[Use of the materials based on partially reduced graphene-oxide with silver nanoparticle as bacteriostatic and bactericidal agent]. / Wykorzystanie materialów opartych na czesciowo redukowanym tlenku grafenu z nanoczastkami srebra jako srodków bakteriostatycznych i bakteriobójczych.

BACKGROUND: Researches have been synthesizing nanocomposites with antibacterial properties for a dozen of years. A lot of study have confirmed a high antibacterial activity of silver nanoparticles and oxygraphene. Silica, titanium dioxide and hydroxyapatites of 1-100 nm are used as carrier for these composites. OBJECTIVES: To synthesise graphene-silver nanocomposites and to determine their antibacterial properties. MATERIALS AND METHODS: The following bacteria strains from the American Type Culture Collection were tested: Staphylococcus aureus ATCC 6538 (Gram-positive bacteria), Escherichia coli ATCC 11229 (Gram-negative bacteria), Klebsiella pneumoniae ATCC 4352 (Gram-negative bacteria). Clinical isolates of bacteria strains (from wounds) were also tested (from species of Staphylococcus aureus, Escherichia coli and Klebsiella pneumoniae). The antibacterial effect of nanocomposites was determined by minimal inhibitory concentration (MIC) and minimal bactericidal concentration (MBC) values according to the reference methods of Clinical and Laboratory Standards Institute (CLSI) for the determination of MICs of aerobic bacteria by broth microdilution. The samples have physical and chemical characteristics. RESULTS: The results showed bacteriostatic (0.4-1.6 microg/ml) and bactericidal (0.4-3.2 microg/ml) efficacy of composities. CONCLUSION: The synthesized nanocomposites of graphene-oxide can be used in biology and medicine as bacteriostatic and bactericidal factor and may be used as an alternative to antibiotics and chemioterapeutics.

Hydroxyapatites and europium(III) doped hydroxyapatites as a carrier of silver nanoparticles and their antimicrobial activity.

Hydroxyapatites (Ca10(PO4)6(OH)2 and Eu3+:Ca10(PO4)6(OH)2) were synthesized by aqueous synthesis route. Hydroxyapatites were impregnated with silver ions that were subsequently reduced. XRD, TEM, and SAED measurements were used in order to determine the crystal structure and morphology of the final products. The results showed the well crystallized hydroxyapatite grains with diameter of about 35 nm and with silver nanoparticles on their surface. The antimicrobial activity of the nanoparticles against: Staphylococcus aureus ATCC 6538 as model of the Gram-positive bacteria, Escherichia coli ATCC 11229, and Klebsiella pneumoniae ATCC 4352 as model of Gram-negative bacteria, were shown with the best activity against K. pneumoniae. These nanocomposite powders can be a promising antimicrobial agent and a fluorescent material for biodetection due to their optical and bioactive properties.

Proteomic analysis of serum of workers occupationally exposed to arsenic, cadmium, and lead for biomarker research: a preliminary study.

The main factor of environmental contamination is the presence of the heavy metals lead, cadmium, and arsenic. The aim of serum protein profile analysis of people chronically exposed to heavy metals is to find protein markers of early pathological changes. The study was conducted in a group of 389 healthy men working in copper foundry and 45 age-matched non-exposed healthy men. Toxicological test samples included whole blood, serum, and urine. Thirty-seven clinical parameters were measured. Based on the parameters values of the healthy volunteers, the centroid in 37-dimensional space was calculated. The individuals in the metal-exposed and control groups were ordered based on the Euclidean distance from the centroid defined by the first component according to Principal Component Analysis (PCA). Serum samples of two individuals, one from the control and one from the metal-exposed group, were chosen for proteomic analysis. In optimized conditions of two-dimensional gel electrophoresis (2-DE), two protein maps were obtained representing both groups. Twenty-eight corresponding protein spots from both protein maps were chosen and identified based on PDQuest analysis and the SWISS-2DPAGE database. From a panel of six proteins with differences in expression greater than a factor of two, three potential markers with the highest differences were selected: hemoglobin-spot 26 (pI 7.05, Mw 10.53), unidentified protein-spot 27 (pI 6.73, Mw 10.17), and unidentified protein-spot 25 (pI 5.75, Mw 12.07). Further studies are required to prove so far obtained results. Identified proteins could serve as potential markers of preclinical changes and could be in the future included in biomonitoring of people exposed to heavy metals.

Sialic acid-containing lipopolysaccharides of Salmonella O48 strains--potential role in camouflage and susceptibility to the bactericidal effect of normal human serum.

Sialic acid (N-acetylneuraminic acid, NeuAc) plays an essential role in protecting gram-negative bacteria against the bactericidal activity of serum and may contribute to the pathogenicity of bacteria by mimicking epitopes that resemble host tissue components (molecular mimicry). The role of sialic acid (NeuAc)-containing lipopolysaccharides (LPS) of Salmonella O48 strains in the complement activation of normal human serum (NHS) was investigated. NeuAc-containing lipooligosaccharides cause a downregulation of complement activation and may serve to camouflage the bacterial surface from the immunological response of the host. Serotype O48 Salmonella strains have the O-antigen structure containing NeuAc while its serovars differ in outer membrane protein composition. In this study, the mechanisms of complement activation responsible for killing Salmonella O48 serum-sensitive rods by NHS were established. Four of such mechanisms involving pathways, which are important in the bactericidal mechanism of complement activation, were distinguished: only the classical/lectin pathways, independent activation of the classical/lectin or alternative pathway, parallel activation of the classical/lectin and alternative pathways, and only the alternative pathway important in the bactericidal action of human serum. To further study the role of NeuAc, its content in bacterial cells was determined by gas-liquid chromatography-mass spectrometry in relation to 3-deoxy-D-manno-2-octulosonic acid (Kdo), an inherent constituent of LPS. The results indicate that neither the presence of sialic acid in LPS nor the length of the O-specific part of LPS containing NeuAc plays a decisive role in determining bacterial resistance to the bactericidal activity of complement and that the presence of sialic acid in the structure of LPS is not sufficient to block the activation of the alternative pathway of complement. We observed that for three strains with a very high NeuAc/Kdo ratio the alternative pathways were decisive in the bactericidal action of human serum. The results indicated that those strains are not capable of inhibiting the alternative pathway very effectively. As the pathogenicity of most Salmonella serotypes remains undefined, research into the interactions between these bacterial cells and host organisms is indispensable.

Use of zwitterionic type of detergent in isolation of Escherichia coli O56 outer membrane proteins improves their two-dimensional electrophoresis (2-DE).

Escherichia coli O56 were originally isolated from infected humans. Here it is reported that using the zwitterionic detergent (Zwittergent Z 3-14) to isolate outer membrane proteins (OMPs) from Escherichia coli O56 is suitable for their separation by two-dimensional electrophoresis (2-DE) using pH 3-10 immobilized pH gradient IPG strips (BIO-RAD).

The occurrence, biodiversity and toxicity of Bacillus thuringiensis strains isolated from the insect pest Lymantria dispar (Poland).

The aim of this investigation was to survey the occurrence, biodiversity, and toxicity of Bacillus thuringiensis strains originating from dead caterpillars of the forest pest, Lymantria dispar (Lepidoptera). Morphological, biochemical, and microscopic identification of isolates from the insects showed the presence of five different Bacillus species, including 2% of B. thuringiensis. Based on the biochemical profiles, the B. thuringiensis were determined to be B. thuringiensis finitimus-like and B. thuringiensis alesti-like bacilli. Both produced spherical inclusions composed of three or five protoxins. The molecular weights of these proteins varied from 20 to ca. 64 kDa. Mixtures of spores/inclusions of the B. thuringiensis were tested for their toxicity against larvae of Drosophila melanogaster. The mortality levels of the larvae caused by these spores and crystalline inclusions varied from 5 to 15%. The lethal doses (LD50) of these isolates against D. melanogaster were 8.8 x 10(12) spores/ml for B. thuringiensis finitimus and 1.3 10(18) spores/ml for B. thuringiensis alesti.

Human complement activation by smooth and rough Proteus mirabilis lipopolysaccharides.

INTRODUCTION: Proteus mirabilis bacilli play an important role in human urinary tract infections, bacteremia, and rheumatoid arthritis. The authors previously studied human complement C3 conversion by smooth-form P. mirabilis O10, O23, O30, and O43 lipopolysaccharides (LPSs) and showed that smooth Proteus LPSs fragmented C3 in a dose- and time-dependent manner. In the present study, one smooth P. mirabilis S1959 and its two polysaccharide-truncated LPSs isolated from an R mutant strain were used to study the C3 conversion. MATERIALS AND METHODS: The conversion of C3 to C3c by smooth and rough P. mirabilis LPSs was studied by capture ELISA and crossed immunoelectrophoresis. Proteins isolated from the outer membrane were analyzed by discontinuous sodium dodecyl sulfate gel electrophoresis. RESULTS: The smooth P. mirabilis S1959 (O3) strain was resistant to the bactericidal activity of human serum, in contrast to the Ra and Re mutant strains. The presence of an exposed core oligosaccharide in R110 LPS was not sufficient to protect the strain from serum-dependent killing. In addition to LPS structure, the outer-membrane proteins may also play roles in protecting the smooth P. mirabilis S1959 (O3) strain from the bactericidal action of serum. It was shown that the Ra P. mirabilis R110 and the Re P. mirabilis R45 mutants possess very different OMP compositions from that of the P. mirabilis S 1959 strain. CONCLUSION: Regardless of the complement resistance of the P. mirabilis strains, the S1959, R110, and R45 LPSs fragmented C3 and induced C3c neo-antigen exposure. The use of complement-deficient human serum allows the conclusion that the Re-type P. mirabilis R45 LPS fragmented C3 by the antibody-independent classical pathway.

Isolation of outer membrane proteins (OMP) from Salmonella cells using zwitterionic detergent and their separation by two-dimensional electrophoresis (2-DE).

We report that using the zwitterionic detergent Zwittergent Z 3-14 to isolate outer membrane proteins (OMPs) from Salmonella 048 is suitable for their separation by two-dimensional electrophoresis (2-DE) in a capillary tube system. Sample preparation is a very crucial step for any bacterial proteomic study. Some modifications were introduced to the 2-DE protocol suggested by O'Farrell and BioRad, which significantly impaired the resolution of proteins. 2-DE analysis of OMPs may be helpful in the interpretation of the variable susceptibility of Salmonella 048 rods to the bactericidal activity of serum.

The interaction between Pseudomonas aeruginosa cells and cationic PC:Chol:DOTAP liposomal vesicles versus outer-membrane structure and envelope properties of bacterial cell.

The interactions between cationic liposomal formulations (PC:Chol:DOTAP 3:4:3) and 23 Pseudomonas aeruginosa strains were tested. The study was undertaken because different antimicrobial results had been obtained by the authors for Pseudomonas aeruginosa strains and liposomal antibiotics (Drulis-Kawa, Z., Gubernator, J., Dorotkiewicz-Jach, A., Doroszkiewicz, W., Kozubek, A., 2006. The comparison of in vitro antimicrobial activity of liposomes containing meropenem and gentamicin. Cell. Mol. Biol. Lett., 11, 360-375; Drulis-Kawa, Z., Gubernator, J., Dorotkiewicz-Jach, A., Doroszkiewicz W., Kozubek, A., 2006. In vitro antimicrobial activity of liposomal meropenem against Pseudomonas aeruginosa strains. Int. J. Pharm., 315, 59-66). The experiments evaluate the roles of the bacterial outer-membrane structure, especially outer-membrane proteins and LPS, and envelope properties (hydrophobicity and electrostatic potential) in the interactions/fusion process between cells and lipid vesicles. The interactions were examined by fluorescent microscopy using PE-rhodamine-labelled liposomes. Some of the strains exhibited red-light emission (fusion with vesicles or vesicles surrounding the cell) and some showed negative reaction (no red-light emission). The main aim of the study was to determine what kinds of bacterial structure or envelope properties have a major influence on the fusion process. Negatively charged cells and hydrophobic properties promote interaction with cationic lipid vesicles, but no specific correlation was noted for the tested strains. A similar situation concerned LPS structure, where parent strains and their mutants possessing identical ladder-like band patterns in SDS-PAGE analysis exhibited totally different results with fluorescent microscopy. Outer-membrane protein analysis showed that an 18-kDA protein occurred in the isolates showing fusion with rhodamine-labelled vesicles and, conversely, strains lacking the 18-kDA protein exhibited no positive reaction (red emission). This suggests that even one protein may be responsible for favouring stronger interactions between Pseudomonas aeruginosa cells and cationic liposomal formulations (PC:Chol:DOTAP 3:4:3).

[Infectivityand resistance to antibiotics of non-fermentative rods isolated from patients hospitalised in pediatric center of Lower Silesia in Wroclaw in the years 2000-2006]. / Wystepowanie i lekoopornosc paleczek niefermentujacych izolowanych od pacjentów hospitalizowanych w dolnoslaskim centrum pediatrycznym we Wroclawiu w latach 2000-2006.

The aim of this study was to evaluate the frequency of isolation and antimicrobial resistance testing of non-fermentative rods isolated from clinical specimens from patients hospitalized in Korczak Pediatric Center of Lower Silesia in Wroclaw. The susceptibility of bacteria to selected antibiotics was determined. The commonest pathogens were Pseudomonas rods (81.8%) isolated from respiratory system and urine of patients hospitalized in unit intensive care. Variety of resistance patterns were detected in bacteria. ESBL were detected the most of S. maltophilia. Strains of Pseudomonas and Acinetobacter resistant to carbapenems were detected with a frequency of 5.5% and 35.9%.

A comparison of the in vitro antimicrobial activity of liposomes containing meropenem and gentamicin.

The antimicrobial activity of eight cationic, two neutral and three anionic liposome compositions containing meropenem and gentamicin was tested in vitro in broth and serum medium. The cationic formulations showed better antibacterial efficacy against both Gram-positive and Gram-negative bacteria than the anionic and neutral ones, regardless of the encapsulated drug. The most effective formulations were the cationic PC/DOPE/DOTAP 3:4:3 and PC/Chol/DOTAP 3:4:3, as the MICs with meropenem were 2 to 4 times lower than those of the free drug.

In vitro antimicrobial activity of liposomal meropenem against Pseudomonas aeruginosa strains.

Twelve lipid formulations of liposomal meropenem were tested on six drug-susceptible and two drug-resistant Pseudomonas aeruginosa strains to determine their antibacterial activity. Cationic liposomes, especially PC/DOPE/SA 4:4:2 and PC/DOTAP/Chol 5:2:3, were more effective than anionic ones against sensitive isolates as the MICs of those formulations were two to four times lower than those of the free drug. None of the studied liposomal forms of meropenem exhibited bactericidal activity against isolates, which are drug-resistant due to low permeability. Even Fluidosomes (liposomes made of DPPC/DMPG 18:1), which demonstrated fusion with P. aeruginosa membranes, showed 4-16 times higher MICs for sensitive and resistant strains than did the free meropenem.

Bactericidal activity of normal bovine serum (NBS) directed against some Enterobacteriaceae with sialic acid-containing lipopolysaccharides (LPS) as a component of cell wall.

The sensitivity of bacteria to the bactericidal activity of serum depends on the structure and organization of the bacterial outer membrane. Sialic acid has been found in the O-specific region of bacterial lipopolysaccharide (LPS) and it plays an essential role in protecting Gram-negative bacteria against the bactericidal activity of human and animal serum. The susceptibility of Gram-negative bacilli with sialic acid-containing LPS to the bactericidal action of normal bovine serum (NBS) was determined. The examined strains (Escherichia coli O104 (PCM 270), E. coli O24 (PCM 195), E. coli O56 (PCM 2372), Citrobacter braakii O37 (PCM 2346) and Salmonella enterica ssp. enterica serovar Toucra O48 (PCM 2359) showed variable sensitivity to the bactericidal effect of the serum. The role of the mechanisms of complement activation in the killing process was also determined.

Survival of Proteus mirabilis O3 (S1959), O9 and O18 strains in normal human serum (NHS) correlates with the diversity of their outer membrane proteins (OMPs).

Urinary tract infections are frequently caused by Proteus mirabilis strains. In the previous studies there were defined the complete structures of O-polysaccharide parts of lipopolysaccharides from strains: P. mirabilis O3 (S1959), P. mirabilis O9 and P. mirabilis O18. In the present study it was investigated bactericidal effect of normal human serum (NHS) to P. mirabilis strains. We also focused on the diversity of outer membrane proteins (OMPs) being separated on a gel isolated from tested strains. Serial passage of P. mirabilis O18 in 90% normal bovine serum (NBS) contributed to over-expressing some classes of OMPs.

Potential possibilities of using phage typing in elimination of multidrug resistant staphylococci.

Coagulase-negative staphylococci (CoNS) have become the most often isolated bacteria from blood culture, spinal fluid and respiratory tracts of neonates. These nosocomial strains are often resistant to oxacillin and other antibiotics (macrolides, aminoglycosides and fluorochinolones). 50 multidrug resistant CoNS strains isolated from bloodstream neonatal infections were tested for sensitivity to 23 lytic staphylococcus bacteriophages. No lytic patterns for 19 of the phages were observed. Phages P4, A3R and 676/Z were active against 46%, 54% and 56% of the strains, respectively. In general, 60% of CoNS isolates were susceptible to one or more of the staphylococcus bacteriophages.

Differentiated pattern of protein composition of crystalline inclusions of newly isolated Bacillus thuringiensis strains from Silesia in Poland.

Protein profiles of crystal delta-endotoxins were determined in twenty nine Bacillus thuringiensis strains-soil and phylloplane isolates--from Poland. Electrophoretic analysis revealed quantatively and qualitatively different patterns of delta-endotoxin crystal preparations of these B. thuringiensis strains. The crystalline parasporal inclusions of B. thuringiensis isolates were composed of two, three, four or five proteins. Molecular weights of these polypeptides varied from 23.4 kDa to 142 kDa. There is lack of correlation between serovars of B. thuringiensis strains, the morphology of crystals and the number and size of proteins in parasporal inclusions.

The susceptibility of anaerobic bacteria isolated from periodontal diseases to photodynamic inactivation with Fotolon (chlorin e6).

Photodynamic inactivation (PDI) may be a very promising alternative method for the antimicrobial treatment of periodontitis. Several studies have demonstrated the sensitivity of subgingival flora to PDI using toluidine blue, methylene blue, and chlorin e6 derivatives. In the present study we report the activity of the Fotolon sensitizer, composed of chlorin e6 and polyvinylpyrrolidone (PVP), against anaerobic bacteria isolated from periodontal diseases. Over 99.9% reduction in colony forming units in 20 Gram-positive and 30 Gram-negative clinical anaerobic strains was obtained.

[Bactericidal activity of normal human serum to gram-negative rods with sialic acid containing lipopolysaccharide]. / Bakteriobójcza aktywnosc surowicy ludzkiej wobec paleczek gram-ujemnych posiadajacych w strukturze lipopolisacharydu kwas sjalowy.

The complement plays the most important role in eliminating bacterial invasion of the host, by facilitating phagocytosis of potential pathogens and by participating in the direct essential role in protecting gram-negative bacteria against bactericidal activity of serum. Sialic acids which are important constitutes of animal tissue glycoconjugates are also present in antigens of some bacterial strains. The susceptibility of gram-negative strains with sialic acid--containing lipopolysaccharides to bactericidal action sera was examined.

The factor C3 conversion in human complement by smooth Shigella flexneri lipopolysaccharides.

Shigella flexneri rods play an important role in human intestinal infections. In the presented studies we have shown that O-acetyl and glucose residues, substituted in main GalNAc-Rha chains of lipopolysaccharide (LPS) are important for the bactericidal effect of human serum. By dot-blot, immunoblotting and ELISA with immobilized LPS we have shown correlation of C3 fragments deposition and serum resistance. LPSs isolated from a serum-sensitive strain deposited more C3 fragments than LPSs from serum-resistant Shigella flexneri strains.