Effect of ferricyanide onEscherichia coli

Ferricyanide, ferrocyanide and phosphates in concentrations greater than 0.07 M inhibit the respiration of NAD-linked substrates and succinate in washed cell suspension ofE. coli. Citrate accumulation in the medium under the inhibiting conditions indicates that these salts bring about a change in the permeability of the cell membrane of this bacterium.In general, ferricyanide does not resemble in action those compounds which uncouple respiratory-energy production. Energy production is prevented only when ferricyanide is the main electron acceptor. The inhibition studies and the addition of co-factors, indicate that cytochromes are not involved in the reduction of ferricyanide by cell-free extracts ofE. coli, either with NADH or succinate. An FAD-containing enzyme is probably involved in the reduction of ferricyanide by NADH, while succinic dehydrogenase seems to react directly with ferricyanide.We wish to thank Group Capt. Tom Gray-Young for technical advice and R. Filzroy for technical assistance.This work was supported by a grant from the Science Research Council.     

Examination of indigenous microbiota and survival of Escherichia coli 0157 and Salmonella in a paper milling environment

Aims: A public beach was frequently cited for health advisories because of high Escherichia coli levels, the source suspected to be a paper mill located upstream. This investigation sought to confirm whether or not the paper mill was the pollution source, and to characterize the risk to recreational bathers imposed by the source. Methods and Results: Quantification of E. coli in river water collected at incremental distances showed that paper mill effluent caused elevated E. coli levels in beach samples. Samples collected throughout the mill were variably positive for heterotrophic bacteria, total coliforms and E. coli, but negative for pathogenic E. coli O157 and Salmonella. Escherichia coli O157 or Salmonella spiked into mill samples (4·2 log₁₀ or 5·6 log₁₀ CFU per 100 ml, respectively) fell below detection levels within 14–24 h in raw (unaltered) samples, while in heat‐sterilized replicates, the counts remained at initial levels or increased over 36 h. Conclusions: Pathogenic E. coli O157 and Salmonella were not isolated from paper mill samples. The absence of native bacteria allowed the survival of pathogens, while their presence accelerated pathogen decline. Significance and Impact of the Study: The co‐existence of paper mill and swimming beach may be reasonable for now in spite of the limitations of an E. coli‐based assay for beach water.     

Multiplicity of phosphorylation sites onEscherichia coli isocitrate dehydrogenase

Several lines of evidence indicate that the in vivo phosphorylation of isocitrate dehydrogenase (EC 1.1.1.42) inEscherichia coli occurs at multiple sites: first, the phosphorylated enzyme can be resolved by two-dimensional electrophoresis into three distinct spots differing in charge; second, the analysis of its phosphoamino acid content shows that it is modified at both serine and threonine residues; third, its extensive hydrolysis by proteolytic enzymes yields several different phosphopeptides.     

The effect of free chlorine onEscherichia coli populations

The effect of free chlorine onEscherichia coli populations was studied by chlorination of a population of 10⁵ cells/ml. This cell density was low enough for the free-to-combined chlorine ratio to be 6.01 or greater. The predominance of free chlorine resulted in rapid and complete population death.Survivors obtained by dechlorination prior to complete population death were recovered equally well on nonselective and selective media. Although this suggests that survivors are not injured, evidence of survivor injury was observed.Colonies resulting from growth of these survivors had a smaller diameter than colonies from unchlornated controls. This suggests that the chlorinated cells have an increased lag and provides indirect evidence of survivor injury. Injury was indicated directly by an increase in the lag time of surviving cells in slide culture. Variability in the severity of free-chlorine-induced injury was indicated by a broadened range in the survivor lag times.     

Survival and viability of nonculturableEscherichia coli andVibrio cholerae in the estuarine and marine environment

Plating methods for estimating survival of indicator organisms, such asEscherichia coli, and water-borne pathogens includingVibrio cholerae, have severe limitations when used to estimate viable populations of these organisms in the aquatic environment. By combining the methods of immunofluorescent microscopy, acridine orange direct counting, and direct viable counting, with culture methods such as indirect enumeration by most probable number (MPN) estimation and direct plating, it was shown that bothE. coli andV. cholerae undergo a nonrecoverable stage of existence, but remain viable. Following 2-week incubations in saltwater (5–25%o NaCl) microcosms, total counts, measured by direct microscopic examination of fluorescent antibody and acridine orange stained cells, remained unchanged, whereas MPN estimates and plate counts exhibited rapid decline. Results of direct viable counting, a procedure permitting estimate of substrate-responsive viable cells by microscopic examination, revealed that a significant proportion of the nonculturable cells were, indeed, viable. Thus, survival of pathogens in the aquatic environment must be re-assessed. The die-off or decay concept may not be completely valid. Furthermore, the usefulness of the coliform and fecal coliform indices for evaluating water quality for public health purposes may be seriously compromised, in the light of the finding reported here.     

Effects of substrate biodegradability on the mass and activity of the associated estuarine microbiota

Multiple biochemical assays of microbial mass and activities were applied to the estuarine detrital microbiota colonizing morphologically similar polyvinyl chloride needles and needles from slash pine (Pinus elliottii). Biodegradable pine needles consistently showed 2- to 10-fold higher values of extractable adenosine 5'-triphosphate, rates of oxygen utilization, activities of alkaline phosphatase and phosphodiesterase, and the mucopeptide cell wall component muramic acid than did the polyvinyl chloride needles, during a 14-week incubation in a semitropical estuary. The higher activities by the microbiota of the biodegradable substrate correlated with estimates of the microbial density from scanning electron microscopy. The microbial community associated with the nondegradable substrate showed minimal activity of beta-d-galactosidase, beta-d-glucosidase, and alpha-d-mannosidase in contrast to the biota of the degradable substrate, which showed 10- to 100-fold higher activities of these glycoesterases. These enzymes logically could be involved in catabolism of the carbohydrate polymers of the detritus. Assuming equivalent rates of predation, a surface that is also a utilizable substrate supports a three- to fivefold more active microbial population.     

Mesophilic chitin-degrading anaerobes isolated from an estuarine environment

Abstract Eight strains of obligately anaerobic, mesophilic, chitinolytic bacteria were isolated from the sediment of an estuarine environment. The isolates were rod-shaped, Gram-negative, and formed terminal spherical spores that swelled the sporangium. The major products from the fermentation of chitin were: acetate, ethanol, formate, CO₂, H₂ and ammonia. Growth of the isolates was possible at pH values ranging from 5.0–9.0. During the fermentation of chitin, N -acetylglucosamine accumulated in the culture fluids and was not metabolized. No organic compounds other than chitin and its oligomers could be demonstrated to support growth of the isolates. Hydrolysis of chitin proceeded at a relatively low rate and was incomplete. Approximately 65% of the initial amount of chitin was hydrolyzed during a period of 5–10 days. Supplementation of the medium with yeast extract, casamino acids or peptone did not enhance the rate of chitin hydrolysis, but reducing agents such as Na₂S₂O₄ and Ti (III)-NTA markedly stimulated the rate of chitin fermentation.
The ecological implications of the high degree of substrate specialization are discussed.     

Effect of the essential oil fromAchillea fragrantissima onEscherichia coli cells

Escherichia coli cells treated with the essential oil from the plantAchillea fragrantissima released five polypeptides as well as K⁺ ions into the incubation medium. The oil also inhibited the respiration ofE. coli cells and reduced their ATP content. Electron micrographs showed that oil-treated cells were permeable to uranyl acetate. The effect of the essential oil on the cell membrane is discussed.     

Inductive effects of environmental concentration of atrazine onEscherichia coli andEnterococcus faecalis

Atrazine solutions (0.1, 1, 10 and 100 microg/L) inoculated with Escherichia coli and Enterococcus faecalis under natural conditions significantly increased (p < or = 0.05) the population levels of both test bacteria; it indicates the ability of bacterial cells to degrade atrazine and to use the original compound or its degradation products as nutrient(s). In some cases, alterations in the morphology of the colonies were also observed on selective solid media. Biochemical differentiation was also found and, on the other hand, a loss of culturability was recorded; this suggests that bacteria have entered in a viable but nonculturable state. A re-appearance of the colonies occurred after inoculation on tryptone-soy agar with atrazine.     

The effect of actinomycin D and flavomycin onEscherichia coli R+ strains

When tested by¹⁴C-uracil incorporation, a higher permeation of actinomycin D into R⁺ Escherichia coli cells was observed. From actinomycin D and flavomycin only flavomycin was found to be effective in R⁺ cells selective growth inhibition. The results indicate that the effect of flavomycin is related to the fertility functions of the strain. The possible practical importance of flavomycin application for R⁺ cells elimination in the bacterial population is discussed.     

Effect of fecal pollution on Vibrio parahaemolyticus densities in an estuarine environment

Vibrio parahaemolyticus densities in the Narragansett Bay, R.I., estuary were found to significantly correlate with the level of fecal pollution in the water. Results of field investigations showed a definite association between the levels of this organism and those of Escherichia coli, Clostridium perfringens, and enterococci. Densities of V. parahaemolyticus were greatest in the near-surface waters of contaminated areas and decreased sharply with both the distance from the sources of fecal pollution and the depth of the water column. A positive association with the amount of particulate matter in the water and specifically with its zooplankton content also was found. No association was obtained with any of the physical or chemical parameters examined except dissolved oxygen. The results of laboratory studies on the growth of V. parahaemolyticus in collected estuarine waters were consistent with the field observations in that nutrients in sewage did not directly produce any increase in natural or inoculated levels of the organism. Of several particulates added to the water, only chitin and net zooplankters (live or dead) supported the growth of V. parahaemolyticus. The addition of sewage to the water had no measurable effect, whether or not the various particulates also were present. The data show that wastewater effluents have an effect on V. parahaemolyticus densities in this estuary and indicate that the effect is indirect, probably mediated by biostimulation of the food chain and manifested at the level of the microfauna.     

Identification and characterization of humic substances-degrading bacterial isolates from an estuarine environment

Bacterial isolates were obtained from enrichment cultures containing humic substances extracted from estuarine water using an XAD-8 resin. Eighteen isolates were chosen for phylogenetic and physiological characterization based on numerical importance in serial dilutions of the enrichment culture and unique colony morphology. Partial sequences of the 16S rRNA genes indicated that six of the isolates were associated with the alpha subclass of Proteobacteria, three with the gamma-Proteobacteria, and nine with the Gram-positive bacteria. Ten isolates degraded at least one (and up to six) selected aromatic single-ring compounds. Six isolates showed ability to degrade [(14)C]humic substances derived from the dominant salt marsh grass in the estuary from which they were isolated (Spartina alterniflora), mineralizing 0.4-1.1% of the humic substances over 4 weeks. A mixture of all 18 isolates did not degrade humic substances significantly faster than any of the individual strains, however, and no isolate degraded humic substances to the same extent as the natural marine bacterial community (3.0%). Similar studies with a radiolabeled synthetic lignin ([beta-(14)C]dehydropolymerisate) showed measurable levels of degradation by all 18 bacteria (3.0-8.8% in 4 weeks), but mineralization levels were again lower than that observed for the natural marine bacterial community (28.2%). Metabolic capabilities of the 18 isolates were highly variable and generally did not map to phylogenetic affiliation.     

Aglycosylantibody. Effects of exoglycosidase treatments on autochthonous antibody survival time in the circulation.

Rabbit anti-hapten antibodies were purified by affinity chromatography and characterized immunochemical for in vivo studies of their blood clearance rate and organ distribution after treatment with various glycosidases. Following sequential removal of sialic acid, galactose, and N-acetylglucosamine with the appropriate cellulose-immobilized exoglycosidases, the antibody populations were recharacterized, radiolabeled, and introduced intravenously into the original animals. Using double radioiodine lables it was possible to demonstrate alterations in purified antibody survival times in the circulation and altered organ distribution after glycolytic cleavage. Removal of terminal sialic acid resulted in rapid blood clearance and enhanced localization of asialoantibody in the liver. Subsequent removal of penultimate galactose residues returned both antibody survival time in the circulation and organ distribution to near normal. Removal of subpenultimate N-acetylglucosamine moieties resulted in aglycosylantibody survival values which were intermediate between asialo- and asialoagalactoantibodies. Removal of the three saccharide also increased kidney localization. The results are evaluated based on current concepts of the biological roles of protein-linked carbohydrate and plasma glycoprotein survival time in the circulation.     

Effects of temperature on growth of Vallisneria americana in a sub-tropical estuarine environment

The submersed aquatic vegetation (SAV) species Vallisneria americana Michx. (tape grass) is a valuable resource in the Caloosahatchee estuary and in many other aquatic systems. Given the variable nature of freshwater inflows and environmental conditions in the Caloosahatchee, it is necessary to understand how tape grass will respond to high and low salinity conditions at different light and temperature levels. Specifically, quantitative information is needed as input to modeling tools that can be applied to predict growth and survival of tape grass under a range of environmental conditions present in the estuary. We determined growth rates for small and medium sized tape grass plants obtained from the Caloosahatchee estuary, southwest coastal Florida, USA in freshwater (0.5 psu) under high (331 μE m^?2 s^?1) and low light (42 μE m^?2 s^?1) and at 10 psu under high light conditions. We ran six treatments at five temperatures spanning 13–32 °C for 8–9 weeks. The optimum temperature for growth was roughly 28 °C, with a minimum threshold temperature of 13 °C and a maximum threshold temperature of 38 °C. Plants grew fastest in freshwater, at high light and temperatures greater than 20 °C. The slowest growth rates were observed at 13 °C regardless of salinity, light or plant size. Our results suggest that tape grass growth is strongly influenced by water temperature and that additional stressors such as low light and elevated salinity can reduce the range of temperature tolerance, especially at colder water temperatures.     

Aquatic insects in an estuarine environment: densities, distribution and salinity tolerance

1. Aquatic insects were quantitatively surveyed at five sites along the tidally influenced section of a river-dominated estuary in North Wales. Site 1 was the furthest upstream and was established as a reference site as it was never inundated by salt water. Site 5 was the furthest downstream and was inundated by all incoming tides. Numerically, insects made up 32% of the estuarine invertebrate fauna. 2. Although the densities of most insect taxa decreased towards the estuary mouth, there were significant numbers present downstream for much of the year; for example, in April at site 4 (which was inundated by 81% of all high tides), a mean of 3514 chironomid larvae were recorded per m² of estuary bed. Even at site 5, which was inundated twice daily, there were 747 larvae per m². Among the larger aquatic insects, caddisfly and elmid beetle larvae, together with stonefly nymphs, were consistently taken at site 4 (e.g. maxima of forty-eight caddisfly larvae m^–2 in December and seventy elmids m^–2 in April), although their densities were lower than upstream. 3. There were seasonal shifts in the longitudinal distribution of several taxa, most notably the extension of chironomids down the estuary in April and July, and the concentration of simuliid larvae and mayfly nymphs at site 2 in July. The total freshwater benthos showed a downstream shift between September and December, which was maintained through April and into the summer. The latter was despite peak saltwater inundation (highest tides) in October, November and April. In June and July, when saltwater intrusion was lowest, the ranges of many aquatic insects had contracted to sites 1 and 2. 4. Laboratory experiments showed that virtually all individuals of nineteen species of insects collected from site 1 (freshwater) survived a 4-h immersion in 8.75‰ saltwater (25% strength seawater). Immersion in progressively more saline solutions reduced the survivorship of first the mayflies, followed by the caddisflies Glossosoma conformis and Hydropsyche instabilis. After 4 h in full strength seawater, all specimens of the stonefly Dinocras cephalotes, over half of the Perla bipunctata, and some individuals of nine species of caddisfly were alive. Four species of caddisfly (Sericostoma personatum, Odontocerum albicorne, Potamophylax cingulatus and Adicella reducta) survived a 24-h simulated tidal cycle of immersion. With the exception of P. cingulatus, a few individuals of these caddisfly species survived immersion in full-strength seawater for 24 h. For some individual species there was good agreement between their observed longitudinal distribution in the estuary and laboratory-measured salinity tolerance; however, there was no significant correlation, overall, for the fauna.     

Relative effects of bacterial and protozoan predators on survival of Escherichia coli in estuarine water samples.

The relative effect of protozoan and bacterial predators on the survival of Escherichia coli in estuarine water samples was examined. Predacious protozoa exerted their major influence on E. coli destruction during the first 2 days of a 10-day-decline period. Inhibition of protozoa after day 2 had little effect on E. coli survival. Bacterial predators also contributed to E. coli destruction but in natural estuarine water samples were maintained at lower levels due to "grazing" by predacious protozoa.     

Modifications of estuarine sedimentary microbiota by exclusion of epibenthic predators

The ability of epibenthic predators (crabs and fishes) to influence biomass and community structure of sedimentary microbiota was investigated in St. George Sound-Apalachicola Bay System, Florida, U.S.A. Replicate areas (4 m²) of mud-flat sediment were caged in the field to confine and exclude predators. Uncaged areas were used as controls. The microbiota (prokaryotes and microeukaryotes) of the sediments was characterized at Weeks 0, 2, and 6 by measuring concentrations of phospholipid and analyzing fatty acids of the microbial lipids extracted from the sediments. Data were analyzed using analysis of variance and step-wise discriminant analysis. After 2 wk, the microbiota of the predator exclusion treatment was significantly different from that in control and predator inclusion treatments. After 6 wk, these differences became more pronounced. There were no demonstrable caging effects that could account for treatment differences. Results indicated that removal of predators had a profound effect on microbial communities in estuarine sediments. Thus, the top trophic level (epibenthic predators) had an important role in regulating the structure of the lowest trophic level (the microbiota).