Evaluation of biosorption potency of <Emphasis Type="Italic">Acinetobacter</Emphasis> sp. for removal of hexavalent chromium from tannery effluent

Two bacterial consortia were developed by continuous enrichment of microbial population of tannery and pulp and paper mill effluent contained Serratia mercascens, Pseudomonas fluorescence, Escherichia coli, Pseudomonas aeruginosa and Acinetobacter sp. identified by 16S rDNA method. The consortia evaluated for removal of chromate [(Cr(VI)] in shake flask culture indicated pulp and paper mill consortium had more potential for removal of chromate. Acinetobacter sp. isolated from pulp and paper mill consortium removed higher amount of chromate [Cr(VI)] under aerobic conditions. Parameters optimized in different carbon, nitrogen sources, and pH, indicated maximum removal of chromate in sodium acetate (0.2%), sodium nitrate (0.1%) and pH 7 by Acinetobacter sp. Bacteria was applied in 2-l bioreactor significantly removed chromate after 3 days. The results of the study indicated removal of more than 75% chromium by Acinetobacter sp. determined by diphenylcarbazide colorimetric assay and atomic absorption spectrophotometer after 7 days. Study of microbial [Cr(VI)] removal and identification of reduction intermediates has been hindered by the lack of analytical techniques. Therefore, removal of chromium was further substantiated by transmission electron microscopy (TEM), scanning electron microscopy (SEM) and energy-dispersive X-ray spectroscopy (EDX) which indicated bioaccumulation of chromium in the bacterial cells.     

Catalase Expression Is Modulated by Vancomycin and Ciprofloxacin and Influences the Formation of Free Radicals in Staphylococcus aureus Cultures

Detection of free radicals in biological systems is challenging due to their short half-lives. We have applied electron spin resonance (ESR) spectroscopy combined with spin traps using the probes PBN (N-tert-butyl-α-phenylnitrone) and DMPO (5,5-dimethyl-1-pyrroline N-oxide) to assess free radical formation in the human pathogen Staphylococcus aureus treated with a bactericidal antibiotic, vancomycin or ciprofloxacin. While we were unable to detect ESR signals in bacterial cells, hydroxyl radicals were observed in the supernatant of bacterial cell cultures. Surprisingly, the strongest signal was detected in broth medium without bacterial cells present and it was mitigated by iron chelation or by addition of catalase, which catalyzes the decomposition of hydrogen peroxide to water and oxygen. This suggests that the signal originates from hydroxyl radicals formed by the Fenton reaction, in which iron is oxidized by hydrogen peroxide. Previously, hydroxyl radicals have been proposed to be generated within bacterial cells in response to bactericidal antibiotics. We found that when S. aureus was exposed to vancomycin or ciprofloxacin, hydroxyl radical formation in the broth was indeed increased compared to the level seen with untreated bacterial cells. However, S. aureus cells express catalase, and the antibiotic-mediated increase in hydroxyl radical formation was correlated with reduced katA expression and catalase activity in the presence of either antibiotic. Therefore, our results show that in S. aureus, bactericidal antibiotics modulate catalase expression, which in turn influences the formation of free radicals in the surrounding broth medium. If similar regulation is found in other bacterial species, it might explain why bactericidal antibiotics are perceived as inducing formation of free radicals.     

Electron Microscopy and X-ray Analysis of Cr-Containing Precipitates Synthesized by Newly Isolated Actinobacterium,Flexivirga alba ST13T

Chromium(Cr) precipitate synthesized by Cr(VI)-reducing bacterium Flexivirga alba ST13^T was examined using transmission electron microscopy (TEM) and the energy dispersive X-ray (EDX). The strain showed altered-morphology after exposing to Cr(VI) in minimal medium. The resultant precipitate included bacterial pellet and needle-like structure which was similar to the structure made from Cr(OH)₃ precipitate. Cr was observed in bacterial cells using TEM–EDX. Bacteria with high electron density showed the precipitation of Ca in addition to Cr. The isolated strain would be useful to precipitate Cr from Cr(VI)-containing environment.     

Sensitivity of Mixed Populations of Staphylococcus aureus and Escherichia coli to Mercurials

Staphylococcus aureus was found to have a higher resistance to merbromin and mercuric chloride in the presence of Escherichia coli. The protective effect of the gram-negative organism on S. aureus was due to the production of extracellular glutathione and hydrogen sulfide and to an unequal distribution of the inhibitor between the two species. S. aureus did not significantly influence the resistance of E. coli to mercurials.     

Evaluation of Ultrasound-Induced Damage to Escherichia coli and Staphylococcus aureus by Flow Cytometry and Transmission Electron Microscopy

As a nonthermal sterilization technique, ultrasound has attracted great interest in the field of food preservation. In this study, flow cytometry and transmission electron microscopy were employed to investigate ultrasound-induced damage to Escherichia coli and Staphylococcus aureus. For flow cytometry studies, single staining with propidium iodide (PI) or carboxyfluorescein diacetate (cFDA) revealed that ultrasound treatment caused cell death by compromising membrane integrity, inactivating intracellular esterases, and inhibiting metabolic performance. The results showed that ultrasound damage was independent of initial bacterial concentrations, while the mechanism of cellular damage differed according to the bacterial species. For the Gram-negative bacterium E. coli, ultrasound worked first on the outer membrane rather than the cytoplasmic membrane. Based on the double-staining results, we inferred that ultrasound treatment might be an all-or-nothing process: cells ruptured and disintegrated by ultrasound cannot be revived, which can be considered an advantage of ultrasound over other nonthermal techniques. Transmission electron microscopy studies revealed that the mechanism of ultrasound-induced damage was multitarget inactivation, involving the cell wall, cytoplasmic membrane, and inner structure. Understanding of the irreversible antibacterial action of ultrasound has great significance for its further utilization in the food industry.     

Deposition pattern of hydrogen peroxide in the leaf sheaths of rice under salt stress

Deposition pattern of hydrogen peroxide (H₂O₂) under salt stress (100 mM NaCl) was examined cytochemically in rice (Oryza sativa L. cv. Pokkali) through the reaction of H₂O₂ with cerium chloride (CeCl₃) to produce electron dense precipitates of cerium perhydroxide. The distribution pattern of cerium perhydroxide precipitates in leaf sheath was considerably different from other parts of rice under salinity stress. Cerium perhydroxide precipitates were mainly accumulated on the tonoplast of leaf sheath under salinity, although they were localized on the cell wall and plasma membrane in all other tissues such as leaf blade and root.     

Bacteria May Cope Differently from Similar Membrane Damage Caused by the Australian Tree Frog Antimicrobial Peptide Maculatin 1.1

Maculatin 1.1 (Mac1) is an antimicrobial peptide from the skin of Australian tree frogs and is known to possess selectivity toward Gram-positive bacteria. Although Mac1 has membrane disrupting activity, it is not known how Mac1 selectively targets Gram-positive over Gram-negative bacteria. The interaction of Mac1 with Escherichia coli, Staphylococcus aureus, and human red blood cells (hRBC) and with their mimetic model membranes is here reported. The peptide showed a 16-fold greater growth inhibition activity against S. aureus (4 μm) than against E. coli (64 μm) and an intermediate cytotoxicity against hRBC (30 μm). Surprisingly, Sytox Green uptake monitored by flow cytometry showed that Mac1 compromised both bacterial membranes with similar efficiency at ∼20-fold lower concentration than the reported minimum inhibition concentration against S. aureus. Mac1 also reduced the negative potential of S. aureus and E. coli membrane with similar efficacy. Furthermore, liposomes mimicking the cell membrane of S. aureus (POPG/TOCL) and E. coli (POPE/POPG) were lysed at similar concentrations, whereas hRBC-like vesicles (POPC/SM/Chol) remained mostly intact in the presence of Mac1. Remarkably, when POPG/TOCL and POPE/POPG liposomes were co-incubated, Mac1 did not induce leakage from POPE/POPG liposomes, suggesting a preference toward POPG/TOCL membranes that was supported by surface plasma resonance assays. Interestingly, circular dichroism spectroscopy showed a similar helical conformation in the presence of the anionic liposomes but not the hRBC mimics. Overall, the study showed that Mac1 disrupts bacterial membranes in a similar fashion before cell death events and would preferentially target S. aureus over E. coli or hRBC membranes.     

Effects of colicin A and staphylococcin 1580 on amino acid uptake into membrane vesicles of Escherichia coli and Staphylococcus aureus

Staphylococcin 1580 increased the relative amount of diphosphatidylglycerol and decreased the amount of phosphatidylglycerol in cells of Staphlococcus aureus, while the amounts of lysylphosphatidylglycerol, phosphatidic acid and total phospholipid remained constant.Treatment of cells of Escherichia coli and S. aureus with colicin A and staphylococcin 1580, respectively, did not affect proton impermeability but subsequent addition of carbonylcyanide-m-chlorophenylhydrazone resulted in a rapid influx of protons into the cells.Bacteriocin-resistant and -tolerant mutants of E. coli and S. aureus were isolated. The bacteriocins caused leakage of amino acids preaccumulated into membrane vesicles of resistant mutants and had no significant effect on membrane vesicles of tolerant mutants.The uptake of amino acids into membrane vesicles was inhibited by both bacteriocins, irrespective of the electron donors applied. The bacteriocin inhibition was noncompetitive. The bacteriocins did not affect oxygen consumption and dehydrogenases in membrane vesicles.Both bacteriocins suppressed the decrease in the fluorescence of 1-anilino-8-naphthalene sulfonate caused by d-lactate or α-glycerol phosphate when added to membrane vesicles.It is concluded that the bacteriocins uncouple the transport function from the electron transport system.     

Effects of Green Tea Compound Epigallocatechin-3-Gallate against Stenotrophomonas maltophilia Infection and Biofilm

We investigated the in vitro and in vivo activities of epigallocatechin-3-gallate (EGCg), a green tea component, against Stenotrophomonas maltophilia (Sm) isolates from cystic fibrosis (CF) patients. In vitro effects of EGCg and the antibiotic colistin (COL) on growth inhibition, survival, and also against young and mature biofilms of S. maltophilia were determined. Qualitative and quantitative changes on the biofilms were assessed by confocal laser scanning microscopy (CLSM). Further, in vivo effects of nebulized EGCg in C57BL/6 and Cftr mutant mice during acute Sm lung infection were evaluated. Subinhibitory concentrations of EGCg significantly reduced not only biofilm formation, but also the quantity of viable cells in young and mature biofilms. CLSM showed that EGCg-exposed biofilms exhibited either a change in total biofilm biovolume or an increase of the fraction of dead cells contained within the biofilm in a dose depended manner. Sm infected wild-type and Cftr mutant mice treated with 1,024 mg/L EGCg by inhalation exhibited significantly lower bacterial counts than those undergoing no treatment or treated with COL. EGCg displayed promising inhibitory and anti-biofilm properties against CF Sm isolates in vitro and significantly reduced Sm bacterial counts in an acute infection model with wild type and CF mice. This natural compound may represent a novel therapeutic agent against Sm infection in CF.     

Antimicrobial activity of histidine-rich peptides is dependent on acidic conditions

Synthetic peptides composed of multiples of the consensus heparin-binding Cardin and Weintraub sequences AKKARA and ARKKAAKA are antimicrobial. Replacement of lysine and arginine by histidine in these peptides completely abrogates their antimicrobial and heparin-binding activities at neutral pH. However, the antibacterial activity against Gram-negative (Escherichia coli, Pseudomonas aeruginosa) and Gram-positive bacteria (Bacillus subtilis and Staphylococcus aureus) as well as the fungus Candida albicans, was restored at acidic conditions (pH 5.5). Fluorescence microscopy and FACS analysis showed that the binding of the histidine-rich peptides to E. coli and Candida was significantly enhanced at pH 5.5. Likewise, fluorescence studies for assessment of membrane permeation as well as electron microscopy analysis of peptide-treated bacteria, paired with studies of peptide effects on liposomes, demonstrated that the peptides induce membrane lysis only at acidic pH. No discernible hemolysis was noted for the histidine-rich peptides. Similar pH-dependent antimicrobial activities were demonstrated for peptides derived from histidine-rich and heparin-binding regions of human kininogen and histidine-rich glycoprotein. The results demonstrate that the presence of an acidic environment is an important regulator of the activity of histidine-rich antimicrobial peptides.     

Bactericidal effect of Naja nigricollis toxin γ is related to its membrane-damaging activity

The aim of the present study is to investigate the causal relationship between membrane-damaging activity and bactericidal activity of Naja nigricollis toxin γ. Toxin γ showed a similar inhibitory activity on the growth of Staphylococcus aureus (Gram-positive bacteria) and Escherichia coli (Gram-negative bacteria). Antibacterial activity of toxin γ correlated positively with increase in membrane permeability of bacterial cells. Morphological examination showed that toxin γ disrupted the integrity of bacterial membrane. Toxin γ showed similar binding capability with lipopolysaccharide (LPS) and lipoteichoic acid (LTA), and destabilization of LPS layer and inhibition of LTA biosynthesis on cell wall increased bactericidal effect of toxin γ on E. coli and S. aureus, respectively. Although the potency of toxin γ on permeabilzing model membrane of E. coli and S. aureus was similar, the mode of interaction between toxin γ and model membrane of E. coli and S. aureus differed. Membrane-damaging activity of toxin γ was inhibited by either LPS or LTA. Nevertheless, LPS and LTA altered differently membrane-bound conformation of toxin γ. Taken together, our data suggest that bactericidal activity of toxin γ depends on its ability to induce membrane permeability, and that LPS and LTA structurally suppresses bactericidal effect of toxin γ.     

Effect of Milk Proteins on Adhesion of Bacteria to Stainless Steel Surfaces

Stainless steel coupons were treated with skim milk and subsequently challenged with individual bacterial suspensions of Staphylococcus aureus, Pseudomonas fragi, Escherichia coli, Listeria monocytogenes, and Serratia marcescens. The numbers of attached bacteria were determined by direct epifluorescence microscopy and compared with the attachment levels on clean stainless steel with two different surface finishes. Skim milk was found to reduce adhesion of S. aureus, L. monocytogenes, and S. marcescens. P. fragi and E. coli attached in very small numbers to the clear surfaces, making the effect of any adsorbed protein layer difficult to assess. Individual milk proteins α-casein, β-casein, κ-casein, and α-lactalbumin were also found to reduce the adhesion of S. aureus and L. monocytogenes. The adhesion of bacteria to samples treated with milk dilutions up to 0.001% was investigated. X-ray photoelectron spectroscopy was used to determine the proportion of nitrogen in the adsorbed films. Attached bacterial numbers were inversely related to the relative atomic percentage of nitrogen on the surface. A comparison of two types of stainless steel surface, a 2B and a no. 8 mirror finish, indicated that the difference in these levels of surface roughness did not greatly affect bacterial attachment, and reduction in adhesion to a milk-treated surface was still observed. Cross-linking of adsorbed proteins partially reversed the inhibition of bacterial attachment, indicating that protein chain mobility and steric exclusion may be important in this phenomenon.     

Lethal effect of chitosan‐Ag (I) films on Staphylococcus aureus as evaluated by electron microscopy

Aim: This article investigated the lethal effect and morphological changes on Staphylococcus aureus strains ATCC 25923 and ATCC 6538P produced by chitosan‐Ag (I) films as observed by electron microscopy. Methods and Results: The antimicrobial activity of films against staphylococci was determined using the broth dilution method and agar diffusion test. Killing curves, transmission and scanning electron microscopy (TEM and SEM) techniques were employed to evaluate the bacterial death and morphological changes in bacterial cells after exposure to chitosan‐Ag (I) films. Films affected the cell structure of Staph. aureus, causing elongation of cells, disaggregation of grape‐like cluster, contraction of bacterial cytoplasm, thickening of cell wall, increase in cell wall roughness, cell disruption with loss of intracellular material, filamentation and bacteriolysis, as seen in the micrographs following 3, 6, 12 and 16 h of incubation. Conclusions: Obtained images clearly show that chitosan‐Ag (I) films have a notable antistaphylococcal activity. Significance and Impact of the Study: Information from this study can be employed in guiding future strategies to improve the design of materials for the food industry packaging.     

Atomic force microscopy and hydrodynamic characterization of the adhesion of <Emphasis Type="Italic">staphylococcus aureus</Emphasis> to hydrophilic and hydrophobic substrata at different pH values

Understanding the mechanism of the bacterial cell adhesion to solid surfaces is of great medical and industrial importance. Bacterial adhesion to inert surfaces, such as a catheter, and other indwelling devices can form biofilm, consequently cause severe morbidity and often fatal infections. Initial bacterial adhesion to the material surfaces is a complicated process that is affected by various physicochemical properties of both bacterial cells and substratum surfaces. The surface properties of the cells were characterized by the sessile drop technique. Moreover, the interfacial free energy of Staphylococcus aureus adhesion to the supporting materials was determined. The results showed that S. aureus examined at different pH levels could be considered hydrophilic. We noted hat the electron-donor character of S. aureus was important at intermediate pH (pH 5, pH 7, and pH 9) and it decreased at both limits acidic and basic conditions. In addition, the adhesion of Staphylococcus aureus ATCC 25923 to the hydrophilic glass and hydrophobic indium tin oxide (ITO)-coated glass surfaces at different pH values (2, 3, 5, 7, 9 and 11) was investigated using atomic force microscopy (AFM) and image analysis was assessed with the Mathlab^® program. The data analysis showed that cells (number of adhering cells to glass and ITO-coated glass surface) adhered strongly at acidic pH and weakly at alkaline pH. Also, S. aureus has the ability to attach to both hydrophobic and hydrophilic surfaces, but the adhesion was higher on hydrophobic surface.     

Biofilm testing of microbiota: An essential step during corneal scrap examination in Egyptian acanthamoebic keratitis cases

Purpose

To detect co-infections in the culture-proven acanthamoebic keratitis (AK) cases, and to test the capability of biofilm formation in the isolated microbiota. The clinical findings, habit of wearing contact lens and in-vitro antibiotic resistance were analyzed further according to the biofilm formation capability.

Experiences with the Coulter Counter in Bacteriology

Viable and killed suspensions of Staphylococcus aureus SM, Escherichia coli, and Serratia marcescens, as well as polystyrene spheres, 0.81 and 2.85 μ in diameter, were counted electronically with a model A Coulter Counter. Simultaneous counts by standard bacteriological methods and microscopy were done for purposes of control and comparison with the data from the Coulter Counter. Results indicated: (i) electrical characteristics of different bacterial populations are different; (ii) electronic counts were consistent for species used; (iii) live S. aureus exhibits a denser pattern of thick bright pulses on the cathode-ray tube than does live E. coli; (iv) killed bacteria resemble inert particles in pulse pattern; and (v) some viable bacteria do not react independently of current flow, as do inert particles and killed bacteria.     

(−)‐Epigallocatechin‐3‐gallate inhibits human angiotensin‐converting enzyme activity through an autoxidation‐dependent mechanism

We investigated the molecular mechanisms involved in the angiotensin‐converting enzyme (ACE) inhibition by (?)‐epigallocatechin‐3‐gallate (EGCg), a major tea catechin. EGCg inhibited both the ACE activity in the lysate of human colorectal cancer cells and human recombinant ACE (rh‐ACE) in a dose‐dependent manner. Co‐incubation with zinc sulfate showed no influence on the rh‐ACE inhibition by EGCg, whereas it completely counteracted the inhibitory effect of ethylenediaminetetraacetic acid, a chelating‐type ACE inhibitor. Although hydrogen peroxide was produced by the autoxidation of EGCg, hydrogen peroxide itself had little effect on the ACE activity. Conversely, the co‐incubation of EGCg with borate or ascorbic acid significantly diminished the EGCg inhibition. A redox‐cycling staining experiment revealed that rh‐ACE was covalently modified by EGCg. A Lineweaver–Burk plot analysis indicated that EGCg inhibited the ACE activity in a non‐competitive manner. These results suggested that EGCg might allosterically inhibit the ACE activity through oxidative conversion into an electrophilic quinone.     

In situ heterogeneity of peroxisomal oxidase activities: An update

Summary Oxidases are a widespread group of enzymes. They are present in numerous organisms and organs and in various tissues, cells, and subcellular compartments, such as mitochondria. An important source of oxidases, which is investigated and discussed in this study, are the (micro)peroxisomes. Oxidases share the ability to reduce molecular oxygen during oxidation of their substrate, yielding an oxidized product and hydrogen peroxide. Besides the hydrogen peroxide-catabolizing enzyme catalase, peroxisomes contain one or more hydrogen peroxide-generating oxidases, which participate in different metabolic pathways. During the last four decades, various methods have been developed and elaborated for the histochemical localization of the activities of these oxidases. These methods are based either on the reduction of soluble electron acceptors by oxidase activity or on the capture of hydrogen peroxide. Both methods yield a coloured and/or electron dense precipitate. The most reliable technique in peroxisomal oxidase histochemistry is the cerium salt capture method. This method is based on the direct capture of hydrogen peroxide by cerium ions to form a fine crystalline, insoluble, electron dense reaction product, cerium perhydroxide, which can be visualized for light microscopy with diaminobenzidine. With the use of this technique, it became clear that oxidase activities not only vary between different organisms, organs, and tissues, but that heterogeneity also exists between different cells and within cells, i.e. between individual peroxisomes. A literature review, and recent studies performed in our laboratory, show that peroxisomes are highly differentiated organelles with respect to the presence of active enzymes. This study gives an overview of thein situ distribution and heterogeneity of peroxisomal enzyme activities as detected by histochemical assays of the activities of catalase, and the peroxisomal oxidasesd-amino acid oxidase,l--hydroxy acid oxidase, polyamine oxidase and uric acid oxidase.