Improving qPCR methodology for detection of foaming bacteria by analysis of broad-spectrum primers and a highly specific probe for quantification of Nocardia spp. in activated sludge.

AIMS: To develop qPCR broad-spectrum primers combined with a Nocardia genus-specific probe for the identification of a broad spectrum of Nocardia spp. and to analyse the effects of using this developed primer and probe set on the ability to quantify Nocardia spp. in mixed DNA. METHODS AND RESULTS: The consequences of using a degenerative primer set and species-specific probe for the genus Nocardia on qPCR assays were examined using DNA extracts of pure cultures and activated sludge. The mixed DNA extracts where the target organism Nocardia flavorosea concentration ranged from 5 × 102 to 5 × 106 copies per reaction, while the background organism's DNA (Mycobacterium bovis) concentration was held at 5 × 106 copies per reaction, only produced comparable cycle threshold florescence levels when N. flavorosea concentration was greater than or equal to the background organism concentration. When concentrations of N. flavorosea were lowered in increments of 1 log, while holding M. bovis concentrations constant at 5 × 106 copies per reaction, all assays demonstrated delayed cycle threshold values with a maximum 34·6-fold decrease in cycle threshold at a ratio of 106 M. bovis: 102 N. flavorosea copies per reaction. CONCLUSIONS: The data presented in this study indicated that increasing the ability of a primer set to capture a broad group of organisms can affect the accuracy of quantification even when a highly specific probe is used. This study examined several applications of molecular tools in complex communities such as evaluating the effect of mispriming vs interference. It also elucidates the importance of understanding the community genetic make-up on primer design. SIGNIFICANCE AND IMPACT OF THE STUDY: Degenerative primers are very useful in amplifying bacterial DNA across genera, but reduce the efficiency of qPCR reactions. Therefore, standards that address closely related background species must be used to obtain accurate qPCR results.

A biomarker for the identification of swine fecal pollution in water, using the STII toxin gene from enterotoxigenic Escherichia coli.

This research developed a PCR method to identify swine fecal pollution in water, using a portion of the STII toxin gene from enterotoxigenic Escherichia coli as the target sequence. This method showed the gene to have a wide-spread geographical distribution and temporal stability; and the primers demonstrated high specificity, sensitivity, and reliability. A total of 110 DNA extracts from different animal fecal and human sewage samples were screened using the primers and no positives resulted. Centrifugation and filtration methods for concentrating E. coli seeded into stream, ocean, secondary effluent, and dairy lagoon waters resulted in detection limits at the femtogram and attogram levels. E. coli with the biomarker seeded into stream, ocean, and secondary effluent waters remained stable for approximately 2 weeks for all water types. Of the farm lagoon and waste samples tested, 94% were positive for the STII trait, regardless of the number of E. coli screened and 100% were positive when > or =35 E. coli isolates were screened. As the PCR product of the target sequence yielded a single band, the method is applicable to dot blot detection methodology, yielding great accuracy in determining the presence of swine fecal sources.

A biomarker for the identification of cattle fecal pollution in water using the LTIIa toxin gene from enterotoxigenic Escherichia coli.

This research describes a method based on PCR to identify cattle fecal pollution in water using a portion of the heat labile toxin IIA (LTIIa) gene from enterotoxigenic Escherichia coli (ETEC). We describe the development of the primers and target. DNA extracts (221) from different animal fecal and human sewage samples were screened and showed no cross-reactivity. Minimum detection limits using centrifugation and filtration methods to concentrate E. coli seeded into stream, ocean, and secondary effluent waters were found to be at femtogram and attogram levels, respectively. Stability of the biomarker in stream, ocean, and secondary effluent waters was 2-4 weeks for all water types. Finally, 33 farm lagoon and waste samples were collected and 31 tested to validate the method; 93% were positive for the LTIIa trait when >1,000 E. coli were screened and 100% positive when >10(5) E. coli were screened. Prevalence of the toxin gene in the E. coli population affected the outcome of the analyses. The cow biomarker can be used in watershed studies to identify cattle waste with great accuracy if the appropriate numbers of E. coli are screened.

Long-term effects of consuming foods containing psyllium seed husk on serum lipids in subjects with hypercholesterolemia.

The effects of consuming foods containing 0 (control), 3.4, 6.8, or 10.2 g psyllium seed husk (PSH)/d for 24 wk on the serum lipid profile were assessed in this randomized, double-blind controlled study. Men and women (n = 286) with LDL-cholesterol concentrations between 3.36 and 5.68 mmol/L (130 and 220 mg/dL) were randomly assigned to one of four treatment groups after following a low-fat diet for > or = 8 wk. At week 24, LDL cholesterol was 3% above baseline in the control group. In the group consuming 10.2 g PSH/d, LDL cholesterol remained below baseline during treatment, with a value 5.3% below that of the control group at week 24 (P < 0.05 compared with the control group). No significant differences were observed in HDL cholesterol or triacylglycerol. Although modest, the effect of 10.2 g PSH/d on LDL cholesterol (relative to the control) persisted throughout the 24-wk treatment period, indicating potential for long-term benefit.

Psyllium-enriched cereals lower blood total cholesterol and LDL cholesterol, but not HDL cholesterol, in hypercholesterolemic adults: results of a meta-analysis.

We conducted a meta-analysis to determine the effect of consumption of psyllium-enriched cereal products on blood total cholesterol (TC), LDL cholesterol (LDL-C) and HDL cholesterol (HDL-C) levels and to estimate the magnitude of the effect among 404 adults with mild to moderate hypercholesterolemia (TC of 5.17-7.8 mmol/L) who consumed a low fat diet. Studies of psyllium cereals were identified by a computerized search of MEDLINE and Current Contents and by contacting United States-based food companies involved in psyllium research. Published and unpublished studies were reviewed by one author and considered eligible for inclusion in the meta-analysis if they were conducted in humans, were randomized, controlled experiments, and included a control group that ate cereal providing /=50 y) on blood lipids. The meta-analysis showed that subjects who consumed a psyllium cereal had lower TC and LDL-C concentrations [differences of 0.31 mmol/L (5%) and 0.35 mmol/L (9%), respectively] than subjects who ate a control cereal; HDL-C concentrations were unaffected in subjects eating psyllium cereal. There was no effect of sex, age or menopausal status on blood lipids. Results indicate that consuming a psyllium-enriched cereal as part of a low fat diet improves the blood lipid profile of hypercholesterolemic adults over that which can be achieved with a low fat diet alone.

Biorthogonally accessed three-dimensional two-photon memory for relational database operations.

Memory bandwidth is a bottleneck for very large database machines. Parallel-access three-dimensional two-photon memories have the potential of achieving enormous throughput (>100 Gbit/s) and capacity (1 Tbit/cm(3)) [Appl. Opt. 29, 2058 (1990)] and, consequently, are well suited for this application. Our analysis shows that some operations can be completed more than 2 orders of magnitude faster with this type of memory than with a system based on serial-access storage. These particular memories have a further feature of being accessible in orthogonal directions. We show that this property, used in conjunction with a three-dimensional data-organization scheme designed for this approach, leads to improved performance by permitting the user a choice of accessing strategies for a given operation.

A food industry perspective on folic acid fortification.

The U.S. Public Health Service (PHS) has recommended that all women of childbearing years, capable of becoming pregnant, consume 400 micrograms folic acid/d to reduce their risk of having a neural tube defect (NTD)-affected pregnancy. The U.S. Food and Drug Administration subsequently proposed a folate fortification scheme for cereal grains, which also allowed the continued fortification of breakfast cereals at 0.1 mg per serving. To determine the contribution of ready-to-eat breakfast cereals (RTEC) to folate intakes in women of childbearing years, data were analyzed from the U.S. Department of Agriculture's 1989-1991 Continuing Survey of Food Intakes by Individuals and 1987-1988 Nationwide Food Consumption Survey. Women consuming RTEC have higher intakes of folate than women reporting no RTEC consumption. Recent reports indicate that most women are unaware of the PHS recommendation to consume more folate, and many health professionals are not advising women of the need to consume adequate folate during childbearing years. The food industry has been an effective communicator of health and nutrition messages and should be encouraged to raise awareness about the role of folate in NTDs. Better analysis also needs to be conducted to identify women at risk of low folate intakes, so that targeted education efforts can be made and appropriate vehicles identified for delivering folate to these women.

Modification of reagents in the EnviroAmp kit to increase recovery of Legionella organisms in water.

Organisms of the bacterial genus Legionella, commonly found in aqueous reservoirs, have been associated with Legionnaires' disease (legionella pneumonia, caused by Legionella pneumophila) and Pontiac fever (nonpneumonic legionellosis). EnviroAmp Legionella sample preparation, polymerase chain reaction amplification, and detection kits (Perkin-Elmer Corp.) were developed for rapid detection of DNA from organisms of the genus Legionella and the species L. pneumophila from environmental water samples. The kits are based on molecular techniques incorporating polymerase chain reaction amplification and detection by reverse dot blot hybridization to particular genus and species probes. The manufacturer states that the EnviroAmp Legionella sample preparation, polymerase chain reaction amplification, and detection kits can detect approximately 100 Legionella organisms/mL (10,000 organisms/100 mL) in the original water sample. The sensitivity of the kits was increased to 0.1 colony-forming units/mL (10 colony-forming units/100 mL), at least for cultured organisms, by modifying the EnviroAmp Legionella sample preparation kit protocol. Data obtained in this study indicated that sample volume could be increased from 100 to 1000 mL (in the absence of interfering substances such as humic acid) and DNA extraction volume could be decreased from 2 to 0.5 mL to increase the ability of the kit to detect lower numbers of Legionella spp. or L. pneumophila per volume.

Partial response precoding for parallel-readout optical memories.

The data density that can be resolved in optical memories is limited by the inherent band-limited nature of optical systems. We show how a precoding technique used in serial communications, called partial response precoding, can be applied to parallel readout optical memories to precompensate for the spatial data broadening that occurs as result of this band limiting. We experimentally demonstrate a factor-of-15 improvement in average worst-case contrast ratio and an order-of-magnitude improvement in average contrast ratio. Over 50% more area was needed to achieve the same contrast ratio in a system without precoding.

Distribution of uidA gene sequences in Escherichia coli isolates in water sources and comparison with the expression of beta-glucuronidase activity in 4-methylumbelliferyl-beta-D-glucuronide media.

The uidA gene, which encodes the beta-glucuronidase enzyme, was detected in 97.7% of 435 Escherichia coli isolates from treated and raw water sources by DNA-DNA hybridization; 92.4% of the strains expressed the translational product in 4-methylumbelliferyl-beta-D-glucuronide-containing media after reinoculation. Upon initial isolation from water samples, the minimal medium o-nitrophenyl-beta-D-galactopyranoside-4-methylum-belliferyl -beta-D-glucuronide preparations failed to detect more than 50% of the E. coli isolates that possessed uidA gene. Treated water gave the lowest recovery, with Colilert producing 26% positive samples and Coliquik producing 48% positive samples. There appears to be no relationship between the intensity of the autoradiographic signals of the uidA gene and the expression of beta-glucuronidase activity. Therefore, another variable such as physiological condition of the bacteria could be responsible for the nonexpression of the enzyme activity.

Physiological studies of chloramine resistance developed by Klebsiella pneumoniae under low-nutrient growth conditions.

This study investigated the physiological mechanisms of resistance to chloramines developed by Klebsiella pneumoniae grown in a nutrient-limited environment. Growth under these conditions resulted in cells that were smaller than cells grown under high-nutrient conditions and extensively aggregated. Cellular aggregates ranged from 10 to more than 10,000 cells per aggregate, with a mean population aggregate size of 90 cells. This aggregation may have been facilitated by the presence of extracellular polymer material. By using glucose as a reference of capsule content, it was determined that growth under low-nutrient conditions produced cells with 8 x 10(-14) to 41 x 10(-14) g of carbohydrate per cell, with a mean +/- standard deviation of 27 x 10(-14) +/- 16 x 10(-14) g of carbohydrate per cell. In comparison, growth under high-nutrient conditions resulted in 2.7 x 10(-14) to 5.9 x 10(-14) g of carbohydrate per cell, with a mean and standard deviation of 4.3 x 10(-14) +/- 1.2 x 10(-14) g of carbohydrate per cell. Cell wall and cell membrane lipids also varied with growth conditions. The ratio of saturated to unsaturated fatty acids in cells grown under low-nutrient conditions was approximately five times greater than that in cells grown under high-nutrient conditions, suggesting possible differences in membrane permeability. An analysis of sulfhydryl (-SH) groups revealed no quantitative difference with respect to growth conditions. However, upon exposure to chloramines, only 33% of the -SH groups of cells grown under low-nutrient conditions were oxidized, compared with 80% oxidization of -SH groups in cells grown under high-nutrient conditions. The reduced effectiveness of chloramine oxidization of -SH groups in cells grown under low-nutrient conditions may be due to restricted penetration of chloramines into the cells, conformational changes of enzymes, or a combination of both factors. The results of this study suggest that chloramine resistance developed under low-nutrient growth conditions may be a function of multiple physiological factors, including cellular aggregation and protection of sulfhydryl groups within the cell.

Detection of virulence factors in culturable Escherichia coli isolates from water samples by DNA probes and recovery of toxin-bearing strains in minimal o-nitrophenol-beta-D-galactopyranoside-4-methylumbelliferyl-beta-D-g luc uronide media.

A total of 449 Escherichia coli isolates in treated and raw water sources were submitted to DNA-DNA hybridization using seven different DNA probes to detect homology to sequences that code for Shiga-like toxins I and II; heat-stabile and heat-labile toxins, adherence factors EAF and eae, and the fimbrial antigen of entero-hemorrhagic E. coli. Fifty-nine (13%) of the isolates demonstrated homology with one or more specific DNA probes. More than 50% of the isolates in treated water were not recovered in MMO-4-methylumbelliferyl-beta-D-glucuronide media designed for detection of this indicator.

Impact of growth conditions on resistance of Klebsiella pneumoniae to chloramines.

The resistance of Klebsiella pneumoniae to inorganic monochloramine (1.5 mg/liter; 3:1 Cl2:N ratio, pH 8.0) was examined in relation to growth phase, temperature of growth, and growth under decreased nutrient conditions. Growth phase did not impact resistance to chloramines. Mid-exponential and stationary-phase cells, grown in a yeast extract-based medium, had CT99 values and standard deviations of 4.8 +/- 0.1 and 4.6 +/- 0.2 mg.min/liter, respectively. Growth temperature did not alter chloramine resistance at short contact times. CT99 values of cells grown at 15 and 23 degrees C were 4.5 +/- 0.2 and 4.6 +/- 0.2 mg.min/liter, respectively. However, at longer contact times, CT99.99 values of cells grown at 15 and 23 degrees C were 14 and 8 mg.min/liter, respectively, suggesting a small resistant subpopulation for cells grown at the lower temperature. Growth under decreased nutrient conditions resulted in a concomitant increase in resistance to chloramines. When K. pneumoniae was grown in undiluted Ristroph medium and Ristroph medium diluted by 1:100 and 1:1,000, the CT99 values were 4.6 +/- 0.2, 9.6 +/- 0.4, and 24 +/- 7.0 mg.min/liter, respectively. These results indicate that nutrient availability has a greater impact than growth phase or growth temperature in promoting the resistance of K. pneumoniae to inorganic monochloramine.

Rapid method for separation of bacterial DNA from humic substances in sediments for polymerase chain reaction.

The polymerase chain reaction (PCR) was used to amplify an Escherichia coli 16S ribosomal gene fragment from sediments with high contents of humic substances. Total DNA was extracted from 1 g of E. coli seeded or unseeded samples by a rapid freeze-and-thaw method. Several approaches (use of Bio-Gel P-6 and P-30 and Sephadex G-50 and G-200 columns, as well as use of the Stoffel fragment) were used to reduce interference with the PCR. The best results were obtained when crude DNA extracts containing humic substances were purified by using Sephadex G-200 spun columns saturated with Tris-EDTA buffer (pH 8.0). Eluted fractions were collected for PCR analyses. The amplified DNA fragment was obtained from seeded sediments containing fewer than 70 E. coli cells per g. Because only 1/100 of the eluted fractions containing DNA extracts from 70 cells per g was used for the PCR, the sensitivity of detection was determined to be less than 1 E. coli cell. Thus, DNA direct extraction coupled with this technique to remove interference by humic substances and followed by the PCR can be a powerful tool to detect low numbers of bacterial cells in environmental samples containing humic substances.

Detection of low numbers of bacterial cells in soils and sediments by polymerase chain reaction.

Polymerase chain reaction was used to amplify the low copy number of two 16S ribosomal gene fragments from soil and sediment extracts. Total DNA for polymerase chain reaction was extracted from 1 g of seeded or unseeded samples by a rapid freeze-and-thaw method. Amplified DNA fragments can be detected in DNA fractions isolated from seeded soil containing less than 3 Escherichia coli cells and from seeded sediments containing less than 10 cells. This research demonstrated that coupling polymerase chain reaction to direct DNA extraction improves sensitivity by 1 and 2 orders of magnitude for sediments and soils, respectively. This technique could become a powerful tool for genetic ecology studies.

A simple technique for electroelution of DNA from environmental samples.

A novel method was developed for the extraction of DNA from environmental material. Soil or sediment samples were encapsulated in agarose blocks and, following treatment with lysis reagents, the DNA was extracted by electroelution.

Effect of 2-hydroxybenzoate on the maintenance of naphthalene-degrading pseudomonads in seeded and unseeded soil.

The addition of specific nontoxic inducers of catabolic operons to contaminated sites is an approach that may enhance the efficiency of in situ biodegradation. We determined the genetic response of six pseudomonads to salicylate (also known as 2-hydroxybenzoate) added directly to 50 g of nonsterile soil samples. The strains, isolated from a polyaromatic hydrocarbon-contaminated soil, metabolized naphthalene as the sole source of available carbon, and their DNA sequences show significant homology to the nahAB genes of the degradative plasmid NAH7. Duplicate nonsterile soil cultures were incubated for up to 30 days. Experimental soil cultures were seeded with naphthalene-degrading strains (10(8) CFU g-1) originally isolated from the soil and amended with salicylate (16 or 160 micrograms g-1). Soil samples were analyzed periodically for the population density of heterotrophic bacteria and naphthalene degraders and for the abundance of the naphthalene-degradative genotype in the bacterial community. At 160 micrograms g-1, salicylate sustained the density of naphthalene degraders at the introduced density for 30 days in addition to producing a two- to sixfold increase in the occurrence in the bacterial community of DNA sequences homologous to the nah operon. No change in recoverable bacterial population densities was observed when soil samples were amended with 16 micrograms of salicylate g-1, but this concentration of salicylate induced a significant increase in the level of nah-related genes in the population.

Comparative study of commercial 4-methylumbelliferyl-beta-D-glucuronide preparations with the Standard Methods membrane filtration fecal coliform test for the detection of Escherichia coli in water samples.

The performance capabilities of two commercial 4-methylumbelliferyl-beta-D-glucuronide preparations were evaluated for the detection of Escherichia coli from water samples. Eighty-three water samples were collected from a treated water reservoir, and 32 samples were collected from untreated surface water. There was a statistically significant difference between the two commercial preparations compared with the Standard Methods membrane filtration fecal coliform (MFC) method for the detection of E. coli from treated water samples. However, there was no difference between the two methods and the MFC test for E. coli detection from the untreated surface water samples. The disagreement between the two commercial products and the MFC method was primarily due to the occurrence of false-negative results with the two commercial products. The data indicate that the occurrence of false-negative samples could be attributed to impaired substrate specificity and sensitivity of the two tests for E. coli detection. There was no apparent relationship between the occurrence of false-negative results and heterotrophic plate counts in samples.

Total coliform detection in drinking water: comparison of membrane filtration with Colilert and Coliquik.

The Colilert (CL) and Coliquik (CQ) systems were compared in a presence-absence format against the Standard Methods membrane filtration (MF) technique to determine whether differences existed in total coliform detection. Approximately 750 water samples were collected from distribution systems, covered and uncovered storage reservoirs, well sites, and the influent to drinking water treatment plants. Samples were analyzed for total coliforms and heterotrophic bacteria with MF, CL, and CQ. The agreements between CL and MF and between CQ and MF were both greater than 94.8%, which indicates that both may be acceptable methods for total coliform detection. Disagreement between the CL and CQ methods was primarily due to false-negative results. Furthermore, laboratory and field inoculation methods were compared for CL, more than 98% agreement was obtained. This finding indicates that sampling and immediate field inoculation may be an alternative to the traditional laboratory inoculation.

Rapid method for direct extraction of DNA from soil and sediments.

A rapid method for the direct extraction of DNA from soil and sediments was developed. The indigenous microorganisms in the soil and sediments were lysed by using lysozyme and a freeze-thaw procedure. The lysate was extracted with sodium dodecyl sulfate and phenol-chloroform. In addition to a high recovery efficiency (greater than 90%), the yields of DNA were high (38 and 12 micrograms/g [wet weight] from sediments and soil, respectively). This method generated minimal shearing of the extracted DNA. The crude DNA could be further purified with an Elutip-d column if necessary. An additional advantage of this method is that only 1 g of sample is required, which allows for the analysis of small samples and the processing of many samples in a relatively short (7 h) period.