Seasonal dynamics of prokaryotic abundance and activities in relation to environmental parameters in a transitional aquatic ecosystem (Cape Peloro, Italy).

This study examines the effects of temporal changes on microbial parameters in a brackish aquatic ecosystem. To this aim, the abundances of prokaryotes and vibrios together with the rates of enzymatic hydrolysis of proteins by leucine aminopeptidase (LAP), polysaccharides by ß-glucosidase (GLU) and organic phosphates by alkaline phosphatase (AP), heterotrophic prokaryotic production (HPP), respiration (R), were seasonally investigated, during a 2-year period in the coastal area of Cape Peloro (Messina, Italy), constituted by two brackish lakes (Faro and Ganzirri). In addition, physical and chemical parameters (temperature, salinity, nutrients) and particulate organic carbon and nitrogen (POC, PN) were measured. The influence of multiple factors on prokaryotic abundances and activities was analysed. The results showed that Cape Peloro area is characterised by high seasonal variability of the microbial parameters that is higher than the spatial one. Combined changes in particulate matter and temperature (T), could explain the variability in vibrios abundance, GLU and R activities in both lakes, indicating a direct stimulation of the warm season on the heterotrophic prokaryotic metabolism. Positive correlations between T (from 13.3 to 29.6 °C) and HPP, LAP, AP, POC, PN are also observed in Ganzirri Lake. Moreover, the trophic status index and most of the microbial parameters show significant seasonal differences. This study demonstrates that vibrios abundance and microbial activities are responsive to the spatial and seasonal changes of examined area. The combined effects of temperature and trophic conditions on the microbial parameters lead us to suggest their use as potential indicators of the prokaryotic response to climate changes in temperate brackish areas.

Assessment of the ecological status of transitional waters in Sicily (Italy): first characterisation and classification according to a multiparametric approach.

A 1-year cycle of observations was performed in four Sicilian transitional water systems (Oliveri-Tindari, Cape Peloro, Vendicari and Marsala) to characterise their ecological status. A panel of variables among which trophic and microbial (enzyme activities, abundance of hetetrophic bacteria and of bacterial pollution indicators) parameters, were selected. Particulate organic carbon (POC) and nitrogen (PON) and chlorophyll-a (Chl-a) contents defined the trophic state, while microbial hydrolysis rates and abundance gave insights on microbial community efficiency in organic matter transformation and on allochthonous inputs. To classify the trophic state of examined waters, the synthetic trophic state index (TRIX) was calculated. Microbial hydrolysis rates correlated positively with POC and Chl-a, which increased along the eutrophication gradient. The significant relationships among TRIX, trophic and microbial parameters suggested the use of leucine aminopeptidase, alkaline phosphatase and POC as suitable parameters to implement the Water Framework Directive when assessing the ecological status of transitional water systems.

Fluorescent antibody-viability staining and beta-glucuronidase assay as rapid methods for monitoring Escherichia coli viability in coastal marine waters.

A faecal pollution monitoring of coastal Messina waters was performed by comparing three (microscopic, enzyme, and culture) methods. Evidence of Escherichia coli cells (29.99 to 96.79% of the total enteropathogenic serotypes) retaining their viability into the marine environment was shown. beta-Glucuronidase activity rates suggested that living cells were also metabolically active. Heavily polluted sites were detected, where improperly treated urban wastes were discharged. Significant relationships between microscopic and enzymatic data proved both methods to be suitable alternatives to the culture method for E. coli detection, improving environmental quality assessment.

Combined fluorescent antibody assay and viability staining for the assessment of the physiological states of Escherichia coli in seawaters.

AIMS: A comparison of methods that combine the use of immune sera with specific fluorescent probes for testing viability at single cell level was performed in order to estimate different living attributes of Escherichia coli in natural seawater samples. METHODS AND RESULTS: Cell culturability was assayed by plate method, respiratory activity and membrane integrity were determined by an indirect fluorescent antibody assay, combined with 5-cyano-2, 3 ditolyl tetrazolium chloride and propidium iodide, respectively. Results showed the coexistence of different physiological states within the E. coli population, of which a large fraction (46%) of cells was actively respiring. CONCLUSIONS: The methodological approach used offer interesting perspectives in water pollution monitoring, particularly when the differentiation between dead and living E. coli cells is required for a more precise assessment of the bacteriological quality of seawaters. SIGNIFICANCE AND IMPACT OF THE STUDY: The study suggests the importance of knowledge of the viability status of faecal bacteria in aquatic environments as a fundamental issue for the preservation of public health; the availability of rapid analytical procedures for this purpose may find significant applications in the evaluation of the sanitary risk consequent to water use.

Immunofluorescence detection of Escherichia coli in seawater: a comparison of various commercial antisera.

Through a microscopical method, relying on the interaction between fluorescent antibodies and target antigen, it is possible to detect and enumerate Escherichia coli in seawaters. Various commercial monoclonal and polyclonal antisera have been tested in an indirect immunofluorescence (IIF) assay developed for microbiological monitoring of coastal waters. Prior to use, they have been titrated and screened for cross-reactions with a collection of clinical and environmental isolates. A comparison among counts obtained on field samples showed higher performance for microscopical than for plate methods, due to the ability of all antisera to label target cells specifically, regardless of their viability. Because of their different specificities, polyclonal antisera yielded better quantitative results than monoclonal antisera. The study further suggested the usefulness of the immunofluorescence assay as a rapid alternative analytical tool for the specific detection of bacterial pathogens in aquatic environments.

Development of an enzyme assay for rapid assessment of Escherichia coli in seawaters.

AIMS: An analytical protocol has been developed and applied for the detection of glucuronidase activity in marine waters as a rapid alternative approach to assess the microbiological quality of seawaters. METHODS AND RESULTS: The fluorogenic substrate 4-methylumbelliferyl-beta-D-glucuronide is cleaved to a fluorescent product, methylumbelliferone, by the enzyme beta-glucuronidase, specific to Escherichia coli and closely related enterobacterial species (Shigella). The results suggest that this test is related to E. coli numbers, as estimated by immunofluorescence, more significantly than to faecal coliform numbers, obtained from culture media. CONCLUSIONS: The determination of the potential rate of glucuronidase activity may be used as a diagnostic tool for the indirect estimation of the presence of E. coli in seawaters. SIGNIFICANCE AND IMPACT OF THE STUDY: The method may be particularly useful in the early warning of seawater pollution, allowing the screening of coastal areas with different contamination levels in reduced time.

Use of the indirect immunofluorescence method for detection and enumeration of Escherichia coli in seawater samples.

The determination of Escherichia coli in marine waters through a rapid method, the microscopic indirect immunofluorescent technique, is evaluated in comparison with the conventional count on m-FC agar medium. The data obtained in seawater samples, collected monthly along the Messina coastline, show good sensitivity of the analysis and agreement between the microscopic and culture technique, with a detection limit of 10(2) cells 100 ml(-1) for immunofluorescence.

Bacterial pollution of Messina coastal waters: a one year study.

A year's monitoring of faecal pollution of marine coastal waters surrounding Messina was carried out in 1996/97. The distribution of faecal coliforms was evaluated in 15 stations located along the Sicilian coastline, sampled monthly in coincidence of the two opposing current phases ("montante" and "scendente" currents) which characterise the Straits of Messina. The data obtained provided a complete picture of hygienic-sanitary conditions of the area and highlighted the presence of heavily polluted sites in correspondence with river outflows. Higher bacterial counts were associated with lower salinity values and higher ammonia concentrations; over an annual study, they occurred during the coldest months, showing the negative impact of continental water inputs on the bacteriological quality of coastal waters.

Microbiological monitoring of Castellammare Gulf (TP) waters for their suitability in marine aquaculture.

A three year investigation into the microbiological conditions of the waters of the Gulf of Castellammare (TP) was carried out in order to evaluate the possible utilization of this area as an aquaculture site. Analysis of a total of 168 samples was aimed at estimating the quantitative distribution of total and fecal coliforms, enterococci and halophilic vibrios. The qualitative distribution of Vibrio species was also studied with particular reference to potentially pathogenic species. Coliforms showed the highest densities (10(2)-10(3) CFU/100 ml) in November 1993 at the coastal stations and mostly negative values during the subsequent samplings; in contrast, halophilic vibrios prevailed in the warm months. V. alginolyticus and V. fluvialis prevailed respectively among the Vibrio population grown at 35 and 24 degrees C, grouped in 13 and in 4 clusters at similarity levels of 81-96%. Quantitative data demonstrate the presence of low levels of microbial contamination, which suggests the general suitability of the area for fish farming.

NMDA receptors play an anti-aggregating role in human platelets.

Receptors for different monoamines, peptides and other neurohormones are present in the plasma membrane of platelets, and the sophisticated process of haemostasis is regulated by the interplay of their physiologic agonists (1). The recent report of a platelet binding site for phencyclidine (2) suggested a possible role of N-methyl-D-aspartate (NMDA) receptors in platelet function. Isotherms of [3H]-glutamate (GLU), [3H]-CGP-39653, [3H]-glycine (GLY) and [3H]-MK-801 carried out in platelet membranes yielded Bmax and Kd values for these ligands similar to those present in neurons, and NMDA only partially displaced [3H]-GLU. In neurons [3H]-MK-801 binding is potentiated by GLU and/or GLY and, being specific for the open NMDA receptor channel species, it has a functional meaning. In platelet membranes neither GLU and/or GLY increased [3H]-MK-801 binding; thus suggesting that NMDA receptors in platelets are different from those present in neurons. GLU or NMDA alone did not induce platelet aggregation. However, both amino acids were antagonistic on the aggregating activity of arachidonic acid (AA), NMDA being 3 orders of magnitude more active than GLU, and NMDA also antagonized adenosine diphosphate (ADP) and platelet aggregating factor (PAF) induced platelet aggregation. Finally, NMDA increased cAMP levels in intact platelets, and such an effect did not occur in a Ca(2+)-free medium; yet, cAMP increase was not antagonized by the calmodulin inhibitor trifluoperazine (TFP). It was concluded that platelet membranes carry an NMDA receptor, functionally distinct from the neuronal one, which seems to play an anti-aggregating role.

Distribution and numerical taxonomy of Vibrionaceae in the waters of the Straits of Messina.

The results of a study carried out by numerical analysis on Vibrio strains isolated from the waters of the Straits of Messina are reported. The quantitative data showed the presence of low bacterial densities (ranging from 9 to 99 CFU/100 ml of water) due to the intense currents which characterize this area; also the highest bacterial counts were generally found during the "montante" current. With regard to the qualitative results, there was a predominance of vibrios belonging to V. mediterranei, V. splendidus II and V. pelagius II species, which represented respectively 25%, 19% and 13% of the total bacterial population. The species distribution did not seem related to sampling stations or depth. The taxonomic structure, obtained using the simple matching coefficient and unweighted average linkage clustering, revealed the presence of 7 main clusters (S = 80-95%), which included strains of various origin because of the particular turbulence of the waters of the Straits.

First data on the distribution and ecology of Vibrio spp. of the Straits of Magellan (South America).

During the austral summer of 1991 a study was carried out on the presence and distribution of the genus Vibrio in the Straits of Magellan. Vibrios strains were isolated using membrane filters and Marine Agar 2216 in anaerobiosis. Variations of the populations of total heterotrophic bacteria and vibrios were observed both on the surface and along the column of water. All vibrios are psychrotrophic and were grouped in 4 cluster among which cluster 1, identified as presumed V. anguillarum, seems the most important including 73% of strains. A certain habitat segregation of clusters was noted. Cluster 4 was found only in a deep and permanently colder water mass. The relations between 20 environmental parameters and the bacterial population were also studied. Significant positive correlations were observed between the vibrios population and various fractions of suspended particulate matter.