Treatment Of Bacterial Cells Research Articles

Antimicrobial Conjugated Oligoelectrolytes Containing Triphenylphosphonium Solubilizing Groups.

Conjugated oligoelectrolytes (COEs) are an emerging class of amphiphilic antimicrobial compounds with a modular molecular framework suitable for simple chemical derivatization. Here, a series of COE derivatives with a stilbene-conjugated segment and triphenylphosphonium (TPP) pendant groups was designed and synthesized to understand how lipophilic cationic groups impact antimicrobial activity. In vitro evaluations against ESKAPE pathogens showed broad-spectrum activity towards multi-drug resistant (MDR) bacteria and mycobacteria, with TPP groups enhancing antimicrobial activity towards clinically relevant Gram-negative strains compared to their ammonium analogues. We studied the interactions of DM6P, the most active TPP-COE compound, with various membrane assays. Treatment of bacterial cells with DM6P showed enhanced permeability of cell membranes without inducing the development of significant bacterial resistance. Moreover, DM6P eliminated 99.99 % of methicillin-resistant Staphyloccocus aureus (MRSA) in an in vivo wound model. These results represent a promising chemical strategy for increasing the activity spectrum of membrane-active COE antibiotics to tackle challenging drug-resistant targets.

Evaluation of the effect of ibuprofen in combination with ciprofloxacin on the virulence-associated traits, and efflux pump genes of Pseudomonas aeruginosa.

Biofilm formation and antibiotic efflux are two determinant factors in the development of drug resistance phenotype by Pseudomonas aeruginosa. Non-steroid anti-inflammatory drugs have shown the antimicrobial potential to be used in combination with antibiotics against bacterial pathogens. In this work, the effect of ibuprofen alone and in combination with ciprofloxacin on some virulence traits and the expression of the alginate synthesis and efflux pump genes of clinical isolates of P. aeruginosa was investigated. The checkerboard titration assay was used to evaluate the synergism of the drugs. P. aeruginosa strains were grown in the presence of sub-inhibitory concentrations of the drug and their biofilm formation level, swarming, swimming, and hemolytic activity were assessed. Also, the relative expression of the alg44, algT/U, mexB, and oprM genes was determined by qPCR assay. The MIC of ibuprofen and ciprofloxacin were measured 2048 and 32µg/mL and the drugs showed synergic antibacterial activity (FIC = 0.4). Moreover, ibuprofen alone and in combination with ciprofloxacin, significantly reduced the expression of alg44 (0.22 and 0.25 folds) and algT/U (0.26 and 0.37 folds) genes, while increased the expression of the mexB (1.64 and 1.83 folds) and oprM (1.36 and 1.92 folds) genes. Simultaneous treatment of bacterial cells with ibuprofen and ciprofloxacin significantly decreased bacterial biofilm formation (65%), swimming, swarming, and hemolytic activity (85%), compared with the control. This work suggests that ibuprofen has considerable anti-virulence potential against P. aeruginosa and could be employed for combination therapy with antibiotics after further characterizations.

Antiinfective properties of ursolic acid-loaded chitosan nanoparticles against Staphylococcus aureus.

The present study aimed to synthesize ursolic acid-loaded chitosan nanoparticles (UA-Ch-NPs) as an antiinfective agent against 21 Staphylococcus aureus isolates. The UA-Ch-NPs were synthesized by a simple method and then characterized by TEM, FTIR, DLS-zeta potential, and XRD analyses. According to the characterization results, highly dispersed spherical nanoparticles with a mean diameter of 258 nm and a zeta potential of + 40.1 mV were developed. The antibacterial properties of UA-Ch-NPs were investigated and their inhibitory effect on biofilm formation was demonstrated by AFM. Finally, the expression levels of icaA and icaD were measured using real-time PCR. Results indicated that the minimum inhibitory concentration (MIC) of UA and UA-Ch-NPs against S. aureus was 64 and 32 µg/mL, respectively. The treatment of bacterial cells with UA-Ch-NPs significantly decreased the expression of icaA and icaD genes which are engaged in biofilm formation. Our results indicated that UA-Ch-NPs could be a promising material for antibacterial and antibiofilm applications.

Lectin-Mediated Bacterial Modulation by the Intestinal Nematode Ascaris suum.

Ascariasis is a global health problem for humans and animals. Adult Ascaris nematodes are long-lived in the host intestine where they interact with host cells as well as members of the microbiota resulting in chronic infections. Nematode interactions with host cells and the microbial environment are prominently mediated by parasite-secreted proteins and peptides possessing immunomodulatory and antimicrobial activities. Previously, we discovered the C-type lectin protein AsCTL-42 in the secreted products of adult Ascaris worms. Here we tested recombinant AsCTL-42 for its ability to interact with bacterial and host cells. We found that AsCTL-42 lacks bactericidal activity but neutralized bacterial cells without killing them. Treatment of bacterial cells with AsCTL-42 reduced invasion of intestinal epithelial cells by Salmonella. Furthermore, AsCTL-42 interacted with host myeloid C-type lectin receptors. Thus, AsCTL-42 is a parasite protein involved in the triad relationship between Ascaris, host cells, and the microbiota.

Characterisation and action mode of Gluconacin, a bacteriocin with antimicrobial activity against Xanthomonas albilineans

AbstractGluconacin, a bacteriocin produced by Gluconacetobacter diazotrophicus PAL5, has previously shown a large spectrum of inhibitory activity against beneficial and phytopathogenic bacteria. The present study proposes a three‐dimensional (3D) structure for Gluconacin based on modelling and describes some physicochemical characteristics and the effect of the peptide on cells of the phytopathogenic bacterium Xanthomonas albilineans ICMP196. The Gluconacin 3D structural model results demonstrated the 7 α‐helices and 11 β‐sheets. Growth inhibition of the indicator microorganism, X. albilineans, occurred in the first moments of contact with Gluconacin (0.25 μg μl−1). Treatment of bacterial cells caused significant loss of inorganic phosphate and UV‐absorbing materials. Scanning electron microscopy images showed that Gluconacin‐treated X. albilineans cells were fully lysed. These results suggest that the mode of action of this peptide involves altered membrane integrity and increased permeability, resulting in complete cell lysis. Physicochemical characterisation demonstrated stability of biological activity under high temperatures, including autoclaving conditions and low pH. The activity was reduced after treatment with proteases and some surfactants and abolished with ethylenediaminetetraacetic acid (EDTA). The 3D conformation model indicates the possibility of a hydrophobic region, which could possibly interact directly with the cell membrane of target bacteria. In conclusion, the ability of Gluconacin to inhibit growth of phytopathogens of agricultural importance opens up new opportunities for biotechnological applications and, consequently, a reduction in the use of pesticides.

Design, synthesis, antibacterial activity and toxicity of novel quaternary ammonium compounds based on pyridoxine and fatty acids.

A diverse series of 43 novel "soft antimicrobials" based on quaternary ammonium pyridoxine derivatives which include six-membered acetals and ketals of pyridoxine bound via cleavable linker moieties (amide, ester) with a fragment of fatty carboxylic acid was designed. Nine compounds exhibited invitro promising antibacterial activity against Gram-positive and Gram-negative bacterial strains with MIC values comparable with reference antiseptics miramistin, benzalkonium chloride and chlorohexidine. On various clinical isolates, the lead compounds 6i and 12a exhibited antibacterial activity comparable with that of benzalkonium chloride while higher than that of miramistin. Moreover, 6i and 12a were able to kill bacteria embedded into the matrix of mono- and dual species biofilms. The treatment of bacterial cells by either 6i and 12a lead to fast depolarization of the membrane suggesting that the membrane is an apparent molecular target of compounds. 6i and 12a were non mutagenic neither in SOS-chromotest nor in Ames test and non-toxic invivo at acute oral (LD50>2000mg/kg) and cutaneous administration (LD50>2500mg/kg) on mice. Taken together, our data allow suggesting described active compounds as promising starting point for the new antibacterial agents development.

Zinc oxide nanoparticles inhibit bacterial biofilm formation via altering cell membrane permeability

In the current scenario nanoparticles (NPs) have gained a breathtaking impetus due to their multidimensional applications in varied fields. In the present effort, biogenic synthesis of Zinc Oxide nanoparticles (ZnO NPs) was carried out using aqueous extract of dried powder of Emblica officinalis (Amla). Physicochemical characterization of nanoparticles was carried out via UV-Visible (UV-Vis) spectroscopy, Fourier transform infrared spectroscopy (FTIR), X-ray diffractometer (XRD), Scanning electron microscopy (SEM) and Transmission electron microscopy (TEM) wherein the particles were found to be quasi spherical and with a size ranging between 3 and 11 nm. The ZnO nanoparticles exhibited significant antibacterial activity against bacteria as Streptococcus pyogenes MTCC 442, Bacillus cereus MTCC 1272, Escherichia coli MTCC 1687 and Pseudomonas aeruginosa MTCC 4673. The nanoparticles displayed high anti-biofilm activity toward all the bacterial strains, when tested against three different base materials viz. glass, plastic and metal (Aluminum). Further, the nanoparticle treatment of bacterial cells caused changes in their cell membrane permeability, leading to leakage of nucleic acid from the bacterial cells, thereby defining it as the most probable mechanism for their anti-biofilm potential.

Possible involvement of ROS generation in vorinostat pretreatment induced enhancement of the antibacterial activity of ciprofloxacin.

The mechanism underlying ciprofloxacin action involves interference with transcription and replication of bacterial DNA and, thus, the induction of double-strand breaks in DNA. It also involves elevated oxidative stress, which might contribute to bacterial cell death. Vorinostat was shown to induce oxidative DNA damage. The current work investigated a possible interactive effect of vorinostat on ciprofloxacin-induced cytotoxicity against a number of reference bacteria. Standard bacterial strains were Escherichia coli ATCC 35218, Staphylococcus aureus ATCC29213, Pseudomonas aeruginosa ATCC 9027, Staphylococcus epidermidis ATCC 12228, Acinetobacter baumannii ATCC 17978, Proteus mirabilis ATCC 12459, Klebsiella pneumoniae ATCC 13883, methicillin-resistant Staphylococcus aureus (MRSA) (ATCC 43300), and Streptococcus pneumoniae (ATCC 25923). The antibacterial activity of ciprofloxacin, with or without pretreatment of bacterial cells by vorinostat, was examined using the disc diffusion procedure and determination of the minimum inhibitory concentration (MIC) and zones of inhibition of bacterial growth. All tested bacterial strains showed sensitivity to ciprofloxacin. When pretreated with vorinostat, significantly larger zones of inhibition and smaller MIC values were observed in all bacterial strains compared to those treated with ciprofloxacin alone. In correlation, generation of reactive oxygen species (ROS) induced by the antibacterial action of ciprofloxacin was enhanced by treatment of bacterial cells with vorinostat. Results showed the possible agonistic properties of vorinostat when used together with ciprofloxacin. This could be related to the ability of these agents to enhance oxidative stress in bacterial cells.

EFFECT OF LOW-INTENSITY RED LASER ON GROWTH OF STAPHYLOCOCCUS AUREUS AND SENSITIZING EFFECT OF PHOTODITAZIN

Study the effect of laser emission in the red spectrum on growth of methicillin- sensitive. and methicillin-resistant strains of Staphylococcus aureus, as well as photodynamic effect of photosensitizer photoditazin. Effect of light of semiconduc- tor red laser (X 660 nm, 100 mW/cm2) at 30,'60, 90 and 180 J/cm2 on growth of S. aureus colonies was determined. Time of exposure 5; 10, 15 and 30 minutes. In certain series of experiments bacterial cells were sensitized in advance by a Water. solution of photoditazin at a concentration of5xl0-6 M. Red laser emission was established to cause a pronounced suppression of bacterial growth. This effect on standard S. aureus strain only took place dur- ing use of relatively high exposure doses (180 J/cm2). Photosensitivity of methicillin-resistant strain turned out to be significantly higher: bacteriostatic effect of red light was noted already at the dose of 60 J/cm2. Treatment of bacterial cells with photoditazin in advance signifi- cantly enhanced growth-inhibiting effect of laser light.

Context-specific inhibition of translation by ribosomal antibiotics targeting the peptidyl transferase center

The first broad-spectrum antibiotic chloramphenicol and one of the newest clinically important antibacterials, linezolid, inhibit protein synthesis by targeting the peptidyl transferase center of the bacterial ribosome. Because antibiotic binding should prevent the placement of aminoacyl-tRNA in the catalytic site, it is commonly assumed that these drugs are universal inhibitors of peptidyl transfer and should readily block the formation of every peptide bond. However, our in vitro experiments showed that chloramphenicol and linezolid stall ribosomes at specific mRNA locations. Treatment of bacterial cells with high concentrations of these antibiotics leads to preferential arrest of translation at defined sites, resulting in redistribution of the ribosomes on mRNA. Antibiotic-mediated inhibition of protein synthesis is most efficient when the nascent peptide in the ribosome carries an alanine residue and, to a lesser extent, serine or threonine in its penultimate position. In contrast, the inhibitory action of the drugs is counteracted by glycine when it is either at the nascent-chain C terminus or at the incoming aminoacyl-tRNA. The context-specific action of chloramphenicol illuminates the operation of the mechanism of inducible resistance that relies on programmed drug-induced translation arrest. In addition, our findings expose the functional interplay between the nascent chain and the peptidyl transferase center.

Ciprofloxacin-Induced Antibacterial Activity Is Atteneuated by Pretreatment with Antioxidant Agents.

Ciprofloxacin works through interfering with replication and transcription of bacterial DNA, which leads to increased oxidative stress, and death of bacterial cells. Drugs with strong antioxidant such as tempol, melatonin and pentoxifylline might interfere with the antibacterial activity of ciprofloxacin. In the current study, the effect of these drugs on the cytotoxicity of ciprofloxacin was investigated against several reference bacteria. Standard bacterial strains included Escherichia coli ATCC 35218, Staphylococcus aureus ATCC29213, Pseudomonas aeruginosa ATCC 9027, Staphylococcus epidermidis ATCC 12228, Acinetobacter baumannii ATCC 17978, Proteus mirabilis ATCC 12459, Klebsiella pneumoniae ATCC 13883, methicillin-resistant Staphylococcus aureus (MRSA) (ATCC 43300), and Streptococcus pneumoniae (ATCC 25923). The antibacterial activity of ciprofloxacin with or without treatment of bacterial cells by tempol, melatonin or pentoxifylline was assessed using the disc diffusion method and by measuring the minimum inhibitory concentration (MIC) and zones of inhibition of bacterial growth. All of the tested bacterial strains were sensitive to ciprofloxacin. When treated with tempol, melatonin or pentoxifylline, all bacterial strains showed significantly smaller zones of inhibition and larger MIC values compared ciprofloxacin alone. In correlation, reactive oxygen species (ROS) generation induced by ciprofloxacin antibacterial action was diminished by treatment of bacterial cells with tempol, melatonin or pentoxifylline. In conclusion, results indicate the possible antagonistic properties for agents with antioxidant properties such as tempol, melatonin and pentoxifylline when they are used concurrently with flouroquinolones. This could be related to the ability of these agents to inhibit oxidative stress in bacterial cells.

Abiotic formation of 32,35-anhydrobacteriohopanetetrol: A geomimetic approach

Treatment of bacterial cells from Zymomonas mobilis in the presence of acidic clay minerals in aqueous medium leads to the formation of 32,35-anhydrobacteriohopanetetrols (anhydroBHTs), confirming that these compounds, widespread in the geological record, are likely formed by diagenetic (abiotic) processes. We determined that anhydroBHTs result not only from the acid-catalyzed transformation of free bacteriohopanetetrol, but also of other composite biohopanoids such as bacteriohopanetetrol (BHT) glycoside and BHT cyclitol ether.

Hydrogen peroxide induces protection against lethal effects of cumene hydroperoxide in Escherichia coli cells: an Ahp dependent and OxyR independent system?

Pretreatment with 2.5 mM H2O2 protects bacterial cells against cumene hydroperoxide killing. This response is independent of the OxyR system, but possibly involves the participation of Ahp protein, since ahp mutants are not protected. Treatment of bacterial cells with high H2O2 concentrations caused an alteration on the electrophoretic profile of the smaller subunit (22-kDa) of Ahp. This alteration does not require novel gene products and is not dependent on the OxyR protein. In this way, we propose that the modification of the 22-kDa subunit of Ahp by high H2O2 concentration may be responsible for the protection against the lethal effects of cumene hydroperoxide.

In vitro attachment and invasion of chicken ovarian granulosa cells by Salmonella enteritidis phage type 8.

The attachment and invasion of chicken ovarian granulosa cells by Salmonella enteritidis was examined in vitro. The attachment was inhibited by preincubation of granulosa cells with anti-chicken fibronectin antibody (approximately 70% reduction in attachment) or preincubation with a 14-kDa fimbrial protein isolated from S. enteritidis (68% reduction in attachment). Treatment of bacterial cells with the tetrapeptide RGDS before addition to granulosa cells resulted in inhibition of attachment (60% inhibition when 2 x 10(7) CFU of bacteria was treated with 500 microg of peptide). Treatment with the peptide GRGD resulted in similar magnitude of inhibition, indicating that extracellular matrix proteins play significant roles in the interaction of S. enteritidis with granulosa cells. In contrast, treatment of the bacterial cells with the peptide GRAD did not result in significant inhibition of attachment to the granulosa cells. S. enteritidis was found to attach specifically to fibronectin, collagen IV, and laminin-coated microtiter plate wells, with the rank order of attachment as follows: fibronectin > laminin > collagen IV. Light and transmission electron micrographs of S. enteritidis invasion of granulosa cells showed organisms with or without a surrounding membrane in the cytoplasm of granulosa cells. In some instances, dividing bacterial cells were observed in the cytoplasm. Results of this study demonstrated that S. enteritidis interacts with granulosa cells in a specific manner and can invade and multiply in these cells. The granulosa cell layer of the preovulatory follicles may be a preferred site for the colonization of the chicken ovaries by invasive strains of S. enteritidis.

A rapid method for the determination of bacterial fatty acid composition

Fatty acid methyl esters suitable for gas chromatographic analysis were prepared by treatment of bacterial cells with sodium methoxide (1 N) in anhydrous methanol. The methanolysis of cells, washed or not with sterile water, was carried out at room temperature for 5 min. This method was effective for Gram-positive and Gram-negative bacteria, but not for growing yeast cells. The coefficient of variation of fatty acid methyl ester values was 4–7% of the mean values (range 0.4–25%).

Oxidative damage to bacterial DNA and evidence for its repair

Oxidative damage to DNA can be caused by excited oxygen species, which are produced by radiation or are by-products of aerobic metabolism. Endogenous levels of 8-hydroxy-2′-deoxyguanosine (8-OH-dG), an adduct that results from the damage of DNA caused by hydroxyl radical, have been detected inE. coli andS. typhimurium. Treatment of bacterial cells with various concentrations of hydrogen peroxide caused a moderate increase in the 8-OH-dG content. The enzymatic release of 8-OH-dG from asocorbate/Cu(II)-treated DNA was effected by an extract ofE. coli cells. These results indicate that 8-OH-dG is formedin vivo in bacterial DNA through endogenous oxidative mechanisms and on treatment with an oxygen radical-producing agent and that it is repairable.

Gyrase inhibitors can increase gyrA expression and DNA supercoiling

Treatment of bacterial cells with inhibitors of gyrase at high concentration leads to relaxation of DNA supercoils, presumably through interference with the supercoiling activity of gyrase. Under certain conditions, however, the inhibitors can also increase supercoiling. In the case of coumermycin A1, this increase occurs at low drug concentrations. Oxolinic acid increases supercoiling in a partially resistant mutant. We found that increases in chromosomal DNA supercoiling, which were blocked by treatment with chloramphenicol, were accompanied by an increased expression rate of gyrA. This result is consistent with gyrase being responsible for the increase in supercoiling. In wild-type cells, increases in gyrA expression were transient, suggesting that when supercoiling reaches sufficiently high levels, gyrase expression declines. Oxolinic acid studies carried out with a delta topA strain showed that drug treatment also increased plasmid supercoiling. The levels of supercoiling and topoisomer heterogeneity were much higher when the plasmid contained one of several promoters fused to galK. Since oxolinic acid causes an increase in gyrA expression, it appears that gyrase levels may be important in transcription-mediated changes in supercoiling even when topoisomerase I is absent.

Enterotoxic, Cytotoxic, Necrotic and Lethal Activities in Cell-Free Extracts of Salmonella Strains Isolated from Humans

Unconcentrated cell-free sonic extracts from thirty Salmonella strains isolated from the faeces and blood of humans were investigated for the production of enterotoxins in various tests (Vero cell, infant mouse, rabbit skin permeability and rabbit ileal loop), as well as for lethal activity in adult mice. Sonic extracts from 23 (76.7%) strains were lethal for mice, 21 (70%) increased skin permeability and 3 (10%) showed necrotizing activity for the rabbit skin. No Salmonella strain producing typical Escherichia coli toxins, such as thermolabile (LT) or thermostable (STa) enterotoxins, Verotoxin (VT) or cytotoxic necrotizing factor (CNF) cytotoxins, was detected. Non-repetitive fluid accumulation in rabbit loops was obtained when unconcentrated sonic extracts from 10 selected strains were assayed in seven rabbits. Growth of Salmonella in casamino acid yeast extract medium, followed by treatment of bacterial cells with polymyxin B, was demonstrated to be a rapid and sensitive method for releasing the delayed permeability factor.

Scanning electron microscopic delineation of bacterial surface topology using cationized ferritin

A novel method is presented for the labelling of negatively charged bacterial cell surface structures which allows their ultrastructural observation at the scanning electron microscopic level of resolution. The method is based on treatment of bacterial cells with cationized ferritin prior to examination. The method is simple to perform, rapid, reproducible and inexpensive. The approach provides a new topographical view of the fine structure of the bacterial cell surface.

Specificity of monoclonal antibodies reactive with Fusobacterium nucleatum: Effect of formalin fixation

Monoclonal antibody specific for Fusobacterium nucleatum was reacted with untreated and formalin fixed F. nucleatum cells by an enzyme-linked immunosorbent assay (ELISA) and by indirect immunofluorescence. Treatment of bacterial cells with formalin destroyed the antigenic determinant responsible for reactivity with this monoclonal antibody in both assays. Formalin fixation had no effect on hemagglutination activity (HA) of F. nucleatum cells or reactivity with polyvalent rabbit antiserum in double diffusion in agar. Scanning electron microscopy demonstrated that formalin fixation did not affect binding of F. nucleatum cells to microtiter plates. When developing monoclonal antibodies to be used as diagnostic reagents, the antigenic form utilized for immunization should be identical to the antigenic form which will eventually be used in the diagnostic assay.