Comparison of two kinds of Biolog microplates (GN and ECO) in their ability to distinguish among aquatic microbial communities.

We compared the abilities of Biolog's GN and ECO plates to distinguish among aerobic and heterotrophic bacterial communities in samples from six aquatic environments. The Biolog system is based on interpreting patterns of sole-carbon substrate utilization indicated by color development in a 96-well microtiter plate. Whether of fresh or saltwater origin, bacterial communities utilized > 95% of substrates in both types of plates. Samples from any one environment exhibited similar time courses of average well color development (AWCD) in both GN and ECO plates. Principal component analysis was performed on data sets resulting from combinations of algorithms (AWCD and curve-integration methods) and levels of color development (end-point and set-point approaches). In all cases, the two types of plates demonstrated an equal capacity to discriminate among the heterotrophic expressions of the six microbial communities. Substantial deviation from an anticipated 1:1 correspondence occurred when color development of 25 substrates common to both types of plates was compared. The discrepancies likely are related to the different formulations of low-nutrient media in GN and ECO plates.     

Bacterial Distribution and Phylogenetic Diversity in the Changjiang Estuary before the Construction of the Three Gorges Dam

The bacterial community structure in the Changjiang estuary was studied for comparison with future changes, related to the construction of the Three Gorges Dam. Population densities of bacteria in the surface water at station C1 estimated by CFU on marine agar plates and by DAPI direct count, were 2.8 x 10(4) ml(-1) and 4.2 x 10(5) ml(-1), respectively. Physicochemical properties of water, such as temperature and salinity, suggested that station C1 was affected by freshwater from the Changjiang River. Cluster analysis of the PCR-RFLP patterns obtained from 9 samples showed that the bacterial community structure at station C1 was different from the structure at the other stations. Bacterial diversity in the surface water at station C1 was studied based on the genotypes of the 250 clones of 16S rRNA, and on the phenotypes generated on Biolog GN plates for 70 isolates. Sequences of bacteria from two common marine groups, alpha- and gamma-Proteobacteria, were frequently observed. Some other divisions, including the beta-Proteobacteria, C/F/B group, low G+C gram positive, high G+C gram positive, chloroplasts, and relatives of Verrucomicrobia were also observed. The putative dominant species based on both genotype and phenotype analyses were close relatives of Alteromonas macleodii or Roseobacter spp. These results reflected the nutrient-rich environment at station C1.     

Monitoring of fecal pollution in coastal waters by use of rapid enzymatic techniques.

Enzyme assays for 4-methylumbelliferyl-beta-D-galactopyranosidase and 4-methylumbelliferyl-beta-D-glucuronidase activities were used for rapid detection (25 min) of fecal water pollution and to determine the impact of sewage discharge in coastal waters. Two coastal areas were investigated: (i) an estuary characterized by a high degree of contamination downstream of a discharge from a sewage treatment plant and a low degree of water renewal and (ii) a fjord with a low degree of pollution and a high degree of water renewal. Statistical analysis showed that a global correlation curve could be used to estimate concentrations of culturable fecal coliform bacteria in the two coastal areas, although environmental factors important for cell physiology (e.g., salinity) varied at different sampling locations. The sensitivity limit for detection of 4-methylumbelliferyl-beta-D-glucuronidase activity corresponded to bacterial concentrations on the order of 10 to 100 CFU/100 ml. The 4-methylumbelliferyl-beta-D-galactopyranosidase assay was less sensitive because of a higher rate of substrate autohydrolysis. The detection limit corresponded to bacterial concentrations on the order of 100 to 1,000 fecal coliforms per 100 ml.     

Interactions between heterotrophic plate count bacteria and coliform organisms

Studies were initiated to investigate the interactions between heterotrophic plate count bacteria and coliform organisms. We used spiked samples to show that heterotrophic plate count bacteria could reduce coliform densities by more than 3 logs within 8 days. Some heterotrophic plate count bacteria were able to cause injury to the coliform population. A significant correlation (r = 0.66; P less than 0.05) was observed between the initial level of heterotrophic plate count bacteria and the rate of coliform decline. Competition for limiting organic carbon was hypothesized to be responsible for the observed effects.     

Interactions between heterotrophic plate count bacteria and coliform organisms.

Studies were initiated to investigate the interactions between heterotrophic plate count bacteria and coliform organisms. We used spiked samples to show that heterotrophic plate count bacteria could reduce coliform densities by more than 3 logs within 8 days. Some heterotrophic plate count bacteria were able to cause injury to the coliform population. A significant correlation (r = 0.66; P less than 0.05) was observed between the initial level of heterotrophic plate count bacteria and the rate of coliform decline. Competition for limiting organic carbon was hypothesized to be responsible for the observed effects.     

Bacteriological water quality effects of hydraulically dredging contaminated upper Mississippi River bottom sediment.

Bacteriological effects of hydraulically dredging polluted bottom sediment in the navigation channel of the Upper Mississippi River (river mile 827.5 [about 1,332 km] to 828.1 [about 1,333 km]) were investigated. Bottom sediment in the dredging site contained high total coliform densities (about 6,800 most-probable-number total coliform index per g [dry weight] and 3,800 membrane filter total coliforms per g [dry weight]), and fecal coliforms comprised an average 32% of each total coliform count. Total coliform and fecal coliform densities in water samples taken immediately below the dredge discharge pipe were each approximately four times corresponding upstream values; fecal streptococcus densities were approximately 50 times corresponding upstream values. Correlation analysis indicated that mean turbidity values downstream to the dredging operation were directly and significantly (r greater than 0.94) related to corresponding total coliform, fecal coliform, and fecal streptococcus densities. Salmonellae and shigellae were not recovered from either upstream or downstream water samples. Turbidity and indicator bacteria levels had returned to predredge values within less than 2 km below the dredge spoil discharge area at the prevailing current velocity (about 0.15 m/s).     

Viability and metabolic features of bacteria indigenous to a contaminated deep aquifer

The quantitation and characterization of indigenous bacteria of a deep aquifer, located in the southwestern United States and contaminated with halogenated aliphatic compounds, was undertaken. Water samples were obtained aseptically from depths of 45 to 151 m from four sites that ranged from 260 to 1,800 m in distance from the location of contaminant release. Sediment samples were also obtained from the proximal and distal sites for analyses. Results for aerobic and anaerobic colony-forming units were obtained on four agar media that were used to retrieve heterotrophs, oligotrophs, and pseudomonads. Most probable number estimates were obtained from a liquid medium favorable for oligotrophs. Representative isolates were tested against Biolog plates (Biolog, Inc., Hayward, Calif.) for patterns of carbon source utilization. Of 103 Gram-negative (GN) isolates, 48 could not be identified and the others were only tentatively identified via the Biolog database, and none of the 35 Gram-positive (GP) isolates were identifiable. However, the metabolic patterns were subjected to average cluster linkage analyses; the GN and GP bacteria were separable into eight and four groups, respectively. The oligotroph group comprised one-third of the GN and one-half of the GP isolates. The consensus carbon source utilization pattern for each group was determined and will be useful in future characterization of additional aquifer bacterial isolates. Although predominantly aerobic and oligotrophic, the microbial community of this aquifer was highly diverse with discernible viability and metabolic features of the microbiota distinctive to each of the four water and two sediment samples. Correspondence to: C.M. McCarthy.     

Bacteria associated with granular activated carbon particles in drinking water

A sampling protocol was developed to examine particles released from granular activated carbon filter beds. A gauze filter/Swinnex procedure was used to collect carbon fines from 201 granular activated carbon-treated drinking water samples over 12 months. Application of a homogenization procedure (developed previously) indicated that 41.4% of the water samples had heterotrophic plate count bacteria attached to carbon particles. With the enumeration procedures described, heterotrophic plate count bacteria were recovered at an average rate of 8.6 times higher than by conventional analyses. Over 17% of the samples contained carbon particles colonized with coliform bacteria as enumerated with modified most-probable-number and membrane filter techniques. In some instances coliform recoveries were 122 to 1,194 times higher than by standard procedures. Nearly 28% of the coliforms attached to these particles in drinking water exhibited the fecal biotype. Scanning electron micrographs of carbon fines from treated drinking water showed microcolonies of bacteria on particle surfaces. These data indicate that bacteria attached to carbon fines may be an important mechanism by which microorganisms penetrate treatment barriers and enter potable water supplies.     

Bacteria associated with granular activated carbon particles in drinking water.

A sampling protocol was developed to examine particles released from granular activated carbon filter beds. A gauze filter/Swinnex procedure was used to collect carbon fines from 201 granular activated carbon-treated drinking water samples over 12 months. Application of a homogenization procedure (developed previously) indicated that 41.4% of the water samples had heterotrophic plate count bacteria attached to carbon particles. With the enumeration procedures described, heterotrophic plate count bacteria were recovered at an average rate of 8.6 times higher than by conventional analyses. Over 17% of the samples contained carbon particles colonized with coliform bacteria as enumerated with modified most-probable-number and membrane filter techniques. In some instances coliform recoveries were 122 to 1,194 times higher than by standard procedures. Nearly 28% of the coliforms attached to these particles in drinking water exhibited the fecal biotype. Scanning electron micrographs of carbon fines from treated drinking water showed microcolonies of bacteria on particle surfaces. These data indicate that bacteria attached to carbon fines may be an important mechanism by which microorganisms penetrate treatment barriers and enter potable water supplies.     

Full-scale studies of factors related to coliform regrowth in drinking water.

An 18-month survey of 31 water systems in North America was conducted to determine the factors that contribute to the occurrence of coliform bacteria in drinking water. The survey included analysis of assimilable organic carbon (AOC), coliforms, disinfectant residuals, and operational parameters. Coliform bacteria were detected in 27.8% of the 2-week sampling periods and were associated with the following factors: filtration, temperature, disinfectant type and disinfectant level, AOC level, corrosion control, and operational characteristics. Four systems in the study that used unfiltered surface water accounted for 26.6% of the total number of bacterial samples collected but 64.3% (1,013 of 1,576) of the positive coliform samples. The occurrence of coliform bacteria was significantly higher when water temperatures were > 15 degrees C. For filtered systems that used free chlorine, 0.97% of 33,196 samples contained coliform bacteria, while 0.51% of 35,159 samples from chloraminated systems contained coliform bacteria. The average density of coliform bacteria was 35 times higher in free-chlorinated systems than in chloraminated water (0.60 CFU/100 ml for free-chlorinated water compared with 0.017 CFU/100 ml for chloraminated water). Systems that maintained dead-end free chlorine levels of < 0.2 mg/liter or monochloramine levels of < 0.5 mg/liter had substantially more coliform occurrences than systems that maintained higher disinfectant residuals. Free-chlorinated systems with AOC levels greater than 100 micrograms/liter had 82% more coliform-positive samples and 19 times higher coliform levels than free-chlorinated systems with average AOC levels less than 99 micrograms/liter. Systems that maintained a phosphate-based corrosion inhibitor and limited the amount of unlined cast iron pipe had fewer coliform bacteria. Several operational characteristics of the treatment process or the distribution system were also associated with increased rates of coliform occurrence. The study concludes that the occurrence of coliform bacteria within a distribution system is dependent upon a complex interaction of chemical, physical, operational, and engineering parameters. No one factor could account for all of the coliform occurrences, and one must consider all of the parameters described above in devising a solution to the regrowth problem.     

Bacterial species in raw and cured compost from a large-scale urban composter

Summary Raw and cured compost samples from a large-scale urban composter were studied over a period of eight months to gain information on bacterial species present. Total viable, aerobic heterotrophic bacteria, lactose-positive bacteria, antibiotic and metal-resistant bacteria and thermophilic bacteria were enumerated. Both raw and cured compost samples contained metal and antibiotic-tolerant bacteria (–1 compost) as well as high numbers (as high as Log 7.4 CFU g^–1 dry weight compost) of thermophilic bacteria isolated by growth at 55 °C. Selected colonies were also identified using the Biolog 95 substrate identification system.Escherichia coli andSalmonella spp. were not detected in compost samples.     

Effect of environmental factors on the relationship between concentrations of coprostanol and fecal indicator bacteria in tropical (Mekong Delta) and temperate (Tokyo) freshwaters

A reliable assessment of microbial indicators of fecal pollution (total coliform, Escherichia coli, and fecal streptococcus) is critical in tropical environments. Therefore, we investigated the relationship between concentrations of indicator bacteria and a chemical indicator, coprostanol (5beta-cholestan-3beta-ol), in tropical and temperate regions. Water samples were collected from the Mekong Delta, Vietnam, during wet and dry seasons, and from Tokyo, Japan, during summer, the aftermath of a typhoon, and winter. During the wet season in the Mekong Delta, higher bacterial densities were observed in rivers, probably due to the higher bacterial inputs from soil particles with runoff. In Tokyo, higher bacterial densities were usually observed during summer, followed by those in the typhoon aftermath and winter. A strong logarithmic correlation between the concentrations of E. coli and coprostanol was demonstrated in all surveys. Distinctive seasonal fluctuations were observed, as concentrations of coprostanol corresponding to 1,000 CFU of E. coli/100 ml were at their lowest during the wet season in the Mekong Delta and the typhoon aftermath in Tokyo (30 ng/liter), followed by the dry season in the Mekong Delta and the summer in Tokyo (100 ng/liter), and they were much higher during the winter in Tokyo (400 ng/liter). These results suggested that E. coli is a specific indicator of fecal contamination in both tropical and temperate regions but that the densities are affected by elevated water temperature and input from runoff of soil particles. The concurrent determination of E. coli and coprostanol concentrations could provide a possible approach to assessing the reliability of fecal pollution monitoring data.     

Distribution of aerobic bacteria,protozoa, algae,and fungi in deep subsurface sediments

The distribution of microorganisms in deep subsurface profiles was determined at three sites at the Savannah River Plant, Aiken, South Carolina. Acridine orange direct counts (AODC) of bacteria were highest in surface soil samples and declined to the 10⁶ to 10⁷ per gram range in the subsurface, but then did not decline further with depth. In the subsurface, AODC values varied from layer to layer, the highest being found in samples from sandy aquifer formations and the lowest in clayey interbed layers. Sandy aquifer sediments also contained the highest numbers of viable bacteria as determined by aerobic spread plate counts (CFU) on a dilute heterotrophic medium. In some of these samples bacterial CFU values approached 100% of the AODC values. Viable protozoa (amoebae and flagellates, but no ciliates) were found in samples with high bacterial CFU values. A variety of green algae, phytoflagellates, diatoms, and a few cyanobacteria were found at low population densities in samples from two of the three boreholes. Low numbers of fungi were evenly distributed throughout the profiles at all three sites. Microbial population density estimates correlated positively with sand content and pore‐water pH, and negatively with clay content and pore‐water metal concentration. A large diversity of prokaryotic and eukaryotic microorganisms was found in samples with high population densities. A survey of bacterial strains isolated from subsurface samples revealed associations of gram‐positive bacteria with high clay sediments and gram‐negative bacteria with sandy sediments. The ability to deposit lipophilic storage material (presumably poly‐ß‐hydroxybutyrate) was found in a high proportion of isolates from sandy sediments, but only rarely in isolates from high clay sediments.     

Monitoring Biolog patterns and r/K-strategists in the intensive culture of Artemia juveniles

Artemia juveniles were cultured under intensive conditions in three series of six tanks at different times. Both plate counts and Biolog GN microtitre plates were used to monitor the microbial community. Repetitions of the Artemia cultures in time revealed significant differences in Biolog patterns and bacterial counts, showing that normal culture practices, including chlorination of the sea water, does not allow control of the associated microbial community. However, the similarity among the Biolog fingerprints of all 18 tanks as determined by the Pearson correlation coefficient was always high. Both observations together indicate that the microbial community which colonizes the culture tanks seems to be determined by both deterministic factors (e.g. salinity, temperature, oxygen concentration, composition of the feed) and stochastic factors (micro-organisms still present in the sea water after chlorination, on the walls of the culture tanks or in the air around the culture tanks). When a high proportion of microbial r -strategists was present in the water at the beginning of the Artemia culture, the smallest differences in Biolog patterns among the tanks of one series throughout the culture period were observed. A parallelism between Artemia rearing success, functional fingerprint of the bacterial community and the proportion of r-strategists present, was observed. This suggests an important role of the bacterial community in the overall Artemia rearing success.     

Differentiation of marine luminous bacteria using commercial identification plates

Differentiation of marine luminous bacteria using Biolog GN plate combined with API 20e or the BBL Crystal ID plate inoculated with cell suspensions in artifical seawater was accomplished by comparison to type species using cluster analysis. Inoculum density affected the results from Biolog GN plates, but had less of an effect on the reactions obtained from API 20e strip or BBL Crystal ID plate. In a few cases, combination of the Biolog GN traits along with either the API 20e or Crystal ID traits was necessary to differentiate some marine luminous bacteria. © 1998 John Wiley & Sons, Ltd.     

Effects of Gypsophila saponins on bacterial growth kinetics and on selection of subterranean clover rhizosphere bacteria

Plant secondary metabolites, such as saponins, have a considerable impact in agriculture because of their allelopathic effects. They also affect the growth of soil microorganisms, especially fungi. We investigated the influence of saponins on rhizosphere bacteria in vitro and in soil conditions. The effects of gypsophila saponins on the growth kinetics of rhizosphere bacteria were studied by monitoring the absorbance of the cultures in microtiter plates. Gypsophila saponins (1%) increased the lag phase of bacterial growth. The impact of gypsophila saponins on subterranean clover rhizosphere was also investigated in a pot experiment. The addition of gypsophila saponins did not modify clover biomass but significantly increased (twofold with 1% saponins) the weight of adhering soil. The number of culturable heterotrophic bacteria of the clover rhizosphere was not affected by the addition of gypsophila saponins. Nevertheless, the phenotypical characterization of the dominant Gram-negative strains of the clover rhizosphere, using the Biolog system, showed qualitative and quantitative differences induced by 1% saponins. With the addition of saponins, the populations of Chryseomonas spp. and Acinetobacter spp., the two dominant culturable genera of control clover, were no longer detectable or were significantly decreased, while that of Aquaspirillum dispar increased and Aquaspirillum spp. became the major genus. Aquaspirillum dispar and Aquaspirillum spp. were also the dominant rhizosphere bacteria of Gypsophila paniculata, which greatly accumulates these saponins in its roots. These results suggest that saponins may control rhizosphere bacteria in soil through rhizodeposition mechanisms.     

Sampling and Pooling Methods for Capturing Herd Level Antibiotic Resistance in Swine Feces using qPCR and CFU Approaches

The aim of this article was to define the sampling level and method combination that captures antibiotic resistance at pig herd level utilizing qPCR antibiotic resistance gene quantification and culture-based quantification of antibiotic resistant coliform indicator bacteria. Fourteen qPCR assays for commonly detected antibiotic resistance genes were developed, and used to quantify antibiotic resistance genes in total DNA from swine fecal samples that were obtained using different sampling and pooling methods. In parallel, the number of antibiotic resistant coliform indicator bacteria was determined in the same swine fecal samples. The results showed that the qPCR assays were capable of detecting differences in antibiotic resistance levels in individual animals that the coliform bacteria colony forming units (CFU) could not. Also, the qPCR assays more accurately quantified antibiotic resistance genes when comparing individual sampling and pooling methods. qPCR on pooled samples was found to be a good representative for the general resistance level in a pig herd compared to the coliform CFU counts. It had significantly reduced relative standard deviations compared to coliform CFU counts in the same samples, and therefore differences in antibiotic resistance levels between samples were more readily detected. To our knowledge, this is the first study to describe sampling and pooling methods for qPCR quantification of antibiotic resistance genes in total DNA extracted from swine feces.     

Detection of small numbers of Campylobacter jejuni and Campylobacter coli cells in environmental water, sewage, and food samples by a seminested PCR assay

A rapid and sensitive assay was developed for detection of small numbers of Campylobacter jejuni and Campylobacter coli cells in environmental water, sewage, and food samples. Water and sewage samples were filtered, and the filters were enriched overnight in a nonselective medium. The enrichment cultures were prepared for PCR by a rapid and simple procedure consisting of centrifugation, proteinase K treatment, and boiling. A seminested PCR based on specific amplification of the intergenic sequence between the two Campylobacter flagellin genes, flaA and flaB, was performed, and the PCR products were visualized by agarose gel electrophoresis. The assay allowed us to detect 3 to 15 CFU of C. jejuni per 100 ml in water samples containing a background flora consisting of up to 8, 700 heterotrophic organisms per ml and 10,000 CFU of coliform bacteria per 100 ml. Dilution of the enriched cultures 1:10 with sterile broth prior to the PCR was sometimes necessary to obtain positive results. The assay was also conducted with food samples analyzed with or without overnight enrichment. As few as 相似文献   

Effects of Pinus sylvestris root growth and mycorrhizosphere development on bacterial carbon source utilization and hydrocarbon oxidation in forest and petroleum-contaminated soils

The hypothesis that Pinus sylvestris L. root and mycorrhizosphere development positively influences bacterial community-linked carbon source utilization, and drives a concomitant reduction in mineral oil levels in a petroleum hydrocarbon- (PHC-) contaminated soil was confirmed in a forest ecosystem-based phytoremediation simulation. Seedlings were grown for 9 months in large petri dish microcosms containing either forest humus or humus amended with cores of PHC-contaminated soil. Except for increased root biomass in the humus/PHC treatment, there were no other significant treatment-related differences in plant growth and needle C and N status. Total cell and culturable bacterial (CFU) densities significantly increased in both rhizospheres and mycorrhizospheres that actively developed in the humus and PHC-contaminated soil. Mycorrhizospheres (mycorrhizas and extramatrical mycelium) supported the highest numbers of bacteria. Multivariate analyses of bacterial community carbon source utilization profiles (Biolog GN microplate) from different rhizosphere, mycorrhizosphere, and bulk soil compartments, involving principal component and correspondence analysis, highlighted three main niche-related groupings. The respective clusters identified contained bacterial communities from (i) unplanted bulk soils, (ii) planted bulk PHC and rhizospheres in PHC-contaminated soils, and (iii) planted bulk humus and rhizosphere/mycorrhizosphere-influenced humus, and mycorrhizosphere-influenced PHC contaminated soil. Correspondence analysis allowed further identification of amino acid preferences and increased carboxylic/organic acid preferences in rhizosphere and mycorrhizosphere compartments. Decreased levels of mineral oil (non-polar hydrocarbons) were detected in the PHC-contaminated soil colonized by pine roots and mycorrhizal fungi. These data further support our view that mycorrhizosphere development and function plays a central role in controlling associated bacterial communities and their degradative activities in lignin-rich forest humus and PHC-contaminated soils.     

Distribution of Vibrio vulnificus in the Chesapeake Bay.

Vibrio vulnificus is a potentially lethal human pathogen capable of producing septicemia in susceptible persons. Disease is almost always associated with consumption of seafood, particularly raw oysters, or with exposure of wounds to seawater. An oligonucleotide DNA probe (V. vulnificus alkaline phosphatase-labeled DNA probe [VVAP]), previously shown to be highly specific for V. vulnificus, was used to enumerate this species in environmental samples collected from the Chesapeake Bay between April 1991 and December 1992. Total aerobic, heterotrophic, culturable bacteria were enumerated by plate counts on nonselective medium. The number of V. vulnificus organisms was determined by colony lifts of spread plates for subsequent hybridization with VVAP. V. vulnificus was not detected in any samples collected during February and March (water temperature of < 8 degrees C) but was found in 80% of the water samples collected during May, July, September, and December (water temperature of > 8 degrees C), with concentrations ranging from 3.0 x 10(1) to 2.1 x 10(2)/ml (ca. 8% of the total culturable heterotrophic bacteria). In a multiple regression analysis, increased V. vulnificus concentrations were correlated with lower salinities and with isolation from samples collected closer to the bottom. Isolation from oysters was demonstrable when water temperatures were 7.6 degrees C, with concentrations ranging from 1.0 x 10(3) to 4.7 x 10(4)/g (ca. 12% of total culturable bacteria). In samples collected in May and July, V. vulnificus was identified in seven of seven plankton samples and four of nine sediment samples. Our data demonstrate that V. vulnificus is a widespread and important component of the bacterial population of the Chesapeake Bay, with counts that are comparable to those reported from the Gulf of Mexico.