Isolation and characterization of an efficient nitro-reducing bacterium, <Emphasis Type="Italic">Streptomyces</Emphasis><Emphasis Type="Italic">mirabils</Emphasis> DUT001, from soil

A bacterial strain, designated as DUT001, was isolated from soil samples collected in Qingpu, Shanghai, China, and found to be capable of reducing nitro polycyclic aromatic compounds and polynitrated aromatic compounds. It was identified as Streptomyces mirabils based on morphological and physiological-biochemical properties, as well as 16S rDNA sequences and phylogenetic analysis. The optimum growth temperature and pH for Streptomyces mirabils DUT001 are 28°C and 7.2, respectively. The best carbon source and nitrogen source are glucose and yeast extract. Streptomyces mirabils DUT001 exhibited highly efficient activity in reduction of nitroaromatic compounds, 4-nitro-1, 8-naphthalic anhydride (4-NNA), 3-nitro-1, 8-naphthalic anhydride, 4-nitrophthalimide, 3-nitrophthalimide 1, 3-dinitrobenzene, and 1, 4-dinitrobenzene, to their amino derivatives with yields ranging from 70–99% under mild conditions. 1% (w/v) of the nonionic surfactant, Triton X-100 and 0.1 mM of redox mediator, anthraquinone-2, 6-disulfonate (AQDS), could accelerate the nitroreduction process. The bioreduction activity of the Streptomyces mirabils DUT001 offers its application potentials in bioremediation of nitroaromatic compounds and synthesis of important amino derivatives.     

Mechanism of Antimicrobial Effect of Quinone Compounds

The qualitative change and quantitative change of p-benzoquinone were followed in yeast suspension to obtain the information on the mechanism of the antimicrobial effect of quinone compounds. When the quinone concentration was below 0.16 mM, the quinone was found to be continuously reduced to hydroquinone by the yeast in the presence of energy substrate, whereas the concentration exceeded 0.16 mM, the reduction ability was rapidly destroyed even in the presence of energy substrate.

The critical concentration for destruction of the reduction ability was considered to be related to the toxic effect of the quinone on the yeast.     

Bacillus halotolerans strain LYSX1-induced systemic resistance against the root-knot nematode Meloidogyne javanica in tomato

Purpose

Determination of the nematicidal potential and mode of action of bacteria isolated from tobacco rhizosphere soil against the root-knot nematode Meloidogyne javanica in tomato plants.

Methods

Antagonistic bacteria were isolated from rhizosphere soil of tobacco infested with root-knot nematodes. Culture filtrate was used to examine nematicidal activity and ovicidal action of bacterial strains. Biocontrol of M. javanica and growth of treated tomato plants were assessed in pot experiments. To clarify whether secondary metabolites of bacteria in tomato roots induced systemic resistance to M. javanica, bacterial culture supernatants and second-stage juvenile nematodes were applied to spatially separated tomato roots using a split-root system. Bacterial strains were identified by 16S rDNA and gyrB gene sequencing and phylogenetic analysis.

Results

Of the 15 bacterial strains isolated, four (LYSX1, LYSX2, LYSX3, and LYSX4) demonstrated nematicidal activity against second-stage juveniles of M. javanica, and strain LYSX1 showed the greatest antagonistic activity; there was dose-dependent variability in nematicidal activity and inhibition of egg mass hatching by strain LYSX1. In vivo application of LYSX1 to tomato seedlings decreased the number of egg masses and galls and increased the root and shoot fresh weight. Treatment of half of the split-root system with LYSX1 reduced nematode penetration to the other half by 41.64%. Strain LYSX1 was identified as Bacillus halotolerans.

Conclusion

Bacillus halotolerans LYSX1 is a potential microbe for the sustainable biocontrol of root-knot nematodes through induced systemic resistance in tomato.

     

Studies on Utilization of Hydrocarbons by Yeasts

The production of microbial cell substances from hydrocarbons has been attracting attention of people for many years. Production of bacterial cell from hydrocarbons is disadvantageous because of the difficulty in separating cell from the broth.

We have tested hydrocarbon-utilizing yeasts isolated from garden soil for cell production. The effect of medium composition on yeast growth and the utilization of individual hydrocarbon by yeast, strain Y-3, were investigated.

As a nitrogen source, urea was more effective than ammonium nitrate. When a very smal! amount of corn steep liquor was added, yeast growth was very improved. Aliphatic series of hydrocarbon lower than C₉ were not or very slightly assimilated by this yeast.

Generally speaking, series of even-number hydrocarbons were more effective than those of odd-number hydrocarbons.

We found that the yeast Y-3 strain reported in the previous paper¹⁾ has a diterminal oxidation system of hydrocarbon.

This yeast capable of growing in mineral-salts solution with hydrocarbons as sole source of carbon produced a series of dioic acid from n-undecane. These acids are 1,11-undecane dioic acid, 1,9-nonane dioic acid (azelaic acid), 1,7-heptane dioic acid (pimelic acid) and 1,5-pentane dioic acid (glutaric acid). 1,10-Decane dioic acid (sebacic acid) was also isolated from n-decane cultures.

Azelaic acid was partially transformed into pimelic acid and glutaric acid by treating it with resting cells of this yeast.

1,11-Undecane dioic was also transformed into azelaic acid pimelic acid, and glutaric acid by the same treatment as described above.     

Studies on Transfer of Antiseptics to Microbes and their Toxic Effect

The adsorption of phenols and esters of acid antiseptics by the bacterial cell (Escherichia coli and Staphylococcus aureus) was investigated in relatoin to their toxic effect, and it has been observed that the definite quantity of antiseptics must be adsorbed on the solid phase of the bacterial cell in order to give the definite toxic effect, and the toxic effect is independent of the quantity dissolved in the inner cell fluid or in the lipid phase of the cell. The result shows that the toxic effect of these antiseptics on either bacteria and yeast, is exclusively limited by the adsorbed quantity.

The adsorbed quantity required for the definite toxic effect was nearly the same as that previously observed in the case of the yeast, and the mechanism of the toxic action of these antiseptics was assumed to be same with each other in any case of microbes.     

In vitro activity of Bacillus firmus against the burrowing nematode Radopholus similis,the root-knot nematode Meloidogyne incognita and the stem nematode Ditylenchus dipsaci

Antagonistic bacteria have been repeatedly shown to be promising microorganisms for the biological control of sedentary and migratory endoparasitic nematodes. Depending on the bacteria involved, the mechanisms of action include: obligate parasitism, reduction in penetration, growth inhibition due to competition for nutrients and antibiosis associated with bioactive metabolites. In the present studies, the mode of action of the antagonist bacteria Bacillus firmus, isolated from a bionematicide, was evaluated. Significant rates of paralysis and mortality were detected after incubation of three nematode species in low concentrations of the pure culture filtrates following removal of the bacterial cells. The same culture filtrates also significantly reduced hatching of Meloidogyne incognita. Pure bacterial cell suspensions added to sand also reduced survival of R. similis in bioassays by 41% over the controls. The mode-of-action responsible for nematode paralysis and mortality was therefore demonstrated to be closely associated with the production of bioactive compounds secondary metabolites by the bacteria.     

Volatile organic compounds from endophytic fungi as innovative postharvest control of Fusarium oxysporum in cherry tomato fruits

To assess their potential as biopesticides, the effect on the growth of phytopathogen Fusarium oxysporum of six volatile organic compounds from endophytic fungi was studied in vivo and in vitro; compounds were used both as a mixture and individually. In vivo studies were performed inoculating the pathogen into cherry tomatoes, while the in vitro antifungal effect was studied using agar dilution and gas phase methods. Also, the morphology of the hyphae exposed to these compounds was analyzed. Moreover, the possible mechanism of action of these compounds was determined by studying the respiration and cell membrane permeability. Results show that the compounds have a significant concentration-dependent antifungal effect individually and act in a synergic manner. Additionally, changes in cell membrane permeability, damage to the hyphal morphology, and an inhibitory effect on the respiration were observed. The mixture of the six compounds may be used for postharvest control of F. oxysporum in tomatoes.

     

In Vitro Amphotericin B Effects on Growth,Viability and Dimorphism of Paracoccidioides brasiliensis: Reversal of the Treatment

The in vitro effects of amphotericin B deoxycholate suspension (fungizone) on Paracoccidioides brasiliensis growth, cell viability and transformation were investigated. We also analyzed the protein synthesis patterns of both cellular forms, yeast and mycelium in the presence of AmB. This drug, at 30 μg/ml, highly inhibited yeast growth, which could be recovered depending on treatment time, where the most effective reversion was observed after 6 hr of incubation. The yeast cell viability, that had been partially affected by the drug, could also be efficiently recovered after AmB was removed. The effect of AmB on the cellular dimorphism process showed a strong reduction in the mycelium to yeast transformation (80% inhibition compared to the control without the drug). On the other hand, the transformation from yeast to mycelium in the presence of AmB was 50% affected, relative to the control. In contrast to the growth and cell viability experiments, the reversion effects on dimorphism were partial when the drug was removed, even with only 6 hr treatment. The two-dimensional gels of ³⁵S-labeled proteins revealed a strong reduction in the three species of 80, 71 and 56 kDa in yeast and mycelium when treated with AmB.     

Use of Gifu Anaerobic Medium for culturing 32 dominant species of human gut microbes and its evaluation based on short-chain fatty acids fermentation profiles

Recently, a “human gut microbial gene catalogue,” which ranks the dominance of microbe genus/species in human fecal samples, was published. Most of the bacteria ranked in the catalog are currently publicly available; however, the growth media recommended by the distributors vary among species, hampering physiological comparisons among the bacteria. To address this problem, we evaluated Gifu anaerobic medium (GAM) as a standard medium. Forty-four publicly available species of the top 56 species listed in the “human gut microbial gene catalogue” were cultured in GAM, and out of these, 32 (72%) were successfully cultured. Short-chain fatty acids from the bacterial culture supernatants were then quantified, and bacterial metabolic pathways were predicted based on in silico genomic sequence analysis. Our system provides a useful platform for assessing growth properties and analyzing metabolites of dominant human gut bacteria grown in GAM and supplemented with compounds of interest.     

Methods to Inhibit Bacterial Pyomelanin Production and Determine the Corresponding Increase in Sensitivity to Oxidative Stress

Pyomelanin is an extracellular red-brown pigment produced by several bacterial and fungal species. This pigment is derived from the tyrosine catabolism pathway and contributes to increased oxidative stress resistance. Pyomelanin production in Pseudomonas aeruginosa is reduced in a dose dependent manner through treatment with 2-[2-nitro-4-(trifluoromethyl)benzoyl]-1,3-cyclohexanedione (NTBC). We describe a titration method using multiple concentrations of NTBC to determine the concentration of drug that will reduce or abolish pyomelanin production in bacteria. The titration method has an easily quantifiable outcome, a visible reduction in pigment production with increasing drug concentrations. We also describe a microtiter plate method to assay antibiotic minimum inhibitory concentration (MIC) in bacteria. This method uses a minimum of resources and can easily be scaled up to test multiple antibiotics in one microtiter plate for one strain of bacteria. The MIC assay can be adapted to test the affects of non-antibiotic compounds on bacterial growth at specific concentrations. Finally, we describe a method for testing bacterial sensitivity to oxidative stress by incorporating H₂O₂ into agar plates and spotting multiple dilutions of bacteria onto the plates. Sensitivity to oxidative stress is indicated by reductions in colony number and size for the different dilutions on plates containing H₂O₂ compared to a no H₂O₂ control. The oxidative stress spot plate assay uses a minimum of resources and low concentrations of H₂O₂. Importantly, it also has good reproducibility. This spot plate assay could be adapted to test bacterial sensitivity to various compounds by incorporating the compounds in agar plates and characterizing the resulting bacterial growth.     

Antibacterial activity of resin based coatings containing marine microbial extracts

Bioactive agents produced by marine surface‐associated bacteria have been incorporated into experimental antifouling coatings. It was found that coating formulation can affect the action spectra of bacterial extracts involved. Little work has been done on the effect of permeability on the leaching of bioactive compounds from antifouling coatings. The formulations with 1.5% and 1.0% alginate produced significantly larger zones (p < 0.05) than with 0.5% alginate. Test formulations continued to leach active compounds after continuous immersion in seawater for 120 h. The bacterial strains used to produce the active compounds were identified by biochemical tests and/or the analysis of the DNA from a 500 bp section of the gene coding for the 16S rRNA subunit. Strains used were tentatively identified as Planococcus citreus, seven Bacillus strains (including three Bacillus pumilus and three Bacillus lautus strains), Cytophaga fucicola, Cytophaga uliginosa and a Pseudoalteromonas spp.     

Identification of rosmarinic acid and sulfated flavonoids as inhibitors of microfouling on the surface of eelgrass Zostera marina

A bioassay-guided approach was used to identify defense compounds that are present on the surface of Zostera marina and which inhibit settlement of microfoulers at natural concentrations. Moderately polar eelgrass surface extracts inhibited the settlement of seven marine bacteria and one yeast that originated from non-living substrata. In contrast, five other bacterial strains that had been directly isolated from eelgrass surfaces were all insensitive, which suggested a selective effect of surface metabolites on the microbial communities present on eelgrass. Bioassay-guided isolation of active compounds from the extracts in combination with UPLC-MS and ¹H-NMR spectroscopy resulted in the identification of rosmarinic acid, luteolin-7-sulfate and diosmetin-7-sulfate or its isomer chrysoeriol-7-sulfate. All three compounds are nontoxic repellents, as they did not inhibit bacterial growth, but prevented bacterial settlement in a dose-dependent manner. Between 15.6 and 106.8 μg ml^?1 of rosmarinic acid were present on the eelgrass surface, enough for half maximal settlement inhibition of bacteria.     

Escherichia coli Growth Changes by the Mediated Effects After Low-Intensity Electromagnetic Irradiation of Extremely High Frequencies

Water is the major constituent of environmental medium and biological systems. The effects occurring in water as a result of low-intensity electromagnetic irradiation (EMI) in extremely high frequencies are supposed to be the primary mechanism to create conditions for biological responses. The EMI effects on Escherichia coli, after irradiation of their suspension, are most probably water-mediated. Indirect effects of EMI at 51.8, 53, 70.6, and 73 GHz frequencies on bacteria, through water, assay buffer (Tris–phosphate buffer with inorganic salts at low or moderate concentrations), or peptone growth medium were studied. The mediated effects of 70.6 and 73 GHz irradiated water, assay buffer, and growth medium on E. coli growth characteristics were insignificant. But the results were different for 51.8 and 53 GHz. EMI mediated effects on bacterial growth were clearly demonstrated. The effects were more strongly expressed with 53 GHz. Moreover, it was shown that 70.6 and 73 GHz similarly suppressed the cell growth after direct irradiation of E. coli in water or on solid medium. Interestingly, for 51.8 and 53 GHz the bacterial growth decreases after suspension irradiation was less, compared to the direct irradiation of bacteria on solid medium. Especially, it was also more expressed in case of 53 GHz. Also with electron microscopy, EMI-induced bacterial cell sizes and structure different changes were detected. In addition, the distinguished changes in surface tension, oxidation–reduction potential and pH of water, assay buffer, growth medium, and bacterial suspension were determined. They depended on EMI frequency used. The differences could be associated with the partial absorbance of EMI energy by the surrounding medium, which depends on a specific frequency. The results are crucial to understand biophysical mechanisms of EMI effects on bacteria.     

Fluorescent assay based on resazurin for detection of activity of disinfectants against bacterial biofilm

A new, quick method, using the resazurin dye test as a bacterial respiration indicator, has been developed to assay the antibacterial activity of various substances used as disinfectants against bacterial biofilm growth on clinical devices. Resazurin was used to measure the presence of active biofilm bacteria, after adding disinfectant, in relation to a standard curve generated from inocula in suspension of the same organism used to grow the biofilm. The biofilm was quantified indirectly by measuring the fluorescent, water-soluble resorufin product produced when resazurin is reduced by reactions associated with respiration. Four products used as disinfectants and the biofilm growth of five bacterial species on carriers made of materials commonly found in clinical devices were studied. Under test conditions, chlorhexidine, NaOCl, ethanol, and Perasafe at concentrations of 0.2, 0.01, 350, and 0.16 mg/ml, respectively, all produced 5-log reductions in biofilm cell numbers on the three different carriers. The redox-driven test depends on bacterial catabolism, for which reason resazurin reduction produces an analytic signal of the bacterial activity in whole cells, and therefore could be used for determining disinfectant efficacy in an assay based on the metabolic activity of microorganisms grown as biofilm or in suspension.     

Transport of bacteria in porous media: I. An experimental investigation

The convective transport of concentrated suspension of bacteria in porous media is of interest for several processes such as microbial enhanced oil recovery and in situ bioremediation. The parameters which affect the transport of the bacterium Bacillus licheniformis JF-2, a candidate microorganism for microbial enhanced oil recovery, were investigated experimentally in sandpacks. Bacteria retention and permeability reduction occurred primarily in the first few centimeters upon entering the porous medium. In downstream sections of the sandpack, the permeability reduction was low, even in cases in which high cell concentrations (10(8) cfu/mL) were detected in the effluent. The effect of (i) addition of a dispersant, (ii) linear velocity of injection, (iii) cell concentration, (iv) salinity (v) temperature, and (vi) the presence of a residual oleic phase were determined experimentally. A lower reduction in permeability and a higher effluent bacterial concentration were obtained in the presence of dispersant, high injection velocities, low salinities, and at a higher temperature. Macroscopic measurements at different linear velocities and in the presence or absence of dispersants suggest that the formation of reversible microaggregates and multiparticle hydrodynamic exclusion may be the primary mechanisms for bacterial retention and permeability reduction. (c) 1994 John Wiley & Sons, Inc.     

Identification of a potent indigoid persister antimicrobial by screening dormant cells

The subpopulation of bacterial cells that survive myriad stress conditions (e.g., nutrient deprivation and antimicrobials) by ceasing metabolism, revive by activating ribosomes. These resuscitated cells can reconstitute infections; hence, it is imperative to discover compounds which eradicate persister cells. By screening 10,000 compounds directly for persister cell killing, we identified 5-nitro-3-phenyl-1H-indol-2-yl-methylamine hydrochloride (NPIMA) kills Escherichia coli persister cells more effectively than the best indigoid found to date, 5-iodoindole, and better than the DNA-crosslinker cisplatin. In addition, NPIMA eradicated Pseudomonas aeruginosa persister cells in a manner comparable to cisplatin. NPIMA also eradicated Staphylococcus aureus persister cells but was less effective than cisplatin. Critically, NPIMA kills Gram-positive and Gram-negative bacteria by damaging membranes and causing lysis as demonstrated by microscopy and release of extracellular DNA and protein. Furthermore, NPIMA was effective in reducing P. aeruginosa and S. aureus cell numbers in a wound model, and no resistance was found after 1 week. Hence, we identified a potent indigoid that kills persister cells by damaging their membranes.     

Synergistic Effect of 5-Nitro-2′-deoxyuridine with Ganciclovir Against Human Cytomegalovirus In Vitro

Abstract

In this paper we describe a practical synthesis of 5-nitro-2′-deoxyuridine (4) and 1-(2-deoxy-2-fluoro-β-D-arabinofuranosyl)-5-nitrouracil (11). These compounds were then evaluated for their ability to inhibit the growth of human cytomegalovirus (HCMV, strain AD169) in MRC-5 cells using a plaque reduction assay. Compound 11 was unable to inhibit the growth of HCMV at the highest concentration tested (100 μg/mL). However, compound 4 (5-NO₂-dU) exhibited marginal activity against HCMV in vitro in a dose-dependent manner with a 50% inhibitory concentrations (IC₅₀) of 1 to 5 μg/mL. Combinations of 5-NO₂-dU with ganciclovir synergistically inhibited HCMV induced cell killing in culture.     

Thymidine incorporation of bacteria sequentially extracted from soil using repeated homogenization-centrifugation

Bacteria were sequentially extracted from soil into a water suspension after shaking soil with water or mixing it in a blender followed by a low-speed centrifugation. Bacteria, which were released only after several cycles of homogenization-centrifugation, had higher growth rates as judged from thymidine and leucine incorporation, whereas bacteria that were more readily released by a gentle shaking procedure had the lowest growth rate. This indicated that bacteria more tightly bound to soil particles were growing faster than those that were more easily released into the water suspension. The same pattern was found both in an agricultural and a forest soil, with contrasting pH and organic matter content, and irrespective of whether the bacteria were labeled before or after the centrifugation steps. The different growth rates of the bacteria could not be explained by different partitioning of label between different macromolecules, different cell size, different viability of the bacteria, or different dilution of the added radioactive substrate in the different homogenization-centrifugation fractions. The total amount of phospholipid fatty acids per bacterial cell was also similar in the different fractions. Different composition of the bacterial communities in the different homogenization-centrifugation fractions was indicated by a gradually altered phospholipid fatty acid pattern of the extracted bacteria, and an increased hydrophobicity of the bacteria released only after several homogenization-centrifugation treatments. Correspondence to: E. Bååth     

Extracellular Metabolites of Hydrocarbon-Oxidizing Bacteria as Substrates for Sulfate Reduction

The relationship between bacterial oxidation of hydrocarbons and sulfate reduction was studied in an experimental system with liquid paraffin used as a source of organic compounds inoculated with silt taken from a reservoir. Pseudomonads dominated in the hydrocarbon-oxidizing silt bacteriocenosis. However, Rodococcusand Arthrobacteria amounted to no more than 3%. Arthrobacteria dominated the microbial association formed in the paraffin film of the model system. Sulfate-reducing bacteria were represented by genera Desulfomonas, Desulfotomaculum, and Desulfovibrio. The growth of sulfate-reducing bacteria in media containing paraffin, successive products of its oxidation (cetyl alcohol, stearate, and acetate), and extracellular metabolites of hydrocarbon-reducing bacteria was studied. The data showed that sulfate-reducing bacteria did not use paraffin or cetyl alcohol as growth substrates. However, active growth of sulfate-reducing bacteria was observed in the presence of stearate and extracellular water-soluble or lipid metabolites of Arthrobacteria.     

Inhibitory Effect of Bacterial Attachment on Candidal Growth Due to Adherence with Mannose-Sensitive Pili

A bacterial strain with affinity to Candida albicans was successfully obtained from a natural environment. An uncovered Petri dish containing a suspension of heat-killed C. albicans cells was allowed to stand in a laboratory for several days. Some bacteria which had adhered to the candidal cells were tested for their ability to agglutinate the cells. A bacterial strain, designated later as CAB-1, was found to agglutinate candidal cells through bridging by mannose-sensitive pili. CAB-1 showed similar bacteriological characteristics to those of Citrobacter freundii by ID test. The adherence of CAB-1 to candidal cell was precisely presented by scanning electron microscopy. The inhibitory effect of CAB-1 attachment to candidal cells on the growth of Candida was also preliminarily confirmed.