Synthesis and characterization of silver nanoparticles using Cynodon dactylon leaves and assessment of their antibacterial activity

Many methods of synthesizing silver nanoparticles (Ag-NPs) by reducing Ag⁺ ions using aqueous/organic extracts of various plants have been reported in the past, but the methods are rather slow. In this investigation, silver nanoparticles were quickly synthesized from aqueous silver nitrate through a simple method using leaf extract of a plant—Cynodon dactylon which served as reducing agent, while sunlight acted as a catalyst. The formation of Ag-NPs was indicated by gradual change in colour and pH and confirmed by ultraviolet–visible spectroscopy. The Ag-NPs showed a surface plasmon resonance at 451 nm. Based on the decrease in pH, a possible mechanism of the synthesis of Ag-NPs involving hydroxyl (OH^?) ions of polyphenols of the leaf extract is postulated. Ag-NPs having (111) and (200) crystal lattices were confirmed by X-ray diffraction. Scanning electron microscopy revealed the spherical nature of the Ag-NPs, while transmission electron microscopy showed that the nanoparticles were polydispersed with a size range of 8–10 nm. The synthesized Ag-NPs also demonstrated their antibacterial activity against Escherichia coli, Pseudomonas aeruginosa, Staphylococcus aureus and Salmonella typhimurium.     

Antibacterial activity and mechanism of action of ε-poly-l-lysine

ε-Poly-l-lysine (ε-PL)² is widely used as an antibacterial agent because of its broad antimicrobial spectrum. However, the mechanism of ε-PL against pathogens at the molecular level has not been elucidated. This study investigated the antibacterial activity and mechanism of ε-PL against Escherichia coli O157:H7 CMCC44828. Propidium monoazide-PCR test results indicated that the threshold condition of ε-PL for complete membrane lysis of E. coli O157:H7 was 10 μg/mL (90% mortality for 5 μg/mL). Further verification of the destructive effect of ε-PL on cell structure was performed by atomic force microscopy and transmission electron microscopy. Results showed a positive correlation between reactive oxygen species (ROS) ³ levels and ε-PL concentration in E. coli O157:H7 cells. Moreover, the mortality of E. coli O157:H7 was reduced when antioxidant N-acetylcysteine was added. Results from real-time quantitative PCR (RT-qPCR) ⁴ indicated that the expression levels of oxidative stress genes sodA and oxyR were up-regulated 4- and 16-fold, respectively, whereas virulence genes eaeA and espA were down-regulated after ε-PL treatment. Expression of DNA damage response (SOS response) ⁵ regulon genes recA and lexA were also affected by ε-PL. In conclusion, the antibacterial mechanism of ε-PL against E. coli O157:H7 may be attributed to disturbance on membrane integrity, oxidative stress by ROS, and effects on various gene expressions, such as regulation of oxidative stress, SOS response, and changes in virulence.     

R-Plasmid Transfer to and from Escherichia coli Strains Isolated from Human Fecal Samples

Strains of Escherichia coli recently isolated from human feces were examined for the frequency with which they accept an R factor (R1) from a derepressed fi⁺ strain of E. coli K-12 and transfer it to fecal and laboratory strains. Colicins produced by some of the isolates rapidly killed the other half of the mating pair; therefore, conjugation was conducted by a membrane filtration procedure whereby this effect was minimized. The majority of fecal E. coli isolates accepted the R factor at lower frequencies than K-12 F⁻, varying from 10⁻² per donor cell to undetectable levels. The frequencies with which certain fecal recipients received the R-plasmid were increased when its R⁺ transconjugant was either cured of the R1-plasmid and remated with the fi⁺ strain or backcrossed into the parental strain. The former suggests the loss of an incompatibility plasmid, and the latter suggests the modification of the R1-plasmid deoxyribonucleic acid (DNA). In general, the fecal R⁺E. coli transconjugants were less effective donors for K-12 F⁻ and heterologous fecal strains than was the fi⁺ K-12 strain, whereas the single strain of Citrobacter freundii examined was generally more competent. Passage of the R1-plasmid to strains of salmonellae reached mating frequencies of 10⁻¹ per donor cell when the recipient was a Salmonella typhi previously cured of its resident R-plasmid. However, two recently isolated strains of Salmonella accepted the R1-plasmid from E. coli K-12 R⁺ or the R⁺E. coli transconjugants at frequencies of 5 × 10⁻⁷ or less.     

Effects of antibacterial mineral leachates on the cellular ultrastructure, morphology, and membrane integrity of Escherichia coli and methicillin-resistant Staphylococcus aureus

Background

We have previously identified two mineral mixtures, CB07 and BY07, and their respective aqueous leachates that exhibit in vitro antibacterial activity against a broad spectrum of pathogens. The present study assesses cellular ultrastructure and membrane integrity of methicillin-resistant Staphylococcus aureus (MRSA) and Escherichia coli after exposure to CB07 and BY07 aqueous leachates.

Methods

We used scanning and transmission electron microscopy to evaluate E. coli and MRSA ultrastructure and morphology following exposure to antibacterial leachates. Additionally, we employed Bac light LIVE/DEAD staining and flow cytometry to investigate the cellular membrane as a possible target for antibacterial activity.

Results

Scanning electron microscopy (SEM) and transmission electron microscopy (TEM) imaging of E. coli and MRSA revealed intact cells following exposure to antibacterial mineral leachates. TEM images of MRSA showed disruption of the cytoplasmic contents, distorted cell shape, irregular membranes, and distorted septa of dividing cells. TEM images of E. coli exposed to leachates exhibited different patterns of cytoplasmic condensation with respect to the controls and no apparent change in cell envelope structure. Although bactericidal activity of the leachates occurs more rapidly in E. coli than in MRSA, LIVE/DEAD staining demonstrated that the membrane of E. coli remains intact, while the MRSA membrane is permeabilized following exposure to the leachates.

Conclusions

These data suggest that the leachate antibacterial mechanism of action differs for Gram-positive and Gram-negative organisms. Upon antibacterial mineral leachate exposure, structural integrity is retained, however, compromised membrane integrity accounts for bactericidal activity in Gram-positive, but not in Gram-negative cells.     

Design,synthesis and antibacterial activities of 5-(pyrazin-2-yl)-4H-1,2,4-triazole-3-thiol derivatives containing Schiff base formation as FabH inhibitory

A series of novel schiff base derivatives (H¹–H²⁰) containing pyrazine and triazole moiety have been designed and synthesized, and their biological activities were also evaluated as potential inhibitors of β-ketoacyl-acyl carrier protein synthase III (FabH). These compounds were assayed for antibacterial activity against Escherichia coli, Pseudomonas aeruginosa, Staphylococcus aureus, Bacillus subtilis and Bacillus amyloliquefaciens and selected compounds among them were tested for their Escherichia coli FabH inhibitory activity. Based on the biological data, compound H¹⁷ showed the most potent antibacterial activity with MIC values of 0.39–1.56 μg/mL against the tested bacterial strains and exhibited the most potent E. coli FabH inhibitory activity with IC₅₀ of 5.2 μM, being better than the positive control Kanamycin B with IC₅₀ of 6.3 μM. Furthermore, docking simulation was performed to position compound H¹⁷ into the E. coli FabH active site to determine the probable binding conformation. This study indicated that compound H¹⁷ has demonstrated significant E. coli FabH inhibitory activity as a potential antibacterial agent and provides valuable information for the design of E. coli FabH inhibitors.     

Antibacterial Evaluation of Novel Organoarsenic Compounds by the Microcalorimetric Method

Antibacterial activities of novel organoarsenic compounds As(III)-containing Schiff bases on Escherichia coli (CCTCCAB91112) were investigated by microcalorimetry in this study. The experimental result showed that the arsenic(III)-containing Schiff bases at micromolar concentration exhibit strong inhibition on the E. coli. Specifically, the growth rate constant k decreased, and the generation time t _G and the inhibitory ratio I (percentage) increased with the increased dose of the arsenicals as inhibitors. All of the arsenicals display the feature of considerable lag phase inhibition on the cell growth. The compound 4-(4-bromobenzaliminyl)phenylarsenoxide makes the lag phase of E. coli cell growth cycles to reach 650 min at 5 μmol/L. The compounds with donating electron groups at aromatic ring B have lower IC₅₀ to present higher antibacterial activity. The compound 4-(4-hydroxyl-3-methoxylbenzaliminyl)phenylarsenoxide has the lowest IC₅₀ (1.82 μmol/L) to show the strongest antibacterial activity among them.     

Drug Resistance of Enteric Bacteria VIII. Chromosomal Location of Nontransferable R Factor in Escherichia coli

The R₂₁(TC) factor, obtained by transduction of the R₁₀(TC.CM.SM.SA) factor with phage ε to group E Salmonella, is not transferable by the normal conjugal process. However, when R₂₁(TC)⁺ transductants are infected with the F₁₃ factor, the nontransferable R₂₁(TC) factor acquires transmissibility by conjugation. R₂₁(TC)⁺ conjugants of Escherichia coli K-12, to which only the R₂₁(TC) factor was transmitted by cell-to-cell contact from an F′ R⁺ donor, were still unable to transfer their R₂₁(TC) factor by conjugation. In crosses between Hfr and F⁻E. coli K-12 strains containing R₂₁(TC), the gene responsible for tetracycline resistance was located on the E. coli K-12 chromosome between lac and pro, near lac.     

Inhibition of Mg2+ binding and DNA religation by bacterial topoisomerase I via introduction of an additional positive charge into the active site region

Among bacterial topoisomerase I enzymes, a conserved methionine residue is found at the active site next to the nucleophilic tyrosine. Substitution of this methionine residue with arginine in recombinant Yersinia pestis topoisomerase I (YTOP) was the only substitution at this position found to induce the SOS response in Escherichia coli. Overexpression of the M326R mutant YTOP resulted in ~4 log loss of viability. Biochemical analysis of purified Y. pestis and E. coli mutant topoisomerase I showed that the Met to Arg substitution affected the DNA religation step of the catalytic cycle. The introduction of an additional positive charge into the active site region of the mutant E. coli topoisomerase I activity shifted the pH for optimal activity and decreased the Mg²⁺ binding affinity. This study demonstrated that a substitution outside the TOPRIM motif, which binds Mg²⁺directly, can nonetheless inhibit Mg²⁺ binding and DNA religation by the enzyme, increasing the accumulation of covalent cleavage complex, with bactericidal consequence. Small molecules that can inhibit Mg²⁺ dependent religation by bacterial topoisomerase I specifically could be developed into useful new antibacterial compounds. This approach would be similar to the inhibition of divalent ion dependent strand transfer by HIV integrase in antiviral therapy.     

Purification and antibacterial mechanism of fish-borne bacteriocin and its application in shrimp (<Emphasis Type="Italic">Penaeus vannamei</Emphasis>) for inhibiting <Emphasis Type="Italic">Vibrio parahaemolyticus</Emphasis>

Vibrio parahaemolyticus: is recognized as the main cause of gastroenteritis associated with consumption of seafood. Bacteriocin-producing Lactobacillus plantarum FGC-12 isolated from golden carp intestine had strong antibacterial activity toward V. parahaemolyticus. The fish-borne bacteriocin was purified by a three-step procedure consisting of ethyl acetate extraction, gel filtration chromatography and high performance liquid chromatography. Its molecular weight was estimated at 4.1 kDa using SDS-PAGE. The fish-borne bacteriocin reached the maximum production at stationary phase after 20 h. It was heat-stable (30 min at 121?°C) and remained active at pH range from 3.0 to 5.5, but was sensitive to nutrasin, papain and pepsin. Its minimum inhibitory concentration for V. parahaemolyticus was 6.0 mg/ml. Scanning electron microscopy analysis showed that the fish-borne bacteriocin disrupted cell wall of V. parahaemolyticus. The antibacterial mechanism of the fish-borne bacteriocin against V. parahaemolyticus might be described as action on membrane integrity in terms of the leakage of electrolytes, the losses of Na⁺K⁺-ATPase, AKP and proteins. The addition of the fish-borne bacteriocin to shrimps leaded V. parahaemolyticus to reduce 1.3 log units at 4?°C storage for 6 day. Moreover, a marked decline in total volatile base nitrogen and total viable counts was observed in bacteriocin treated samples than the control. It is clear that this fish-borne bacteriocin has promising potential as biopreservation for the control of V. parahaemolyticus in aquatic products.     

Characterization and antibacterial activity of the nanocomposite of half-fin anchovy (Setipinna taty) hydrolysates/zinc oxide nanoparticles

Half-fin anchovy (Setipinna taty) hydrolysates (HAHp) was conjugated with zinc oxide nanoparticles (ZnO NPs) using a hydrothermal method to develop a novel antibacterial nanocomposite. The generated supernatants of the conjugate, designated as HAHp(3.0)/ZnO NPs, were characterized by transmission electron microscopy, high resolution transmission electron microscopy, and inductively coupled plasma-optical emission spectrometer. Results showed that HAHp(3.0) was absorbed on the surface of the ZnO NPs. The total content of zinc element was 9127.4 mg/kg in HAHp(3.0)/ZnO NPs. The increased antibacterial effects were observed for the HAHp(3.0)/ZnO NPs with the minimal inhibitory concentration of 3.5 μgprotein/mL against Escherichia coli (E. coli), Pseudomonas fluorescens, Salmonella and Staphylococcus aureus, compared to the bare HAHp(3.0). The antibacterial activity of HAHp(3.0)/ZnO NPs was further evaluated using E. coli as the model strain. The incubation of HAHp(3.0)/ZnO NPs increased the outer and inner membrane permeability in E. coli cells, and the leakages of potassium ions and the cytoplasmic β-galactosidase were detected during the process. Furthermore, porous structures were observed on the membrane of E. coli cells by scanning electron microscopy. In addition, the formation of intracellular reactive oxygen species was detected using fluorescence microscopy. The results suggested that the HAHp(3.0)/ZnO NPs could be a promising antibacterial nanocomposite.     

Polysaccharide-based silver nanoparticles synthesized by <Emphasis Type="Italic">Klebsiella oxytoca</Emphasis> DSM 29614 cause DNA fragmentation in <Emphasis Type="Italic">E. coli</Emphasis> cells

Silver nanoparticles (AgNPs), embedded into a specific exopolysaccharide (EPS), were produced by Klebsiella oxytoca DSM 29614 by adding AgNO₃ to the cultures during exponential growth phase. In particular, under aerobic or anaerobic conditions, two types of silver nanoparticles, named AgNPs-EPS^aer and the AgNPs-EPS^anaer, were produced respectively. The effects on bacterial cells was demonstrated by using Escherichia coli K12 and Kocuria rhizophila ATCC 9341 (ex Micrococcus luteus) as Gram-negative and Gram-positive tester strains, respectively. The best antimicrobial activity was observed for AgNPs-EPS^aer, in terms of minimum inhibitory concentrations and minimum bactericidal concentrations. Observations by transmission electron microscopy showed that the cell morphology of both tester strains changed during the exposition to AgNPs-EPS^aer. In particular, an electron-dense wrapped filament was observed in E. coli cytoplasm after 3 h of AgNPs-EPS^aer exposition, apparently due to silver accumulation in DNA, and both E. coli and K. rhizophila cells were lysed after 18 h of exposure to AgNPs-EPS^aer. The DNA breakage in E. coli cells was confirmed by the comparison of 3-D fluorescence spectra fingerprints of DNA. Finally the accumulation of silver on DNA of E. coli was confirmed directly by a significant Ag⁺ release from DNA, using the scanning electrochemical microscopy and the voltammetric determinations.     

Metagenomic cloning and characterization of Na+/H+ antiporter genes taken from sediments in Chaerhan Salt Lake in China

A metagenomic library containing 8,000 clones was constructed by using genomic DNA obtained from Chaerhan Salt Lake in northwest China. Three Na⁺/H⁺ antiporters, C4-NhaG, C47-NhaG and C49-NhaG that grouped to the NhaG family, were screened and cloned from this metagenome by complementing Escherichia coli strain KNabc (ΔnhaA ΔnhaB ΔchaA) in medium containing 0.2 M NaCl. The three putative Na⁺/H⁺ antiporters were membrane proteins with 10, 11 and 11 transmembrane segments, respectively. They enabled E. coli KNabc to grow in medium containing 0.2–0.6 M Na⁺ or 7–14 mM Li⁺. Everted membrane vesicles prepared from E. coli KNabc cells carrying C49-NhaG exhibited Na⁺/H⁺ and Li⁺/H⁺ antiport activities.     

Protection against oxidative stress in Escherichia coli stationary phase by a phosphate concentration-dependent genes expression

Escherichia coli gradually decline the capacity to resist oxidative stress during stationary phase. Besides the aerobic electron transport chain components are down-regulated in response to growth arrest. However, we have previously reported that E. coli cells grown in media containing at least 37 mM phosphate maintained ndh expression in stationary phase, having high viability and low NADH/NAD⁺ ratio. Here we demonstrated that, in the former condition, other aerobic respiratory genes (nuoAB, sdhC, cydA, and ubiC) expression was maintained. In addition, reactive oxygen species production was minimal and consequently the levels of thiobarbituric acid-reactive substances and protein carbonylation were lower than the expected for stationary cells. Interestingly, defense genes (katG and ahpC) expression was also maintained during this phase. Our results indicate that cells grown in high phosphate media exhibit advantages to resist endogenous and exogenous oxidative stress in stationary phase.     

Kinetics of luminescence in the 10−6–10−4-s range in Chlorella

The decay of luminescence in the 6–600-μs range following a microsecond flash has been studied in Chlorella. The decay is highly polyphasic; three kinetic components are outlined, in confirmation of the results of K. L. Zankel (1971, Biochim. Biophys. Acta 245, 373–385).Extrapolation of the decay to zero dark time suggests that a unique metastable species C^?₊, resulting from photochemical charge separation in the System II reaction center, is the substrate of the recombination reaction which gives rise to luminescence.The fast (5–10 μs) and medium (50–70 μs) phases of the decay denote different stabilization steps, preceding relaxation of the centers by electron and proton transduction to the photosynthetic chain.NH₂OH specifically inhibits the fast phase and enhances the medium phase. This effect is explained by assuming that the fast phase results from electron transfer from the water splitting system Z to the oxidized primary donor Y.3-(3,4-Dichlorophenyl)-1,1-dimethylurea (DCMU), in the presence of NH₂OH elicits another fast phase. It is believed that DCMU affords a parasitic stabilization of C^?₊ by forming a complex with Q^?.     

Antimutagenic effects of 5-fluorouracil and 5-fluorodeoxyuridine on UV-induced mutagenesis in Escherichia coli

Inhibitors of UV induction of the SOS function were screened. A log phase culture of E. coli PQ37 (sulA::lacZ, rfa, uvrA, Pho^c) was irradiated with UV and then immediately subjected to culture for 2 h in a liquid LB medium containing each test compound. Expression of the SOS gene (sulA) was assayed by monitoring the levels of β-galactosidase. In order to examine the inhibitory effects of test compounds on protein synthesis, the levels of the constitutive alkaline phosphatase were assayed in parallel.The total number of compounds tested was 233, including 44 food and feed additives, 23 naturally occurring compounds and derivatives, 21 antibiotics, 61 pesticides, 33 inorganics and 51 other chemicals. As a result, 5-fluorouracil and 5-fluorodeoxyuridine were found to inhibit considerably the UV induction of the SOS gene without any inhibition of protein synthesis. Mutagenesis induced by UV irradiation was depressed by the addition of either compound at non-toxic concentrations.     

Electron microscopy of the K2 killer effect of Saccharomyces cerevisiae T206 on a mesophilic wine yeast

A mesophilic wine yeast, Saccharomyces cerevisiae CSIR Y217 K ^– R ^– was subjected to the K₂ killer effect of Saccharomyces cerevisiae T206 K ⁺ R ⁺ in a liquid grape medium. The lethal effect of the K₂ mycoviral toxin was confirmed by methylene blue staining. Scanning electron microscopy of cells from challenge experiments revealed rippled cell surfaces, accompanied by cracks and pores, while those unaffected by the toxin, as in the control experiments, showed a smooth surface. Transmission electron microscopy revealed that the toxin damaged the cell wall structure and perturbed cytoplasmic membranes to a limited extent.     

Visible light responsive photocatalyst induces progressive and apical-terminus preferential damages on Escherichia coli surfaces

Background

Recent research shows that visible-light responsive photocatalysts have potential usage in antimicrobial applications. However, the dynamic changes in the damage to photocatalyzed bacteria remain unclear.

Methodology/Principal Findings

Facilitated by atomic force microscopy, this study analyzes the visible-light driven photocatalyst-mediated damage of Escherichia coli. Results show that antibacterial properties are associated with the appearance of hole-like structures on the bacteria surfaces. Unexpectedly, these hole-like structures were preferentially induced at the apical terminus of rod shaped E. coli cells. Differentiating the damages into various levels and analyzing the percentage of damage to the cells showed that photocatalysis was likely to elicit sequential damages in E. coli cells. The process began with changing the surface properties on bacterial cells, as indicated in surface roughness measurements using atomic force microscopy, and holes then formed at the apical terminus of the cells. The holes were then subsequently enlarged until the cells were totally transformed into a flattened shape. Parallel experiments indicated that photocatalysis-induced bacterial protein leakage is associated with the progression of hole-like damages, further suggesting pore formation. Control experiments using ultraviolet light responsive titanium-dioxide substrates also obtained similar observations, suggesting that this is a general phenomenon of E. coli in response to photocatalysis.

Conclusion/Significance

The photocatalysis-mediated localization-preferential damage to E. coli cells reveals the weak points of the bacteria. This might facilitate the investigation of antibacterial mechanism of the photocatalysis.     

Possibilities of the conjugation process in mycobacteria

Results obtained when studying conjugation in mycobacteria by means of different methods are summarized. The method of conjugation on surface of a solid complete medium was tested with different auxotrophic mutants of different strains ofMycobacterium smegmatis. It was not possible to obtain positive results even by means of the above method. This was probably due to unsuitability of the chosen strains ofMycobacterium smegmatis. Preparation of the donor strain by transfer of the F factor fromEscherichia coli F’ORF 1ade ⁺ lac⁺ pro⁺ toMycobacterium phlei PA adeStm ^r by means of sexduction is described. Frequency of the phenotype PAade ⁺ Stm^r increased in the average by two and a half orders of magnitude with respect to the control, however, a further transfer from cultures of the cellsade ⁺ Stm^r to cells ade could not be demonstrated. Experiments aimed at transferring the R factor from strainsEscherichia coli K-12 toMycobacterium phlei were unsuccessful.