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Modelling the unidentified mortality burden from thirteen infectious pathogenic microorganisms in infants.

Official statistics routinely underestimate mortality from specific microorganisms and deaths are assigned to non-specific syndromes. Here we estimate mortality attributed to specific pathogens by modelling non-specific infant deaths from laboratory reports and codes on death certificates for these pathogens, 1993-2000 in England and Wales using a generalized linear model. In total, 22.4-59.8% of non-specific deaths in infants (25-66 deaths a year) are attributable to specific pathogens. Yearly deaths from Bordetella pertussis in neonates are 6.8 [95% confidence interval (CI) 1.5-11.9]. In post-neonates 9.4 (95% CI 2.3-16.6) deaths a year are attributable to Neisseria meningitidis, 7.3 (95% CI 2.4-12.3) to Streptococcus pneumoniae, from 2.8 (95% CI 0.8-4.9) to 15.1 (95% CI 9.4-20.9) to respiratory syncytial virus (RSV) and 3 (95% CI 0.3-5.9) to parainfluenza type 2. Our results suggest there is substantial hidden mortality for a number of pathogens in infants. A considerable proportion of deaths classified to infectious syndromes are non-infectious, suggesting low specificity of death certification. Laboratory reports were the more reliable source, reinforcing the asset of strong surveillance systems.

Bacteria↗

A simple device for the exposure of animals to infectious microorganisms by the airborne route.

In order to evaluate prophylaxis and therapy for individuals infected with pathogens by the airborne route, we have designed and built a simple apparatus in which small laboratory animals may be exposed to aerosols of infectious microorganisms. Animals are kept in a chamber closed by a HEPA filter and exposed to the pathogen aerosolized using a Collison nebulizer. Air in the exposure chamber may be sampled to show that the infectious agent is present but the dose of agent must be expressed as 50% effective doses determined by titration. An effective dose may be defined by whatever criteria are chosen to judge disease. Using this apparatus we have shown that St Louis encephalitis (SLE) virus is infectious for mice by the airborne route. These data support the idea that there may be significant hazard to personnel exposed to aerosols of infectious SLE after a laboratory accident.

Administration, Inhalation↗

Seroprevalence of IgG antibodies to the chlamydia-like microorganism 'Simkania Z' by ELISA.

The newly described microorganism 'Simkania Z', related to the Chlamydiae, has been shown to be associated with bronchiolitis in infants and community acquired pneumonia in adults. The prevalence of infection in the general population is unknown. A simple ELISA assay for the detection of serum IgG antibodies to 'Simkania Z' was used to determine the prevalence of such antibodies in several population samples in southern Israel (the Negev). The groups tested included 94 medical and nursing students, 100 unselected blood donors, 106 adult members of a Negev kibbutz (communal agricultural settlement), and 45 adult Bedouin, residents of the Negev. IgG antibodies to 'Simkania Z' were found in 55-80% of these presumably healthy individuals, independently of antibodies to Chlamydia trachomatis and Chlamydia pneumoniae. The Bedouin had a seropositivity rate of 80%, while all other groups had rates of between 55 and 64%. These results indicate that 'Simkania Z' infection is probably common in southern Israel.

Adolescent↗

Characterization of underivatized lipid biomarkers from microorganisms with pyrolysis short-column gas chromatography/ion trap mass spectrometry.

A microvolume Curie-point pyrolysis short-column (5 m) gas chromatography/mass spectrometry (Py-GC/MS) procedure was developed for the characterization of various lipid moieties in microorganisms. High linear flow rates (approximately 175 cm/s) characterized the GC conditions in order to effect an efficient chromatographic transfer and elution of the underivatized diglycerides and monoglycerides, and small modifications were necessary to the ion trap MS system in order for it to accommodate the relatively high gas load. During a typical analysis run anhydrodiacylglycerides eluted within a 5-6-min time frame. Gram-positive bacilli and Gram-negative species were differentiated from each other by the pyrolysis patterns of their lipid components. In spite of the complexity of the analyte, a straightforward visual analysis was achieved with the aid of simple computerized data display procedures. These procedures included examination of (1) total ion current (TIC) profiles of the lipid region of the reconstructed chromatogram, (2) the integrated mass spectrum of this region, (3) selected reconstructed ion chromatograms (RICs), (4) RIC intensity distributions, and (5) corresponding mass spectra. An appealing aspect of the lipid data reduction procedure is that most of it can be accomplished visually without requiring computerized pattern recognition techniques.

Bacteria↗

Electrochemical biosensor array for the identification of microorganisms based on lectin-lipopolysaccharide recognition.

Rapid identification of bacterial strains remains a well-known problem in applied medicine and, for viable pathogens, is an important diagnostic goal. We have investigated an electrochemical biosensor array, in which transduction is based on respiratory cycle activity measurements, where the microorganism's native respiratory chain is interrupted with non-native external oxidants. The selective biochemical recognition agents employed in this study are lectins that, once immobilized, recognize and bind to cell surface lipopolysaccharides. Porous membranes with different surface properties were examined as potential immobilization supports for these lectins. Optimizations performed using concanavalin A and E. coli JM105 show that immobilization methods involving pre-activated membranes significantly reduce the time required to create a functional lectin layer on the membrane surface. Overall, we found general agreement between agglutination test results and the electrochemical assessment of lectin-cell binding. Chronocoulometric measurements were made for cells captured on lectin-modified Immunodyne ABC membranes physically affixed to Pt working electrodes. This lectin-based sensor array was exposed to viable cells of gram-negative and gram-positive bacteria as well as yeast, and chronocoulometric measurements were used to generate a pattern of responses for each organism toward each lectin. Principal component analysis was used to classify the chronocoulometric results for the different microbial strains. With this new method, six microbial species (Baccilus cereus, Staphylococcus aureus, Proteus vulgaris, Escherichia coli, Enterobacter aerogenes, Saccharomyces cerevisiae) were readily distinguished.

Animals↗

On-line dual microdialysis with ESI-MS for direct analysis of complex biological samples and microorganism lysates.

A novel dual-microdialysis approach has been developed for fast and efficient fractionation and cleanup for ESI-MS and ESI MS/MS analyses of biological samples. A modified dynamic microdialyzer utilizing two mirror-image serpentine channels, which sandwich a regenerated cellulose membrane of selected molecular weight cutoff, serves as the first stage for the removal of high-molecular-weight components and cellular residue. The second stage employs a hollow microdialysis capillary to remove low-molecular-weight species (e.g., salts) which can degrade or preclude analysis ESI-MS. A protein mixture consisting of 30 microM bovine serum albumin (BSA), 4.0 microM cytochrome c, 2.3 microM ubiquitin, and 9.4 microM bradykinin in 0.5 M NaCl was used to evaluate the performance of this system. Essentially complete removal of both BSA and NaCl was achieved, resulting in high-quality mass spectra containing only the lower molecular weight proteins. After passing through the on-line dual-microdialysis system, a crude bacteria cell lysate yielded clean ESI-mass spectra in approximately 20 min. MS/MS of selected ions demonstrated abundant fragment ions and provided a second-dimension "fingerprint" of the complex cellular fraction. Preliminary application of this technique for direct characterization of microorganism lysates is presented.

Escherichia coli↗

Differentiation of microorganisms based on pyrolysis-ion trap mass spectrometry using chemical ionization.

The ability to differentiate microorganisms using pyrolysision trap mass spectrometry was demonstrated for five Gram-negative disease-causing organisms: Brucella melitensis, Brucella suis, Vibrio cholera, Yersinia pestis, and Francisella tularensis. Bacterial profiles were generated for gamma-irradiated bacterial samples using pyrolytic methylation and compared for electron ionization and chemical ionization using several liquid reagents with increasing proton affinities. Electron ionization combined with pyrolysis caused extensive fragmentation, resulting in a high abundance of lower mass ions and diminishing the diagnostic value of the technique for compound identification and bacterial profiling. Chemical ionization reduced the amount of fragmentation due to ionization while enhancing the molecular ion region of the fatty acids. As the proton affinity of the reagent increased, the protonated molecular ions of the fatty acids became the predominant ions observed in the mass spectrum. As a result, chemical ionization was shown to be more effective than electron ionization in bacterial profiling. Whereas the bacteria could be distinguished at the Genera level using electron ionization, further differentiation to the subspecies level was possible using chemical ionization. The greatest separation among the five test organisms, in terms of Euclidean distances, was obtained using ethanol as the chemical ionization reagent and using pooled masses representing specific fatty acid biomarkers rather than total ion profiles.

Fatty Acids↗

Syntheses of Curcumin Bioconjugates and Study of Their Antibacterial Activities against beta-Lactamase-Producing Microorganisms.

In the present study curcumin bioconjugates, viz. di-O-glycinoylcurcumin (I), di-O-glycinoyl-C(4)-glycylcurcumin (II), 5'-deoxy-5'-curcuminylthymidine (5'-cur-T) (IV), and 2'-deoxy-2'-curcuminyluridine (2'-cur-U) (V) have been synthesized and characterized by elemental analysis and (1)H NMR. The turmeric peptide (Tp) was isolated from the aqueous turmeric extract of the turmeric rhizome. The antibacterial activity of these four bioconjugates and also of the turmeric peptide and sodium salt of curcumin (III) have been tested particularly for beta-lactamase-producing microorganisms.

Bacteria↗

Structural and functional properties of calmodulin from the eukaryotic microorganism Dictyostelium discoideum.

Calmodulin was purified from the eukaryotic microorganism Dictyostelium discoideum and characterized in terms of its nearly complete primary structure and quantitative activator activity. The strategy for amino acid sequence analysis took advantage of the highly conserved structure of calmodulin and employed a new procedure for limited cleavage of calmodulin that uses a protease from mouse submaxillary gland. Fourteen amino acid sequence differences between Dictyostelium and bovine calmodulin were identified unequivocally, as well as an unmethylated lysine at residue 115 instead of N epsilon, N epsilon, N epsilon-trimethyllysine. Seven of the amino acid substitutions in Dictyostelium calmodulin are novel in that the residues at these positions are invariant in all calmodulin sequences previously examined, most notably an additional residue at the carboxy terminus. Comparison of the Dictyostelium calmodulin sequence with other calmodulin sequences shows that the region with the greatest extended sequence identity includes parts of the first and second structural domains and the interdomain region between domains 1 and 2. Dictyostelium calmodulin activated bovine brain cyclic nucleotide phosphodiesterase in a manner indistinguishable from that of bovine brain calmodulin. However, Dictyostelium calmodulin activated pea NAD kinase to a maximal level 4.6-fold greater than that produced by bovine brain calmodulin. This functional difference demonstrates the potential biological importance of the limited number of amino acid sequence differences between Dictyostelium calmodulin and other calmodulins and provides further insight into the structure, function, and evolution of the calmodulin family of proteins.

2',3'-Cyclic-Nucleotide Phosphodiesterases↗

A simple strategy for the purification of large thermophilic proteins overexpressed in mesophilic microorganisms: application to multimeric enzymes from Thermus sp. strain T2 expressed in Escherichia coli.

The heating of protein preparations of mesophilic organism (e.g., E. coli) produces the obliteration of all soluble multimeric proteins from this organism. In this way, if a multimeric enzyme from a thermophilic microorganism is expressed in these mesophilic hosts, the only large protein remaining soluble in the preparation after heating is the thermophilic enzyme. These large proteins may be then selectively adsorbed on lowly activated anionic exchangers, enabling their full purification in just these two simple steps. This strategy has been applied to the purification of an alpha-galactosidase and a beta-galactosidase from Thermus sp. strain T2, both expressed in E. coli, achieving the almost full purification of both enzymes in only these two simple steps. This very simple strategy seems to be of general applicability to the purification of any thermophilic multimeric enzyme expressed in a mesophilic host.

Anion Exchange Resins↗

Metal speciation dynamics and bioavailability. 2. Radial diffusion effects in the microorganism range.

The free ion activity model for the biouptake of metals from complex media is limited to cases where mass transfer is not flux determining. This paper follows a previous paper (Van Leeuwen, H. P. Environ. Sci. Technol. 1999, 33, 3743) where speciation dynamics and bioavailability of metals are analyzed in terms of bioconversion kinetics and simultaneous metal transport in the medium coupled with dissociation kinetics. Such analysis shows under what conditions labile complex species contribute to the biouptake process or, equivalently, under what conditions the free ion activity model is not obeyed. The present work addresses the theoretical extension of the expressions for the metal flux in the medium by a radial diffusion term so that these are also applicable in the microorganism size range. The transition from macroscopic to microscopic surfaces affects not only the nature of the flux but also the extent of lability of complex species (Van Leeuwen, H. P.; Pinheiro, J. P. J. Electroanal. Chem. 1999, 471, 55), and this can have a dramatic influence on the rate of biouptake of metal ions. Labilities of metal complexes and the ensuing limiting metal fluxes are therefore systematically analyzed for various dimensions of the uptaking surface. Different conditions of bioaffinities and bioconversion capacities are considered, and a number of examples of metal complexes with specified kinetic features are discussed.

Biological Availability↗

Utilization and transformation of aquatic humic substances by autochthonous microorganisms.

Aquatic humic substances (HS) from a bog lake water, a riverwater, and a groundwater were isolated after enrichment on XAD 8 columns and added to a Czapek-Dox nutrient broth which was used either in full strength or without glucose and/or NaNO3. The individual flasks were inoculated with natural microbial populations of corresponding water samples or with a Pseudomonas fluorescens strain isolated from groundwater. The presence of HS resulted in an increase of bacterial numbers in nearly all cultures incubated for 3 weeks at 25 degrees C on a shaker. HS reisolated from cultures without glucose or NaNO3 showed no or only minor quantitative differences as compared to those from sterile controls. In full strength nutrient broth up to 27% of HS were utilized. Data obtained by spectroscopic methods (UV/vis/FTIR) and elemental analysis indicated a decrease in particle size and a loss in aromaticity and aliphatic carbon in HS reisolated from the microbial cultures. Simultaneously an increase in the N content of HS was observed, which probably originated from some constituents of microbial biomass such as proteins and amino sugars. The NMR data also documented that significant transformations of HS occurred in the individual microbial cultures. After incubation, increased amounts of aromatic acids were detected in some liquid media and residual HS by GC/MS or capillary electrophoresis. 1H NMR spectroscopy was less effective in indicating structural differences in the HS than 13C NMR but revealed considerable detail of the microbial degradation of riverine HS, when limited sample was available. The newly developed NMR increment analysis provided substantial detail of aromatic structures in a microbially altered HS. The microbial degradation of HS strongly depended on the composition of the HS, the species selection of the microorganisms, and to a lesser extent on the culture conditions. For any series of identical inoculum and HS, full broth media initiated the most extensive alteration of HS.

Biological Availability↗

Anaerobic oxidation of crude oil hydrocarbons by the resident microorganisms of a contaminated anoxic aquifer.

The biodegradation of two crude oils by microorganisms from an anoxic aquifer previously contaminated by natural gas condensate was examined under methanogenic and sulfate-reducing conditions. Artificially weathered Alaska North Slope crude oil greatly stimulated both methanogenesis and sulfate reduction. Gas chromatographic analysis revealed the entire n-alkane fraction of this oil (C13-C34) was consumed under both conditions. Naphthalene, 2-methylnaphthalene, and 2-ethylnaphthalene were also biodegraded but only in the presence of sulfate. Alba crude oil, which is naturally depleted in n-alkanes, resulted in a relatively modest stimulation of methanogenesis and sulfate reduction. Polycyclic aromatic hydrocarbon biodegradation was similar to that found for the Alaska North Slope crude oil, but a broader range of compounds was metabolized, including 2,6-dimethylnaphthalene and 2,7-dimethylnaphthalene in the presence of sulfate. These results indicate that n-alkanes are relatively labile, and their biodegradation in terrestrial environments is not necessarily limited by electron acceptor availability. Polycyclic aromatic hydrocarbons are relatively more recalcitrant, and the biodegradation of these substrates appeared to be sulfate-dependent and homologue-specific. This information should be useful for assessing the limits of in situ crude oil biodegradation in terrestrial environments and for making decisions regarding risk-based corrective actions.

Anaerobiosis↗

Inactivation of microorganisms using electrostatic fields.

This research investigated whether electrostatic fields can be used to inactivate surface-borne and airborne microorganisms. Vegetative cells of Pseudomonas fluorescens and Bacillus subtilis var. niger were deposited on filters and subjected to electrostatic fields of different strengths and polarities for controlled time periods. In addition, P. fluorescens bacteria, which represent sensitive species, were aerosolized and exposed to electrostatic fields of up to +/- 10 kV/cm. The results have shown that more than 90% of the P. fluorescens cells deposited on the surface of nonconductive filters are inactivated when fields of 15 kV/cm are applied for 30 min or longer. Similar effects were observed when P. fluorescens were exposed to fields of 5 and 10 kV/cm for 2 h. In contrast, the culturability of B. subtilisvar. niger cells exposed under the same conditions did not substantially decrease. Exposure of airborne P. fluorescens to +/- 10 kV/cm for 30 s also did not result in a significant reduction of culturability. This research has shown that specific combinations of electrostatic field strength and exposure time can be used to effectively inactivate certain bacterial cells deposited on nonconductive surfaces. For the investigated conditions, the treatment was not effective for bacteria in the airborne state.

Air Pollution↗

The role of reactive oxygen species in the electrochemical inactivation of microorganisms.

Electrochemical disinfection has emerged as one of the most promising alternatives to the conventional disinfection of water in many applications. Although the mechanism of electrochemical disinfection has been largely attributed to the action of electro-generated active chlorine, the role of other oxidants, such as the reactive oxygen species (ROS) *OH, O3, H2O2, and *O2- remains unclear. In this study, we examined the role of ROS in the electrochemical disinfection using a boron-doped diamond (BDD) electrode in a chloride-free phosphate buffer medium, in order to avoid any confusion caused by the generation of chlorine. To determine which species of ROS plays the major role in the inactivation, the effects of several operating factors, such as the presence of *OH scavenger, pH, temperature, and the initial population of microorganisms, were systematically investigated. This study clearly showed that the *OH is the major lethal species responsible for the E. coli inactivation in the chloride-free electrochemical disinfection process, and that the E. coli inactivation was highly promoted at a lower temperature, which was ascribed to the enhanced generation of O3.

Boron↗

Occurrence of 12-methyltridecanal in microorganisms and physiological samples isolated from beef.

12-Methyltridecanal (MT) smelling tallowy, beef-like was formed from plasmalogens when beef was boiled. To clarify the origin of MT, its concentration was determined by a stable isotope dilution assay in bacteria and protozoa isolated from the rumen of bovine animals as well as in the plasma, erythrocytes, and other physiological samples. The highest amounts of MT were found in bacteria followed by protozoa. The MT content of the erythrocytes was small. The results support the hypothesis that microorganisms are the main source of MT of which a small amount is resorbed by the animal and transported to the muscular tissue where MT is incorporated into plasmalogens.

Abomasum↗

An investigation of chlorophenol proton affinities and their influence on the biological activity of microorganisms.

The proton affinities of 15 chlorophenols are calculated by ab initio methods. Straight correlation between proton affinities and changes in the electronic structure is observed. The proton affinities decrease linearly with the electronic density gain on the chlorine atoms, as the liberation of the proton increases. To confirm the importance of the proton affinities on the toxicity of chlorophenols, calorimetric responses of these molecules and related ones where the acid proton is changed to a methyl group (anisol and its chlorinated derivatives) were used to verify their effects on Chromobacterium violaceum. The results confirmed that the chlorophenols are more toxic than the respective chloroanisols and suggest that high proton affinities are associated with low toxic activity. The toxicity of the chlorophenols can be associated with the respiratory mechanism in some microorganisms.

Anti-Bacterial Agents↗