Monoclonal antibody detection of Pseudomonas spp. in refrigerated meat by an indirect ELISA

Monoclonal antibodies generated against live cells of Pseudomonas fluorescens have been used in an indirect ELISA format for the detection of Pseudomonas spp. in refrigerated meat. The detection threshold for the ELISA assay developed in this work was 10⁴ cfu cm⁻².     

Experimental Infection of Nosema sp. Ghose in an Unnatural Host Lesioderma sericorne

ABSTRACT. Various doses of a microsporan parasite, Nosema sp., were fed to third and fourth instar larvae of Lesioderma sericorne that infested different types of stored grains. A spore dose of 3 × 10³ spores/individual resulted in a 39% infection rate, reduction in larval and adult weights, and mean spore concentrations of 1.28 ± 0.2 × 10⁸ spores/larva and 1.1 ± 0.2 × 10⁸ spores/adult. At the above dose, mortality was not well marked (about 35% in larvae and 25% in adults). At 3 × 10⁴ spores/individual, the rate of mortality increases to 80% in larvae and 60% in adults. However, more of the pest population (88% of larvae and 73% of adults) died at a dose of 3 × 10⁵ spores/individual. This dose produced mean spore concentrations of 3.91 ± 0.2 × 10⁸ spores/larva and 2.89 ± 0.2 × 10⁸ spores/adult. Insect death was caused by heavy damage to gut epithelia and fat bodies.     

The effect of essential oils of basil on the growth of Aeromonas hydrophila and Pseudomonas fluorescens

Basil essential oils, including basil sweet linalool (BSL) and basil methyl chavicol (BMC), were screened for antimicrobial activity against a range of Gram-positive and Gram-negative bacteria, yeasts and moulds using an agar well diffusion method. Both essential oils showed antimicrobial activity against most of the micro-organisms examined except Clostridium sporogenes , Flavimonas oryzihabitans , and three species of Pseudomonas . The minimum inhibitory concentration (MIC) of BMC against Aeromonas hydrophila and Pseudomonas fluorescens in TSYE broth (as determined using an indirect impedance method) was 0·125 and 2% (v/v), respectively; the former was not greatly affected by the increase of challenge inoculum from 10³ to 10⁶ cfu ml⁻¹. Results with resting cells demonstrated that BMC was bactericidal to both Aer. hydrophila and Ps. fluorescens . The growth of Aer. hydrophila in filter-sterilized lettuce extract was completely inhibited by 0·1% (v/v) BMC whereas that of Ps. fluorescens was not significantly affected by 1% (v/v) BMC. In addition, the effectiveness of washing fresh lettuce with 0·1 or 1% (v/v) BMC on survival of natural microbial flora was comparable with that effected by 125 ppm chlorine.     

Potassium Fluxes on Hyperosmotic Shock and the Effect of Phenol and Bronopol (2-bromo-2-nitropropan-1,3-diol) on Deplasmolysis of Pseudomonas aeruginosa

Potassium fluxes and the effect of phenol and bronopol on deplasmolysis of Pseudomonas aeruginosa were followed in sucrose and glycerol plasmolysing systems.
In sucrose, K⁺ uptake related to the solute concentration. Proline increased the rate and overall K⁺ uptake, the latter by a factor of three. It was concluded that there was no rigid maximum in the accumulation of intracellular K⁺ as long as intracellular neutrality in electrical charges was maintained.
In glycerol, K⁺ uptake was parallel with glycerol penetration. The process was reversed, however, on equilibration of glycerol. This suggested that glycerol inhibited K⁺ retention against a concentration gradient rather than that K⁺ was excluded as a consequence of the osmotic established steady state. This view was enforced by the fact that the reversal of K⁺ uptake occurred in 20 and 30% glycerol but not in 10%.
Phenol and bronopol did not affect deplasmolysis in glycerol significantly, although some effect on K⁺ uptake and glycerol permeability could be seen. In the sucrose system, phenol acted according to its mode of action generally accepted, i.e. inhibiting deplasmolysis at low and allowing solute penetration at higher concentrations, whereas very high concentrations caused coagulation of the cytoplasm. Bronopol inhibited deplasmolysis, except at very low concentrations. Proline did not prevent the inhibition of deplasmolysis in either of the solute systems, except at the very low bronopol concentrations where the deplasmolysis rate only was affected.     

Exo-β-1,3-Glucanase from Yeast Inhibits Colletotrichum lupini and Botrytis cinerea Spore Germination

Yeast exo-β-1,3-glucanase (EXG1) was evaluated as an inhibitory agent of Colletotrichum lupini and Botrytis cinerea. Extracts obtained from yeast transformed with the exg1 gene, expressing high levels of EXG1 activity, or control untransformed yeast cultures that lacked EXG1 activity, were added to different starting concentrations of C. lupini fungal spore suspensions (2.5 × 10³ to 80 × 10³ spores per flask), and mycelial dry weight was measured after 5 days. Inhibition of C. lupini mycelial growth by EXG1 compared with control extracts ranged from 41 to 20% when added to starting fungal spore concentrations of 2.5 × 10³ to 80 × 10³, respectively. EXG1 activity in the extracts from the transformed yeast remained high over the 5-day incubation period. Addition of the EXG1 extract after C. lupini spore germination resulted in lower inhibition, indicating that the EXG1 targets the β-glucan in the cell walls of the fungal spores at an early stage of germination. Furthermore, the yeast EXG1 extracts were also shown to inhibit Botrytis cinerea spore germination and growth. Thus, the use of the yeast exg1 gene for protection of crops, such as lupin and pear in transgenic strategies against C. lupini and B. cinerea , respectively, could be considered.     

The use of positron emission tomography for studies of long-distance transport in plants: uptake and transport of 18F

Abstract. Positron emission tomography (PET) has been utilized to obtain dynamic images of long distance nutrient translocation in plants. Positron emitting ¹⁸F, produced by a Van de Graaff accelerator using the reaction ¹⁸O(p,n)¹⁸F, was fed in solution to excised stems of Glycine max positioned vertically in a large-aperture PET detector system. Images of tracer activity were recorded with a time resolution of 0.5 min and a spatial resolution of 4 mm. Maximum tracer activities at stem sites were obtained within 3 min of the pulse feed. A model is presented enabling evaluation of regional values for tracer flow, tracer binding, flow speed and flow volume. Analysis of data for one stem position yielded a flow volume of 2.1mm³ min⁻¹ and a flow speed of 36cm min⁻¹. Comparison with the distribution of ¹⁴C-inulin, which was simultaneously fed to the cut stems, indicates the ¹⁸F is suitable for use as an apoplastic tracer; 92% of the tracer activity accumulated in the leaves. The fraction of ¹⁸F that remained bound was most concentrated at stem nodal regions, an observation consistent with the existence of transfer cells at these sites. Advantages and limitations of PET applied to plant physiological investigations are discussed.     

Bacteria Antagonistic to Pseudomonas tolaasii and their Control of Brown Blotch of the Cultivated Mushroom Agaricus bisporus

S ummary : Pseudomonas tolaasii was isolated from casing peat of healthy and diseased mushroom beds, compost of diseased mushroom beds and from soils round a mushroom farm. It was not isolated from fresh peat or compost from healthy mushroom beds. Three bacteria antagonistic to Ps. tolaasii were isolated from soil and peat. These were a nonfluorescent Pseudomonas sp. (closest to Ps. multivorans ) from soil; and strains of Ps. fluorescens and Enterobacter aerogenes from peat. When the antagonists and the pathogen were added in the ratio of 8 × 10⁷: 10⁶ cells/ml to unsterilized peat and applied to mushroom trays, infection of mushroom sporophores by the pathogen was effectively controlled. In vitro studies failed to show lysis or growth inhibition of Ps. tolaasii by the antagonists.     

Heterotrophic bacterial populations in the mineral waters of thermal springs in Spain

The microbiological quality and heterotrophic bacterial populations of 26 thermal mineral water springs in Spain were studied. In most of the springs the number of viable aerobes was less than 10³ cfu ml^-1 and the number of sporulated bacteria less than 10² cfu ml^-1. No significant differences were foundin the counts obtained with Plate Count Agar (PCA) and PCA diluted 1 : 10 and incubated at 22°, 37° and 45°C. Total coliforms were found in 14 springs, faecal streptococci in three, spores of sulphite-reducing Clostridium and Pseudomonas aeruginosa in seven. Neither Escherichia coli nor Staphylococcus aureus were found. A total of 665 strains were isolated and 85·4% of these identified; 329 were Gram-positive and 239 were Gram-negative. The genera most prevalent present in the springs were Pseudomonas (in 92.3%), Bacillus (65.4%), Enterobacter, Micrococcus and Staphylococcus (50%), Acinetobacter (42.3%), Arthrobacter (38.4%), Clostridium (27%) and Xanthomonas (23%). Gram-negative bacteria predominated in the mesothermal springs and Gram-positive bacteria in the hyper- and hypothermal springs. The most common Gram-negative rod species isolated were Ps. fluorescens, Ps. aeruginosa, Ps. putida, Ent. agglomerans, Ent. sakazakii, Ac. calcoaceticus and Ent. amnigenus.     

Uptake of 14C-gibberellic acid by mature grapefruit (Citrus paradisi Macf.) as affected by relative humidity and method of application

Effects of the method of application and relative humidity on the uptake of ¹⁴C-gibberellic acid (¹⁴C-GA₃) by mature grapefruit (Citrus paradisi Macf.) were examined. Uptake was higher when ¹⁴C-GA₃ was applied as a 'drying-out' solution than as a 'non-drying' solution. When ¹⁴C-GA, was applied by the 'drying-out' method, which closely imitates field conditions, rates of uptake were very high while the solution was drying out and during the first few hours after drying. Uptake from the dry residue continued in decreasing rates till the end of the experiment (72 h). Uptake from the dry residue was higher when fruits were incubated at 100% than at 50% relative humidity (RH). Transfer of fruits from 50% to 100% RH as late as 48 h after drying still increased the rate of uptake. Drying-out treatment solutions produced higher uptake rates with neutral (pH 7) as well as acid (pH 4) treatment solutions, and in the presence of triton B-1956, Triton X-100 or L-77 surfactants.     

Runoff losses of Pseudomonas aureofaciens (lacZY) from soil

Abstract: Leaching of genetically modified microorganisms (GMMs) through soil profiles is generally not a significant concern, since GMMs typically remain near the soil surface following application. The presence of high numbers of GMMs at the soil surface, however, suggests that losses via runoff may occur. Traditional methods of plating nonlabeled bacteria lack precision and are thus seldom used to monitor runoff losses. To examine whether lac ZY, a common genetic marker, could be used to evaluate bacterial runoff from soil, a lac ZY⁺ strain of Pseudomonas aureofaciens 3732 RN-L11 was used at three different pH levels, with and without wheat ( Triticum aestivum L.) cover in a greenhouse experiment. Twelve times over a 245 day period, soils were subjected to simulated rainfall of 84 mm h⁻¹ for a 15 min duration. Runoff losses and survival were quantified at each time. Pseudomonas aureofaciens survived for the longest period at a soil pH of 7; survival was reduced at lower pHs. The number of cells in runoff were usually related to the number of cells surviving in the soil. When high soil populations were present, runoff losses often exceeded 10¹⁰ cfu event⁻¹. When the soil population declined to low or undetectable levels, the runoff contained fewer organisms. Runoff losses of 10⁸ cfu event⁻¹, however, were observed during one runoff event even when the soil population was below the detection limit. This study indicates that lac ZY is an effective marker, and that runoff of GMMs may be an important mechanism for movement to nontarget environments.     

Tracer dose and availability time of thymidine and bromodeoxyuridine: application of bromodeoxyuridine in cell kinetic studies

Abstract. The present experiments with [¹⁴C]-thymidine (TdR) and [³H]-bromo-deoxyuridine (BrdU) using mouse jejunal crypt cells show that the upper limit of the tracer dose of TdR is about 0.5 µg g body weight^-1 and that of BrdU is about 5·0 µg g body weight^-1. Applying these doses, the proportions of the endogenous DNA synthesis attributed to the exogenous DNA precursor are 2% and 9% respectively. For [³H]-TdR doses commonly used in cell kinetic studies this proportion is only 0-1-1.0%, a negligible quantity that does not influence the endogenous DNA synthesis. The maximum availability time of tracer doses of TdR as well as BrdU is 40 to 60 min, the majority of the precursors being incorporated after 20 min. The availability time is the same for TdR doses exceeding the tracer dose by a factor of 80, whereas it is prolonged in the case of BrdU doses exceeding the tracer dose by a factor of 50. BrdU is suitable to replace radioactively labelled TdR in short term cell kinetic studies, i.e. determination of the labelling index or of the S phase duration by double labelling. However, more studies are needed to elucidate how far BrdU can replace TdR in long term studies as shown by differences between the fraction of labelled mitoses (FLM) curves of a human renal cell carcinoma measured with BrdU and [³H]-TdR.     

Real-time quantification of Pseudomonas fluorescens cell removal from glass surfaces due to bacteriophage varphiS1 application

Aims:  To study the efficacy of the lytic phage φS1 in eliminating Pseudomonas fluorescens in the early stage of biofilm formation, using an in situ and real time methodology for cell quantification.
Methods and Results:  Cell adhesion and phage infection studies were carried out in a parallel plate flow chamber under laminar conditions. Cells were allowed to adhere until reaching 1·7–1·8 × 10⁶ cells cm⁻² and phage infection was performed with two different phage concentrations (2 × 10⁹ PFU ml⁻¹ and 1 × 10¹⁰ PFU ml⁻¹). Phage concentration clearly affects the speed of infection. The less concentrated phage solution promoted a three times slower rate of cell removal but did not affect the overall percentage of cell removal. In fact, after a longer infection period the less concentrated phage solution reached the same 93% cell removal value.
Conclusions:  Phages are efficient in the eradication of bacterial cells at the early stage of biofilm formation and their presence at the surface did not allow bacterial recolonization of the surface.
Significance and Impact of the Study:  To date, no published studies have been made concerning in situ and real time quantification of cell removal from surfaces due to phage action.     

Efficacy of Beauveria bassiana (Bals.) Vuill. against the spruce bark beetle, Ips typographus L., in the laboratory under various conditions

Abstract:  In laboratory bioassays, the efficacy of the entomopathogenic fungus Beauveria bassiana against the spruce bark beetle, Ips typographus , was tested under various conditions. Four of the tested isolates and the commercial product Boverol^® caused 99–100% mortality when tested at a concentration of 1.0 × 10⁷ conidia/ml at 25°C. Using B. bassiana isolate 138 at a concentration of 1.0 × 10⁶, the median survival time (MST) was 6.1 d and significantly longer compared with the MST of 4.2 and 4.0 d at 1.0 × 10⁷ and 1.0 × 10⁸ conidia/ml, respectively. In the next experiment, the beetles were maintained on spruce bark, filter paper or artificial diet during the bioassay with Boverol^®, and significant differences in the MST of 3.6, 2.5 and 5.3 d, respectively, were noticed. The experiment with Boverol^® at different temperatures showed that the beetles lived significantly longer at 15°C (MST 8.7 d) than at 20, 25, 30 and 35°C. At 25°C, the beetles died most rapidly (MST 3.5 d). At different relative humidities (RH) of 40, 70 and 100%, nearly all beetles were dead after treatment with a suspension of Boverol^® at 1.0 × 10⁷ conidia/ml. At 40% RH, 49% of the untreated beetles died after 7 d. The best effects were achieved with the following bioassay: beetles were fed for three days on artificial diet, then dipped into a solution of 1.0 × 10⁷ conidia/ml and transferred on a piece of spruce bark in Petri dishes at 25°C and 70% RH.     

Interaction between fish spoilage bacteria Pseudomonas sp. and Shewanella putrefaciens in fish extracts and on fish tissue

The interaction between fish spoilage bacteria, Pseudomonas sp. and Shewanella putrefaciens , was investigated using fish extract and fish tissue as model systems. Isolates of Pseudomonas that produced iron chelators, siderophores, inhibited growth of S. putrefaciens in a fish-extract-agar diffusion assay but no, or only weak, antagonistic activity was seen when the medium was supplemented with iron. Sterile-filtered supernatant fluid from a siderophore-producing Pseudomonas grown in fish extract was inhibitory to S. putrefaciens if the number of Pseudomonas was above 10⁸ cfu ml⁻¹. In contrast, supernatant fluids from siderophore-negative Pseudomonas isolates did not inhibit growth of S. putrefaciens. The inhibitory effect was, except for one strain of Pseudomonas , not seen in supernatant fluids from iron-enriched cultures of Pseudomonas sp. Finally, siderophore-producing Pseudomonas sp. lowered the maximum cell level of S. putrefaciens 1–2 log units from 10⁹ to 10¹⁰ cfu g⁻¹ when the strains were grown on fish muscle blocks at 0°C but the growth rate of S. putrefaciens was not affected.     

Plasmid and chromosomally encoded luminescence marker systems for detection of Pseudomonas fluorescens in soil

Luminescent strains of Pseudomonas fluorescens 10586 were constructed in which luciferase production was constitutive by introduction of Vibrio fischeri luxABE genes on the chromosome and on a multicopy plasmid. Light production in liquid batch culture was directly proportional to biomass concentration during exponential growth and enabled detection by luminometry of 1.7 × 10³ and 8.9 × 10⁴ cells/ml for the plasmid and chromosomally marked strains, respectively. Luminescent colonies of both strains were detectable by eye, enabling viable cell enumeration on solid media against a background of non-luminescent strains. Following inoculation into sterile and non-sterile soil lower levels of detection were increased but detection of 8.1–59 × 10³and 2.2–30 × 10³ cells per g of soil was possible for plasmid and chromosomally marked strains. Maximum specific growth rate in liquid culture was unaffected by introduction of lux marker genes on the chromosome, but was reduced in the plasmid marked strain. The chromosomally encoded marker was stable in both liquid culture and in soil, but the plasmid was unstable during continuous subculturing in liquid medium and during growth in soil. The chromosomally encoded luminescence-marker system therefore provides a convenient, non-extractive technique for quantification of genetically modified soil microbial inocula.     

Quantification of pathogenic micro-organisms in the sludge from treated hospital wastewater

The sludge from hospital waste treatment facilities is a potential source of infectious organisms. The average numbers of micro-organisms in the sludge of hospital wastewater in Taiwan were as follows: total count 8·1 × 10⁷ cfu g⁻¹ (dry weight of sludge), and 1·4 × 10⁶, 3·6 × 10⁵, 1·6 × 10⁵, 2·2 × 10⁵ and 5·5 × 10⁴ cfu g⁻¹ (dry weight of sludge) for total coliforms, faecal coliforms, faecal streptococci, Pseudomonas aeruginosa and Salmonella spp., respectively . Salmonella spp. were detected in 37% (10 of 27) of the sludges from hospital wastewaters. Therefore, the treatment of such sludge to reduce pathogenic micro-organisms should be considered.     

Beauveria bassiana infection of eggs of stored-product beetles

Beauveria bassiana (Balsamo) Vuillemin was tested under maximum challenge conditions with an estimated dose of 1.1 × 10⁵ conidia/mm² for its effects on eggs of four of the major beetle pests of stored grain and grain products. When ambient relative humidity (RH) was 92%, hatch of fungus-treated Rhyzopertha dominica (Fabricius) eggs was 13% versus 58% for control eggs, and hatch of treated Tribolium castaneum (Herbst) was 17% versus 51% for controls. There was no fungus effect at RH of 48 and 73%. Fungus treatment of Cryptolestes ferrugineus (Stephens) and Oryzaephilus surinamensis (Linnaeus) eggs had no effect. Sectioned T. castaneum eggs demonstrated that the fungus penetrates and infects them.     

Differing effects of suspended sediments on the performance of native and exotic Daphnia

1. Although large cladocerans are usually uncommon in large rivers, Daphnia lumholtzi (an exotic species in North America) is widespread and occasionally abundant. We collected zooplankton on the Illinois River (Illinois, U.S.A.) in 1995 and 1996 and found that the peak density of D. lumholtzi (25 L⁻¹) typically exceeded that of all native species combined. Maximum density occurred during warm periods (up to 27 °C) when concentrations of inorganic suspended sediments were high (>50 mg L⁻¹).
2. Using a life table experiment, we examined the effects of variation in suspended sediment (0 and 80 mg L⁻¹) and food (10⁴ and 10⁵ Ankistrodesmus cells mL⁻¹) on fitness of D. lumholtzi and the native Daphnia parvula. Daphnia lumholtzi had greater survivorship than D. parvula in most treatments and higher life-time fertility than D. parvula in all treatments. Both species achieved their fastest intrinsic rates of growth in treatments with high food, but their responses to suspended solids differed. The growth rate of D. lumholtzi in high food was slightly increased by higher turbidity, whereas that of D. parvula was depressed.
3. Results suggest that the ability of D. lumholtzi to tolerate suspended solids is an important factor contributing to its success in invading North American rivers.     

Properties of the Sodium Extrusion Mechanism Controlled by Nerve Growth Factor in Chick Embryo Dorsal Root Ganglionic Cells

Abstract: Cell dissociates from embryonic chick dorsal root ganglia, incubated for 6 h with ²²Na⁺, accumulated four to six times more radioactivity in the absence than in the presence of Nerve Growth Factor (NGF). The accumulation of radioactivity paralleled the external Na⁺ concentration, indicating that the cells may have been reaching equilibrium with the medium. Delayed presentation of NGF to ²²Na⁺-loaded cells caused a rapid loss of radioactivity, even with extracellular ²²Na⁺ still present, demonstrating that NGF caused an overall efflux of Na⁺ rather than an accelerated equilibration. The Na⁺ exclusion from ²²Na⁺-loaded cells was dependent upon NGF concentration. Use of nutrient-rich medium, serum, and certain hormones and other proteins did not prevent the Na+ accumulation in the absence of NGF or its reversion by delayed NGF administration. Incubation of the ganglionic cells with ouabain or dinitrophenol during the ²²Na⁺ loading period (no NGF) increased the rate, but not the magnitude, of loading. The same incubation carried out in a Na+-free medium and followed by ²²Na⁺ presentation resulted in fast radioactive loading that was identical to that occurring in drug-free, NGF-deprived cells and was not prevented by presentation of NGF together with the ²²Na⁺. These data are consistent with a model in which NGF acts through a Na⁺ pump rather than by restricting Na+ influxes.     

Osmoregulation of fission yeast: cloning of two distinct genes encoding glycerol-3-phosphate dehydrogenase, one of which is responsible for osmotolerance for growth

Many types of microorganisms, including both prokaryotes and eukaryotes, have developed mechanisms to adapt to severe osmotic stress. In this study, we isolated multicopy suppressor genes for a Schizosaccharomyces pombe mutant, which exhibited the clear phenotype of being osmosensitive for growth (Osm^s) on agar plates containing high concentrations of either non-ionic or ionic osmotic solutes. Two genes were thus identified, and each was suggested to encode an NADH-dependent glycerol-3-phosphate dehydrogenase (GPD), which is required for glycerol synthesis. The nucleotide sequences, determined for these genes (named gpd1 ⁺ and gpd2 ⁺, respectively), revealed that S. pombe has two distinct GPD isozymes. They are only 60% identical to each other in their amino acid sequences. One such isozyme, GPD1, was shown to be directly involved in osmoregulation, based on the following observations. (i) Expression of gpd1 ⁺ was regulated at the mRNA level in response to osmotic upshift, (ii) It was demonstrated that wild-type cells markedly accumulated internal glycerol under high-osmolarity growth conditions. (iii) Δ gpd1 mutants, however, failed to do so even in a high-osmolarity medium, and thus exhibited an Osm^s phenotype. On the other hand, the gpd2 ⁺ gene was constitutively expressed at a particular low level, regardless of the osmolarity of the medium.