Identification of Staphylococcus from French dry sausage

Fifty-one strains of staphylococci isolated from French dry sausages were mainly identified with Staphylococcus carnosus, S. xylosus, S. warneri and S. saprophyticus. The API Staphylococcus identification system proved to be reliable for S. xylosus and S. carnosus. The identification of S. warneri and S. saprophyticus was performed by DNA-DNA hybridization. These species are better identified by taking into account not only the API Staphylococcus system but also the following characters: novobiocin and lysozyme susceptibilities, production of D-lactate. hydrolysis of tri-olein.     

Characterization of universal small-subunit rRNA hybridization probes for quantitative molecular microbial ecology studies.

Universal oligonucleotide hybridization probes targeting the small-subunit rRNA are commonly used to quantify total microbial representation in environmental samples. Universal probes also serve to normalize results obtained with probes targeting specific phylogenetic groups of microorganisms. In this study, six universal probes were evaluated for stability of probe-target duplexes by using rRNA from nine organisms representing the three domains of Bacteria, Archaea, and Eucarya. Domain-specific variations in dissociation temperatures were observed for all probes. This could lead to a significant bias when these probes are used to quantify microbial populations in environmental samples. We suggest lowering the posthybridization wash stringency for two of the universal probes (S-*-Univ-1390-a-A-18 and S-*-Univ-1392-a-A-15) examined. These two probes were evaluated with traditional and modified hybridization conditions to characterize defined mixtures of rRNAs extracted from pure cultures and rRNA samples obtained from anaerobic digester samples. Probe S-*-Univ-1390-a-A-18 provided excellent estimations of domain-level community composition of these samples and is recommended for future use in microbial ecology studies.     

A Staphylococcus aureus-specific oligonucleotide probe derived from 16S rRNA gene sequences

Genes encoding 16S rRNA were sequenced from 16 species of Staphylococcus. Sequence analysis highlighted a potential Staph. aureus -specific region and a complementary oligonucleotide probe was synthesized and its specificity tested. Northern blotting indicated molecular specificity, and dot blots to RNA from Staph. aureus, Staph. capitis, Staph. caprae, Staph. carnosus, Staph. caseolyticus, Staph. cohnii, Staph. epidermidis, Staph. gallinarum, Staph. haemolyticus, Staph. hominis, Staph. hyicus, Staph. saprophyticus, Staph. sciuri, Staph. simulans, Staph. warneri and Staph. xylosus indicated species-specificity.     

Fusobacterium prausnitzii and related species represent a dominant group within the human fecal flora

The human gut microflora plays a key role in nutrition and health. It has been extensively studied by conventional culture techniques. However these methods are difficult, time consuming and their results not always consistent. Furthermore microscopic counts indicate that only 20 to 40% of the total flora can be cultivated. Among the predominant species of the human gut, Fusobacterium prausnitzii was reported either as one of the most frequent and numerous species or was seldom retrieved. We designed and validated a specific rRNA-targeted oligonucleotide probe, called S-*-Fprau-0645-a-A-23, to accurately detect and quantify F. prausnitzii and relatives within the human fecal microflora. The target group accounted for 5.3 +/- 3% of total bacterial 16S rRNA using dot blot hybridization (10 human fecal samples) and 16.5 +/- 7% of cells stained with Dapi using in situ hybridization (10 other human fecal samples). A specific morphology seemed to be typical and dominant: two cells forming an asymmetrical double droplet. This work showed that F. prausnitzii and phylogenetically related species represent a dominant group within the human fecal flora.     

Quantification of Hyphomicrobium populations in activated sludge from an industrial wastewater treatment system as determined by 16S rRNA analysis

The bacterial community structure of the activated sludge from a 25 million-gal-per-day industrial wastewater treatment plant was investigated using rRNA analysis. 16S ribosomal DNA (rDNA) libraries were created from three sludge samples taken on different dates. Partial rRNA gene sequences were obtained for 46 rDNA clones, and nearly complete 16S rRNA sequences were obtained for 18 clones. Seventeen of these clones were members of the beta subdivision, and their sequences showed high homology to sequences of known bacterial species as well as published 16S rDNA sequences from other activated sludge sources. Sixteen clones belonged to the alpha subdivision, 7 of which showed similarity to Hyphomicrobium species. This cluster was chosen for further studies due to earlier work on Hyphomicrobium sp. strain M3 isolated from this treatment plant. A nearly full-length 16S rDNA sequence was obtained from Hyphomicrobium sp. strain M3. Phylogenetic analysis revealed that Hyphomicrobium sp. strain M3 was 99% similar to Hyphomicrobium denitrificans DSM 1869(T) in Hyphomicrobium cluster II. Three of the cloned sequences from the activated sludge samples also grouped with those of Hyphomicrobium cluster II, with a 96% sequence similarity to that of Hyphomicrobium sp. strain M3. The other four cloned sequences from the activated sludge sample were more closely related to those of the Hyphomicrobium cluster I organisms (95 to 97% similarity). Whole-cell fluorescence hybridization of microorganisms in the activated sludge with genus-specific Hyphomicrobium probe S-G-Hypho-1241-a-A-19 enhanced the visualization of Hyphomicrobium and revealed that Hyphomicrobium appears to be abundant both on the outside of flocs and within the floc structure. Dot blot hybridization of activated sludge samples from 1995 with probes designed for Hyphomicrobium cluster I and Hyphomicrobium cluster II indicated that Hyphomicrobium cluster II-positive 16S rRNA dominated over Hyphomicrobium cluster I-positive 16S rRNA by 3- to 12-fold. Hyphomicrobium 16S rRNA comprised approximately 5% of the 16S rRNA in the activated sludge.     

Quantification of Methanosaeta Species in Anaerobic Bioreactors Using Genus- and Species-Specific Hybridization Probes

A BSTRACTTo evaluate the role of Methanosaeta spp. in a variety of anaerobic environments, small-subunit rRNA targeted oligonucleotide hybridization probes were developed and experimentally characterized. The probes were designed to be genus specific for Methanosaeta and species specific for Methanosaeta concilii and Methanosaeta thermophila. The temperature of dissociation was determined for each probe. Probe specificities were determined using a diverse collection of Archaea and through an evaluation of probe nesting using samples from a variety of anaerobic bioreactors. Cell fixation and hybridization conditions for fluorescence in situ hybridizations were also evaluated. Although permeability of methanogens was variable, M. concilii cells could be permeabilized using a range of paraformaldehyde and ethanol based fixation conditions. Using the newly designed probes together with previously designed probes for methanogens, it was determined that Methanosaeta spp. were the dominant aceticlastic methanogens in a variety of anaerobic bioreactors when acetate concentrations were low. Their levels were higher in bioreactors with granular sludge than in those with flocculent sludge. In lab-scale upflow anaerobic sludge blanket reactors, the levels of M. concilii rRNA were as high as 30% of the total rRNA.     

Staphylococcus pasteuri-specific oligonucleotide probes derived from a random amplified DNA fragment

Abstract A rapid polymerase chain reaction method was developed to differentiate Staphylococcus pasteuri from other staphylococcal species, especially the phenotypically similar S. warneri . The oligonucleotide probes used as primers were designed from the sequence of a S. pasteuri random amplified polymorphic DNA fragment.     

Development of alternate ssu-rRNA probing strategies for characterizing aquatic microbial communities

Plastids in phytoplankton retain prokaryote-like DNA sequences that may generate false-positive signals from eubacterial small subunit (ssu) rRNA oligonucleotide probes, resulting in the overestimation of bacterial activity in aquatic microbial communities. To assess the extent of possible plastid-associated binding to eubacterial signals, we performed an extensive database search, flask experiments using algal and cyanobacterial pure cultures, and field trials on five common eubacterial probes: S-D-Bact-008-a-A-19, S-D-Bact-338-a-A-18, S-D-Bact-785-a-A-19, S-D-Bact-927-a-A-17, and S-D-Bact-1088-a-A-20. The database search and laboratory tests showed significant potential for binding among most bacterial probes and organelle ssu-rRNA. However, we propose two probing strategies to overcome this problem. First, one could use Bact-785 and Bact-338 in tandem, with the plastid component being estimated as the difference between the two signals (Bact-338 has approximately 70% overlap with known plastid sequences). Alternately, one might use Bact-338 as the primary eubacterial probe, but then use Cyan-785-a-A-19 (a probe that binds significantly to plastid rRNA) to correct for the plastid-associated false-positive signal. Both strategies would use a eukaryotic probe (S-D-Euca-1379-a-A-16) and Cyan-785-b-A-19 (a probe for most cyanobacteria) to further segregate rRNA signals. Trials were successfully performed using the strategies on samples from a recent field study.     

In situ probing of Xylella fastidiosa in honeydew of a xylem sap-feeding insect using 16S rRNA-targeted fluorescent oligonucleotides

Xylella fastidiosa is a plant pathogen that threatens a US$ 4.6 billion worldwide wine and citrus industry. Monitoring its presence and distribution in plants and vectors is crucial for designing control strategies, as well as for understanding its ecological role and fate. We developed two fluorescent oligonucleotide probes complementary to different regions of the 16S rRNA gene of X. fastidiosa. The specificity of the newly designed probes S-S-X.fas-0067-a-A-18 and S-S-X.fas-1439-a-A-18 was demonstrated using fluorescence in situ hybridization (FISH) for 12 Xylella isolates, 15 closely related microorganisms and three plant endophytes. These probes were used to detect and quantify X. fastidiosa in plant sap (average value of 2.9 +/- 0.3 x 10(6) cells ml(-1)) from three different citrus orchards. In a second experiment, cells were quantified in honeydew (2.2 +/- 0.2 x 10(4) cells ml(-1)) collected from the insect vector Bucephalogonia xanthophis during the acquisition access period on an infected plant. The number of pathogen cells retained or digested by the insect is 10,000 times greater than the estimated minimum value to ensure an efficient transmission. Polymerase chain reaction (PCR) amplification using specific primers with plant sap and honeydew samples, followed by sequencing, confirmed the presence of the plant pathogen. This is the first demonstration of FISH being used for environmental samples, such as plant sap and insect honeydew, to estimate the abundance of a plant pathogen during infection.     

Specific Neisseria gonorrhoeae DNA-probes derived from ribosomal RNA

Eighteen sequences complementary to less-conserved regions within the 16S and 23S ribosomal ribonucleic acid (rRNA) of Neisseria gonorrhoeae were subcloned or chemically synthesized and used as probes in a dot-spot deoxyribonucleic acid (DNA): DNA hybridization format. Some of these probes exclusively detected Neisseria gonorrhoeae nucleic acid, whereas others also showed hybridization signals with nucleic acid from other bacterial species. Our results indicate that rRNA-derived DNA-probes can be used to differentiate between very closely related taxa without the use of Southern-blot analysis.     

Mycobacteria possess a surprisingly small number of ribosomal RNA genes in relation to the size of their genome

DNAs from Mycobacterium tuberculosis, M. intracellulare, M. phlei and M. smegmatis were digested by restriction enzymes and hybridized with three probes consisting of the 5' (16S rRNA), the middle (16S and 23S rRNA), and the 3' (23S and 5S rRNA) portions of the Escherichia coli rrnB operon. The resulting hybridization patterns indicate that slow-growing Mycobacteria species (i.e., M. tuberculosis and M. intracellulare), with genome size 3.13 - 4.29 X 10(9) daltons, appear to possess only one rRNA operon, whereas fast-growing species (i.e., M. phlei and M. smegmatis), with genome size 4.30 - 5.20 X 10(9) daltons, appear to possess two rRNA operons.     

Designation of Streptomycete 16S and 23S rRNA-based target regions for oligonucleotide probes.

The 16S and 23S rRNA of various Streptomyces species were partially sequenced and screened for the presence of stretches that could define all members of the genus, groups of species, or individual species. Nucleotide 929 (Streptomyces ambofaciens nomenclature [J.L. Pernodet, M.T. Alegre, F. Boccard, and M. Guerineau, Gene 79:33-46, 1989]) is a nucleotide highly unique to Streptomyces species which, in combination with flanking regions, allowed the designation of a genus-specific probe. Regions 158 through 203 of the 16S rRNA and 1518 through 1645 of the 23S rRNA (helix 54 [Pernodet et al., Gene 79:33-46, 1989]) have a high potential to define species, whereas the degree of variation in regions 982 through 998 and 1102 through 1122 of the 16S rRNA is less pronounced but characteristic for at least certain species. Alone or in combination with each other, these regions may serve as target sites for synthetic oligonucleotide probes and primers to be used in the determination of pure cultures and in the characterization of community structures. The specificity of several probes is demonstrated by dot blot hybridization.     

Designation of Streptomycete 16S and 23S rRNA-based target regions for oligonucleotide probes

The 16S and 23S rRNA of various Streptomyces species were partially sequenced and screened for the presence of stretches that could define all members of the genus, groups of species, or individual species. Nucleotide 929 (Streptomyces ambofaciens nomenclature [J.L. Pernodet, M.T. Alegre, F. Boccard, and M. Guerineau, Gene 79:33-46, 1989]) is a nucleotide highly unique to Streptomyces species which, in combination with flanking regions, allowed the designation of a genus-specific probe. Regions 158 through 203 of the 16S rRNA and 1518 through 1645 of the 23S rRNA (helix 54 [Pernodet et al., Gene 79:33-46, 1989]) have a high potential to define species, whereas the degree of variation in regions 982 through 998 and 1102 through 1122 of the 16S rRNA is less pronounced but characteristic for at least certain species. Alone or in combination with each other, these regions may serve as target sites for synthetic oligonucleotide probes and primers to be used in the determination of pure cultures and in the characterization of community structures. The specificity of several probes is demonstrated by dot blot hybridization.     

A nested array of rRNA targeted probes for the detection and identification of enterococci by reverse hybridization

Complete 23S and almost complete 16S rRNA gene sequences were determined for the type strains of the validly described Enterococcus species, Melissococcus pluton and Tetragenococcus halophilus. A comprehensive set of rRNA targeted specific oligonucleotide hybridization probes was designed according to the multiple probe concept. In silico probe design and evaluation was performed using the respective tools of the ARB program package in combination with the ARB databases comprising the currently available 16S as well as 23S rRNA primary structures. The probes were optimized with respect to their application for reverse hybridization in microplate format. The target comprising 16S and 23S rDNA was amplified and labeled by PCR (polymerase chain reaction) using general primers targeting a wide spectrum of bacteria. Alternatively, amplification of two adjacent rDNA fragments of enterococci was performed by using specific primers. In vitro evaluation of the probe set was done including all Enterococcus type strains, and a selection of other representatives of the gram-positive bacteria with a low genomic DNA G+C content. The optimized probe set was used to analyze enriched drinking water samples as well as original samples from waste water treatment plants.     

Oligonucleotide microarray for identification of Enterococcus species

For detection of most members of the Enterococcaceae, the specificity of a novel oligonucleotide microarray (ECC-PhyloChip) consisting of 41 hierarchically nested 16S or 23S rRNA gene-targeted probes was evaluated with 23 pure cultures (including 19 Enterococcus species). Target nucleic acids were prepared by PCR amplification of a 4.5-kb DNA fragment containing large parts of the 16S and 23S rRNA genes and were subsequently labeled fluorescently by random priming. Each tested member of the Enterococcaceae was correctly identified on the basis of its unique microarray hybridization pattern. The evaluated ECC-PhyloChip was successfully applied for identification of Enterococcus faecium and Enterococcus faecalis in artificially contaminated milk samples demonstrating the utility of the ECC-PhyloChip for parallel identification and differentiation of Enterococcus species in food samples.     

Identification and differentiation of Staphylococcus carnosus and Staphylococcus simulans by species-specific PCR assays of sodA genes

The aim of this study was to design species-specific PCR assays for rapid and reliable identification and differentiation of Staphylococcus (S.) carnosus and S. simulans strains. Two different sets of primers, targeting the manganese-dependent superoxide dismutase (sodA) gene of S. carnosus and S. simulans, respectively, were designed. Species-specificity of both sets of primers was evaluated by using 93 strains, representing 26 different species of the genus Staphylococcus, 3 species of the genus Kocuria (K.), 1 species of the genus Micrococcus (Mic.) and 1 species of the genus Macrococcus (Mac.) as reference. By using primers simF and simR the expected PCR fragment was obtained only when purified DNA from S. simulans strains was used. Amplification performed by using primers carF and carR produced a PCR fragment of the expected length, when DNA from strains of S. carnosus and S. condimenti were used as template. Nevertheless, DraI digestion of the carF/carR PCR fragment allowed a clear differentiation of strains of these two species. Species-specific PCR assays designed during this study, overcoming many of the limitations of the traditional identification procedures, can be considered a valid strategy for detection and identification of S. carnosus and S. simulans strains. The rapidity (about 4h from DNA isolation to results), the reliability and low cost of the PCR procedures established suggests that the methods may be profitably applied for specific detection and identification of S. carnosus, S. condimenti and S. simulans strains in starter cultures and meat products.     

16S-23S rDNA intergenic spacer and 23S rDNA of anaerobic ammonium-oxidizing bacteria: implications for phylogeny and in situ detection

Recently, anaerobic ammonium-oxidizing bacteria (AAOB) were identified by comparative 16S rDNA sequence analysis as a novel, deep-branching lineage within the Planctomycetales . This lineage consists currently of only two, not yet culturable bacteria which have been provisionally described as Candidatus 'Brocadia anammoxidans' and Candidatus 'Kuenenia stuttgartiensis'. In this study, a large fragment of the rDNA operon, including the 16S rDNA, the intergenic spacer region (ISR) and approximately 2 000 bases of the 23S rDNA, was polymerase chain reaction (PCR) amplified, cloned and sequenced from both AAOB. The retrieved 16S rDNA sequences of both species contain an insertion at helix 9 with a previously overlooked pronounced secondary structure (new subhelices 9a and 9b). This insertion, which is absent in all other known prokaryotes, is detectable by fluorescence in situ hybridization (FISH) and thus present in the mature 16S rRNA. In contrast with the genera Pirellula , Planctomyces and Gemmata that possess unlinked 16S and 23S rRNA genes, both AAOB have the respective genes linked together by an ISR of approximately 450 bp in length. Phylogenetic analysis of the obtained 23S rRNA-genes confirmed the deep branching of the AAOB within the Planctomycetales and allowed the design of additional specific FISH probes. Remarkably, the ISR of the AAOB also could be successfully detected by FISH via simultaneous application of four monolabelled oligonucleotide probes. Quantitative FISH experiments with cells of Candidatus 'Brocadia anammoxidans' that were inhibited by exposure to oxygen for different time periods demonstrated that the concentration of transcribed ISR reflected the activity of the cells more accurately than the 16S or 23S rRNA concentration. Thus the developed ISR probes might become useful tools for in situ monitoring of the activity of AAOB in their natural environment.     

The development of specific rRNA-derived oligonucleotide probes for Haemophilus ducreyi, the causative agent of chancroid.

Part of a ribosomal ribonucleic acid (rRNA) cistron of Haemophilus ducreyi was enzymically amplified using conserved primers within the rRNA molecules, cloned in a plasmid vector, and sequenced. From the nucleotide sequence, eight oligonucleotides complementary to different regions in the 16S and 23S rRNA molecules were selected, chemically synthesized, and used as hybridization probes. Hybridization experiments with at least 41 H. ducreyi strains and 13 or 14 non-H. ducreyi strains revealed that all eight oligonucleotide probes were highly reliable and completely specific for H. ducreyi strains. Comparisons of 16S rRNA sequences confirm that H. ducreyi is a member of the Pasteurellaceae though not closely related to other species in this family.     

A species-specific probe and a PCR assay for the marine bacterium,Pseudomonas stutzeri strain Zobell

The cloning, sequencing, and analysis of a Pseudomonas stutzeri Zobell 23S rRNA gene is described. Three variable regions were identified, and oligonucleotides homologous to portions of these regions were synthesized. The oligonucleotides were used as probes to screen DNA from various cultured bacteria to identify a species-specific probe. All probes were found to hybridize strongly with P. stutzeri Zobell DNA under stringent conditions and did not hybridize with other Pseudomonas species. One probe showed slight cross-reactivity with DNA from four other bacteria under the hybridization conditions used. Finally, PCR conditions were optimized for detection of P. stutzeri Zobell in mixed culture with a detection limit of 400 cells. The assay detected P. stutzeri Zobell rDNA in coastal seawater samples sampled over a 20-month period. In the future, these probes could be used to quantify the 23S rRNA and rDNA from P. stutzeri Zobell in mixed culture and in environmental samples.