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Lyotropic liquid crystal as a real-time detector of microbial immune complexes.

AIMS: To design a simple method for the detection of microbe-immune complexes exploiting the optical and elastic properties of a biocompatible liquid crystalline material. METHODS AND RESULTS: Aqueous solution of disodium cromoglycate (DSCG), a lyotropic chromonic liquid crystal (LCLC), was aligned in a glass cell so as to be optically dark in polarized light. Immune complexes of at least three to four organisms altered the DSCG alignment such that polarized light was subsequently transmitted to reveal the presence of pathogens as optically bright regions around the immune complexes. CONCLUSIONS: This work describes the first method to detect viable micro-organisms in real time using LCLC. SIGNIFICANCE AND IMPACT OF THE STUDY: This technique provides a powerful tool for the detection of microbes in minutes, exploiting the optical and elastic properties of LC.

Antibodies, Bacterial↗

Dextran degrading activity of oral microbial flora.

Dextran degrading activity of the human oral microflora was detected by culturing in TYD broth (Tryptose 1.0%, Yeast extract 0.3%, Dextran T-150 (Pharmacia, MW 150,000) 0.15%). All of the plaue and saliva samples collected from 10 subjects showed a dextran degrading activity, both cultured aerobically and anaerobically, while the anaerobic culture was more active than the aerobic. Furthermore, some individual differences were observed in their activity. Crude enzyme(s) was extracted from the supernatant of a mixed culture of plaque sample by adding ammonium-sulfate to 0.6 and 0.8 saturation (called E-1 and E-2, respectively). E-1 contained 2 dextran-degrading enzymes, one being thought to be an endo-enzyme, with the optimal of H being 5.0 and the other an exo-enzyme with the optimal pH being 7.0-7.5. On the other hand, E-2 contained 1 enzyme of the endo-type. Thirteen strains producing dextranase were isolated from the plaque and were identified as Bacteroides oralis-like organisms. Several other organisms were thought to produce dextranase, although we failed to isolate them in this experiment.

Adult↗

Shigella flexneri strains produce bacteriocins active against members of the human microbial intestinal flora.

The principal aim of this work was to detect the bacteriocinogenic capacity of S. flexneri strains on members of the human intestinal flora. A total of 49 bacteriocinogenic S. flexneri strains were isolated from individuals of both sexes and different ages. The bacteriocins were detected by means of the drop method using E. coli and B. fragilis as target strains. The serotypes of the S. flexneri were determined. The producer capacity of bacteriocins was analysed in 10 different colonies of the same cellular clone and also the arbitrary units were determined. The highest number of bacteriocinogenic S. flexneri strains were obtained from diarrhoeal individuals from 0-10 years old and the S. flexneri serotype 2a was the most abundant. It was demonstrated that E. coli and B. fragilis isolated from the normal intestinal flora of healthy individuals were susceptible to the bacteriocinogenic S. flexneri strains. By means of the determination of arbitrary units per ml of the bacteriocin, it was demonstrated that colonies from a single colony isolate of a same clone of bacteriocinogenic S. flexneri produce different quantities of bacteriocin.

Adolescent↗

[The isolation of Helicobacter pylori from the gastric juice].

Gastric contents was examined in 49 patients with duodenal bulb ulcers. Helicobacter pylori were isolated in 83.7% of cases. H. pylori density was found to be 10(3)-10(5) microbial cells per ml. Bacterioscopic studies with gram staining helped detect H. pylori in 79.6% of cases, cultural method revealed the bacteria in 63.3%, and urease test in 4.1% of cases. Poor efficacy of urease test is explained by its poor sensitivity (10(6) microbial cells per ml or higher). The authors analyze the frequency of H. pylori isolation in relation to the month of the examination and gastric contents coloring.

Adolescent↗

Succession of bacterial and fungal communities during a traditional pot fermentation of rice vinegar assessed by PCR-mediated denaturing gradient gel electrophoresis.

Denaturing gradient gel electrophoresis (DGGE) based on small subunit rRNA gene was applied to a traditional rice vinegar fermentation process in which the conversion of rice starch into acetic acid proceeded in a pot. The fungal DGGE profile indicated that the transition from Aspergillus oryzae to Saccharomyces sp. took place at the initial stage at which alcohol production was observed. The early stage was characterized by the coexistence of Saccharomyces sp. and lactic acid bacteria. Almost all of the bacterial DGGE bands related to lactic acid bacteria were replaced by bands derived from Lactobacillus acetotolerance and Acetobacter pasteurianus at the stage at which acetic acid started to accumulate. The microbial succession, tested in three different pots, was found to be essentially identical. Among the bacteria isolated at the early stage, some species differed from those detected by DGGE. This is the first report to reveal the microbial community succession that occurs during a unique vinegar fermentation process, as determined by a culture-independent method.

Acetic Acid↗

Utilization and transformation of aquatic humic substances by autochthonous microorganisms.

Aquatic humic substances (HS) from a bog lake water, a riverwater, and a groundwater were isolated after enrichment on XAD 8 columns and added to a Czapek-Dox nutrient broth which was used either in full strength or without glucose and/or NaNO3. The individual flasks were inoculated with natural microbial populations of corresponding water samples or with a Pseudomonas fluorescens strain isolated from groundwater. The presence of HS resulted in an increase of bacterial numbers in nearly all cultures incubated for 3 weeks at 25 degrees C on a shaker. HS reisolated from cultures without glucose or NaNO3 showed no or only minor quantitative differences as compared to those from sterile controls. In full strength nutrient broth up to 27% of HS were utilized. Data obtained by spectroscopic methods (UV/vis/FTIR) and elemental analysis indicated a decrease in particle size and a loss in aromaticity and aliphatic carbon in HS reisolated from the microbial cultures. Simultaneously an increase in the N content of HS was observed, which probably originated from some constituents of microbial biomass such as proteins and amino sugars. The NMR data also documented that significant transformations of HS occurred in the individual microbial cultures. After incubation, increased amounts of aromatic acids were detected in some liquid media and residual HS by GC/MS or capillary electrophoresis. 1H NMR spectroscopy was less effective in indicating structural differences in the HS than 13C NMR but revealed considerable detail of the microbial degradation of riverine HS, when limited sample was available. The newly developed NMR increment analysis provided substantial detail of aromatic structures in a microbially altered HS. The microbial degradation of HS strongly depended on the composition of the HS, the species selection of the microorganisms, and to a lesser extent on the culture conditions. For any series of identical inoculum and HS, full broth media initiated the most extensive alteration of HS.

Biological Availability↗

Fungal spores as such do not cause nasal inflammation in mold exposure.

The increased frequency of respiratory symptoms and diseases among sawmill workers has been linked to occupational exposure to airborne contaminants, especially to different fungi. Similar adverse health effects, together with elevated levels of inflammatory mediators in the nasal lavage (NAL) fluid, have been detected in people working in mold-damaged buildings. However, the indoor fungal spore concentrations in moldy houses are much lower than those in sawmills. To study the effect of fungal spores as such on the inflammatory markers in the NAL fluid, we conducted a similar study in sawmill workers who had an intense exposure to fungal spores. NAL was performed in 11 sawmill workers both during high occupational microbial exposure and during their vacation. Concentrations of nitric oxide (NO), tumor necrosis factor-alpha (TNFalpha), interleukin (IL)-4, IL-5, and IL-6 in NAL fluid were analyzed, and occupational exposure to inhalable dust, fungal spores, endotoxins and terpenes was confirmed by personal monitoring during the day of sampling. In addition, health data were collected with a questionnaire. Personal exposures to fungal spores varied from 2 x 10(5) spores/m(3) to 1.5 x 10(6) spores/m(3) (mean 7 x 10(5) spores/m(3)). Concentrations of inhalable dust, endotoxins, and terpenes were relatively low. There were no differences between work and vacation in the concentrations of NO and proinflammatory cytokines in NAL fluid or in the reporting of symptoms, and the levels were lower than in workers at a moldy school building. However, the concentration of TNFalpha in the NAL fluid was positively correlated with the concentration of terpenes in the working environment (r =.768; p =.006). These results show that microbial exposure as such does not invariably result in inflammatory changes detectable with the NAL method. We suggest that the type of microbial flora or microbial products in the occupational environment determines the proinflammatory potency of microbial exposure.

Adult↗

Genotypic and phenotypic responses of a riverine microbial community to polycyclic aromatic hydrocarbon contamination.

The phenotypic and genotypic adaptation of a freshwater sedimentary microbial community to elevated (22 to 217 microgram [dry weight] of sediment-1) levels of polycyclic aromatic hydrocarbons (PAHs) was determined by using an integrated biomolecular approach. Central to the approach was the use of phospholipid fatty acid (PLFA) profiles to characterize the microbial community structure and nucleic acid analysis to quantify the frequency of degradative genes. The study site was the Little Scioto River, a highly impacted, channelized riverine system located in central Ohio. This study site is a unique lotic system, with all sampling stations having similar flow and sediment characteristics both upstream and downstream from the source of contamination. These characteristics allowed for the specific analysis of PAH impact on the microbial community. PAH concentrations in impacted sediments ranged from 22 to 217 microgram (dry weight) of sediment-1, while PAH concentrations in ambient sediments ranged from below detection levels to 1.5 microgram (dry weight) of sediment-1. Total microbial biomass measured by phospholipid phosphate (PLP) analysis ranged from 95 to 345 nmol of PLP g (dry weight) of sediment-1. Nucleic acid analysis showed the presence of PAH-degradative genes at all sites, although observed frequencies were typically higher at contaminated sites. Principal component analysis of PLFA profiles indicated that moderate to high PAH concentrations altered microbial community structure and that seasonal changes were comparable in magnitude to the effects of PAH pollution. These data indicate that this community responded to PAH contamination at both the phenotypic and the genotypic level.

Adaptation, Physiological↗

Effects of the antibiotic ciprofloxacin on stream microbial communities and detritivorous macroinvertebrates.

Microbial communities play an important role in stream ecosystem processes, such as breakdown of senescent leaf litter, and as a primary nutritional source for detritivorous macroinvertebrates. Antibiotics may affect stream microbial communities and associated ecosystem processes, especially because recent stream and river monitoring programs have indicated the presence of antibiotics downstream of wastewater treatment plants. In the current study, effects of chronic exposure to the fluoroquinolone antibiotic ciprofloxacin (Cipro) were examined on stream microbial community-level physiological profiles and growth indices of detritivorous amphipods (Gammarus spp.) and caddisflies (Lepidostoma liba). Microcosm experiments were conducted using stream sediments and water, senesced leaf material (Acer saccharum), and macroinvertebrates. A shift in function of leaf-associated microbial communities (based on carbon source utilization) was observed for samples exposed to 100 microg/L of Cipro for 12 d compared to control and treatments exposed to 1 and 10 microg/L of Cipro. This was attributable to carbohydrate substrates, which had 2.7- to 3.5-fold lower microbial respiration than the lower concentrations and control (p < 0.001). For detritivores, Gammarus spp. condition index did not differ among control, 0.1, and 1.0 microg/L treatments after 30-d exposures (p > 0.05). Similarly, L. liba growth rate did not vary among control, 10, and 100 microg/L treatments after 45-d exposures (p > 0.05). These results suggest that Cipro may affect leaf-associated microbial communities, but at concentrations four orders of magnitude above those detected in streams. However, effects of the antibiotic on growth and condition of detritivores were not observed. Future work should focus on identifying specific changes in stream microbial communities as a result of Cipro exposure and impacts on other aquatic species.

Animals↗

Long-term performance and microbial dynamics of an up-flow fixed bed reactor established for the biodegradation of fluorobenzene.

An up-flow fixed bed reactor (UFBR) was established to investigate the biodegradation of fluorobenzene (FB) under a number of operating conditions, which included variation in the concentration of FB in the feed stream (up to 180 mg l(-1)) and temporary suspension of feeding. Degradation of FB was followed for a period of 8 months under a continuous flow regime. During the operation of the UFBR, FB was never detected in the reactor effluent, being biodegraded by the microbial biofilm or adsorbed to the granular activated carbon (GAC). Biodegradation of FB was observed from the beginning of the reactor operation, and overall, it accounted for 50% of the total amount fed to the bioreactor. High organic loads of FB (210-260 mg d(-1) dm(-3)) were found to affect the biological removal efficiency, possibly due to an inhibitory effect caused by the higher FB concentrations fed to the bioreactor (149-179 mg l(-1)). When FB feeding was suspended for 1 month, biodegradation continued, indicating that the adsorbed FB became bioavailable. Biofilm bacterial dynamics were followed throughout the UFBR operation by denaturing gradient gel electrophoresis and plate-counting techniques, showing that a quite stable community was found in the bioreactor, and this was mainly attributed to the high selective pressure exerted by the presence of FB.

Biofilms↗

Early detection of secondary caries using quantitative, light-induced fluorescence.

The authors hypothesize that the arrestment and remineralization of these lesions could be improved if secondary caries could be detected and monitored at earlier stages. Traditional diagnostic techniques detect secondary caries when it is relatively advanced and when significant tissue has been lost. This in vitro study evaluated the Quantitative Light-induced Fluorescence (QLF) system for detecting and monitoring demineralization surrounding tooth-colored restorations. This investigation was divided into three studies. The first study evaluated QLF for the detection and measurement of chemically induced lesions surrounding resin composite restorations. The second study evaluated QLF for the detection of demineralization around different tooth-colored restorations (glass ionomer, resin composite, compomer and smart material) created in a microbial caries model. Finally, the third study tested whether QLF was effective at detecting early wall lesions adjacent to resin composite restorations. Data from Study 1 demonstrated the potential for QLF to detect very early secondary caries and to distinguish between the different stages of early demineralization. Study 2 confirmed the potential for QLF to detect early secondary caries created by cariogenic bacteria and concluded that the four types of materials differed in their ability to prevent secondary caries in this model, with the glass ionomer being the most effective and the non-fluoride releasing composite performing the worst, which allowed for the development of larger lesions. The results of Study 3 suggest a potential application of the QLF system to detect early wall lesions. Data from this investigation strongly suggest that QLF is a potentially viable technology to detect and monitor early secondary caries.

Analysis of Variance↗

Streptomycin as a selective agent to facilitate recovery and isolation of introduced and indigenous Sphingomonas from environmental samples.

Sphingomonas is an organism of major interest for the degradation of organic contaminants in soils and other environments. A medium based on the aminoglycoside antibiotic streptomycin (Sm) was developed, which, together with the yellow pigmentation of Sphingomonas, facilitated the detection, recovery and quantification of culturable Sphingomonas from soils. All 29 previously described bacterial strains belonging to 17 different Sphingomonas species were able to grow on mineral media containing 200 microg ml(-1) streptomycin, showing that the capacity to resist high concentrations of Sm is a common characteristic within Sphingomonas. Incorporation of Sm into the mineral medium led to a significant reduction in the background microbial population and a concomitant 100 times more sensitive detection of Sphingomonas inoculated in non-sterile soil matrices. The Sm-containing medium was used to examine a variety of hydrocarbon-contaminated soils for the presence and biodiversity of Sphingomonas. Incorporation of Sm in the medium led to a significant increase in the number of yellow-pigmented colonies. Comparison of contaminated and non-contaminated soils derived from the same site revealed colonization by culturable yellow-pigmented Sm-resistant bacteria of the polluted location solely. Both yellow and non-yellow-pigmented colonies were purified from plates containing glucose and Sm, and BOX-polymerase chain reaction (PCR) was used to sort out clonally related strains. Representative strains from the major BOX-PCR clusters were identified using FAME and partial 16S rRNA gene sequencing. Forty-eight of 58 Sm-resistant isolates were identified as Sphingomonas sp. Streptomycin-resistant Sphingomonas isolates generated BOX-PCR diversity patterns that were site dependent and represented different species mainly belonging to Sphingomonas subgroups containing species formerly designated as Sphingopyxis and Sphingobium. The ability to degrade phenanthrene was only found in a minority of the Sphingomonas isolates, which all originated from soils containing high phenanthrene concentrations.

Anti-Bacterial Agents↗

Cell-to-cell signalling in Escherichia coli and Salmonella enterica.

Cell-to-cell signalling in prokaryotes that leads to co-ordinated behaviour has been termed quorum sensing. This type of signalling can have profound impacts on microbial community structure and host-microbe interactions. The Gram-negative quorum-sensing systems were first discovered and extensively characterized in the marine Vibrios. Some components of the Vibrio systems are present in the classical genetic model organisms Escherichia coli and Salmonella enterica. Both organisms encode a signal receptor of the LuxR family, SdiA, but not a corresponding signal-generating enzyme. Instead, SdiA of Salmonella detects and responds to signals generated only by other microbial species. Conversely, E. coli and Salmonella encode the signal-generating component of a second system (a LuxS homologue that generates AI-2), but the sensory apparatus for AI-2 differs substantially from the Vibrio system. The only genes currently known to be regulated by AI-2 in Salmonella encode an active uptake and modification system for AI-2. Therefore, it is not yet clear whether Salmonella uses AI-2 as a signal molecule or whether AI-2 has some other function. In E. coli, the functions of both SdiA and AI-2 are unclear due to pleiotropy. Genetic strategies to identify novel signalling systems have been performed with E. coli and Providencia stuartii. Several putative signalling systems have been identified, one that uses indole as a signal and another that releases what appears to be a peptide. The latter system has homologues in E. coli and Salmonella, as well as other bacteria, plants and animals. In fact, the protease components from Providencia and Drosophila are functionally interchangeable.

Bacterial Proteins↗

Neutralization of the inhibitory effect of sodium polyanetholesulfonate on Neisseria meningitidis in blood cultures processed with the Du Pont Isolator System.

The inhibitory and bactericidal effects of sodium polyanetholesulfonate on Neisseria meningitidis were neutralized by blood lysis which occurs within the pediatric Isolator 1.5 Microbial tube (E. I. du Pont de Nemours & Co., Inc., Wilmington, Del.). Lysed blood was more effective than 2.5% gelatin in preventing the inhibitory effect. All but 1 of 16 N. meningitidis strains were killed by 0.06% sodium polyanetholesulfonate in the absence of lysed blood, whereas none were killed by 1.0% sodium polyanetholesulfonate when lysed blood was present. The possible clinical relevance of these results was reflected in the improved detection of meningococcemia in children when the Isolator 1.5 Microbial tube was compared with a conventional broth system.

Bacteriological Techniques↗

[Epidemiologic studies of the microbial colonization of severely burned patients].

Bacteria isolated between 1/1/1983 and 12/31/1984 from the burns of 122 patients in a burns unit at the Ruhr-Universität Bochum were studied. Grampositive bacteria were predominant in colonizing the burn wounds (62.5% of all strains isolated). In the patients with more than 40% of total body surface area (TBSA) burn, isolation of Staph aureus was most frequent. The exogenous colonization rate with Staph. aureus was 86%. Coagulase negative Staphylococci were identified in 89.6% of all patients (71.4% of the patients with more than 40% TBSA burn). There was a constant decline in detecting Pseudomonas aeruginosa from the second half of 1983. Microbial sensitivity testing was performed in 834 cases. Gramnegative strains of bacteria resistant to Ampicillin, Mezlocillin, Piperacillin and Ticarcillin were found in 10 up to 97% of the tested strains. Acinetobacter calcoaceticus var. antitratus and Enterobacter cloacae usually displayed a wide resistant pattern. Some strains were resistant as to 16 antibiotics. The incidence of multiresistant Staph. aureus was studied. The time course of multiresistance was paralleled by the incidence of a 6-fold resistance to Benzylpenicillin, Oxacillin, Tetracyclin(T), Gentamycin(G), Erythromycin(E), and Sulfadiacin(S). The probability of simultaneous resistance to 2 of the 4 antibiotics (T, G, E, S) ranged between 0.7 and 0.98. 98 out of 336 Staph. aureus isolates showed a simultaneous resistance to T, G, E and S (29%).

Adolescent↗

Inhibition of lymphocyte proliferation induced in vitro by microbial antigens in HIV-infected subjects.

In the course of a study of lymphocyte responses to microbial antigens in HIV-infected patients, we detected a previously unrecognized phenomenon of inhibition of lymphocyte baseline proliferation, induced by the presence of tetanus toxoid and Escherichia coli in the cultures. The effects of tetanus toxoid and Escherichia coli on lymphocyte proliferation in vitro were assessed by comparing the 3H-thymidine uptake by lymphocytes cultured without stimulant with the uptake of lymphocytes cultured in the presence of the antigens. Twenty-six patients with HIV infection (20 asymptomatic/persistent generalized lymphadenopathy, 2 AIDS-related complex, 4 AIDS) were investigated and the controls were 33 healthy individuals without evidence of HIV infection. Eight out of 22 asymptomatic/PGL and ARC patients progressed to full-blown AIDS in the mean follow-up of 26 months. The inhibition of proliferation was considered to be significant when the uptake of 3H-thymidine was reduced by 20% in the presence of the antigens. Using these criteria, 50% of the patients studied with tetanus toxoid and 36% of those studied with E. coli had evidence of the inhibitory phenomenon. Seven of the eight patients who developed AIDS during the observation period had the inhibitory phenomenon. In the group of patients without the inhibitory signs only one evolved to AIDS during the follow-up. The possibility of this phenomenon being related to an induction of suppressor cell activity by the antigens is discussed.

AIDS-Related Complex↗

Changes in androgenic steroid profile due to urine contamination by microorganisms: a prospective study in the context of doping control.

Urine contamination by microorganisms may affect the interpretation of urinalysis in different areas of clinical diagnosis. This is particularly relevant in doping control. A prospective study was designed to assess the effects of urine contamination by selected pathogens on the endogenous androgenic steroid profile. Pooled urine from a healthy male volunteer with standard steroid profile compared with reference values for the Caucasian population was sterilized by filtration and stored in sterile glass tubes. Aliquots were inoculated with known amounts of 15 different organisms (bacteria, fungi, and moulds) and incubated at 37 degrees C for 2 weeks. Different markers of urine contamination, such as pH, deconjugation of steroids, and metabolic by-products, were determined. Alkalization of urinary pH was not a reliable indicator of urine contamination as several organisms grew in this medium and no alteration of this parameter was found. In uncontaminated urine, less than 10% of steroid glucuronide conjugates were spontaneously hydrolyzed. Higher rates of hydrolysis for sulfate conjugates were found. An unconjugated fraction higher than 10% of the total amount of testosterone was a reliable indicator of urine contamination. However, microbial production of testosterone or epitestosterone was not detected. In contrast, a few organisms were able to synthesize 5alpha-androstanedione, 5beta-androstanedione, and androstenedione using endogenous steroids as substrates.

Androgens↗

Control of ammonia formation during Bacillus subtilis fermentation of legumes.

The control of ammonia formation during the Bacillus subtilis fermentation of autoclaved, roasted soybean cotyledons (Glycine max) and of autoclaved African locust bean cotyledons (Parkia spp.) was investigated. Addition of NaCl, 1.5 mol (kg wet cotyledons)-1, part way through the fermentation inhibited ammonia formation and softening of the cotyledons. Addition of glycerol, 1.7 mol (kg wet cotyledons)-1 part way through the fermentation inhibited alkalinisation and ammonia formation while allowing enzymic activity and softening of the cotyledons to continue. Restriction of the oxygen supply by incubating the cotyledons in a sealed container also prevented excessive ammonia production and increase in pH value. Fermentations conducted in sealed containers with an air to cotyledons ratio of approximately 130-175 ml air (g wet cotyledons)-1 supported good microbial growth and proteolysis without the formation of detectable ammonia aroma.

Ammonia↗