Widespread Occurrence of Bacterial Human Virulence Determinants in Soil and Freshwater Environments

The occurrence of 22 bacterial human virulence genes (encoding toxins, adhesins, secretion systems, regulators of virulence, inflammatory mediators, and bacterial resistance) in beech wood soil, roadside soil, organic agricultural soil, and freshwater biofilm was investigated by nested PCR. The presence of clinically relevant bacterial groups known to possess virulence genes was tested by PCR of 16S and 23S rRNA genes. For each of the virulence genes detected in the environments, sequencing and NCBI BLAST analysis confirmed the identity of the PCR products. The virulence genes showed widespread environmental occurrence, as 17 different genes were observed. Sixteen genes were detected in beech wood soil, and 14 were detected in roadside and organic agricultural soils, while 11 were detected in the freshwater biofilm. All types of virulence traits were represented in all environments; however, the frequency at which they were detected was variable. A principal-component analysis suggested that several factors influenced the presence of the virulence genes; however, their distribution was most likely related to the level of contamination by polycyclic aromatic hydrocarbons and pH. The occurrence of the virulence genes in the environments generally did not appear to be the result of the presence of clinically relevant bacteria, indicating an environmental origin of the virulence genes. The widespread occurrence of the virulence traits and the high degree of sequence conservation between the environmental and clinical sequences suggest that soil and freshwater environments may constitute reservoirs of virulence determinants normally associated with human disease.     

Direct Method for Determining the Viability of a Freshly Generated Mixed Bacterial Aerosol

A direct method was developed to determine the viability of a freshly generated mixed bacterial aerosol. A mixed suspension of (32)P-labeled Staphylococcus aureus and (35)S-labeled Proteus mirabilis was nebulized, and the aerosol was collected and separated according to particle size with an Andersen sampler. Quantitative and qualitative bacteriological and radioisotopic techniques were used to obtain ratios of bacterial to radioactive counts for each organism in samples of the nebulizer suspension and aerosol. Loss of viability was calculated from the change that occurred between the ratio of the nebulizer suspension and the ratio of the aerosol. The viability of S. aureus was unaffected by aerosolization, whereas the viability of P. mirabilis declined by 20 to 60% and was inversely proportional to particle size. The advantages of this method over present indirect methods, as well as potential applications of the method, are discussed.     

Direct Measurements of Natural Planktonic Bacterial Community Viability by Flow Cytometry

A range of fluorescent viability dyes were used in conjunction with flow cytometry to rapidly enumerate viable bacteria from freshwater environments. Optimal labelling was achieved by using carboxyfluorescein diacetate or chemchrome B with a detergent-mediated permeabilization step. The viable bacterial count under optimal conditions was 7% in oligotrophic lake water and 75% in polluted river water.     

Estimation of Bacterial Nitrate Reduction Rates at In Situ Concentrations in Freshwater Sediments

A method was developed to follow bacterial nitrate reduction in freshwater sediments by using common high-performance liquid chromatographic equipment. The low detection limit (14 pmol) of the method enabled us to study concentration profiles and reaction kinetics under natural conditions. Significant nitrate concentrations (1 to 27 μM) were observed in the sediment of Lake Vechten during the nonstratified period; the concentration profiles showed a successive depletion of oxygen, nitrate, and sulfate with depth. The profiles were restricted to the upper 3 cm of the sediment which is rich in organics and loosely structured. Nitrate reduction in the sediment-water interface followed first-order reaction kinetics at in situ concentrations. Remarkably high potential nitrate-reducing activity was observed in the part of the sediment in which nitrate did not diffuse. This activity was also observed throughout the whole year. Estimates of K_m varied between 17 and 100 μM and V_max varied between 7.2 and 36 μmol cm⁻³ day⁻¹ for samples taken at different depths. The diffusion coefficient of nitrate ([10 ± 0.4] × 10⁻⁶ cm² s⁻¹) across the sediment-water interface was estimated by a constant-source technique and applied to a mathematical model to estimate the net nitrate reduction during the nonstratified period. In this period, observed nitrate reduction rates by the model, 0.2 to 0.4 mmol m⁻² day⁻¹, were lower than those found for oxygen (27 mmol m⁻² day⁻¹) and sulfate (0.4 mmol m⁻² day⁻¹). During the summer stratification, nitrate was absent in the sediment and reduction could not be estimated by the model.     

Direct Determination of Carbon and Nitrogen Contents of Natural Bacterial Assemblages in Marine Environments

In order to better estimate bacterial biomass in marine environments, we developed a novel technique for direct measurement of carbon and nitrogen contents of natural bacterial assemblages. Bacterial cells were separated from phytoplankton and detritus with glass fiber and membrane filters (pore size, 0.8 μm) and then concentrated by tangential flow filtration. The concentrate was used for the determination of amounts of organic carbon and nitrogen by a high-temperature catalytic oxidation method, and after it was stained with 4′,6-diamidino-2-phenylindole, cell abundance was determined by epifluorescence microscopy. We found that the average contents of carbon and nitrogen for oceanic bacterial assemblages were 12.4 ± 6.3 and 2.1 ± 1.1 fg cell⁻¹ (mean ± standard deviation; n = 6), respectively. Corresponding values for coastal bacterial assemblages were 30.2 ± 12.3 fg of C cell⁻¹ and 5.8 ± 1.5 fg of N cell⁻¹ (n = 5), significantly higher than those for oceanic bacteria (two-tailed Student’s t test; P < 0.03). There was no significant difference (P > 0.2) in the bacterial C:N ratio (atom atom⁻¹) between oceanic (6.8 ± 1.2) and coastal (5.9 ± 1.1) assemblages. Our estimates support the previous proposition that bacteria contribute substantially to total biomass in marine environments, but they also suggest that the use of a single conversion factor for diverse marine environments can lead to large errors in assessing the role of bacteria in food webs and biogeochemical cycles. The use of a factor, 20 fg of C cell⁻¹, which has been widely adopted in recent studies may result in the overestimation (by as much as 330%) of bacterial biomass in open oceans and in the underestimation (by as much as 40%) of bacterial biomass in coastal environments.     

Microtiter Method for the Evaluation of Viable Cells in Bacterial Cultures

A microtiter technique was investigated as a means of evaluating viable cells in bacterial cultures. Parallel experiments were performed employing the conventional agar plate method along with the microtiter procedure. Statistical analyses showed that the correlation between the two methods was highly significant. With this new method, many samples were analyzed simultaneously, and readable results were obtained in 12 to 15 hr. Other advantages of this method were substantial savings of time, space, and materials. Also, the applicability of this method to estimates of mixed bacterial populations was demonstrated by studying the associative growth of two bacterial cultures.     

Sensitive Procedure for Detecting Residual Viable Virus in Inactivated Rabies Vaccine

A procedure for testing inactivated rabies vaccines of tissue culture origin for residual viable virus is reported in which the vaccine to be tested is passed in primary hamster kidney cell culture (PHK) before mouse inoculation. In preliminary experiments, titrations of rabies virus in which each dilution was passed in PHK before inoculating mice yielded titers 100 to 10,000 times higher than the titers obtained for the same virus by direct mouse inoculation. This rabies virus amplification procedure was evaluated by testing 18 lots of inactivated rabies vaccine of tissue culture origin. No viable virus was found in these vaccine lots when tested by direct intracerebral inoculation of mice. Eight of these 18 lots were found to contain viable virus, however, when tested by passage in PHK cell culture. The significance of low levels of viable virus in rabies vaccines is discussed. It is recommended that the amplification procedure described in this report be used in the safety testing of rabies vaccines of tissue culture origin and that it be evaluated for use in testing other rabies vaccines of low tissue content.     

有机物料腐熟剂产品中芽孢杆菌含量的检测

目的对于有机物料腐熟剂中可能存在的芽孢杆菌进行确证和含量测定。采用MYP作为芽孢杆菌的选择性培养基,观察芽孢杆菌在该培养基上的菌落形态和生长状况,并通过厌氧培养、丙酸盐利用,V-P反应,淀粉水解等生理生化确证实验可以分辨和确定腐熟剂中的枯草芽孢杆菌、地衣芽孢杆菌、巨大芽孢杆菌和短小芽孢杆菌含量。方法要优于现有的腐熟剂有效活菌检测方法。     

Effectiveness of SYTOX Green Stain for Bacterial Viability Assessment

The effectiveness of SYTOX Green nucleic acid stain for measuring bacterial viability was tested on starved populations of Escherichia coli and Salmonella typhimurium. This stain underestimates the fraction of dead cells within starved populations containing cells with damaged nucleic acids or membranes. Its application to natural samples should be considered with caution.     

Rate of Bacterial Mortality in Aquatic Environments

A method is proposed which provides a minimum estimate of the rate of bacterial mortality in growing natural populations of planktonic bacteria. This estimate is given by the rate of decrease of radioactivity from the DNA of a [³H]thymidine-labeled natural assemblage of bacteria after all added thymidine has been exhausted from the medium. Results obtained from river water, estuarine water, and seawater show overall bacterial mortality rates in the range 0.010 to 0.030 h⁻¹, in good agreement with the range of growth rates measured in the same environments. Use of selective filtration through Nuclepore filters (pore size, 2 μm) allowed us to determine the contribution of microzooplankton grazing to overall bacterial mortality. Grazing rates estimated by this method ranged from 0 to 0.02 h⁻¹.     

Effect of Lysozyme on Enterococcal Viability in Low Ionic Environments

The action of lysozyme on the enterococcal cell differed markedly as a function of the ionic strength of the environment. In high ionic environments (I 0.3), the traditionally slow lytic response and decrease in viability were noted. In a low ionic environment the majority of the cell wall was hydrolyzed, but cellular integrity was preserved and almost all cellular protein, deoxyribonucleic acid and ribonucleic acid remained with the lysozyme-cell complex. However, under these conditions, lysozyme inactivated energy-yielding metabolism, and a rapid extensive loss of viability was observed. Some other basic compounds without lytic activity on the cell wall also effected a substantial reduction in viability. The data suggest that lysozyme acts on the cell membrane to effect disruption of cellular metabolism.     

A Comparison of Alternative Methods to Viable Count for Indicating Growth of Mycoplasma gallisepticum in Liquid Culture

In an attempt to find a more rapid method than a viable count for estimating growth of Mycoplasma gallisepticum in broth culture, seven alternative methods were each examined for their correlation with viable counts, reproducibility and time taken to obtain results. Opacity measurement was the quickest and showed closest correlation with viable count and also high reproducibility. The other methods examined showed either lack of sensitivity or reproducibility or poorer correlation with viable count.     

淡水涡虫染色体的制备方法

以涡虫的再生组织为材料,用秋水仙素处理长有再生组织的涡虫片段,并通过改善各种实验条件,得到高清晰度的染色体图谱。结果表明,本方法简便易行,制成的染色体分散好,形态清晰,适用于对涡虫染色体的进一步研究。     

Improved Climate Risk Simulations for Rice in Arid Environments

We integrated recent research on cardinal temperatures for phenology and early leaf growth, spikelet formation, early morning flowering, transpirational cooling, and heat- and cold-induced sterility into an existing to crop growth model ORYZA2000. We compared for an arid environment observed potential yields with yields simulated with default ORYZA2000, with modified subversions of ORYZA2000 and with ORYZA_S, a model developed for the region of interest in the 1990s. Rice variety ‘IR64’ was sown monthly 15-times in a row in two locations in Senegal. The Senegal River Valley is located in the Sahel, near the Sahara desert with extreme temperatures during day and night. The existing subroutines underestimated cold stress and overestimated heat stress. Forcing the model to use observed spikelet number and phenology and replacing the existing heat and cold subroutines improved accuracy of yield simulation from EF = −0.32 to EF =0.70 (EF is modelling efficiency). The main causes of improved accuracy were that the new model subversions take into account transpirational cooling (which is high in arid environments) and early morning flowering for heat sterility, and minimum rather than average temperature for cold sterility. Simulations were less accurate when also spikelet number and phenology were simulated. Model efficiency was 0.14 with new heat and cold routines and improved to 0.48 when using new cardinal temperatures for phenology and early leaf growth. The new adapted subversion of ORYZA2000 offers a powerful analytic tool for climate change impact assessment and cropping calendar optimisation in arid regions.     

Correlation of Direct Viable Counts with Heterotrophic Activity for Marine Bacteria

Viable-bacteria counts, heterotrophic activity, and substrate responsiveness of viable bacteria have been used to measure microbial activity. However, the relationship between these parameters is not clear. Thus, the direct viable count (DVC) method was used to analyze seawater samples collected from several different geographical locations. Samples collected from offshore waters of the South China Sea and western Pacific Ocean yielded DVC that indicated the presence of surface and subsurface peaks of viable, substrate-responsive bacteria which could be correlated with turnover rates of amino acids obtained by using uniformly ¹⁴C-labeled amino acids. DVC were always less than total viable counts (acridine orange direct counts), and the DVC subsurface peak occurred close to and within the chlorophyll a zone, suggesting algal-bacterial interactions within the layer. For comparison with the open-ocean samples, selected substrates were used to determine the response of viable bacteria present in seawater samples collected near an ocean outfall of the Barceloneta Regional Waste Treatment Plant, Barceloneta, Puerto Rico. The number of specific substrate-responsive bacteria at the outfall stations varied depending on the substrate used and the sampling location. Changes in the population size or physiological condition of the bacteria were detected and found to be associated with the presence of pharmaceutical waste.     

Estimation of Provisional Seed Viability Constants for Lupinus polyphyllus Lindley

A limited amount of data on the hermetic storage of Lupinuspolyphyllus Lindley seeds in several different environmentshas been used to provide estimates of seed viability constants.These allow reasonably accurate predictions of seed storagelife under known conditions (orthodox storage), especially inthe short or medium term. Lupinus polyphyllus Lindley, seed storage, viability constants, influence of seed moisture content and temperature     

Radioassay for Hydrogenase Activity in Viable Cells and Documentation of Aerobic Hydrogen-Consuming Bacteria Living in Extreme Environments

An isotopic tracer assay based on the hydrogenase-dependent formation of tritiated water from tritium gas was developed for in life analysis of microbial hydrogen transformation. This method allowed detection of bacterial hydrogen metabolism in pure cultures or in natural samples obtained from aquatic ecosystems. A differentiation between chemical-biological and aerobic-anaerobic hydrogen metabolism was established by variation of the experimental incubation temperature or by addition of selective inhibitors. Hydrogenase activity was shown to be proportional to the consumption or production of hydrogen by cultures of Desulfovibrio vulgaris, Clostridium pasteurianum, and Methanosarcina barkeri. This method was applied, in connection with measurements of free hydrogen and most-probable-number enumerations, in aerobic natural source waters to establish the activity and document the ecology of hydrogen-consuming bacteria in extreme acid, thermal, or saline environments. The utility of the assay is based in part on the ability to quantify bacterial hydrogen transformation at natural hydrogen partial pressures, without the use of artificial electron acceptors.