Pigments influence the tolerance of Pseudomonas aeruginosa PAO1 to photodynamically induced oxidative stress.

Pseudomonas aeruginosa is an opportunistic pathogen known to be resistant to different classes of antibiotics and disinfectants. P. aeruginosa also displays a certain degree of tolerance to photodynamic therapy (PDT), an alternative antimicrobial approach exploiting a photo-oxidative stress induced by exogenous photosensitizers and visible light. To evaluate whether P. aeruginosa pigments can contribute to its relative tolerance to PDT, we analysed the response to this treatment of isogenic transposon mutants of P. aeruginosa PAO1 with altered pigmentation. In general, in the presence of pigments a higher tolerance to PDT-induced photo-oxidative stress was observed. Hyperproduction of pyomelanin makes the cells much more tolerant to stress caused by either radicals or singlet oxygen generated by different photosensitizers upon photoactivation. Phenazines, pyocyanin and phenazine-1-carboxylic acid, produced in different amounts depending on the cultural conditions, are able to counteract both types of PDT-elicited reactive oxygen species. Hyperproduction of pyoverdine, caused by a mutation in a quorum-sensing gene, rendered P. aeruginosa more tolerant to a photosensitizer that generates mainly singlet oxygen, although in this case the observed tolerance to photo-oxidative stress cannot be exclusively attributed to the presence of the pigment.

AIF-1 and RNASET2 Play Complementary Roles in the Innate Immune Response of Medicinal Leech.

Recent studies demonstrated that allograft inflammatory factor-1 (AIF-1) and RNASET2 act as chemoattractants for macrophages and modulate the inflammatory processes in both vertebrates and invertebrates. The expression of these proteins significantly increases after bacterial infection; however, the mechanisms by which they regulate the innate immune response are still poorly defined. Here, we evaluate the effect of bacterial lipopolysaccharide injection on the expression pattern of these genes and the interrelation between them during innate immune response in the medicinal leech, an invertebrate model with a simple anatomy and a marked similarity with vertebrates in inflammatory processes. Collectively, prokaryotic-eukaryotic co-cultures and in vivo infection assays suggest that RNASET2 and AIF-1 play a crucial role in orchestrating a functional cross-talk between granulocytes and macrophages in leeches, resulting in the activation of an effective response against pathogen infection. RNASET2, firstly released by granulocytes, likely plays an early antibacterial role. Subsequently, AIF-1+ RNASET2-recruited macrophages further recruit other macrophages to potentiate the antibacterial inflammatory response. These experimental data are in keeping with the notion of RNA-SET2 acting as an alarmin-like molecule whose role is to locally transmit a "danger" signal (such as a bacterial infection) to the innate immune system in order to trigger an appropriate host response.

Methylene Blue Doped Films of Wool Keratin with Antimicrobial Photodynamic Activity.

In this work, keratin films doped with different amounts of methylene blue (MB) were developed in order to prepare new biodegradable and biocompatible materials for tissue engineering and wound healing, able to exert antimicrobial photodynamic activity upon irradiation with visible light. Preliminary results indicated that the swelling ratio, as well as the MB release, increases by increasing the pH. Moreover, the generation of reactive oxygen species (ROS) and singlet oxygen can be easily triggered and controlled by a fine-tuning of the irradiation time and MB concentration in the films. As concerns the photodynamic effects on keratin, the ROS attack does not induce any significant photodegradation on the protein, even if a slight photo-oxidation of sulfonated amino acids occurs. Finally, the film with the highest MB concentration (400 µg per gram of keratin) displays a significant photobactericidal activity against Staphylococcus aureus with a bacterial reduction that increases by increasing the irradiation time. In particular, the irradiation of KFMB400 film incubated with S. aureus at a concentration of 10(8) cfu mL(-1) determined the 99.9% killing rate and the killing effect increased proportionally with irradiation time.

Photoinduced antibacterial activity of two dicationic 5,15-diarylporphyrins.

Antimicrobial photodynamic treatment combines the use of photosensitizers (PSs) and visible light to kill bacterial cells. Cationic porphyrins are PSs largely used against bacteria and, among them, those featuring one positive charge on each of the 5,10,15,20-tetraaryl substituent (tetracationic) are the most used. The aim of this study was to synthesize two dicationic 5,15-di(N-alkyl-4-pyridyl)porphyrins, bearing methyl (PS 3) and benzyl (PS 4) N-alkylating groups, and to compare the efficiency in antibacterial photodynamic treatment, upon irradiation with a halogen-tungsten white lamp. The killing efficiency of the PS 4 was constantly found higher than that of the PS 3 against both pure and mixed cultures of laboratory model microorganisms as well as against wild wastewater microflora. The two PSs are comparable as regards singlet oxygen generation, but show a different repartition coefficient; the more lipophilic benzylated PS 4 shows a better interaction with the bacterial cells than the methylated one (PS 3). The data support the hypothesis that an efficient PS-cell binding is required to obtain significant effects. A correlation among cell binding, photoinactivation and PS lipophilicity is suggested.

Effect of organic matter on the in vitro photoeradication of Pseudomonas aeruginosa by means of a cationic tetraaryl-porphyrin.

Photodynamic therapy is emerging as an antimicrobial alternative approach; the concomitant presence of a photosensitizer (PS), O(2) and visible light induces lethal oxidative damages to bacterial cells. Among Gram-negative bacteria, Pseudomonas aeruginosa seems to be one of the least susceptible to photodynamic treatment. In this study, we evaluated the influence of several experimental conditions on photoeradication of a planktonic culture of P. aeruginosa PAO1 by means of a tetracationic meso-arylsubstituted porphyrin (RM24). Our findings suggest that the photo-oxidative stress induced by RM24 is strictly correlated to the amount of PS bound to the cells that in turn decreases with the increasing concentrations of organic compounds in the medium. The photoeradication is dependent on PS concentrations, cellular density and light dose. RM24 was able to induce oxidative stress by means of singlet oxygen formation, although ROS formation cannot be ruled out. The standardized experimental conditions of the photospot test allowed us to evidence intraspecific PDT sensitivity differences among three strains of P. aeruginosa.