Metal Distribution and Characterization of Cultivable Lead-Resistant Bacteria in Shooting Range Soils

Shooting ranges represent about 10% of polluted sites in Switzerland. This pollution, mainly consisting of lead (Pb) and antimony (Sb), can spread into the local groundwater, wildlife and plants. In this study, shooting range stop butt soils (elevated soil mount behind the target) of three sites (Versoix, Cartigny and Nyon, Western Switzerland) were investigated for metal contamination and culturable lead-resistant bacteria in contaminated soils. The inductively coupled plasma mass spectrometry analysis of surface stop butt soils (0–5 cm) indicated high metal concentrations especially for Pb and Sb, e.g., the Pb concentration ranging from 61,135 to 395,651, 23,821 to 201,268 and 120 to 27,517 mg kg^?1 in the soil from the sites of Versoix, Cartigny and Nyon, respectively. Molecular analysis of 16S rRNA gene demonstrates the presence of bacteria from diverse classes: Flavobacteriia, α-Proteobacteria, γ-Proteobacteria, Actinobacteria and Bacilli that exhibited high Pb minimum inhibitory concentration value from 1,036 to 2,694 mg kg^?1. The isolated strains could be the subject of further studies to evaluate their role in bioremediation of lead-contaminated soil.     

Aquatic Plants Stimulate the Growth of and Biofilm Formation by Mycobacterium ulcerans in Axenic Culture and Harbor These Bacteria in the Environment

Mycobacterium ulcerans is the causative agent of Buruli ulcer, one of the most common mycobacterial diseases of humans. Recent studies have implicated aquatic insects in the transmission of this pathogen, but the contributions of other elements of the environment remain largely unknown. We report here that crude extracts from two green algae added to the BACTEC 7H12B culture medium halved the doubling time of M. ulcerans and promoted biofilm formation. Using the 7H12B medium, modified by the addition of the algal extract, and immunomagnetic separation, we also demonstrate that M. ulcerans is associated with aquatic plants in an area of the Ivory Coast where Buruli ulcer is endemic. Genotype analysis showed that plant-associated M. ulcerans had the same profile as isolates recovered in the same region from both aquatic insects and clinical specimens. These observations implicate aquatic plants as a reservoir of M. ulcerans and add a new potential link in the chain of transmission of M. ulcerans to humans.     

Molecular Identification and Biofilm-Forming Ability of Culturable Aquatic Bacteria In Microbial Biofilms Formed in Drinking Water Distribution Networks

Drinking water distribution networks are known to harbor microbial biofilms. The aim of the present work is to (i) identify the culturable bacteria presented in the drinking-water distribution network, (ii) investigate the ability of isolated bacteria to form biofilm under some environmental stress conditions and some eliminating or removing treatments. To achieve it, 57 strains were isolated from biofilm (43 isolates) and water samples (14 isolates) collected from five stations in drinking-water distribution network in Taif city, Kingdom of Saudi Arabia (KSA). Partial sequences of 16S rRNA gene in the 57 isolates ensured the presence of only 22 different strains in biofilm samples. Among these strains, only 14 strains were also detected in water samples. Gram-negative Aeromonas hydrophila was the most occurred bacterium in the microbial biofilm obtained from the purified-water storage tanks followed by Gram-negative Pseudomonas sp. Gram-positive Bacillus subtilis was the most occurred bacterium in the microbial biofilm collected from the ends of the distribution pipes. Among the 22 isolated strains, 13 strains were strong biofilm producers at 30 and 37°C. The effects of environmental stresses including nutrient starvation (diluted TSB, 20:1), heating (100°C for 10 min), UV-treatment (240 nm for 10 min) and dynamic incubation (150 rpm min^?1) on the formation of biofilm were also investigated. These conditions affected the biofilm formation ability of the isolated strains at different levels. Nutrient starvation enhanced biofilm formation by most of the isolates. Among some biofilm deforming treatments, SDS and trypsin had considerable effects on preventing biofilm formation by most of the isolated strains. In conclusion, the results of the present work indicated that not all biofilm strains released from biofilm to the drinking water. Also, not all biofilm strains were able to form biofilm. Most of isolated bacteria had ability to form biofilm at suboptimum temperature of growth. These results may provide basic information on formation of microbial biofilms and overcome the problem of deteriorating of water quality in the drinking-water distribution networks.     

Tryptophanase-Positive Bacteria in the Marine Environment

The prevalence of indole-positive organisms in the gut and environment of marine animals was studied. Indole formation by a group of the isolates was found to occur only in the presence of tryptophan. The isolates examined were all assigned to the genus Vibrio.     

Degradation of 2-Methylisoborneol by Aquatic Bacteria

2-Methylisoborneol (MIB) is a musty- or muddy-smelling compound which occurs in some natural waters and which is difficult to remove by conventional water treatment methods. Bacterial degradation of MIB was examined in batch culture experiments. Cultures able to metabolize MIB were enriched in a mineral salts medium supplemented with milligram-per-liter levels of the compound and were inoculated with water and sediment samples from reservoirs where MIB is seasonally produced. Bacteria from degrading cultures were isolated on R2A agar and identified as predominantly Pseudomonas spp. Degradation occurred only in cultures consisting of three or more different bacteria. MIB supported growth as the sole added carbon source at 1 to 6.7 mg/liter. MIB was also degraded at microgram-per-liter levels in sterile filtered lake water inoculated with washed bacteria and in synthetic medium supplemented with various sugars or acetate. Complete degradation of MIB took from 5 days to more than 2 weeks. Enrichment with isoborneol, a structural analog of MIB, failed as a preenrichment for MIB degraders. Isoborneol at 20 to 40 mg/liter readily supported bacterial growth, whereas MIB at 12 to 20 mg/liter took months to degrade. The relative recalcitrance of MIB compared with isoborneol may be a result of the additional methyl group in MIB.     

Identification of Anaerobic Selenate-Respiring Bacteria from Aquatic Sediments

The diversity population of microorganisms with the capability to use selenate as a terminal electron acceptor, reducing it to selenite and elemental selenium by the process known as dissimilatory selenate reduction, is largely unknown. The overall objective of this study was to gain an in-depth understanding of anaerobic biotransformation of selenium in the environment, particularly anaerobic respiration, and to characterize the microorganisms catalyzing this process. Here, we demonstrate the isolation and characterization of four novel anaerobic dissimilatory selenate-respiring bacteria enriched from a variety of sources, including sediments from three different water bodies in Chennai, India, and a tidal estuary in New Jersey. Strains S5 and S7 from India, strain KM from the Meadowlands, NJ, and strain pn1, categorized as a laboratory contaminant, were all phylogenetically distinct, belonging to various phyla in the bacterial domain. The 16S rRNA gene sequence shows that strain S5 constitutes a new genus belonging to Chrysiogenetes, while strain S7 belongs to the Deferribacteres, with greater than 98% 16S rRNA gene similarity to Geovibrio ferrireducens. Strain KM is related to Malonomonas rubra, Pelobacter acidigallici, and Desulfuromusa spp., with 96 to 97% 16S rRNA gene similarity. Strain pn1 is 99% similar to Pseudomonas stutzeri. Strains S5, S7, and KM are obligately anaerobic selenate-respiring microorganisms, while strain pn1 is facultatively anaerobic. Besides respiring selenate, all these strains also respire nitrate.     

Ocean Warming and Spread of Pathogenic Vibrios in the Aquatic Environment

Vibrios are among the most common bacteria that inhabit surface waters throughout the world and are responsible for a number of severe infections both in humans and animals. Several reports recently showed that human Vibrio illnesses are increasing worldwide including fatal acute diarrheal diseases, such as cholera, gastroenteritis, wound infections, and septicemia. Many scientists believe this increase may be associated with global warming and rise in sea surface temperature (SST), although not enough evidence is available to support a causal link between emergence of Vibrio infections and climate warming. The effect of increased SST in promoting spread of vibrios in coastal and brackish waters is considered a causal factor explaining this trend. Field and laboratory studies carried out over the past 40 years supported this hypothesis, clearly showing temperature promotes Vibrio growth and persistence in the aquatic environment. Most recently, a long-term retrospective microbiological study carried out in the coastal waters of the southern North Sea provided the first experimental evidence for a positive and significant relationship between SST and Vibrio occurrence over a multidecadal time scale. As a future challenge, macroecological studies of the effects of ocean warming on Vibrio persistence and spread in the aquatic environment over large spatial and temporal scales would conclusively support evidence acquired to date combined with studies of the impact of global warming on epidemiologically relevant variables, such as host susceptibility and exposure. Assessing a causal link between ongoing climate change and enhanced growth and spread of vibrios and related illness is expected to improve forecast and mitigate future outbreaks associated with these pathogens.     

An Ecological Risk Assessment of Ammonia in the Aquatic Environment

Ammonia is released in the environment by many industries and other human activities. The major quantifiable sources of ammonia released to aquatic ecosystems across Canada are municipal wastewater treatment plants, at an estimated total quantity of 62,000 tonnes per year. Given the sources of ammonia releases in the environment and the properties of the substance, terrestrial plants and aquatic organisms are potential risk targets. A tiered assessment approach has been used to determine the ecological risk in the aquatic environment from ammonia released in municipal wastewater effluents. The results obtained for two case studies with the probabilistic risk analysis used in the highest tier support the conclusion that the conditions encountered in these two locations can lead to ammonia concentrations capable of producing an adverse ecological impact.     

An Ecological Risk Assessment of Phenol in the Aquatic Environment

A probabilistic ecological risk assessment of phenol was undertaken to determine the risks posed to biota as a result of phenol release to the Canadian environment. A three-tiered approach was used to estimate risks, with progressively more realistic assumptions being applied at each tier. In Canada, the major sources of phenol are municipal wastewater treatment plants, pulp, paper and wood products mills, steel and metal products facilities and refineries. Thus, the highest exposures will occur in receiving waters near these point sources, primarily due to the short half-life of phenol in the aquatic environment. Sensitive aquatic organisms include salmonids (e.g., rainbow trout Oncorhynchus mykiss) and amphibians (e.g., leopard frog Rana pipiens). The results of the risk assessment indicate that species are exposed to elevated levels of phenol near point sources, but these levels represent only a minor risk to aquatic biota.     

Molecular Characterization of Endophytic Bacteria from Metal Hyperaccumulator Aquatic Plant (Eichhornia crassipes) and Its Role in Heavy Metal Removal

In this study, among a collection of heavy metals resistant endophytic bacterial strains isolated from aquatic hyperaccumulator plant (Eichhornia crassipes), one plant growth promoting endophytic bacteria (PGPE), SVUB4 was selected for its ability to utilize 1-aminocyclopropane-1-carboxylic acid (ACC) as the sole N source and accumulate different heavy metals. The SVUB4 strain was characterized as Enterobacter sp. on the basis of its 16S rDNA sequences. Assessment of the parameters of plant growth promotion revealed the intrinsic ability of the strain for the production of IAA, siderophore and solubilization of insoluble phosphate. Furthermore, plasmid DNA analysis of Enterobacter sp. strain SVUB4 indicated the presence of a single large plasmid element. The results of plasmid curing experiments demonstrated that the ability of this strain to grow in presence of Cd and Zn was encoded by the 98 kb plasmid, whereas the ability to grow in the presence of Pb appeared to be encoded by the chromosome. The Cd and Zn removal capacity of the respective metal sensitive strain (plasmidless) were about 36 and 45 μg/g-1 DW, respectively, while the removal capacity of the both metal by metal resistant strain (p SVUB4) showed a significantly higher Cd and Zn removal capacity of 153 and 228 μg/g^?1 DW, respectively. However, both strains exhibited a similar pattern of Pb accumulation. The present observation also showed that for wild-type strain SVUB4 (pSVUB4), the overall level of IAA production in the absence and in the presence of Cd²⁺ or Zn²⁺was approximately the same. Nevertheless, strain SVUB4M in this respect appeared to be more sensitive to heavy metals: a noticeable decrease in IAA production was observed under the effect of both metals, especially with Cd²⁺.     

Distribution of l-Leucine-pyruvate Transaminase in Bacteria

Dodecadien-1-ols, tetradecadien-1-ols and hexadecadien-1-ols with a conjugated (E,E)- or (E)-diene system between the ωl,ω3- and ω5, ω7-positions, their acetates, and aldeh?de derivatives (lepidopterous sex pheromones and candidates) were analyzed by electron impact mass spectrometry, which was operated at 70 eV ionization voltage. Three functional derivatives with a same diene system presented a similar spectral pattern, except for the molecular ions (M⁺), [M ? H₂O]⁺ of the alcohols and [M ? CH₃CO₂H]⁺ of the acetates. Each isomer showed a characteristic fragment ion series of C_nH_2n?2⁺ ~C_nH_2n?5⁺ (C₄~C₉), which reflected the double-bond position in the molecule, indicating a method for determining the position of a natural diene pheromone by comparing its mass spectrum with those of the synthetic dienes. By this method, the natural pheromone of Hellula undalis was confirmed to be a ω3, ω5-diene. Furthermore, the fitness indexes proposed by Kuwahara et al. were calculated for some pheromone components, using the mass spectra of synthetic dienes, in order to examine the possibility and limitation for applications of those mass spectra to natural pheromone studies.     

Distribution of ATP-inhibited Ribonuclease in Bacteria

ATP-inhibited ribonuclease was first found in Bacillus amyloliquefaciens cells and was isolated by the authors previously. The distribution of the ATP-inhibited ribonuclease was surveyed on various bacteria. ATP-inhibited ribonuclease was found in only several species of Bacillus genus, i.e., B. subtilis (i), B. amyloliquefaciens (ii), B. cereus (iii), B. cereus var. mycoides (iv), and B. thiiringiensis (v). ATP-inhibited ribonuclease of Bacillus genus was subdivided into two types. The first type of enzyme found in (i) and (ii) cells had optimum pH at 5.7 and was sensitive to 5 mM EDTA. The second type of enzyme found in (iii), (iv), and (v) cells had optimum pH at 6.8 and was partially inhibited by 5 mM EDTA.     

Airborne Bacteria in an Urban Environment

Samples were taken at random intervals over a 2-year period from urban air and tested for viable bacteria. The number of bacteria in each sample was determined, and each organism isolated was identified by its morphological and biochemical characteristics. The number of bacteria found ranged from 0.013 to 1.88 organisms per liter of air sampled. Representatives of 19 different genera were found in 21 samples. The most frequently isolated organisms and their percent of occurence were Micrococcus (41%), Staphylococcus (11%), and Aerococcus (8%). The bacteria isolated were correlated with various weather and air pollution parameters using the Pearson product-moment correlation coefficient method. Statistically significant correlations were found between the number of viable bacteria isolated and the concentrations of nitric oxide (−0.45), nitrogen dioxide (+0.43), and suspended particulate pollutants (+0.56). Calculated individually, the total number of Micrococcus, Aerococcus, and Staphylococcus, number of rods, and number of cocci isolated showed negative correlations with nitric oxide and positive correlations with nitrogen dioxide and particulates. Statistically significant positive correlations were found between the total number of rods isolated and the concentration of nitrogen dioxide (+0.54) and the percent relative humidity (+0.43). The other parameters tested, sulfur dioxide, hydrocarbons, and temperature, showed no significant correlations.     

Aquatic hyphomycetes of Wisconsin: Distribution and ecology

Summary 1. Eighteen species in fourteen genera of aquatic hyphomycetes were found in a survey of Wisconsin streams and lakes. No clear pattern of geographic distribution was evident but several species were more frequently encountered in northern areas.2. Moderate to swift currents were found to support the most abundant growth and greatest variety of species. Type of bottom and relative water temperature showed no detectable effect.3. Most of the commonly-occurring species were indifferent to the oxygen regime. Nitrogen levels did not appear to affect the abundance of these fungi and no pH preference was discernible throughout the relatively narrow range that was recorded.4. Carbon dioxide level and abundance of aquatic hyphomycetes varied inversely. Total hardness and total alkalinity appeared to have a limiting effect at concentrations in excess of 250 ppm.     

Luminescence-Based Systems for Detection of Bacteria in the Environment

Abstract

The development of techniques for detection and tracking of microorganisms in natural environments has been accelerated by the requirement for assessment of the risks associated with environmental release of genetically engineered microbial inocula. Molecular marker systems are particularly appropriate for such studies and luminescence-based markers have the broadest range of applications, involving the introduction of prokaryotic (lux) or eukaryotic (luc) genes for the enzyme luciferase.

Lux or luc genes can be detected on the basis of unique DNA sequences by gene probing and PCR amplification, but the major advantage of luminescence-based systems is the ability to detect light emitted by marked organisms or by luciferase activity in cell-free extracts. Luminescent colonies can be detected by eye, providing distinction from colonies of indigenous organisms, and the sensitivity of plate counting can be increased greatly by CCD imaging. Single cells or microcolonies of luminescent organisms can also be detected in environmental samples by CCD image-enhanced microscopy, facilitating study of their spatial distribution. The metabolic activity of luminescence-marked populations can be quantified by luminometry and does not require extraction of cells or laboratory growth. Metabolic activity, and potential activity, of marked organisms therefore can be measured during colonization of soil particles and plant material in real time without disturbing the colonization process.

In comparison with traditional activity techniques, luminometry provides significant increases in sensitivity, accuracy, and, most importantly, selectivity, as activity can be measured in the presence of indigenous microbial communities. The sensitivity, speed, and convenience of luminescence measurements make this a powerful technique that is being applied to the study of an increasingly wide range of ecological problems. These include microbial survival and recovery, microbial predation, plant pathogenicity, phylloplane and rhizosphere colonization and reporting of gene expression in environmental samples.     

Role of Productivity and Protozoan Abundance for the Occurrence of Predation-resistant Bacteria in Aquatic Systems

Top-down control of lower trophic levels, e.g., bacteria, has been suggested to increase along aquatic productivity gradients. The response by the bacterial community may be to become more predation resistant in highly productive environments. To test this hypothesis, samples were taken from 20 aquatic systems along a productivity gradient (dissolved organic carbon from 7 to 71 mg/L), during late summer. The results showed that the biomass of bacteria, phytoplankton, and ciliates increased along the gradient (r2 = 0.532, 0.426, and 0.758, P < 0.01, respectively). However, the organisms did not increase equally, and the ratio of protozoan to bacterial biomass showed a 100-fold increase along the gradient. Ciliates dominated the protozoan biomass in the more nutrient-rich waters. The edibility of colony-forming bacteria was tested using a ciliate predator, Tetrahymena pyriformis. Bacterial edibility was found to decrease with increases in nutrient richness and ciliate biomass in the aquatic systems (r2 = 0.358, P < 0.01; r2 = 0.242, P < 0.05, respectively). Quantile regression analysis indicated that the selection pressures on edible bacteria were increasing along the productivity gradient. Thus, inedible forms of bacteria were selected for in the transition from oligotrophic to eutrophic conditions. Isolated bacteria were distributed among the alpha-, beta-, and gamma- Proteobacteria and the Actinobacteria and Firmicutes taxa. We conclude that bacterial predation resistance increases in nutrient-rich waters with high protozoan predation.     

Toxigenic Vibrio cholerae in the Aquatic Environment of Mathbaria, Bangladesh

Toxigenic Vibrio cholerae, rarely isolated from the aquatic environment between cholera epidemics, can be detected in what is now understood to be a dormant stage, i.e., viable but nonculturable when standard bacteriological methods are used. In the research reported here, biofilms have proved to be a source of culturable V. cholerae, even in nonepidemic periods. Biweekly environmental surveillance for V. cholerae was carried out in Mathbaria, an area of cholera endemicity adjacent to the Bay of Bengal, with the focus on V. cholerae O1 and O139 Bengal. A total of 297 samples of water, phytoplankton, and zooplankton were collected between March and December 2004, yielding eight V. cholerae O1 and four O139 Bengal isolates. A combination of culture methods, multiplex-PCR, and direct fluorescent antibody (DFA) counting revealed the Mathbaria aquatic environment to be a reservoir for V. cholerae O1 and O139 Bengal. DFA results showed significant clumping of the bacteria during the interepidemic period for cholera, and the fluorescent micrographs revealed large numbers of V. cholerae O1 in thin films of exopolysaccharides (biofilm). A similar clumping of V. cholerae O1 was also observed in samples collected from Matlab, Bangladesh, where cholera also is endemic. Thus, the results of the study provided in situ evidence for V. cholerae O1 and O139 in the aquatic environment, predominantly as viable but nonculturable cells and culturable cells in biofilm consortia. The biofilm community is concluded to be an additional reservoir of cholera bacteria in the aquatic environment between seasonal epidemics of cholera in Bangladesh.     

A Human Health Risk Assessment of Pharmaceuticals in the Aquatic Environment

Analyses were conducted on four pharmaceutical compounds, representing different therapeutic classes, to evaluate the presence and potential adverse human health effects of trace levels of these substances in aqueous environmental media. Acetylsalicylic acid, clofibrate, cyclophosphamide, and indomethacin have been detected in aqueous environmental media including sewage treatment plant effluent, surface water, drinking water, and groundwater. An extensive literature search and chemical-specific risk assessments were performed to assess the potential human health significance of each compound's individual presence in environmental media. Safe water quality limits were estimated for each pharmaceutical by following the USEPA Methodology for Deriving Ambient Water Quality Criteria for the Protection of Human Health and were compared to the concentrations found in the environment. The calculation of the provisional ambient water quality criteria involved estimation of human exposure to contaminated water, including intake via bioaccumulation in fish, and calculation of cancer risk and non-cancer hazard indices. Parameters detailing the toxicological and pharmacological nature, exposure assessment, and environmental fate and transport of each pharmaceutical were also considered. The overall conclusion was that based on available data, no appreciable risk to humans exists, as the detected concentrations of each of these pharmaceutical compounds found in aqueous media were far below the derived safe limits