Chip-calorimetric evaluation of the efficacy of antibiotics and bacteriophages against bacteria on a minute-timescale

Rapid detection of antibiotic resistances of clinical bacterial strains would allow an early selective antibiotic therapy and a faster intervention and implementation of infection control measurements. In clinical practice, however, conventional antibiotic susceptibility tests of bacteria often need 24 h until the results are obtained. The metabolic heat production of bacteria is an excellent possibility to record their physiological activities and could therefore be used for a rapid discrimination of bacterial strains which are resistant or non-resistant to antibiotics and also to lytic bacteriophages, respectively. Unfortunately, conventional calorimeters suffer from need of comparably large volumes of bacterial suspensions are characterised by slow operation and high costs which restrict their application in clinical laboratories. The present paper demonstrates that a new type of calorimeters developed on silicon-chip technology enables the detection of antibiotic resistances on a minute-timescale. For this reasons, a prototype chip calorimeter was used which sensitivity is 20 nW related to the heat production of about 10⁴ bacteria. For a clear discrimination of antibiotic resistance about 10⁵ bacteria are required. The antibiotic resistances and susceptibilities of different strains of Staphylococcus aureus to cefoxitin and the sensitivities of S. aureus DSM 18421 and E. coli DSM 498 to a mixture of two bacteriophages were studied. Comparing the heat productions of cultures incubated with antibiotics or bacteriophages to those without these antibacterial preparations enabled a clear discrimination of resistant and non-resistant strains already after totally 2 h.     

Screening of potential antibiotic action of cellulolytic fungi

Twenty different strains of filamentous fungi were initially selected for evaluation of cellulolytic activity using a single test in a simple mineral salts culture medium with filter paper as the only carbon source. Those fungi strains that were capable of completely breaking the filter paper strip within 4–8 d were assayed also for antimicrobial action, using Staphyloccocus a ureus ATCC 6538P according to the so-called agar piece method. We screened three different strains with both capacities: the production of cellulolytic activity and antibiotic action. The experimental results suggest that the fungi Pinicillium sp. FOPCO1, Aspergillus sp. F0Q001, and Cephalosporium sp. F03800 have both capabilities because they grew rapidly on cellulose as the only carbon source and were able to produce an area of growth inhibition in S. aureus of approx 2.04, 1.57, and 2.39 cm, respectively, on agar plates using the agar piece method. Subsequently, the antibiotic production obtained with those cellulolytic strains was evaluated by submerged fermentation at the flask level, in a simple culture medium containing lactose without biosynthesis precursor, obtaining 3670, 2830, and 4060 antibiotic units/mL, referred to as penicillin G, whereas for cellulolytic activity, the results were 1.34, 1.81 and 0.57 FPU/mL, respectively.     

Extraction of Graphene Nanostructures from Colocasia esculenta and Nelumbo nucifera Leaves and Surface Functionalization with Tin Oxide: Evaluation of Their Antibacterial Properties

Expeditious evolution of antimicrobial resistance in recent years has been identified as a growing concern by various health organizations around the world. Herein, facile and environmentally benign production of highly antibacterial carbonaceous nanomaterials from Colocasia esculenta and Nelumbo nucifera leaves is reported. After carbonization and oxidative treatment, smaller graphene domains are formed in Colocasia esculenta derivatives, whereas larger sheetlike structures are observed in the case of Nelumbo nucifera. Smaller particle size makes quantum confinement effects more prominent, as is evident in fine-tuning of the photoluminescence emission after each stage of treatment. The influence of precursor materials on the antibacterial properties of the nanosystems is also demonstrated. When microbiocidal activity was tested against model bacteria Pseudomonas aeruginosa, the nanocomposite derived from Colocasia esculenta leaves showed higher activity than the antibiotic drug clarithromycin (control) with a measured zone of inhibition of 40±0.5 mm. This is one of the highest values reported in comparison with plant-based carbon–silver nanosystems. Quantitative analysis revealed that the nanocomposite obtained from Colocasia esculenta leaves has antimicrobial efficacy equivalent to those of commercial antibiotic drugs and is able to eradicate bacteria at much lower concentrations than that obtained from Nelumbo nucifera leaves.     

Towards Photochromic Azobenzene-Based Inhibitors for Tryptophan Synthase

Light regulation of drug molecules has gained growing interest in biochemical and pharmacological research in recent years. In addition, a serious need for novel molecular targets of antibiotics has emerged presently. Herein, the development of a photocontrollable, azobenzene-based antibiotic precursor towards tryptophan synthase (TS), an essential metabolic multienzyme complex in bacteria, is presented. The compound exhibited moderately strong inhibition of TS in its E configuration and five times lower inhibition strength in its Z configuration. A combination of biochemical, crystallographic, and computational analyses was used to characterize the inhibition mode of this compound. Remarkably, binding of the inhibitor to a hitherto-unconsidered cavity results in an unproductive conformation of TS leading to noncompetitive inhibition of tryptophan production. In conclusion, we created a promising lead compound for combatting bacterial diseases, which targets an essential metabolic enzyme, and whose inhibition strength can be controlled with light.     

Microcalorimetric investigation of the effect of La3+ on mitochondria isolated from avian chicken liver tissue cells

A microcalorimetric technique was used to evaluate the influence of La³⁺ on mitochondria isolated from the liver tissue of Avian chicken. By means of LKB-2277 bioactivity monitor, ampoule method at 37°C, we obtained the thermogenic curves of the metabolism of mitochondria. After isolation from the chicken liver tissue, mitochondria still have metabolic activity and can live for a long time depending on the stored nutrients. In order to analyze the results, the maximum power (P _m) and the decline rate constants (k _d) were obtained. The addition of La³⁺ results in an increase of the maximum heat production and decline rate constants. Furthermore, values of P _m and k _d are linked to the concentration of La³⁺. According to the thermogenic curves under different conditions, it is clear that metabolic mechanism of mitochondria has been changed with the addition of La³⁺. This revised version was published online in August 2006 with corrections to the Cover Date.     

Semibatch production of fructo-oligosaccharides from sucrose by immobilized cells ofAureobasidium pullulans

Aureobasidium pullulans cells with fructosyltransferase activity were immobilized in 2% calcium alginate beads, and the production of fructo-oligosaccharides from sucrose was studied in a stirred tank bioreactor. It was found that cells ofA. pullulans were entrapped evenly on the alginate matrix of 2.2 mm in diameter, and an effectiveness factor of the beads was determined to be 0.3. By comparison with the system of free cells in batch operation, the total amount of fructo-oligosaccharides produced by immobilized cells was similar although the composition of fructo-oligosaccharides was found to be different. In semibatch operation with immobilized cells, reproducible results up to 60 cycles were obtained at 50 °C and this operation resulted in no loss of activity of immobilized cells.

     

Bacitracin production by a new strain ofBacillus subtilis

A new strain ofBacillus subtilis C 126 was isolated from sugar cane fermentation and produced an antibiotic that inhibited the growth ofMicrococcus flavus. The production of the antibiotic in culture medium followed to extraction withn-butanol, thin layer chromatography, and microbiological tests indicated that a polypeptide antibiotic was produced. The fraction obtained by Sephadex G-25 column and analyzed by HPLC indicated that bacitracin complex was produced.     

Optimization of the Production Medium for Biosynthesis of Antifungal Antibiotic Ak-111-81 by Phosphate-Deregulated Mutant of <Emphasis Type="Italic">Streptomyces hygroscopicus</Emphasis>

Experimental mathematical designs were applied for optimization of a nutrient medium for biosynthesis of the antifungal antibiotic AK-111-81 by phosphate-deregulated mutant of Streptomyces hygroscopicus 111-81. Antifungal antibiotic AK-111-81 possesses well-expressed activity against Fusarium graminearum and other phytopathogenic fungi. The level of the production of the antibiotic AK-111-81 on this medium is more than three times higher than on the initial medium. The optimized quantitative composition of the nutrient culture media is (g/l): glucose, 20; soy meal, 18; ammonium succinate, 3; CaCO₃, 1.     

UPLC-MS-Based Metabolomic Study of Streptomyces Strain HCCB10043 Under Different pH Conditions Reveals Important Pathways Affecting the Biosynthesis of A21978C Compounds

The A21978C family of compounds includes precursors of daptomycin, an important antibiotic for the treatment of diseases infected by Gram-positive resistant bacteria. Focusing on these valuable compounds, the differences in metabolites obtained with or without pH control in their producing strain Streptomyces parvus HCCB10043 were investigated by comparative metabolomics analysis based on UPLC-TOF-MS technology. According to principal component analysis, there were fourteen biomarker compounds selected under the two pH culture conditions. The ten known compounds were divided into two types: a glycoside family participating in the primary metabolism (daidzein, glycitein, genistein, and soyasaponin Bb) and a peptide family of secondary metabolites (valistatin, bestatin, 3-amino-2-hydroxy-4-phenylbutanoylvalylisoleucine, and arylomycins A2, A4, and A5). Through orthogonal partial least squares-discriminant analysis, three compounds, soyasaponin Bb and arylomycins A2 and A4 were identified as the most relevant compounds to A21978C1-3 production, the glycolytic pathway, and the NRPS synthesis pathway. The competitive relationship between arylomycin and A21978C was verified. These results have demonstrated the usefulness of the metabolomic strategy based on UPLC-MS in studying significant metabolic changes in actinomycetes. Moreover, this metabolomic strategy can provide new ideas and guidance for the regulation and improvement of secondary metabolites production.     

Additive effect of Lygodium venustum SW. in association with gentamicin

The aim of this work was to evaluate the interactions between gentamicin and the ethanol extract of the fern Lygodium venustum SW (EELV). The ethanol extract of L. venustum was obtained, the phytocompounds were identified and the EELV was assayed by the checkerboard method with gentamicin against two bacterial strains multiresistant to antibiotics. The antibiotic activity of gentamicin, when associated with the extract, was enhanced in an additive manner against both strains. The results indicated that L. venustum can be a source of secondary metabolites to be used in association with antibiotics as aminoglycosides in the antibiotic chemotherapy against resistant bacteria.     

The influence of inert solids on ethanol production by Saccharomyces cerevisiae

The catalytic role of various inert solid supports on acceleration of alcohol fermentation by Saccharomyces cerevisiae was investigated. The enhanced rate of alcohol production was dependent on the nature of the support as well as on the amount used. Among all the tested supports, chitosan flakes showed the maximum yield of alcohol (93% of theoretical yield). This higher rate of alcohol production was associated with the twofold increase in the activity of alcohol dehydrogenase over control. Our results suggest that the addition of a small fraction of solids in submerged fermentations to facilitate cell anchorage for enhanced metabolic activity is easier and more economical compared to cell immobilization processes. IICT Communication No. 4266. Some of the results in this article are covered under a patent.     

The Potential of Immobilized Biocatalysts for Production of Industrial Chemicals

The successful translation from conception to practice of processes based on immobilized biocatalyst technology has been slower than anticipated. There are severe barriers, both technical and economic, limiting the introduction of immobilized biocatalyst technology to replace conventional processing procedures and processes for the production of chemicals by synthetic or fermentative routes. A small number of immobilized enzyme processes are in operation commercially, the most noteworthy being in food-related processes and in the pharmaceutical industry, where they are used for carbohydrate conversions and antibiotic transformations, respectively. There does not, as yet, appear to be any large-scale industrial application of immobilized cell technology. Examples from our laboratory—immobilized yeast for ethanol production andAspergillus niger for citric acid synthesis—illustrate the problems that have to be overcome.     

Preliminary Studies for Cephamycin C Purification Technique

A study was made for purification of cephamycin C from fermentation of Streptomyces clavuligerus. Initially, the culture broth was clarified by microfiltration and ultrafiltration, after which the resulting permeates were subjected to nonspecific adsorption and ion-exchange chromatography on resin columns. The antibiotic activity was measured by the biological method at each stage by assaying its activity against the Escherichia coli ESS, super sensitive to β-lactam antibiotic. The purification processes were assessed in relation to the variables affecting each step. The purification efficiency by nonspecific adsorption was monitored by UV spectrophotometry, while the ion-exchange adsorption fractions were assessed by NMR spectroscopy. Some of the fractions obtained during purification were also analyzed by mass spectrometry (LC/MS and LC/MS/MS) to identify the cephamycin C molecule. These preliminary results proved the process feasibility.     

Stoffwechselprodukte von Mikroorganismen. 253. Mitteilung. Hormaomycin,ein neues Peptid-lacton mit morphogener Aktivität auf Streptomyceten

Hormaomycin, a Novel Peptide Lactone with Morphogenetic Activity on Streptomyces A culture identified as Streptomyces griseoflavus (strain W-384) has been found to produce a novel peptide-lactone antibiotic designated hormaomycin ( 6 ). The empirical molecular formula of the compound is established to be C₅₅H₆₉ClN₁₀O₁₄. The constituent amino acids of the antibiotic are suggested to be allothreonine ( 1 ; 1), isoleucine ( 2 ; 1), 3-methyl-phenylalanine ( 3 ; 2), and, for the first time identified from a natural source, 4-[(Z)-prop-1-enyl]-proline ( 4 ; 1) and 3-(2-nitrocyclopropyl)-alanine ( 5 ; 2). The amino acids were delivered by acidic hydrolysis and assigned by high-resolution- GC/MS analysis (after transformation to derivatives) in combination with extended 2D-NMR experiments of the antibiotic itself. From the latter, it became plausible that the N-terminus of the peptide chain is acylated by a Cl-containing derivative of 1H-pyrrol-2-carboxylic acid. Hormaomycin is active against some Gram-positive bacteria. In addition, the antibiotic exhibits potent aerial mycelium-inducing activity and effects the production of antibiotics.     

Production of Lactic Acid from Raw Sweet Potato Powders by Rhizopus Oryzae Immobilized in Sodium Alginate Capsules

Rhizopus oryzae immobilized in calcium alginate was applied in lactic acid fermentation with unhydrolyzed raw sweet potato powders as the sole carbon source. The effects of sodium alginate concentration, calcium chloride concentration, and the immobilized bead diameter on lactic acid production were investigated. Increase in sodium alginate concentration during the gelation process would harden the immobilized capsule, which led to a decrease in lactic acid production. The increase in calcium chloride would increase the thickness of the immobilized capsule, which would increase the mass transfer resistance. Nevertheless, while the calcium chloride was lower than 15%, it would not have obvious effects on lactic acid production. A larger bead could have more space for cell growth, which led to the maximum lactic acid production observed at the 5-mm bead diameter. Moreover, results of repeated-batch operation suggested that immobilized cells could have higher stability in lactic acid production than free cells. The total cumulative lactic acid in immobilized-cell operation could increase by 55% as compared with free-cell operation after 216 h (seven repeated-batches), and no loss of amylolytic activity was observed. The results indicated that immobilized R. oryzae by Ca-alginate could be suitable for lactic acid production from unhydrolyzed raw potato powders.     

Modulation of Melanogenesis and Antioxidant Status of Melanocytes in Response to Phototoxic Action of Doxycycline

Doxycycline is a commonly used tetracycline antibiotic showing the broad spectrum of antibacterial action. However, the use of this antibiotic is often connected with the risk of phototoxic reactions that lead to various skin disorders. One of the factors influencing the photosensitivity reactions is the melanin content in melanocytes. In this study, the impact of doxycycline and UVA irradiation on cell viability, melanogenesis and antioxidant defense system in cultured normal human epidermal melanocytes (HEMn‐DP) was examined. The exposure of cells to doxycycline and UVA radiation resulted in concentration‐dependent loss in melanocytes viability and induced melanin biosynthesis. Significant changes were stated in cellular antioxidant enzymes activity: SOD, CAT and GPx, which indicates alterations of antioxidant defense system. The results obtained in vitro may explain the mechanisms of phototoxic reactions that occur in normal human epidermal melanocytes in vivo after exposure of skin to doxycycline and UVA radiation.     

Strong Antibiotic Activity of the Myxocoumarin Scaffold in vitro and in vivo

The increasing emergence of resistances against established antibiotics is a substantial threat to human health. The discovery of new compounds with potent antibiotic activity is thus of utmost importance. Within this work, we identify strong antibiotic activity of the natural product myxocoumarin B from Stigmatella aurantiaca MYX-030 against a range of clinically relevant bacterial pathogens, including clinical isolates of MRSA. A focused library of structural analogs was synthesized to explore initial structure-activity relationships and to identify equipotent myxocoumarin derivatives devoid of the natural nitro substituent to significantly streamline synthetic access. The cytotoxicity of the myxocoumarins as well as their potential to cure bacterial infections in vivo was established using a zebrafish model system. Our results reveal the exceptional antibiotic activity of the myxocoumarin scaffold and hence its potential for the development of novel antibiotics.     

Antimicrobial Activity of Water‐Soluble Triazole Phenazine Clickamers against E. coli

The antimicrobial potency of phenazine derivatives is attenuated by their inherently hydrophobic nature, complicating their use as antibiotic drugs. We have analyzed the cytotoxicity and mode of action of water‐soluble bis‐triazolyl phenazines against E. coli and a human epithelial (HaCat) cell line. We observed complete inhibition of bacterial growth over concentration ranges that do not affect the viability of human epithelial cells. Confocal fluorescence microscopy revealed a high degree of interaction between the phenazine compounds and E. coli, as well as evidence of membrane damage in phenazine‐treated E. coli. Additional data suggests that the potency of these particular water‐soluble phenazine compounds does not result from the production of reactive oxygen species, but rather from cytotoxic interference with metabolic electron‐transfer cascades.