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.     

Microflotation: New low gas-flow rate foam separation technique for bacteria and algae

Foam separation of microorganisms has been investigated with varying success by many workers, usually at high rates of gas flow. Microflotation was developed to overcome some of the disadvantages inherent in these high gas-flow-rate processes and is introduced in this paper as a new technique for the foam separation of microorganisms at low gas-flow rates. With microflotation, a stable surface phase is produced by adding an insoluble collector such as a long-chain fatty acid or amine. The formation of an insoluble surface phase eliminates the need for high foaming. Low rates of gas flow are used resulting in a more efficient separation and a less voluminous and drier surface phase upon which to collect the microorganisms. The efficiency of this technique is also improved by using flotation aids such as frothers and flocculents. Frothers are used to improve the collector properties of the surfactant and to refine further the small bubbles produced by a very fine sparger. Small concentrations of flocculents, such as alum, are used to partially agglomerate the organisms and provide sites for adsorption of collector. The work described in this paper is preliminary in nature, designed to illustrate that a low flow-rate process may be used to separate microorganisms and to stimulate further research. The applications discussed are removal of the bacterium, Escherichia coli, and alum, and two species of algae, Chlamydomonas reinhardtii and Chlorella ellipsoidea, using stearylamine without alum. The frother used was ethanol.     

Thiosulfate and tetrathionate degradation as well as biofilm generation by Thiobacillus intermedius and Thiobacillus versutus studied by microcalorimetry,HPLC, and ion-pair chromatography

The growth of Thiobacillus (T.) intermedius strain K12 and Thiobacillus versutus strain DSM 582 on thiosulfate and tetrathionate was studied combining on-line measurements of metabolic activity and sulfur compound analysis. Most results indicate that T. intermedius oxidized thiosulfate via tetrathionate to sulfate. Concomittantly, sulfur compound intermediates like triand pentathionate were detectable. The formation is probably the result of highly reactive sulfane monosulfonic acids. The formation of tetrathionate allows the cells to buffer temporarily the proton excretion from sulfuric acid production. With T. versutus intermediate sulfur compounds were not detectable, however, sulfur was detectable. The possibility of a thiosulfate oxidation via dithionate, S₂O _inf6 ^sup2- , is discussed. The on-line measurement of metabolic activity by microcalorimetry enabled us to detect that cells of T. intermedius adhere to surfaces and produce a biofilm by a metabolic process whereas those of T. versutus fail to do so. The importance of the finding is discussed.     

基于流式细胞仪高通量分选的深海微生物单细胞培养

【目的】建立适用于海洋微生物的流式细胞分选与高通量单细胞培养的方法,通过该方法从印度洋深海样品中分离微生物纯培养菌株。【方法】利用流式细胞仪单细胞分选功能,以前向角(FSC)和侧向角(SSC)散射光信号代替荧光信号作为分选逻辑,对深海水体和沉积物样品中微生物进行单细胞高通量分选和培养。【结果】确定了流式细胞分选的区域和条件,发现所建立方法适于分离海洋水体微生物,而不是沉积物微生物。从印度洋深海水体样品中获得61个潜在新菌株,分属于6个新属种,占分离菌株总数的26.29%,其16S rRNA基因序列与已培养的模式菌株相似性为89.79%–95.37%。【结论】本研究所建立的方法有助于提高发现海洋微生物新物种的效率,获得更多新的海洋微生物资源。     

Unfolding of tertiary structure ofHalobacterium halobium flagellins does not result in flagella destruction

The structure ofHalobacterium halobium R₁M₁ flagella is investigated by the methods of scanning microcalorimetry, circular dichroism, and electron microscopy. It is shown that melting curves of flagella in solutions with a different concentration of NaCl display only one peak of heat capacity that corresponds to one cooperatively melting domain. It is found that flagella do not dissociate after melting. The possible structural organization of archaebacterial flagella is discussed.     

Molecular tools for speciation and epidemiological studies of Acanthamoeba

Current diagnostic methods for Acanthamoeba identification rely heavily on light microscopic techniques that do not provide sufficient information about the identification of Acanthamoeba at the species level, thus delaying accurate identification of the infective agent. Here we report the use of polymerase chain reaction (PCR)-based restriction enzyme analyses to detect and speciate Acanthamoeba from both clinical and environmental sources by comparing their restriction endonuclease patterns. Significant diversity was observed between and within morphologically defined Acanthamoeba species. The usefulness of PCR-based assays and other available diagnostic methods is discussed. Received: 15 August 2001 / Accepted: 15 October 2001     

Flow Microcalorimetric Studies of Yeast Growth: Fundamental Aspects

The importance of factors such as cultural conditions e.g. organism background, inoculum density and glucose concentration are explored and their effects on the experimental recording discussed. The relevance of these findings to microbial microcalorimetry in general and to microcalorimetric identification of organisms in particular is described. The conclusions drawn are (i) that microbial identification by microcalorimetry is not possible and (ii) that great caution must be exercised in interpreting metabolic data derived from microcalorimetric studies.     

A calorimetric and fluorescence study of batch cultures of Saccharomyces cerevisiae

Summary Batch growth of Saccharomyces cerevisiae was studied using a fluorescence probe as well as a microcalorimeter. The biphasic growth pattern previously demonstrable by microcalorimetry was also seen using the fluorescence probe. Acid production rate changes in the transition phase could be detected by pH measurements. Qualitative similarities between calorimetric and fluorescence measurements are discussed.     

Continuous culture of Candida lipolytica on n-hexadecane

Candida lipolytica was grown continuously on n-hexadecane as the main source of carbon. A transient continuous-culture experiment was also conducted to investigate hydrocarbon-limited growth; the hydrocarbon feed flow rate was stopped for several hours and then resumed at a reduced steady-state flow rate. Interfacial tension, Sauter mean diameter, pseudosolubility, fraction of cells in the aqueous phase, oil-phase volume fraction, and cell concentration were measured to characterize the system. The microorganisms appear to utilize both the submicron drops and the microscopic drops. The effects of interfacial tension, pseudosolubility, and unoccupied interfacial area on the kinetics of hydrocarbon fermentation are discussed here. A conceptual model for hydrocarbon uptake is presented and discussed.     

Development of a method based on surface enhanced laser desorption and ionization time of flight mass spectrometry for rapid identification of <Emphasis Type="Italic">Klebsiella pneumoniae</Emphasis>

A method based on surface enhanced laser desorption and ionization time of flight mass spectrometry (SELDI-TOF MS) was developed for the rapid identification of Klebsiella pneumoniae by directly applying bacterial colonies without further protein extraction. A total of 40 K. pneumoniae and 114 other related microorganisms isolated clinically were analyzed by SELDI-TOF MS. An identification model for K. pneumoniae was established by artificial neural networks (ANNs) with classification accuracy of 100%. The model was blindly tested with 43 K. pneumoniae and 53 control bacteria again. The results showed that the model was successful with accuracy of 96.9%, sensitivity of 100% and specificity of 943%. This strategy is potential for rapid identification of K. pneumoniae.     

Studies on Biosynthesis on Biotin by Microorganisms

The accumulation of biotin-vitamers in the culture media of a large number of microorganisms (about 700 strains) was studied. The contents of the biotin-vitamers were quantitatively determined by microbiological assays with Lactobacillus arabinosus and Saccharomyces cerevisiae.

It was found that large amounts of biotin-vitamers were accumulated by various microorganisms such as Streptomyces, molds and bacteria, and that the yield of biotin-vitamers was enhanced by the addition of pimelic acid or azelaic acid to the media. It was also found that the main portion of the vitamers accumulated by many microorganisms did not support the growth of Lactobacillus arabinosus, while it did support that of Saccharomyces cerevisiae. The small amounts of true biotin were observed in the culture media of various Streptomyces and molds, but hardly in the culture media of bacteria.

The identification of biotin-vitamers accumulated by various microorganisms is described, and the distribution of the vitamers in microorganisms is also described.

The results presented in this paper show that the main component of the vitamers accumulated by many microorganisms is identified as desthiobiotin by anion exchange column chromatography, paper chromatography and chemical analysis. Small amounts of fraction B (unidentified vitamers) and Fraction D (biotin) were also detected in the culture media of various molds and Streptomyces. However, these fractions were not observed in the culture media of any bacteria tested.

It was also found that large amounts of an unknown biotin-vitamer was accumulated by various bacteria. The vitamer was avidin-uncombinable, and, from the paper electrophoretic studies, it was assumed that the vitamer might be an analogue of pelargonic acid.     

Hydrodynamics and mass transfer in miniaturized bubble column bioreactors

Miniaturized bubble columns (MBCs) have different hydrodynamics in comparison with the larger ones, but there is a lack of scientific data on MBCs. Hence, in this study, the effect of gas hold-up, flow regimes, bubble size distribution on volumetric oxygen mass transfer coefficient at different pore size spargers and gas flow rates in MBCs in the presence and absence of microorganisms were investigated. It was found that flow regime transition occurred around low gas flow rates of 1.18 and 0.85 cm/s for small (16–40 µm) and large (40–100 µm) pore size spargers, respectively. Gas hold-up and K_La in MBC with small size sparger were higher than those with larger one, with an increasing effect in the presence of microorganisms. A comparison revealed that the wall effect on the flow regime and gas hold-up in MBCs was greater than bench-scale bubble columns. The K_La values significantly increased up to tenfold using small pore size sparger. In the MBC and stirred tank bioreactors, the maximum obtained cell concentrations were OD₆₀₀ of 41.5 and 43.0, respectively. Furthermore, it was shown that in MBCs, higher K_La and lower turbulency could be achieved at the end of bubbly flow regime.

     

Calorimetric studies of p-nitrophenol binding to α- and β-cyclodextrin

The thermodynamics of the binding of p-nitrophenol to both α- and β-cyclodextrin has been investigated as a function of pH and solvent composition using the technique of flow microcalorimetry. A preferential binding of the anionic form of the ligand is found for both cyclodextrins. It is postulated that the additional affinity for p-nitrophenolate is due to a dispersion interaction between the cyclodextrin cavity and the delocalized charge of the ligand. Studies of the binding of p-nitrophenol analogs and of the binding of p-nitrophenol as a function of ionic strength and DMSO cosolvent composition are consistent with this theory.     

草酸青霉的植物多糖降解酶基因表达调控的研究进展

植物生物质是地球上最丰富的可再生资源,对其生物炼制可生产高附加值的生物基产品。生物炼制需要使用植物多糖降解酶(plant-polysaccharide-degrading enzymes,PPDEs),如纤维素酶、木聚糖酶和生淀粉酶。丝状真菌草酸青霉(Penicillium oxalicum)能分泌完整的具有高活力的植物多糖降解酶,但其产量低限制了大规模生产及应用。草酸青霉中植物多糖降解酶的生物合成受到多种调控因子包括转录因子的严格调控。本文主要介绍在以植物生物质甘蔗渣和木薯生淀粉为原料的生物炼制中,涉及的一些关键微生物方面的问题,如从高产植物多糖降解酶的真菌菌株的筛选、育种,到草酸青霉植物多糖降解酶合成及其基因表达的调控基因的鉴定,以及酶产量提高的工程菌株的构建等,为丝状真菌资源的开发与利用提供理论指导。     

Modeling and analysis of hydrodynamic and physico-chemical effects in bacterial deposition on surfaces

The parallel-plate flow chamber (PFC) is often used for characterizing the propensity of microorganisms to attachment to surfaces. The model presented quantitatively analyzes the complex interplay of diffusion, convection, inertial lift, buoyancy, and surface forces in the PFC, which make it difficult to separate the surface- and microorganism-specific effects from the hydrodynamics. An empirical dimensionless factor K entering the boundary condition expresses enhancement of adhesion diffusion of microorganisms across a thin fluid layer adjacent to the surface by adhesion forces. The model examines the role of various factors (eg shear rate, size of bacterium, and strength of adhesion) on the rate of bacterial deposition. Using no adjustable parameter for strongly adhesive surfaces and K as the only adjustable parameter for repulsive or weakly adhesive surfaces, the model explains the observed decrease in deposition flux at high flow rates and compares reasonably with reported experimental results. The results suggest that the fitted value of K may be used for ‘rating’ the propensity of bacteria to deposit on surfaces and separating this from hydrodynamic effects.     

Identification of microbes from the surfaces of food-processing lines based on the flow cytometric evaluation of cellular metabolic activity combined with cell sorting

In this study the design of a flow cytometry-based procedure to facilitate the detection of adherent bacteria from food-processing surfaces was evaluated. The measurement of the cellular redox potential (CRP) of microbial cells was combined with cell sorting for the identification of microorganisms. The procedure enhanced live/dead cell discrimination owing to the measurement of the cell physiology. The microbial contamination of the surface of a stainless steel conveyor used to process button mushrooms was evaluated in three independent experiments. The flow cytometry procedure provided a step towards monitoring of contamination and enabled the assessment of microbial food safety hazards by the discrimination of active, mid-active and non-active bacterial sub-populations based on determination of their cellular vitality and subsequently single cell sorting to isolate microbial strains from discriminated sub-populations. There was a significant correlation (r = 0.97; p < 0.05) between the bacterial cell count estimated by the pour plate method and flow cytometry, despite there being differences in the absolute number of cells detected. The combined approach of flow cytometric CRP measurement and cell sorting allowed an in situ analysis of microbial cell vitality and the identification of species from defined sub-populations, although the identified microbes were limited to culturable cells.     

MALDI-TOF mass spectrometry rapid pathogen identification and outcomes of patients with bloodstream infection: A systematic review and meta-analysis

There was inconsistent evidence regarding the use of matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) for microorganism identification with/without antibiotic stewardship team (AST) and the clinical outcome of patients with bloodstream infections (BSI). In a systematic review and meta-analysis, we evaluated the effectiveness of rapid microbial identification by MALDI-TOF MS with and without AST on clinical outcomes. We searched PubMed and EMBASE databases from inception to 1 February 2022 to identify pre–post and parallel comparative studies that evaluated the use of MALDI-TOF MS for microorganism identification. Pooled effect estimates were derived using the random-effects model. Twenty-one studies with 14,515 patients were meta-analysed. Compared with conventional phenotypic methods, MALDI-TOF MS was associated with a 23% reduction in mortality (RR = 0.77; 95% CI: 0.66; 0.90; I² = 35.9%; 13 studies); 5.07-h reduction in time to effective antibiotic therapy (95% CI: −5.83; −4.31; I² = 95.7%); 22.86-h reduction in time to identify microorganisms (95% CI: −23.99; −21.74; I² = 91.6%); 0.73-day reduction in hospital stay (95% CI: −1.30; −0.16; I² = 53.1%); and US$4140 saving in direct hospitalization cost (95% CI: $-8166.75; $-113.60; I² = 66.1%). No significant heterogeneity sources were found, and no statistical evidence for publication bias was found. Rapid pathogen identification by MALDI-TOF MS with or without AST was associated with reduced mortality and improved outcomes of BSI, and may be cost-effective among patients with BSI.     

Aerobiological monitoring of the 'Sistine Chapel': airborne bacteria and microfungi trends

The present paper reports the results of a bacteriological and mycological monitoring carried out on the airborne microflora of the Sistine Chapel. The general aim of the study was to evaluate the impact of the flow of visitors, as well seasonal effects, on the qualitative and quantitative variations of microorganisms. Two sampling campaigns were carried out in May and November 1997. A Surface Air System (SAS) sampler (active system) and a sedimentation based sampler (passive system), supported by an original plinth, were used. Temperature, humidity and carbon dioxide were detected. VITEK SYSTEMS jr. for Staphylococcus spp. and microscopic observation for microfungi were the identification methods. In spite of the conditioning and filtration air system, initial results with both samplers, show a positive correlation between the airborne microorganisms and presence and number of visitors. The SAS samples showed higher microbial load, for both bacteria and fungi, than the passive ones, but the epidemiological meaning of the differently collected data varies. The increase during visiting hours of human Staphylococcus spp. is stronger than the airborne bacterial load increase. The microfungi most frequently isolated were Cladosporium spp. and Penicillium spp. These preliminary data underline the significance of the survey for the protection of such a precious environment, and encourage the Authors to continue the ongoing monitoring. This revised version was published online in June 2006 with corrections to the Cover Date.     

UNIQEM, a microbiological database: Problems and prospects

The UNIQEM database, designed to accumulate general microbiological data, is currently used to store and make available information about microorganisms studied and maintained at the Institute of Microbiology, Russian Academy of Sciences. UNIQEM can accumulate and maintain list-form information on a wide range of microorganisms (a property database) and facilitates collecting, processing, and publishing diverse data having to do with these microorganisms and their properties (a catalogue database). The database supports the retrieval of microorganisms by specifying an arbitrary set of their properties and has the potential for eventually evolving into a computer instrument for unattended identification of microorganisms. UNIQEMAkhlynin, D.S. and Gal’chenko, V.F., 1998.     

Biological control of Dutch elm disease: Larvicidal activity of Trichoderma harzianum, T. polysporum and Scytalidium lignicola in Scolytus scolytus and S. multistriatus reared in artificial culture

A method is described for the culture of larvae of Scolytus multistriatus and Scolytus scolytus on an artificial medium following exposure to cultures of microorganisms. In control cultures, a natural mortality rate of 21.2% was found for S. multistriatus and 17.6% for S. scolytus. The effects of Trichoderma harzianum, T. polysporum and Scytalidium lignicola on fifth instar larvae of S. scolytus and S. multistriatus reared on the artificial medium were studied. The fungi were larvicidal and larval mortality was increased to more than 80% by inoculation of the larvae with the fungi. Another fungus, Phomopsis oblonga, had little effect on larvae of S. scolytus. The results are discussed in relation to mechanism of pathogenicity of the fungi and their potential use in the control of Dutch elm disease. It is proposed that with modifications, the method is applicable to other bark beetle pests.