Studies on the antibacterial activity of phanquone: chelating properties in relation to mode of action against Escherichia coli and Staphylococcus aureus

Phanquone is active against a wide range of Gram-positive and Gram-negative organisms. Its activity is affected by the nature of the suspending fluid, pH and anaerobic growth conditions. Its ability to chelate metal ions was examined and found to be related to its antibacterial activity, which was reduced by the presence of added metal ions, e.g. Co (II), Cu(II), Fe(II) and Fe(III) in nutrient media for both E. coli and S. aureus. When antibacterial activity was examined in dis-nutrient media for both E. coli and S. aureus. When antibacterial activity was examined in distilled water, then certain added metal ions, whilst antagonizing activity was examined in distilled water, then certain added metal ions, whilst antagonizing the activity of Phanquone against E. coli, exerted a co-operative effect in the case of S. aureus. The addition of EDTA and NTA lowered the activity of Phanquone against S. aureus, but not E. coli, while the addition of thiol-containing compounds lowered its activity against E. coli but not S. aureus. concentration quenching was observed for S. aureus but not for E. coli, while overnight pre-incubation at 4 degrees C resulted in the appearance of a growth zone inside the zone of inhibition in the case of S. aureus but not E. coli. Phanquone may have a different mode of action against the two organisms.     

壳聚糖抑菌机制的初步研究

壳聚糖在医学、食品、环保、日化用品等领域有着广泛而重要的应用.近年来,壳聚糖由于对不同的菌类都具有良好的抑菌效果而被研究者们密切关注.然而,有关壳聚糖抑菌机制的研究却并不多,其抑菌机制也没有被完全阐明.在本研究中,我们发现很多金属离子可以对壳聚糖的抑菌效果产生影响,高浓度金属离子(0.5%)可以使壳聚糖完全丧失抑菌活性.还发现金黄色葡萄球菌和白色念珠菌在壳聚糖的作用下会发生钾离子和ATP的渗漏,而且五万分子量的壳聚糖引起钾离子和ATP的渗漏大约比五千分子量壳聚糖多2到4倍.不同分子量的壳聚糖对金黄色葡萄球菌和白色念珠菌都具有较好的抑菌效果,但是引起钾离子和ATP的渗漏量却存在很大差异,这说明小分子量壳聚糖很可能存在与大分子量壳聚糖不同的抑菌机制.     

Preparation and important functional properties of water-soluble chitosan produced through Maillard reaction

The objective of this research was to improve the solubility of chitosan at neutral or basic pH using the Maillard-type reaction method. To prepare the water-soluble chitosans, various chitosans and saccharides were used under various operating conditions. Biological and physicochemical properties of the chitosan-saccharide derivatives were investigated as well. Results indicated that the solubility of modified chitosan is significantly greater than that of native chitosan, and the chitosan-maltose derivative remained soluble when the pH approached 10. Among chitosan-saccharide derivatives, the solubility of chitosan-fructose derivative was highest at 17.1 g/l. Considering yield, solubility and pH stability, the chitosan-glucosamine derivative was deemed the optimal water-soluble derivative. Compared with the acid-soluble chitosan, the chitosan-glucosamine derivative exhibited high chelating capacity for Zn(2+), Fe(2+) and Cu(2+) ions. Relatively high antibacterial activity against Escherichia coli and Staphylococcus aureus was noted for the chitosan-glucosamine derivative as compared with native chitosan. Results suggest that the water-soluble chitosan produced using the Maillard reaction may be a promising commercial substitute for acid-soluble chitosan.     

Preparation and antibacterial activity of chitosan nanoparticles

Chitosan nanoparticles, such as those prepared in this study, may exhibit potential antibacterial activity as their unique character. The purpose of this study was to evaluate the in vitro antibacterial activity of chitosan nanoparticles and copper-loaded nanoparticles against various microorganisms. Chitosan nanoparticles were prepared based on the ionic gelation of chitosan with tripolyphosphate anions. Copper ions were adsorbed onto the chitosan nanoparticles mainly by ion-exchange resins and surface chelation to form copper-loaded nanoparticles. The physicochemical properties of the nanoparticles were determined by size and zeta potential analysis, atomic force microscopy (AFM), FTIR analysis, and XRD pattern. The antibacterial activity of chitosan nanoparticles and copper-loaded nanoparticles against E. coli, S. choleraesuis, S. typhimurium, and S. aureus was evaluated by calculation of minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC). Results show that chitosan nanoparticles and copper-loaded nanoparticles could inhibit the growth of various bacteria tested. Their MIC values were less than 0.25 microg/mL, and the MBC values of nanoparticles reached 1 microg/mL. AFM revealed that the exposure of S. choleraesuis to the chitosan nanoparticles led to the disruption of cell membranes and the leakage of cytoplasm.     

Activation of DNA-hydrolyzing antibodies from the sera of autoimmune-prone MRL-lpr/lpr mice by different metal ions

We have shown previously that electrophoretically and immunologically homogeneous polyclonal IgGs from the sera of autoimmune-prone MRL mice possess DNase activity. Here we have analyzed for the first time activation of DNase antibodies (Abs) by different metal ions. Polyclonal DNase IgGs were not active in the presence of EDTA or after Abs dialysis against EDTA, but could be activated by several externally added metal (Me(2+)) ions, with the level of activity decreasing in the order Mn(2+)> or =Mg(2+)>Ca(2+)> or =Cu(2+)>Co(2+)> or =Ni(2+)> or =Zn(2+), whereas Fe(2+) did not stimulate hydrolysis of supercoiled plasmid DNA (scDNA) by the Abs. The dependencies of the initial rate on the concentration of different Me(2+) ions were generally bell-shaped, demonstrating one to four maxima at different concentrations of Me(2+) ions in the 0.1-12 mM range, depending on the particular metal ion. In the presence of all Me(2+) ions, IgGs pre-dialyzed against EDTA produced only the relaxed form of scDNA and then sequence-independent hydrolysis of relaxed DNA followed. Addition of Cu(2+), Zn(2+), or Ca(2+) inhibited the Mg(2+)-dependent hydrolysis of scDNA, while Ni(2+), Co(2+), and Mn(2+) activated this reaction. The Mn(2+)-dependent hydrolysis of scDNA was activated by Ca(2+), Ni(2+), Co(2+), and Mg(2+) ions but was inhibited by Cu(2+) and Zn(2+). After addition of the second metal ion, only in the case of Mg(2+) and Ca(2+) or Mn(2+) ions an accumulation of linear DNA (single strand breaks closely spaced in the opposite strands of DNA) was observed. Affinity chromatography on DNA-cellulose separated DNase IgGs into many subfractions with various affinities to DNA and very different levels of the relative activity (0-100%) in the presence of Mn(2+), Ca(2+), and Mg(2+) ions. In contrast to all human DNases having a single pH optimum, mouse DNase IgGs demonstrated several pronounced pH optima between 4.5 and 9.5 and these dependencies were different in the presence of Mn(2+), Ca(2+), and Mg(2+) ions. These findings demonstrate a diversity of the ability of IgG to function at different pH and to be activated by different optimal metal cofactors. Possible reasons for the diversity of polyclonal mouse abzymes are discussed.     

Preparation of chitosan-nylon-6 blended membranes containing silver ions as antibacterial materials

Chitosan-nylon-6 blended membranes were prepared by combining solvent evaporation and a phase inversion technique, and then used to chelate silver ions. Gram-positive bacteria (Staphylococcus aureus) and Gram-negative bacteria (Escherichia coli) were used to study the antibacterial properties of the membranes. Fourier-transform infrared spectroscopy (FTIR) and differential scanning calorimetry (DSC) indicated hydrogen-bond interactions between chitosan and nylon-6. From the scanning electron microscopy (SEM) pictures, it was observed that with the increase of nylon-6 content, the blended membrane gradually became a material with porous morphology. After chelating silver ions, the tensile strength of the membranes increased. The antibacterial activity with the variation of chitosan content, the pH value and the concentration of the silver nitrate solution used to prepare Ag(+)-loaded membranes were investigated systematically. The results indicated that the chitosan-nylon-6 blended membranes with Ag(+) were antibacterial to both Gram-positive bacteria and Gram-negative bacteria. The antibacterial activity improved with the increased chitosan content due to the larger amount of silver ions loaded. The antibacterial property of the chitosan-nylon-6 blended membranes could be primarily attributed to the content of chitosan and silver ions as well as the surface morphology of the membranes.     

Chitosan-cobalt(II) and nickel(II) chelates as antibacterial agents

Antibacterial behavior of chitosan-bivalent metal chelates (Co and Ni) was investigated in vitro against standard bacteria, Staphylococcus aureus ATCC 4533, S. faecalis ATCC 8043 and Escherichia coli ATCC 25923. The chitosan-metal chelates were prepared by varying the molar ratio of metal ions to a fixed amount of chitosan. The metal ion contents, structural properties and morphology of the chelates were respectively determined using ICP-OES, FT-IR and SEM. All the chitosan-metal chelates showed wide spectrum of effective antibacterial activities better than free chitosan and the individual metal ions. The results indicated that inhibitory effects of the chelates were dependent not only on the property of the coordinated metal ion, but also on the molar ratio of the metal ion. Consequently, the ideal inhibitory effects could be obtained with metal ion of high charge intensity and when the molar ratio of chitosan to metal was above 1:1. These chelates are promising materials for novel antibacterial agents.     

Chitosan�CEDTA New Combination is a Promising Candidate for Treatment of Bacterial and Fungal Infections

Chitosan is an attractive preparation widely used as a pharmaceutical excipient. This study aimed to evaluate the antimicrobial activities of chitosan derivatives, EDTA, and the newly developed chitosan-EDTA combination against Gram-negative and Gram-positive bacteria as well as Candida albicans. Antimicrobial activity was studied. Both minimal Inhibitory Concentrations (MIC) and minimal biocidal concentrations (MBC) were determined. Chitosan acetic acid recorded lower MIC values against Enterococcus faecalis, Escherichia coli, Staphylococcus aureus, Pseudomonas aeruginosa, and Candida albicans than those exhibited by EDTA. EDTA failed to have inhibitory activity against Enterococcus faecalis as well as MBC against any of the studied microorganisms. Chitosan acetic acid's MBC were recorded to all examined species. Checkerboard assay results indicated a synergistic antimicrobial activity of the new combination against Staphylococcus aureus and an additive effect against other microorganisms. Moreover, a short microbial exposure to chitosan-EDTA (20-30 min) caused complete eradication. Due to the continuous emergence of resistant strains, there is an urgent need to discover new antimicrobial agents. Our findings suggest the use of chitosan as an enhancing agent with antibacterial and antifungal properties in combination with EDTA in pharmaceutical preparations.     

Antibacterial characteristics and activity of acid-soluble chitosan

The antibacterial activity of chitosan was investigated by assessing the mortality rates of Escherichia coli and Staphylococcus aureus based on the extent of damaged or missing cell walls and the degree of leakage of enzymes and nucleotides from different cellular locations. Chitosan was found to react with both the cell wall and the cell membrane, but not simultaneously, indicating that the inactivation of E. coli by chitosan occurs via a two-step sequential mechanism: an initial separation of the cell wall from its cell membrane, followed by destruction of the cell membrane. The similarity between the antibacterial profiles and patterns of chitosan and those of two control substances, polymyxin and EDTA, verified this mechanism. The antibacterial activity of chitosan could be altered by blocking the amino functionality through coupling of the chitosan to active agarose derivatives. These results verify the status of chitosan as a natural bactericide.     

壳聚糖中胺基对其抑菌性能的影响及与DNA的作用

采用抑菌圈法研究了壳聚糖对大肠杆菌(E.coli)和金黄色葡萄球菌(St.aureus)的抑菌活性。利用壳聚糖的席夫碱反应,对壳聚糖的胺基进行保护后,研究了壳聚糖中胺基对其抑菌性能的影响。同时,运用紫外吸收光谱和电化学的方法,研究了壳聚糖与DNA的相互作用,提出了壳聚糖对E.coli和St.aureus的抑菌机理。研究结果表明,壳聚糖对E.coli和St.aureus具有很好的抑制作用,且抑菌活性与其胺基有关;壳聚糖能与细胞内带负电的核酸结合,使细胞正常DNA复制生理功能受到影响,抑制细菌的繁殖,从而达到抑菌的目的。     

Role of divalent metal ions in serum complement activity of the American alligator (Alligator mississippiensis)

Treatment of alligator serum with different concentrations of EDTA resulted in a concentration-dependent inhibition of serum-mediated sheep red blood cell (SRBC) hemolysis. This inhibition of serum-dependent hemolysis was observed for other chelators of divalent metal ions, such as phosphate and citrate. Treatment of alligator serum with 5 mM EDTA completely inhibited SRBC hemolysis, which could be totally restored by the addition of 5 mM Ca(2+) or Mg(2+), but not Cu(2+) or Ba(2+). These data indicate a specific need for Ca(2+) and/or Mg(2+) in the serum-mediated hemolysis of SRBCs. Kinetic analyses revealed that the addition of 30 mM EDTA 1 min after incubation of SRBCs with serum resulted in only 30% inhibition of hemolytic activity. However, addition of EDTA as early as 3 min post-incubation resulted in complete SRBC hemolysis. Pretreatment of serum with EDTA inhibited the hemolytic activity, but the activity could be restored in a time-dependent manner by the addition of Ca(2+)or Mg(2+). These data indicate that, as in human serum, the need for divalent metal ions occurs early in the alligator serum complement cascade.     

Antibacterial activity of pyrrolidine dithiocarbamate

Pyrrolidine dithiocarbamate (PDTC), an antioxidant with a metal-chelating activity, has been used widely to inhibit the expression of inflammatory genes in vitro and in vivo. This study investigated whether PDTC has an antimicrobial activity against various bacteria. The antibacterial activity of PDTC and other compounds was evaluated in vitro by the broth microdilution method against Porphyromonas gingivalis, Actinobacillus actinomycetemcomitans, Staphylococcus aureus, and Escherichia coli. Bacterial growth was inhibited by PDTC, where a wide range of sensitivity was demonstrated among the tested bacteria. The antibacterial activity of PDTC was reduced by the addition of copper chloride; in contrast, it was enhanced considerably by zinc chloride. Two different zinc chelators, Ca-saturated EDTA (Ca-EDTA) and N,N,N',N'-tetrakis (2-pyridylmethyl) ethylenediamine, blocked the antibacterial activity of PDTC, whereas Zn-EDTA failed to reduce the activity of PDTC. These results demonstrate for the first time that PDTC possesses an antibacterial activity, for which zinc is required, and suggest that PDTC, possessing a dual anti-inflammatory and antibacterial activity, may be considered for topical use for inflammatory diseases of bacterial origin.     

Enzymatic characterization of a serralysin-like metalloprotease from the entomopathogen bacterium, Xenorhabdus

We investigated the enzymatic properties of a serralysin-type metalloenzyme, provisionally named as protease B, which is secreted by Xenorhabdus bacterium, and probably is the ortholog of PrA peptidase of Photorhabdus bacterium. Testing the activity on twenty-two oligopeptide substrates we found that protease B requires at least three amino acids N-terminal to the scissile bond for detectable hydrolysis. On such substrate protease B was clearly specific for positively charged residues (Arg and Lys) at the P1 substrate position and was rather permissive in the others. Interestingly however, it preferred Ser at P1 in the oligopeptide substrate which contained amino acids also C-terminal to the scissile bond, and was cleaved with the highest k(cat)/K(M) value. The pH profile of activity, similarly to other serralysins, has a wide peak with high values between pH 6.5 and 8.0. The activity was slightly increased by Cu(2+) and Co(2+) ions, it was not sensitive for serine protease inhibitors, but it was inhibited by 1,10-phenanthroline, features shared by many Zn-metalloproteases. At the same time, EDTA inhibited the activity only partially even either after long incubation or in excess amount, and Zn(2+) was inhibitory (both are unusual among serralysins). The 1,10-phenanthroline inhibited activity could be restored with the addition of Mn(2+), Cu(2+) and Co(2+) up to 90-200% of its original value, while Zn(2+) was inefficient. We propose that both the Zn inhibition of protease B activity and its resistance to EDTA inhibition might be caused by an Asp in position 191 where most of the serralysins contain Asn.     

Enzymatic characterization of a maltogenic amylase from Lactobacillus gasseri ATCC 33323 expressed in Escherichia coli

A gene corresponding to a maltogenic amylase (MAase) in Lactobacillus gasseri ATCC 33323 (lgma) was cloned and expressed in Escherichia coli. The recombinant LGMA was efficiently purified 24.3-fold by one-step Ni-NTA affinity chromatography. The final yield and specific activity of the purified recombinant LGMA were 68% and 58.7 U/mg, respectively. The purified enzyme exhibited optimal activity for beta-CD hydrolysis at 55 degrees C and pH 5. The relative hydrolytic activities of LGMA to beta-CD, soluble starch or pullulan was 8:1:1.9. The activity of LGMA was strongly inhibited by most metal ions, especially Zn(2+), Fe(2+), Co(2+) and by EDTA. LGMA possessed some unusual properties distinguishable from typical MAases, such as being in a tetrameric form, having hydrolyzing activity towards the alpha-(1,6)-glycosidic linkage and being inhibited by acarbose.     

Structure-activity relationship study of anoplin.

Anoplin is a decapeptide amide, GLLKRIKTLL-NH2 derived from the venom sac of the solitary spider wasp, Anoplius samariensis. It is active against Gram-positive and Gram-negative bacteria and is not hemolytic towards human erythrocytes. The present paper reports a structure-activity study of anoplin based on 37 analogues including an Ala-scan, C- and N-truncations, and single and multiple residue substitutions with various amino acids. The analogues were tested for antibacterial activity against both S. aureus ATCC 25923 and E. coli ATCC 25922, and several potent antibacterial analogues were identified. The cytotoxicity of the analogues against human erythrocytes was assessed in a hemolytic activity assay. The antibacterial activity and selectivity of the analogues against S. aureus and E. coli varied considerably, depending on the hydrophobicity and position of the various substituted amino acids. In certain cases the selectivity for Gram-positive and Gram-negative bacteria was either reversed or altogether eliminated. In addition, it was generally found that antibacterial activity coincided with hemolytic activity.     

Enzymes of Trichomonas foetus. Separation and properties of two β-galactosidases

The beta-galactosidase activity in extracts of Trichomonas foetus is separable into two fractions by gel filtration on Sephadex G-200. When o-nitrophenyl beta-d-galactoside is used as substrate the first fraction to be eluted, beta-galactosidase 1, has 50 times the activity (units per mg of protein) of the crude preparation. This fraction is activated by Mn(2+) and Co(2+) and inhibited by Hg(2+) and EDTA. In the presence of Mn(2+) the pH optimum for the hydrolysis of o-nitrophenyl beta-d-galactoside or lactose is 5.8-6.0. beta-Galactosidase 1 is an exoglycosidase that releases beta-linked galactose joined to aliphatic and various carbohydrate aglycones. Hydrolysis is prevented, however, by a substituent on either the subterminal sugar or the terminal non-reducing beta-galactosyl residue in an oligosaccharide. The second fraction, beta-galactosidase 2, is not activated by metal ions or inhibited by EDTA and has a broad pH optimum from 4.5 to 6.0.     

Antibacterial activity of chitosan tripolyphosphate nanoparticles loaded with various metal ions

The aim of this study was to prepare and select chitosan nanoparticles loaded metal ions with high antibacterial activities. Chitosan nanoparticles were prepared based on ionic gelation between chitosan and sodium tripolyphosphate. Then, Ag⁺, Cu²⁺, Zn²⁺, Mn²⁺, or Fe²⁺ was individually loaded onto chitosan nanoparticles. Their particle sizes and zeta potentials were measured. Their antibacterial activities were evaluated by determination of minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) against Escherichia coli 25922, Salmonella choleraesuis ATCC 50020 and Staphylococcus aureus 25923 in vitro. Results showed that antibacterial activity was significantly enhanced by the metal ions loaded, except for Fe²⁺. Especially for chitosan nanoparticles loaded Cu²⁺, the MIC and MBC against E. coli 25922, S.choleraesuis ATCC 50020 and S. aureus 25923 were 21–42 times lower than that of Cu²⁺, respectively. Moreover, it was found that antibacterial activity was directly proportional to zeta potential.     

Effect of pH on preferential antibacterial-activity of ethylenediaminetetraacetic acid (EDTA)]

Ethylenediaminetetraacetic acid (EDTA), a chelating agent, was examined for the antibacterial activity against 15 species of bacteria by treating with a 10mM solution at pH adjusted to 5.0, 7.0 or 9.0. All bacterial species tested were classified into three groups; tentatively named the pH5 EDTA-sensitive group comprising Vibrio cholerae and Staphylococcus aureus, the pH9 EDTA-sensitive group comprising Escherichia coli and Pseudomonas aeruginosa and the EDTA-nonsensitive group comprising Proteus mirabilis. The EDTA-sensitivity grouping may be used as a tool for preferential decontamination of certain bacteria in live edible fishes, although further experiments are needed to characterize more strains and also species of bacteria.     

Binding of transition metal ions [cobalt, copper, nickel and zinc] with furanyl-, thiophenyl-, pyrrolyl-, salicylyl- and pyridyl-derived cephalexins as potent antibacterial agents

A method is described for the preparation of novel cephalexin-derived furanyl-, thiophenyl-, pyrrolyl-, salicylyl- and pyridyl-containing compounds showing potent antibacterial activity. The binding of these newly synthesized antibacterial agents with metal ions such as cobalt(II), copper(II), nickel(II) and zinc(II) has been studied and their inhibitory properties against various bacterial species such as Escherichia coli, Staphylococcus aureus, Pseudomonas aeruginosa, and Klebsiella pneumoniae are also reported. These results suggest that metal ions to possess an important role in the designing of metal-based antibacterials and that such complexes are more effective against infectious diseases compared to the uncomplexed drugs.     

Purification and characterization of a novel chitinase gene from Paecilomyces thermophila expressed in Escherichia coli

A novel chitinase gene (PtChiA) from the thermophilic fungus Paecilomyces thermophila was cloned and expressed in Escherichia coli as an intracellular soluble protein. The gene sequence alignment indicates that PtChiA belongs to glycoside hydrolase (GH) family 18 and has an open reading frame comprising of 1473 bp nucleotide sequences with five introns. PtChiA encodes 400 amino acids without any predicted signal peptide. PtChiA was purified by Ni-IDA chromatography. It displayed an acidic optimum pH of 4.5 and broad pH stability (pH 4.0-10.5). The enzyme exhibited an optimal temperature of 50°C and was stable up to 40°C. PtChiA was strongly inhibited by anionic detergent SDS, and also by metal ions Hg(2+) and Mn(2+). It did not exhibit any antifungal activity against pathogenic fungi. It has the ability to hydrolyze colloidal chitin into chito-oligomers suggesting its use in conversion of chitin waste into chito-oligosaccharides.