.

Fifty-three southern USA Borrelia isolates were characterized using randomly amplified polymorphic DNA fingerprinting analysis (RAPD). Twenty-nine types were recognized among 37 B. andersonii strains, seven types among eight B. bissettii strains, and seven types among seven B. burgdorferi sensu stricto strains. Strain TXW-1 formed a separate RAPD type. Nearly complete sequences of the rrs genes from 17 representative southern Borrelia were determined. The similarity values were found to be 96-100% within the B. burgdorferi sensu lato (s.l.) complex, 94-99% among the relapsing fever borreliae, and 93-99% between the two complexes. Phylogenetic analysis indicated that all the Borrelia strains we analyzed could be divided into two parts: the B. burgdorferi s.l. complex and the relapsing fever borreliae complex. TXW-1 segregated with the North American relapsing fever borreliae and formed a separate subbranch.

Animals↗
PubMed1997

Western immunoblot analysis of Haemobartonella muris and comparison of 16S rRNA gene sequences of H. muris, H. felis, and Eperythrozoon suis.

Infectious agents were isolated from the spleens of three wild mice (Apodemus argenteus) by intraperitoneal inoculation of the spleen homogenate into laboratory mice. The laboratory mice developed clinical signs and splenomegaly, and three isolates were maintained by passage in mice. Tetracyclines were effective in preventing infection of mice with these agents, but streptomycin and penicillin were ineffective. The agents did not grow in bacterial growth media or chicken embryos. In smears of blood from infected mice stained by the Giemsa or the indirect immunofluorescence method, numerous organisms were found on the surfaces of erythrocytes. Electron microscopy revealed cell wall-less pleomorphic cocci of 350 to 700 nm in diameter. On the basis of these results, the isolates were identified as Haemobartonella muris. There was no antigenic cross-reactivity with Rickettsia or Ehrlichia spp. or other related organisms. Western immunoblot analysis of three strains of H. muris with mouse antisera to H. muris revealed identical major antigens of 118, 65, 53, 45, and 40 kDa. By heteroduplex analysis of the three PCR-amplified segments of the 16S rRNA genes, the three strains of H. muris were found to be identical. The 16S rRNA genes of one of the H. muris strains, four strains of H. felis, and two strains of Eperythrozoon suis were sequenced and compared. The sequences of two strains of H. felis from cats in California were identical, as were the sequences of a strain from a cat in Ohio and a strain from a cat in Florida, but the similarity of sequences between the California and the Ohio-Florida strains was only 85%. The sequence of an H. muris strain was unique and was more closely related to that of the Ohio-Florida strain of H. felis (89%) than to that of the California strain of H. felis (84%). The sequence of E. suis from a pig in Illinois was identical to that from another pig from Taiwan. The similarity of the 16S rRNA gene sequence of E. suis with those of three Haemobartonella strains was 84 to 92%, with that of E. suis being most similar to that of the H. felis strain from California. In the phylogenetic analysis based on 16S rRNA gene sequences, the Haemobartonella spp. and E. suis formed a distinct clade more closely related to Mycoplasma spp. (79 to 83% similarity) than to Anaplasma marginale (72 to 75% similarity). Our results suggest that the Haemobartonella spp. and E. suis may be reclassified in the same genus in the family Mycoplasmataceae.

Anaplasmataceae↗
PubMed2005

Pusillimonas noertemannii gen. nov., sp. nov., a new member of the family Alcaligenaceae that degrades substituted salicylates.

The taxonomic position of a Pseudomonas-like strain, designated BN9(T), was investigated. This strain had previously been isolated as a 5-aminosalicylate-degrading organism from a 6-aminonaphthalene-2-sulphonate-degrading mixed bacterial culture. Previously, detection of ubiquinone Q-8, a polyamine pattern with putrescine, spermidine and 2-hydroxyputrescine as the major polyamines, and partial 16S rRNA gene sequencing had suggested that strain BN9(T) belongs to the 'Betaproteobacteria'. This was supported by sequencing the 16S rRNA gene, which demonstrated 94-96 % sequence similarity to different species of the genera Achromobacter, Alcaligenes and Bordetella, and suggested that strain BN9(T) represents a member of the family Alcaligenaceae. Different methods for the construction of phylogenetic dendrograms placed the strain separately from all other genera within the Alcaligenaceae. Fatty acid analysis demonstrated the presence of high concentrations of C(19 : 0) cyclo omega8c. On the basis of low 16S rRNA gene sequence similarity to other members of the Alcaligenaceae, fatty acid and polar lipid profiles, and other unique phenotypic properties of strain BN9(T), the creation of a new genus and species with the name Pusillimonas noertemannii gen. nov., sp. nov. is proposed. The type strain is BN9(T) (= DSM 10065(T) = NCIMB 14020(T)).

Alcaligenaceae↗
PubMed1993

Ileal symbiont intracellularis, an obligate intracellular bacterium of porcine intestines showing a relationship to Desulfovibrio species.

A new genus and species of obligate intracellular-bacteria found in porcine intestines are described. Growth on any bacteriological medium deprived of living cells has not been demonstrated. The organism has been grown intracellularly in cell culture. The 16S rRNA gene sequence data, DNA probe results, and microscopic observations provide evidence that these bacteria differ from those in other described genera and that they belong to the delta subdivision of the class Proteobacteria. We have amplified and sequenced the 16S ribosomal DNA of four preparations of the intracellular bacterium from pigs. For this, intracellular organisms were released and purified from the infected cells without culture techniques. After DNA purification, the polymerase chain reaction with primers complementary to highly conserved eubacterial sequences was used to amplify regions of 16S ribosomal DNA which were subsequently cloned (in some cases) and sequenced directly by standard techniques. The sequences obtained from each preparation were identical and were most similar to that of a sulfate-reducing proteobacterium, Desulfovibrio desulfuricans ATCC 27774 (91% similarity). An oligonucleotide probe complementary to a hypervariable region of the 16S rRNA sequence of the bacterium hybridized with intracellular organisms obtained from porcine intestines. The bacterium is a gram-negative, curved rod with tapered ends. It multiplies intracellularly in the cytoplasm of ileal epithelial cells by septation. The vernacular name Ileal symbiont (IS) intracellularis is proposed for this bacterium. The type strain of IS intracellularis is strain 1482/89 grown in cell culture from a pig affected by proliferative enteropathy. It is deposited in the National Collection of Type Cultures, Colindale, London, as NCTC 12656.

Animals↗
PubMed2001

An alternative to the accepted phylogeny of purple bacteria based on 16S rRNA: analyses of the amino acid sequences of cytochromes C2 and C556 from Rhodobacter (Rhodovulum) sulfidophilus.

It is becoming increasingly apparent from complete genome sequences that 16S rRNA data, as currently interpreted, does not provide an unambiguous picture of bacterial phylogeny. In contrast, we have found that analysis of insertions and deletions in the amino acid sequences of cytochrome c2 has some advantages in establishing relationships and that this approach may have broad utility in acquiring a better understanding of bacterial relationships. The amino acid sequences of cytochromes c2 and c556 have been determined in whole or in part from four strains of Rhodobacter sulfidophilus. The cytochrome c2 contains three- and eight-residue insertions as well as a single-residue deletion in common with the large cytochromes c2 but in contrast to the small cytochromes c2 and mitochondrial cytochromes. In addition, the Rb. sulfidophilus protein shares a rare six- to seven-residue insertion with other Rhodobacter cytochromes c2. The cytochrome c556 is a low-spin class II cytochrome c homologous to the greater family of cytochromes c', which are usually high-spin. The similarity of cytochrome c556 to other species of class II cytochromes is consistent with the relationships deduced from comparisons of cytochromes c2. Thus, our results do not support placement of Rb. sulfidophilus in a separate genus, Rhodovulum, which was proposed primarily on the basis of 16S rRNA sequences. Instead, the Rhodobacter cytochromes c2 are distinct from those of other genera and species of purple bacteria and show a different pattern of relationships among species than reported for 16S rRNA.

Amino Acid Sequence↗
PubMed2005

Development of a real-time PCR-based system targeting the 16S rRNA gene sequence for rapid detection of Alicyclobacillus spp. in juice products.

The thermophilic, aciduric Alicyclobacillus spp. are becoming an increasing spoilage concern in the beverage industry. Rapid methods to detect their presence in both raw materials and final products are desirable for industrial quality control. The objective of this study was to develop a real-time TaqMan-based polymerase chain reaction (PCR) system for rapid and specific detection of thermophilic spoilage bacteria, mainly Alicyclobacillus spp., for food industry applications. Two primers and a fluorogenic probe targeting 16S rRNA encoding gene sequences, capable of detecting the genus Alicyclobacillus and a few other closely related thermophiles, were developed, and the efficiency of the detection system was evaluated in both bacterial medium and juice products. Using this system, the presence of less than 100 Alicyclobacillus cells could be detected without cross-reactivity with other common food-borne bacteria.

Beverages↗
PubMed2026

Indoor Air Quality (IAQ) analysis and 16S rRNA gene sequencing of indoor air pollutants in a 3000-capacity religious auditorium.

A thorough molecular and environmental evaluation of microbial isolates and indoor air quality (IAQ) at a place of worship is presented in this work. The study used a dual-methodological approach, including systematic environmental monitoring to assess occupant health concerns and 16S rRNA gene sequencing for taxonomic identification. Four different bacterial isolates were successfully identified by molecular analysis using BLAST and phylogenetic reconstruction: Bacillus tropicus (A11), Leclercia adecarboxylata (A12), Acinetobacter sp. (A13), and Escherichia coli (A14). The evolutionary position of isolate A13 indicated possible horizontal gene transfer, underscoring the existence of flexible, opportunistic pathogens within the indoor environment, whereas isolates A11 and A14 demonstrated significant genetic stability. Concurrently, six sessions of air quality monitoring showed a thermally demanding environment, with humidity (56.00-70.51%) and temperatures (29.21-33.79 °C) continuously surpassing ASHRAE guidelines. The average pollutant concentrations are within WHO and USEPA safety criteria, however, there were outliers that may suggest sporadic pollutant penetration (outdoor) or resuspension of dust. The results demonstrate a clear relationship between increased chemical pollutants (TVOCs) and reduced thermal comfort. To reduce the congregation's acute respiratory and cardiovascular risks, optimal ventilation systems and source-control measures are urgently needed, as evidenced by the presence of clinically relevant microorganisms combined with dangerous pollution levels.

Analysis↗
PubMedDate not supplied

Insights on the diversity within a "species" of Thalassicolla (Spumellarida) based on 16S-like ribosomal RNA gene sequencing.

We compared 16S-like ribosomal RNA (rRNA) coding regions of samples of the solitary spumellarian radiolarian Thalassicolla nucleata collected from the Sargasso Sea and the Pacific Ocean. Sequences derived from these locations showed variability in both length and base-pair composition. This level of sequence variability is similar to the degree of variability reported in the literature for species- or even genus-level distinctions. Explanations for our results include multiple alleles for the rRNA gene, or the existence of multiple species of Thalassicolla that are morphologically indistinguishable. The seven existing descriptions of Thalassicolla species, including T. nucleata, are discussed in view of these molecular findings and with reference to our current understanding of the physiology and life cycle of the spumellarian radiolaria.

Animals↗