Matrix-assisted laser desorption ionisation time-of-flight mass spectrometry (MALDI-TOF-MS) of intact microorganisms, also known as intact cell MALDI-TOF-MS (ICM-MS), has been shown to produce ...characteristic mass spectral fingerprints of moieties desorbed from the cell surface. ICM-MS spectra can be obtained in minutes after removal of a colony from a culture plate. The similarity of ICM-MS spectra of replicate samples and of two different batches of the same bacterial strain demonstrates, in this study, the reproducibility of the technique. We have developed the Manchester Metropolitan University Search Engine (MUSE™) to rapidly build and search databases of ICM-MS spectra. A database of 35 strains, representing 20 species and 12 genera, was built with MUSE™ and used to identify 212 isolates. The database was created in 26 s and loaded in 10 s, ready for searching, which took less than 1 s per isolate. Correct matches were made in 79%, 84% and 89% of the 212 samples at strain, species and genus levels, respectively. At least 50% of the replicates of 42 of the 45 isolates matched the correct strain, and the most commonly identified species for 43 of the 45 isolates was the correct one. The close match of the
Escherichia coli strains containing the O157 antigen and the
E. coli strains containing the K1 antigen suggests that these antigens may have a dominating influence on the ICM-MS fingerprints of these strains. We now have the ability to acquire ICM-MS fingerprints of bacteria and to search a database of these fingerprints within minutes, so that the rapid identification of bacteria to the strain level can be realised.
Slow degradation of organic matter in acidic Sphagnum peat bogs suggests a limited activity of organotrophic microorganisms. Monitoring of the Sphagnum debris decomposition in a laboratory simulation ...experiment showed that this process was accompanied by a shift in the water color to brownish due to accumulation of humic substances and by the development of a specific bacterial community with a density of 2.4 x 10 super(7) cells ml super(-1). About half of these organisms are metabolically active and detectable with rRNA-specific oligonucleotide probes. Molecular identification of the components of this microbial community showed the numerical dominance of bacteria affiliated with the phyla Alphaproteobacteria, Actinobacteria, and Planctomycetes. The population sizes of the Firmicutes and Bacteroidetes, which are believed to be the main agents of bacterially-mediated decomposition in eutrophic wetlands, were low. The numbers of planctomycetes increased at the final stage of Sphagnum decomposition. The representative isolates of the Alphaproteobacteria were able to utilize galacturonic acid, the only low-molecular-weight organic compound detected in the water samples; the representatives of the Planctomycetes were able to decompose some heteropolysaccharides, which points to the possible functional role of these groups of microorganisms in the community under study. Thus, the composition of the bacterial community responsible for Sphagnum decomposition in acidic and low-mineral oligotrophic conditions seems to be fundamentally different from that of the bacterial community which decomposes plant debris in eutrophic ecosystems at neutral pH.
The rapid identification of bacteria in many kinds of samples, i.e., clinical, food, water, and material, is important from a hygienic standpoint. In this paper, we describe the development of a ...convenient bacterial identification system using bacterial cells by a plastic DNA array. The small plastic base, which was cut from an S-BIO(R) PrimeSurface(R) plastic base developed for the covalent immobilization of aminomodified DNA, was used as a substitute for a glass base. The species-specific primers were immobilized on the small plastic base and the spots specific to each bacterial species were observed after 2 types of thermal cycles in a single PCR tube using bacterial culture broth as a sample. The results obtained using the culture broth were the same as those obtained with total DNA extracted from bacterial cells. The detection limits of Staphlococcus aureus (S. aureus) ATCC25923 and Escherichia coli (E. coli) ATCC25922 were 8.7×103 cells/μl and 2.1×102 cells/μl, respectively. This system is useful and convenient for the identification of bacteria in many types of samples. Moreover, further improvements in conditions, such as the ingredients in the reaction mixture, thermal cycles, and the steps of visualization would result in a more efficient system of identification.
Diagnostic tools for identification of bacteria have developed dramatically in the last decade. Sequencing of genes coding for rRNA has led to revolutionary insights into the phylogeny and taxonomy ...of bacteria, and to new demands on the service provided by national reference laboratories for identification of bacteria. At the Danish Reference Laboratory for Identification of Bacteria, partial 16S rDNA sequencing has been used since 2001 to identify “difficult” strains submitted for taxonomic elucidation. Experiences relating to phenotypic as well as 16S rDNA sequencing of the first 175 strains examined are presented. Approximately 2/3 of the strains were Gram‐positive and 1/3 Gram‐negative. One fifth of the strains were anaerobic, while 4/5 were either facultatively anaerobic or aerobic. Methodological agreement was seen for most strains at species and/or genus level. Methodological disagreement was relatively rare. In 1/6 of the strains valuable information was obtained from sequencing results, while for some strains identification was based primarily on the phenotypic results. Only a few strains could not be clearly identified by either method. A very large number of strains representing taxons ranging from facultatively anaerobic to aerobic and anaerobic species and genera, Gram‐positive as well as Gram‐negative, were successfully examined. Of the submitted strains many have only rarely been encountered as human pathogens. Thus, genotypic identification may result in recognition of hitherto seldom recognized or unrecognized bacteria as human pathogens, which will lead to a better understanding of the nature of human infections. It is self evident that we should focus on slowly growing, fastidious or ‘difficult’ organisms when using sequencing for national reference purposes. Short sequences (450–650 base pairs) seem sufficient for most identifications. Molecular bacterial identification is a powerful tool for national reference laboratories, enhancing both the speed and validity of examinations performed.
Phylogenetic estimation method without determination of DNA sequence was developed. By this method, fragment length polymorphism separately digested with multiple restriction enzymes was measured ...using microchip electrophoresis and affiliated with those calculated from corresponding DNA sequence in the theoretical database. The phylogenies of 129 NO
3
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reducing bacteria newly isolated from field soils were estimated by this method, and were compared to those by carbon source utilization profiles and by comparative sequence analysis of 16S rDNA. Various bacteria such as Micrococcus sp. (one isolate), Acidovorax delofieldii (one isolate), Cupriavidus necator (one isolate), Burkholderia sp., (seven isolates), Commamonas acidovorans (two isolates), Herbaspirillum seropedicae (two isolates), Ralstonia sp. (six isolates), Pseudomonas spp. (14 isolates), and Acinetobacter spp. (one isolate), were affiliated as similar to those by sequence analysis of 16S rDNA, while exact affiliation of genus was difficult for those belonging to Enterobacteriaceae.
Twenty seven arsenic-transforming bacterial strains were isolated from three different arsenic polluted areas in Bulgaria. The bacterial strains were taxonomical identified and characterized. It was ...determined that they belong to the genera: Pseudomonas, Alcaligenes, Azoarcus and Thauera. Using PCR-RFLP of 16S DNA and of ITS fragment, it was determined that the investigated bacteria are different strains of the species: Ps. pseudoalcaligenes, Ps. mendocina and identity strains of the species: Ps. stutzeri, Ps. putida, Ps. aeruginosa and Ps. fulva.
Recently a new method, SEnsing of Phage-Triggered Ion Cascade
(SEPTIC) was proposed for the rapid detection and identification
of bacteria via the electrical field caused by the stochastic
emission ...of ions during phage infection. In this Letter, we present
linear network theoretical considerations about the detection limits
of the method. The considerations are based on our published data
of the E. coli detection experiments and on the assumption
of a linear response between the number of bacteria and the measured
power density spectrum of the fluctuation-signal. Some practical
limits of the detectability of the present agents with possible
noise measurement arrangements are discussed in this paper. The
calculations indicate that the detection and identification of a
single bacterium can be achieved with natural (wild) phages
with reasonable efforts within a time window of 10 minutes.
The rapid identification of bacteria in many kinds of samples, i.e., clinical, food, water, and material, is important from a hygienic standpoint. In this paper, we describe the development of a ...convenient bacterial identification system using bacterial cells by a plastic DNA array. The small plastic base, which was cut from an S-BIO® PrimeSurface® plastic base developed for the covalent immobilization of amino-modified DNA, was used as a substitute for a glass base. The species-specific primers were immobilized on the small plastic base and the spots specific to each bacterial species were observed after 2 types of thermal cycles in a single PCR tube using bacterial culture broth as a sample. The results obtained using the culture broth were the same as those obtained with total DNA extracted from bacterial cells. The detection limits of Staphlococcus aureus (S. aureus) ATCC25923 and Escherichia coli (E. coli) ATCC25922 were 8.7×103 cells/μl and 2.1×102 cells/μl, respectively. This system is useful and convenient for the identification of bacteria in many types of samples. Moreover, further improvements in conditions, such as the ingredients in the reaction mixture, thermal cycles, and the steps of visualization would result in a more efficient system of identification.
In this report, we describe the use of universal primer PCR (UPPCR) for the detection of 16S ribosomal RNA (rRNA) genes from
Aeromonas hydrophila captured by anti-
A. hydrophila antibody coupled to a ...microplate. The approach combining immuno-capture with UPPCR provides a quick, sensitive, and reproducible way for the detection of bacterial cells.
In this Letter, we propose a simple method to strongly enhance
the sensitivity of the detection of the new technique called
"SEnsing of Phage-Triggered Ion Cascade (SEPTIC)". The method
accumulates ...those phage infected bacteria which are emitting ions
and releases those which have stopped the ion emission activity.
The estimated increasing of the sensitivity can reach several
orders of magnitude at practical conditions.
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