To see the other types of publications on this topic, follow the link: Bacteria; Oligonucleotideos.
Journal articles on the topic 'Bacteria; Oligonucleotideos'
Create a spot-on reference in APA, MLA, Chicago, Harvard, and other styles
Consult the top 50 journal articles for your research on the topic 'Bacteria; Oligonucleotideos.'
Next to every source in the list of references, there is an 'Add to bibliography' button. Press on it, and we will generate automatically the bibliographic reference to the chosen work in the citation style you need: APA, MLA, Harvard, Chicago, Vancouver, etc.
You can also download the full text of the academic publication as pdf and read online its abstract whenever available in the metadata.
Browse journal articles on a wide variety of disciplines and organise your bibliography correctly.
1
Anthony, R. M., T. J. Brown, and G. L. French. "Rapid Diagnosis of Bacteremia by Universal Amplification of 23S Ribosomal DNA Followed by Hybridization to an Oligonucleotide Array." Journal of Clinical Microbiology 38, no. 2 (2000): 781–88. http://dx.doi.org/10.1128/jcm.38.2.781-788.2000.
Full textAbstract:
The rapid identification of bacteria in blood cultures and other clinical specimens is important for patient management and antimicrobial therapy. We describe a rapid (<4 h) detection and identification system that uses universal PCR primers to amplify a variable region of bacterial 23S ribosomal DNA, followed by reverse hybridization of the products to a panel of oligonucleotides. This procedure was successful in discriminating a range of bacteria in pure cultures. When this procedure was applied directly to 158 unselected positive blood culture broths on the day when growth was detected, 125 (79.7%) were correctly identified, including 4 with mixed cultures. Nine (7.2%) yielded bacteria for which no oligonucleotide targets were present in the oligonucleotide panel, and 16 culture-positive broths (10.3%) produced no PCR product. In seven of the remaining eight broths, streptococci were identified but not subsequently grown, and one isolate of Staphylococcus aureus was misidentified as a coagulase-negative staphylococcus. The accuracy, range, and discriminatory power of the assay can be continually extended by adding further oligonucleotides to the panel without significantly increasing complexity or cost.
2
Chizhikov, Vladimir, Avraham Rasooly, Konstantin Chumakov, and Dan D. Levy. "Microarray Analysis of Microbial Virulence Factors." Applied and Environmental Microbiology 67, no. 7 (2001): 3258–63. http://dx.doi.org/10.1128/aem.67.7.3258-3263.2001.
Full textAbstract:
ABSTRACT Hybridization with oligonucleotide microchips (microarrays) was used for discrimination among strains of Escherichia coli and other pathogenic enteric bacteria harboring various virulence factors. Oligonucleotide microchips are miniature arrays of gene-specific oligonucleotide probes immobilized on a glass surface. The combination of this technique with the amplification of genetic material by PCR is a powerful tool for the detection of and simultaneous discrimination among food-borne human pathogens. The presence of six genes (eaeA, slt-I,slt-II, fliC, rfbE, andipaH) encoding bacterial antigenic determinants and virulence factors of bacterial strains was monitored by multiplex PCR followed by hybridization of the denatured PCR product to the gene-specific oligonucleotides on the microchip. The assay was able to detect these virulence factors in 15 Salmonella,Shigella, and E. coli strains. The results of the chip analysis were confirmed by hybridization of radiolabeled gene-specific probes to genomic DNA from bacterial colonies. In contrast, gel electrophoretic analysis of the multiplex PCR products used for the microarray analysis produced ambiguous results due to the presence of unexpected and uncharacterized bands. Our results suggest that microarray analysis of microbial virulence factors might be very useful for automated identification and characterization of bacterial pathogens.
3
Verri, A., P. Mazzarello, S. Spadari, and F. Focher. "Uracil-DNA glycosylases preferentially excise mispaired uracil." Biochemical Journal 287, no. 3 (1992): 1007–10. http://dx.doi.org/10.1042/bj2871007.
Full textAbstract:
We have investigated the substrate specificity of human, viral and bacterial uracil-DNA glycosylases employing as substrate double-stranded oligonucleotides containing in the same position of the 5′-32P-labelled strand an uracil residue facing, on the complementary strand, guanine (mimicking cytosine deamination) or adenine (mimicking dUTP misincorporation). The enzyme removal of uracil was monitored and quantified by the generation of alkali-sensitive apyrimidinic sites. All three uracil-DNA glycosylases excise uracil from mispaired oligonucleotides (U/G) more efficiently than from paired oligonucleotides (U/A). The enzymes also remove uracil from single-stranded oligonucleotide with an efficiency similar to that observed with U/A paired oligonucleotide. The efficient recognition of U/G mispair by uracil-DNA glycosylase is important in minimizing miscoding transcripts and C/G-->T/A transitions in proliferating cells.
4
Bertilsson, Stefan, Colleen M. Cavanaugh, and Martin F. Polz. "Sequencing-Independent Method To Generate Oligonucleotide Probes Targeting a Variable Region in Bacterial 16S rRNA by PCR with Detachable Primers." Applied and Environmental Microbiology 68, no. 12 (2002): 6077–86. http://dx.doi.org/10.1128/aem.68.12.6077-6086.2002.
Full textAbstract:
ABSTRACT Oligonucleotide probes targeting the small-subunit rRNA are commonly used to detect and quantify bacteria in natural environments. We developed a PCR-based approach that allows synthesis of oligonucleotide probes targeting a variable region in the 16S rRNA without prior knowledge of the target sequence. Analysis of all 16S rRNA gene sequences in the Ribosomal Database Project database revealed two universal primer regions bracketing a variable, population-specific region. The probe synthesis is based on a two-step PCR amplification of this variable region in the 16S rRNA gene by using three universal bacterial primers. First, a double-stranded product is generated, which then serves as template in a linear amplification. After each of these steps, products are bound to magnetic beads and the primers are detached through hydrolysis of a ribonucleotide at the 3′ end of the primers. This ultimately produces a single-stranded oligonucleotide of about 30 bases corresponding to the target. As probes, the oligonucleotides are highly specific and could discriminate between nucleic acids from closely and distantly related bacterial strains, including different species of Vibrio. The method will facilitate rapid generation of oligonucleotide probes for large-scale hybridization assays such as screening of clone libraries or strain collections, ribotyping microarrays, and in situ hybridization. An additional advantage of the method is that fluorescently or radioactively labeled nucleotides can be incorporated during the second amplification, yielding intensely labeled probes.
5
Jelinkova, Pavlina, Jakub Hrdy, Jirina Markova, et al. "Development and Inter-Laboratory Validation of Diagnostics Panel for Detection of Biothreat Bacteria Based on MOL-PCR Assay." Microorganisms 9, no. 1 (2020): 38. http://dx.doi.org/10.3390/microorganisms9010038.
Full textAbstract:
Early detection of biohazardous bacteria that can be misused as biological weapons is one of the most important measures to prevent the spread and outbreak of biological warfare. For this reason, many instrument platforms need to be introduced into operation in the field of biological warfare detection. Therefore the purpose of this study is to establish a new detection panel for biothreat bacteria (Bacillus anthracis, Yersinia pestis, Francisella tularensis, and Brucella spp.) and confirm it by collaborative validation by using a multiplex oligonucleotide ligation followed by polymerase chain reaction and hybridization to microspheres by MagPix detection platform (MOL-PCR). Appropriate specific sequences in bacterial DNA were selected and tested to assemble the detection panel, and MOLigo probes (short specific oligonucleotides) were designed to show no cross-reactivity when tested between bacteria and to decrease the background signal measurement on the MagPix platform. During testing, sensitivity was assessed for all target bacteria using serially diluted DNA and was determined to be at least 0.5 ng/µL. For use as a diagnostic kit and easier handling, the storage stability of ligation premixes (MOLigo probe mixes) was tested. This highly multiplex method can be used for rapid screening to prevent outbreaks arising from the use of bacterial strains for bioterrorism, because time of analysis take under 4 h.
6
Tian, Lingyang, Takuichi Sato, Kousuke Niwa, Mitsuo Kawase, Anne C. R. Tanner, and Nobuhiro Takahashi. "Rapid and Sensitive PCR-Dipstick DNA Chromatography for Multiplex Analysis of the Oral Microbiota." BioMed Research International 2014 (2014): 1–10. http://dx.doi.org/10.1155/2014/180323.
Full textAbstract:
A complex of species has been associated with dental caries under the ecological hypothesis. This study aimed to develop a rapid, sensitive PCR-dipstick DNA chromatography assay that could be read by eye for multiplex and semiquantitative analysis of plaque bacteria. Parallel oligonucleotides were immobilized on a dipstick strip for multiplex analysis of target DNA sequences of the caries-associated bacteria,Streptococcus mutans,Streptococcus sobrinus,Scardovia wiggsiae,Actinomycesspecies, andVeillonella parvula. Streptavidin-coated blue-colored latex microspheres were to generate signal. Target DNA amplicons with an oligonucleotide-tagged terminus and a biotinylated terminus were coupled with latex beads through a streptavidin-biotin interaction and then hybridized with complementary oligonucleotides on the strip. The accumulation of captured latex beads on the test and control lines produced blue bands, enabling visual detection with the naked eye. The PCR-dipstick DNA chromatography detected quantities as low as 100 pg of DNA amplicons and demonstrated 10- to 1000-fold higher sensitivity than PCR-agarose gel electrophoresis, depending on the target bacterial species. Semiquantification of bacteria was performed by obtaining a series of chromatograms using serial 10-fold dilution of PCR-amplified DNA extracted from dental plaque samples. The assay time was less than 3 h. The semiquantification procedure revealed the relative amounts of each test species in dental plaque samples, indicating that this disposable device has great potential in analysis of microbial composition in the oral cavity and intestinal tract, as well as in point-of-care diagnosis of microbiota-associated diseases.
7
He, Ping-An, and Li Xia. "Oligonucleotide Profiling for Discriminating Bacteria in Bacterial Communities." Combinatorial Chemistry & High Throughput Screening 10, no. 4 (2007): 247–55. http://dx.doi.org/10.2174/138620707780636646.
Full text
8
Pernthaler, Annelie, Jakob Pernthaler, and Rudolf Amann. "Fluorescence In Situ Hybridization and Catalyzed Reporter Deposition for the Identification of Marine Bacteria." Applied and Environmental Microbiology 68, no. 6 (2002): 3094–101. http://dx.doi.org/10.1128/aem.68.6.3094-3101.2002.
Full textAbstract:
ABSTRACT Fluorescence in situ hybridization (FISH) with horseradish peroxidase (HRP)-labeled oligonucleotide probes and tyramide signal amplification, also known as catalyzed reporter deposition (CARD), is currently not generally applicable to heterotrophic bacteria in marine samples. Penetration of the HRP molecule into bacterial cells requires permeabilization procedures that cause high and most probably species-selective cell loss. Here we present an improved protocol for CARD-FISH of marine planktonic and benthic microbial assemblages. After concentration of samples onto membrane filters and subsequent embedding of filters in low-gelling-point agarose, no decrease in bacterial cell numbers was observed during 90 min of lysozyme incubation (10 mg ml−1 at 37°C). The detection rates of coastal North Sea bacterioplankton by CARD-FISH with a general bacterial probe (EUB338-HRP) were significantly higher (mean, 94% of total cell counts; range, 85 to 100%) than that with a monolabeled probe (EUB338-mono; mean, 48%; range, 19 to 66%). Virtually no unspecific staining was observed after CARD-FISH with an antisense EUB338-HRP. Members of the marine SAR86 clade were undetectable by FISH with a monolabeled probe; however, a substantial population was visualized by CARD-FISH (mean, 7%; range, 3 to 13%). Detection rates of EUB338-HRP in Wadden Sea sediments (mean, 81%; range, 53 to 100%) were almost twice as high as the detection rates of EUB338-mono (mean, 44%; range, 25 to 71%). The enhanced fluorescence intensities and signal-to-background ratios make CARD-FISH superior to FISH with directly labeled oligonucleotides for the staining of bacteria with low rRNA content in the marine environment.
9
Fessehaie, Anania, Solke H. De Boer, and C. André Lévesque. "An Oligonucleotide Array for the Identification and Differentiation of Bacteria Pathogenic on Potato." Phytopathology® 93, no. 3 (2003): 262–69. http://dx.doi.org/10.1094/phyto.2003.93.3.262.
Full textAbstract:
Oligonucleotides, 16 to 24 bases long, were selected from the 3′ end of the 16S gene and the 16S–23S intergenic spacer regions of bacteria pathogenic on potato, including Clavibacter michiganensis subsp. sepedonicus, Ralstonia solanacearum, and the pectolytic erwinias, including Erwinia carotovora subsp. atroseptica and carotovora and E. chrysanthemi. Oligonucleotides were designed and formatted into an array by pin spotting on nylon membranes. Genomic DNA from bacterial cultures was amplified by polymerase chain reaction using conserved ribosomal primers and labeled simultaneously with digoxigenin-dUTP. Hybridization of amplicons to the array and subsequent serological detection of digoxigenin label revealed different hybridization patterns that were distinct for each species and subspecies tested. Hybridization of amplicons generally was restricted to appropriate homologous oligonucleotides and cross-hybridization with heterologous oligonucleotides was rare. Hybridization patterns were recorded as separate gray values for each hybridized spot and revealed a consistent pattern for multiple strains of each species or subspecies isolated from diverse geographical regions. In preliminary tests, bacteria could be correctly identified and detected by hybridizing to the array amplicons from mixed cultures and inoculated potato tissue.
10
Fischer, Kathrin, Dittmar Hahn, Otto Daniel, Josef Zeyer, and Rudolf I. Amann. "In situ analysis of the bacterial community in the gut of the earthwormLumbricus terrestrisL. by whole-cell hybridization." Canadian Journal of Microbiology 41, no. 8 (1995): 666–73. http://dx.doi.org/10.1139/m95-092.
Full textAbstract:
The bacterial community in the gut of the earthworm Lumbricus terrestris was analyzed by whole-cell hybridization with 16S rRNA targeted oligonucleotide probes. Whole-cell hybridization protocols using fluorescence-, peroxidase-, or digoxigenin-labeled oligonucleotide probes facilitated detection of significant fractions of bacterial cells stained with 4′,6-diamidino-2-phenylindole (DAPI) in the fore-, mid-, and hind-gut and cast of the earthworm. The application of peroxidase- and digoxigenin-labeled probes, however, was hampered by several methodological drawbacks: the requirement of enzymatic permeabilization, the diffuse images of stained cells, and the incompatibility with DAPI staining used as control. Quantitative analysis of the bacterial community was also influenced by its considerable variability in different individual earthworms. Though the number of bacteria detected by DAPI staining as well as by whole-cell hybridization with the fluorescent eubacterial probe Eub338 generally showed a significant increase in the number of bacteria towards the end of the gut, a decrease in bacterial numbers could be found in some earthworms. In situ analysis of the bacterial community in the fore-, mid-, and hind-gut of one individual earthworm by whole-cell hybridization with the fluorescent eubacterial probe Eub338 recorded 15, 30, and 25% of DAPI-stained bacteria, respectively. In the cast 37% of the bacteria were detected. Similar to counts obtained by DAPI and by whole-cell hybridization with probe Eub338, the number of bacteria belonging to the α-, β-, and γ-subgroups of proteobacteria increased significantly towards the end of the gut and remained high in the cast. While the most significant difference in the counts of bacteria belonging to the α-subgroup was obtained between the hind-gut and cast, bacterial populations of the β- and γ- subgroups of proteobacteria increased most prominently between the fore- and hind-gut.Key words: digoxigenin, fluorescent probes, in situ detection, Lumbricus terrestris, rRNA, whole-cell hybridization.
11
Wagner, M., R. Amann, H. Lemmer, W. Manz, and K. H. Schleifer. "Probing Activated Sludge with Fluorescently Labeled rRNA Targeted Oligonucleotides." Water Science and Technology 29, no. 7 (1994): 15–23. http://dx.doi.org/10.2166/wst.1994.0294.
Full textAbstract:
Activated sludge samples from municipal sewage treatment plants were characterized using 16S and 23S ribosomal RNA targeted oligonucleotide probes specific for defined phylogenetic groups of bacteria. Comparison of in situ community structures as determined by molecular biological methods with the composition of the heterotrophic saprophyte flora isolated on nutrient rich medium revealed large discrepancies. These are caused by the selectivity of media and culture conditions. The most significant effect of cultivation on nutrient rich medium is an underestimation of bacteria belonging to the beta-subclass of Proteobacteria and an overestimation of bacteria belonging to the gamma-subclass of Proteobacteria. Therefore, culture dependent enumerations of the gamma-subclass bacteria of the genus Acinetobacter in plants with enhanced biological phosphate removal (EBPR) resulted in significant overestimations. In situ identification by fluorescent oligonucleotide probing revealed that Acinetobacter numbers were below 8% of the active bacterial cells in the examined EBPR-plants. In situ hybridization techniques also bear the potential for the early and correct identification of filamentous bacteria as indicators for sludge bulking and foaming. A 16S ribosomal RNA targeted oligonucleotide probe specific for Sphaerotilus spec, was developed and successfully applied for in situ investigation of this filamentous bacterium.
12
Pereira, Sara, Rita Sobral Santos, Luís Moreira, et al. "Lipoplexes to Deliver Oligonucleotides in Gram-Positive and Gram-Negative Bacteria: Towards Treatment of Blood Infections." Pharmaceutics 13, no. 7 (2021): 989. http://dx.doi.org/10.3390/pharmaceutics13070989.
Full textAbstract:
Bacterial resistance to antibiotics threatens the ability to treat life-threatening bloodstream infections. Oligonucleotides (ONs) composed of nucleic acid mimics (NAMs) able to inhibit essential genes can become an alternative to traditional antibiotics, as long as they are safely transported in human serum upon intravenous administration and they are carried across the multilayered bacterial envelopes, impermeable to ONs. In this study, fusogenic liposomes were considered to transport the ONs and promote their internalization in clinically relevant bacteria. Locked nucleic acids and 2′-OMethyl RNA were evaluated as model NAMs and formulated into DOTAP–DOPE liposomes followed by post-PEGylation. Our data showed a complexation stability between the post-PEGylated liposomes and the ONs of over 82%, during 24 h in native human serum, as determined by fluorescence correlation spectroscopy. Quantification by a lipid-mixing assay showed that liposomes, with and without post-PEGylation, fused with all bacteria tested. Such fusion promoted the delivery of a fraction of the ONs into the bacterial cytosol, as observed by fluorescence in situ hybridization and bacterial fractionation. In short, we demonstrated for the first time that liposomes can safely transport ONs in human serum and intracellularly deliver them in both Gram-negative and -positive bacteria, which holds promise towards the treatment of bloodstream infections.
13
Jolly, Pawan, Pedro Estrela, and Michael Ladomery. "Oligonucleotide-based systems: DNA, microRNAs, DNA/RNA aptamers." Essays in Biochemistry 60, no. 1 (2016): 27–35. http://dx.doi.org/10.1042/ebc20150004.
Full textAbstract:
There are an increasing number of applications that have been developed for oligonucleotide-based biosensing systems in genetics and biomedicine. Oligonucleotide-based biosensors are those where the probe to capture the analyte is a strand of deoxyribonucleic acid (DNA), ribonucleic acid (RNA) or a synthetic analogue of naturally occurring nucleic acids. This review will shed light on various types of nucleic acids such as DNA and RNA (particularly microRNAs), their role and their application in biosensing. It will also cover DNA/RNA aptamers, which can be used as bioreceptors for a wide range of targets such as proteins, small molecules, bacteria and even cells. It will also highlight how the invention of synthetic oligonucleotides such as peptide nucleic acid (PNA) or locked nucleic acid (LNA) has pushed the limits of molecular biology and biosensor development to new perspectives. These technologies are very promising albeit still in need of development in order to bridge the gap between the laboratory-based status and the reality of biomedical applications.
14
Vuoriranta, P., M. Männistö, and H. Soranummi. "Occurrence of Sphingomonas sp. bacteria in cold climate drinking water supply system biofilms." Water Supply 3, no. 1-2 (2003): 227–32. http://dx.doi.org/10.2166/ws.2003.0108.
Full textAbstract:
Members of the bacterial genus Sphingomonas (recently split into four genera), belonging to α-4-subclass of Proteobacteria, were isolated and characterised from water distribution network biofilms. Water temperature in the studied network, serving 200,000 people, is less than 5°C for about five months every winter. Sphingomonads, characterised using fluorescent oligonucleotide probes and fatty acid composition analysis (FAME), were a major group of bacteria among the distribution network biofilm isolates. Intact biofilms, grown on steel slides in a biofilm reactor fed with tap water, were detected in situ using fluorescence labelled oligonucleotide probes (FISH). Hybridisation with probes targeted on α-proteobacteria and sphingomonads was detected, but FISH on intact biofilms suffered from non-specific hybridisation and intensive autofluorescence, possibly due to extracellular material around the bacterial cells attached to biofilm. These preliminary results indicate that bacteria present in the distribution network biofilms in this study phylogenetically differ from those detected in more temperate regions.
15
Peplies, Jörg, Frank Oliver Glöckner, and Rudolf Amann. "Optimization Strategies for DNA Microarray-Based Detection of Bacteria with 16S rRNA-Targeting Oligonucleotide Probes." Applied and Environmental Microbiology 69, no. 3 (2003): 1397–407. http://dx.doi.org/10.1128/aem.69.3.1397-1407.2003.
Full textAbstract:
ABSTRACT The usability of the DNA microarray format for the specific detection of bacteria based on their 16S rRNA genes was systematically evaluated with a model system composed of six environmental strains and 20 oligonucleotide probes. Parameters such as secondary structures of the target molecules and steric hindrance were investigated to better understand the mechanisms underlying a microarray hybridization reaction, with focus on their influence on the specificity of hybridization. With adequate hybridization conditions, false-positive signals could be almost completely prevented, resulting in clear data interpretation. Among 199 potential nonspecific hybridization events, only 1 false-positive signal was observed, whereas false-negative results were more common (17 of 41). Subsequent parameter analysis revealed that this was mainly an effect of reduced accessibility of probe binding sites caused by the secondary structures of the target molecules. False-negative results could be prevented and the overall signal intensities could be adjusted by introducing a new optimization strategy called directed application of capture oligonucleotides. The small number of false-positive signals in our data set is discussed, and a general optimization approach is suggested. Our results show that, compared to standard hybridization formats such as fluorescence in situ hybridization, a large number of oligonucleotide probes with different characteristics can be applied in parallel in a highly specific way without extensive experimental effort.
16
Curti, Elena, Stephen J. Smerdon, and Elaine O. Davis. "Characterization of the Helicase Activity and Substrate Specificity of Mycobacterium tuberculosis UvrD." Journal of Bacteriology 189, no. 5 (2006): 1542–55. http://dx.doi.org/10.1128/jb.01421-06.
Full textAbstract:
ABSTRACT UvrD is a helicase that is widely conserved in gram-negative bacteria. A uvrD homologue was identified in Mycobacterium tuberculosis on the basis of the homology of its encoded protein with Escherichia coli UvrD, with which it shares 39% amino acid identity, distributed throughout the protein. The gene was cloned, and a histidine-tagged form of the protein was expressed and purified to homogeneity. The purified protein had in vitro ATPase activity that was dependent upon the presence of DNA. Oligonucleotides as short as four nucleotides were sufficient to promote the ATPase activity. The DNA helicase activity of the enzyme was only fueled by ATP and dATP. UvrD preferentially unwound 3′-single-stranded tailed duplex substrates over 5′-single-stranded ones, indicating that the protein had a duplex-unwinding activity with 3′-to-5′ polarity. A 3′ single-stranded DNA tail of 18 nucleotides was required for effective unwinding. By using a series of synthetic oligonucleotide substrates, we demonstrated that M. tuberculosis UvrD has an unwinding preference towards nicked DNA duplexes and stalled replication forks, representing the likely sites of action in vivo. The potential role of M. tuberculosis UvrD in maintenance of bacterial genomic integrity makes it a promising target for drug design against M. tuberculosis.
17
Laramée, Louise, John R. Lawrence, and Charles W. Greer. "Molecular analysis and development of 16S rRNA oligonucleotide probes to characterize a diclofop-methyl-degrading biofilm consortium." Canadian Journal of Microbiology 46, no. 2 (2000): 133–42. http://dx.doi.org/10.1139/w99-129.
Full textAbstract:
Genomic DNA from nine individual bacteria, isolated from a diclofop-methyl-degrading biofilm consortium, was extracted for genetic characterization. The degradation of diclofop-methyl produces metabolites that are known intermediates or substrates for bacteria that degrade a variety of chlorinated aromatic compounds. Accordingly, oligonucleotide primers were designed from specific catabolic genes for chlorinated organic degradation pathways, and tested by PCR to determine if these genes are involved in diclofop-methyl degradation. DNA homology between the PCR products and the known catabolic genes investigated by Southern hybridization analysis and by sequencing, suggested that novel catabolic genes are functioning in the isolates. Specific fluorescent oligonucleotides were designed for two of the isolates, following 16S rDNA sequencing and identification of each of the isolates. These probes were successfully used for fluorescent in situ hybridization (FISH) studies of the two isolates in the biofilm consortium. Key words: consortium, catabolic gene, diclofop-methyl, 16S rDNA, FISH, SCLM.
18
Jin, Sheng-Quan, Bin-Cheng Yin, and Bang-Ce Ye. "Multiplexed Bead-Based Mesofluidic System for Detection of Food-Borne Pathogenic Bacteria." Applied and Environmental Microbiology 75, no. 21 (2009): 6647–54. http://dx.doi.org/10.1128/aem.00854-09.
Full textAbstract:
ABSTRACT In the present study, a simple and rapid multiplexed bead-based mesofluidic system (BMS) was developed for simultaneous detection of food-borne pathogenic bacteria, including Staphylococcus aureus, Vibrio parahaemolyticus, Listeria monocytogenes, Salmonella, Enterobacter sakazakii, Shigella, Escherichia coli O157:H7, and Campylobacter jejuni. This system is based on utilization of isothiocyanate-modified microbeads that are 250 μm in diameter, which were immobilized with specific amino-modified oligonucleotide probes and placed in polydimethylsiloxane microchannels. PCR products from the pathogens studied were pumped into microchannels to hybridize with the oligonucleotide-modified beads, and hybridization signals were detected using a conventional microarray scanner. The short sequences of nucleic acids (21 bases) and PCR products characteristic of bacterial pathogens could be detected at concentrations of 1 pM and 10 nM, respectively. The detection procedure could be performed in less than 30 min with high sensitivity and specificity. The assay was simple and fast, and the limits of quantification were in the range from 500 to 6,000 CFU/ml for the bacterial species studied. The feasibility of identification of food-borne bacteria was investigated with samples contaminated with bacteria, including milk, egg, and meat samples. The results demonstrated that the BMS method can be used for effective detection of multiple pathogens in different foodstuffs.
19
Bertaux, J., M. Schmid, N. Chemidlin Prevost-Boure, et al. "In Situ Identification of Intracellular Bacteria Related to Paenibacillus spp. in the Mycelium of the Ectomycorrhizal Fungus Laccaria bicolor S238N." Applied and Environmental Microbiology 69, no. 7 (2003): 4243–48. http://dx.doi.org/10.1128/aem.69.7.4243-4248.2003.
Full textAbstract:
ABSTRACT Bacterial proliferations have recurrently been observed for the past 15 years in fermentor cultures of the ectomycorrhizal fungus Laccaria bicolor S238N, suggesting the presence of cryptic bacteria in the collection culture of this fungus. In this study, intracellular bacteria were detected by fluorescence in situ hybridization in combination with confocal laser scanning microscopy in several collection subcultures of L. bicolor S238N. They were small (0.5 μm in diameter), rare, and heterogeneously distributed in the mycelium and were identified as Paenibacillus spp. by using a 16S rRNA-directed oligonucleotide probe initially designed for bacteria isolated from a fermentor culture of L. bicolor S238N.
20
de las RIVAS, BLANCA, ÁNGELA MARCOBAL, ALFONSO V. CARRASCOSA, and ROSARIO MUÑOZ. "PCR Detection of Foodborne Bacteria Producing the Biogenic Amines Histamine, Tyramine, Putrescine, and Cadaverine." Journal of Food Protection 69, no. 10 (2006): 2509–14. http://dx.doi.org/10.4315/0362-028x-69.10.2509.
Full textAbstract:
This study describes an easy PCR method for the detection of foodborne bacteria that potentially produce histamine, tyramine, putrescine, and cadaverine. Synthetic oligonucleotide pairs for the specific detection of the gene coding for each group of bacterial histidine, tyrosine, ornithine, or lysine decarboxylases were designed. Under the conditions used in this study, the assay yielded fragments of 372 and 531 bp from histidine decarboxylase–encoding genes, a 825-bp fragment from tyrosine decarboxylases, fragments of 624 and 1,440 bp from ornithine decarboxylases, and 1,098- and 1,185-bp fragments from lysine decarboxylases. This is the first PCR method for detection of cadaverine-producing bacteria. The method was successfully applied to several biogenic amine–producing bacterial strains.
21
Hou, Xiao-li, Han-liang Jiang, Qing-yi Cao, Li-ying Zhao, Barbara J. Chang, and Zhi Chen. "Using oligonucleotide suspension arrays for laboratory identification of bacteria responsible for bacteremia." Journal of Zhejiang University SCIENCE B 9, no. 4 (2008): 291–98. http://dx.doi.org/10.1631/jzus.b0710470.
Full text
22
Jani, Saumya, Maria Soledad Ramirez, and Marcelo E. Tolmasky. "Silencing Antibiotic Resistance with Antisense Oligonucleotides." Biomedicines 9, no. 4 (2021): 416. http://dx.doi.org/10.3390/biomedicines9040416.
Full textAbstract:
Antisense technologies consist of the utilization of oligonucleotides or oligonucleotide analogs to interfere with undesirable biological processes, commonly through inhibition of expression of selected genes. This field holds a lot of promise for the treatment of a very diverse group of diseases including viral and bacterial infections, genetic disorders, and cancer. To date, drugs approved for utilization in clinics or in clinical trials target diseases other than bacterial infections. Although several groups and companies are working on different strategies, the application of antisense technologies to prokaryotes still lags with respect to those that target other human diseases. In those cases where the focus is on bacterial pathogens, a subset of the research is dedicated to produce antisense compounds that silence or reduce expression of antibiotic resistance genes. Therefore, these compounds will be adjuvants administered with the antibiotic to which they reduce resistance levels. A varied group of oligonucleotide analogs like phosphorothioate or phosphorodiamidate morpholino residues, as well as peptide nucleic acids, locked nucleic acids and bridge nucleic acids, the latter two in gapmer configuration, have been utilized to reduce resistance levels. The major mechanisms of inhibition include eliciting cleavage of the target mRNA by the host’s RNase H or RNase P, and steric hindrance. The different approaches targeting resistance to β-lactams include carbapenems, aminoglycosides, chloramphenicol, macrolides, and fluoroquinolones. The purpose of this short review is to summarize the attempts to develop antisense compounds that inhibit expression of resistance to antibiotics.
23
Davenport, Colin F., and Burkhard Tümmler. "Abundant Oligonucleotides Common to Most Bacteria." PLoS ONE 5, no. 3 (2010): e9841. http://dx.doi.org/10.1371/journal.pone.0009841.
Full text
24
DUFLOS, GUILLAUME, LAURENCE THERAULAZ, GERARD GIORDANO, VINCENT MEJEAN, and PIERRE MALLE. "Quantitative PCR Method for Evaluating Freshness of Whiting (Merlangius merlangus) and Plaice (Pleuronectes platessa)." Journal of Food Protection 73, no. 7 (2010): 1344–47. http://dx.doi.org/10.4315/0362-028x-73.7.1344.
Full textAbstract:
We have developed a method for rapid quantification of fish spoilage bacteria based on quantitative PCR with degenerated oligonucleotides that hybridize on the torA gene coding for trimethylamine N-oxide reductase, one of the major bacterial enzymes in fish spoilage. To show the utility of this gene, we used a regular PCR with DNA extracts from whiting (Merlangius merlangus) and plaice (Pleuronectes platessa) stored in ice. Quantitative PCR showed that the number of copies of the torA gene, i.e., the number of spoilage bacteria, increases with length of storage. This approach can therefore be used to evaluate freshness for the two fish species studied (whiting and plaice).
25
Fuchs, Bernhard M., Kazuaki Syutsubo, Wolfgang Ludwig, and Rudolf Amann. "In Situ Accessibility of Escherichia coli 23S rRNA to Fluorescently Labeled Oligonucleotide Probes." Applied and Environmental Microbiology 67, no. 2 (2001): 961–68. http://dx.doi.org/10.1128/aem.67.2.961-968.2001.
Full textAbstract:
ABSTRACT One of the main causes of failure of fluorescence in situ hybridization with rRNA-targeted oligonucleotides, besides low cellular ribosome content and impermeability of cell walls, is the inaccessibility of probe target sites due to higher-order structure of the ribosome. Analogous to a study on the 16S rRNA (B. M. Fuchs, G. Wallner, W. Beisker, I. Schwippl, W. Ludwig, and R. Amann, Appl. Environ. Microbiol. 64:4973–4982, 1998), the accessibility of the 23S rRNA of Escherichia coli DSM 30083T was studied in detail with a set of 184 CY3-labeled oligonucleotide probes. The probe-conferred fluorescence was quantified flow cytometrically. The brightest signal resulted from probe 23S-2018, complementary to positions 2018 to 2035. The distribution of probe-conferred cell fluorescence in six arbitrarily set brightness classes (classes I to VI, 100 to 81%, 80 to 61%, 60 to 41%, 40 to 21%, 20 to 6%, and 5 to 0% of the brightness of 23S-2018, respectively) was as follows: class I, 3%; class II, 21%; class III, 35%; class IV, 18%; class V, 16%; and class VI, 7%. A fine-resolution analysis of selected areas confirmed steep changes in accessibility on the 23S RNA to oligonucleotide probes. This is similar to the situation for the 16S rRNA. Indeed, no significant differences were found between the hybridization of oligonucleotide probes to 16S and 23S rRNA. Interestingly, indications were obtained of an effect of the type of fluorescent dye coupled to a probe on in situ accessibility. The results were translated into an accessibility map for the 23S rRNA ofE. coli, which may be extrapolated to other bacteria. Thereby, it may contribute to a better exploitation of the high potential of the 23S rRNA for identification of bacteria in the future.
26
Pereira, Sara, Ruwei Yao, Mariana Gomes, et al. "Can Vitamin B12 Assist the Internalization of Antisense LNA Oligonucleotides into Bacteria?" Antibiotics 10, no. 4 (2021): 379. http://dx.doi.org/10.3390/antibiotics10040379.
Full textAbstract:
The emergence of bacterial resistance to traditional small-molecule antibiotics is fueling the search for innovative strategies to treat infections. Inhibiting the expression of essential bacterial genes using antisense oligonucleotides (ASOs), particularly composed of nucleic acid mimics (NAMs), has emerged as a promising strategy. However, their efficiency depends on their association with vectors that can translocate the bacterial envelope. Vitamin B12 is among the largest molecules known to be taken up by bacteria and has very recently started to gain interest as a trojan-horse vector. Gapmers and steric blockers were evaluated as ASOs against Escherichia coli (E. coli). Both ASOs were successfully conjugated to B12 by copper-free azide-alkyne click-chemistry. The biological effect of the two conjugates was evaluated together with their intracellular localization in E. coli. Although not only B12 but also both B12-ASO conjugates interacted strongly with E. coli, they were mostly colocalized with the outer membrane. Only 6–9% were detected in the cytosol, which showed to be insufficient for bacterial growth inhibition. These results suggest that the internalization of B12-ASO conjugates is strongly affected by the low uptake rate of the B12 in E. coli and that further studies are needed before considering this strategy against biofilms in vivo.
27
Guz, Leszek, and Krzysztof Puk. "Molecular detection of Renibacterium salmoninarum in rainbow trout (Oncorhynchus mykiss) from Poland." Fisheries & Aquatic Life 28, no. 4 (2020): 234–37. http://dx.doi.org/10.2478/aopf-2020-0028.
Full textAbstract:
Abstract Renibacterium salmoninarum causes bacterial kidney disease mainly in salmonid fish. Oligonucleotide primers incorporating R. salmoninarum unique sequences were designed to amplify a 501 bp region of the gene encoding a 57 kDa soluble extra-cellular protein. The primers did not amplify other wide varieties of aquatic or piscine bacteria Aeromonas salmonicida or Yersinia ruckeri. This assay provides a molecular description and definitive identification of R. salmoninarum in Poland.
28
Llobet-Brossa, Enric, Ramon Rosselló-Mora, and Rudolf Amann. "Microbial Community Composition of Wadden Sea Sediments as Revealed by Fluorescence In Situ Hybridization." Applied and Environmental Microbiology 64, no. 7 (1998): 2691–96. http://dx.doi.org/10.1128/aem.64.7.2691-2696.1998.
Full textAbstract:
ABSTRACT The microbial community composition of Wadden Sea sediments of the German North Sea coast was investigated by in situ hybridization with group-specific fluorescently labeled, rRNA-targeted oligonucleotides. A large fraction (up to 73%) of the DAPI (4′,6-diamidino-2-phenylindole)-stained cells hybridized with the bacterial probes. Nearly 45% of the total cells could be further identified as belonging to known phyla. Members of theCytophaga-Flavobacterium cluster were most abundant in all layers, followed by the sulfate-reducing bacteria.
29
Jürgens, Klaus, Jakob Pernthaler, Sven Schalla, and Rudolf Amann. "Morphological and Compositional Changes in a Planktonic Bacterial Community in Response to Enhanced Protozoan Grazing." Applied and Environmental Microbiology 65, no. 3 (1999): 1241–50. http://dx.doi.org/10.1128/aem.65.3.1241-1250.1999.
Full textAbstract:
ABSTRACT We analyzed changes in bacterioplankton morphology and composition during enhanced protozoan grazing by image analysis and fluorescent in situ hybridization with group-specific rRNA-targeted oligonucleotide probes. Enclosure experiments were conducted in a small, fishless freshwater pond which was dominated by the cladoceran Daphnia magna. The removal of metazooplankton enhanced protozoan grazing pressure and triggered a microbial succession from fast-growing small bacteria to larger grazing-resistant morphotypes. These were mainly different types of filamentous bacteria which correlated in biomass with the population development of heterotrophic nanoflagellates (HNF). Small bacterial rods and cocci, which showed increased proportion after removal of Daphnia and doubling times of 6 to 11 h, belonged nearly exclusively to the beta subdivision of the classProteobacteria and the Cytophaga-Flavobacteriumcluster. The majority of this newly produced bacterial biomass was rapidly consumed by HNF. In contrast, the proportion of bacteria belonging to the gamma and alpha subdivisions of theProteobacteria increased throughout the experiment. The alpha subdivision consisted mainly of rods that were 3 to 6 μm in length, which probably exceeded the size range of bacteria edible by protozoa. Initially, these organisms accounted for less than 1% of total bacteria, but after 72 h they became the predominant group of the bacterial assemblage. Other types of grazing-resistant, filamentous bacteria were also found within the beta subdivision ofProteobacteria and the Cytophaga-Flavobacteriumcluster. We conclude that the predation regimen is a major structuring force for the bacterial community composition in this system. Protozoan grazing resulted in shifts of the morphological as well as the taxonomic composition of the bacterial assemblage. Grazing-resistant filamentous bacteria can develop within different phylogenetic groups of bacteria, and formerly underepresented taxa might become a dominant group when protozoan predation is the major selective pressure.
30
Kenzaka, Takehiko, Ai Ishidoshiro, Nobuyasu Yamaguchi, Katsuji Tani, and Masao Nasu. "rRNA Sequence-Based Scanning Electron Microscopic Detection of Bacteria." Applied and Environmental Microbiology 71, no. 9 (2005): 5523–31. http://dx.doi.org/10.1128/aem.71.9.5523-5531.2005.
Full textAbstract:
ABSTRACT A new scanning electron microscopic method was developed for gaining both phylogenetic and morphological information about target microbes using in situ hybridization with rRNA-targeted oligonucleotide probes (SEM-ISH). Target cells were hybridized with oligonucleotide probes after gold labeling. Gold enhancement was used for amplification of probe signals from hybridized cells. The hybridized cells released a strong backscatter electron signal due to accumulation of gold atoms inside cells. SEM-ISH was applied to analyze bacterial community composition in freshwater samples, and bacterial cell counts determined by SEM-ISH with rRNA-targeted probes for major phyla within the domain Bacteria were highly correlated to those by fluorescent in situ hybridization (FISH). The bacterial composition on surface of river sediment particles before and after cell dispersion treatment by sonication was successfully revealed by SEM-ISH. Direct enumeration of bacterial cells on the surface of sonicated sediment particles by SEM-ISH demonstrated that members of Cytophaga-Flavobacterium existed tightly on the surface of particles. SEM-ISH allows defining the number and distribution of phylogenetically defined cells adherent to material surfaces, which is difficult in FISH, and it gives new insight into electron microscopic studies of microorganisms in their natural environment.
31
Al-Khaldi, Sufian F., Scott A. Martin, Avraham Rasooly, and Jeff D. Evans. "DNA Microarray Technology Used for Studying Foodborne Pathogens and Microbial Habitats: Minireview." Journal of AOAC INTERNATIONAL 85, no. 4 (2002): 906–10. http://dx.doi.org/10.1093/jaoac/85.4.906.
Full textAbstract:
Abstract Microarray analysis is an emerging technology that has the potential to become a leading trend in bacterial identification in food and feed improvement. The technology uses fluorescent-labeled probes amplified from bacterial samples that are then hybridized to thousands of DNA sequences immobilized on chemically modified glass slides. The whole gene or open reading frame(s) is represented by a polymerase chain reaction fragment of double-strand DNA, approximately 1000 base pair (bp) or 20–70 bp single-strand oligonucleotides. The technology can be used to identify bacteria and to study gene expression in complex microbial populations, such as those found in food and gastrointestinal tracts. Data generated by microarray analysis can be potentially used to improve the safety of our food supply as well as ensure the efficiency of animal feed conversion to human food, e.g., in meat and milk production by ruminants. This minireview addresses the use of microarray technology in bacterial identification and gene expression in different microbial systems and in habitats containing mixed populations of bacteria.
32
Fuchs, Bernhard Maximilian, Günter Wallner, Wolfgang Beisker, Ines Schwippl, Wolfgang Ludwig, and Rudolf Amann. "Flow Cytometric Analysis of the In Situ Accessibility of Escherichia coli 16S rRNA for Fluorescently Labeled Oligonucleotide Probes." Applied and Environmental Microbiology 64, no. 12 (1998): 4973–82. http://dx.doi.org/10.1128/aem.64.12.4973-4982.1998.
Full textAbstract:
ABSTRACT In situ identification of whole fixed bacterial cells by hybridization with fluorescently labeled, rRNA-targeted oligonucleotide probes is often limited by low signal intensities. In addition to an impermeability of the cell periphery and a low cellular rRNA content, the three-dimensional structure of the ribosome may hinder the access of oligonucleotides to their target sites. Until now, a systematic study on the accessibility of 16S rRNA target sites had not been done. Here, we report fluorescence intensities obtained with more than 200 oligonucleotide probes (mostly 18-mers) used with whole fixed cells ofEscherichia coli DSM 30083T. Two overlapping sets of adjacent oligonucleotides, 171 in total, were designed to cover the full length of the 16S rRNA. The two sets are shifted by 5 to 13 nucleotides. The probes were labeled with carboxyfluorescein, and signal intensities of hybridized cells were quantified by flow cytometry. Care was taken that the signal intensity of cells was dependent solely on the in situ accessibility of probe target sites. The brightest signal resulted from probe Eco1482, complementary to positions 1482 to 1499. With this probe, the fluorescence was 1.7 times brighter than that of the standard bacterial probe EUB338 and 44 times brighter than that of the worst probe, Eco468. The distribution of probe-conferred cell fluorescence in six arbitrarily set brightness classes (classes I to VI; 100 to 81%, 80 to 61%, 60 to 41%, 40 to 21%, 20 to 6%, and 5 to 0% of the brightness with Eco1482, respectively) was as follows: I, 4%; II, 14%; III, 21%; IV, 29%, V, 19%; and VI, 13%. A more detailed analysis of helices 6, 18, and 23 with additional probes demonstrated that a shift of the target region by only a few bases could result in a decline of cell fluorescence from >80 to <10%. Considering the high evolutionary conservation of 16S rRNA, the in situ accessibility map of E. coli should facilitate a more rational selection of probe target sites for other species as well.
33
Sekiguchi, Yuji, Yoichi Kamagata, Kazunori Nakamura, Akiyoshi Ohashi, and Hideki Harada. "Fluorescence In Situ Hybridization Using 16S rRNA-Targeted Oligonucleotides Reveals Localization of Methanogens and Selected Uncultured Bacteria in Mesophilic and Thermophilic Sludge Granules." Applied and Environmental Microbiology 65, no. 3 (1999): 1280–88. http://dx.doi.org/10.1128/aem.65.3.1280-1288.1999.
Full textAbstract:
ABSTRACT 16S rRNA-targeted in situ hybridization combined with confocal laser scanning microscopy was used to elucidate the spatial distribution of microbes within two types of methanogenic granular sludge, mesophilic (35°C) and thermophilic (55°C), in upflow anaerobic sludge blanket reactors fed with sucrose-, acetate-, and propionate-based artificial wastewater. The spatial organization of the microbes was visualized in thin sections of the granules by using fluorescent oligonucleotide probes specific to several phylogenetic groups of microbes. In situ hybridization with archaeal- and bacterial-domain probes within granule sections clearly showed that both mesophilic and thermophilic granules had layered structures and that the outer layer harbored mainly bacterial cells while the inner layer consisted mainly of archaeal cells. Methanosaeta-,Methanobacterium-, Methanospirillum-, andMethanosarcina-like cells were detected with oligonucleotide probes specific for the different groups of methanogens, and they were found to be localized inside the granules, in both types of which dominant methanogens were members of the genusMethanosaeta. For specific detection of bacteria which were previously detected by whole-microbial-community 16S ribosomal DNA (rDNA)-cloning analysis (Y. Sekiguchi, Y. Kamagata, K. Syutsubo, A. Ohashi, H. Harada, and K. Nakamura, Microbiology 144:2655–2665, 1998) we designed probes specific for clonal 16S rDNAs related to unidentified green nonsulfur bacteria and clones related toSyntrophobacter species. The probe designed for the cluster closely related to Syntrophobacter species hybridized with coccoid cells in the inner layer of the mesophilic granule sections. The probe for the unidentified bacteria which were clustered with the green nonsulfur bacteria detected filamentous cells in the outermost layer of the thermophilic sludge granule sections. These results revealed the spatial organizations of methanogens and uncultivated bacteria and their in situ morphologies and metabolic functions in both mesophilic and thermophilic granular sludges.
34
Ouverney, Cleber C., Gary C. Armitage, and David A. Relman. "Single-Cell Enumeration of an Uncultivated TM7 Subgroup in the Human Subgingival Crevice." Applied and Environmental Microbiology 69, no. 10 (2003): 6294–98. http://dx.doi.org/10.1128/aem.69.10.6294-6298.2003.
Full textAbstract:
ABSTRACT Specific oligonucleotide hybridization conditions were established for single-cell enumeration of uncultivated TM7 and IO25 bacteria by using clones expressing heterologous 16S rRNA. In situ analysis of human subgingival crevice specimens revealed that a greater proportion of samples from sites of chronic periodontitis than from healthy sites contained TM7 subgroup IO25. In addition, IO25 bacterial cells from periodontitis site samples were more abundant and fourfold longer than IO25 cells from healthy site samples.
35
da Silva, Serge Gomes, David C. Gillan, Nicole Dubilier, and Chantal De Ridder. "Characterization by 16S rRNA gene analysis and in situ hybridization of bacteria living in the hindgut of a deposit-feeding echinoid (Echinodermata)." Journal of the Marine Biological Association of the United Kingdom 86, no. 5 (2006): 1209–13. http://dx.doi.org/10.1017/s0025315406014202.
Full textAbstract:
The hindgut caecum of the deposit-feeding echinoid Echinocardium cordatum harbours a symbiotic bacterial microflora, organized into layered mats around detrital particles owing to the proliferation of filamentous bacteria. The bacterial community was analysed using 16S rRNA gene analysis and fluorescence in situ hybridization. The purpose was to characterize its biodiversity and to identify its predominant members. The majority of the 16S sequences belong to the δ-Proteobacteria (61.5%), the Bacteroidetes and the Firmicutes constitute the two other main bacterial groups (respectively 23.1% and 15.4%). A total of 41% of the δ-Proteobacteria clones isolated belong to a bacterium of the genus Desulfonema for which a specific oligonucleotide probe was designed, enabling identification of its distribution in the nodules.
36
Morris, Cindy E., Jean-Michel Monier, and Marie-Agnès Jacques. "A Technique To Quantify the Population Size and Composition of the Biofilm Component in Communities of Bacteria in the Phyllosphere." Applied and Environmental Microbiology 64, no. 12 (1998): 4789–95. http://dx.doi.org/10.1128/aem.64.12.4789-4795.1998.
Full textAbstract:
ABSTRACT The presence of microbial biofilms in the phyllosphere of terrestrial plants has recently been demonstrated, but few techniques to study biofilms associated with living plant tissues are available. Here we report a technique to estimate the proportion of the bacterial population on leaves that is assembled in biofilms and to quantitatively isolate bacteria from the biofilm and nonbiofilm (solitary) components of phyllosphere microbial communities. This technique is based on removal of bacteria from leaves by gentle washing, separation of biofilm and solitary bacteria by filtration, and disintegration of biofilms by ultrasonication. The filters used for this technique were evaluated for their nonspecific retention rates of solitary bacteria and for the efficiency of filtration for different concentrations of solitary bacteria in the presence of biofilms and other particles. The lethality and efficiency of disintegration of the sonication conditions used here were also evaluated. Isolation and quantification of bacteria by this technique is based on use of culture media. However, oligonucleotide probes, sera, or epifluorescent stains could also be used for direct characterization of the biofilm and solitary bacteria in the suspensions generated by this technique. Preliminary results from estimates of biofilm abundance in phyllosphere communities show that bacteria in biofilms constitute between about 10 and 40% of the total bacterial population on broad-leaf endive and parsley leaves.
37
Prado, Mario Duran, Rafael De Prado, and Antonio R. Franco. "Design and optimization of degenerated universal primers for the cloning of the plant acetolactate synthase conserved domains." Weed Science 52, no. 4 (2004): 487–91. http://dx.doi.org/10.1614/ws-03-098r.
Full textAbstract:
A set of universal and degenerate primers has been designed to clone (by polymerase chain reaction [PCR]) the conserved domains of the acetolactate synthase (ALS) gene where mutations confer resistance to ALS herbicides in plants. These primers were successful in cloning conserved domains of ALS in all monocotyledonous and dicotyledonous plants tested to date, as well as that of bacteria. Total genomic DNA was used as the source of target DNA because no introns were found in the sequences to be amplified. The design of the universal primers was performed after subtle modifications of the consensus degenerate hybrid oligonucleotide primers approach, which implies the synthesis of hybrid oligonucleotide primers containing fixed clamp 5′ and degenerate core 3′ sequences. Optimizations of PCR reactions were done according to a Taguchy approach described for the first time with degenerate oligonucleotides. This method optimizes a PCR reaction using four variables (deoxynucleoside triphosphate, DNA, primers, and Mg2+) under three different concentrations per variable using just nine reactions. The ALS herbicide-binding domains from many susceptible and resistant plants can be cloned and sequenced in a few hours by using only 100 mg of starting plant material, like one leaf or several small seedlings or seeds.
38
Carroll, Nora M., Emma E. M. Jaeger, Sarah Choudhury, et al. "Detection of and Discrimination between Gram-Positive and Gram-Negative Bacteria in Intraocular Samples by Using Nested PCR." Journal of Clinical Microbiology 38, no. 5 (2000): 1753–57. http://dx.doi.org/10.1128/jcm.38.5.1753-1757.2000.
Full textAbstract:
A nested PCR protocol has been developed for the detection of and discrimination between 14 species of gram-positive and -negative bacteria in samples of ocular fluids. First-round PCR with pan-bacterial oligonucleotide primers, based on conserved sequences of the 16S ribosomal gene, was followed by a gram-negative-organism-specific PCR, which resulted in a single 985-bp amplification product, and a multiplex PCR which resulted in two PCR products: a 1,025 bp amplicon (all bacteria) and a 355 bp amplicon (gram-positive bacteria only). All products were detected by gel electrophoresis. The sensitivity of the assay was between 10 fg and 1 pg of bacterial DNA, depending on the species tested, equivalent to between 24 and 4 live bacteria spiked in water. The identification was complete in 3.5 h. The molecular techniques were subsequently applied to four samples of intraocular fluid, (three vitreous and one aqueous) from three patients with clinical signs of bacterial endophthalmitis (test samples) and two samples of vitreous from a patient with chronic intraocular inflammation (control samples). In all culture-positive samples (two of three vitreous and one of one aqueous), a complete concordance was observed between molecular methods and culture results. PCR correctly identified the gram stain classification of the organisms. The bacterial etiology was also identified in a culture-negative patient with clinical history and signs highly suggestive of bacterial endophthalmitis. Furthermore, control samples from a patient with chronic intraocular inflammation remained PCR negative. In summary, this protocol has demonstrated potential as a rapid diagnostic test in confirming the diagnosis of infection and also determining the Gram status of bacteria with high specificity and sensitivity.
39
Schramm, A., L. H. Larsen, N. P. Revsbech, and R. I. Amann. "Structure and function of a nitrifying biofilm as determined by microelectrodes and fluorescent oligonucleotide probes." Water Science and Technology 36, no. 1 (1997): 263–70. http://dx.doi.org/10.2166/wst.1997.0062.
Full textAbstract:
Microelectrodes for O2 and NO2−/NO3− and fluorescently labelled 16S rRNA-targeted oligonucleotide probes were combined to examine the activity and stratification of nitrifying bacteria in a trickling filter biofilm. Microprofiles showed that O2 consumption and NO3−/NO2− production were restricted to the upper 50-100 μm of the biofilm. The vertical distribution of the nitrifying bacteria Nitrosomonas sp. and Nitrobacter sp. was investigated by fluorescent in situ hybridisation (FISH) with specific oligonucleotides. Nitrifiers formed a dense layer of cells and cell clusters in the upper part of the biofilm. This correlates well with the measured activity profiles. Ammonia- and nitrite-oxidisers occurred in close vicinity to each other supporting a fast sequential metabolism from ammonia to nitrate. Both species were not restricted to the oxic part of the biofilm, but also appeared -in lower numbers- in the anoxic layers on the bottom of the biofilm. A short term decrease in the O2 concentration of the bulk water resulted in a quick decrease in O2 penetration and metabolic rates inside the biofilm. However, neither the stratification nor the cellular ribosome content of nitrifiers changed within a few hours.
40
Swingle, Bryan, Eric Markel, Nina Costantino, Mikhail G. Bubunenko, Samuel Cartinhour, and Donald L. Court. "Oligonucleotide recombination in Gram-negative bacteria." Molecular Microbiology 75, no. 1 (2010): 138–48. http://dx.doi.org/10.1111/j.1365-2958.2009.06976.x.
Full text
41
Liu, Yi, Jin-Xiang Han, Hai-Yan Huang, and Bo Zhu. "Development and Evaluation of 16S rDNA Microarray for Detecting Bacterial Pathogens in Cerebrospinal Fluid." Experimental Biology and Medicine 230, no. 8 (2005): 587–91. http://dx.doi.org/10.1177/153537020523000810.
Full textAbstract:
The rapid identification of bacteria in cerebrospinal fluid (CSF) is very Important for patient management and antimicrobial therapies. We developed a 16S DNA microarray–based method that targets 16S rDNA and can directly detect bacteria from CSF without cultivation. Universal primers and specific probes were designed from the 16S rDNA sequence data retrieved directly from the GenBank database. The specificity of the assay is obtained through a combination of microarray hybridization and enzymatic labeling of the constructed specific probes. Cultivation-dependent assays were used as reference methods in the development and evaluation of the method. With the exception of Mycobacterium tuberculosis and Proteus mirabilis, forty-five positive blood culture media were successfully differentiated. When this procedure was applied directly to 100 CSF specimens, 29 specimens from 16 patients were positive by bacterial culture and 3 culture-positive CSF specimens produced no hybridized signals. The remaining 26 specimens were correctly identified, including one with mixed infection. The accuracy, sensitivity, and specificity of the assay can be increased further by designing more oligonucleotides for the microarray. This method is versatile and makes it possible to detect more bacteria in a single assay and discriminate different bacterial genera.
42
Lehtola, Markku J., Eila Torvinen, Ilkka T. Miettinen, and C. William Keevil. "Fluorescence In Situ Hybridization Using Peptide Nucleic Acid Probes for Rapid Detection of Mycobacterium avium subsp. avium and Mycobacterium avium subsp. paratuberculosis in Potable-Water Biofilms." Applied and Environmental Microbiology 72, no. 1 (2006): 848–53. http://dx.doi.org/10.1128/aem.72.1.848-853.2006.
Full textAbstract:
ABSTRACT Here, we present for the first time a high-affinity peptide nucleic acid (PNA) oligonucleotide sequence for detecting Mycobacterium avium bacteria, including the opportunistically pathogenic subspecies M. avium subsp. avium, M. avium subsp. paratuberculosis, and M. avium subsp. silvaticum, by the fluorescence in situ hybridization (FISH) method. There is evidence that M. avium subsp. avium especially is able to survive and grow in drinking-water biofilms and possibly transmit via drinking water. The designed PNA probe (MAV148) specificity was tested with several bacterial species, including other mycobacteria and mycolic acid-containing bacteria. From the range of bacterial strains tested, only M. avium subsp. avium and M. avium subsp. paratuberculosis strains were hybridized. The PNA FISH method was applied successfully to detect M. avium subsp. avium spiked in water samples and biofilm established within a Propella biofilm reactor fed with potable water from a distribution supply.
43
Rudi, Knut, Olav M. Skulberg, Frank Larsen, and Kjetill S. Jakobsen. "Quantification of Toxic Cyanobacteria in Water by Use of Competitive PCR Followed by Sequence-Specific Labeling of Oligonucleotide Probes." Applied and Environmental Microbiology 64, no. 7 (1998): 2639–43. http://dx.doi.org/10.1128/aem.64.7.2639-2643.1998.
Full textAbstract:
ABSTRACT A complete nucleic-acid-based assay which consists of sample preparation, DNA amplification, and chromogenic detection was developed for quantifying potential toxin-producing cyanobacteria of interest to the public. The sample preparation strategy involves the same solid phase for cell concentration and DNA purification. For the detection step, we used a combination of competitive PCR amplification, sequence-specific labeling of oligonucleotide probes, hybridization of the labeled oligonucleotides to immobilized complements and, finally, chromogenic detection. The complete assay was tested with water containing toxin-producing cyanobacteria belonging to the genusMicrocystis. A detection limit of 100 cells/ml and a quantitative range of more than 3 orders of magnitude were obtained. This approach can easily be adapted to a wide range of bacterial species and has the potential for simultaneous detection and quantitation of several different target organisms by a single assay.
44
Zeninskaya, N. A., A. V. Kolesnikov, A. K. Ryabko, I. G. Shemyakin, I. A. Dyatlov, and A. V. Kozyr. "Aptamers in the Treatment of Bacterial Infections: Problems and Prospects." Annals of the Russian academy of medical sciences 71, no. 5 (2016): 350–58. http://dx.doi.org/10.15690/vramn591.
Full textAbstract:
Aptamers are short single-stranded oligonucleotides which are selected via targeted chemical evolution in vitro to bind a molecular target of interest. The aptamer selection technology is designated as SELEX (Systematic evolution of ligands by exponential enrichment). SELEX enables isolation of oligonucleotide aptamers binding a wide range of targets of interest with little respect for their nature and molecular weight. A number of applications of aptamer selection were developed ranging from biosensor technologies to antitumor drug discovery. First aptamer-based pharmaceutical (Macugen) was approved by FDA for clinical use in 2004, and since then more than ten aptamer-based drugs undergo various phases of clinical trials. From the medicinal chemist’s point of view, aptamers represent a new class of molecules suitable for the development of new therapeutics. Due to the stability, relative synthesis simplicity, and development of advanced strategies of target specific molecular selection, aptamers attract increased attention of drug discovery community. Difficulties of the development of next-generation antibiotics basing on the conventional basis of combinatorial chemistry and high-throughput screening have also amplified the interest to aptamer-based therapeutic candidates. The present article reviews the investigations focused on the development of antibacterial aptamers and discusses the potential and current limitations of the use of this type of therapeutic molecules.
45
Wolf-Jäckel, Godelind A., Mette Boye, Øystein Angen, Matthias Müller, and Tim K. Jensen. "Fluorescence in situ hybridization in species-specific diagnosis of ovine Campylobacter abortions." Journal of Veterinary Diagnostic Investigation 32, no. 3 (2020): 413–19. http://dx.doi.org/10.1177/1040638720915678.
Full textAbstract:
Campylobacter infection is a leading cause of ovine abortion worldwide. Campylobacter fetus and C. jejuni are the major species involved. We report herein on abortion storms in 4 Danish sheep flocks. Initially, no pathogenic bacteria were isolated from placental and fetal tissues on aerobic and selective media despite the presence of severe suppurative and necrotizing placentitis with numerous bacteria located intracellularly in trophoblasts. Fluorescence in situ hybridization (FISH) was then applied on abortion material from 13 cases; species-specific oligonucleotide probes directed against either C. fetus or C. jejuni were used in combination with a general bacterial probe. C. fetus was detected as the only lesion-associated bacterial species in 4 cases from 2 flocks, and C. jejuni in 6 cases from the other 2 flocks, thereby establishing the likely etiology of the abortion storms in all 4 flocks. FISH is a useful detection tool in culture-negative cases with tissue lesions suggestive of bacterial infection. Furthermore, FISH is a fast and economical method to detect and identify the zoonotic agent Campylobacter within ovine abortion material.
46
Cottrell, Matthew T., and David L. Kirchman. "Natural Assemblages of Marine Proteobacteria and Members of the Cytophaga-Flavobacter Cluster Consuming Low- and High-Molecular-Weight Dissolved Organic Matter." Applied and Environmental Microbiology 66, no. 4 (2000): 1692–97. http://dx.doi.org/10.1128/aem.66.4.1692-1697.2000.
Full textAbstract:
ABSTRACT We used a method that combines microautoradiography with hybridization of fluorescent rRNA-targeted oligonucleotide probes to whole cells (MICRO-FISH) to test the hypothesis that the relative contributions of various phylogenetic groups to the utilization of dissolved organic matter (DOM) depend solely on their relative abundance in the bacterial community. We found that utilization of even simple low-molecular-weight DOM components by bacteria differed across the major phylogenetic groups and often did not correlate with the relative abundance of these bacterial groups in estuarine and coastal environments. The Cytophaga-Flavobacter cluster was overrepresented in the portion of the assemblage consuming chitin,N-acetylglucosamine, and protein but was generally underrepresented in the assemblage consuming amino acids. The amino acid-consuming assemblage was usually dominated by the α subclass of the class Proteobacteria, although the representation of α-proteobacteria in the protein-consuming assemblages was about that expected from their relative abundance in the entire bacterial community. In our experiments, no phylogenetic group dominated the consumption of all DOM, suggesting that the participation of a diverse assemblage of bacteria is essential for the complete degradation of complex DOM in the oceans. These results also suggest that the role of aerobic heterotrophic bacteria in carbon cycling would be more accurately described by using three groups instead of the single bacterial compartment currently used in biogeochemical models.
47
Hess, Sebastian, Andreas Suthaus, and Michael Melkonian. "“Candidatus Finniella” (Rickettsiales, Alphaproteobacteria), Novel Endosymbionts of Viridiraptorid Amoeboflagellates (Cercozoa, Rhizaria)." Applied and Environmental Microbiology 82, no. 2 (2015): 659–70. http://dx.doi.org/10.1128/aem.02680-15.
Full textAbstract:
ABSTRACTTheRickettsiales(Alphaproteobacteria) are obligate intracellular bacteria that colonize a wide range of eukaryotic hosts, including diverse metazoa and protists. Here, we characterize rickettsial endosymbionts discovered in the cytoplasm of the algivorous amoeboflagellatesViridiraptor invadensandOrciraptor agilis(Viridiraptoridae, Cercozoa, Rhizaria), supplying evidence of free-living, phagotrophic members of the Cercozoa serving as hosts forRickettsiales. According to 16S rRNA gene phylogenies, the bacteria represent two closely related but distinct genotypes within a deep-branching rickettsial clade, which contains the genera “CandidatusOdyssella,” “CandidatusParacaedibacter,” and “CandidatusCaptivus.” Using the full-cycle rRNA approach, we detected the novel bacteria in four of nine viridiraptorid strains tested. Furthermore, two specific oligonucleotide probes with a single-nucleotide-difference discriminated both bacterial genotypes by fluorescencein situhybridization (FISH). We establish the candidate species “CandidatusFinniella inopinata” (found inViridiraptor invadens) and “CandidatusFinniella lucida” (found inOrciraptor agilis) for the novel bacteria and propose a new, provisional family ofRickettsiales, “CandidatusParacaedibacteraceae.”
48
Uehara, Taketo, Atsuhiko Kushida, and Yoji Momota. "Rapid and sensitive identification of Pratylenchus spp. using reverse dot blot hybridization." Nematology 1, no. 5 (1999): 549–55. http://dx.doi.org/10.1163/156854199508441.
Full textAbstract:
AbstractA reverse dot blot assay for identification of Pratylenchus spp. has been developed using specific oligonucleotides designed from the sequence of the internal transcribed spacer (ITS) region. The reverse dot blot is a technique which can be especially used for the simultaneous identification of various bacteria. The target fragment was amplified, and labelled with digoxigenine by a polymerase chain reaction (PCR). The amplified fragment was hybridised with the membrane-immobilised oligonucleotide and the hybridization was detected non-radioactively. By this assay, it was possible to identify P. penetrans, P. coffeae, P. vulnus, P. loosi, P. brachyurus, P. crenatus and P. zeae in a single hybridization. Identification rapide et fiable de Pratylenchus spp. a l'aide de l'hybridation retro dot blot - Une technique retro dot blot pour l'identification de Pratylenchus spp. a ete mise au point en utilisant des oligonucleotides specifiques derives de la sequence de l'espaceur transcrit interne (ITS). La technique dot blot est une technique qui peut etre specialement utilisee pour l'identification simultanee de nombreuses bacteries. Le fragment cible a ete amplifie et marque par la digoxigenine a l'aide de la reaction de polymerisation en chaine (PCR). Le fragment amplifie a ete hybride avec l'oligonucleotide immobilise par membrane et l'hybridation detectee non-radioactivement. Grace a cette technique, il a ete possible d'identifier P. penetrans, P. coffeae, P. vulnus, P. loosi, P. brachyurus, P. crenatus et P. zeae en une seule hybridation.
49
Apprill, Amy, Heather Q. Marlow, Mark Q. Martindale, and Michael S. Rappé. "Specificity of Associations between Bacteria and the Coral Pocillopora meandrina during Early Development." Applied and Environmental Microbiology 78, no. 20 (2012): 7467–75. http://dx.doi.org/10.1128/aem.01232-12.
Full textAbstract:
ABSTRACTRelationships between corals and specific bacterial associates are thought to play an important role in coral health. In this study, the specificity of bacteria associating with the coralPocillopora meandrinawas investigated by exposing coral embryos to various strains of cultured marine bacteria, sterile seawater, or raw seawater and examining the identity, density, and location of incorporated cells. The isolates utilized in this experiment included members of the Roseobacter and SAR11 clades of theAlphaproteobacteria, aPseudoalteromonasspecies of theGammaproteobacteria, and aSynechococcusspecies of theCyanobacteriaphylum. Based on terminal restriction fragment length polymorphism analysis of small-subunit rRNA genes, similarities in bacterial communities associated with 170-h-old planulae were observed regardless of treatment, suggesting that bacteria may have been externally associated from the outset of the experiment. Microscopic examination ofP. meandrinaplanulae by fluorescencein situhybridization with bacterial and Roseobacter clade-specific oligonucleotide probes revealed differences in the densities and locations of planulae-associated cells. Planulae exposed to either raw seawater or strains ofPseudoalteromonasand Roseobacter harbored the highest densities of internally associated cells, of which 20 to 100% belonged to the Roseobacter clade. Planulae exposed to sterile seawater or strains of the SAR11 clade andSynechococcusdid not show evidence of prominent bacterial associations. Additional analysis of the raw-seawater-exposed planulae via electron microscopy confirmed the presence of internally associated prokaryotic cells, as well as virus-like particles. These results suggest that the availability of specific microorganisms may be an important factor in the establishment of coral-bacterial relationships.
50
Perche, Federico, Tony Le Gall, Tristan Montier, Chantal Pichon, and Jean-Marc Malinge. "Cardiolipin-Based Lipopolyplex Platform for the Delivery of Diverse Nucleic Acids into Gram-Negative Bacteria." Pharmaceuticals 12, no. 2 (2019): 81. http://dx.doi.org/10.3390/ph12020081.
Full textAbstract:
Antibiotic resistance is a growing public health concern. Because only a few novel classes of antibiotics have been developed in the last 40 years, such as the class of oxazolidinones, new antibacterial strategies are urgently needed (Coates, A.R. et al., 2011). Nucleic acid-based antibiotics are a new type of antimicrobials. However, free nucleic acids cannot spontaneously cross the bacterial cell wall and membrane; consequently, their intracellular delivery into bacteria needs to be assisted. Here, we introduce an original lipopolyplex system named liposome polymer nucleic acid (LPN), capable of versatile nucleic acid delivery into bacteria. We characterized LPN formed with significant therapeutic nucleic acids: 11 nt antisense single-stranded (ss) DNA and double-stranded (ds) DNA of 15 and 95 base pairs (bp), 9 kbp plasmid DNA (pDNA), and 1000 nt ssRNA. All these complexes were efficiently internalized by two different bacterial species, i.e., Escherichia coli and Pseudomonas aeruginosa, as shown by flow cytometry. Consistent with intracellular delivery, LPN prepared with an antisense oligonucleotide and directed against an essential gene, induced specific and important bacterial growth inhibition likely leading to a bactericidal effect. Our findings indicate that LPN is a versatile platform for efficient delivery of diverse nucleic acids into Gram-negative bacteria.