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.     

Enumeration of polysaccharide-degrading Bacteroides species in human feces by using species-specific DNA probes

DNA probes that are specific for each of five predominant species of human colonic Bacteroides (B. thetaiotaomicron, B. uniformis, B. distasonis, "Bacteroides group 3452-A", and B. ovatus) were used to detect and enumerate these species in fecal samples from two adult volunteers. These five species are capable of fermenting many of the complex polysaccharides that are thought to be sources of carbon and energy for bacteria in the colon. Estimates for the concentrations of some of these species in feces have not been previously available because of the difficulties in differentiating colonic Bacteroides spp. by conventional biochemical tests. Our results indicate that all the species except B. ovatus were present in high numbers (greater than 10(9)/g [dry weight]) in the feces of both volunteers. However, the concentrations of the more versatile polysaccharide-degrading species within this group of organisms (7.6 X 10(9) to 12.0 X 10(9)/g [dry weight] for B. thetaiotaomicron; 2.9 X 10(9) to 6.3 X 10(9)/g [dry weight] for "Bacteroides group 3452-A") did not differ significantly from the concentrations of less versatile polysaccharide-degrading species (1.2 X 10(10) to 2.0 X 10(10)/g [dry weight] for B. uniformis; 5.8 X 10(9) to 8.4 X 10(9)/g [dry weight] for B. distasonis). B. ovatus was not detectable by our method. Since our lower limit of detection is approximately 1 X 10(9) to 2 X 10(9)/g (dry weight) of feces, this is consistent with earlier estimates that indicated that the concentration of B. ovatus in feces is near or below this value.(ABSTRACT TRUNCATED AT 250 WORDS)     

Enumeration of polysaccharide-degrading Bacteroides species in human feces by using species-specific DNA probes.

DNA probes that are specific for each of five predominant species of human colonic Bacteroides (B. thetaiotaomicron, B. uniformis, B. distasonis, "Bacteroides group 3452-A", and B. ovatus) were used to detect and enumerate these species in fecal samples from two adult volunteers. These five species are capable of fermenting many of the complex polysaccharides that are thought to be sources of carbon and energy for bacteria in the colon. Estimates for the concentrations of some of these species in feces have not been previously available because of the difficulties in differentiating colonic Bacteroides spp. by conventional biochemical tests. Our results indicate that all the species except B. ovatus were present in high numbers (greater than 10(9)/g [dry weight]) in the feces of both volunteers. However, the concentrations of the more versatile polysaccharide-degrading species within this group of organisms (7.6 X 10(9) to 12.0 X 10(9)/g [dry weight] for B. thetaiotaomicron; 2.9 X 10(9) to 6.3 X 10(9)/g [dry weight] for "Bacteroides group 3452-A") did not differ significantly from the concentrations of less versatile polysaccharide-degrading species (1.2 X 10(10) to 2.0 X 10(10)/g [dry weight] for B. uniformis; 5.8 X 10(9) to 8.4 X 10(9)/g [dry weight] for B. distasonis). B. ovatus was not detectable by our method. Since our lower limit of detection is approximately 1 X 10(9) to 2 X 10(9)/g (dry weight) of feces, this is consistent with earlier estimates that indicated that the concentration of B. ovatus in feces is near or below this value.(ABSTRACT TRUNCATED AT 250 WORDS)     

Group-specific 16S rRNA targeted probes for the detection of type I and type II methanotrophs by fluorescence in situ hybridisation

The study of methane-oxidising bacteria (methanotrophs) is of special interest, because of their role in the natural reduction of methane emissions from many different sources. Therefore new probes were developed to detect specifically either type I (Methylococcaceae) or type II methanotrophs (Methylocystaceae). The probes have shown high specificity in fluorescence in situ hybridisations (FISH), as demonstrated by parallel hybridisation of target and reference strains as well as sequence data analysis. With these probes, methanotrophs were detected in soil and root samples from rice microcosms, demonstrating their applicability even in a complex environmental matrix.     

16S rRNA PCR鉴定脆弱类杆菌

目的:应用16SrRNA序列设计PCR引物鉴别脆弱类杆菌。方法:通过脆弱类杆菌16SrRNA序列特异性位点设计引物,对4株脆弱类杆菌及大肠杆菌、乳酸杆菌、嗜热链球菌等进行PCR扩增。应用琼脂糖电泳法对PCR扩增产物进行特异性检测。结果:脆弱类杆菌在176bp左右出现特异性条带,而其他细菌均未出现特异性条带。结论:通过16SrRNA序列中特异位点设计引物进行PCR,可特异性鉴定脆弱类杆菌。     

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.     

Assessment of Candida shehatae viability by flow cytometry and fluorescent probes

Quantification of different physiological states of Candida shehatae cells was performed by flow cytometry associated with two fluorescent probes. Propidium iodide and carboxyfluorescein diacetate acetoxymethyl ester fluorescent dyes were chosen based on data from the literature. A staining procedure, developed from the previous works was applied to the yeast. Then, the protocol was improved to fit with fermentation constraints such as no physiological interference between the staining procedure and the cells, shortest preparation time and small amounts of dyes. From this optimisation, propidium iodide was included in the sample at 8mg/L whereas carboxyfluorescein was first diluted in Pluronic? agent and used at 3mg/L, samples were incubated for 10min at 40°C. Repeatability and accuracy were evaluated to validate this flow cytometry procedure for viability determination.     

Development of Faecalibacterium 16S rRNA gene marker for identification of human faeces

Aims:  The focus of this study was to identify a bacterial 16S rRNA gene sequence, unique to microbiota in the human gut, for use in development of a dependable PCR assay to detect human faecal pollution in water.
Methods and Results:  Suppression subtractive hybridization (SSH) and bioinformatics were used to identify a genetic marker, within the 16S rRNA gene of Faecalibacterium , for the detection of human faeces. DNA sequencing analysis demonstrated that a majority (16) of 74 clones of the SSH library contained insertion sequences identified as Faecalibacterium 16S rRNA genes . Human faeces-specific sequences were derived and six PCR primer sets designed and tested against faecal DNA samples from human and nonhuman sources. One PCR primer set, HFB-F3 and HFB-R5, was exclusively associated with human faeces. These primers generated a human faeces-specific amplicon of 399 bp from 60·2% of human faecal samples and 100% of sewage samples.
Conclusions:  The subject Faecalibacterium marker is specific for sewage.
Significance and Impact of the Study:  This study represents the initial report of a Faecalibacterium marker for human faeces, which may prove useful for microbial source tracking.     

Clostridium difficile and Clostridium perfringens species detected in infant faecal microbiota using 16S rRNA targeted probes

Clostridium perfringens and Clostridium difficile are pathogenic clostridia potentially associated with gastrointestinal infections and allergy in infants. To enable the molecular detection and quantification of these species in the infant gut, two 16S rRNA oligonucleotide probes were developed: Cdif198 for C. difficile and Cperf191 for C. perfringens. We defined the probes in silico using the RDP sequence database. The probes were then validated using FISH combined with flow cytometry and a collection of target and non-target strains, and faecal samples inoculated with dilutions of C. difficile and C. perfringens strains. These new probes were used to assess the composition of the intestinal microbiota of 33 infants of 1.5 to 18.5 months of age, associated with a panel of 8 probes targeting the predominant faecal bacterial groups of humans. The probes designed allowed detection and quantification of the relative proportions of C. difficile (0.5+/-1.0%) and C. perfringens (2.1+/-2.3%) in the microbiota of infants.     

Quantifying bacterial population dynamics in compost using 16S rRNA gene probes

Composting provides a dynamic setting for studying ecological topics such as succession, competition, and community stability in a relatively short period of time. This study used hierarchical small sub-unit-based rRNA gene probes to quantify the change in the relative abundance of phylogenetic groups common to compost in laboratory scale reactors. Bacterial 16S rRNA gene targets accounted for only 37% of all small subunit (SSU) rRNA genes initially, but increased to a maximum of 83% of the total at 84 h. The sum of rRNA genes detected using probes specific to Pseudomonas and low-G+C Gram-positive rRNA genes represented between 16% and 87% of the total. The lack of hybridization to the taxon-specific probes was most pronounced between 36 h and 60 h, when the pH was between 4.6 and 4.8. During this period the relative abundance of taxon-specific gene targets accounted for only 17–33% of the total bacterial rRNA gene targets. Pseudomonas-type 16S rRNA genes were the most abundant of the groups measured until 72 h. Those genes had their highest relative abundance at 12 h (78% of bacterial rRNA genes; 30% of all rRNA genes), after which time their relative abundance began to decline as the temperature increased. Prior to 72 h, 16S rRNA genes from low-G+C Gram-positive bacteria (LGC-GPB) represented less than 7% of the bacterial rRNA genes. However, by 84 h the relative abundance of LGC-GPB and Bacillus rRNA genes had increased to 60% and 18% of the bacterial rRNA gene targets, respectively (50% and 15% of all rRNA genes, respectively).     

High-temperature fluorescent in situ hybridization for detecting Escherichia coli in seawater samples, using rRNA-targeted oligonucleotide probes and flow cytometry

Fluorescence in situ hybridization (FISH) is a widely used method to detect environmental microorganisms. The standard protocol is typically conducted at a temperature of 46 degrees C and a hybridization time of 2 or 3 h, using the fluorescence signal intensity as the sole parameter to evaluate the performance of FISH. This paper reports our results for optimizing the conditions of FISH using rRNA-targeted oligonucleotide probes and flow cytometry and the application of these protocols to the detection of Escherichia coli in seawater spiked with E.coli culture. We obtained two types of optimized protocols for FISH, which showed rapid results with a hybridization time of less than 30 min, with performance equivalent to or better than the standard protocol in terms of the fluorescence signal intensity and the FISH hybridization efficiency (i.e., the percentage of hybridized cells giving satisfactory fluorescence intensity): (i) one-step FISH (hybridization is conducted at 60 to 75 degrees C for 30 min) and (ii) two-step FISH (pretreatment in a 90 degrees C water bath for 5 min and a hybridizing step at 50 to 55 degrees C for 15 to 20 min). We also found that satisfactory fluorescence signal intensity does not necessarily guarantee satisfactory hybridization efficiency and the tightness of the targeted population when analyzed with a flow cytometer. We subsequently successfully applied the optimized protocols to E. coli-spiked seawater samples, i.e., obtained flow cytometric signatures where the E. coli population was well separated from other particles carrying fluorescence from nonspecific binding to probes or from autofluorescence, and had a good recovery rate of the spiked E. coli cells (90%).     

Detection of Legionella species in potting mixes using fluorescent in situ hybridisation (FISH)

This study used Fluorescent in situ Hybridisation (FISH) with rRNA targeted oligonucleotide probes combined with scanning confocal laser microscopy to successfully detect Legionella spp. in commercially available potting mix. A range of techniques were explored to optimise the FISH method by reducing background fluorescence and preventing non-specific binding of probes. These techniques included the use of a blocking agent, UV light treatment, image subtraction of a nonsense probe and spectral unmixing of specific probes fluorescence and autofluorescence dependent on the specific emission spectra of probe fluorophores.Spectral unmixing was the best microscopy technique for reducing background fluorescence and non-specific binding of probes was not observed. The rapid turnaround time and increased sensitivity of the FISH provides as an alternative to traditional culture methods, which are tedious and often give varied results. FISH is also advantageous compared to PCR methods as it provides information on the structure of the microbial community the bacteria is situated in. This study demonstrates that FISH could provide an alternative method for Legionella detection and enumeration in environmental samples.     

Identification of Comamonas species using 16S rRNA gene sequence

A bacterial strain Bz02 was isolated from a water sample collected from river Gomti at the Indian city of Lucknow. We characterized the strain using 16S rRNA sequence. Phylogenetic analysis showed that the strain formed a monophyletic clade with members of the genus Comamonas. The closest phylogenetic relative was Comamonas testosteroni with 95% 16S rRNA gene sequence similarity. It is proposed that the identified strain Bz02 be assigned as the type strain of a species of the genus Comamonas (Comamonas sp Bz02) based on 16S rRNA gene sequence search in Ribosomal Database Project, small subunit rRNA and large subunit rRNA databases together with the phylogenetic tree analysis. The sequence is deposted in GenBank with the accession number {"type":"entrez-nucleotide","attrs":{"text":"FJ211417","term_id":"209164434"}}FJ211417.     

Quantification of bacteria in human feces using 16S rRNA-hybridization, DNA-staining and flow cytometry

Hybridization of bacteria with fluorescent probes targeting 16S rRNA and inspection of hybridized bacteria with fluorescence microscopy (microscopy-FISH, i.e. fluorescence in situ hybridization) have constituted an accessible method for the analysis of mixed bacterial samples such as feces. However, microscopy-FISH is a slow method and prone to errors. Flow cytometry (FCM) enables analysis of bacteria more rapidly, accurately and reliably than microscopy. In this study, a FCM method for the analysis of 16S rRNA-hybridized and DNA-stained fecal bacteria was developed. The results of FCM-FISH were comparable to those of microscopy-FISH, and the coefficients of variation of the FCM analyses were extraordinarily low. In previous FCM-FISH studies, the Eub 338 probe, which is supposed to hybridize all bacteria, has been used to detect all bacteria present in the sample. We found that Eub 338 did not bind to all bacteria, which could be detected by DNA-staining; while SYTOX Orange DNA-stain detected all bacterial species tested and produced high fluorescence intensities enabling clear separation of bacteria from non-bacterial material. Thus, DNA-staining is a method of choice for the detection of all bacteria in FCM-FISH. We conclude that FCM of 16S rRNA-hybridized and DNA-stained bacteria is a rapid and reliable method for the analysis of mixed bacterial samples including feces.     

Estimation of aneuploidy levels in human spermatozoa using chromosome specific probes and in situ hybridisation

Summary The paper describes an attempt to estimate the frequency of aneuploid human spermatozoa with disomic Y chromosome and disomic chromosome 1 complements, using chromosome specific probes and in situ hybridisation. This approach was used as an alternative to the differential staining techniques that have been applied to spermatozoa in previous studies aimed at estimating levels of aneuploidy for chromosome 1 and the Y chromosome. A frequency of 1.8 per 1000 YY-bearing spermatozoa and 3.5 per 1000 disomy 1 spermatozoa was found, both figures being in excess of those found by sperm genome karyotyping. The technical limitations of the method are discussed.     

Combination of 16S rRNA-targeted oligonucleotide probes with flow cytometry for analyzing mixed microbial populations.

Fluorescent oligonucleotide hybridization probes were used to label bacterial cells for analysis by flow cytometry. The probes, complementary to short sequence elements within the 16S rRNA common to phylogenetically coherent assemblages of microorganisms, were labeled with tetramethylrhodamine and hybridized to suspensions of fixed cells. Flow cytometry was used to resolve individual target and nontarget bacteria (1 to 5 microns) via probe-conferred fluorescence. Target cells were quantified in an excess of nontarget cells. The intensity of fluorescence was increased additively by the combined use of two or three fluorescent probes complementary to different regions of the same 16S rRNA.     

Multiparametric analysis of cell membrane permeability by two colour flow cytometry with complementary fluorescent probes

We describe an improved twin-probe multiparameter flow cytometric technique to examine cell membrane permeability. Ability to retain preloaded intracellular bis-carboxyethyl carboxy fluorescein (BCECF, green fluorescence) and to exclude extracellular propidium (red fluorescence) is measured, simultaneously with forward and right-angle scatter. This has significant advantages over an earlier method using fluorescein together with ethidium. In addition to the two expected cell populations which were stained green positive, red negative (by convention membrane "intact" and "viable," Region 1) and green negative, red positive ("membrane-damaged" and "non-viable," Region 3), a third population was seen which fluoresced neither green nor red and displayed intermediate light scatter characteristics (Region 2). This was true for each of 9 cell types in vitro. For EMT6 mouse mammary tumour cells held under sub-optimal conditions or treated with membrane-active drugs, progression from Region 1 to Region 2 was observed, followed by further progression from Region 2 to Region 3. Cells eventually accumulated in Region 3. These results suggest that sequential changes in membrane structure lead to increased permeability, first with respect to intracellular BCECF and in turn to extracellular propidium.     

Quantification of whole-cell in situ hybridization with oligonucleotide probes by flow cytometry of Escherichia coli cells

The use of fluorescence in situ hybridization (FISH) in conjunction with flow cytometry is a popular method of analysing environmental microbial populations. However, false-positive results can be produced if the specificity of oligonucleotide probe binding is not considered. An aim of this research was to evaluate the specificity of labelled oligonucleotide probe binding in FISH by flow cytometry. An excess of unlabelled probe was used to competitively inhibit the specific binding of labelled probe. Comparisons were made between the mean cell fluorescence and the number of fluorescently stained cells in a pure culture of Escherichia coli ATCC 53323. Specific binding of species-specific probes for the detection of E. coli was in the range 47–70% of total binding. A eukaryote probe and a nonsense probe, used as negative controls, had no specific binding with cells of E. coli. The significance of the results obtained is that the enumeration of specifically probe-bound microbial cells by FISH and flow cytometry must be made by an application of labelled and unlabelled probes to distinguish specifically stained cells. This is also a more practical method for the analysis of environmental samples compared to washing of excess non-specifically bound probe, due to the reduction of cell loss from the analysis.     

Unlabeled helper oligonucleotides increase the in situ accessibility to 16S rRNA of fluorescently labeled oligonucleotide probes

Target site inaccessibility represents a significant problem for fluorescence in situ hybridization (FISH) of 16S rRNA with oligonucleotide probes. Here, unlabeled oligonucleotides (helpers) that bind adjacent to the probe target site were evaluated for their potential to increase weak probe hybridization signals in Escherichia coli DSM 30083(T). The use of helpers enhanced the fluorescence signal of all six probes examined at least fourfold. In one case, the signal of probe Eco474 was increased 25-fold with the use of a single helper probe, H440-2. In another case, four unlabeled helpers raised the FISH signal of a formerly weak probe, Eco585, to the level of the brightest monolabeled oligonucleotide probes available for E. coli. The temperature of dissociation and the mismatch discrimination of probes were not significantly influenced by the addition of helpers. Therefore, using helpers should not cause labeling of additional nontarget organisms at a defined stringency of hybridization. However, the helper action is based on sequence-specific binding, and there is thus a potential for narrowing the target group which must be considered when designing helpers. We conclude that helpers can open inaccessible rRNA regions for FISH with oligonucleotide probes and will thereby further improve the applicability of this technique for in situ identification of microorganisms.