Discriminant analysis of microcalorimetric data of bacterial growth

In this work a bacterial classification method based on the discriminant analysis of the microcalorimetric data provided by the growth power-time (p-t) curves is developed. This method is applied to classify several species of Enterobacteria of different origins, and the results are compared with those obtained by conventional techniques. The proposed analysis allows us to classify bacteria into species and discriminate among strains of the same species. The classification is carried out using one run of each isolate after standardization of inocula and growth conditions. The discrimination power of available microcalorimetric data is also discussed, and the most discriminant set of data is proposed as the input variables of the analysis. Finally, the advantages of microcalorimetry as a taxonomical technique are discussed.     

Enthalpy of interaction between coaggregating and non-coaggregating oral bacterial pairs--a microcalorimetric study

Bacterial adhesion and coaggregation are involved in the development of oral biofilms, called dental plaque. Although various techniques have already been used to study different aspects of these bacterial interactions, microcalorimetry has not yet been applied. This paper describes how isothermal reaction calorimetry can be employed to determine the enthalpy of coaggregation between two oral bacterial pairs. For most biological processes, the enthalpy tends to reach a minimum value, reflecting the most stable state, which is directly related to the heat content of the system. The calorimeter consists of four measuring units where reaction ampoules are filled with 1.5 ml of an Actinomyces naeslundii 147 suspension, while reference ampoules are filled with buffer only. After equilibration at 25 degrees C, 80 microl of a streptococcal suspension was titrated into the reaction ampoules. To study possible saturation of the binding sites on the actinomyces surface, three consecutive injections with streptococcal suspensions were done. Following each injection, a 20-microl aliquot was taken from the ampoule kept outside the calorimeter and the number of free (S(f)) and bound (S(b)) streptococci was determined microscopically. Experiments were carried out with a coaggregating streptococcal strain (Streptococcus oralis J22) and a non-coaggregating strain (Streptococcus sanguis PK1889), serving as a control. The coaggregation enthalpy was exothermic, that is, heat was released in the reaction ampoule upon coaggregation and the heat released by the coaggregating pair minus the heat released by the non-coaggregating pair yielded a coaggregation enthalpy of -0.015 x 10(-6) mJ/bound streptococcus for the first injection. Upon consecutive injections, the coaggregation enthalpy decreased to -0.0004 x 10(-6) mJ/bound streptococcus. Comparison with enthalpy changes reported for lectin-carbohydrate binding suggests that a huge number of binding sites are involved in the formation of one bacterial coaggregate.     

Utilization of tmRNA sequences for bacterial identification

Background  

Ribosomal RNA molecules are widely used for phylogenetic and in situ identification of bacteria. Nevertheless, their use to distinguish microorganisms within a species is often restricted by the high degree of sequence conservation and limited probe accessibility to the target in fluorescence in situ hybridization (FISH). To overcome these limitations, we examined the use of tmRNA for in situ identification. In E. coli, this stable 363 nucleotides long RNA is encoded by the ssrA gene, which is involved in the degradation of truncated proteins.     

Estimations on the degradability of ores and bacterial leaching activity using short-time microcalorimetric tests

Abstract: This work is based on calorimetric measurements on batch cultures of ferrous iron-oxidizing thiobacilli and leaching cultures degrading solid substrates in the titration vessel under aerobic conditions. It has been demonstrated that heat-flow levels are a direct measure for oxidation rates of known reactions under non-limiting conditions. The total loss of heat-energy per unit of ferrous iron oxidized is identical under similar incubation conditions with different Thiobacillus ferrooxidans strains and different cell-densities in culture suspension. Under limiting conditions a reduced heat-loss occurred. This may have been due to either a more effective use of the substrate or to a shift to different redox reactions. Oxygen was proved to be a limiting factor in dense bacterial cultures with high substrate concentrations in the titration vessel. The experimental duration decreased significantly with the use of increasing cell densities. Therefore cell densities in degradation experiments with solid substrates were adjusted to between 5X10⁹ and 1x10¹⁰ cells/ml in short-term experiments. T. ferrooxidans is the most important species for short-term leaching experiments. Calorimetric measurements allow the best strains for a degradation of unknown substrates to be found. The calculated heat energy is a measure of the amount of converted substrate. High heat-flow only occurs if catabolic reactions take place and maintenance reactions and phenomena like adhesion of cells to surface do not cause significant loss of heat.     

Broad spectrum microarray for fingerprint-based bacterial species identification

Background  

Microarrays are powerful tools for DNA-based molecular diagnostics and identification of pathogens. Most target a limited range of organisms and are based on only one or a very few genes for specific identification. Such microarrays are limited to organisms for which specific probes are available, and often have difficulty discriminating closely related taxa. We have developed an alternative broad-spectrum microarray that employs hybridisation fingerprints generated by high-density anonymous markers distributed over the entire genome for identification based on comparison to a reference database.     

Robustness of two MALDI-TOF mass spectrometry systems for bacterial identification

MALDI-TOF-MS systems (Microflex-Bruker Daltonics/BioTyper? and Axima-Assurance-Shimadzu/SARAMIS-AnagnosTec) were assessed for bacterial identification. Focusing on bacteria difficult to identify routinely, 296 strains were identified by molecular biology techniques as gold standard. MALDI-TOF-MS identification provided correct results at genus and species level for 94.9%, 83.4% and 83.8%, 65.9% with Biotyper and Saramis respectively.     

Assay for identification of inhibitors for bacterial MraY translocase or MurG transferase

Bacterial peptidoglycan synthesis is a well-characterized system for targeting new antimicrobial drugs. Formation of the peptidoglycan precursors Lipid I and Lipid II is catalyzed by the gene products of mraY and murG, which are involved in the first and second steps of the lipid cycle reactions, respectively. Here we describe the development of an assay specific for identifying inhibitors of MraY or MurG, based on the detection of radiolabeled [(14)C]GlcNAc incorporated into Lipid II. Assay specificity is achieved with the biotin tagging of the Lipid I precursor UDP-MurNAc-pentapeptide. This allows for the separation and identification of lipid products produced by the enzymatic activity of the MraY and MurG proteins, and thus identification of specific inhibitors.     

猕猴桃细菌性溃疡病生防放线菌的筛选与鉴定

对分离自新疆巴里坤盐湖和达坂城盐湖的179株放线菌进行拮抗丁香假单胞杆菌猕猴桃致病变种筛选,从中筛选到8株具有拮抗作用的放线菌,其中ZBW8-1、E14-3和ZB5-6抗菌活性最显著,其抑菌圈直径分别为27.5 mm、23.6 mm和26.2 mm。针对这3株放线菌进行菌落形态观察,16s rRNA扩增、测序及系统进化分析,并对其进行抗生素合成相关基因的PCR鉴定。结果表明,放线菌ZBW8-1、E14-3和ZB5-6分别与糖霉菌属（Glycomyces）的Glycomyces fuscus TRM 49117、原小单孢菌属（Promicromonospora）的维也纳原小单孢菌（Promicromonospora vindobonensis）V45^T以及拟诺卡氏菌属（Nocardiopsis）的Nocardiopsis terrae YIM 90022^T 16s rRNA序列同源性最高,分别为99.86%、99.10%和99.86%。在系统进化关系上,这3株菌也分别与上述菌株聚类在同一个分支上。ZBW8-1、E14-3和ZB5-6菌株中均携带有NRPS基因,其中ZBW8-1和ZB5-6菌株中还含有PKS-II基因,且ZBW8-1菌株中还含有PKS-I基因。对这3株抗菌活性放线菌进行进化地位分析及抗生素合成相关功能基因的鉴定,将为研究这些菌株中的活性次生代谢产物奠定理论基础。     

人工神经原网络处理PCR数据在细菌鉴定中的应用

目的16SrRNA和16S-23SrRNA间区片段是常用细菌分类鉴定靶点,本研究探讨人工神经原网络（ANN）对上述位点PCR扩增产物数据分析在细菌快速鉴定方面的价值。方法2对15SrRNA基因荧光引物和1对16S-23SrRNA区间基因引物用于扩增血液标本中分离出的317株细菌。相关毛细管电泳（CE）限制性片段长度多态性（RFLP）和单链构象多态性（SSCP）数据进行人工神经原网络分析。结果16S-23SrRNA基因的RFLP数据对未知菌鉴定的准确率高于16SrRNA基因的SSCP数据,分别为98．0％和79．6％。结论实验证明了人工神经原网络作为一种模式识别方法对于简化细菌鉴定十分有价值。     

rpoB sequence analysis as a novel basis for bacterial identification

Comparison of the sequences of conserved genes, most commonly those encoding 16S rRNA, is used for bacterial genotypic identification. Among some taxa, such as the Enterobacteriaceae, variation within this gene does not allow confident species identification. We investigated the usefulness of RNA polymerase beta-subunit encoding gene ( rpoB  ) sequences as an alternative tool for universal bacterial genotypic identification. We generated a database of partial rpoB for 14 Enterobacteriaceae species and then assessed the intra- and interspecies divergence between the rpoB and the 16S rRNA genes by pairwise comparisons. We found that levels of divergence between the rpoB sequences of different strains were markedly higher than those between their 16S rRNA genes. This higher discriminatory power was further confirmed by assigning 20 blindly selected clinical isolates to the correct enteric species on the basis of rpoB sequence comparison. Comparison of rpoB sequences from Enterobacteriaceae was also used as the basis for their phylogenetic analysis and demonstrated the genus Klebsiella to be polyphyletic. The trees obtained with rpoB were more compatible with the currently accepted classification of Enterobacteriaceae than those obtained with 16S rRNA. These data indicate that rpoB is a powerful identification tool, which may be useful for universal bacterial identification.     

An overlay gel method for identification and isolation of bacterial beta-lactamases

A modification of the iodometric technique using an overlay gel was employed for fast identification and isolation of beta-lactamase types TEM, SHV and AmpC from non-denaturing gels. Osmotic shock preparations of the three beta-lactamases were run on polyacrylamide gels without SDS and ampicillin containing overlay gels were flooded with the iodine solution before being placed on polyacrylamide gel strips. Distinct clear bands appeared in dark blue backgrounds indicating beta-lactamase activity.     

Evaluating amplified rDNA restriction analysis assay for identification of bacterial communities

Amplified ribosomal DNA restriction analysis (ARDRA) and restriction fragment length polymorphism were originally used for strain typing and for screening clone libraries to identify phylogenetic clusters within a microbial community. Here we used ARDRA as a model to examine the capacity of restriction-based techniques for clone identification, and the possibility of deriving phylogenetic information from ARDRA-based dendrograms. ARDRA was performed in silico on 48,759 sequences from the Ribosomal Database Project, and it was found that the fragmentation profiles were not necessarily unique for each sequence in the database, resulting in different species sharing fragmentation profiles. Although ARDRA-based clusters separated clones into different genera, these phylogenetic clusters did not overlap with trees constructed according to sequence alignment, calling into question the intra-genus ARDRA-based phylogeny. It is thus suggested that the prediction power of ARDRA clusters in identifying clone phylogeny be regarded with caution.     

A novel approach for the identification of bacterial taxa-specific molecular markers

AIMS: To develop and establish a methodology for an oriented and fast identification of species taxa-specific molecular markers useful for the identification of micro-organisms. METHODS AND RESULTS: From the complete microbial genomes available in Pfam database, taxa-specific protein domains were identified which lead to the selection of taxa-specific loci. This strategy was used to identify six genetic markers: four specific for Pseudomonas syringae pv. tomato, one specific for P. syringae pv. syringae and one specific for P. putida. The discriminatory potential of these loci was evaluated by Southern hybridization using several pseudomonad species and pathovars, by dot-blot hybridization and by multiplex PCR optimized for the simultaneous detection of P. putida, P. syringae pv. syringae and P. syringae pv. tomato. Sensitivity assays indicated a detection limit of approximately 10 pg of chromosomal DNA template needed for each bacterium. CONCLUSIONS: The proposed methodology was efficient on the selection of six Pseudomonas-specific markers able to discriminate Pseudomonas at the species and pathovar level. SIGNIFICANCE AND IMPACT OF THE STUDY: The oriented search of taxa-specific molecular probes described in this work, which can be easily extended to other groups of bacteria, will improve the accuracy and expedite the identification of micro-organisms by DNA-based molecular methods.     

Methods for the identification and analysis of l-altrose in bacterial polysaccharides

Abstract The two genes ( cfxP ) for phosphoribulokinase (PRK) in Alcaligenes eutrophus H16 are simultaneously expressed, resulting in the formation of PRK isoenzymes. The isoenzymes are structurally and immunoligically closely related. Their subunits differ only slightly in size. M _rs of 33 000 and 32 500 were determined for the chromosomally and megaplasmid pHF1-encoded subunits, respectively. The pHG1-encoded gene, cfxP , was cloned in Eschirichia coli and expressed under the cloned in Escherichia coli and expressed under the control of the lac promoter of pUC9 vectors. Native PRK with subunits of M _r 32 500 was formed, confirming the identity and functionality of cfxP _p . However, the recombinant PRK had a significantly lower specific activity than the authentic enzyme.     

The identification of transport parameters from truncated physiological tracer curves

The primary data curves from the injection of tracers into the circulation are often obscured by the appearance of a recirculation hump. Previous techniques for parameter identification in the presence of such a complication have been extended from compartmental models to include circulatory models consisting of partial differential equations of the Taylor dispersion and Turner capacitance types. A comparative analysis of the efficiency of parameter identification by several computational strategies is presented.