Species-specific oligonucleotide probes for the detection and identification of Lactobacillus isolated from mouse faeces

AIMS: To develop species-specific monitoring techniques for rapid detection and identification of Lactobacillus isolated from mouse faeces. METHODS AND RESULTS: The specificity of oligonucleotide probes was evaluated by dot blot hybridization to 16S rDNA and 23S rDNA amplified by PCR from 12 Lactobacillus type strains and 100 strains of Lactobacillus isolated from mouse faeces. Oligonucleotide probes specific for each Lactobacillus species hybridized only with targeted rDNA. The Lactobacillus strains isolated from mouse faeces were identified mainly as Lactobacillus intestinalis, L. johnsonii, L. murinus and L. reuteri using species-specific probes. 16S rDNA of eight unidentified isolates were sequenced and two new probes were designed. Four of eight strains of unhybridized Lactobacillus were identified as L. johnsonii/gasseri group, and the remaining four strains as L. vaginalis. CONCLUSIONS: The species-specific probe set of L. intestinalis, L. johnsonii, L. murinus, L. reuteri and L. vaginalis in this study was efficient for rapid identification of Lactobacillus isolated from mouse faeces. SIGNIFICANCE AND IMPACT OF THE STUDY: The oligonucleotide probe set for Lactobacillus species harboured in the mouse intestine, can be used for rapid identification of lactobacilli and monitoring of the faecal Lactobacillus community.     

Application of rRNA-targeted oligonucleotide probes in biotechnology

Ribosomal RNA-targeted oligonucleotide probes have become valuable tools for the detection of microorganisms involved in important biotechnological processes. Microorganisms which are of major importance for processes such as wastewater treatment, microbial leaching or methane production can be detected and quantified in situ within a complex microbial community. For certain processes, such as nitrification or biological phosphate removal, new microorganisms have become the focus of interest and have led to an improved understanding of these bioremediation techniques. Hybridization techniques have become fast and reliable alternatives to conventional cultivation techniques in the food industry as a control method for starter cultures for fermentation processes or product control. Recent analytical tools such as flow cytometry and digital image processing have improved the efficiency of these techniques. This review is intended to present a summary of methodological aspects of rRNA-based hybridization techniques and their application in biotechnology.     

probeBase: an online resource for rRNA-targeted oligonucleotide probes

Ribosomal RNA-(rRNA)-targeted oligonucleotide probes are widely used for culture-independent identification of microorganisms in environmental and clinical samples. ProbeBase is a comprehensive database containing more than 700 published rRNA-targeted oligonucleotide probe sequences (status August 2002) with supporting bibliographic and biological annotation that can be accessed through the internet at http://www.probebase.net. Each oligonucleotide probe entry contains information on target organisms, target molecule (small- or large-subunit rRNA) and position, G+C content, predicted melting temperature, molecular weight, necessity of competitor probes, and the reference that originally described the oligonucleotide probe, including a link to the respective abstract at PubMed. In addition, probes successfully used for fluorescence in situ hybridization (FISH) are highlighted and the recommended hybridization conditions are listed. ProbeBase also offers difference alignments for 16S rRNA-targeted probes by using the probe match tool of the ARB software and the latest small-subunit rRNA ARB database (release June 2002). The option to directly submit probe sequences to the probe match tool of the Ribosomal Database Project II (RDP-II) further allows one to extract supplementary information on probe specificities. The two main features of probeBase, 'search probeBase' and 'find probe set', help researchers to find suitable, published oligonucleotide probes for microorganisms of interest or for rRNA gene sequences submitted by the user. Furthermore, the 'search target site' option provides guidance for the development of new FISH probes.     

Identification of individual prokaryotic cells by using enzyme-labeled, rRNA-targeted oligonucleotide probes.

A method to microscopically detect and identify individual cells of members of the domains Bacteria and Archaea is presented. rRNA-targeted oligonucleotides were 5' end labeled with the enzyme horseradish peroxidase and used for whole-cell hybridization. Specifically bound probe was visualized by the enzymatic formation of an intracellular precipitate from the substrate diaminobenzidine. Permeation of the enzyme-labeled probe into whole fixed cells of gram-negative bacteria required their pretreatment with lysozyme-EDTA, whereas permeability of some archaebacterial cells was improved by addition of detergent to the hybridization buffer. Hitherto we had not achieved penetration of enzyme-labeled probe into gram-positive bacteria and yeast cells. This method should be a valuable tool for identification of suitable prokaryotic cells in environments with elevated background fluorescence or in situations in which an epifluorescence microscope is not available.     

Identification of individual prokaryotic cells by using enzyme-labeled, rRNA-targeted oligonucleotide probes.

A method to microscopically detect and identify individual cells of members of the domains Bacteria and Archaea is presented. rRNA-targeted oligonucleotides were 5' end labeled with the enzyme horseradish peroxidase and used for whole-cell hybridization. Specifically bound probe was visualized by the enzymatic formation of an intracellular precipitate from the substrate diaminobenzidine. Permeation of the enzyme-labeled probe into whole fixed cells of gram-negative bacteria required their pretreatment with lysozyme-EDTA, whereas permeability of some archaebacterial cells was improved by addition of detergent to the hybridization buffer. Hitherto we had not achieved penetration of enzyme-labeled probe into gram-positive bacteria and yeast cells. This method should be a valuable tool for identification of suitable prokaryotic cells in environments with elevated background fluorescence or in situations in which an epifluorescence microscope is not available.     

Identification of lactococci and enterococci by colony hybridization with 23S rRNA-targeted oligonucleotide probes

Specific sequences of 23S rRNA of Lactococcus lactis, Enterococcus faecalis, Enteroccus faecium, and Enterococcus malodoratus/Enterococcus avium were identified, and complementary oligonucleotide probes were synthesized. The specificity of the probes was evaluated by dot blot and colony hybridizations. The probes can be used for the specific detection and identification of colonies of the corresponding species in mixed cultures.     

Identifying members of the domain Archaea with rRNA-targeted oligonucleotide probes.

Two 16S rRNA-targeted oligonucleotide probes were designed for the archaeal kingdoms Euryachaeota and Crenarchaeota. Probe specificities were evaluated by nonradioactive dot blot hybridization against selected reference organisms. The successful application of fluorescent-probe derivatives for whole-cell hybridization required organism-specific optimizations of fixation and hybridization conditions to assure probe penetration and morphological integrity of the cells. The probes allowed preliminary grouping of three new hyperthermophilic isolates. Together with other group-specific rRNA-targeted oligonucleotide probes, these probes will facilitate rapid in situ monitoring of the populations present in hydrothermal systems and support cultivation attempts.     

Identification of lactococci and enterococci by colony hybridization with 23S rRNA-targeted oligonucleotide probes.

Specific sequences of 23S rRNA of Lactococcus lactis, Enterococcus faecalis, Enteroccus faecium, and Enterococcus malodoratus/Enterococcus avium were identified, and complementary oligonucleotide probes were synthesized. The specificity of the probes was evaluated by dot blot and colony hybridizations. The probes can be used for the specific detection and identification of colonies of the corresponding species in mixed cultures.     

Design and application of new 16S rRNA-targeted oligonucleotide probes for the Azospirillum-Skermanella-Rhodocista-cluster

The genera Azospirillum, Skermanella and Rhodocista form a phylogenetic subgroup within the alfa subclass of Proteobacteria. Based on comparative 16S rRNA sequence analysis a nested set of new oligonucleotide probes was designed. It comprises probes for the whole genus cluster Azospirillum-Skermanella-Rhodocista, for the Azospirilli subcluster I including A. lipoferum, A. doebereinerae, A. largimobile, A. brasilense and A. halopraeferens, for the Azospirilli subcluster II including A. amazonense, A. irakense and the genus Skermanella, for the genus Rhodocista as well as for all Azospirilli species or species cluster. The new probes allow a fast and reliable in situ identification of bacteria belonging to the Azospirillum-Skermanella-Rhodocista-cluster at different phylogenetic levels. The specificity of the new probes was tested with 56 strains of the Azospirillum-Rhodocista-Skermanella-cluster and selected reference cells from other genera by hybridising with the complete probe set. In addition, applications of the fluorescently labelled probes for in situ identification of isolates and for the in situ localisation of A. brasilense on maize roots were demonstrated using confocal laser scanning microscopy.     

In situ identification of cyanobacteria with horseradish peroxidase-labeled, rRNA-targeted oligonucleotide probes

Individual cyanobacterial cells are normally identified in environmental samples only on the basis of their pigmentation and morphology. However, these criteria are often insufficient for the differentiation of species. Here, a whole-cell hybridization technique is presented that uses horseradish peroxidase (HRP)-labeled, rRNA-targeted oligonucleotides for in situ identification of cyanobacteria. This indirect method, in which the probe-conferred enzyme has to be visualized in an additional step, was necessary since fluorescently monolabeled oligonucleotides were insufficient to overstain the autofluorescence of the target cells. Initially, a nonfluorescent detection assay was developed and successfully applied to cyanobacterial mats. Later, it was demonstrated that tyramide signal amplification (TSA) resulted in fluorescent signals far above the level of autofluorescence. Furthermore, TSA-based detection of HRP was more sensitive than that based on nonfluorescent substrates. Critical points of the assay, such as cell fixation and permeabilization, specificity, and sensitivity, were systematically investigated by using four oligonucleotides newly designed to target groups of cyanobacteria.     

Quantification of Enterococcus faecalis and Enterococcus faecium in different foods using rRNA-targeted oligonucleotide probes

The abundance of Enterococcus faecalis and Enterococcus faecium in different Spanish foods was evaluated by using taxon-specific oligonucleotide probes targeted against extracted rRNA. Two satisfactory methods were developed for RNA extraction. Although the yield and purity of total RNA obtained largely depended on the type of food, method 1 should be recommended. The quantitative results obtained with the oligonucleotide probes DB6 for E. faecium and DB8 for E. faecalis showed that these two species accounted for less than 0.5% of the active microflora in all the food samples tested. These results suggest that enterococci form only a minor portion of the microflora of these products.     

Family- and genus-level 16S rRNA-targeted oligonucleotide probes for ecological studies of methanotrophic bacteria

Methanotrophic bacteria play a major role in the global carbon cycle, degrade xenobiotic pollutants, and have the potential for a variety of biotechnological applications. To facilitate ecological studies of these important organisms, we developed a suite of oligonucleotide probes for quantitative analysis of methanotroph-specific 16S rRNA from environmental samples. Two probes target methanotrophs in the family Methylocystaceae (type II methanotrophs) as a group. No oligonucleotide signatures that distinguish between the two genera in this family, Methylocystis and Methylosinus, were identified. Two other probes target, as a single group, a majority of the known methanotrophs belonging to the family Methylococcaceae (type I/X methanotrophs). The remaining probes target members of individual genera of the Methylococcaceae, including Methylobacter, Methylomonas, Methylomicrobium, Methylococcus, and Methylocaldum. One of the family-level probes also covers all methanotrophic endosymbionts of marine mollusks for which 16S rRNA sequences have been published. The two known species of the newly described genus Methylosarcina gen. nov. are covered by a probe that otherwise targets only members of the closely related genus Methylomicrobium. None of the probes covers strains of the newly proposed genera Methylocella and "Methylothermus," which are polyphyletic with respect to the recognized methanotrophic families. Empirically determined midpoint dissociation temperatures were 49 to 57 degrees C for all probes. In dot blot screening against RNA from positive- and negative-control strains, the probes were specific to their intended targets. The broad coverage and high degree of specificity of this new suite of probes will provide more detailed, quantitative information about the community structure of methanotrophs in environmental samples than was previously available.     

Potential of Lactobacillus gasseri isolated from infant faeces to produce bacteriocin

Thirty strains of Lactobacillus gasseri isolated from infant faeces were examined for production of an antimicrobial agent against 10 strains of the seven species of the genus Lactobacillus. Each of the strains inhibited the growth of at least one of the indicator strains. The agent was sensitive to proteolytic enzymes and stable for 20 min at 120°C. It is a bacteriocin designated as gassericin A.     

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.     

Order-specific 16S rRNA-targeted oligonucleotide probes for (hyper)thermophilic archaea and bacteria

New oligonucleotide probes were designed and evaluated for application in fluorescence in situ hybridization (FISH) studies on (hyper)thermophilic microbial communities—Arglo32, Tcoc164, and Aqui1197 target the 16S rRNA of Archaeoglobales, Thermococcales, and Aquificales, respectively. Both sequence information and experimental evaluation showed high coverage and specificity of all three probes. The signal intensity of Aqui1197 was improved by addition of a newly designed, unlabeled helper oligonucleotide, hAqui1045. It was shown that in addition to its function as a probe for Aquificales, Aqui1197 is suitable as a supplementary probe to extend the coverage of the domain-specific bacterial probe EUB338. In sediments from two hydrothermal seeps on Vulcano Island, Italy, the microbial community structure was analyzed by FISH with both established and the new oligonucleotide probes, showing the applicability of Arglo32, Tcoc164, and Aqui1197/hAqui1045 to natural samples. At both sites, all major groups of (hyper)thermophiles, except for methanogens, were detected: Crenarchaeota (19%, 16%), Thermococcales (14%, 22%), Archaeoglobales (14%, 12%), Aquificales (5%, 8%), Thermotoga/Thermosipho spp. (12%, 9%), Thermus sp. (12%, none), and thermophilic Bacillus sp. (12%, 8%).     

rRNA-targeted寡核苷酸探针识别活性污泥微生物群落结构与功能研究进展

活性污泥中微生物群落内部关系非常复杂 ,及时对活性污泥中优势菌群和群落内部关系进行监测是污水处理中采取正确措施的关键。历史研究表明传统培养方法经常导致活性污泥优势菌群检测的失败 ,而r RNA- targeted寡核苷酸探针作为一种快速原位监测活性污泥微生物群落结构和功能的新工具被引入 ,使我们对参与污水净化的微生物群落结构和优势菌群能有较全面的了解。就该方法在识别除磷污泥、脱氮污泥、污泥泡沫和膨胀污泥中微生物群落结构和功能的典型应用进行综述 ,分析了该方法存在的优点和缺点 ,并对目前已建立且应用于活性污泥微生物检测的 r RNA- targeted寡核苷酸探针进行了详细总结     

Fluorescent in situ hybridization with rRNA-targeted oligonucleotide probes to identify small phytoplankton by flow cytometry.

Because of their tiny size (0.2 to 2 microns), oceanic picophytoplanktonic cells (either cultured strains or natural communities) are difficult to identify, and some basic questions concerning their taxonomy, physiology, and ecology are still largely unanswered. The present study was designed to test the suitability of in situ hybridization with rRNA fluorescent probes detected by flow cytometry for the identification of small photosynthetic eukaryotes. Oligonucleotide probes targeted against regions of the 18S rRNAs of Chlorophyta lineage (CHLO probe) and of non-Chlorophyta (NCHLO probe) algal species were designed. The CHLO and NCHLO probes, which differed by a single nucleotide, allowed discrimination of chlorophyte from nonchlorophyte cultured strains. The sensitivity of each probe was dependent upon the size of the cells and upon their growth stage. The mean fluorescence was 8 to 80 times higher for specifically labeled than for nonspecifically labeled cells in exponential growth phase, but it decreased sharply in stationary phase. Such taxon-specific probes should increase the applicability of flow cytometry for the rapid identification of cultured pico- and nanoplanktonic strains, especially those that lack taxonomically useful morphological features.     

The effect of nucleobase-specific fluorescence quenching on in situ hybridization with rRNA-targeted oligonucleotide probes

Oligonucleotide probes labeled with fluorescent dyes are used in a variety of in situ applications to detect specific DNA or RNA molecules. It has been described that probe fluorescence might be quenched upon hybridization in a sequence specific way. Here, a set of 17 oligonuleotides labeled with 6-carboxyfluorescein was used to examine the relevance of nucleotide specific quenching for fluorescence in situ hybridization (FISH) to whole fixed bacterial cells. Probes quenched upon hybridization to a guanine-rich region of purified RNA in solution were not quenched upon FISH. Among other factors the high protein concentration within cells may prevent quenching of probe fluorescence in situ.