Quantification of Uncultured Ruminococcus obeum-Like Bacteria in Human Fecal Samples by Fluorescent In Situ Hybridization and Flow Cytometry Using 16S rRNA-Targeted Probes

A 16S rRNA-targeted probe was designed and validated in order to quantify the number of uncultured Ruminococcus obeum-like bacteria by fluorescent in situ hybridization (FISH). These bacteria have frequently been found in 16S ribosomal DNA clone libraries prepared from bacterial communities in the human intestine. Thirty-two reference strains from the human intestine, including a phylogenetically related strain and strains of some other Ruminococcus species, were used as negative controls and did not hybridize with the new probe. Microscopic and flow cytometric analyses revealed that a group of morphologically similar bacteria in feces did hybridize with this probe. Moreover, it was found that all hybridizing cells also hybridized with a probe specific for the Clostridium coccoides-Eubacterium rectale group, a group that includes the uncultured R. obeum-like bacteria. Quantification of the uncultured R. obeum-like bacteria and the C. coccoides-E. rectale group by flow cytometry and microscopy revealed that these groups comprised approximately 2.5 and 16% of the total community in fecal samples, respectively. The uncultured R. obeum-like bacteria comprise about 16% of the C. coccoides-E. rectale group. These results indicate that the uncultured R. obeum-like bacteria are numerically important in human feces. Statistical analysis revealed no significant difference between the microscopic and flow cytometric counts and the different feces sampling times, while a significant host-specific effect on the counts was observed. Our data demonstrate that the combination of FISH and flow cytometry is a useful approach for studying the ecology of uncultured bacteria in the human gastrointestinal tract.     

Variations of Bacterial Populations in Human Feces Measured by Fluorescent In Situ Hybridization with Group-Specific 16S rRNA-Targeted Oligonucleotide Probes

Six 16S rRNA-targeted oligonucleotide probes were designed, validated, and used to quantify predominant groups of anaerobic bacteria in human fecal samples. A set of two probes was specific for species of the Bacteroides fragilis group and the species Bacteroides distasonis. Two others were designed to detect species of the Clostridium histolyticum and the Clostridium lituseburense groups. Another probe was designed for the genera Streptococcus and Lactococcus, and the final probe was designed for the species of the Clostridium coccoides-Eubacterium rectale group. The temperature of dissociation of each of the probes was determined. The specificities of the probes for a collection of target and reference organisms were tested by dot blot hybridization and fluorescent in situ hybridization (FISH). The new probes were used in initial FISH experiments to enumerate human fecal bacteria. The combination of the two Bacteroides-specific probes detected a mean of 5.4 × 10¹⁰ cells per g (dry weight) of feces; the Clostridium coccoides-Eubacterium rectale group-specific probe detected a mean of 7.2 × 10¹⁰ cells per g (dry weight) of feces. The Clostridium histolyticum, Clostridium lituseburense, and Streptococcus-Lactococcus group-specific probes detected only numbers of cells ranging from 1 × 10⁷ to 7 × 10⁸ per g (dry weight) of feces. Three of the newly designed probes and three additional probes were used in further FISH experiments to study the fecal flora composition of nine volunteers over a period of 8 months. The combination of probes was able to detect at least two-thirds of the fecal flora. The normal biological variations within the fecal populations of the volunteers were determined and indicated that these variations should be considered when evaluating the effects of agents modulating the flora.     

Specific Detection of Dehalococcoides Species by Fluorescence In Situ Hybridization with 16S rRNA-Targeted Oligonucleotide Probes

Dehalococcoides ethenogenes is the only known cultivated organism capable of complete dehalogenation of tetrachloroethene (PCE) to ethene. The prevalence of Dehalococcoides species in the environment and their association with complete dehalogenation of chloroethenes suggest that they play an important role in natural attenuation of chloroethenes and are promising candidates for engineered bioremediation of these contaminants. Both natural attenuation and bioremediation require reliable and sensitive methods to monitor the presence, distribution, and fate of the organisms of interest. Here we report the development of 16S rRNA-targeted oligonucleotide probes for Dehalococcoides species. The two designed probes together encompass 28 sequences of 16S rRNA genes retrieved from the public database. Except D. ethenogenes and CBDB1, all the others are environmental clones obtained from sites contaminated with chlorinated ethenes. They are all closely related and form a unique cluster of Dehalococcoides species. In situ hybridization of probe Dhe1259t with D. ethenogenes strain 195 and two enrichment cultures demonstrated the applicability of the probe to monitoring the abundance of active Dehalococcoides species in these enrichment samples.     

Automated Design of Probes for rRNA-Targeted Fluorescence In Situ Hybridization Reveals the Advantages of Using Dual Probes for Accurate Identification

Fluorescence in situ hybridization (FISH) is a common technique for identifying cells in their natural environment and is often used to complement next-generation sequencing approaches as an integral part of the full-cycle rRNA approach. A major challenge in FISH is the design of oligonucleotide probes with high sensitivity and specificity to their target group. The rapidly expanding number of rRNA sequences has increased awareness of the number of potential nontargets for every FISH probe, making the design of new FISH probes challenging using traditional methods. In this study, we conducted a systematic analysis of published probes that revealed that many have insufficient coverage or specificity for their intended target group. Therefore, we developed an improved thermodynamic model of FISH that can be applied at any taxonomic level, used the model to systematically design probes for all recognized genera of bacteria and archaea, and identified potential cross-hybridizations for the selected probes. This analysis resulted in high-specificity probes for 35.6% of the genera when a single probe was used in the absence of competitor probes and for 60.9% when up to two competitor probes were used. Requiring the hybridization of two independent probes for positive identification further increased specificity. In this case, we could design highly specific probe sets for up to 68.5% of the genera without the use of competitor probes and 87.7% when up to two competitor probes were used. The probes designed in this study, as well as tools for designing new probes, are available online (http://DECIPHER.cee.wisc.edu).     

Detection of Ralstonia solanacearum, Which Causes Brown Rot of Potato, by Fluorescent In Situ Hybridization with 23S rRNA-Targeted Probes

During the past few years, Ralstonia (Pseudomonas) solanacearum race 3, biovar 2, was repeatedly found in potatoes in Western Europe. To detect this bacterium in potato tissue samples, we developed a method based on fluorescent in situ hybridization (FISH). The nearly complete genes encoding 23S rRNA of five R. solanacearum strains and one Ralstonia pickettii strain were PCR amplified, sequenced, and analyzed by sequence alignment. This resulted in the construction of an unrooted tree and supported previous conclusions based on 16S rRNA sequence comparison in which R. solanacearum strains are subdivided into two clusters. Based on the alignments, two specific probes, RSOLA and RSOLB, were designed for R. solanacearum and the closely related Ralstonia syzygii and blood disease bacterium. The specificity of the probes was demonstrated by dot blot hybridization with RNA extracted from 88 bacterial strains. Probe RSOLB was successfully applied in FISH detection with pure cultures and potato tissue samples, showing a strong fluorescent signal. Unexpectedly, probe RSOLA gave a less intense signal with target cells. Potato samples are currently screened by indirect immunofluorescence (IIF). By simultaneously applying IIF and the developed specific FISH, two independent targets for identification of R. solanacearum are combined, resulting in a rapid (1-day), accurate identification of the undesired pathogen. The significance of the method was validated by detecting the pathogen in soil and water samples and root tissue of the weed host Solanum dulcamara (bittersweet) in contaminated areas.     

Improved Fluorescent In Situ Hybridization Method for Detection of Bacteria from Activated Sludge and River Water by Using DNA Molecular Beacons and Flow Cytometry

Fluorescent in situ hybridization (FISH) remains a key technique in microbial ecology. Molecular beacons (MBs) are self-reporting probes that have potential advantages over linear probes for FISH. MB-FISH strategies have been described using both DNA-based and peptide nucleic acid (PNA)-based approaches. Although recent reports have suggested that PNA MBs are superior, DNA MBs have some advantages, most notably cost. The data presented here demonstrate that DNA MBs are suitable for at least some FISH applications in complex samples, providing superior discriminatory power compared to that of corresponding linear DNA-FISH probes. The use of DNA MBs for flow cytometric detection of Pseudomonas putida resulted in approximately double the signal-to-noise ratio of standard linear DNA probes when using laboratory-grown cultures and yielded improved discrimination of target cells in spiked environmental samples, without a need for separate washing steps. DNA MBs were also effective for the detection and cell sorting of both spiked and indigenous P. putida from activated sludge and river water samples. The use of DNA MB-FISH presents another increase in sensitivity, allowing the detection of bacteria in environmental samples without the expense of PNA MBs or multilaser flow cytometry.     

Grazing of a Tetrahymena sp. on Adhered Bacteria in Percolated Columns Monitored by In Situ Hybridization with Fluorescent Oligonucleotide Probes

Predation of attached Pseudomonas putida mt2 by the small ciliate Tetrahymena sp. was investigated with a percolated column system. Grazing rates were examined under static and dynamic conditions and were compared to grazing rates in batch systems containing suspended prey. The prey densities were 2 × 10⁸ bacteria per ml of pore space and 2 × 10⁸ bacteria per ml of suspension, respectively. Postingestion in situ hybridization of bacteria with fluorescent oligonucleotide probes was used to quantify ingestion. During 30 min, a grazing rate of 1,382 ± 1,029 bacteria individual⁻¹ h⁻¹ was obtained with suspended prey; this was twice the grazing rate observed with attached bacteria under static conditions. Continuous percolation at a flow rate of 73 cm h⁻¹ further decreased the grazing rate to about 25% of the grazing rate observed with suspended prey. A considerable proportion of the protozoans fed on neither suspended bacteria nor attached bacteria. The transport of ciliates through the columns was monitored at the same time that predation was monitored. Less than 20% of the protozoans passed through the columns without being retained. Most of these organisms ingested no bacteria, whereas the retained protozoans grazed more efficiently. Retardation of ciliate transport was greater in columns containing attached bacteria than in bacterium-free columns. We propose that the correlation between grazing activity and retardation of transport is a consequence of the interaction between active predators and attached bacteria.     

Visualization and Enumeration of Marine Planktonic Archaea and Bacteria by Using Polyribonucleotide Probes and Fluorescent In Situ Hybridization

Fluorescent in situ hybridization (FISH) using rRNA-specific oligonucleotide probes has emerged as a popular technique for identifying individual microbial cells. In natural samples, however, the signal derived from fluor-labeled oligonucleotide probes often is undetectable above background fluorescence in many cells. To circumvent this difficulty, we applied fluorochrome-labeled polyribonucleotide probes to identify and enumerate marine planktonic archaea and bacteria. The approach greatly enhanced the sensitivity and applicability of FISH with seawater samples, allowing confident identification and enumeration of planktonic cells to ocean depths of 3,400 m. Quantitative whole-cell hybridization experiments using these probes accounted for 90 to 100% of the total 4′,6-diamidino-2-phenylindole (DAPI)-stained cells in most samples. As predicted in a previous study (R. Massana, A. E. Murray, C. M. Preston, and E. F. DeLong, Appl. Environ. Microbiol. 63:50–56, 1997), group I and II marine archaea predominate in different zones in the water column, with maximal cell densities of 10⁵/ml. The high cell densities of archaea, extending from surface waters to abyssal depths, suggest that they represent a large and significant fraction of the total picoplankton biomass in coastal ocean waters. The data also show that the vast majority of planktonic prokaryotes contain significant numbers of ribosomes, rendering them easily detectable with polyribonucleotide probes. These results imply that the majority of planktonic cells visualized by DAPI do not represent lysed cells or “ghosts,” as was suggested in a previous report.     

In Situ Localization of Azospirillum brasilense in the Rhizosphere of Wheat with Fluorescently Labeled, rRNA-Targeted Oligonucleotide Probes and Scanning Confocal Laser Microscopy

The colonization of wheat roots by Azospirillum brasilense was used as a model system to evaluate the utility of whole-cell hybridization with fluorescently labeled, rRNA-targeted oligonucleotide probes for the in situ monitoring of rhizosphere microbial communities. Root samples of agar- or soil-grown 10- and 30-day-old wheat seedlings inoculated with different strains of A. brasilense were hybridized with a species-specific probe for A. brasilense, a probe hybridizing to alpha subclass proteobacteria, and a probe specific for the domain Bacteria to identify and localize the target bacteria. After hybridization, about 10 to 25% of the rhizosphere bacteria as visualized with 4(prm1),6-diamidino-2-phenylindole (DAPI) gave sufficient fluorescence signals to be detected with rRNA-targeted probes. Scanning confocal laser microscopy was used to overcome disturbing effects arising from autofluorescence of the object or narrow depth of focus in thick specimens. This technique also allowed high-resolution analysis of the spatial distribution of bacteria in the rhizosphere. Occurrence of cells of A. brasilense Sp7 and Wa3 was restricted to the rhizosphere soil, mainly to the root hair zone. C-forms of A. brasilense were demonstrated to be physiologically active forms in the rhizosphere. Strain Sp245 also was found repeatedly at high density in the interior of root hair cells. In general, the combination of fluorescently labeled oligonucleotide probes and scanning confocal laser microscopy provided a very suitable strategy for detailed studies of rhizosphere microbial ecology.     

In Situ Hybridization of Prochlorococcus and Synechococcus (Marine Cyanobacteria) spp. with rRNA-Targeted Peptide Nucleic Acid Probes

A simple method for whole-cell hybridization using fluorescently labeled rRNA-targeted peptide nucleic acid (PNA) probes was developed for use in marine cyanobacterial picoplankton. In contrast to established protocols, this method is capable of detecting rRNA in Prochlorococcus, the most abundant unicellular marine cyanobacterium. Because the method avoids the use of alcohol fixation, the chlorophyll content of Prochlorococcus cells is preserved, facilitating the identification of these cells in natural samples. PNA probe-conferred fluorescence was measured flow cytometrically and was always significantly higher than that of the negative control probe, with positive/negative ratio varying between 4 and 10, depending on strain and culture growth conditions. Prochlorococcus cells from open ocean samples were detectable with this method. RNase treatment reduced probe-conferred fluorescence to background levels, demonstrating that this signal was in fact related to the presence of rRNA. In another marine cyanobacterium, Synechococcus, in which both PNA and oligonucleotide probes can be used in whole-cell hybridizations, the magnitude of fluorescence from the former was fivefold higher than that from the latter, although the positive/negative ratio was comparable for both probes. In Synechococcus cells growing at a range of growth rates (and thus having different rRNA concentrations per cell), the PNA- and oligonucleotide-derived signals were highly correlated (r = 0.99). The chemical nature of PNA, the sensitivity of PNA-RNA binding to single-base-pair mismatches, and the preservation of cellular integrity by this method suggest that it may be useful for phylogenetic probing of whole cells in the natural environment.     

Detection of Neuropeptide mRNA in Rat Brain by In Situ Hybridization with Radiolabeled Synthetic Oligonucleotide Probes

Abstract

Radiolabeled synthetic oligonucleotide probes were used for detection of somatostatin and vasopressin mRNA in rat brain by in situ hybridization.     

Design and Performance of a 16S rRNA-Targeted Oligonucleotide Probe for Detection of Members of the Genus Bdellovibrio by Fluorescence In Situ Hybridization

A 16S rRNA-targeted, Cy3-labeled oligonucleotide probe was designed to detect members of the genus Bdellovibrio by fluorescence in situ hybridization. Specific hybridization conditions were established; however, the detection of bdellovibrios in environmental samples required enrichment, confirming that Bdellovibrio spp. are not present in large numbers in the environment.     

Double Labeling of Oligonucleotide Probes for Fluorescence In Situ Hybridization (DOPE-FISH) Improves Signal Intensity and Increases rRNA Accessibility

Fluorescence in situ hybridization (FISH) with singly labeled rRNA-targeted oligonucleotide probes is widely applied for direct identification of microbes in the environment or in clinical specimens. Here we show that a replacement of singly labeled oligonucleotide probes with 5′-, 3′-doubly labeled probes at least doubles FISH signal intensity without causing specificity problems. Furthermore, Cy3-doubly labeled probes strongly increase in situ accessibility of rRNA target sites and thus provide more flexibility for probe design.Since its introduction almost 20 years ago (2, 7), the identification of microorganisms by fluorescence in situ hybridization (FISH) with singly labeled rRNA-targeted probes has found widespread application in environmental and medical microbiology (1, 16). Despite being a methodologically robust technique, standard FISH suffers from several limitations (26) that may prevent successful detection of target microorganisms. One of the most frequently reported FISH problems is a low signal intensity of the detected microbes.Dim signals can be caused by a low cellular concentration of the target molecules (16S rRNA or 23S rRNA), a feature typically found in microorganisms thriving in oligotrophic environments (19). In order to increase the sensitivity of FISH and make it suitable for the detection of microbes with a low ribosome content, several strategies have been developed (6, 13, 18, 19, 21, 25), of which catalyzed reporter deposition (CARD)-FISH has found the most widespread application. The CARD-FISH technique (18, 21) uses horseradish peroxidase (HRP)-labeled oligonucleotide probes and tyramide signal amplification and achieves a 26- to 41-fold-higher sensitivity than standard FISH (11). However, CARD-FISH is rather expensive, requires enzymatic pretreatment to allow the large horseradish peroxidase-labeled probes to penetrate the target cells (17), and causes a dramatically altered melting behavior of the probes (11).Another frequently encountered FISH problem is the low in situ accessibility of many regions of the 16S and 23S rRNA for singly labeled probes (9, 10). Probes targeting such regions, which comprise about one-third of the Escherichia coli 16S rRNA (10), confer signals which are very dim or even below the detection limit. In order to avoid the selection of poorly accessible target sites for FISH probe design, a consensus 16S rRNA accessibility map for prokaryotes has been established based on detailed accessibility maps of two bacterial model organisms and one archaeal model organism (3). Considering this consensus map during probe design is recommended, but it excludes many probes with useful specificities from FISH applications. Furthermore, accessibility of many 16S and 23S rRNA target sites varies between different microorganisms and thus cannot yet be reliably predicted in silico. Accessibility of target sites to probes can be improved by the following: (i) use of unlabeled helper probes (8), (ii) elongation of the hybridization time up to 96 h (30), (iii) elongation of the probes, resulting in an altered ΔG°_overall (30), or (iv) use of peptide nucleic acid probes (reference 26 and references therein). However, all these strategies have specific limitations. For example, the design of helper probes is often impossible for probes with broader specificities, the extension of the hybridization time might lead to unspecific probe or dye binding in complex samples, probe elongation is often not possible without narrowing its specificity, and previously published oligonucleotide probes cannot simply be converted into the expensive peptide nucleic acid probes without a dramatically changed specificity (26).In principle, using oligonucleotide probes labeled with multiple fluorescent dyes should provide a simple means to increase the FISH signal intensity. The first experiments with multilabeled oligonucleotides were performed briefly after the introduction of the technique in microbiology but resulted in a pronounced increase in unspecific staining of nontarget organisms and/or an unexpected decrease in the signal intensity of the target organism which was attributed to quenching effects (28). Inconsistent with these data, Spear et al. (23) reported a successful increase in the signal-to-noise ratio of FISH-detected fungal cells by application of a multilabeled 18S rRNA-targeted oligonucleotide probe. In this work, we systematically evaluated the effect of 5′- and 3′-doubly labeled oligonucleotide probes (which were not included in the previously published study of multilabeled probes [28]) on the FISH signal intensity of Gram-negative and Gram-positive cells and studied the influence of double labeling on the in situ accessibility of rRNA target sites.The double-labeling-of-oligonucleotide-probes (DOPE)-FISH approach was initially tested with four bacterial pure cultures. These included the gamma- and betaproteobacterial Gram-negative species Escherichia coli (DSM 498) and Burkholderia cepacia (DSM 7288), respectively. In addition, the two Gram-positive bacteria Bacillus subtilis (DSM 10) and Listeria monocytogenes (strain LO28) were used. E. coli, B. cepacia, and B. subtilis were grown according to the DSMZ instructions until they reached their stationary growth phase and were fixed with paraformaldehyde (E. coli and B. cepacia) or ethanol (B. subtilis) as described elsewhere (5). L. monocytogenes strain LO28 was grown on brain heart infusion for 5 h and fixed with ethanol as outlined previously (27). The oligonucleotide probes EUB338 (targeting the 16S rRNA of most but not all bacteria), NonEUB338 (a nonsense probe), GAM42a (targeting the 23S rRNA of many members of the Gammaproteobacteria), and five E. coli-targeted probes with a low 16S rRNA accessibility (10) were obtained as singly and doubly labeled derivatives from Thermo Hybaid (Interactiva Division, Ulm, Germany). More information about the applied probes can be found at probeBase (14) and in the publication by Fuchs et al. (10). FISH was performed by following the standard protocol (5) under the conditions recommended for each probe (14). If not stated otherwise, all hybridizations were carried out with identical hybridization (4 h) and washing times (10 min), respectively. Probe-conferred signal intensities were quantified using a confocal laser scanning microscope (CLSM) (LSM 510 Meta; Zeiss, Oberkochen, Germany) and the software program daime (4) by analyzing at least 1,000 single cells per experiment. For these measurements, individual cells were detected by image segmentation via edge detection.Regardless of the dye used (Cy3, Cy5, or FLUOS) and the respective target organism analyzed, hybridization with the doubly labeled probe EUB338 resulted in an increase in the FISH signal compared to the use of the singly labeled probe EUB338. For three of the four reference organisms, this increase was about 2-fold, while an even stronger signal amplification was observed for B. cepacia (Fig. ​(Fig.1A).1A). Consequently, the distance between the two dye molecules in 18-nucleotide probes labeled at both ends is sufficient to avoid quenching. This is in contrast to oligonucleotides which are multiply labeled at one end or within the probe (28). Hybridization of the four reference organisms with the singly and doubly labeled derivatives of probe GAM42a confirmed these results and demonstrated that double labeling does not increase the background fluorescence of nontarget microorganisms (Fig. ​(Fig.1B).1B). Consistent with these findings, hybridization of all reference organisms with a doubly labeled nonsense probe (with FLUOS, Cy3, and Cy5) resulted in signals below the detection limit of the CLSM if standard FISH settings were applied (data not shown). We also evaluated the influence of the hybridization time on the signal intensity achievable by DOPE-FISH by varying the hybridization time between 1 and 6 h. In these experiments, no significant difference in DOPE-FISH signal intensities of the Gram-positive and Gram-negative reference organisms were observed, indicating that the additional label of the DOPE-FISH probes does not dramatically influence the hybridization kinetics (data not shown).Open in a separate windowFIG. 1.(A) Effect of double labeling of the EUB338 probe on the FISH signal intensity of four reference organisms. For each organism, the signal intensity conferred by a doubly labeled EUB338 probe was normalized to the signal intensity obtained with the same probe as a singly labeled derivative. Hatched, light-gray, and dark-gray bars depict results with the Cy3-, Cy5- and FLUOS-labeled probe EUB338, respectively. (B) Effect of double labeling of the probe Gam42a (in the presence of the unlabeled competitor probe Bet42a, specific for most members of the Betaproteobacteria [14]) on the FISH signal intensities of four reference organisms. For each organism, the signal intensity conferred by the doubly labeled probe Gam42a was normalized to the signal intensity obtained for E. coli with the same probe as a singly labeled derivative. Hatched, light-gray, and dark-gray bars depict results with the Cy3-, Cy5-, and FLUOS-labeled probe, respectively. The weak unspecific signals observed with some DOPE-FISH probes for B. subtilis are also detectable at comparable intensities with singly labeled probes (data not shown). (C) Cy3-doubly labeled but not FLUOS-doubly labeled probes improve in situ accessibility of E. coli 16S rRNA target sites. E. coli was hybridized with five probes representing brightness classes V and VI (3). FISH signals were recorded for Cy3-singly and -doubly labeled probes and normalized to the FISH signal obtained for E. coli with the singly labeled probe EUB338. Light-gray and dark-gray bars depict results with Cy3-singly and doubly labeled probes, respectively. FLUOS-singly and -doubly labeled probes showed no signal. For all panels, all experiments were performed in triplicate. Error bars indicate the standard deviation. ND, not detectable.Although DOPE-FISH worked well with all tested reference organisms, it should be noted that the Gram-positive species L. monocytogenes showed a signal amplification only if it was treated with lysozyme (according to Wagner et al. [27]) prior to the application of the doubly labeled probe. Without this enzymatic pretreatment, application of the singly labeled probe EUB338 resulted in a stronger signal than was seen with the doubly labeled probe EUB338, indicating that for some Gram-positive microorganisms with dense cell walls, double labeling impairs probe penetration. The observation that lysozyme treatment of L. monocytogenes also enhanced the probe-conferred signal of the singly labeled EUB338 probe confirmed that the cell wall of this organism after ethanol fixation is also not freely permeable to singly labeled probes (27; also data not shown). Enzymatic pretreatment of fixed microbial cells is also routinely applied for successful application of CARD-FISH, but since this method uses peroxidase-labeled probes which are much larger than DOPE-FISH probes, such treatments are also used for Gram-negative bacteria (11). Although many microorganisms are detectable by CARD-FISH with enhanced signal intensities, appropriate pretreatment protocols are not yet available for all microbes. For example, the sheathed filamentous methane oxidizer Crenothrix polyspora can easily be detected by standard FISH (24), but only very few cells within the filaments show a signal after CARD-FISH even if harsh permeabilization pretreatments are applied (see Fig. S1A in the supplemental material), a phenomenon known for sheathed microorganisms (12). In contrast, detection of all Crenothrix cells with more than 2-fold-increased signal intensity is readily possible by DOPE-FISH (see Fig. S1B).Prior research has demonstrated that probe labeling with horseradish peroxidase for CARD-FISH dramatically alters the melting behavior of oligonucleotide probes (11). Therefore, we recorded melting curves for Cy3-, Cy5-, and FLUOS-singly and -doubly labeled probes EUB338 and Gam42a by applying increasingly stringent conditions in the hybridization and wash steps (5). Interestingly, these experiments showed that the FLUOS singly labeled probes formed less-stable duplexes with their target sequences than the respective Cy3- and Cy5-labeled probes (Fig. ​(Fig.2).2). This effect, which is consistent with recent data on the stabilizing effect of various fluorophores on model probe-target duplexes (15), indicates that FLUOS-labeled FISH probes are generally applied under more-stringent conditions than Cy3- or Cy5-labeled probes. Cy3- and Cy5-doubly labeled probes displayed with their target organisms probe dissociation profiles similar to those of the respective FLUOS singly labeled probes, demonstrating that Cy3 or Cy5 double labeling does not further stabilize but rather moderately weakens the probe-target hybrid. Consistent with these findings, doubly FLUOS-labeled probes showed the lowest T_m (Fig. ​(Fig.2).2). Importantly, double labeling of probe GAM42a did not adversely affect mismatch discrimination, as shown by its dissociation profiles if in situ hybridizations were performed at various stringencies with B. cepacia containing a single mismatch in the probe target site of its 23S rRNA. (Fig. ​(Fig.2B).2B). These results indicate that the specificities of DOPE-FISH probes can be regarded as identical to those of standard singly labeled FISH probes.Open in a separate windowFIG. 2.Comparison of probe dissociation profiles of singly and doubly labeled probes. For each profile, the microscopic settings were adjusted for the lowest formamide concentration and subsequently kept constant. Dashed and solid lines represent sigmoid fittings for singly and doubly labeled probes, respectively. (A) Dissociation profiles of the singly and doubly labeled probe Gam42a with E. coli as the target organism. Empty circles, squares, and triangles represent data obtained with the Cy3-, Cy5-, and FLUOS-singly labeled probe GAM42a, respectively. Filled circles, squares, and triangles depict the data measured for the Cy3-, Cy5-, and FLUOS-doubly labeled probe GAM42a, respectively. (B) Dissociation profiles of the singly and doubly labeled probe Gam42a with B. cepacia as a nontarget organism having a single mismatch to probe GAM42a. Empty circles, squares, and triangles represent data obtained with the Cy3-, Cy5-, and FLUOS-singly labeled probe GAM42a, respectively. Filled circles, squares, and triangles depict the data measured for the Cy3-, Cy5-, and FLUOS-doubly labeled probe GAM42a, respectively. The melting curves for FLUOS-singly labeled and Cy5-doubly labeled probes are almost identical and thus overlap. In the presence of the unlabeled probe Bet42a as a competitor, no probe-conferred signal was recordable for both singly and doubly labeled GAM42a probes. (C) Dissociation profiles of the singly and doubly labeled probe EUB338 with E. coli as the target organism. Empty circles, squares, and triangles represent data obtained with the Cy3-, Cy5-, and FLUOS-singly labeled probe EUB338, respectively. Filled circles, squares, and triangles depict the data measured for the Cy3-, Cy5-, and FLUOS-doubly labeled probe EUB338, respectively. For all panels, error bars are not shown since they were always smaller than the symbols.In order to analyze whether the in situ accessibility of rRNA target sites to doubly labeled probes differs from that to those labeled with only one dye, we tested five probes targeting E. coli 16S rRNA. These probes were described as yielding only very dim signals with standard FISH as a consequence of limited target site accessibility and were thus assigned to the lowest brightness classes, V or VI (3, 10). Consistently, standard FISH with these five singly labeled probes (Cy3 and FLUOS) gave no or very weak signals (Fig. ​(Fig.1C).1C). Unexpectedly, however, Cy3-doubly labeled derivatives of these probes produced much brighter signals (Fig. ​(Fig.1C),1C), and for some of the probes (Eco468 and Eco1310), the DOPE-FISH signal intensity was higher than that measured for the Cy3-singly labeled probe EUB338 (Fig. ​(Fig.1C).1C). One could speculate that a Cy3 label at the 3′ end and not the double labeling might be responsible for the improved accessibility of rRNA target sites for Cy3 DOPE-FISH probes. However, since selected probes (Eco262, Eco468, and Eco1310) labeled with a single Cy3 molecule at the 3′ end did not result in increased fluorescence, this hypothesis can be rejected (data not shown). Interestingly, FLUOS-labeled DOPE-FISH probes did not show increased fluorescence, strongly indicating that the improved accessibility of Cy3 DOPE-FISH probes depends on the chemical structure of the fluorophore. This is consistent with the observation that Cy5 double labeling of the five E. coli probes also resulted in improved probe accessibilities (data not shown). While Cy3 and Cy5 double labeling decreases the probe-target duplex stability (Fig. ​(Fig.2),2), it apparently helps to resolve secondary or tertiary structures responsible for poor in situ accessibility of rRNA target sites. It is tempting to speculate that binding of Cy3 or Cy5 to double-stranded rRNA regions, analogous to the previously reported intercalation of certain cyanine class dyes in DNA (29) or other modes of nucleic acid binding by cyanine dyes (15), contributes to this phenomenon.The improved accessibility of rRNA target sites for Cy3 DOPE-FISH probes offers more flexibility for probe design because it enables the use of probes with excellent specificity but low standard FISH signal intensity for the successful in situ detection of microbes. This advantage of DOPE-FISH is nicely demonstrated by the probe Ntspa175 (5′-GAC CAG GAG CCG TAT GCG-3′), which targets the 16S rRNA (GenBank accession no. {"type":"entrez-nucleotide","attrs":{"text":"GU229885","term_id":"281398158","term_text":"GU229885"}}GU229885) of an uncultured nitrite oxidizer of the genus Nitrospira thriving in activated sludge. At 25% formamide in the standard FISH hybridization buffer (5), this probe is highly specific as demonstrated by Clone-FISH (22) using another activated sludge-derived 16S rRNA Nitrospira-like sequence with a single mismatch to probe Ntspa175 as a nontarget control (data not shown). Standard FISH of activated sludge with the Cy3-labeled probe Ntsp175, which targets the 175-to-193 region in the 16S rRNA, resulted in the detection of Nitrospira microcolonies with very variable FISH signal intensities. A considerable number of stained microcolonies had extremely dim FISH signals, indicating that these cells had a ribosome content too low to be reliably detectable by a standard FISH probe of a low brightness class. DOPE-FISH of the same sample with the Cy3-doubly labeled probe Ntspa175 led to a pronounced increase in signal intensity of the target cells (see Fig. S2 in the supplemental material) without causing increased background fluorescence if standard confocal-microscope settings were applied. In accordance with this observation, the relative biovolume-abundance of the detectable Ntspa175-stained population compared to the biovolume of those cells labeled by the Nitrospira genus-specific probe Ntspa662 (14) in the activated sludge increased by a factor of 1.81 ± 0.1 if a doubly labeled Ntspa175 probe was used (measurements made by the software package daime using confocal-microscope images as described previously [4]).In summary, DOPE-FISH with commercially available doubly labeled oligonucleotide probes is a straightforward modification of the standard FISH procedure which increases the signal intensity of standard FISH probes by at least a factor of 2 without causing specificity problems. Importantly, the influence of DOPE-FISH on the dissociation profile of probes is not larger than that caused by a dye switch from Cy3 to FLUOS if singly labeled probes are used for FISH. Thus, previously optimized hybridization and washing conditions for published probes can be applied for DOPE-FISH. Since DOPE-FISH unlocks previously inaccessible target sites on the rRNA, this new FISH approach offers more options for the design of specific probes, a task which becomes increasingly difficult with the rapid growth of rRNA databases (20).      

Simultaneous Quantification of Active Carbon- and Nitrogen-Fixing Communities and Estimation of Fixation Rates Using Fluorescence In Situ Hybridization and Flow Cytometry

Understanding the interconnectivity of oceanic carbon and nitrogen cycles, specifically carbon and nitrogen fixation, is essential in elucidating the fate and distribution of carbon in the ocean. Traditional techniques measure either organism abundance or biochemical rates. As such, measurements are performed on separate samples and on different time scales. Here, we developed a method to simultaneously quantify organisms while estimating rates of fixation across time and space for both carbon and nitrogen. Tyramide signal amplification fluorescence in situ hybridization (TSA-FISH) of mRNA for functionally specific oligonucleotide probes for rbcL (ribulose-1,5-bisphosphate carboxylase/oxygenase; carbon fixation) and nifH (nitrogenase; nitrogen fixation) was combined with flow cytometry to measure abundance and estimate activity. Cultured samples representing a diversity of phytoplankton (cyanobacteria, coccolithophores, chlorophytes, diatoms, and dinoflagellates), as well as environmental samples from the open ocean (Gulf of Mexico, USA, and southeastern Indian Ocean, Australia) and an estuary (Galveston Bay, Texas, USA), were successfully hybridized. Strong correlations between positively tagged community abundance and ¹⁴C/¹⁵N measurements are presented. We propose that these methods can be used to estimate carbon and nitrogen fixation in environmental communities. The utilization of mRNA TSA-FISH to detect multiple active microbial functions within the same sample will offer increased understanding of important biogeochemical cycles in the ocean.     

Detection of Bacteria in Phagocyte-Smears from Septicemia-Suspected Blood by In Situ Hybridization Using Biotinylated Probes

We report herein the detection of intracellular bacteria in phagocyte-smears obtained from septicemia-suspected blood samples by in situ hybridization. This was obtained by using nick-translated biotin-11-dUTP-labeled DNA probes and streptavidin-alkaline phosphatase conjugates for visualization of the hybridized signals. The probes were made from random genomic DNA clones of bacteria which are frequently the causative agents of bacteremia, such as Staphylococcus spp., Pseudomonas aeruginosa, Enterococcus faecalis, Escherichia coli, Klebsiella spp. and Enterobacter spp. When our in situ hybridization method was compared with conventional culture protocols for the ability to detect bacteria from the blood of patients suspected of having septicemia, 30 positive results were obtained in 50 specimens by in situ hybridization methods. In contrast, only 7 positive results were obtained by blood cultures. Thus, even if bacteria cannot be detected by conventional blood cultures and histology, our in situ hybridization method allows for direct observation of bacterial foci in circulating phagocytes and identification of the bacteria. Our investigations suggest that in septicemia, circulating polymorphonuclear neutrophils carry some surviving bacteria as well as metabolized bacterial DNA and RNA for a considerable period of time. Thus, our in situ hybridization method using the phagocyte-smears have diagnostic value for detecting most bacteria which cause septicemia.     

The Microscopic Examination of Phytophthora cinnamomi in Plant Tissues Using Fluorescent In Situ Hybridization

The microscopic examination of Phytophthora cinnamomi in plant tissues is often difficult as structures such as hyphae, chlamydospores and oospores are frequently indistinguishable from those of other fungi and oomycetes, with histological stains not enabling species differentiation. This lack of staining specificity makes the localization of P. cinnamomi hyphae and reproductive structures within plant tissues difficult, especially in woody tissues. This study demonstrates that with the use of a species‐specific fluorescently labelled DNA probe, P. cinnamomi can be specifically detected and visualized directly using fluorescent in situ hybridization (FISH) without damage to plant or pathogen cell integrity or the need for subculturing. This approach provides a new application for FISH with potential use in the detailed study of plant–pathogen interactions in plants.     

Identification and Localization of Frankia Strains in Alnus Nodules by In Situ Hybridization of nif H mRNA with Strain-Specific Oligonucleotide Probes

In situ hybridization of Frankia mRNA with specific probes wasused to localize the strains Arl3 and AcoN24d in Alnus nodulesobtained after inoculation with one or both strains. The probesconsisted of 18-mer oligonucleotides, complementary to strain-specificsequences located within the nif H gene. Sections of nodulesinoculated with only one strain revealed a specific hybridizationbetween the probe and the corresponding Frankia strain mRNA.In sections of dually-inoculated nodules the presence of thestrain AcoN24d in the nodule was clearly shown whereas thoseof the strain Arl3 could not be detected. This suggests thatthe strain Arl3 is less infective than the strain AcoN24d andis not present within the nodule. Key words: Nitrogen fixation, actinorhizae, autoradiography, histochemistry     

Rapid Detection, Identification, and Enumeration of Escherichia coli Cells in Municipal Water by Chemiluminescent In Situ Hybridization

A new chemiluminescent in situ hybridization (CISH) method provides simultaneous detection, identification, and enumeration of culturable Escherichia coli cells in 100 ml of municipal water within one working day. Following filtration and 5 h of growth on tryptic soy agar at 35°C, individual microcolonies of E. coli were detected directly on a 47-mm-diameter membrane filter using soybean peroxidase-labeled peptide nucleic acid (PNA) probes targeting a species-specific sequence in E. coli 16S rRNA. Within each microcolony, hybridized, peroxidase-labeled PNA probe and chemiluminescent substrate generated light which was subsequently captured on film. Thus, each spot of light represented one microcolony of E. coli. Following probe selection based on 16S ribosomal DNA (rDNA) sequence alignments and sample matrix interference, the sensitivity and specificity of the probe Eco16S07C were determined by dot hybridization to RNA of eight bacterial species. Only the rRNA of E. coli and Pseudomonas aeruginosa were detected by Eco16S07C with the latter mismatch hybridization being eliminated by a PNA blocker probe targeting P. aeruginosa 16S rRNA. The sensitivity and specificity for the detection of E. coli by PNA CISH were then determined using 8 E. coli strains and 17 other bacterial species, including closely related species. No bacterial strains other than E. coli and Shigella spp. were detected, which is in accordance with 16S rDNA sequence information. Furthermore, the enumeration of microcolonies of E. coli represented by spots of light correlated 92 to 95% with visible colonies following overnight incubation. PNA CISH employs traditional membrane filtration and culturing techniques while providing the added sensitivity and specificity of PNA probes in order to yield faster and more definitive results.     

Detection and Enumeration of Methanotrophs in Acidic Sphagnum Peat by 16S rRNA Fluorescence In Situ Hybridization, Including the Use of Newly Developed Oligonucleotide Probes for Methylocella palustris

Two 16S rRNA-targeted oligonucleotide probes, Mcell-1026 and Mcell-181, were developed for specific detection of the acidophilic methanotroph Methylocella palustris using fluorescence in situ hybridization (FISH). The fluorescence signal of probe Mcell-181 was enhanced by its combined application with the oligonucleotide helper probe H158. Mcell-1026 and Mcell-181, as well as 16S rRNA oligonucleotide probes with reported group specificity for either type I methanotrophs (probes M-84 and M-705) or the Methylosinus/Methylocystis group of type II methanotrophs (probes MA-221 and M-450), were used in FISH to determine the abundance of distinct methanotroph groups in a Sphagnum peat sample of pH 4.2. M. palustris was enumerated at greater than 10⁶ cells per g of peat (wet weight), while the detectable population size of type I methanotrophs was three orders of magnitude below the population level of M. palustris. The cell counts with probe MA-221 suggested that only 10⁴ type II methanotrophs per g of peat (wet weight) were present, while the use of probe M-450 revealed more than 10⁶ type II methanotroph cells per g of the same samples. This discrepancy was due to the fact that probe M-450 targets almost all currently known strains of Methylosinus and Methylocystis, whereas probe MA-221, originally described as group specific, does not detect a large proportion of Methylocystis strains. The total number of methanotrophic bacteria detected by FISH was 3.0 (±0.2) × 10⁶ cells per g (wet weight) of peat. This was about 0.8% of the total bacterial cell number. Thus, our study clearly suggests that M. palustris and a defined population of Methylocystis spp. were the predominant methanotrophs detectable by FISH in an acidic Sphagnum peat bog.