Green synthesis of zinc oxide nanoparticles using novel bacterium strain (Bacillus subtilis NH1-8) and their in vitro antibacterial and antibiofilm activities against Salmonellatyphimurium.

Zinc oxide nanoparticles (ZnO NPs) are used in a range of applications, including food packaging, preservation, and storage. In the current investigation, extracellular green synthesis of ZnO NPs through an simple, eco-friendly, and rapid approach using a novel bacterial strain (Bacillus subtilis NH1-8) was studied. To assess the morphological, optical, and structural properties of ZnO NPs, transmission electron microscopy (TEM), energy-dispersive X-ray (EDX), scanning electron microscopy (SEM), fourier transform infrared (FTIR) spectroscopy, ultraviolet-visible (UV-vis) spectroscopy, and X-ray diffraction (XRD) techniques were carried out. In addition, disk diffusion, minimum bactericidal concentration (MBC), and minimum inhibitory concentration (MIC) methods were performed to determine the antibacterial activity of ZnO NPs. The average size of biosynthesized ZnO NPs was 39 nm, exhibiting semi-spherical, which was confirmed by TEM analyses. The UV-vis spectroscopy exhibited the absorption peak at 200-800nm. The ZnO NPs have shown effective antimicrobial and antibiofilm activities against S. typhimurium. Thus, biosynthesized ZnO NPs could be exploited as a breakthrough technology in the surface coating of food containers and cans to minimize contamination by S. typhimurium.

Multifunctional Acidocin 4356 Combats Pseudomonas aeruginosa through Membrane Perturbation and Virulence Attenuation: Experimental Results Confirm Molecular Dynamics Simulation.

A longstanding awareness in generating resistance to common antimicrobial therapies by Gram-negative bacteria has made them a major threat to global health. The application of antimicrobial peptides as a therapeutic agent would be a great opportunity to combat bacterial diseases. Here, we introduce a new antimicrobial peptide (∼8.3 kDa) from probiotic strain Lactobacillus acidophilus ATCC 4356, designated acidocin 4356 (ACD). This multifunctional peptide exerts its anti-infective ability against Pseudomonas aeruginosa through an inhibitory action on virulence factors, bacterial killing, and biofilm degradation. Reliable performance over tough physiological conditions and low hemolytic activity confirmed a new hope for the therapeutic setting. Antibacterial kinetic studies using flow cytometry technique showed that the ACD activity is related to the change in permeability of the membrane. The results obtained from molecular dynamic (MD) simulation were perfectly suited to the experimental data of ACD behavior. The structure-function relationship of this natural compound, along with the results of transmission electron microscopy analysis and MD simulation, confirmed the ability of the ACD aimed at enhancing bacterial membrane perturbation. The peptide was effective in the treatment of P. aeruginosa infection in mouse model. The results support the therapeutic potential of ACD for the treatment of Pseudomonas infections.IMPORTANCE Multidrug-resistant bacteria are a major threat to global health, and the Pseudomonas bacterium with the ability to form biofilms is considered one of the main causative agents of nosocomial infections. Traditional antibiotics have failed because of increased resistance. Thus, finding new biocompatible antibacterial drugs is essential. Antimicrobial peptides are produced by various organisms as a natural defense mechanism against pathogens, inspiring the possible design of the next generation of antibiotics. In this study, a new antimicrobial peptide was isolated from Lactobacillus acidophilus ATCC 4356, counteracting both biofilm and planktonic cells of Pseudomonas aeruginosa A detailed investigation was then conducted concerning the functional mechanism of this peptide by using fluorescence techniques, electron microscopy, and in silico methods. The antibacterial and antibiofilm properties of this peptide may be important in the treatment of Pseudomonas infections.

Ecological role of Acinetobacter calcoaceticus GSN3 in natural biofilm formation and its advantages in bioremediation.

This study assessed the role of a new Acinetobacter calcoaceticus strain, GSN3, with biofilm-forming and phenol-degrading abilities. Three biofilm reactors were spiked with activated sludge (R1), green fluorescent plasmid (GFP) tagged GSN3 (R2), and their combination (R3). More than 99% phenol removal was achieved during four weeks in R3 while this efficiency was reached after two and four further operational weeks in R2 and R1, respectively. Confocal scanning electron microscopy revealed that GSN3-gfp strains appeared mostly in the deeper layers of the biofilm in R3. After four weeks, almost 7.07 × 107 more attached sludge cells were counted per carrier in R3 in comparison to R1. Additionally, the higher numbers of GSN3-gfp in R2 were unable to increase the efficiency as much as measured in R3. The presence of GSN3-gfp in R3 conveyed advantages, including enhancement of cell immobilization, population diversity, metabolic cooperation and ultimately treatment efficiency.

Paenibacillus xanthanilyticus sp. nov., a xanthan-degrading bacterium isolated from soil.

A xanthan-degrading bacterium, strain AS7T, was isolated from soil and its taxonomic position was determined using a polyphasic approach. Strain AS7T was a Gram-stain-variable, spore-forming, motile, aerobic, rod-shaped bacterium. Phylogenetic analysis based on 16S rRNA gene sequence analysis revealed that strain AS7T belongs to the genus Paenibacillus, sharing the highest level of sequence similarity with Paenibacillus phyllosphaerae PALXIL04T (98.0 %). The cell-wall peptidoglycan contained meso-diaminopimelic acid. MK-7 was the dominant isoprenoid quinone and the major fatty acid was anteiso-C15 : 0. Polar lipids consisted of diphosphatidylglycerol, phosphatidylglycerol, phosphatidylethanolamine and two unknown phospholipids. These chemotaxonomic characteristics were consistent with the isolate belonging to the genus Paenibacillus. The G+C content of the genomic DNA was 51.0 mol% and the DNA-DNA hybridization value between strain AS7T and P. phyllosphaerae PALXIL04T was only 14.4±2.5 %. On the basis of phylogenetic analyses, phenotypic and chemotaxonomic characteristics, and DNA-DNA relatedness value, strain AS7T represents a novel species of the genus Paenibacillus, for which the name Paenibacillus xanthanilyticus sp. nov. is proposed. The type strain is AS7T (=IBRC M 10987T=LMG 29451T).

Production of nanocellulose in miniature-bioreactor: Optimization and characterization.

Bacterial cellulose (BC) is a very fascinating microbial biopolymer which is mainly produced by Gluconacetobacter xylinum. Optimization of BC production by G. xylinum was performed based on scale-down studies in miniature-bioreactor and response surface methodology in which the optimum pH value (6.5) and shaking rate (50 rpm) were obtained. The static culture condition for BC production has newly been defined. Nanostructure of BC includes nanofibers up to (60 nm) and nanoporosity up to (265 nm) was observed by scanning electron microscopy. By Fourier transform infrared spectroscopy study, the most expected BC interaction is nucleophilic interaction. MTT assay showed high biocompatibility. Appropriate mechanical strength (0.37 MPa) and Young's modulus (3.36 MPa) evinced BC scaffold utilization for skin tissue. The results indicate that BC sheets can be utilized in biomedical application and nanotechnology approaches.

Starmerella orientalis f.a., sp. nov., an ascomycetous yeast species isolated from flowers.

Four strains of a novel ascomycetous yeast species were isolated from flowers in Iran and China. Phylogenetic analysis of the sequences of the ITS region (including 5.8S rRNA gene) and the LSU rRNA gene D1/D2 domains indicated that these strains belong to the Starmerella clade and show divergence from previously described species in this clade. Growth reactions on carbon and nitrogen sources were similar to those observed in related species of the Starmerella clade. Sexual reproduction was not observed after mating tests on different sporulation media. Based on physiological characteristics and phylogeny of rRNA gene sequences, the novel species is most closely related to Candida (iter. nom. Starmerella) powellii and Candida (iter. nom. Starmerella) floricola. It is therefore assigned to the genus Starmerella and described as Starmerella orientalis f.a., sp. nov. The type strain is SAM09T ( = IBRC-M 30204T = CBS 14142T). The MycoBank accession number is MB 814379.

Wickerhamomyces orientalis f.a., sp. nov.: an ascomycetous yeast species belonging to the Wickerhamomyces clade.

Five closely related yeast strains were isolated from soil in Kharg Island, Persian Gulf, Iran, and from fallen fruits in Galle, Sri Lanka, during separate projects. Morphologically, the strains produced white-coloured yeast colonies, with cells that were ovoid to ellipsoidal, making branched, true hyphae and pseudohyphae. Ascospore formation was not observed. Biochemically, the strains were able to ferment d-glucose and weakly ferment d-galactose. The strains could use a wide variety of carbon sources except methanol and hexadecane. Phylogenetic analyses using combined sequences of the small ribosomal subunit and the D1/D2 domains of the LSU, as well as the internal transcribed spacer regions, suggested that these strains belong to the Wickerhamomyces clade and that together they form one strongly supported phylogenetic clade. Differences in their sequences, biochemistry and morphology suggest they are representatives of distinct species of the genus Wickerhamomyces. Therefore, the name Wickerhamomyces orientalis f.a., sp. nov. is proposed to accommodate these novel strains; the type strain is IBRC-M 30103T (=CBS 13306T). The MycoBank number is MB 807323.

Basidioascus persicus sp. nov., a yeast-like species of the order Geminibasidiales isolated from soil.

A novel species of basidiomycetes was isolated from kitchen garden soil in Shahryar city, Tehran province, Iran. Molecular and conventional methods were employed to identify and classify this single isolate. Morphologically, the isolate was considered yeast-like with hyaline and oval cells reproducing by monopolar budding, forming ballistoconidia, hyphae, arthroconidia and didymospores. Basidia and basidiospores resembling those produced by Basidioascus species were observed. Sequencing and Bayesian phylogenetic analysis of rRNA genes and the internal transcribed spacer region revealed its sister relationship to described species of the genus Basidioascus. Assimilation and fermentation tests, cell-wall carbohydrate analysis and enzyme activity tests were performed to provide insight into the metabolism of the isolate. Based on morphology, physiology and phylogeny of rRNA gene sequences, the isolate was shown to represent a novel species of the genus Basidioascus, described as Basidioascus persicus sp. nov. (holotype IBRC P1010180(T) = ex-type IBRC M30078(T) = isotype CBS 12808(T)). The MycoBank number of the novel species is MB 804703. An emended description of the genus Basidioascus is also provided.

Phytate-Induced Dose-Response Auto-Activation of Enzyme in Commercial Recombinant Phytase From Escherichia coli.

Background: Microbial phytase is one of the most widely used enzymes in food industries like cattle, poultry, and aquaculture food. Therefore, understanding the kinetic properties of the enzyme is very important to evaluate and predict its behavior in the digestive system of livestock. Working on phytase is one of the most challenging experiments because of some problems, including free inorganic phosphate (FIP) impurity in phytate (substrate) and interference reaction of the reagent with both phosphates (product and phytate impurity). Objective: In the present study, FIP impurity of phytate was removed, and then it was shown that the substrate (phytate) has a dual role in enzyme kinetics: substrate and activator. Material and Methods: phytate impurity was decreased by two-step recrystallization prior to the enzyme assay. The impurity removal was estimated by the ISO30024:2009 method and confirmed by Fourier-transform infrared (FTIR) spectroscopy. The kinetic behavior of phytase activity was evaluated using the purified phytate as substrate by non-Michaelis-Menten analysis, including Eadie-Hofstee, Clearance, and Hill plots. The possibility of an allosteric site on phytase was assessed by molecular docking. Results: The results showed a 97.2% decrease in FIP due to recrystallization. The phytase saturation curve had a sigmoidal appearance, and Lineweaver-Burk plot with a negative y-intercept indicated the positive homotropic effect of the substrate on the enzyme activity. A right-side concavity of Eadie-Hofstee plot confirmed it. Hill coefficient was calculated to be 2.26. Molecular docking also showed that Escherichia coli phytase molecule has another binding site for phytate very close to the active site, called "allosteric site". Conclusions: The observations strongly propose the existence of an intrinsic molecular mechanism in Escherichia coli phytase molecules to be promoted for more activity by its substrate, phytate (positive homotropic allosteric effect). In silico analysis showed that phytate binding to the allosteric site caused new substrate-mediated inter-domain interactions, which seems to lead to a more active conformation of phytase. Our results provide a strong basis for animal feed development strategies, especially in the case of poultry food and supplements, regarding a short food passage time in their gastrointestinal tract and variable concentration of phytate along with it. Additionally, the results strengthen our understanding of phytase auto-activation as well as allosteric regulation of monomeric proteins in general.

Production of Childinan SF-2 as Bioactive Compound from Daldinia childiae: A Survey on Antioxidant, Antibacterial and Antitumor Properties.

Background: Fungal extracts have received increased attention due to their great medicinal applications including antitumor, immune-modulating, antioxidant, radical scavenging, antiviral, antibacterial, antifungal and detoxificating properties. Objectives: This study is the first report on a novel bioactive compound, namely Childinan SF-2 which was isolated from soil ascomycete fungus. The significant antibacterial, antioxidant and cytotoxic properties of the extract may lead to development of novel, safe and useful substances. Materials and Methods: The isolate was identified on the basis of molecular approach. Spore suspension was inoculated in the culture medium and the bioactive compound was isolated from the viscous fermented broth via ethanol precipitation of the extracellular compound. The basic chemical composition of the extract including protein, carbohydrate, sulfate radical and uronic acid content were measured. FTIR (Fourier-transform infrared spectroscopy) and GC-MS (Gas chromatography-mass spectrometry) analysis were used for further structural characterization. The extract was utilized for treatment of AGS and MDA cell lines to assess the cell cycle and apoptosis. The antioxidant activity was examined using DPPH, hydroxyl radicals scavenging, ß-carotene bleaching inhibition and ferric reducing power assay methods. The extract was tested for evaluation of antibacterial activity using different Gram-positive and Gram-negative bacterial strains. Results: The fungal isolate was identified as the new strain Daldinia childiae SF-2. Initial biochemical characterization of the extract showed that the fungal biopolymer was composed of total sugars, protein, uronic acids and sulfated groups with values of 91.6%, 2.15%, 2.25% and 1.05% (w/w), respectively. FTIR and GC-MS analysis revealed that Childinan SF-2 might be mainly constructed from D-glucose, D-mannitol and D-galactofuranose. The in vitro experiments revealed that Childinan SF-2 enhanced the percentage of necrosis and apoptosis of cancer cells and blocked the cell cycle progression as shown by flowcytometry. Childinan SF-2 represented a considerable antioxidant and antibacterial activity. Conclusions: These results indicated that Childinan SF-2 can serve as a potential source in medicinal applications. Keywords Exopolysaccharides; Fungal Biopolymers; Mycelial Secretions; Natural Pharmaceuticals; Xylariaceae.

Biotransformation and removal of arsenic oxyanions by Alishewanella agri PMS5 in biofilm and planktonic states.

Arsenic oxyanions are toxic chemicals that impose a high risk to humans and other living organisms in the environment. The present study investigated indigenous heterotrophic bacteria in the tailings dam effluent (TDE) of a gold mining factory. Thirty-seven arsenic resistant bacteria were cultured on Reasoner's 2A agar supplemented with arsenic salts through filtration. One strain encoded as PMS5 with the highest resistance to 140-mM sodium arsenite and 600-mM sodium arsenate in tryptic soy broth was selected for further investigations. According to phenotypic examinations and 16S rDNA sequence analysis, PMS5 belonged to the genus Alishewanella and was sensitive to most of the examined antibiotics. The biosorption and bioaccumulation abilities of arsenic salts were observed in this isolate based on Scanning Electron Microscopy (SEM) with Energy Dispersive X-Ray Analysis (EDX) and biosorption and bioaccumulation data. PMS5 was also found to cause the volatilization and biotransformation of arsenic oxyanions through their oxidation and reduction. Moreover, the contribution of PMS5 to arsenic (3+, 5+) bioprocessing under oligotrophic conditions was confirmed in fixed-bed reactors fed with the TDE of the gold factory (R1) and synthetic water containing As5+ (R2). According to biofilm assays such as biofilm staining, cell count, detachment assay and SEM, the arsenic significantly reduced the biofilm density of the examined reactors compared to that of the control (R3). Arsenate reduction and arsenite oxidation under bioreactor conditions were respectively obtained as 75.5-94.7% and 8%. Furthermore, negligible arsenic volatilization (1.2 ppb) was detected.

Considerable increase in Poly(3-hydroxybutyrate) production via phbC gene overexpression in Ralstonia eutropha PTCC 1615.

Introduction: Poly(3-hydroxybutyrate) (PHB) is a well-known biodegradable polymer produced by some microorganisms and can be a suitable alternative for petrochemical plastics. PHB synthase encoded by phb C gene is the main enzyme in PHB biosynthesis pathway in Ralstonia eutropha. The aim of current study was the transformation of R. eutropha PTCC 1615 with its own phb C gene and evaluation of the overexpression effect on PHB accumulation. Methods: DNA fragment including phbC gene and its promoter and terminator regions, was isolated from R. eutropha PTCC 1615, inserted into pET28a(+) vector, and transferred to the competent bacteria using calcium chloride and heat shock method. The effect of the cloned gene expression on PHB production was investigated with absorption of crotonic acid produced through PHB dehydration. Statistical analyses were carried out by SPSS software. Results: PHB content of cells of the engineered strain was 1.4 times more than that of the native bacteria. This significant difference can be an important finding for improvement of biopolymer production. Conclusion: Overexpression of phb C, the critical gene in PHB biosynthesis pathway, in R. eutropha PTCC 1615 had considerable effect on PHB accumulation.

A Comparative Taxonomic Profile of Microbial Polyethylene and Hydrocarbon-Degrading Communities in Diverse Environments.

BACKGROUND: Polyethylene (PE) is one of the most abundant plastic wastes which accumulates in marine and terrestrial environments. As microbial degradation has been a promising approach for the bioremediation of polluted environments, identification of the microbial community profile where these pollutants accumulate, has recently been in focus. OBJECTIVE: We have investigated the taxonomic and functional characteristics of polyethylene- degrading microorganisms in a plastic waste recycling site in Tehran, Iran. MATERIALS AND METHODS: We have analyzed and compared a 16S rRNA dataset from this study with 15 datasets from 4 diverse plastic and oil polluted habitats to identify and evaluate bacterial communities involved in bioremediation. RESULTS: Our findings reveal that Proteobacteria, Actinobacteria, Acidobacteria and Cloroflexi were the dominant phyla and Actinobacteria, Alphaproteobacteria, Gammaproteobacteria and Acidimicrobia were dominant classes in these samples. The most dominant Kegg Orthology associated with PE bioremediation in these samples are related to peroxidases, alcohol dehydrogenases, monooxygenases and dioxygenases. CONCLUSIONS: Long-term presence of contaminants in soil could lead to changes in bacterial phyla abundance, resulting in metabolic adaptations to optimize biological activity and waste management in a diverse group of bacteria.

Biological function and molecular properties of Pyrenaican SF-1 as biological macromolecule extracted from Daldinia pyrenaica.

Molecular properties and biological functions of Pyrenaican SF-1 as a novel biological macromolecule extracted from a fungal isolate were studied. The isolate was identified as Daldinia pyrenaica on the basis of 5.8S rDNA sequencing. Pyrenaican SF-1 was obtained from the culture filtrate of the fungal isolate. The partial characterization of biochemical structure of Pyrenaican SF-1 was conducted. The fungal extract was also tested for the treatment of AGS, MDA and HeLa cell lines to assess cells proliferation, cells cycle and apoptosis. Furthermore, Pyrenaican SF-1 extract was tested for its antibacterial and antioxidant activity. Initial chemical analysis revealed that Pyrenaican SF-1 extract was composed of various monosaccharides such as d-glucose, D- mannitol, D-arabinose and ß-D-ribopyranose. In vitro study indicated that Pyrenaican SF-1 could effectively elevate percentage of apoptosis and necrosis of cancer cells and block cell cycle phase of the control group. The fungal extract could inhibit proliferation of Hela and MDA cell up to 67% and 56%, respectively. Moreover, Pyrenaican SF-1 represented a strong antioxidant activity compared to that one obtained from vitamin C. On the other hand, Pyrenaican SF-1 exhibited growth inhibitory effects against different Gram-negative and Gram-positive bacterial strains. Pyrenaican SF-1 can be considered as a bioactive macromolecule with promising application in pharmaceutical and medical sectors.

Isolation and characterization of a novel gamma-radiation-resistant bacterium from hot spring in Iran.

30 pure colonies with various colors and shapes were isolated from the samples of water and mud which were collected from the Abe-Siah hot springs in Ramsar, a city in north of Iran, with high-level of radiation (up to 4 Gy). Colonies were irradiated by 22 KGy radiations and only one of them survived. The survived bacterium with an optimum growth of 30 to 37 degrees C, pH 6 to 7, and at minimum of 5% NaCl concentration showed promising characteristics. This strain is aerobic, mesophilic, white colony, with catalase-positive, oxidase-negative, non motile, spore forming, rod-shape and finally Gram positive. Strain Bacillus sp. WHO represented a strain of genus Bacillus megaterium according to a phylogenetic analysis of the 16S rDNA and biochemical features. Its radio-resistancy was compared with E. coli and B. megaterium which were inactivated in 2 KGy and 5.9 KGy respectively. In order to reveal the mechanisms of this extreme radio-resistancy and WHO DNA repair system we examined its proteomic map, following gamma-irradiation, using two-dimensional polyacrylamide gel electrophoresis and silver-staining. The expression levels of majority number of protein spots showed significant changes under radiation stress.

Bioactive exopolysaccharide from Neopestalotiopsis sp. strain SKE15: Production, characterization and optimization.

Fungal exopolysaccharides are powerful resources of medicinal applications. Neopestalotiopsis sp. SKE15 was isolated and identified according to phenotypical and genotypical analyses (GenBank Accession No. MG649986). The exopolysaccharide (EPS) was produced by cultivation of mycelia in broth culture and extracted. The production was optimized to 2.02 g/l after selection of agitation, temperature, FeSO4 and K2HPO4 concentrations as the most influencing factors using Placket-Burman design and then by applying response surface methodology. Analytical Tools showed that the EPS is composed of a polysaccharide (1.5-2.1 × 106 Da) and its probable low molecular weight derivatives, in a wide range of chain lengths, among them an oligosaccharide of about 1970 Da was dominant. GC-MS (Gas chromatography-mass spectrometry) analysis revealed the EPS was mainly constructed from d-glucose, sorbitol and D-galactose. The EPS showed antibacterial activity against Staphylococcus aureus ATCC 25923, Bacillus subtilis ATCC 6633 and Pseudomonas aeruginosa ATCC 27853. DPPH (1,1-diphenyl-2-picryl-hydrazyl) and hydroxyl radical scavenging activity assays showed strong antioxidant activity of the EPS. A challenge with three different cancerous cell lines showed cytotoxic activity of the EPS at final concentration of 100 and 200 µg/ml. Further investigation on medicinal applications of the biopolymer is promising.

Effects of Nonionic Surfactants on Xanthan Gum Production: a Survey on Cellular Interactions.

BACKGROUND: Xanthomonas campestris is a biopolymer producing gram negative bacterium. Production of xanthan biopolymer can be affected by different extrinsic factors as well as surfactants. Hitherto, effects of nonionic surfactants on xanthan production have been studied in a limited number of articles. OBJECTIVE: In the present study, nonionic surfactants were used to pursue their effects on improvement of xanthan production. Moreover, a number of cellular consequences upon the treatment were investigated with impacts on gum production. MATERIALS AND METHODS: Effects of different nonionic surfactants (Tween 20, Tween 80 and Triton X100) on xanthan production and Xanthomonas campestris cells were assessed by ultramicroscopy (SEM), changes in culture turbidity, leakage of sugars and ATP, and quality of xanthan (i.e. pyruvate content and determination of polymer molecular weight). RESULTS: The nonionic surfactant Tween 20 increased ATP (3.2 folds) and sugar leakage (3.1 folds). Furthermore, they caused cell shape alteration. Tween 80 improved both xanthan production (11 g.L-1) and viscosity of the product (1368 cP), while the total biomass remained unchanged (2.2 g.L-1). Molecular weight of xanthan was enhanced (from 23 to 59 million Da). Toxic effect of 5% (v/v) Triton X 100 decreased the turbidity of culture to 120 NTU and total biomass was diminished to 1 g.L-1. Tween 20 caused the loss of ATP and sugar leakage and led to lower xanthan production. It had no effect on biomass content. CONCLUSIONS: In general, amounts of surfactants that bacterial cells can tolerate seem to be helpful in substrate and metabolite transportation, and enzyme activities involved in xanthan biosynthesis and release. Surfactants induce harsh damages to cell barriers, preventing the growth and adversely affecting quantity and quality of xanthan gum.

Production of Xanthanases by Paenibacillus spp.: Complete Xanthan Degradation and Possible Applications.

Background: A number of microorganisms and their enzymes have been reported as xanthan depolymerizers. Paenibacillus species are well-known polysaccharide hydrolyzing bacteria. However, Paenibacillus alginolyticus and Paenibacillus sp. XD are the only species in the genus which are now known to degrade xanthan. Objectives: Complete biodegradation of the xanthan exopolysaccharide is a rarely found capability among microorganisms. The aim of this study is to survey xanthanase producing bacteria with an appropriate bioactivity for the biopolymer degradation under different environmental conditions. Materials and Methods: The bacteria were isolated based on viscosity reduction of the xanthan solution. Bacterial isolates were identified using rep-PCR (repetitive element-based genomic fingerprinting) and 16S rDNA sequencing. Xanthanases were identified using rep-PCR (repetitive element-based genomic fingerprinting) and 16S rDNA sequencing. Xanthanases were characterized by measuring their activity at different temperatures, pH values, and NaCl concentrations. Degradation of other polysaccharides and xanthan degradation products were investigated based on the screening plate method and TLC (thin-layer chromatography), respectively. Results:Six isolates from different Paenibacillus species with a complete xanthan degrading capability were isolated from Urmia Lake. Phylogenetic analysis placed these strains within the genus Paenibacillus with the closest relatives that were found to be P. nanensis, P. phyllosphaerae, P. agaridevorans, P. agarexedens, and P. taohuashanense. These isolates displayed different levels of the xanthan biodegradation activity in temperatures ranging from 15 to 55°C and pH values from 4 to 11. Xanthanolytic activity was generally prevented in presence of NaCl (> 0.1 mol.L-1). Furthermore, the isolated Paenibacillus spp. could degrade several other polysaccharides including xylan, CMC (carboxymethyl cellulose), starch, alginate, and pectin. Conclusion: Novel strains of the six different Paenibacillus species that were introduced in the present study are able to produce xanthanases with interesting characteristics. In light of the results from this study, special applications, particularly in healthcare, medicine, and the environment is hereby proposed for these enzymes.

Improved effect of amikacin-loaded poly(D,L-lactide-co-glycolide) nanoparticles against planktonic and biofilm cells of Pseudomonas aeruginosa.

PURPOSE: Amikacin is one of the most effective antibiotics against Pseudomonas aeruginosa infections, but because of its high toxicity, the use of this antibiotic has been clinically limited. In the present study, amikacin was successfully loaded into a new formulation of nanoparticles (NPs) based on poly(d,l-lactide-co-glycolide) 50 : 50 in order to enhance the treatment efficacy. The synthetized amikacin-loaded PLGA nanoparticles with high drug loading and stability were used to eliminate P. aeruginosa cells in planktonic and biofilm states. METHODOLOGY: P. aeruginosa PAO1 biofilm susceptibility studies were done using the minimum biofilm eradication concentration assay. The association of fluorescently labeled amikacin-loaded nanoparticles (A-NPs) with mouse monocyte macrophage cells (RAW 264.7), and the nanoparticles ability to interact and eradicate the bacterial cells even in the form of biofilms, was investigated using Flow cytometric studies and confocal laser scanning microscopy. RESULTS: Flow cytometric studies showed that these NPs were able to interact with planktonic and biofilm bacterial cells. Moreover, following 1 h of incubation of A-NPs with 1-day-old biofilm, it was found that particles penetrate through the entire biofilm thickness. Live/dead fluorescent staining followed by CLSM analysis showed that the A-NPs were more effective than free drug in biofilm eradication. CONCLUSION: The good antibacterial and antibiofilm activities of A-NPs, in addition to their ability to enter macrophages without any cytotoxicity for these cells, make them a potential candidate to treat P. aeruginosa infections.

Ex situ study of Enterococcus faecalis survival in the recreational waters of the southern coast of the Caspian Sea.

BACKGROUND AND OBJECTIVES: The US Environmental Protection Agency has suggested faecal enterococci as the primary bacterial indicators. Of more importance is their direct correlation with swimmer-associated gastroenteritis in recreation water quality monitoring. In contrast to other seawater bodies with 3.5% salinity, the recreational waters in the southern coast of the Caspian Sea possess its own salinity (about 1% w/v) and thus require further investigations to determine the capacity of Enterococcus faecalis as the sole primary microbial index in this unique aquatic environment. MATERIALS AND METHODS: The survey of the presence and survival of E. faecalis as a microbial index in the recreational waters of the southern Caspian Sea was carried out using a microcosm as an experimental model. The concentration of E. faecalis cells in samples of seawater were estimated by a standard membrane filtration method using m-Enterococcus agar as the selective culture medium. As the current standard culture-based methods are not reliable enough for the detection of non-growing, damaged and under-tension bacteria, PCR was used to identify the possible VBNC form of the bacterium after disappearance of the culturable cells. RESULTS AND CONCLUSION: A continuous decline in the number of culturable E. faecalis cells resulted in apparent elimination of the bacteria from seawater in a defined period. Detection of intact DNA was possible in the following 60 days. The salinity of about 1% and the self-purification properties of the Caspian Sea make the conditions feasible for the use of this microorganism as a measure of water quality throughout the region. The results confirmed the presence of damaged bacterial cells, namely VBNC forms, indicating the necessity of examining of the sea water samples by using molecular approaches or repair procedures.