Ecological Implications of an Improved Direct Viable Count Method for Aquatic Bacteria

The direct viable count method first described by Kogure et al. (Can. J. Microbiol. 25:415-420, 1979) was improved by using an antibiotic cocktail instead of nalidixic acid alone. We screened 100 marine isolates from two coastal areas for their sensitivities to five replication-inhibiting antibiotics, including four quinolones (nalidixic, piromidic, and pipemidic acids and ciprofloxacin) and one (beta)-lactam (cephalexin). It was shown that growth inhibition of all isolates cannot be readily achieved by using a single antibiotic. Inhibition was much more efficient when all the antibiotics were combined, making it possible to use this method with natural communities. In combination, the concentration of each antibiotic could be lowered and the incubation time could be increased without any growth. Under such conditions, it was shown that the fraction of substrate-responsive cells within natural marine communities is much greater (1 to 2 orders of magnitude) than those reported by traditional procedures. Furthermore, the new procedure made substrate-responsive cells more clearly distinguishable. These improvements resulted in an increased incubation time and were related to metabolic expression of slow-growing cells and/or to the recovery of starved cells. The increased fraction of viable cells within marine communities has ecological implications on the metabolic role of nonculturable cells.     

Viability and Estimation of Shelf-Life of Bacterial Populations

Mathematical concepts associated with the exponential and probit models are developed, and the similarity of the two methods is discussed. Because of its greater flexibility in design, the probit method was used to estimate the shelf-life for four bacterial populations, wet and dry spores of Bacillus anthracis and wet and dry cells of Pasteurella tularensis. On the basis of data gained by storing these organisms at high temperature, the probit method was used to predict the time at which 50% viability would occur for cells stored at 3 C. A plane passing through a three-space showing change in percentage viability of bacteria was formulated by a multiple regression method. With this functional technique, the percentage viability, expressed as a probit, was linearily related to a logarithm of storage time and storage temperature. The use of this method to study the effect of controlled variables on the viability of cells is demonstrated by comparing the effect of viability associated with three additives used prior to drying. The results of the test gave shelf-life estimates which were too low for all additives; however, the order of stability was ranked properly as confirmed by long-term tests.     

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 Bacterial Count as a Rapid Freshness Test for Fish Fillets

Comparison of various indices of deterioration of refrigerated fish fillets showed that the direct bacterial count can be used to predict the storage life of the foodstuff. For direct counts, a thin film made from fillet surface material was spread on a microscope slide, stained, and read. Initial counts were found to correlate well with keeping quality; a period of freshness of 24 or 48 hr at 5 C could be reliably predicted. Preliminary data indicated that hypoxanthine estimation could probably also be used for the prediction of shelf life but that the relative complexity of the procedure detracted from its usefulness.     

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.     

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.     

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.     

Use of Quantitative Real-Time PCR for Direct Detection of Serratia marcescens in Marine and Other Aquatic Environments

Serratia marcescens is the etiological agent of acroporid serratiosis, a distinct form of white pox disease in the threatened coral Acropora palmata. The pathogen is commonly found in untreated human waste in the Florida Keys, which may contaminate both nearshore and offshore waters. Currently there is no direct method for detection of this bacterium in the aquatic or reef environment, and culture-based techniques may underestimate its abundance in marine waters. A quantitative real-time PCR assay was developed to detect S. marcescens directly from environmental samples, including marine water, coral mucus, sponge tissue, and wastewater. The assay targeted the luxS gene and was able to distinguish S. marcescens from other Serratia species with a reliable quantitative limit of detection of 10 cell equivalents (CE) per reaction. The method could routinely discern the presence of S. marcescens for as few as 3 CE per reaction, but it could not be reliably quantified at this level. The assay detected environmental S. marcescens in complex sewage influent samples at up to 761 CE ml⁻¹ and in septic system-impacted residential canals in the Florida Keys at up to 4.1 CE ml⁻¹. This detection assay provided rapid quantitative abilities and good sensitivity and specificity, which should offer an important tool for monitoring this ubiquitous pathogen that can potentially impact both human health and coral health.     

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.     

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。     

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.