Occurrence of Potential Pathogens in Water Containing Ornamental Fishes

The bacterial population of the water supplied with ornamental fish purchased from retail outlets was examined qualitatively and quantitatively. As many as 10(9) viable aerobic organisms per 100 ml were present, with fecal coliform counts as high as 10(5) per 100 ml. Citrobacter, Escherichia, Pseudomonas, and Vibrio were isolated from 75% or more of the samples, whereas Aeromonas, Alcaligenes, Enterobacter, Flavobacterium, and Streptococcus were isolated from 45 to 65% of the samples. Pseudomonas aeruginosa, Edwardsiella tarda, and Klebsiella pneumoniae were also isolated.     

Performance characteristics of a new photometer with a moving filter tape for luminescence assay.

The performance characteristics of a new photometer that incorporated a reaction system consisting of a movable filtration tape have been studied for use with the firefly luciferase assay for adenosine 5'-triphosphate. Precision, linearity, and sensitivity are given for measuring a graded series of constant-light emitters, concentrations of adenosine 5'-triphosphate, and washed bacterial cells. Precision ranged from 1 to 10% coefficient of variation. The coefficient of correlation was 0.9985 for 7 X 10(8) to 7 X 10(3) bacteria per ml; however, the accuracy decreased at the low levels. The sample processing time was 2 min for a 1-ml sample. This instrument shows potential for many applications in quantitating small numbers of organisms, e.g., pollution, water monitoring, and clinical infection detection.     

Performance characteristics of a new photometer with a moving filter tape for luminescence assay.

The performance characteristics of a new photometer that incorporated a reaction system consisting of a movable filtration tape have been studied for use with the firefly luciferase assay for adenosine 5'-triphosphate. Precision, linearity, and sensitivity are given for measuring a graded series of constant-light emitters, concentrations of adenosine 5'-triphosphate, and washed bacterial cells. Precision ranged from 1 to 10% coefficient of variation. The coefficient of correlation was 0.9985 for 7 X 10(8) to 7 X 10(3) bacteria per ml; however, the accuracy decreased at the low levels. The sample processing time was 2 min for a 1-ml sample. This instrument shows potential for many applications in quantitating small numbers of organisms, e.g., pollution, water monitoring, and clinical infection detection.     

The chemistry of insect hemolymph; organic components of the hemolymph of the silkworm,Bombyx mori,and two other species

1. Hemolymph was collected for analysis from the silkworm, Bombyx mori, in a series of developmental stages ranging from the second molt to the late pupa. The mean pH of larval hemolymph after collection was found to be 6.45, that of pupal hemolymph, 6.57; in vivo values may be slightly lower. Total dry solids ranged from 5.4 to 10.6 per cent. Total protein ranged from 1.2 to 5.3 per cent, increasing rapidly during the fifth instar. 2. Free amino acids were separated chromatographically and estimated. Of 19 amino acids identified, amounting collectively to 823 to 1497 mg. per 100 ml., glutamine, histidine, and lysine generally occurred in greatest amount. Tryptophan was not detected, and cystine (or cysteine) was found in only one sample. The total free amino acids account for 35 to 55 per cent of the non-protein nitrogen of the plasma. 3. Free sugars, estimated semiquantitatively on chromatograms, comprise glucose, fructose, and sucrose in total amount ranging from about 5 to 40 mg. per 100 ml. Total acid-soluble, ultrafiltrable carbohydrate, estimated as glucose by the anthrone reaction, ranged from 166 to 635 mg. per 100 ml., indicating the presence of low molecular weight sugar derivatives. 4. Inorganic phosphate amounted to 5 to 15 mg. per 100 ml., and acid-soluble organic phosphate to 100 to 200 mg. per 100 ml. The latter fraction includes several substances, of which one was tentatively identified as glucose-6-phosphate and the remainder are as yet unidentified. 5. Single samples of hemolymph were also taken from larvae of the wax moth, Galleria mellonella, and the spruce sawfly, Diprion hercyniae. These contained even higher concentrations of solutes than the silkworm samples, but with a generally similar distribution. The proportions of the free amino acids were different in each species.     

Evaluation of parameters affecting quantitative detection of Escherichia coli O157 in enriched water samples using immunomagnetic electrochemiluminescence

We report here the use of immunomagnetic (IM) electrochemiluminescence (ECL) for quantitative detection of Esherichia coli O157:H7 in water samples following enrichment in minimal lactose broth (MLB). IM beads prepared in-house with four commercial anti-O157 monoclonal antibodies were compared for efficiency of cell capture. IM-ECL responses for E. coli O157:H7 (strain SEA13B88) were similar for all four commercial anti-O157 LPS monoclonal antibodies. The ECL signal was linearly correlated with E. coli O157:H7 cell concentration, indicating a constant ECL response per cell. Twenty-two strains of E. coli O157:H7 or O157:NM gave comparable ECL signals using IM beads prepared in-house. To assess the potential for interference from background bacteria in MLB-enriched water samples, 10(4) cells of E. coli O157:H7 (strain SEA13B88) were added to enriched samples prior to analysis. There was considerable variability in recovery of E. coli O157:H7 cells; net ECL signals ranged from 1% to 100% of expected values (i.e., percent inhibition from 0% to 99%). Cultures of Klebsiella pneumoniae, Klebsiella oxytoca, and Enterobacter cloacae, subsequently isolated from MLB-enriched water samples via IM separation (IMS), were observed to interfere with the binding of E. coli O157:H7 cells to IM beads. Recoveries of 10(4) E. coli O157:H7 cells were 相似文献   

Selective cytotoxicity of 3-amino-l-tyrosine correlates with peroxidase activity

Summary In the presence of 3-amino-l-tyrosine (3-AT), abundant brown pigment forms in human HL-60 cells, but not in a variety of other cell lines, which are reported to be lower in mean myeloperoxidase (MPO) content than HL-60. Cells were assessed for peroxidase activity with an ABTS-based colorimetric assay and compared to values obtained with known amounts of human myeloperoxidase. HL-60 cells were estimated to contain the equivalent of 37.1 ng myeloperoxidase/10⁶ cells versus 26.1 and 5.0 ng/10⁶ cells for human K562 and murine RAW 264.7 cell lines, respectively. HL-60 cells exhibited a nearly 60% inhibition of proliferation and >70% reduction in cell viability after 4 d of culture in the presence of 100 μg 3-AT per ml. Higher concentrations of 3-AT (up to 400 μg/ml) for 4 d reduced HL-60 proliferation by 80% and decreased viability to 1–3%. Comparable levels of cytotoxicity were achieved in KG-1 cells after 7 d with 200 or 400 μg 3-AT per ml. K562 cells exhibited a 40% reduction in cell number after 7 d with 400 μg 3-AT per ml, but concentrations less than 400 μg/ml did not significantly affect K562 proliferation. K562 viability remained unchanged with doses of 3-AT up to 400 μg/ml. RAW 264.7 cells exhibited unchanged viability and proliferation in the presence of 3-AT at concentrations up to 400 μg 3-AT per ml. K562, KG-1, and RAW 264.7 cells exhibited no evidence of brown pigment formation in the presence of 3-AT and medium containing 10% fetal bovine serum. However, RAW 264.7 cells that were converted to protein-free medium and exposed to 3-AT exhibited intense brown pigment in some cell nuclei. A high percentage of HL-60 cells treated with 3-AT exhibited membrane blebbing, pyknosis, and nuclear fragmentation, which was not observed among other 3-AT-treated cell lines. A mechanism involving toxic intermediates of peroxidase-mediated “aminomelanin” formation is hypothesized.     

Growth kinetics of coliform bacteria under conditions relevant to drinking water distribution systems.

The growth of environmental and clinical coliform bacteria under conditions typical of drinking water distribution systems was examined. Four coliforms (Klebsiella pneumoniae, Escherichia coli, Enterobacter aerogenes, and Enterobacter cloacae) were isolated from an operating drinking water system for study; an enterotoxigenic E. coli strain and clinical isolates of K. pneumoniae and E. coli were also used. All but one of the coliforms tested were capable of growth in unsupplemented mineral salts medium; the environmental isolates had greater specific growth rates than did the clinical isolates. This trend was maintained when the organisms were grown with low levels (less than 1 mg liter-1) of yeast extract. The environmental K. pneumoniae isolate had a greater yield, higher specific growth rates, and a lower Ks value than the other organisms. The environmental E. coli and the enterotoxigenic E. coli strains had comparable yield, growth rate, and Ks values to those of the environmental K. pneumoniae strain, and all three showed significantly more successful growth than the clinical isolates. The environmental coliforms also grew well at low temperatures on low concentrations of yeast extract. Unsupplemented distribution water from the collaborating utility supported the growth of the environmental isolates. Growth of the K. pneumoniae water isolate was stimulated by the addition of autoclaved biofilm but not by tubercle material. These findings indicate that growth of environmental coliforms is possible under the conditions found in operating municipal drinking water systems and that these bacteria could be used in tests to determine assimilable organic carbon in potable water.     

Growth kinetics of coliform bacteria under conditions relevant to drinking water distribution systems.

The growth of environmental and clinical coliform bacteria under conditions typical of drinking water distribution systems was examined. Four coliforms (Klebsiella pneumoniae, Escherichia coli, Enterobacter aerogenes, and Enterobacter cloacae) were isolated from an operating drinking water system for study; an enterotoxigenic E. coli strain and clinical isolates of K. pneumoniae and E. coli were also used. All but one of the coliforms tested were capable of growth in unsupplemented mineral salts medium; the environmental isolates had greater specific growth rates than did the clinical isolates. This trend was maintained when the organisms were grown with low levels (less than 1 mg liter-1) of yeast extract. The environmental K. pneumoniae isolate had a greater yield, higher specific growth rates, and a lower Ks value than the other organisms. The environmental E. coli and the enterotoxigenic E. coli strains had comparable yield, growth rate, and Ks values to those of the environmental K. pneumoniae strain, and all three showed significantly more successful growth than the clinical isolates. The environmental coliforms also grew well at low temperatures on low concentrations of yeast extract. Unsupplemented distribution water from the collaborating utility supported the growth of the environmental isolates. Growth of the K. pneumoniae water isolate was stimulated by the addition of autoclaved biofilm but not by tubercle material. These findings indicate that growth of environmental coliforms is possible under the conditions found in operating municipal drinking water systems and that these bacteria could be used in tests to determine assimilable organic carbon in potable water.     

Rates of Mineralization of Trace Concentrations of Aromatic Compounds in Lake Water and Sewage Samples

The rates of mineralization of phenol, benzoate, benzylamine, p-nitrophenol, and di(2-ethylhexyl) phthalate added to lake water at concentrations ranging from a few picograms to nanograms per milliliter were directly proportional to chemical concentration. The rates were still linear at levels of <1 pg of phenol or p-nitrophenol per ml, but it was less than the predicted value at 1.53 pg of 2,4-dichlorophenoxyacetate per ml. Mineralization of 2,4-dichlorophenoxyacetate was not detected in samples of lake water containing 200 ng of the chemical per ml. The slope of a plot of the rate of phenol mineralization in samples of three lakes as a function of its initial concentration was lower at levels of 1 to 100 μg/ml than at higher concentrations. In lake water and sewage supplemented with <60 ng of ¹⁴C-labeled benzoate or phenylacetate per ml, 95 to 99% of the radioactivity disappeared from solution, indicating that the microflora assimilated little or none of the carbon. The extent of mineralization of some compounds in samples of two lakes and sewage was least in the water with the lowest nutrient levels. No mineralization of 2,4-dichlorophenoxyacetate and the phthalate ester was observed in samples of an oligotrophic lake. These data suggest that mineralization of some chemicals at concentrations of <1 μg/ml is the result of activities of organisms different from those functioning at higher concentrations or of organisms that metabolize the chemicals at low concentrations but assimilate little or none of the substrate carbon.     

Enumeration of phenanthrene-degrading bacteria by an overlayer technique and its use in evaluation of petroleum-contaminated sites.

Bacteria that are capable of degrading polycyclic aromatic hydrocarbons were enumerated by incorporating soil and water dilutions together with fine particles of phenanthrene, a polycyclic aromatic hydrocarbon, into an agarose overlayer and pouring the mixture over a mineral salts underlayer. The phenanthrene-degrading bacteria embedded in the overlayer were recognized by a halo of clearing in the opaque phenanthrene layer. Diesel fuel- or creosote-contaminated soil and water that were undergoing bioremediation contained 6 x 10(6) to 100 x 10(6) phenanthrene-degrading bacteria per g and ca. 5 x 10(5) phenanthrene-degrading bacteria per ml, respectively, whereas samples from untreated polluted sites contained substantially lower numbers. Unpolluted soil and water contained no detectable phenanthrene degraders (desert soil) or only very modest numbers of these organisms (garden soil, municipal reservoir water).     

Enumeration of phenanthrene-degrading bacteria by an overlayer technique and its use in evaluation of petroleum-contaminated sites.

Bacteria that are capable of degrading polycyclic aromatic hydrocarbons were enumerated by incorporating soil and water dilutions together with fine particles of phenanthrene, a polycyclic aromatic hydrocarbon, into an agarose overlayer and pouring the mixture over a mineral salts underlayer. The phenanthrene-degrading bacteria embedded in the overlayer were recognized by a halo of clearing in the opaque phenanthrene layer. Diesel fuel- or creosote-contaminated soil and water that were undergoing bioremediation contained 6 x 10(6) to 100 x 10(6) phenanthrene-degrading bacteria per g and ca. 5 x 10(5) phenanthrene-degrading bacteria per ml, respectively, whereas samples from untreated polluted sites contained substantially lower numbers. Unpolluted soil and water contained no detectable phenanthrene degraders (desert soil) or only very modest numbers of these organisms (garden soil, municipal reservoir water).     

Infection of Acanthamoeba castellanii by Chlamydia pneumoniae.

Chlamydia pneumoniae is an intracellular respiratory pathogen, which, similar to Legionella, might have developed mechanisms to escape the intracellular bactericidal activity of both human host cells and amoeba. We therefore investigated the intracellular growth and survival of C. pneumoniae in Acanthamoeba castellanii by using cell culture, immunofluorescence microscopy, and electron microscopy. A castellanii was incubated with purified elementary bodies of C. pneumoniae TW 183 at a concentration of 10(6) inclusion-forming units (IFU)/ml to give a ratio of approximately 1 IFU of C. pneumoniae per amoeba. Quantitative determination of chlamydial growth within A. castellanii revealed viable and infective C. pneumoniae in the range of 10(4) to 10(5) IFU/ml between days 7 and 14 postinfection. Immunofluorescence analysis and transmission electron microscopy with subsequent immunogold staining confirmed evidence of infection of the amoebae by C. Pneumoniae and additionally revealed that C. pneumoniae entered the typical growth cycle. Our results show that amoebae allow the survival of C. pneumoniae, suggesting that amoebae may serve as an additional reservoir for Chlamydia or Chlamydia-related organisms.     

VIRAL HEPATITIS—A Study of Hyperbilirubinemia with Acholuria in Convalescence

In 42 patients convalescing from viral hepatitis, the total and 1-minute serum bilirubin levels were measured on the day bilirubin was first demonstrated to be absent from the urine.The levels of total bilirubin ranged from 0.5 to 6.2 per 100 ml. of blood (mean 2.8 mg.), while the levels of the 1-minute bilirubin ranged from 0.3 to 3.3 mg. per 100 ml. of blood (mean 1.5 mg.).The reason for acholuria in the presence of the elevated 1-minute direct van den Bergh measurements is not clear, but may be due to the failure of the van den Bergh reaction to accurately measure the exact concentrations of free and conjugated bilirubin present in the plasma.     

Bacterial population in Russian space station "Mir"

We had the opportunity to investigate the bacterial population in air samples, condensation water, and inner wall swabs from the Russian space station Mir. From the first and second air samples during the mission, 29 and 7 bacterial colonies were collected, respectively. The values were equivalent to 16.8 and 4.0 cfu/100 liter air, respectively. Condensation water was collected from three different sites. The total viable bacterial counts were 2.1 x 10(6), 5.2 x 10(2), and 3.0 x 10(1) cfu/ml. The phylogenetic position of each isolate was determined by total 16S rDNA sequencing. Bacteria from air samples were mainly Gram-positive (35/36 colonies), and staphylococci occupied dominant specifically (23/36 colonies). On the other hand, Gram-negative bacteria were mainly isolated from condensation water samples. Most strains were thought to be opportunistic pathogens or environmental bacteria (such as those that inhabit soil, water, or air) found on earth. However, 6 of 23 isolates were suspected to be new species according to phylogenetic analysis and quantitative DNA-DNA hybridization data. The isolation of the other levels 3 and 2 bacteria, using specific selective media, was unsuccessful because all samples were heavily contaminated with fungi. To overcome this situation, PCR methods were applied to survey most levels 3 and 2 pathogenic bacteria in the condensation water samples. Up to 380 different primers for bacterial pathogens were used in this study. Only Mycobacterium avium 16S DNA sequences, however, could be amplified from the three water samples. The average bacteria count was estimated to be about 10(4) organisms/ml water.     

Growth patterns and substrate requirements of naturally occurring obligate oligotrophs

Eighteen strains of obligately oligotrophic bacteria that grow in a medium containing 1 mg of organic carbon per liter and do not grow in a rich medium (5 g/liter of nutrient) were isolated as dominant organisms from the oligotrophic water of Lake Biwa. The growth properties of these, especially of five strains, were examined. The maximum cell yield ranged from 8.5×10⁴/ml to 2.3×10⁶/ml, and their doubling times ranged from 6.6/h to 11.8/h in LT10^–4 medium (0.5 mg trypticase and 0.05 mg yeast extract in 1 liter of filtered and aged lake water). They also showed good growth in lake water medium without adding nutrients. The optimum concentrations for their growth were 5 mg/1, 5–50 mg/1, 50 mg/1, or 500 mg/1, depending on the strains. They utilized glutamate, glycine, serine, and glycolate, but not acetate, proline, or leucine. Several properties were examined. Their growth properties were very different from those of oligotrophs or oligocarbophiles isolated by other researchers.     

New Water Disinfectant: an Insoluble Quaternary Ammonium Resin-Triiodide Combination that Releases Bactericide on Demand

Strongly basic anion-exchange resins form stable, water-insoluble combinations with triiodide ions. The combinations have remarkable antibacterial properties: 3.0 x 10(5)Escherichia coli cells per ml were killed when passed through a 3.8-g column of commercially available resin treated with triiodide (volume 4 ml after treatment). In an attempt to deplete the resin-triiodide complex, 1.14 x 10(9)E. coli cells in 15 liters were passed through the column with no significant loss of effectiveness. The antibacterial capabilities of the resin-triiodide columns ranged from 10(6)Salmonella typhimurium per ml to 1.1 x 10(4)Streptococcus faecalis per ml. Staphylococcus aureus and Pseudomonas aeruginosa were also tested and killed at concentrations of 1.8 x 10(4) and 1.3 x 10(5) per ml, respectively. The cells were not filtered from the water. They emerged from the column in nonviable form. This was demonstrated by using (14)C-labeled bacteria. The irreversible nature of the antibacterial action was revealed when attempts to wash the damaged cells did not restore viability.     

A real-time polymerase chain reaction assay for quantitative detection of the human-specific enterococci surface protein marker in sewage and environmental waters

A real-time polymerase chain reaction (PCR) assay using SYBR Green I dye was developed to quantify the Enterococcus faecium enterococci surface protein (esp) marker in sewage (n = 16) and environmental waters (n = 16). The concentration of culturable enterococci in raw sewage samples ranged between 1.3 x 10(5) and 5.6 x 10(5) colony-forming units (cfu) per 100 ml. The real-time PCR detected 9.8 x 10(3)-3.8 x 10(4) gene copies of the esp marker per 100 ml of sewage. However, the concentration of culturable enterococci and the esp marker in secondary effluent was two orders of magnitude lower than raw sewage. Surface water samples were collected from a non-sewered catchment after storm events and the real-time PCR was applied to quantify the esp marker. Of the 16 samples tested, 6 (38%) were PCR-positive and the concentration of the esp marker ranged between 1.1 x 10(2) and 5.3 x 10(2) gene copies per 100 ml of water samples. The newly developed real-time PCR method was successfully used to quantify the esp marker in samples collected from sewage and environmental waters. The presence of the esp marker in water samples immediately after storm events not only indicated human faecal pollution but also provided evidence of the degree of human faecal pollution. To our knowledge, this is the first study that reports the use of a real-time PCR assay to quantify the esp marker in sewage and surface waters. Such study would provide valuable information for managers for the improved management of water quality.     

Fluorescence microscopy procedure for quantitation of yeasts in beverages.

Existing methods for quantitating yeasts in beverages include time-consuming plate counts that detect only living cells and hemacytometer counts that are reliable only at very high concentrations (e.g., 10(6) to 20 X 10(6) cells per ml). The new method described here involves the use of fluorescence microscopy with the fluorescent stain aniline blue to differentiate yeasts (and other fungi) from backgrounds for easy counting and also may be used in conjunction with membrane filtration to concentrate yeasts from liquids before cell enumeration. Recoveries averaged 91.5% for beverages spiked with levels of 500 to 600,000 organisms per ml. The correlation coefficient of count to spike level was 0.996.     

Fluorescence microscopy procedure for quantitation of yeasts in beverages

Existing methods for quantitating yeasts in beverages include time-consuming plate counts that detect only living cells and hemacytometer counts that are reliable only at very high concentrations (e.g., 10(6) to 20 X 10(6) cells per ml). The new method described here involves the use of fluorescence microscopy with the fluorescent stain aniline blue to differentiate yeasts (and other fungi) from backgrounds for easy counting and also may be used in conjunction with membrane filtration to concentrate yeasts from liquids before cell enumeration. Recoveries averaged 91.5% for beverages spiked with levels of 500 to 600,000 organisms per ml. The correlation coefficient of count to spike level was 0.996.     

Membrane potentials, electrolyte contents, cell pH, and some enzyme activities of fibroblasts

The resting membrane potential of the cultured fibroblasts derived from rabbit subcutaneous tissues was -10.2 +/- 0.20 mV (n = 390). This potential was affected by the potassium concentration in the culture medium, but not by other chemical or hormonal preparations, such as dibutyryladenosine 3',5'-cyclic monophosphate (0.5 to 5.0 mmol/l), sodium fluoride (10(-5) to 10(-4) M), hydrocortisone (10(-7) to 10(-6) M), parathyroid extract (0.5 to 1.0 U/ml), or thyrotrophin (5 to 10 mU/ml). The Na+, K+, and Cl- concentrations of the cultured fibroblasts were 35.4, 85.7, and 22.6 mmol/l cell water, respectively. The water and protein contents of these cells were 82.1 and 9.18 g/100-g cells, respectively. The intracellular pH of fibroblasts as determined by [14C] dimethyloxazolidine-2, 4-dione, and 3H2O ranged between 6.9 and 7.1 when the pH of the culture medium was maintained at 7.4. The activities of Na+, K+-, HCO3(-)-, and Ca++, Mg++-ATPases in these cultured cells were 19.0 +/- 2.1, 13.6 +/- 2.1, and 6.6 +/- 1.2 nmol pi/mg protein per minute, respectively, and the carbonic anhydrase activity was 0.054 U/mg protein. Calculations based on the values for the membrane potential and the electrolyte concentrations observed in this study indicate that Na+, K+, Cl-, and H+ are not distributed according to their electrochemical gradients across the cell membrane. Na+, Cl-, and H+ are actively transported out of the cells and K+ into the cells.