Bacteria In Environmental Samples Research Articles

Quantitative detection of pentachlorophenol-degrading Sphingomonas sp. UG30 in soil by a most-probable-number/polymerase chain reaction protocol

Traditionally, biodegradation of pentachlorophenol (PCP) in soil is studied by disappearance of the parent compound and its intermediates. Because of the lack of methods to enumerate PCP-degrading bacteria in environmental samples, little is known about the dynamics of these organisms in soil. In this study, the efficiency of a modified most-probable-number/polymerase chain reaction (MPN/PCR) protocol was compared to the traditional MPN/[ 14C]PCP mineralization assay to quantify the density of PCP-degrading Sphingomonas sp. UG30 cells inoculated in an agricultural soil. A 753-bp tetrachlorohydroquinone reductive dehalogenase gene ( pcpC) fragment of UG30 was targeted for the MPN/PCR amplification. The MPN/PCR protocol had a detection limit of 3 CFU/g dry soil. A good correlation was established between the MPN/PCR estimations and initial inoculum densities ranging from 30 to 2×10 9 CFU/g of soil. However, the MPN/[ 14C]PCP mineralization protocol underestimated the inoculum density by 70 to 740-fold. Survival of UG30 in soil was monitored by the MPN/PCR assay. Cell density of the UG30 inoculum decreased from 1.8×10 8 to 1.9×10 5 cells/g of soil in the first 20 days of incubation and stabilized at 1.9×10 4 cells/g of soil after 50 days. When the soil was autoclaved prior to inoculation, UG30 cell density remained at 6.7×10 7 cells/g of soil after 50 days of incubation.

Increased species diversity and extended habitat range of sulfur-oxidizing Thiomicrospira spp.

We combined traditional cultivation methods and new molecular techniques to study the diversity and habitat range of bacteria of the genus Thiomicrospira. Specific primers were designed and used in the PCR to amplify the 16S ribosomal DNA (rDNA) of Thiomicrospira spp. and thus detect the presence of these bacteria in environmental samples and enrichment cultures. By using this genus-specific PCR, we were able to amplify 722-bp-long 16S rDNA fragments from different saltwater habitats as well as from a freshwater ecosystem. Furthermore, we were able to isolate most of these bacteria in pure culture by using enrichment cultures for chemolithoautotrophic sulfur-oxidizing bacteria. With denaturing gradient gel electrophoresis (DGGE) of PCR-amplified 16S rDNA fragments followed by hybridization analysis with one of the primers as a genus-specific probe, it was possible to monitor the success of isolation. The combined approach resulted in the isolation of several chemolithoautotrophic bacteria from different habitats: e.g., a coastal sediment along the coast of Chile, a microbial mat of the hypersaline pond Solar Lake (Sinai, Egypt), and the saline spring Artern (Thuringia, Germany). In addition, four different isolates were obtained from sediment and water samples taken at Jadebusen, which is part of the German Waddensea. Comparative analysis of the nearly complete 16S rRNA sequences of these isolates indicated several new species, all grouping with the Thiomicrospira species of the gamma subdivision of the class Proteobacteria. A freshwater Thiomicrospira species could not be isolated, but sequence analysis of the PCR product obtained after amplification of the environmental DNA with the Thiomicrospira-specific primers revealed its phylogenetic affiliation. The study indicates an increased species diversity of Thiomicrospira and the ubiquity of this sulfur-oxidizing bacterium in habitats with reduced sulfur compounds.

Development and application of 16S rRNA-targeted probes for detection of iron- and manganese-oxidizing sheathed bacteria in environmental samples

Comparative sequence analysis of the 16S rRNA genes from several Leptothrix and Sphaerotilus strains led to the design of an oligonucleotide probe (PS-1) based on a sequence within the hypervariable region 1 specific for four Leptothrix strains and for one of the four Sphaerotilus natans strains examined. Another probe (PSP-6) was based on a sequence within the hypervariable region 2. PSP-6 was specific for one of the two evolutionary lineages previously described for Leptothrix spp. (P. L. Siering and W. C. Ghiorse, Int. J. Syst. Bacteriol. 46:173-182, 1996). Fluorescein-labeled oligonucleotide probes were synthesized, and their specificity for fluorescence in situ hybridization identification was confirmed by a laser scanning microscopy technique (W. C. Ghiorse, D. N. Miller, R. L. Sandoli, and P. L. Siering, Microsc. Res. Tech. 33:73-86, 1996) to compare whole-cell hybridizations of closely related bacteria. Probe specificity was also tested in dot blot against total RNA isolated from four Leptothrix strains, four Sphaerotilus strains, and 15 other members of the class Proteobacteria. When the probes were tested on samples from the Sapsucker Woods wetland habitat where Leptothrix spp. are thought to play a role in manganese and iron oxidation, positive signals were obtained from several sheathed filamentous bacteria including some that were morphologically similar to previously isolated strains of "Leptothrix discophora." Other unknown filamentous sheathed bacteria also gave strong positive signals. This work provides a foundation for future studies correlating the presence of members of the Leptothrix-Sphaerotilus group of sheathed bacteria with manganese and iron oxidation activity in habitats where biological iron and manganese oxidation are important environmental processes.

Lux gene technology – a strategy to optimize biological control of soil‐borne diseases

SUMMARY Lux gene technology is based on the transformation of environmental bacteria with genes of the lux operon from the marine bacteria Vibrio fischeri and V. hurveyi. Transformed strains are bioluminescent and thus provide a rapid and very accurate tool for the study of population dynamics, metabolic activity and spatial distribution of specific bacteria in environmental samples. This review summarizes the recent progress in using lux‐marked rhizobacteria to optimize biological control methods for soil‐borne diseases. Special emphasis has been placed on the advantages and limitations of the new technology.

Microscopic detection of Giardia trophozoites in archival pathology specimens of human small intestine: light microscopy, low voltge SEM, and confocal scanning laser microscopy using fluorescent in situ hybridization

In situ hybridization methods have been widely used for the analysis of chromosomal markers, and for the detection and identification of various species of bacteria in environmental samples. In situ hybridization probes to ribosomal RNA have been used as a diagnostic tool for the study of various parasitic protozoa, and fluorescent in situ hybridization (FISH) probes have been used to study the intracellular distribution of mRNA.The intestinal protozoan, Giardia, colonizes the small intestine of a variety of different animals, including humans, and forms an infective cyst stage which is responsible for host-to-host transmission. Light microscopic detection in environmental or fecal samples has relied on the detection of cysts whose presence may be infrequent. In human giardiasis, confirmation of diagnosis has often required microscopic analysis of intestinal biopsies for detection of trophozoites. Analysis of the molecular sequence of 16s rDNA (small subunit) in Giardia isolates has permitted the development of species specific probes for Giardia.

Differential Effects of Growth Temperature on Ice Nuclei Active at Different Temperatures That Are Produced by Cells of Pseudomonas syringae

The temperature at which ice-nucleating bacteria are grown causes differences of 100- to 10,000-fold in the fraction of cells that nucleate ice at a given temperature (ice nucleation frequency). Ice nucleation frequencies of cells of Pseudomonas syringae grown at temperatures that ranged from 9 to 33°C were examined in order to more accurately characterize physiological effects on ice nuclei active at temperatures of from about -2 to -10°C, the temperature range for this phenotype. Large differences in ice nucleation frequency occurred at all but the lowest assay temperatures in cells of P. syringae grown in the temperature range of 15 to 33°C. These differences in ice nucleation frequency may be attributed, at least in part, to post-translational factors. Because other studies have indicated that ice nuclei active at the lowest assay temperatures may reflect the amount of ice nucleation protein produced, while higher nucleation temperatures reflect aggregates of this ice nucleation protein, data was normalized to the frequency of ice nuclei active at the lowest ice nucleation temperatures (which also correspond to the most abundant nuclei). This was done in order to develop a baseline of comparison for cells grown at different temperatures that more clearly shows possible posttranslational effects such as aggregation of the nucleation protein. After this normalization was performed, and in contrast to the results noted above, the number of ice nuclei in cells grown at 9, 15, and 20°C that were active at different assay temperatures was very similar. Differences in ice nucleation frequency that occurred over all assay temperatures in cells grown between 9 and 20°C may be attributed to differences in the total number of nuclei present in the population of cells. The large effects of growth temperature on nucleation frequency have important implications for estimating numbers of ice nucleating bacteria in environmental samples by determining the number of bacterial ice nuclei in such samples.

Detection of methanotrophic bacteria in environmental samples with the PCR.

We designed PCR primers by using the DNA sequences of the soluble methane monooxygenase gene clusters of Methylosinus trichosporium OB3b and Methylococcus capsulatus (Bath), and these primers were found to be specific for four of the five structural genes in the soluble methane monooxygenase gene clusters of several methanotrophs. We also designed primers for the gram-negative methylotroph-specific methanol dehydrogenase gene moxF. The specificity of these primers was confirmed by hybridizing and sequencing the PCR products obtained. The primers were then used to amplify methanotroph DNAs in samples obtained from various aquatic and terrestrial environments. Our sequencing data suggest that a large number of different methanotrophs are present in peat samples and also that there is a high level of variability in the mmoC gene, which codes for the reductase component of the soluble methane monooxygenase, while the mmoX gene, which codes for the alpha subunit of the hydroxylase component of this enzyme complex, appears to be highly conserved in methanotrophs.

Use of fluorochromes for direct enumeration of total bacteria in environmental samples: past and present.

Understanding the role of bacteria in microbial food webs is intimately connected to the methods applied in the direct enumeration of bacteria. We have examined over 220 papers describing studies in which fluorochrome staining followed by epifluorescent microscopic direct counts was used to estimate total bacterial abundances. In this review, we summarize patterns in the use of 3,6-bis[dimethylamino]acridinium chloride (acridine orange) and 4',6-diamidino-2-phenylindole (DAPI), the two stains most frequently used in bacterial enumeration. The staining of samples with these fluorochromes, followed by filtration and direct counting of bacterial cells on filter surfaces, has become routine over the past 10 years. We examine trends in features of the standard direct count methods, such as sample preservation and preparation techniques, membrane filter types used, applied stain concentrations, duration of staining, and counting strategies, in relation to the types of samples being examined. The high variability in bacterial counts observed within similar sample types may be partially accounted for by differences in methods. Synthesizing review findings, we include a recommended method for the direct enumeration of bacteria in environmental samples.

How to monitor released rhizobia

Existing methods for detection and identification of rhizobia are reviewed. Some perspectives for development of new and more effective techniques for monitoring of rhizobia in soil and in inoculants are presented. The advantages of the recently developed approach — PCR-genome fingerprinting, by use of arbitrary and repetitive primers, for precise bacterial identification are described. The possible application of this technique for developing taxon-specific rhizobial probes for direct detection of these bacteria in environmental samples is discussed.

The use of fluorogenic esters to detect viable bacteria by flow cytometry

The ability of flow cytometry (FCM) to detect viable bacteria after staining with a range of fluorogenic esters was investigated with several bacterial species. The dyes studied were the fluorescein diacetate (FDA) derivatives carboxyfluorescein diacetate, 2′,7′‐bis‐(2‐carboxyethyl)‐5(6)‐carboxyfluorescein acetoxymethyl ester and calcein acetoxymethyl ester, as well as ChemChrome B, a commercially‐available stain for the detection of viable bacteria in suspension. No one dye was found to be universal but ChemChrome B dye stained the widest number of Gram‐positive and Gram‐negative species, whereas the FDA derivatives preferentially stained Gram‐positive bacteria. The use of ChemChrome B to detect viable bacteria in environmental samples was investigated further by studying the survival of Klebsiella pneumoniae in lakewater. During survival studies, a higher number of viable bacteria were detected both by direct viable counts and FCM after staining with rhodamine 123 and ChemChrome B than by colony‐forming units, suggesting the presence of viable but nonculturable cells. These results demonstrate the potential use of FCM to enumerate viable bacteria in natural waters.

Use of fluorochromes for direct enumeration of total bacteria in environmental samples: past and present.

Understanding the role of bacteria in microbial food webs is intimately connected to the methods applied in the direct enumeration of bacteria. We have examined over 220 papers describing studies in which fluorochrome staining followed by epifluorescent microscopic direct counts was used to estimate total bacterial abundances. In this review, we summarize patterns in the use of 3,6-bis[dimethylamino]acridinium chloride (acridine orange) and 4',6-diamidino-2-phenylindole (DAPI), the two stains most frequently used in bacterial enumeration. The staining of samples with these fluorochromes, followed by filtration and direct counting of bacterial cells on filter surfaces, has become routine over the past 10 years. We examine trends in features of the standard direct count methods, such as sample preservation and preparation techniques, membrane filter types used, applied stain concentrations, duration of staining, and counting strategies, in relation to the types of samples being examined. The high variability in bacterial counts observed within similar sample types may be partially accounted for by differences in methods. Synthesizing review findings, we include a recommended method for the direct enumeration of bacteria in environmental samples.

Survival of Staphylococcus aureus in lakewater monitored by flow cytometry.

The ability of flow cytometry to detect and enumerate viable bacteria during survival in a lakewater microcosm was assessed using Staphylococcus aureus as a model organism. Counts of colony-forming units (c.f.u.) on nutrient agar were not significantly different from those obtained by flow cytometric detection of rhodamine 123 stained bacteria and there was no evidence for a viable but nonculturable state using these methods. However c.f.u. were significantly lower when estimated using mannitol salts agar compared with nutrient agar. S. aureus was also enumerated immunofluorescently after staining with FITC-IgG. There was no significant difference between the population estimated immunofluorescently and by acridine orange direct counting, and unlike estimations of viability, only slight reductions in total cell numbers were observed. Changes in the protein and nucleic acid content of S. aureus during survival were also measured by flow cytometry to investigate any potential heterogeneity arising within the starved population. Flow cytometric determinations were found to correlate significantly with their respective chemical determinations. These results demonstrate the ability of flow cytometry to detect viable bacteria during starvation and to study changes in macromolecular content. They also illustrate the importance of using appropriate methods for the detection of viable bacteria in environmental samples.

Detection of methylotrophic bacteria in natural samples by molecular probing techniques

To allow a full understanding of the global methane cycle, the contribution made by microbes as a sink for methane must be investigated. Many methane-oxidizing bacteria present in environmental samples are not easily cultured by using normal microbiological techniques, leading to an underestimate of the numbers of such bacteria in the marine and terrestrial environment. We have used DNA sequence information from cloned genes coding for the methane-oxidizing system in bacteria to develop highly specific methane-gene probes in order to detect methane oxidizing bacteria in environmental samples without the need for laboratory cultivation of these organisms. Considerable enhancement of detection by these probes has been achieved using the polymerase chain reaction technique.

Use of a fluorescent redox probe for direct visualization of actively respiring bacteria.

The redox dye 5-cyano-2,3-ditolyl tetrazolium chloride (CTC) was employed for direct epifluorescent microscopic enumeration of respiring bacteria in environmental samples. Oxidized CTC is nearly colorless and is nonfluorescent; however, the compound is readily reduced via electron transport activity to fluorescent, insoluble CTC-formazan, which accumulates intracellularly. Bacteria containing CTC-formazan were visualized by epifluorescence microscopy in wet-mount preparations, on polycarbonate membrane filter surfaces, or in biofilms associated with optically opaque surfaces. Counterstaining of CTC-treated samples with the DNA-specific fluorochrome 4',6-diamidino-2-phenylindole allowed enumeration of active and total bacterial subpopulations within the same preparation. Municipal wastewater, groundwater, and seawater samples supplied with exogenous nutrients yielded CTC counts that were generally lower than total 4',6-diamidino-2-phenylindole counts but typically equal to or greater than standard heterotrophic (aerobic) plate counts. In unsupplemented water samples, CTC counts were typically lower than those obtained with the heterotrophic plate count method. Reduction of CTC by planktonic or biofilm-associated bacteria was suppressed by formaldehyde, presumably because of inhibition of electron transport activity and other metabolic processes. Because of their bright red fluorescence (emission maximum, 602 nm), actively respiring bacteria were readily distinguishable from abiotic particles and other background substances, which typically fluoresced at shorter wavelengths. The use of CTC greatly facilitated microscopic detection and enumeration of metabolically active (i.e., respiring) bacteria in environmental samples.

Reverse Sample Genome Probing, a New Technique for Identification of Bacteria in Environmental Samples by DNA Hybridization, and Its Application to the Identification of Sulfate-Reducing Bacteria in Oil Field Samples

A novel method for the identification of bacteria in environmental samples by DNA hybridization is presented. It is based on the fact that, even within a genus, the genomes of different bacteria may have little overall sequence homology. This allows the use of the labeled genomic DNA of a given bacterium (referred to as a "standard") to probe for its presence and that of bacteria with highly homologous genomes in total DNA obtained from an environmental sample. Alternatively, total DNA extracted from the sample can be labeled and used to probe filters on which denatured chromosomal DNA from relevant bacterial standards has been spotted. The latter technique is referred to as reverse sample genome probing, since it is the reverse of the usual practice of deriving probes from reference bacteria for analyzing a DNA sample. Reverse sample genome probing allows identification of bacteria in a sample in a single step once a master filter with suitable standards has been developed. Application of reverse sample genome probing to the identification of sulfate-reducing bacteria in 31 samples obtained primarily from oil fields in the province of Alberta has indicated that there are at least 20 genotypically different sulfate-reducing bacteria in these samples.

DNA amplification to enhance detection of genetically engineered bacteria in environmental samples.

The polymerase chain reaction (PCR) was performed to amplify a 1.0-kilobase (kb) probe-specific region of DNA from the herbicide-degrading bacterium Pseudomonas cepacia AC1100 in order to increase the sensitivity of detecting the organism by dot-blot analysis. The 1.0-kb region was an integral portion of a larger 1.3-kb repeat sequence which is present as 15 to 20 copies on the P. cepacia AC1100 genome. PCR was performed by melting the target DNA, annealing 24-base oligonucleotide primers to unique sequences flanking the 1.0-kb region, and performing extension reactions with DNA polymerase. After extension, the DNA was again melted, and the procedure was repeated for a total of 25 to 30 cycles. After amplification the reaction mixture was transferred to nylon filters and hybridized against radiolabeled 1.0-kb fragment probe DNA. Amplified target DNA was detectable in samples initially containing as little as 0.3 pg of target. The addition of 20 micrograms of nonspecific DNA isolated from sediment samples did not hinder amplification or detection of the target DNA. The detection of 0.3 pg of target DNA was at least a 10(3)-fold increase in the sensitivity of detecting gene sequences compared with dot-blot analysis of nonamplified samples. PCR performed after bacterial DNA was isolated from sediment samples permitted the detection of as few as 100 cells of P. cepacia AC1100 per 100 g of sediment sample against a background of 10(11) diverse nontarget organisms; that is, P. cepacia AC1100 was positively detected at a concentration of 1 cell per g of sediment. This represented a 10(3)-fold increase in sensitivity compared with nonamplified samples.

Biomass measurement of methane forming bacteria in environmental samples

Methane-forming bacteria contain unusual phytanylglycerol ether phospholipids which can be extracted from the bacteria in sediments and assayed quantitatively by high performance liquid chromatography (HPLC). In this procedure the lipids were extracted, the phospholipids recovered, hydrolyzed, purified by thin layer chromatography, derivatized and assayed by HPLC. Ether lipids were recovered quantitatively from Methanobacterium thermoautotrophicum and sediments at levels as low as 8 × 10 −14 moles. In freshwater and marine sediments the flux of methane to the atmosphere and the methane levels in the pore water reflects the recovery of the phytanyl glycerol ether lipid ‘signature’. The proportion of the ether phospholipid to the total recoverable phospholipid was highest in anaerobic digester sewage sludge and deeper subsurface freshwater sediment horizons.