Influence of aquatic microbiota on the survival in water of the human and eel pathogen Vibrio vulnificus serovar E

The eel and human pathogen Vibrio vulnificus serovar E (biotype 2) is seldom isolated from natural waters, although it can survive in sterilized artificial seawater microcosms for years. The main objective of the present study was to investigate whether aquatic microbiota can limit its survival and recovery from water samples. A set of preliminary experiments of survival in microcosms containing natural seawater and water from eel farms showed that the persistence of this pathogen was mainly controlled by grazing, and secondarily by bacterial competition. The bacterial competition was further analysed in artificial seawater microcosms co-inoculated with selected virulent serovar E (VSE) strains and potential competitors. Competitors included V. vulnificus biotype 1 isolates and strains of selected species that can grow on the selective media designed for V. vulnificus isolation from water samples. Evidences of bacterial competition that was detrimental for VSE recovery were recorded. Thus, some species produced a deleterious effect on VSE strains under starvation, and others were able to use the resources more efficiently under nutrient input. These results suggest that an overgrowth of more efficient competitor bacteria in conventional media used for isolation of V. vulnificus could mask the recovery of VSE strains and explain the scarcity of reports on the isolation of this human and eel pathogen from natural waters.     

Mold challenge study in bottled natural mineral waters and spring waters

Microbiological challenge study was carried out to verify the microbial stability of bottled waters against four different mold species isolated from bottled water (Fusarium sp.; Cladosporium sp.; Penicillium chrysogenum and Aspergillus fumigatus) and to follow the growth of the molds in bottled water. Twelve types of bottled water with different mineralization and CO2 level in PET and glass packages were collected from 4 European countries. Three different inoculation levels of spore suspensions were used to contaminate bottled water samples. The surviving colony forming unit (CFU) numbers and visual growth were monitored during the investigation period (26 weeks). The results of surviving CFU showed that the fungal growth is mostly determined by the carbonation level and the type of the mold strain. Neither the inoculation level nor the mineral content had any significant effect on the survival of the different mold strains. Results showed decreased CFU numbers in carbonated waters, while slow decreasing, stagnation or even some growth in still waters. A. fumigatus was the most resistant test species. None of the other tested mold strains survived the first 12-week test period in carbonated water. Visual growth was not detected in carbonated water samples, in contrast to all of the non-carbonated samples.     

Settlement and growth of the green mussel Perna viridis (L.) in coastal waters: influence of water velocity

Green mussels Perna viridis were observed to be a major foulant in the seawater intake tunnel of a coastal power station. Field experiments were carried out to ascertain what factors were responsible for the successful colonisation by mussels. Two adjacent stations (25 m apart) were selected, one representing the coastal waters and the other representing the intake screens (with higher water velocity). Gonadal activity, larval abundance, spat settlement and growth rate of the mussels were monitored at monthly intervals for a total period of two years. The results showed that the breeding activity of the mussels at the study area is influenced largely by temporal distribution of seawater temperature. However, ensuing larval availability in the coastal waters is more dependent on food availability. On the other hand, spat settlement and growth rate are predominantly influenced by water flow, probably as a result of increased propagule and food flux rate at higher water velocities. Higher water velocity at the intake screens also contributed to mussel dominance by preventing settlement of many potential competitors.     

Effects of soil pH,temperature and water content on the growth of<Emphasis Type="Italic">Burkholderia pseudomallei</Emphasis>

Optimum conditions were determined for the growth of Burkholderia pseudomallei in natural soils or waters. It grows better in paddy soil, crop-covered and fallow field than in fresh and salty water. Although the optimal temperature and pH for the growth were 37 or 42 degrees C, and 6.5 or 7.5 in an environmental-mimicking soil medium, this bacterium can still grow at 4 degrees C, which was suggested to be related with the occurrence of melioidosis in some cold areas. In soil media with water content < 15. B. pseudomallei did not grow until 60 d of incubation, suggesting that water contents of soils in which it dwelled would be one important factor in determining the growth rate.     

Agar Plate Method for Detection and Enumeration of Alkylbenzenesulfonate-Degrading Microorganisms

A simple method for detection and enumeration of alkylbenzenesulfonate (ABS)-degrading microorganisms by using agar plates was developed and used in microbiological studies of coastal marine and polluted river waters. The method depends upon the color responses of neutral red in alkaline medium. Neutral red changes from pink, when it enters into ABS micelles, to yellow, when the ABS is degraded, and does not form micelles. When neutral red-tris(hydroxymethyl)-aminomethane buffer solution and then cationic surfactant solution were sprayed onto the agar surface of ABS-nutrient agar cultures, transparent haloes appeared around the colonies of ABS-degrading microorganisms against a pink background. Viable counts of ABS-degrading bacteria isolated from both seawater and freshwater environments were considerably higher in polluted waters than in less polluted areas. Viable counts of ABS-degrading bacteria averaged 1.5 x 105/ml in samples from the surface water of polluted Tokyo Bay and 3.0 x 104/ml in samples from the surface water of polluted Tamagawa River but were fewer in number in samples from less polluted waters.     

Effect of poly(3-hydroxybutyrate) (PHB) content on the starvation-survival of bacteria in natural waters

Abstract The effect of poly(3-hydroxybutyrate) (PHB) content on the survival of wild-type strains and PHB negative mutants of Bacillus megaterium and Alcaligenes eutrophus in natural waters was studied. The survival strategy of B. megaterium was dominated by the development of resistant forms, but the number of the wild-type vegetative cells was higher than that of PHB mutant strain. In some environmental conditions the mutant spores needed a heat shock for germination, a fact that suggests, for the first time, that PHB plays a role in this phenomenon. Survival of A. eutrophus wild-type strain in all experiments was higher compared to the PHB mutant, and differences were significant. In raw river water, survival of both species was lower than in sterile river water.     

Distribution of Clostridium perfringens in polluted lake environments

Many of the directives that relate to the prevention of pollution or the improvement of fresh water also relate to lake waters since lake waters ultimately inherit much of the pollution that enters into fresh water. In order to determine the influence of the water depth on Clostridium perfringens, we utilised a new medium, lactose-sulfite (LS) broth, suggested for rapid enumeration and identification of C. perfringens. Duplicate samples were collected at each one of the following sites of the polluted station: surface, 60 cm, 90 cm and bottom (1.18 cm). Membrane filtration equipment was used. All samples were alternatively passed through two membrane filters, the first (20-25 microm pore size) was used for retention of the abundant phytoplankton and the second (porosity 0.45 microm) for C. perfringens. Membranes were placed into the first tube of ten-fold dilutions from 10(1) to 10(4) and incubated aerobically in a waterbath at 46 degrees C for 24 h. The numbers of C. perfringens fluctuated depending on the water depth. Vegetative forms were found only in the bottom sampling; they were never found in surface, 60 cm and 90 cm sampling sites. Sporulated forms were found in all sampling sites with the exception of the surface sampling. Clostridium perfringens as an anaerobic bacterium never occurred in the surface waters in vegetative or spore forms, even if the waters were extremely polluted by domestic or industrial activities. Vegetative forms occurred only in the bottom samples but spore forms which are more resistant to various environmental effects occurred in all depths except for the surface.     

Polyphosphate stores enhance the ability of Vibrio cholerae to overcome environmental stresses in a low-phosphate environment

Vibrio cholerae, the causative agent of Asiatic cholera, has been reported to make large quantities of polyphosphate. Inorganic polyphosphate is a ubiquitous molecule with a variety of functions in prokaryotic and eukaryotic cells. We constructed a V. cholerae mutant with a deletion in the polyphosphate kinase (ppk) gene. The mutant was defective in polyphosphate biosynthesis. Deletion of ppk had no significant effect on production of cholera toxin, hemagglutinin/protease, motility, biofilm formation, and colonization of the suckling mouse intestine. The wild type and mutant had similar growth rates in rich and minimal medium and exhibited similar phosphate uptake and alkaline phosphatase induction. In contrast to ppk mutants from other gram-negative bacteria, the V. cholerae mutant survived prolonged starvation in LB medium and artificial seawater basal salts. The ppk mutant was significantly more sensitive to low pH, high salinity, and oxidative stress when it was cultured in low-phosphate minimal medium. The ppk mutant failed to induce catalase when it was downshifted to phosphorus-limiting conditions. Furthermore, the increased sensitivity of the ppk mutant to environmental stressors in phosphate-limited medium correlated with a diminished capacity to synthesize ATP from intracellular reservoirs. We concluded that polyphosphate protects V. cholerae from environmental stresses under phosphate limitation conditions. It has been proposed that toxigenic V. cholerae can survive in estuaries and brackish waters in which phosphorus and/or nitrogen can be a limiting nutrient. Thus, synthesis of large polyphosphate stores could enhance the ability of V. cholerae to survive in the aquatic environment.     

Influence of phosphate ions and alkaline phosphatase activity of cells on survival ofEscherichia coli in seawater

When grown in a minimal medium and suspended for 2 hours in distilled water, seawater, phosphate buffer or a polyphosphate solution,E. coli MC4100 cells with high alkaline phosphatase activity survived in seawater for longer periods than cells with low or no activity. However, mutant cells totally deprived of alkaline phosphatase activity held in phosphate-containing media before transfer to seawater showed survival almost as high as the wild type strain, indicating that alkaline phosphatase activity is not the only factor influencing survival. Alkaline phosphatase activity also increased the protection of cells provided by glycine betaine. Survival was enhanced when cells were preincubated in the presence of phosphate or polyphosphate. Thus, the transfer of cells in wastewater could influence their subsequent survival in seawater.     

Inhibition of common fouling organisms by marine bacterial isolates ith special reference to the role of pigmented bacteria

Two questions of relevance to the establishment of marine biofouling communities were addressed, viz (1) what is the frequency with which bacterial strains isolated from living and inanimate surfaces in the marine environment show inhibitory activity against the settlement of common fouling organisms, and (2) is the antifouling bacterium, D2, an inhabitant of different marine waters, and how unique is this bacterium, in its mode of action against different target organisms? With respect to the first question, ninety three marine bacteria isolated from various rock surfaces from the marine environment were tested against larvae of Balanus amphitrite and spores of Ulva lactuca. Settlement assays against the diatom Amphora sp. were also performed on 10 of these strains. Nine bacterial isolates were shown to be inhibitory against larval settlement and eight of these strains were also inhibitory against algal spores. Altogether 16 strains were inhibitory against the settlement of algal spores while none of the bacterial strains inhibited diatom settlement. With respect to the second question, D2, a dark green pigmented bacterium, isolated from an adult tunicate off the Swedish west coast, has been found to be a very effective inhibitor against common fouling organisms. In order to see if this bacterium can be found in other marine waters, bacteria from living surfaces of marine plants and animals from waters around Sydney, Australia, were isolated and screened for inhibitory activity against barnacle larvae. Seventy four percent of the 23 plant isolates were shown to be inhibitory against larval settlement while only 30% of the 23 isolates from marine animals reduced settlement. Twenty two of the isolates from different seaweeds were dark pigmented and 20 of these strains inhibited settlement of barnacle larvae and algal spores. Three of the strains showed the same phenotypic expression as D2, and the results indicate that these strains may be D2 or closely related strains, suggesting that D2 may be a common inhabitant in the marine environment.     

RESPONSES OF GYMNODINIUM BREVE DAVIS TO NATURAL WATERS OF DIVERSE ORIGIN1

The effect on the growth of Gymnosdinium breve of river waters and seawater samples collected in different seasons and locations was investigated with and without differential enrichments. Growth in natural seawater was most clearly enhanced by addition of EDTA-Fe, sulfide, N and P. The seasonal variation in growth-promoting properties of seawater and river water dominates variations due to differences in location.     

Effect of sediments on the survival of Escherichia coli in marine waters.

Escherichia coli, a fecal coliform, was found to survive for longer periods of time in unsterile natural seawater when sediment material was present than in seawater alone, and at least on one occasion growth was observed to occur. This enteric bacterium was found to increase rapidly in number in autoclaved natural seawater and autoclaved sediment taken from areas receiving domestic wastes, even when the seawater had salinities as high as 34 g/kg. However, in autoclaved seawater, growth was always more gradual and never reached numbers as high as those observed when sediment was present. It was found that nutrients were easily eluted from the sediment after autoclaving or upon addition to artificial seawater, but little elution occured during mixing of the sediments with unsterile natural seawater. The longer survival of E. coli in the sediment is attributed to the greater content of organic matter present in the sediment than the sweater. These laboratory results, in part, could explain why on a volume basis larger numbers of coliforms and fecal coliforms and fecal coliforms were found in estuarine sediments than the overlaying water at field sites.     

A selective medium for the rapid detection by an impedance technique of Pseudomonasspp. associated with poultry meat

A new medium for detecting and enumerating Pseudomonas spp. associated withpoultry meat spoilage by a rapid impedance technique was developed, after testing potentialgrowth promoters for eight Pseudomonas strains and inhibitors against eight competingstrains (Enterobacteriaceae) able to grow on the medium of Mead and Adams (1977). Four basalmedia (brain heart infusion, brucella broth, Shaedler broth and Whitley impedance broth (WIB))and a synthetic medium were evaluated. Whitley impedance broth was the best basal medium fordetecting variations in impedance in relation to Pseudomonas growth. The efficiency ofWIB was improved by adding compounds which enhanced the growth of Pseudomonas onthe synthetic medium. Among the incubation temperatures tested, 22°C proved to be the bestcompromise between growth of Pseudomonas associated with poultry meat spoilage andinhibition of competitors. Among the 15 inhibitory substances evaluated against Pseudomonas competitors, five were chosen for inclusion in the final medium : metronidazole,carbenicilline, cetrimide, cycloheximide and diamide (MCCCD medium). Preliminary resultsobtained from experiments with beef and pork meat showed that this medium could also be usedwithout diamide and at an incubation temperature of 25°C. The impedance technique usingMCCCD medium was then compared with an official method which uses the medium of Meadand Adams (1977) on 106 samples of poultry neck skin. The linear regression coefficient betweenthe two techniques was approximately r = 0·85. Impedance was able to detect 10³ Pseudomonas g⁻¹ within less than 19 h making it a promisingtechnique for predicting poultry meat spoilage.     

Detection of marine birnavirus in the Japanese pearl oyster Pinctada fucata and seawater from different depths

This study examines the seasonal changes of marine birnavirus (MABV) in seawater and the Japanese pearl oyster Pinctada fucata reared at different depths (2 and 15 m). Oysters and seawater were collected in 1998, and a 2-step PCR was carried out to detect MABV. Virus isolation was performed on the PCR-positive samples in the oyster. The detection rate of the MABV genome in the oyster was low during June, but increased after July at both 2 and 15 m depths. MABV was not isolated until after September, when isolation rates of 10 to 28.6% were recorded. The results suggest that growth of MABV in the oyster is similar at 2 and 15 m depth. In contrast, the MABV genome in seawater was present through the year at 15 m depth, but was not detected in summer at 2 m. This suggests that the virus is destroyed by UV and/or other factors at 2 m in summer, but is stable in deeper waters.     

Vibrio sp. strain NM 10, isolated from the intestine of a Japanese coastal fish, has an inhibitory effect against Pasteurella piscicida.

Vibrio sp. strain NM 10 with an inhibitory activity against Pasteurella piscicida K-III was isolated from the intestine of a spotnape ponyfish (Leiognathus nuchalis). This bacterium efficiently produced an antibacterial substance after growth at 20 degrees C for 24 h on 1/5 PYBG agar prepared with 50% seawater at pHs of 7.5 to 9.0. The antibacterial substance was heat labile and proteinaceous, with a molecular mass of less than 5 kDa, possibly a bacteriocin or a bacteriocin-like substance.     

The physiological role of the carotenoid pigments of halobacterium salinarium

Summary A colorless mutant of the extremely halophilic bacterium, Halobacterium salinarium, has been isolated. In comparative experiments with the carotenoid-containing wild type, it was shown that neither type was killed upon exposure to bright sunlight under aerobic conditions. Exposure to tungsten light of an incident intensity comparable to that of bright sunlight inhibited strongly the growth of the colorless mutant; the growth of the carotenoid-containing wild type was unaffected.In their natural habitats, the extremely halophilic bacteria are usually exposed to bright sunlight. It seems likely that such light will affect the growth of these bacteria as does tungsten light. It is therefore inferred that in their natural habitats, carotenoid-containing forms will develop more freely than colorless forms, because the pigments protect the cells against an inhibitory effect of light upon growth. Carotenoid-containing forms have therefore come to dominate the population of extremely halophilic bacteria in nature.     

Bacteriophages active against Bacteroides fragilis in sewage-polluted waters

Twelve strains of different Bacteroides species were tested for their efficiency of detection of bacteriophages from sewage. The host range of several isolated phages was investigated. The results indicated that there was a high degree of strain specificity. Then, by using Bacteroides fragilis HSP 40 as the host, which proved to be the most efficient for the detection of phages, feces from humans and several animal species and raw sewage, river water, water from lagoons, seawater, groundwater, and sediments were tested for the presence of bacteriophages that were active against B. fragilis HSP 40. Phages were detected in feces of 10% of the human fecal samples tested and was never detected in feces of the other animal species studied. Moreover, bacteriophages were always recovered from sewage and sewage-polluted samples of waters and sediments, but not from nonpolluted samples. The titers recovered were dependent on the degree of pollution in analyzed waters and sediments.     

Bacteriophages active against Bacteroides fragilis in sewage-polluted waters.

Twelve strains of different Bacteroides species were tested for their efficiency of detection of bacteriophages from sewage. The host range of several isolated phages was investigated. The results indicated that there was a high degree of strain specificity. Then, by using Bacteroides fragilis HSP 40 as the host, which proved to be the most efficient for the detection of phages, feces from humans and several animal species and raw sewage, river water, water from lagoons, seawater, groundwater, and sediments were tested for the presence of bacteriophages that were active against B. fragilis HSP 40. Phages were detected in feces of 10% of the human fecal samples tested and was never detected in feces of the other animal species studied. Moreover, bacteriophages were always recovered from sewage and sewage-polluted samples of waters and sediments, but not from nonpolluted samples. The titers recovered were dependent on the degree of pollution in analyzed waters and sediments.     

Examination of thirteen petroliferous formations for hydrocarbon-utilizing sulfate-reducing microorganisms

Virgin cores and production fluids were obtained from seven wells, ranging in depth from 805 ft to 14 492 ft, and examined for the presence of sulfate-reducing bacteria (SRB) using Rosenfeld's sulfate-reducing medium modified by using crude oil in place of lactate. Cores from an additional six wells, ranging in depth from 1160 ft to 13 337 ft were tested for SRB using the modified Rosenfeld medium and API-sulfate-reducing medium. Produced waters from five of the six wells were tested also. All of the eleven produced water samples were positive for SRB while H₂S production was not detected from the core samples.