Influence of salinity and organic nutrient concentration on survival and growth of Vibrio cholerae in aquatic microcosms

Laboratory microcosms were employed to evaluate the influence of selected environmental parameters, organic nutrient concentration, and salinity on the growth and survival of a toxigenic strain of Vibrio cholerae LA4808. Over the range conditions tested, this strain of V. cholerae showed maximum response as determined by increased plate counts and direct microscopic counts in microcosms prepared with a chemically defined sea salts solution at a salinity of 25%, but with lower or higher salinity levels, the maximum population size declined. When added organic concentrations of less than 1,000 micrograms/liter were present, a marked salinity effect on the growth of V. cholerae was detected. However, at or above an organic nutrient concentration of 1,000 micrograms/liter, the need for an optimum salinity level was spared. From the results of this study, it is concluded that V. cholerae can grow under conditions of organic nutrient concentration and salinity typical of estuaries. Results obtained support the hypothesis that V. cholerae is an autochthonous member of the estuarine microbial community.     

Effects of microcosm salinity and organic substrate concentration on production of Vibrio cholerae enterotoxin

The effects of aquatic processes on production of cholera toxin by Vibrio cholerae were studied with seawater microcosms. Several salinity and organic nutrient concentrations were employed. At 10 g of organic nutrient per liter of seawater, toxin production increased as the salinity was increased. At lower organic nutrient concentrations, toxin production was markedly enhanced when the salinity was 20 and 25%. Toxin concentration increased with salinity, independent of cell concentration and toxin stability. From the results obtained in this study, it is concluded that physical and chemical parameters of the aquatic environment affect not only the physiological state of V. cholerae, but also its potential pathogenicity.     

Effects of microcosm salinity and organic substrate concentration on production of Vibrio cholerae enterotoxin.

The effects of aquatic processes on production of cholera toxin by Vibrio cholerae were studied with seawater microcosms. Several salinity and organic nutrient concentrations were employed. At 10 g of organic nutrient per liter of seawater, toxin production increased as the salinity was increased. At lower organic nutrient concentrations, toxin production was markedly enhanced when the salinity was 20 and 25%. Toxin concentration increased with salinity, independent of cell concentration and toxin stability. From the results obtained in this study, it is concluded that physical and chemical parameters of the aquatic environment affect not only the physiological state of V. cholerae, but also its potential pathogenicity.     

Influence of water temperature, salinity, and pH on survival and growth of toxigenic Vibrio cholerae serovar 01 associated with live copepods in laboratory microcosms.

The influence of water temperature, salinity, and pH on the multiplication of toxigenic Vibrio cholerae serovar O1 cells and their attachment to live planktonic crustaceans, i.e., copepods, was investigated by using laboratory microcosms. By increasing water temperatures up to 30 degrees C, a pronounced effect on the multiplication of V. cholerae was demonstrated, as was attachment of the cells to live copepods. These were measured by culturable counts on agar plates and direct observation by scanning electron microscopy, respectively. Of the three salinities examined (5, 10, and 15%), maximum growth of V. cholerae and attachment to copepods occurred at 15%. An alkaline pH (8.5) was optimal both for attachment and multiplication of V. cholerae, as compared with pH 6.5 and 7.5. It is concluded that conditions affecting attachment of V. cholerae serovar O1 to live copepods observed under laboratory conditions may also occur in the natural estuarine environment and, thereby, are significant in the epidemiology of cholera.     

Nutritional versatility and growth kinetics of an Aeromonas hydrophila strain isolated from drinking water

The nutritional versatility and growth kinetics of Aeromonas hydrophila were studied to determine the nature and the growth-promoting properties of organic compounds which may serve as substrates for the growth of this organism in drinking water during treatment and distribution. As an initial screening, a total of 69 different organic compounds were tested at a concentration of 2.5 g/liter as growth substrates for 10 A. hydrophila strains. Of these strains, strain M800 attained the highest maximum colony counts in various types of drinking water and river water and was therefore used in further measurements of growth at low substrate concentrations. A mixture of 21 amino acids and a mixture of 10 long-chain fatty acids, when added to drinking water, promoted growth of strain M800 at individual compound concentrations as low as 0.1 microgram of C per liter. Mixtures of 18 carbohydrates and 18 carboxylic acids clearly enhanced growth of the organism at individual compound concentrations above 1 microgram of C per liter. Growth measurements with 63 individual substrates at a concentration of 10 micrograms of C per liter gave growth rates of greater than or equal to 0.1/h with two amino acids, nine carbohydrates, and six long-chain fatty acids. Ks values were determined for arginine (less than or equal to 0.3 micrograms of C per liter), glucose (15.9 micrograms of C per liter), acetate (11.1 micrograms of C per liter), and oleate (2.1 micrograms of C per liter). The data obtained indicate that biomass components, such as amino acids and long-chain fatty acids, can promote multiplication of aeromonads in drinking water distribution systems at concentrations as low as a few micrograms per liter.     

Nutritional versatility and growth kinetics of an Aeromonas hydrophila strain isolated from drinking water.

The nutritional versatility and growth kinetics of Aeromonas hydrophila were studied to determine the nature and the growth-promoting properties of organic compounds which may serve as substrates for the growth of this organism in drinking water during treatment and distribution. As an initial screening, a total of 69 different organic compounds were tested at a concentration of 2.5 g/liter as growth substrates for 10 A. hydrophila strains. Of these strains, strain M800 attained the highest maximum colony counts in various types of drinking water and river water and was therefore used in further measurements of growth at low substrate concentrations. A mixture of 21 amino acids and a mixture of 10 long-chain fatty acids, when added to drinking water, promoted growth of strain M800 at individual compound concentrations as low as 0.1 microgram of C per liter. Mixtures of 18 carbohydrates and 18 carboxylic acids clearly enhanced growth of the organism at individual compound concentrations above 1 microgram of C per liter. Growth measurements with 63 individual substrates at a concentration of 10 micrograms of C per liter gave growth rates of greater than or equal to 0.1/h with two amino acids, nine carbohydrates, and six long-chain fatty acids. Ks values were determined for arginine (less than or equal to 0.3 micrograms of C per liter), glucose (15.9 micrograms of C per liter), acetate (11.1 micrograms of C per liter), and oleate (2.1 micrograms of C per liter). The data obtained indicate that biomass components, such as amino acids and long-chain fatty acids, can promote multiplication of aeromonads in drinking water distribution systems at concentrations as low as a few micrograms per liter.     

Effects of temperature and salinity on Vibrio cholerae growth.

Laboratory microecosystems (microcosms) prepared with a chemically defined sea salt solution were used to study effects of selected environmental parameters on growth and activity of Vibrio cholerae. Growth responses under simulated estuarine conditions of 10 strains of V. cholerae, including clinical and environmental isolates as well as serovars O1 and non-O1, were compared, and all strains yielded populations of approximately the same final size. Effects of salinity and temperature on extended survival of V. cholerae demonstrated that, at an estuarine salinity (25%) and a temperature of 10 degrees C, V. cholerae survived (i.e., was culturable) for less than 4 days. Salinity was also found to influence activity, as measured by uptake of 14C-amino acids. Studies on the effect of selected ions on growth and activity of V. cholerae demonstrated that Na+ was required for growth. The results of this study further support the status of V. cholerae as an estuarine bacterium.     

Effects of temperature and salinity on Vibrio cholerae growth

Laboratory microecosystems (microcosms) prepared with a chemically defined sea salt solution were used to study effects of selected environmental parameters on growth and activity of Vibrio cholerae. Growth responses under simulated estuarine conditions of 10 strains of V. cholerae, including clinical and environmental isolates as well as serovars O1 and non-O1, were compared, and all strains yielded populations of approximately the same final size. Effects of salinity and temperature on extended survival of V. cholerae demonstrated that, at an estuarine salinity (25%) and a temperature of 10 degrees C, V. cholerae survived (i.e., was culturable) for less than 4 days. Salinity was also found to influence activity, as measured by uptake of 14C-amino acids. Studies on the effect of selected ions on growth and activity of V. cholerae demonstrated that Na+ was required for growth. The results of this study further support the status of V. cholerae as an estuarine bacterium.     

Entry of Vibrio harveyi and Vibrio fischeri into the viable but nonculturable state

AIMS: Physiological responses of marine luminous bacteria, Vibrio harveyi (ATCC 14216) and V. fischeri (UM1373) to nutrient-limited normal strength (35 ppt iso-osmolarity) and low (10 ppt hypo-osmolarity) salinity conditions were determined. METHODS AND RESULTS: Plate counts, direct viable counts, actively respiring cell counts, nucleoid-containing cell counts, and total counts were determined. Vibrio harveyi incubated at 22 degrees C in nutrient-limited artificial seawater (ASW) became nonculturable after approximately 62 and 45 d in microcosms of 35 ppt and 10 ppt ASW, respectively. In contrast, V. fischeri became nonculturable at approximately 55 and 31 d in similar microcosms. Recovery of both culturability and luminescence of cells in the viable but nonculturable state was achieved by addition of nutrient broth or nutrient broth supplemented with a carbon source, including luminescence-stimulating compounds. Temperature upshift from 22 degrees C to 30 degrees C or 37 degrees C did not result in recovery from nonculturability. CONCLUSIONS: The study confirms entry of V. harveyi and V. fischeri into the viable but nonculturable state under low-nutrient conditions and demonstrates nutrient-dependent resuscitation from this state. SIGNIFICANCE AND IMPACT OF THE STUDY: This study confirms loss of luminescence of V. harveyi and V. fischeri on entry into the viable but nonculturable state and suggests that enumeration of luminescent cells in water samples may be a rapid method to deduce the nutrient status of a water sample.     

Inhibition of type A and type B (proteolytic) Clostridium botulinum by sorbic acid

The effect of sorbic acid in the pH range 4.9 to 7.0 on the probability P of growth of a single vegetative bacterium of proteolytic strains of Clostridium botulinum has been determined by comparison of the most probable number count of the bacteria in media at pH 4.9 to 7.0 containing a series of concentrations of potassium sorbate and in a nutrient medium at pH 6.8 to 7.0. The media were maintained under strictly anaerobic conditions at a redox potential equivalent to lower than -350 mV at pH 7. In medium adjusted to the required pH with HCl, P for strain ZK3 (type A) at pH 5.1 or 5.5 after 2 days at 30 degrees C was similar to that at pH 6.8 to 7.0 but was slightly lower at pH 4.9. Potassium sorbate inhibited growth, the inhibition being a function of the concentration of undissociated sorbic acid. A calculated undissociated sorbic acid concentration of 156 mg/liter delayed growth of strain ZK3 (type A) but did not result in a significant decrease in P after an incubation time of 14 days. Higher concentrations of undissociated sorbic acid caused longer delays before maximum most probable number counts developed, and a calculated undissociated sorbic acid concentration of 282 mg/liter decreased log P for strain ZK3 after an incubation time of 14 days by a factor of 5.5 to 7.5. Four additional type A strains and five type B strains were inhibited to an extent comparable to inhibition of strain ZK3.(ABSTRACT TRUNCATED AT 250 WORDS)     

Inhibition of type A and type B (proteolytic) Clostridium botulinum by sorbic acid.

The effect of sorbic acid in the pH range 4.9 to 7.0 on the probability P of growth of a single vegetative bacterium of proteolytic strains of Clostridium botulinum has been determined by comparison of the most probable number count of the bacteria in media at pH 4.9 to 7.0 containing a series of concentrations of potassium sorbate and in a nutrient medium at pH 6.8 to 7.0. The media were maintained under strictly anaerobic conditions at a redox potential equivalent to lower than -350 mV at pH 7. In medium adjusted to the required pH with HCl, P for strain ZK3 (type A) at pH 5.1 or 5.5 after 2 days at 30 degrees C was similar to that at pH 6.8 to 7.0 but was slightly lower at pH 4.9. Potassium sorbate inhibited growth, the inhibition being a function of the concentration of undissociated sorbic acid. A calculated undissociated sorbic acid concentration of 156 mg/liter delayed growth of strain ZK3 (type A) but did not result in a significant decrease in P after an incubation time of 14 days. Higher concentrations of undissociated sorbic acid caused longer delays before maximum most probable number counts developed, and a calculated undissociated sorbic acid concentration of 282 mg/liter decreased log P for strain ZK3 after an incubation time of 14 days by a factor of 5.5 to 7.5. Four additional type A strains and five type B strains were inhibited to an extent comparable to inhibition of strain ZK3.(ABSTRACT TRUNCATED AT 250 WORDS)     

Determination of the concentration of maltose- and starch-like compounds in drinking water by growth measurements with a well-defined strain of a Flavobacterium species

The growth kinetics of Flavobacterium sp. strain S12 specialized in the utilization of glycerol, and a number of oligo- and polysaccharides were determined in batch-culture experiments at 15 degrees C in pasteurized tap water supplied with very low amounts of substrates. Kss for the growth on maltotriose, maltotetraose, maltopentaose, and maltohexaose were 0.03 microM or less and below those for glucose (1.5 microM) and maltose (0.16 microM). Kss for starch, amylose, and amylopectin were 8.4, 25.6, and 11.0 micrograms of C per liter, respectively. A yield of 2.3 X 10(7) CFU/micrograms of C on the oligo- and polysaccharides was calculated from the linear relationships observed between maximum colony counts in pasteurized tap water and the concentrations (usually below 25 micrograms of C per liter) of supplied compounds. The maximum colony counts of strain S12 grown in various types of raw water and tap water revealed that raw water contained only a few micrograms of maltose- and starch-like compounds per liter; in tap water the concentrations were all below 1 microgram of C and usually below 0.1 microgram of C per liter. The application of starch-based coagulant aids gave increased concentrations of maltose- and starch-like compounds in the water during treatment, but these concentrations were greatly reduced by coagulation and sedimentation, rapid sand filtration, and slow sand filtration.     

Determination of the concentration of maltose- and starch-like compounds in drinking water by growth measurements with a well-defined strain of a Flavobacterium species.

The growth kinetics of Flavobacterium sp. strain S12 specialized in the utilization of glycerol, and a number of oligo- and polysaccharides were determined in batch-culture experiments at 15 degrees C in pasteurized tap water supplied with very low amounts of substrates. Kss for the growth on maltotriose, maltotetraose, maltopentaose, and maltohexaose were 0.03 microM or less and below those for glucose (1.5 microM) and maltose (0.16 microM). Kss for starch, amylose, and amylopectin were 8.4, 25.6, and 11.0 micrograms of C per liter, respectively. A yield of 2.3 X 10(7) CFU/micrograms of C on the oligo- and polysaccharides was calculated from the linear relationships observed between maximum colony counts in pasteurized tap water and the concentrations (usually below 25 micrograms of C per liter) of supplied compounds. The maximum colony counts of strain S12 grown in various types of raw water and tap water revealed that raw water contained only a few micrograms of maltose- and starch-like compounds per liter; in tap water the concentrations were all below 1 microgram of C and usually below 0.1 microgram of C per liter. The application of starch-based coagulant aids gave increased concentrations of maltose- and starch-like compounds in the water during treatment, but these concentrations were greatly reduced by coagulation and sedimentation, rapid sand filtration, and slow sand filtration.     

Substrate Utilization by an Oxalate-Consuming Spirillum Species in Relation to Its Growth in Ozonated Water

The nutritional versatility of a vibrio-shaped, oxalate-utilizing isolate, strain NOX, obtained from tap water supplied with low concentrations of formate, glyoxylate, and oxalate, was determined by growth experiments with low-molecular-weight carbon compounds at high (grams per liter) and very low (micrograms per liter) concentrations. The organism, which was identified as a Spirillum species, appeared to be specialized in the utilization of a number of carboxylic acids. Yields of 2.9 × 10⁶ CFU/μg of oxalate C and 1.2 × 10⁷ CFU/μg of acetate C were obtained from growth experiments in tap water supplied with various low amounts of either oxalate or acetate. A substrate saturation constant of 0.64 μM oxalate was calculated for strain NOX from the relationship between growth rate and concentration of added oxalate. Maximum colony counts of strain NOX grown in ozonated water (dosages of 2.0 to 3.2 mg of O₃ per liter) were 15 to 20 times larger than the maximum colony counts of strain NOX grown in water before ozonation. Based on the nutritional requirements of strain NOX, it was concluded that carboxylic acids were produced by ozonation. Oxalate concentrations were calculated from the maximum colony counts of strain NOX grown in samples of ozonated water in which a non-oxalate-utilizing strain of Pseudomonas fluorescens had already reached maximum growth. The oxalate concentrations obtained by this procedure ranged from 130 to 220 μg of C/liter.     

Growth of Pseudomonas aeruginosa in tap water in relation to utilization of substrates at concentrations of a few micrograms per liter.

Five Pseudomonas aeruginosa strains were tested for the utilization of 47 low-molecular-weight compounds as their sole sources of carbon and energy for growth at a concentration of 2.5 g/liter. Of these compounds, 31 to 35 were consumed. Growth experiments in tap water at 15 degrees C were carried out with one particular strain (P1525) isolated from drinking water. This strain was tested for the utilization of 30 compounds supplied at a concentration of 25 microgram of C per liter. The growth rate (number of generations per hour) of strain P1525 in this tap water was approximately 0.005 h-1, and with 10 compounds it was larger than 0.03 h-1. An average yield of 6.2 x 10(9) colony-forming units per mg of C was obtained from the maximum colony counts (colony-forming units per milliliter). The average yield and maximum colony count of strain P1525 grown in tap water supplied with a mixture of 45 compounds, each at a concentration of 1 microgram of C per liter, enabled us to calculate that 28 compounds were utilized. Growth rates of two P. aeruginosa strains (including P1525) in various types of water at 15 degrees C were half of those of a fluorescent pseudomonad. The concentrations of assimilable organic carbon calculated from maximum colony counts and average yield values amounted to 0.1 to 0.7% of the total organic carbon concentrations in five types of tap water. The assimilable organic carbon percentages were about 10 times larger in river water and in water after ozonation.     

Growth response of Vibrio cholerae and other Vibrio spp. to cyanobacterial dissolved organic matter and temperature in brackish water

Environmental control of growth and persistence of vibrios in aquatic environments is poorly understood even though members of the genus Vibrio are globally important pathogens. To study how algal-derived organic matter and temperature influenced the abundance of different Vibrio spp., Baltic Sea microcosms inoculated with Vibrio cholerae, Vibrio vulnificus, Vibrio parahaemolyticus, Vibrio alginolyticus and native bacterioplankton, were exposed to different temperatures (12-25 degrees C) and amended with dissolved organic matter from Nodularia spumigena (0-4.2 mg C L(-1)). Vibrio abundance was monitored by culture-dependent and molecular methods. Results suggested that Vibrio populations entered a viable but nonculturable state during the incubations. Abundance of Vibrio spp. and total bacterioplankton were orders of magnitude higher in microcosms amended with organic matter compared with reference microcosms. Vibrio cholerae abundances ranged from 0.9 to 1.9 x 10(5) cells mL(-1) in treatments amended with 4.2 mg C L(-1). Vibrio cholerae abundance relative to total bacterioplankton and other Vibrio spp. also increased >10-fold. In addition, V. vulnificus abundance increased in mesocosms with the highest organic matter addition (0.9-1.8 x 10(4) cells mL(-1)). Temperature alone did not significantly affect abundances of total bacterioplankton, total Vibrio spp. or individual Vibrio populations. By contrast, cyanobacterial-derived organic matter represented an important factor regulating growth and abundance of V. cholerae and V. vulnificus in brackish waters.     

Ecology, serology, and enterotoxin production of Vibrio cholerae in Chesapeake Bay.

A total of 65 isolates of Vibrio cholerae, serotypes other than O--1, have been recovered from water, sediment, and shellfish samples from the Chesapeake Bay. Isolations were not random, but followed a distinct pattern in which salinity appeared to be a controlling factor in V. cholerae distribution. Water salinity at stations yielding V. cholerae (13 out of 21 stations) was 4 to 17 0/00, whereas the salinity of water at stations from which V. cholerae organisms were not isolated was less than 4 or greater than 17 0/00. From results of statistical analyses, no correlation between incidence of fecal coliforms and V. cholerae could be detected, whereas incidence of Salmonella species, measured concurrently, was clearly correlated with fecal coliforms, with Salmonella isolated only in areas of high fecal coliform levels. A seasonal cycle could not be determined since strains of V. cholerae were detectable at low levels (ca. 1 to 10 cells/liter) throughout the year. Although none of the Chesapeake Bay isolates was agglutinable in V. cholerae O group 1 antiserum, the majority for Y-1 adrenal cells. Furthermore, rabbit ileal loop and mouse lethality tests were also positive for the Chesapeake Bay isolates, with average fluid accumulation in positive ileal loops ranging from 0.21 to 2.11 ml/cm. Serotypes of the strains of V. cholerae recovered from Chesapeake Bay were those of wide geographic distribution. It is concluded from the data assembled to date, that V. cholerae is an autochthonous estuarine bacterial species resident in Chesapeake Bay.     

Seasonal Abundance and Distribution of Vibrio cholerae in Coastal Waters Quantified by a 16S-23S Intergenic Spacer Probe

Vibrio cholerae is the causative agent of the severe diarrheal disease cholera and is indigenous to brackish waters. To advance our understanding of the ecology of this bacterium, we have developed a molecular probing method for detection of V. cholerae in coastal waters. Water samples from 7 locations in the Newport Bay watershed, California were sampled monthly for a whole year. V. cholerae concentrations were determined by membrane filtration-colony hybridization using an oligonucleotide probe targeting the 16S-23S intergenic spacer (ITS) region. In addition to V. cholerae concentrations, environmental parameters, including temperature, salinity, total bacterial direct counts, total viable counts, and chlorophyll a concentrations, were determined for each site. V. cholerae was detected year-round throughout the watershed. Regression analysis indicated that the concentration of V. cholerae inversely correlated with salinity (p <0.001). The sampling sites located nearest to the Pacific Ocean had lower concentrations, whereas sites located along the brackish San Diego Creek (salinity 0-12 per thousand) routinely had higher concentrations. V. cholerae concentrations also correlated with temperature (p <0.01) in the watershed, with concentrations ranging from less than 1 CFU mL-1 to 2,930 CFU mL-1 of water. The results of this study indicate that the dynamics of V. cholerae is mainly influenced, out of the parameters measured, by the temperature and salinity of the environment. This information is valuable for understanding the ecology of V. cholerae.     

Increased toxin production by Vibrio cholerae O1 during survival with a green alga, Rhizoclonium fontanum, in an artificial aquatic environment

In the aquatic environment, the physiological state of Vibrio cholerae can be affected by various environmental conditions (e.g., sunlight, pH, temperature, competition with other bacteria for nutrients, etc.). The effect of these factors on the toxigenicity of V. cholerae was investigated. Toxin production by 5 toxigenic strains of V. cholerae incubated in laboratory microcosms containing Rhizoclonium fontanum was tested at different time intervals. The microcosms were exposed to sunlight, and the V. cholerae were in competition for nutrients with the resident bacterial flora of R. fontanum. The increase or decrease in toxin production by V. cholerae recovered at different time intervals was measured by ELISA and compared with the parent strains. Results of the study demonstrated an increase in toxin production by V. cholerae O1 during survival with R. fontanum. It is concluded that various environmental conditions in the aquatic environment affect toxin production by V. cholerae.     

Role of cyanobacteria in the persistence of Vibrio cholerae O139 in saline microcosms

Recently, a new strain of cholera, Vibrio cholerae O139, has emerged as an epidemic strain, but there is little information about its environmental reservoir. The present investigation was aimed to determine the role of cyanobacteria in the persistence of V. cholerae O139 in microcosms. An environmental isolate of V. cholerae O139 and three cyanobacteria (Anabaena sp., Nostoc sp., and Hapalosiphon sp.) were used in this study. Survival of culturable V. cholerae O139 in microcosms was monitored using taurocholate-tellurite gelatin agar medium. Viable but nonculturable V. cholerae O139 were detected using a fluorescent antibody technique. Vibrio cholerae O139 could be isolated for up to 12 days in a culturable form in association with cyanobacteria but could not be isolated in the culturable form after 2 days from control water without cyanobacteria. The viable but nonculturable V. cholerae O139 could be detected in association with cyanobacteria for up to 15 months. These results, therefore, suggest that cyanobacteria can act as a long-term reservoir of V. cholerae O139 in an aquatic environment.