Bacterial community composition in Brazilian Anthrosols and adjacent soils characterized using culturing and molecular identification.

Microbial community composition was examined in two soil types, Anthrosols and adjacent soils, sampled from three locations in the Brazilian Amazon. The Anthrosols, also known as Amazonian dark earths, are highly fertile soils that are a legacy of pre-Columbian settlement. Both Anthrosols and adjacent soils are derived from the same parent material and subject to the same environmental conditions, including rainfall and temperature; however, the Anthrosols contain high levels of charcoal-like black carbon from which they derive their dark color. The Anthrosols typically have higher cation exchange capacity, higher pH, and higher phosphorus and calcium contents. We used culture media prepared from soil extracts to isolate bacteria unique to the two soil types and then sequenced their 16S rRNA genes to determine their phylogenetic placement. Higher numbers of culturable bacteria, by over two orders of magnitude at the deepest sampling depths, were counted in the Anthrosols. Sequences of bacteria isolated on soil extract media yielded five possible new bacterial families. Also, a higher number of families in the bacteria were represented by isolates from the deeper soil depths in the Anthrosols. Higher bacterial populations and a greater diversity of isolates were found in all of the Anthrosols, to a depth of up to 1 m, compared to adjacent soils located within 50-500 m of their associated Anthrosols. Compared to standard culture media, soil extract media revealed diverse soil microbial populations adapted to the unique biochemistry and physiological ecology of these Anthrosols.

Analysis of T-RFLP data using analysis of variance and ordination methods: a comparative study.

The analysis of T-RFLP data has developed considerably over the last decade, but there remains a lack of consensus about which statistical analyses offer the best means for finding trends in these data. In this study, we empirically tested and theoretically compared ten diverse T-RFLP datasets derived from soil microbial communities using the more common ordination methods in the literature: principal component analysis (PCA), nonmetric multidimensional scaling (NMS) with Sørensen, Jaccard and Euclidean distance measures, correspondence analysis (CA), detrended correspondence analysis (DCA) and a technique new to T-RFLP data analysis, the Additive Main Effects and Multiplicative Interaction (AMMI) model. Our objectives were i) to determine the distribution of variation in T-RFLP datasets using analysis of variance (ANOVA), ii) to determine the more robust and informative multivariate ordination methods for analyzing T-RFLP data, and iii) to compare the methods based on theoretical considerations. For the 10 datasets examined in this study, ANOVA revealed that the variation from Environment main effects was always small, variation from T-RFs main effects was large, and variation from T-RFxEnvironment (TxE) interactions was intermediate. Larger variation due to TxE indicated larger differences in microbial communities between environments/treatments and thus demonstrated the utility of ANOVA to provide an objective assessment of community dissimilarity. The comparison of statistical methods typically yielded similar empirical results. AMMI, T-RF-centered PCA, and DCA were the most robust methods in terms of producing ordinations that consistently reached a consensus with other methods. In datasets with high sample heterogeneity, NMS analyses with Sørensen and Jaccard distance were the most sensitive for recovery of complex gradients. The theoretical comparison showed that some methods hold distinct advantages for T-RFLP analysis, such as estimations of variation captured, realistic or minimal assumptions about the data, reduced weight placed on rare T-RFs, and uniqueness of solutions. Our results lead us to recommend that method selection be guided by T-RFLP dataset complexity and the outlined theoretical criteria. Finally, we recommend using binary or relativized peak height data with soil-based T-RFLP data for ordination-based exploratory microbial analyses.

Soil microbial community analysis using two-dimensional polyacrylamide gel electrophoresis of the bacterial ribosomal internal transcribed spacer regions.

Two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) of digested genomic DNA has been previously used in comparative genomics studies of closely related bacteria species. However, a two-dimensional gel electrophoresis approach for examining microbial community structures in environmental samples has not yet been developed. We determined that it is theoretically possible to separate internal transcribed spacer regions (ITS) of bacterial communities into hundreds of operational taxonomic units (OTUs) using 2D-PAGE. Application of 2D-PAGE for separating Bacterial ITS sequences that have been PCR-amplified from replicate soil samples taken from along a Zn gradient resulted in reproducible gels containing hundreds of spots. Clear differences in spot patterns were observed between soil samples that differed in both sampling location and Zn content. The number of OTUs detected using 2D-PAGE of ITS regions was much greater than that observed using Automated Ribosomal Internal Transcribed Spacer Analysis (ARISA), Terminal Restriction Fragment Length Polymorphism (T-RFLP), or Denaturing Gradient Gel Electrophoresis (DGGE). Principal Component Analysis (PCA) of community spot patterns resulted in similar groupings of samples as those obtained using other molecular methods, however, excised spots were found to contain a far lower diversity of different sequences than excised ITS bands of the same length, as determined by RFLP analysis of excision clone libraries and subsequent sequencing of DNA eluted from excised spots. This increase in resolution makes 2D-PAGE of Bacteria ITS fragments from complex microbial communities a viable method for detecting differences between highly similar communities, as well as in streamlining follow-on sequencing efforts by reducing the level of homoplasy (co-migration of heterogeneous sequences) often seen in band-based community fingerprinting methods.

Effect of Cry3Bb transgenic corn and tefluthrin on the soil microbial community: biomass, activity, and diversity.

Transgenic Bt corn expressing the Cry3Bb insecticidal protein active against corn rootworm (CRW) (Diabrotica spp.; Coleoptera: Chrysomelidae) was released for commercial use in 2003 and is expected to be widely adopted. Yet, the direct and indirect risks to soil microorganisms of growing this CRW-resistant Bt corn versus applying insecticides to control the rootworm have not been assessed under field conditions. The effects of CRW Bt corn and the insecticide tefluthrin [2,3,5,6-tetrafluoro-4-methylbenzyl (Z)-(1RS)-cis-3-(2-chloro-3,3,3-trifluoroprop-1-enyl)-2,2-dimethylcyclopropanecarboxylate] on soil microbial biomass, activity (N mineralization potential, short-term nitrification rate, and soil respiration), and bacterial community structure as determined by terminal restriction fragment length polymorphism (T-RFLP) analysis were assessed over two seasons in a field experiment. Bt corn had no deleterious effects on microbial activity or bacterial community measures compared with the non-transgenic isoline. The T-RFLP analysis indicated that amplifiable bacterial species composition and relative abundance differed substantially between years, but did not differ between rhizosphere and bulk soils. The application of tefluthrin also had no effect on any microbial measure except decreased soil respiration observed in tefluthrin-treated plots compared with Bt and non-transgenic isoline (NoBt) plots in 2002. Our results indicate that the release of CRW Bt corn poses little threat to the ecology of the soil microbial community based on parameters measured in this study.

A randomized trial of polyurethane and silicone percutaneous endoscopic gastrostomy catheters.

BACKGROUND: No data are available on differences in complication rate and long-term functioning between polyurethane and silicone percutaneous endoscopic gastrostomy (PEG) catheters. METHODS: We randomized patients who qualified for PEG placement to receive either a polyurethane or silicone PEG catheter. Patients were prospectively monitored for 28 days after placement for the occurrence of complications. Data on long-term PEG survival were obtained retrospectively from the Hospital and general practitioner's medical records. RESULTS: One hundred and six patients were randomized (polyurethane 50, silicone 56). During the first four weeks of follow-up, major complications occurred twice with both polyurethane and silicone PEGs (relative risk 1.1, 95% confidence interval: 0.11-11). Overall complications occurred four times with polyurethane and 17 times with silicone PEGs (relative risk 3.8, 95% confidence interval: 1.37-10.5). Long-term follow-up was available in 96 patients. Seven polyurethane PEGs and 10 silicone PEGs were removed because of PEG malfunctioning, the remainder functioned well until death or the reinstitution of oral feeding. The median complication-free survival was 916 days for the polyurethane PEG and 354 days for the silicone PEG (Log rank test: P=0.24). CONCLUSION: Polyurethane PEG catheters were associated with less short-term complications than silicone catheters, but major complications and long-term function were comparable.

The constipated stomach. An underdiagnosed problem in patients with abdominal pain?

The number of dyspeptic patients with upper abdominal pain that are referred for investigation is increasing and will undoubtedly continue to increase, because these days peptic ulcer disease is increasingly becoming a primary care management issue, the specialist being left to deal with the patients who cannot be helped by antibiotics and antisecretory drugs prescribed by their general practitioner. Many of these patients are referred for an upper endoscopy to rule out organic disease. Carefully taken history, however, shows that a great number of those dyspeptics, on the basis of their clinical manifestations, do have a functional gastrointestinal disorder, representing the 'irritable gut'. A probable better term reflecting the direct relation is the syndrome of 'the constipated stomach'. In our opinion these patients are an important and increasing clinical problem for general practitioners, gastroenterologists, surgeons and physicians. The aim of this article is to make the practitioner aware of advancements in understanding pathophysiologic and psychosocial processes, as well as to give an overview of the great overlap between many functional gastrointestinal disorders, the important role of history-taking and some insights into the functional rectal outlet syndrome.

Argon plasma coagulation in flexible gastrointestinal endoscopy: pilot experiences.

BACKGROUND AND STUDY AIMS: In argon plasma coagulation (APC), high-frequency energy is transmitted to tissue by ionized gas, thus reducing contact with the tissue to a minimum. Successful endoscopic APC was initially reported in the palliative treatment of gastrointestinal neoplasms. The main objectives in this pilot study were to evaluate the treatment indications, efficacy and safety of the use of APC. PATIENTS AND METHODS: Between September 1994 and January 1996, APC was used to treat 125 patients with various forms of gastrointestinal pathology. RESULTS: For local palliative treatment, APC was successfully used alongside snare loop coagulation, dilation, stenting and/or radiotherapy to treat the following conditions: carcinoma of the esophagus: 15 patients, mean number of treatment sessions (MTS) 3.3; gastric carcinoma: 10 patients, MTS 4.9; rectosigmoid carcinoma: seven patients, MTS 2.7; carcinoma of the papilla of Vater: two patients, MTS 1.5. Repeated treatment was also effective for tubulovillous adenoma of the rectum (20 patients, MTS 2.5), stomach (three patients, MTS 2.0), duodenum (two patients, MTS 1.5) and papilla of Vater (two patients, MTS 3.0). In addition, APC proved helpful in coagulating the remaining tissue and achieving hemostasis after polypectomy in the colon (18 patients, MTS 1.2) and in endoscopic treatment of Zenker's diverticulum, for coagulation of the tissue bridge and hemostasis (31 patients, MTS 2.5). Finally, APC was helpful in coagulation of multiple gastric polyps (one patient, one session), hemostasis in superficial ulceration of the duodenal bulb (one patient, one session), after dilation of benign stenoses of anastomoses in the esophagus (one patient, one session) and colon (one patient, one session) and for vascular malformations in the colon (three patients, MTS 1.3), duodenum (one patient, one session), antrum (one patient, two sessions), and watermelon stomach (six patients, MTS 2.8). We recognized signs of perforation in six patients after treatment of Zenker's diverticulum (n = 3), polypectomy in the colon (n = 2) and coagulation of angiodysplasia in the cecum. Laparotomy was carried out in two patients; in one, a perforation was sutured, and in the other no focus of leakage was seen. All six patients recovered without further complications. No complications were observed in any other patients. CONCLUSIONS: These initial experiences indicate that APC seems to be effective in a number of indications, and relatively safe. Objective evaluation, a longer follow-up period, and comparative trials with other treatment modalities should follow.

Hysterectomy: the anorectal pitfall. A guideline for evaluation.

Constipation following routine hysterectomy seems to occur more frequently than originally thought. Treatment depends on whether the patient is referred and to whom. Physical examination seems of limited value. Proper protocols for evaluation of complaints after hysterectomy are mandatory. Colonic transit studies and dynamic rectal examination could be useful. We found an overrepresentation of enteroceles in the hysterectomy group. Management of these abnormalities seems much more complicated than was previously thought. Prospective studies are needed to investigate anorectal disorders after hysterectomy.

No additional value of bismuth subcitrate to combination omeprazole/amoxicillin therapy in the eradication of Helicobacter pylori.

BACKGROUND: We investigated whether addition of bismuth subcitrate (BSC, 4 x 120 mg) to a two week therapy scheme of omeprazole (OME, 2 x 40 mg)/amoxicillin (AMO, 4 x 500 mg) increases Helicobacter pylori eradication. MATERIALS AND METHODS: Patients with dyspepsia underwent upper endoscopy. H. pylori positive patients were randomized to treatment A (OME/AMO, 83 patients) or treatment B (OME/AMO/BSC, 84 patients). RESULTS: In 65 patients of group A (78%) H. pylori was eradicated as determined from the histological assessment (Sydney classification) of antrum and corpus biopsies. In comparison, in 68 patients of group B (81%) H. pylori was eradicated (p = NS between groups). H. pylori eradication in both groups was associated similarly with a decrease of inflammation and activity whereas atrophy and intestinal metaplasia were not affected. A positive association was revealed between the decrease of H. pylori score and the decrease of both inflammation and activity scores for antrum as well as corpus biopsies. CONCLUSIONS: Addition of BSC to OME/AMO does not increase H. pylori eradication in patients with dyspepsia. Eradication of H. pylori is associated with disappearance of epithelial damage (inflammation and activity) in antral and corpus mucosa.

Flexible endoscopic treatment of Zenker's diverticulum: a new approach.

BACKGROUND AND STUDY AIMS: In the past, a number of treatment modalities have been used for the management of a Zenker's diverticulum. These include transcervical diverticulectomy, myotomy, or laser or diathermy treatment via rigid endoscopes. Up to the present, no reports of the treatment of a Zenker's diverticulum with a flexible endoscope have been published. PATIENTS AND METHODS: In this study, we present the results of our first 20 patients (13 male; mean age 82 years) prospectively treated with a flexible endoscope applied through diathermy. All patients had significant symptoms such as dysphagia, recurrent aspiration pneumonia and/or weight loss. RESULTS: Treatment using a mean of three sessions per patient was successful, with a good symptomatic response in all subjects. There were no severe complications associated with the therapy. Four patients complained about a sore throat for a few days. During follow-up (mean 6-7 months), three patients died due to unrelated causes, and 17 remained asymptomatic. CONCLUSIONS: An important advantage is that it is possible to perform the treatment without general anesthesia, and therefore also in patients whose general health is poor. The technique can easily be performed in an interventional endoscopy unit. Our first experiences show that flexible endoscopic treatment is an effective and relatively safe method.

[Successful electroconvulsive therapy in a pregnant woman with neuroleptic malignant syndrome]. / Succesvolle elektroconvulsietherapie bij een zwangere vrouw met het maligne neuroleptica-syndroom.

Neuroleptic malignant syndrome was diagnosed in a 27-year-old pregnant female with a psychiatric disorder, after treatment with haloperidol. The syndrome did not respond to treatment with dantrolene. Serious vegetative instability weighed against the use of electroconvulsive treatment. After 29 3/7 weeks of amenorrhoea electroconvulsive treatment was started, and the clinical picture gradually improved. Mother and child were discharged in good clinical condition.

Coeliac disease. Three cases of delayed diagnosis after a sojourn in the tropics.

Three patients are described in whom the diagnosis of coeliac disease was missed because of a recent sojourn in the tropics. The differences between coeliac disease and tropical sprue, as well as the diagnostic pathways, are discussed.

Subgroups of the Cowpea Miscellany: Symbiotic Specificity within Bradyrhizobium spp. for Vigna unguiculata, Phaseolus lunatus, Arachis hypogaea, and Macroptilium atropurpureum.

Rhizobia classified as Bradyrhizobium spp. comprise a highly heterogeneous group of bacteria that exhibit differential symbiotic characteristics on hosts in the cowpea miscellany cross-inoculation group. To delineate the degree of specificity exhibited by four legumes in the cowpea miscellany, we tested the symbiotic characteristics of indigenous cowpea bradyrhizobia on cowpea (Vigna unguiculata), siratro (Macroptilium atropurpureum), lima bean (Phaseolus lunatus), and peanut (Arachis hypogaea). The most-probable-number counts of indigenous bradyrhizobia at three sites on Maui, Hawaii, were substantially different on the four hosts: highest on siratro, intermediate on cowpea, and significantly lower on both lima bean and peanut. Bradyrhizobia from single cowpea nodules from the most-probable-number assays were inoculated onto the four hosts. Effectiveness patterns of these rhizobia on cowpea followed a normal distribution but were strikingly different on the other legumes. The effectiveness profiles on siratro and cowpea were similar but not identical. The indigenous cowpea-derived bradyrhizobia were of only moderate effectiveness on siratro and were in all cases lower than the inoculant-quality reference strain. Between 5 and 51% of the bradyrhizobia, depending on site, failed to nodulate peanut, whereas 0 to 32% failed to nodulate lima bean. No significant correlation was observed between the relative effectiveness of the bradyrhizobia on cowpea and their corresponding effectiveness on either lima bean or peanut. At all sites, bradyrhizobia that were ineffective on cowpea but that effectively nodulated lima bean, peanut, or both were found. Eighteen percent or fewer of the bradyrhizobia were as effective on lima bean as the reference inoculant strain; 44% or fewer were as effective on peanut as the reference strain. Only 18% of all cowpea-derived bradyrhizobia tested were able to form N(2)-fixing nodules on both lima bean and peanut. These results indicate the need to measure indigenous bradyrhizobial population characteristics directly with the crop of interest to obtain an accurate assessment of the need to inoculate.

Influence of the size of indigenous rhizobial populations on establishment and symbiotic performance of introduced rhizobia on field-grown legumes.

Indigenous rhizobia in soil present a competition barrier to the establishment of inoculant strains, possibly leading to inoculation failure. In this study, we used the natural diversity of rhizobial species and numbers in our fields to define, in quantitative terms, the relationship between indigenous rhizobial populations and inoculation response. Eight standardized inoculation trials were conducted at five well-characterized field sites on the island of Maui, Hawaii. Soil rhizobial populations ranged from 0 to over 3.5 x 10 g of soil for the different legumes used. At each site, no less than four but as many as seven legume species were planted from among the following: soybean (Glycine max), lima bean (Phaseolus lunatus), cowpea (Vigna unguiculata), bush bean (Phaseolus vulgaris), peanut (Arachis hypogaea), Leucaena leucocephala, tinga pea (Lathyrus tingeatus), alfalfa (Medicago sativa), and clover (Trifolium repens). Each legume was (i) inoculated with an equal mixture of three effective strains of homologous rhizobia, (ii) fertilized at high rates with urea, or (iii) left uninoculated. For soybeans, a nonnodulating isoline was used in all trials as the rhizobia-negative control. Inoculation increased economic yield for 22 of the 29 (76%) legume species-site combinations. While the yield increase was greater than 100 kg ha in all cases, in only 11 (38%) of the species-site combinations was the increase statistically significant (P

Modeling symbiotic performance of introduced rhizobia in the field by use of indices of indigenous population size and nitrogen status of the soil.

The ability to predict the symbiotic performance of rhizobia introduced into different environments would allow for a more judicious use of rhizobial inoculants. Data from eight standardized field inoculation trials were used to develop models that could be used to predict the success of rhizobial inoculation in diverse environments based on indices of the size of indigenous rhizobial populations and the availability of mineral N. Inoculation trials were conducted at five diverse sites on the island of Maui, Hawaii, with two to four legumes from among nine species, yielding 29 legume-site observations. The sizes of indigenous rhizobial populations were determined at planting. Soil N mineralization potential, total soil N, N accumulation and seed yield of nonnodulating soybean, and N derived from N(2) fixation in inoculated soybean served as indices of available soil N. Uninoculated, inoculated, and fertilizer N treatments evaluated the impact of indigenous rhizobial populations and soil N availability on inoculation response and crop yield potential. The ability of several mathematical models to describe the inverse relationship between numbers of indigenous rhizobia and legume inoculation responses was evaluated. Power, exponential, and hyperbolic functions yielded similar results; however, the hyperbolic equation provided the best fit of observed to estimated inoculation responses (r = 0.59). The fact that 59% of the observed variation in inoculation responses could be accounted for by the relationship of inoculation responses to numbers of indigenous rhizobia illustrates the profound influence that the size of soil rhizobial populations has on the successful use of rhizobial inoculants. In the absence of indigenous rhizobia, the inoculation response was directly proportional to the availability of mineral N. Therefore, the hyperbolic response function was subsequently combined with several indices of soil N availability to generate models for predicting legume inoculation response. Among the models developed, those using either soil N mineralization potential or N derived from N(2) fixation in soybean to express the availability of mineral N were most useful in predicting the success of legume inoculation. Correlation coefficients between observed and estimated inoculation responses were r = 0.83 for the model incorporating soil N mineralization potential and r = 0.96 for the model incorporating N derived from N(2) fixation. Several equations collectively termed "soil N deficit factors" were also found to be useful in estimating inoculation responses. In general, models using postharvest indices of soil N were better estimators of observed inoculation responses than were those using laboratory measures of soil N availability. However, the latter, while providing less precise estimates, are more versatile because all input variables can be obtained through soil analysis prior to planting. These models should provide researchers, as well as regional planners, with a more precise predictive capability to determine the inoculation requirements of legumes grown in diverse environments.

Synthesis and pharmacological evaluation of a series of dibenzo[a,d]cycloalkenimines as N-methyl-D-aspartate antagonists.

A series of 73 dibenzo[a,d]cycloalkenimines were synthesized and evaluated for their ability to displace (+)-10,11-dihydro-5-methyl-5H-dibenzo[a,d]cyclohepten-5,10-imine ([3H]-(+)-10) from its specific binding site on rat cortical membranes. A number of the more active compounds (Ki ranging from 0.006 to 0.21 microM) were evaluated for N-methyl-D-aspartate (NMDA) antagonist activity in the rat cortical slice (Kb ranging from 0.08 to 0.9 microM) and anticonvulsant activity in the mouse against NMDA induced convulsions. The ED50 values ranged from 0.22 to 7.76 mg/kg and correlated reasonably well with the Kb determination. In the dibenzo[a,d]cyclohepten-5,10-imine series, the (+)-5S,10R enantiomer displayed consistently higher levels of biological activity. While substitution at the 3-position of (+)-10 with electronegative atoms generally increased in vitro activity, a loss of potency relative to (+)-10 (MK-801) was observed in vivo for all of the compounds tested.