Enantioselective analysis of ofloxacin enantiomers by partial-filling capillary electrophoresis with bacteria as chiral selectors

The enantiomeric separation of ofloxacin enantiomers (OFLX) was achieved by using capillary electrophoresis partial-filled with Escherichia coli, Pseudomonas aeruginosa (Gram-negative), and Staphylococcus aureus (Gram-positive) as chiral selectors. Experimental parameters, including the concentration of background electrolyte, applied voltage, length of the filled bacteria plug, and pH of the buffer, were intensively investigated. Baseline separation of OFLX could be achieved within 7 min by using E. coli and P. aeruginosa as chiral selectors under the following conditions: electrophoretic buffer composed of 10 mM phosphate buffer at pH 7.4, applied voltage at 15 kV, and the bacteria (6.0 × 10(8) cells/mL) were injected into the capillary by gravity with injection height of 17.5 cm for 180 s (E. coli), 300 s (P. aeruginosa), and 300 s (S. aureus), respectively. E. coli and P. aeruginosa had better chiral selectivity for OFLX than S. aureus, which was in good agreement with OFLX having better antimicrobial activity on Gram-negative rather than Gram-positive bacteria. A novel method was developed for the enantioselective separation of enantiomers using bacteria as chiral selectors, which provides a new approach for antimicrobials enantioselective analysis, chiral pharmacodynamics, and chiral pharmacokinetics studies.     

STRUCTURE-ACTIVITY RELATIONSHIP OF PORPHINES FOR PHOTOINACTIVATION OF BACTERIA

Abstract— The antibacterial photodynamic effects of uncharged ( o -tetrahydroxyphenyl porphine [THPP], m -THPP and p -THPP), cationic (5,10,15,20-tetra[4- N -methylpyridyllporphine [TMPyP]) and anionic (5,10,15,20-tetra[4-sulfonatophenyl porphine] [TPPS₄]) porphines on Staphylococcus aureus and Escherichia coli bacteria inactivation were examined. The results show that uncharged porphines provoked antibacterial photodynamic activity on S. aureus, and also on E. coli in the presence of the membrane-disorganizing peptide polymixin B nonapeptide (PMNP). The TMPyP compound was highly photoactive toward gram-positive bacteria but only marginally effective on gram-negative cells, whereas TPPS₄ showed no activity on either gram-positive or gram-negative bacteria. The photoactivity of TMPyP is due to the electrostatic attraction between the positively charged sensitizer molecule and the negatively charged membrane of the gram-positive target cells. For TPPS₄, the inactivity toward gram-positive bacteria is due to electrostatic repulsion between the charged sensitizer molecule and the cell membrane. For gram-negative bacteria, the inactivity is conceivably due to preferential (electrostatic) binding to the positively charged PMNP, which is an adjuvant for membrane disorganization, but has no effect on cell viability. For hydrophobic sensitizers, the photoactivity depends on the state of aggregation. The extent of deaggregation of the different THPP isomers was determined by fluorescence measurements of bound sensitizers and could be positively correlated with their photoinactivation capacity. We conclude that the structure-activity relationships of these porphines are affected by their net charge and by aggregation.     

Flow cytometric viability assessment and transmission electron microscopic morphological study of bacteria in glycerol.

Human cadaveric skin allografts are used in the treatment of burns and can be preserved in glycerol at high concentrations. Previously, glycerol has been attributed some antimicrobial effect. In an experimental set-up, we aimed at investigating this effect of prolonged incubation of bacteria in 85% glycerol. Staphylococcus epidermidis, Staphylococcus aureus, Pseudomonas aeruginosa, and Bacillus subtilis were incubated in 85% glycerol. The influence of duration of incubation and temperature on ultrastructure and viability were investigated. Unstressed cultures served as controls. Survival was studied after 24-36 h and 10 days incubation in 85% glycerol at 4 degrees C and 36 degrees C with transmission electron microscopy (TEM) and flow cytometry using viability stains indicating membrane damage (SYTO9, propidium iodide) or esterase activity (carboxyfluorescein diacetate). TEM clearly demonstrated variability in morphological changes of bacteria suggesting different mechanisms of damage. Viability stains supported these findings with faster declining viable cell populations in 85% glycerol at 36 degrees C compared with 4 degrees C. Both methods demonstrated that Gram-negative species were more susceptible than Gram-positive species. In conclusion, 85% glycerol may have some additional antimicrobial effect. Temperature is an important factor herein and Gram-negatives are most susceptible. The latter finding probably reflects the difference in cell wall composition between Gram-positive and Gram-negative bacteria.     

Vertical immobilization of viable bacilliform bacteria into polypyrrole films

We have successfully developed a novel technique for inserting viable bacilliform bacteria into polypyrrole films. All of the five different bacterial cells (Pseudomonas aeruginosa, Acinetobacter calcoaceticus, Serratia marcescens, Bacillus subtilis and Escherichia coli) studied in this work were inserted normal to the film surface, and the viability of P. aeruginosa was unaffected by this immobilization procedure. It was also found that the polypyrrole layer was important to keep the cells alive.     

Essential oil analysis and antibacterial activity of Rosmarinus tournefortii from Algeria

The volatile compounds obtained by hydrodistillation of the aerial parts of Rosmarinus tournefortii De Noé. growing wild in the occidental region of Algeria were analyzed by GC/MS. Thirty-six compounds were characterized representing 95.6% of the essential oil, with camphor (37.6%), 1,8-cineole (10.0%), p-cymene-7-ol (7.8%), and borneol (5.4%) as the major components. The antimicrobial activity was evaluated against three pathogenic bacteria: Gram-negative (Escherichia coli and Pseudomonas aeruginosa) and Gram-positive (Staphylococcus aureus). The minimum inhibitory concentration (MIC; mg/mL) was determined by sub-culture on Muller Hinton agar plates. The essential oil exhibited strong antibacterial activity against E. coli and P. aeruginosa, and was also active against Staphylococcus aureus.     

Chemical constituents of the methanolic extract of leaves of Leiothrix spiralis Ruhland and their antimicrobial activity

Chemical fractionation of the methanolic extract of leaves of Leiothrix spiralis Ruhland afforded the flavonoids luteolin-6-C-β-D-glucopyranoside (1), 7-methoxyluteolin-6-C-β-D-glucopyranoside (2), 7-methoxyluteolin-8-C-β-D-glucopyranoside (3), 4'-methoxyluteolin-6-C-β-D-glucopyranoside (4), and 6-hydroxy-7-methoxyluteolin (5), and the xanthones 8-carboxymethyl-1,5,6-trihydroxy-3-methoxyxanthone (6), 8-carboxy-methyl-1,3,5,6-tetrahydroxyxanthone (7). Methanolic extract, fractions, and isolated compounds of the leaves of L. spiralis were assayed against Gram-positive (Staphylococcus aureus, Bacillus subtilis and Enterococcus faecalis) and Gram-negative bacteria (Escherichia coli, Pseudomonas aeruginosa, Salmonella setubal and Helicobacter pylori) and fungi (the yeasts Candida albicans, C. tropicalis, C. krusei and C. parapsilosis). We observed the best minimum inhibitory concentration values for the methanolic extract against Candida parapsilosis, for the fraction 5 + 6 against Gram-negative bacteria E. coli and P. aeruginosa, and compound 7 against all tested Candida strains. The methanolic extract contents suggest that this species may be a promising source of compounds to produce natural phytomedicines.     

Antimicrobial properties of some bis(iminoacenaphthene (BIAN)-supported N-heterocyclic carbene complexes of silver and gold

The AgCl, AgOAc, AuCl, and AuOAc complexes of the new bis(imino)acenaphthene(BIAN)-supported N-heterocyclic carbene ligand and the precursor imidazolium salt have been investigated with respect to their antimicrobial activities against Staphylococcus aureus, Bacillus subtilis, Escherichia coli and Psudomonas aeruginosa. The most active antimicrobial is the precursor imidazolium salt, which has a minimum inhibitory concentration (MIC) value of <40 μg/mL. The MIC values for the silver complexes IPr(BIAN)AgCl and IPr(BIAN)AgOAc against Gram-positive S. aureus are comparable to that for AgNO?, while those against Gram-negative E. coli and P.aeroginosa are significantly larger. Similar behavior was evident for the gold acetate complex IPr(BIAN)AuOAc. However, in the case of the gold chloride analogue, the MIC values are virtually identical for both the Gram-positive and the Gram-negative bacteria.     

Adaptation to UVA radiation of E. coli growing in continuous culture

Adaptive responses of bacteria to physical or chemical stresses in the laboratory or in the environment are of great interest. Here we investigated the ability of Escherichia coli growing in continuous culture to adapt to UVA radiation. It was shown that E. coli indeed expressed an adaptive response to UVA irradiation at an intensity of 50W/m(2). Cells grown in continuous culture with complex medium (diluted Luria Bertani broth) at dilution rates of 0.7h(-1), 0.5h(-1) and 0.3h(-1) were able to maintain growth under UVA irradiation after a transient reduction of specific growth rate and recovery. In contrast, slow-growing cells (D=0.05h(-1)) were unable to induce enough protection capacity to maintain growth under UVA irradiation. We propose that faster growing E. coli cells have a higher adaptive flexibility to UVA light-stress than slow-growing cells. Furthermore it was shown with flow cytometry and viability stains that at a dilution rate of 0.3h(-1) only a small fraction (1%) of the initial cell population survived UVA light-stress. Adapted cells were significantly larger (30%) than unstressed cells and had a lower growth yield. Furthermore, efflux pump activity was diminished in adapted cells. In a second irradiation period (after omitting UVA irradiation for 70h) adapted cells were able to trigger the adaptive response twice as fast. Additionally, this study shows that continuous cultivation with direct stress application allows reproducible investigation of the physiological and possibly also molecular mechanisms during adaptation of E. coli populations to UVA light.     

Availability of 6-hydroxynicotinic acid for rapid identification of Pseudomonas aeruginosa and Serratia marcescens

Gas chromatography--mass spectrometry has been used to identify specific metabolites produced by Gram-negative bacteria such as Pseudomonas aeruginosa, Serratia marcescens, Klebsiella pneumoniae and Escherichia coli in a defined medium. 6-Hydroxynicotinic acid was detected in spent culture media of Pseudomonas aeruginosa and Serratia marcescens, but could not be detected in those of Klebsiella pneumoniae and Escherichia coli. The production of 6-hydroxynicotinic acid was recognized by the addition of nicotinic acid in urine with Pseudomonas aeruginosa or Serratia marcescens, but not without the addition of nicotinic acid. Among 10(5) Pseudomonas aeruginosa per 1 ml of urine (criteria for the diagnosis of urinary tract infection), 0.15 microgram of 6-hydroxynicotinic acid was detected in urine at 4 h incubation with nicotinic acid at the optimum pH of 6.9, 38 degrees C. The production of 6-hydroxynicotinic acid was proportional to the number of the bacteria and displayed a time dependency. These results suggest that the availability of 6-hydroxynicotinic acid might make for more rapid identification of bacteria than current methods.     

A new antibacterial compound produced by an indigenous marine bacteria--fermentation, isolation, and biological activity

The use of microorganisms for biological purpose has become an effective alternative to control pathogens. A marine bacterium Pseudomonas aeruginosa was isolated from Eal fish of Baluchistan coast of Pakistan. This strain produced a bactericidal antibiotic against environmental and clinical isolates. In this study, we purified bactericidal antibiotic from the ethyl acetate extract of the cells of P. aeruginosa and analyzed its chemical structure. Based on spectrometric analysis, this compound 1 is proposed to be 1-methyl-1,4 dihydroquinoline and is active against methicillin-resistant Staphylococcus aureus (MRSA), methicillin-sensitive S. aureus (MSSA), Salmonella typhi, Shigella flexneri, Escherichia coli, Proteus mirabilis, Vibrio aliginolyticus, Micrococcus luteus, Enterococcus faecalis, Enterobacter faecium but it is not active against G streptococci, Candida albicans, Aspergillus niger. Minimal inhibitory concentration for Gram-positive bacteria was between 50 and 75 microg mL(-1) and for Gram-negative bacteria 75-100 microg mL(-1).     

Effect of organic matter on the in vitro photoeradication of Pseudomonas aeruginosa by means of a cationic tetraaryl-porphyrin

Photodynamic therapy is emerging as an antimicrobial alternative approach; the concomitant presence of a photosensitizer (PS), O(2) and visible light induces lethal oxidative damages to bacterial cells. Among Gram-negative bacteria, Pseudomonas aeruginosa seems to be one of the least susceptible to photodynamic treatment. In this study, we evaluated the influence of several experimental conditions on photoeradication of a planktonic culture of P. aeruginosa PAO1 by means of a tetracationic meso-arylsubstituted porphyrin (RM24). Our findings suggest that the photo-oxidative stress induced by RM24 is strictly correlated to the amount of PS bound to the cells that in turn decreases with the increasing concentrations of organic compounds in the medium. The photoeradication is dependent on PS concentrations, cellular density and light dose. RM24 was able to induce oxidative stress by means of singlet oxygen formation, although ROS formation cannot be ruled out. The standardized experimental conditions of the photospot test allowed us to evidence intraspecific PDT sensitivity differences among three strains of P. aeruginosa.     

Identification of Escherichia coli by detection of hydroquinone and uracil in the urine system

For rapid identification of Escherichia coli, changes of urinary metabolites incubated with E. coli were investigated by gas chromatography--mass spectrometry. Hydroquinone and uracil were detected and the normal urinary constituent 4-deoxythreonic acid was found to diminish in urine incubated with E. coli. Hydroquinone could not be detected in urine incubated with Klebsiella pneumoniae, Serratia marcescens or Pseudomonas aeruginosa. Although uracil was detected in normal urine, urine incubated with E. coli showed an increased uracil level. Urine incubated with K. pneumoniae, S. marcescens or P. aeruginosa evidenced no such change. A decrease of 4-deoxythreonic acid was noted in urine incubated with S. marcescens or P. aeruginosa. In 7.0 X 10(7) cells of E. coli, 0.33-2.36 micrograms of hydroquinone and 13.4-42.0 micrograms of uracil were detected after 3 h of incubation at 38 degrees C, and production was not changed after 4, 5 or 8 h of incubation. These results suggest that the detection of hydroquinone and uracil in urine is useful for rapid identification of E. coli.     

Study on the synthesis and antimicrobial activity of novel cationic porphyrins

A novel series of quaternary ammonium cationic derivatives based on tetrapyridyl-porphyrin was synthesized.All the compounds were evaluated for their in vitro antibacterial activities against S.aureus,E.coli and P.aeruginosa,and antifungal activities against C.albicans,where microorganisms were exposed and unexposed to the irradiation.The results revealed that some of these compounds,especially,3a and 4a displayed satisfactory antibacterial activity against Gram-positive bacteria S.aureus and moderate an...     

Improvement of antibacterial activity of copper nanoclusters for selective inhibition on the growth of gram-positive bacteria

In general, copper nanoclusters (CuNCs) possess very low or even virtually no bactericidal effect. Herein,we report a novel CuNCs possessing significantly high antibacterial activity, that is tannic acid (TA)capped CuNCs (TA-CuNCs). TA-CuNCs exhibit strong absorption and excitation-dependent fluorescence within pH 2-12, resulting from the functional groups of TA-CuNCs due to two prototropic equilibria,phenolphenolate and carboxyliccarboxylate. There exists synergistic effect of TA and copper nanoclusters which endows TA-CuNCs remarkable antibacterial capability as a microbicide, as characterized by the effective inhibition on the growth of gram-positive bacteria by damaging the cell membrane. By incubating 1 x 10~7 CFU/mL of gram-positive bacteria Staphylococcus aureus and Bacillus subtilis with 30 μg/mL of TA-CuNCs for 10 min, the bacteria are completely inhibited, while under same conditions the viabilities of gram-negative bacteria Escherichia coli 0157:H7 and Pseudomonas aeruginosa remain 85.0%, 72.0%, respectively. In addition, TA-CuNCs exhibit low cytotoxicity and favorable biocompatibility demonstrated by standard methyl thiazolyl tetrazolium (MTT) assay with HepG2 and 293 Tcells, giving rise to cell viability of 94.2% for HepG2 and 96.7% for 293 T by incubating 10~6 cell/mL with 200 μg/mL of TA-CuNCs for 24 h. These results make TA-CuNCs a potential alternative as bactericide for infection treatment caused by gram-positive bacteria.     

重组质粒对大肠杆菌HB101生长影响的微量热研究

用微量热方法研究了嗜麦芽假单胞菌AT18, 受体菌大肠杆菌HB101, mel基因工程菌——大肠杆菌HB101/pWSY8和携带克隆载体pUC18质粒的大肠杆菌HB101等的生长代谢过程. 实验结果从热化学和热动力学上阐明了细菌的生长速率常数与其所含质粒的大小呈负相关. 探讨了低温处理对含不同质粒大肠杆菌生长的影响, 发现低温处理对工程菌生长影响最大.     

Highly effective contact antimicrobial surfaces via polymer surface modifiers

Contact antimicrobial coatings with poly(alkylammonium) compositions have been a subject of increasing interest in part because of the contribution of biocide release coatings to antibiotic resistance. Herein, a concept for antimicrobial coatings is developed on the basis of the thermodynamically driven surface concentration of soft block side chains. The concept incorporates structural and compositional guidance from naturally occurring antimicrobial proteins and achieves compositional economy via a polymer-surface modifier (PSM). To implement this concept, polyurethanes were prepared having random copolymer 1,3-propylene oxide soft blocks with alkylammonium and either trifluoroethoxy or PEGlyted side chains. Six carbon (C6) and twelve carbon (C12) alkylammonium chain lengths were used. The PSMs were first tested as 100% coatings and were highly effective against aerosol challenges of Gram-negative bacteria (Pseudomonas aeruginosa and Escherichia coli). To evaluate the surface concentration, solutions containing 2 wt % PSM with a conventional polyurethane were evaporatively coated onto glass slides. These 2% PSM coatings were tested against aerosol challenges of Gram-negative (Pseudomonas aeruginosa and Escherichia coli) and Gram-positive (Staphylococcus aureus) bacteria (107 CFU/mL/30 min). A copolymer soft block containing trifluorethoxy (89 mol %) and C-12 alkylammonium (11 mol %) side chains gave the highest biocidal effectiveness in 30 min: 2 wt %, Gram(+/-) bacteria, 100% kill, and 3.6-4.4 log reduction. A zone of inhibition test showed no biocide release for PSMs and PSM-modified compositions. Characteristics that contribute to concept validation include good hard block/soft block phase separation, a cation/co-repeat group ratio mimicking natural biocidal proteins, a semifluorinated "chaperone" aiding in alkylammonium surface concentration, and a low Tg for the alkylammonium soft block.     

Novel des-fatty acyl-polymyxin B derivatives with Pseudomonas aeruginosa-specific antimicrobial activity

Polymyxin B (PMB) is a cationic cyclic decapeptide antibiotic with a fatty acyl (FA) modification at the α-amino group of Dab1 (Dab: L-α,γ-diaminobutyric acid). In this study, which is part of a series of PMB structure-activity relationship investigations focused on identifying clinically useful peptide antibiotics, we synthesized ten des-FA PMB derivatives whose N-terminal moieties were changed to basic or hydrophilic amino acids. The antimicrobial and lipopolysaccharide (LPS) binding activities of these synthetic analogs were tested. The analogs showed more potent antimicrobial activity against Pseudomonas aeruginosa (P. aeruginosa) compared with the PMB nonapeptide. In particular, [Ser2-Dap3]-PMB(2-10), Guanyl-[Thr2-Dab3]-PMB(2-10), Guanyl-[Dab1-Thr2-Dab3]-PMB(1-10), and N(α,γ)-diguanyl-[Dap3]-PMB(3-10) had antimicrobial activity equivalent to PMB. In LPS binding assays, the displacement curves shifted in a manner proportional to the number of positive charges available to bind to Escherichia coli (E. coli) and P. aeruginosa. Furthermore, peptides with basic side chains were comparable to PMB in binding activity assays against E. coli and P. aeruginosa. The acute toxicities of the peptides were evaluated by intravenously administering the peptides to mice through the tail vein. The toxicities of [Ser2-Dap3]-PMB(2-10), [Dap3]-PMB(3-10), and [Ser3]-PMB(3-10) were lower that of PMB (LD??, 4.8 μmol/kg).     

Composition and antimicrobial activity of the essential oil of Actinidia macrosperma from China

Actinidia macrosperma is a medicinal plant in China and has been well known for its attraction to cats and activities against leprosy and cancers. The compositions and the antimicrobial activity of its leaf oil were reported for the first time. The oil obtained by hydrodistillation and analyzed by GC and GC-MS, was characterized by the high content of monoterpenes. Linalool (48.14%) is the major component identified, followed by 1,2-dimethyl-lindoline (7.94%), linolenic acid methylester (6.57%) and (E)-phytol (5.29%). The antimicrobial activity of the oil was evaluated against four bacterial and three fungal species. The results showed that it exhibited a mild antibacterial activity against two Gram-positive bacteria (Staphylococcus aureus and Bacillus subtilis), a significant activity against Gram-negative bacteria (Escherichia coli), and no activity on Pseudomonas aeruginosa. The test fungi were more sensitive to the oil, with a MIC range of 0.78-1.56 microL mL(-1) than bacteria in the range which were significantly higher from 0.78 to 25.50 microL mL(-1).     

Antimicrobial activity and pollen composition of honey samples collected from different provinces in Turkey

The antibacterial activity of honey samples from different sources were collected and investigated against Bacillus cereus, Staphylococcus aureus ATCC 25923, Pseudomonas aeruginosa ATCC 27853, Klebsiella pneumoniae ATCC 27736, Morganella morganii, Micrococcus luteus NRRL B-4375, Escherichia coli ATCC 35218, and Candida albicans. Pathogens exhibited different sensitivities towards the honey samples. The results showed that majority of the honey samples (75%) generally inhibitied the bacteria tested. The honey samples which were obtained from Izmir (samples 1 and 2) proved more effective as inhibitors against P. aeruginosa, E. coli, and S. aureus. The honey which was obtained from Mu?la (sample 5) exhibited high anticandidal activity on C. albicans. A comparison of the honey samples on the basis of pollen content revealed that they were heterofloral, and samples which had highest antibacterial activity against P. aeruginosa, E. coli, and S. aureus were dominated by pollen from Chenopodiaceae/Amaranthaceae (sample 1), and Trifolium, Trigonella, Cyperaceae, Zea mays and Anthemis taxa (sample 2). The honey proved more effective on bacteria than antibiotics.     

DCAP: a broad-spectrum antibiotic that targets the cytoplasmic membrane of bacteria

Persistent infections are frequently caused by dormant and biofilm-associated bacteria, which often display characteristically slow growth. Antibiotics that require rapid cell growth may be ineffective against these organisms and thus fail to prevent reoccurring infections. In contrast to growth-based antimicrobial agents, membrane-targeting drugs effectively kill slow-growing bacteria. Herein we introduce 2-((3-(3,6-dichloro-9H-carbazol-9-yl)-2-hydroxypropyl)amino)-2-(hydroxymethyl)propane-1,3-diol (DCAP), a potent broad-spectrum antibiotic that reduces the transmembrane potential of Gram-positive and Gram-negative bacteria and causes mislocalization of essential membrane-associated proteins, including MinD and FtsA. Importantly, DCAP kills nutrient-deprived microbes and sterilizes bacterial biofilms. DCAP is lethal against bacterial cells, has no effect on red blood cell membranes, and only decreases the viability of mammalian cells after ≥6 h. We conclude that membrane-active compounds are a promising solution for treating persistent infections. DCAP expands the limited number of compounds in this class of therapeutic small molecules and provides new opportunities for the development of potent broad-spectrum antimicrobial agents.