The activity of silver nanoparticles against microalgae of the Prototheca genus.

AIM: To investigate the in vitro activity of silver NPs (AgNPs) against pathogenic microalgae of the Prototheca genus. MATERIALS & METHODS: The antialgal potential of AgNPs against Prototheca species of both clinical and environmental origin was assessed from minimum inhibitory (algistatic) and algicidal concentrations. The in vitro cytotoxicity of AgNPs against bovine mammary epithelial cell line was evaluated by means of the standard MTT assay. RESULTS: AgNPs showed a strong killing activity toward Prototheca algae, as the minimal algicidal concentration (MAC) values matched perfectly the corresponding minimum inhibitory concentration (MIC) values for all species (MAC = MIC, 1-4 mg/l), except P. stagnora (MIC > 8 mg/l). The concentrations inhibitory to pathogenic Prototheca spp. (MIC, 1-4 mg/l) were below the concentrations at which any toxicity in epithelial cells could be observed (CC20 > 6 mg/l). CONCLUSION: The study emphasizes the potential of AgNPs as a new therapeutic tool for the management of Prototheca infections.

Effects of null mutation of the heat-shock gene htpG on the production of virulence factors by Pseudomonas aeruginosa.

AIM: Pseudomonas aeruginosa is one of the most clinically important opportunistic pathogen in humans. The aim of the project was to study effects of HtpG on the selected virulence factors responsible for pathogenesis and biofilm formation of P. aeruginosa. METHODOLOGY: By characterizing a htpG null mutant of P. aeruginosa, we have identified the role of HtpG in the production of selected factors. RESULTS: We showed that ΔhtpG mutant affects many physiological processes containing: decreased activity of the LasA protease, reduction of biofilm formation, decreased motility, and diminished amount of rhamnolipids and pyoverdine/pyocyanin. These defects were most evident when the ΔhtpG strain was cultured at 42°C. CONCLUSION: Our findings demonstrate the unexplored role of HtpG in the pathogenicity of P. aeruginosa, and indicate potential targets for antibacterial therapeutics. [Formula: see text].

Significance of polymethylmethacrylate (PMMA) modification by zinc oxide nanoparticles for fungal biofilm formation.

The objective of this study was to obtain a material composite with antifungal properties for dentures to be used as an alternative protocol in denture stomatitis treatment and prevention. Denture stomatitis is still a clinical problem in patients particularly vulnerable to this disease. Composites of PMMA and doped ZnO-NPs (weight concentrations, 2.5%, 5%, 7.5%) and PMMA with sprayed solvothermal and hydrothermal ZnO-NPs were tested. The following investigations of newly formed biomaterials were undertaken: influence on Candida albicans solution, biofilm staining, XTT analysis and a quantitative analysis of adhered C. albicans. These studies evidenced the antifungal activity of both nanocomposites PMMA-ZnO-NPs and the efficacy of sputtering of zinc oxide nanoparticles on the PMMA. The study of the biofilm deposition on the surface showed that antifungal properties increase with increasing concentration of ZnO-NPs. The XTT assay in conjunction with testing the turbidity of solutions may indicate the mechanism by which ZnO-NPs exert their effect on the increased induction of antioxidative stress in microorganism cells. The denture base made of the aforesaid materials may play a preventive role in patients susceptible to fungal infections. Based on the results obtained a modified treatment of stomatitis Type II (Newton's classification) complicated by fungal infection was proposed.

Genetic control of bacterial biofilms.

Nearly all bacterial species, including pathogens, have the ability to form biofilms. Biofilms are defined as structured ecosystems in which microbes are attached to surfaces and embedded in a matrix composed of polysaccharides, eDNA, and proteins, and their development is a multistep process. Bacterial biofilms constitute a large medical problem due to their extremely high resistance to various types of therapeutics, including conventional antibiotics. Several environmental and genetic signals control every step of biofilm development and dispersal. From among the latter, quorum sensing, cyclic diguanosine-5'-monophosphate, and small RNAs are considered as the main regulators. The present review describes the control role of these three regulators in the life cycles of biofilms built by Pseudomonas aeruginosa, Staphylococcus aureus, Salmonella enterica serovar Typhimurium, and Vibrio cholerae. The interconnections between their activities are shown. Compounds and strategies which target the activity of these regulators, mainly quorum sensing inhibitors, and their potential role in therapy are also assessed.

Chaperone DnaJ Influences the Formation of Biofilm by Escherichia coli.

DnaJ chaperone, a member of the so called DnaK-DnaJ-GrpE chaperone machine plays an important role in cell physiology. The ability of Escherichia coli ΔdnaJ mutant to form biofilm was studied. It was shown that this mutant is impaired in biofilm development when exposed to 42 degrees C for 2 h. The impairment in biofilm development was observed when the heat shock was applied either at the onset of biofilm formation or 2 h later. The biofilm formed was thinner and its structure was changed as compared to wild-type strain. This defect could be complemented by the introduction of a wild-type gene on a low-copy plasmid.

Interactions of Escherichia coli molecular chaperone HtpG with DnaA replication initiator DNA.

The bacterial chaperone high-temperature protein G (HtpG), a member of the Hsp90 protein family, is involved in the protection of cells against a variety of environmental stresses. The ability of HtpG to form complexes with other bacterial proteins, especially those involved in fundamental functions, is indicative of its cellular role. An interaction between HtpG and DnaA, the main initiator of DNA replication, was studied both in vivo, using a bacterial two-hybrid system, and in vitro with a modified pull-down assay and by chemical cross-linking. In vivo, this interaction was demonstrated only when htpG was expressed from a high copy number plasmid. Both in vitro assays confirmed HtpG-DnaA interactions.

The effect of oleanolic and ursolic acids on the hemolytic properties and biofilm formation of Listeria monocytogenes.

Oleanolic acid and ursolic acid are pentacyclic triterpenoids isolated from a variety of medicinal plants, which have antibacterial activity. Listeria monocytogenes is a Gram-positive facultative pathogen, being the causative agent of listeriosis. The present study was carried out to evaluate the in vitro effect of sub-inhibitory concentrations of both triterpene acids on the pathogenicity determinants of L. monocytogenes: their hemolytic activity and biofilm forming ability. Oleanolic and ursolic acids inhibited listeriolysin O activity without influencing toxin secretion. Biofilm formation, and the viability of L. monocytogenes cells in biofilms was diminished by both compounds. Thus, both acids affected L. monocytogenes virulence. It was also demonstrated that oleanolic acid bound to the peptidoglycan of L. monocytogenes and this interaction was influenced by teichoic acids.

Modulation of antibiotic resistance and induction of a stress response in Pseudomonas aeruginosa by silver nanoparticles.

The objective of this study was to characterize the effects of silver nanoparticles on Pseudomonas aeruginosa. Their interactions with several conventional antibiotics and ability to induce a stress response were examined. Interactions between silver nanoparticles (AgNPs) and antibiotics against free-living cells and biofilm of P. aeruginosa were studied using the chequerboard method and time-kill assays. The ability of AgNPs to induce a stress response was determined by evaluation of cellular levels of the DnaK and HtpG chaperones using SDS-PAGE and Western blot analysis. Synergistic activity against free-living P. aeruginosa between AgNPs and ampicillin, streptomycin, rifampicin and tetracycline, but not oxacillin, ciprofloxacin, meropenem or ceftazidime, was demonstrated by the chequerboard method. No such interactions were observed against P. aeruginosa biofilm. The results of time-kill assays confirmed synergy only for the AgNPs-streptomycin combination. AgNPs induced the expression of chaperone DnaK. No induction of the HtpG chaperone was detected. In conclusion, AgNPs not only display potent bactericidal activity against P. aeruginosa, but also act synergistically with several conventional antibiotics to enhance their effect against free-living bacteria as determined by the chequerboard method. The time-kill assay proved synergy between AgNPs and streptomycin only. The ability of AgNPs to induce the major chaperone protein DnaK may influence bacterial resistance to antimicrobials.

Physiological consequences of mutations in the htpG heat shock gene of Escherichia coli.

Mutation of the heat shock gene, htpG, causes severe defects of several cellular functions in Escherichia coli. A null htpG mutant constructed by gene replacement was impaired in the biosynthesis and secretion of several enzymes, and in biofilm formation and proteolysis. A significant decrease in the activity of ß-lactamase in the ΔhtpG mutant was observed at 42°C. The alkaline phosphatase activity in sonicates of cells propagated at this raised temperature was lower in the ΔhtpG mutant than in the wild-type strain. The ability of the ΔhtpG mutant to degrade abnormal proteins was also impaired compared with the wild-type, but was increased at 42°C. Assays based on bioluminescence and crystal violet staining demonstrated that biofilm formation was diminished in the ΔhtpG mutant at the elevated temperature. All these defects can be complemented upon introducing htpG wild allele.

Silver nanoparticles as an alternative strategy against bacterial biofilms.

Biofilms are complex bacterial communities that resist the action of antibiotics and the human immune system. Bacteria within biofilms are the cause of numerous, almost impossible to eradicate, persistent infections. Biofilms can form on many medical devices and implants, and so have an enormous impact on medicine. Due to the lack of effective anti-biofilm antibiotics, novel alternative compounds or strategies are urgently required. This review describes some of the latest approaches in the field of biofilm treatment. New anti-biofilm technologies target different stages in the biofilm formation process. Some act to modify the colonized biomaterials to make them resistant to biofilm formation. One potentially important candidate treatment uses silver nanoparticles that show anti-bacterial and anti-biofilm activity. The biological action of nano-silver is complex and seems to involve a number of pathways. However, there have been few reports on the anti-biofilm activity of silver nanoparticles and the precise mechanism underlying their action remains unresolved. Here, we describe some anti-biofilm approaches employing AgNPs and consider the challenges and problems that need to be addressed in order to make silver nanoparticles a part of an effective anti-biofilm strategy.

New antibacterial therapeutics and strategies.

Studies on new antibacterial therapeutics and strategies are currently being conducted in many microbiological, pharmaceutical and biochemical laboratories. The antibacterial activity of plant-derived compounds as well as silver and gold nanoparticles is the subject of this minireview. The application of photodynamic therapy is also discussed.

Oleanolic and ursolic acids influence affect the expression of the cysteine regulon and the stress response in Escherichia coli.

The pentacyclic triterpenoids, oleanolic, and ursolic acids, affect peptidoglycan metabolism, altering bacterial morphology, and inhibit the growth and survival of several bacterial species, including pathogenic ones. We investigated the effect of subinhibitory concentrations of these compounds on the expression of three operons from the E. coli cysteine regulon, cysPTWA, cysJIH, and cysB, by using transcriptional fusions with the lacZ reporter gene. An inhibitory effect on ß-galactosidase expression directed by all three chromosomal fusions was observed with both compounds. In addition, oleanolic acid, but not ursolic acid, caused a weak increase in DnaK synthesis, suggesting moderate ability of inducing heat-shock response.

Oleanolic acid and ursolic acid affect peptidoglycan metabolism in Listeria monocytogenes.

The plant pentacyclic triterpenoids, oleanolic and ursolic acids, inhibit the growth and survival of many bacteria, particularly Gram-positive species, including pathogenic ones. The effect of these compounds on the facultative human pathogen Listeria monocytogenes was examined. Both acids affected cell morphology and enhanced autolysis of the bacterial cells. Autolysis of isolated cell walls was inhibited by oleanolic acid, but the inhibitory activity of ursolic acid was less pronounced. Both compounds inhibited peptidoglycan turnover and quantitatively affected the profile of muropeptides obtained after digestion of peptidoglycan with mutanolysin. These results suggest that peptidoglycan metabolism is a cellular target of oleanolic and ursolic acids.

Conjugal transfer of plasmid R6K gamma ori minireplicon derivatives from Escherichia coli to various genera of pathogenic bacteria.

Three R6K-derived gamma ori minireplicons were successfully transferred by conjugation from Escherichia coli to several species of pathogenic bacteria. The pFL129 replicon encodes the wild-type initiation replication protein pi, while plasmids pFL130 and pAG101 encode mutant forms of the pi protein conferring the plasmid copy-up phenotype. Plasmids could be transferred to all recipient species tested, although high efficiency conjugal transfer was only obtained with genera of the Enterobacteriaceae. The efficiency of plasmid transfer to all recipients was lower for the copy-up derivatives, pFL130 and pAG101, than for pFL129. The three gamma ori replicons were stably maintained in all transconjugants except pFL129 in Listeria monocytogenes. The two mutant plasmids retained their copy-up phenotype in the new bacterial hosts.

Genetic and physiological regulation of bacterial endospore development.

Bacterial endospores are complex structures residing inside endospore-forming, mainly gram-positive bacteria. The process of sporulation is considered a simple example of cell differentiation. Endospores enable the organism to resist environmental stresses. Sporulation can be divided into several stages, from axial DNA filamentation to mother cell lysis. The structure and formation of an endospore is an attractive model for the assembly of complex macromolecular structures during development. The expression of genes involved in sporulation is compartmentalized and different sets of genes are expressed in the prespore and mother cell, this being associated with the subsequent activation of four sporulation-specific sigma factors. Their synthesis and activity are tightly regulated and the regulatory mechanisms have overlapping roles.

Role of Escherichia coli DnaK and DnaJ chaperones in spontaneous and induced mutagenesis and their effect on UmuC stability.

The frequency of spontaneous as well as induced reversions of auxotrophic mutations in Escherichia coli AB1157 and its DeltadnaK and DeltadnaKdnaJ derivatives was estimated. The obtained results demonstrate that both mutants tested are characterized by elevated frequency of spontaneous reversions compared to their AB1157 parent. In contrast, the frequency of reversions induced by UV and MMS, i.e. agents inducing the SOS response, is reduced in DeltadnaJ and DeltadnaKdnaJ mutants, pointing to the possible defect of these mutants in error prone repair. Due to the fact that UmuC protein is one of the main players executing the error prone repair, its stability in DeltadnaJ and DeltadnaKdnaJ mutants was also studied. Reduced UmuC stability was demonstrated only in the DeltadnaKdnaJ mutant.

New methods of pathogenic bacteria elimination.

The growing bacterial resistance to antibiotics calls for the elaboration of new pathogens elimination strategies. Some of these methods are based on the conjugative transfer of recombinant plasmids able to eliminate pathogenic recipients by plasmid run-away replication or by killing activity of plasmid-encoded bacteriocins. Using live bacteria as donors of plasmid vectors carrying killing determinants requires meeting many safety restrictions in order to eliminate potential biohazard.