The impact of agarose immobilization on the activity of lytic Pseudomonas aeruginosa phages combined with chemicals.

The implementation of non-traditional antibacterials is currently one of the most intensively explored areas of modern medical and biological sciences. One of the most promising alternative strategies to combat bacterial infections is the application of lytic phages combined with established and new antibacterials. The presented study investigates the potential of agarose-based biocomposites containing lytic Pseudomonas phages (KT28, KTN4, and LUZ19), cupric ions (Cu2+), strawberry furanone (HDMF), and gentamicin (GE) as antibacterials and anti-virulent compounds for novel wound dressings. Phages (KT28, KTN4, LUZ19, and triple-phage cocktail) alone and in combination with a triple-chemical mixture (Cu + GE + HDMF) when applied as the liquid formulation caused a significant bacterial count reduction and biofilm production inhibition of clinical P. aeruginosa strains. The immobilization in the agarose scaffold significantly impaired the bioavailability and diffusion of phage particles, depending on virion morphology and targeted receptor specificity. The antibacterial potential of chemicals was also reduced by the agarose scaffold. Moreover, the Cu + GE + HDMF mixture impaired the lytic activity of phages depending on viral particles' susceptibility to cupric ion toxicity. Therefore, three administration types were tested and the optimal turned out to be the one separating antibacterials both physically and temporally. Taken together, the additive effect of phages combined with chemicals makes biocomposite a good solution for designing new wound dressings. Nevertheless, the phage utilization should involve an application of aqueous cocktails directly onto the wound, followed by chemicals immobilized in hydrogel dressings which allow for taking advantage of the antibacterial and anti-virulent effects of all components. KEY POINTS: • The immobilization in the agarose impairs the bioavailability of phage particles and the Cu + GE + HDMF mixture. • The cupric ions are toxic to phages and are sequestrated on phage particles and agarose matrix. • The elaborated TIME-SHIFT administration effectively separates antibacterials both physically and temporally.

Culture Media Composition Influences the Antibacterial Effect of Silver, Cupric, and Zinc Ions against Pseudomonas aeruginosa.

Different metals, such as silver (Ag), copper (Cu), and zinc (Zn), have been broadly investigated as metals and cations used both in medicine and everyday life due to their broad spectrum of antibacterial activity. Although the antibacterial action of those metals and their ions is well known and studied, the main problem remains in the standardization of experimental procedures to determine the antimicrobial activity as bacteriological media composition might significantly influence the outcome. The presented study aimed to evaluate the appropriability of different culture media (four nutritionally rich and four minimal) in the testing of the antibacterial activity of Ag+, Cu2+, and Zn2+ ions against Pseudomonas aeruginosa. Our investigation revealed the influence of medium ingredients and the presence of phosphates, which significantly reduced the activity of tested metal ions. Moreover, the precipitate formation and decrease in pH in the minimal media were additionally observed. It was assumed that the most favorable medium for metal ion activity testing was Luria-Bertani complex medium and MOPS minimal medium.

The In Vitro Anti-Pseudomonal Activity of Cu2+, Strawberry Furanone, Gentamicin, and Lytic Phages Alone and in Combination: Pros and Cons.

In this study, we investigated the anti-pseudomonal activity of cupric ions (Cu2+), strawberry furanone (HDMF), gentamicin (GE), and three lytic Pseudomonas aeruginosa bacteriophages (KT28, KTN4, LUZ19), separately and in combination. HDMF showed an anti-virulent effect but only when applied with Cu2+ or GE. GE, at a sub-minimal inhibitory concentration, slowed down phage progeny production due to protein synthesis inhibition. Cu2+ significantly reduced both the bacterial cell count and the number of infective phage particles, likely due to its genotoxicity or protein inactivation and cell membrane disruption effects. Furthermore, Cu2+'s probable sequestration by phage particles led to the reduction of free toxic metal ions available in the solution. An additive antibacterial effect was only observed for the combination of GE and Cu2+, potentially due to enhanced ROS production or to outer membrane permeabilization. This study indicates that possible interference between antibacterial agents needs to be carefully investigated for the preparation of effective therapeutic cocktails.

Multifunctionality of Nanosized Calcium Apatite Dual-Doped with Li+/Eu3+ Ions Related to Cell Culture Studies and Cytotoxicity Evaluation In Vitro.

Li+/Eu3+ dual-doped calcium apatite analogues were fabricated using a microwave stimulated hydrothermal technique. XRPD, FT-IR, micro-Raman spectroscopy, TEM and SAED measurements indicated that obtained apatites are single-phased, crystallize with a hexagonal structure, have similar morphology and nanometric size as well as show red luminescence. Lithium effectively modifies the local symmetry of optical active sites and, thus, affects the emission efficiency. Moreover, the hydrodynamic size and surface charge of the nanoparticles have been extensively studied. The protein adsorption (lysozyme, LSZ; bovine serum albumin, BSA) on the nanoparticle surface depended on the type of cationic dopant (Li+, Eu3+) and anionic group (OH-, Cl-, F-) of the apatite matrix. Interaction with LSZ resulted in a positive zeta potential, and the nanoparticles had the lowest hydrodynamic size in this protein medium. The cytotoxicity assessment was carried out on the human osteosarcoma cell line (U2OS), murine macrophages (J774.E), as well as human red blood cells (RBCs). The studied apatites were not cytotoxic to RBCs and J774.E cells; however, at higher concentrations of nanoparticles, cytotoxicity was observed against the U2OS cell line. No antimicrobial activity was detected against Gram-negative bacteria with one exception for P. aeruginosa treated with Li+-doped fluorapatite.

Structural modification of nanohydroxyapatite Ca10(PO4)6(OH)2 related to Eu3+ and Sr2+ ions doping and its spectroscopic and antimicrobial properties.

The Eu3+ and Sr2+ ions co-doped hydroxyapatite nanopowders (Ca10(PO4)6(OH)2) were synthesized via a precipitation method and post heat-treated at 500 °C. The concentration of Eu3+ ions was established in the range of 0.5-5 mol% to investigate the site occupancy preference. The concentration of Sr2+ ions was set at 5 mol%. The structural and morphological properties of the obtained materials were studied by an X-ray powder diffraction, a transmission electron microscopy techniques and infrared spectroscopy. As synthesized nanoparticles were in the range of 11-17 nm and annealed particles were in the range of 20-26 nm. The luminescence properties in dependence of the dopant concentration and applied temperature were investigated. The 5D0 → 7F0 transition shown the abnormally strong intensity for annealed materials connected with the increase of covalency character of Eu3+-O2- bond, which arise as an effect of charge compensation mechanism. The Eu3+ ions occupied three possible crystallographic sites in these materials revealed in emission spectra: one Ca(1) site with C3 symmetry and two Ca(2) sites with Cs symmetry arranged as cis and trans symmetry. The antibacterial properties of Eu3+ and Sr2+ ions doped and co-doped hydroxyapatite nanopowders were also determined against Gram-negative pathogens such as Pseudomonas aeruginosa, Klebsiella pneumoniae and Escherichia coli. Obtained results suggest that both europium and strontium ions may implement antibacterial properties for hydroxyapatites. In the most cases, better antibacterial effect we noticed for dopants at 5 mol% ratio. However, the effect is strongly species- and strain-dependent feature.

Complex Signaling Networks Controlling Dynamic Molecular Changes in Pseudomonas aeruginosa Biofilm.

The environment exerts strong influence on microbes. Adaptation of microbes to changing conditions is a dynamic process regulated by complex networks. Pseudomonas aeruginosa is a life-threating, versatile opportunistic and multi drug resistant pathogen that provides a model to investigate adaptation mechanisms to environmental changes. The ability of P. aeruginosa to form biofilms and to modify virulence in response to environmental changes is coordinated by various mechanisms including two-component systems (TCS), and secondary messengers involved in quorum sensing (QS) and c-di-GMP networks (diguanylate cyclase systems, DGC). In this review, we focus on the role of c-di-GMP during biofilm formation. We describe TCS and QS signal cascades regulated by c-di-GMP in response to changes in the external environment. We present a complex signaling network dynamically changing during the transition of P. aeruginosa from the free-living to sessile mode of growth.

Outer Membrane Proteins of Salmonella as Potential Markers of Resistance to Serum, Antibiotics and Biocides.

Salmonellosis continues to be a significant worldwide health problem. Despite rapid progress in identifying mechanisms of Salmonella virulence and resistance to chemicals, our knowledge of these mechanisms remains limited. Furthermore, it appears that the resistance to antibiotics can be amplified by ubiquitous usage of the disinfectants (biocides), both by industry and by ordinary households. Salmonella, as other Gram-negative bacteria possess outer membrane proteins (OMPs), which participate in maintaining cell integrity, adapting to environment, and interacting with infected host. Moreover, the OMPs may also contribute to resistance to antibacterials. This review summarizes the role of OMPs in Salmonella serum resistance, antibiotics resistance and cross-resistance to biocides. Although collected data do not allow to assign OMPs as markers of the Salmonella susceptibility to the above-mentioned factors, some of these proteins retain a dominant presence in certain types of resistance.

Defensive and Offensive Cross-Reactive Antibodies Elicited by Pathogens: The Good, the Bad and the Ugly.

Understanding how immunity to pathogens develops is crucial for progress in the quest for effective vaccines. Intraspecies and interspecies cross-reacting antibodies are produced in high frequency against immune-relevant and shared microbial epitopes. It has been confirmed that cross-reactive antigens may have a crucial role in natural epidemiology to a particular infection and that cross-protection may influence the outcome of natural infections. On the other hand, the action of cross-reactive antibodies may be very harmful for the host. In this review we discuss both the defensive and offensive capabilities of cross-reactive antibodies. The defensive properties are discussed with regard to the beneficial cross-protective interaction of these antibodies against various microorganisms including viruses, bacteria, fungi and protozoan parasites. We summarize the current knowledge of numerous effector functions of these antibodies such as agglutination, neutralization of infectivity, complement activation, phagocytosis enhancement, and antibody-dependent cellular cytotoxicity. We also discuss the offensive action of cross-reactive antibodies including their detrimental effects in exacerbation of the infective diseases, as well as autoimmune diseases and allergy as a result of inappriopriate or deleterious inflammatory response associated with host tissue destruction. The factors influencing cross-protective capacity of antibodies are also presented.

Presumable role of outer membrane proteins of Salmonella containing sialylated lipopolysaccharides serovar Ngozi, sv. Isaszeg and subspecies arizonae in determining susceptibility to human serum.

BACKGROUND: The O48 group comprises Salmonella bacteria containing sialic acid in the lipopolysaccharide (LPS). Bacteria with sialylated surface structures are described as pathogens that avoid immunological response of the host by making similar their surface antigens to the host's tissues (molecular mimicry). It is known that the smooth-type LPS of Salmonella enterica and outer membrane proteins (OMP) PgtE, PagC and Rck mediate serum resistant phenotype by affecting complement system (C). The aim of this study was to investigate C3 component activation by Salmonella O48 LPS and OMP. FINDINGS: In the present study, we examined C3 component deposition on the three Salmonella O48 strains: S. enterica subspecies enterica serovar Ngozi, S. enterica subsp. enterica sv. Isaszeg, and S. enterica subsp. arizonae containing sialic acid in the O-specific part of LPS. The greatest C3 deposition occurred on Salmonella sv. Isaszeg cells (p < 0.005) as well as on their LPS (low content of sialic acid in LPS) (p < 0.05) after 45 min of incubation in 50% human serum. Weaker C3 deposition ratio on the Salmonella sv. Ngozi (high content of sialic acid in LPS) and Salmonella subsp. arizonae (high content of sialic acid in LPS) cells correlated with the lower C3 activation on their LPS. Immunoblotting revealed that OMP isolated from the tested strains also bound C3 protein fragments. CONCLUSIONS: We suggest that activation of C3 serum protein is dependent on the sialic acid contents in the LPS as well as on the presence of OMP in the range of molecular masses of 35-48 kDa.

Modern Therapeutic Approaches Against Pseudomonas aeruginosa Infections.

Despite the enormous progress that has been made in the last few decades in the field of drug design as well as virulence of pathogenic bacteria, the gradual spread of drug resistance can be observed. Only two new classes of antibiotics have been brought to medicine in the last 30 years. The need for novel antibacterial drugs is especially pressing when considering infections caused by multidrug-resistant (MDR) pathogens such as Pseudomonas aeruginosa. The discovery and development of new anti-pseudomonal therapies is one of the main challenges of modern pharmaceutical sciences. The great variety of innovative approaches presented in the current literature is astonishing. In this review, modern, promising strategies against P. aeruginosa infections are described. Antimicrobials, including new antibiotics, ß-lactamase and efflux pump inhibitors, quorum quenching molecules and nanoparticles with antibacterial activity are currently being intensively studied. Methods of prevention of infection through vaccines, therapeutic antibodies and development of antimicrobial peptides are discussed as approaches that support the human immunological system. Finally, development of alternative/ supportive therapies such as phage therapy and photodynamic therapy, in which the mechanism of action is completely different from current antibiotic therapy, is of great importance.

Bactericidal activities of GM flax seedcake extract on pathogenic bacteria clinical strains.

BACKGROUND: The antibiotic resistance of pathogenic microorganisms is a worldwide problem. Each year several million people across the world acquire infections with bacteria that are antibiotic-resistant, which is costly in terms of human health. New antibiotics are extremely needed to overcome the current resistance problem. RESULTS: Transgenic flax plants overproducing compounds from phenylpropanoid pathway accumulate phenolic derivatives of potential antioxidative, and thus, antimicrobial activity. Alkali hydrolyzed seedcake extract containing coumaric acid, ferulic acid, caffeic acid, and lignan in high quantities was used as an assayed against pathogenic bacteria (commonly used model organisms and clinical strains). It was shown that the extract components had antibacterial activity, which might be useful as a prophylactic against bacterial infection. Bacteria topoisomerase II (gyrase) inhibition and genomic DNA disintegration are suggested to be the main reason for rendering antibacterial action. CONCLUSIONS: The data obtained strongly suggest that the seedcake extract preparation is a suitable candidate for antimicrobial action with a broad spectrum and partial selectivity. Such preparation can be applied in cases where there is a risk of multibacterial infection and excellent answer on global increase in multidrug resistance in pathogenic bacteria.

Isolation and characterisation of KP34--a novel φKMV-like bacteriophage for Klebsiella pneumoniae.

Bacteriophage KP34 is a novel virus belonging to the subfamily Autographivirinae lytic for extended-spectrum ß-lactamase-producing Klebsiella pneumoniae strains. Its biological features, morphology, susceptibility to chemical and physical agents, burst size, host specificity and activity spectrum were determined. As a potential antibacterial agent used in therapy, KP34 molecular features including genome sequence and protein composition were examined. Phylogenetic analyses and clustering of KP34 phage genome sequences revealed its clear relationships with "phiKMV-like viruses". Simultaneously, whole-genome analyses permitted clustering and classification of all phages, with completely sequenced genomes, belonging to the Podoviridae.

Liposomes as delivery systems for antibiotics.

Liposomes are currently in common use as universal drug carriers in the cosmetic and pharmaceutical industries. The manipulation of different physicochemical properties of liposomes enables the design of particular carriers with the desired pharmacokinetic and pharmacodynamic properties. Most studies regarding liposomal antibiotics deal with aminoglycosides, quinolones, polypeptides, and betalactames. Some of the studies focused on improving pharmacokinetics and reducing toxicity, while others involved enhancing antibacterial activity. In an era of an avalanche of increasing bacterial resistance and severe problems in treating bacterial infections, the application of liposomal antibiotic carriers could be useful, but the high cost of liposome preparation and treatment should also be considered.

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).

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.