Antibiofilm Activity of Combretum micranthum G. Don Catechin-Sugar Phytocomplex on Pseudomonas aeruginosa.

Clinicians often have to face infections caused by microorganisms that are difficult to eradicate due to their resistance and/or tolerance to antimicrobials. Among these pathogens, Pseudomonas aeruginosa causes chronic infections due to its ability to form biofilms on medical devices, skin wounds, ulcers and the lungs of patients with Cystic Fibrosis. In this scenario, the plant world represents an important reservoir of natural compounds with antimicrobial and/or antibiofilm properties. In this study, an extract from the leaves of Combretum micranthum G. Don, named Cm4-p, which was previously investigated for its antimicrobial activities, was assayed for its capacity to inhibit biofilm formation and/or to eradicate formed biofilms. The model strain P. aeruginosa PAO1 and its isogenic biofilm hyperproducer derivative B13 were treated with Cm4-p. Preliminary IR, UV-vis, NMR, and mass spectrometry analyses showed that the extract was mainly composed of catechins bearing different sugar moieties. The phytocomplex (3 g/L) inhibited the biofilm formation of both the PAO1 and B13 strains in a significant manner. In light of the obtained results, Cm4-p deserves deeper investigations of its potential in the antimicrobial field.

Screening Antibacterial Photodynamic Effect of Monascus Red Yeast Rice (Hong-Qu) and Mycelium Extracts.

The fungus Monascus is a well-known source of secondary metabolites with interesting pharmaceutical and nutraceutical applications. In particular, Monascus pigments possess a wide range of biological activities (e.g. antimicrobial, antioxidant, anti-inflammatory or antitumoral). To broaden the scope of their possible application, this study focused on testing Monascus pigment extracts as potential photosensitizing agents efficient in antimicrobial photodynamic therapy (aPDT) against bacteria. For this purpose, eight different extracts of secondary metabolites from the liquid- and solid-state fermentation of Monascus purpureus DBM 4360 and Monascus sp. DBM 4361 were tested against Gram-positive and Gram-negative model bacteria, Bacillus subtilis and Escherichia coli and further screened for ESKAPE pathogens, Staphylococcus aureus and Pseudomonas aeruginosa. To the bacterial culture, increasing concentration of extracts was added and it was found that all extracts showed varying antimicrobial activity against Gram-positive bacteria in dark, which was further increased after irradiation. Gram-negative bacteria were tolerant to the extracts' exposure in the dark but sensitivity to almost all extracts that occurred after irradiation. The Monascus sp. DBM 4361 extracts seemed to be the best potential candidate for aPDT against Gram-positive bacteria, being efficient at low doses, i.e. the lowest total concentration of Monascus pigments exhibiting aPDT effect was 3.92 ± 1.36 mg/L for E. coli. Our results indicate that Monascus spp., forming monascuspiloin as the major yellow pigment and not-forming mycotoxin citrinin, is a promising source of antimicrobials and photoantimicrobials.

Bacterial pigments: A colorful palette reservoir for biotechnological applications.

Synthetic derivatives are currently used instead of pigments in many applicative fields, from food to feed, from pharmaceutical to diagnostic, from agronomy to industry. Progress in organic chemistry allowed to obtain rather cheap compounds covering the whole color spectrum. However, several concerns arise from this chemical approach, as it is mainly based on nonrenewable resources such as fossil oil, and the toxicity or carcinogenic properties of products and/or precursors may be harmful for personnel involved in the productive processes. In this scenario, microorganisms and their pigments represent a colorful world to discover and reconsider. Each living bacterial strain may be a source of secondary metabolites with peculiar functions. The aim of this review is to link the physiological role of bacterial pigments with their potential use in different biotechnological fields. This enormous potential supports the big challenge for the development of strategies useful to identify, produce, and purify the right pigment for the desired application. At the end of this ideal journey through the world of bacterial pigments, the attention will be focused on melanin compounds, whose production relies upon different techniques ranging from natural producers, heterologous hosts, or isolated enzymes. In a green workflow, the microorganisms represent the starting and final point of pigment production.

Photoinactivation of Pseudomonas aeruginosa Biofilm by Dicationic Diaryl-Porphyrin.

In recent years, antimicrobial photodynamic therapy (aPDT) has received increasing attention as a promising tool aimed at both treating microbial infections and sanitizing environments. Since biofilm formation on biological and inert surfaces makes difficult the eradication of bacterial communities, further studies are needed to investigate such tricky issue. In this work, a panel of 13 diaryl-porphyrins (neutral, mono- and di-cationic) was taken in consideration to photoinactivate Pseudomonas aeruginosa. Among cationic photosensitizers (PSs) able to efficiently bind cells, in this study two dicationic showed to be intrinsically toxic and were ruled out by further investigations. In particular, the dicationic porphyrin (P11) that was not toxic, showed a better photoinactivation rate than monocationic in suspended cells. Furthermore, it was very efficient in inhibiting the biofilms produced by the model microorganism Pseudomonas aeruginosa PAO1 and by clinical strains derived from urinary tract infection and cystic fibrosis patients. Since P. aeruginosa represents a target very difficult to inactivate, this study confirms the potential of dicationic diaryl-porphyrins as photo-activated antimicrobials in different applicative fields, from clinical to environmental ones.

Nitric Oxide Photo-Donor Hybrids of Ciprofloxacin and Norfloxacin: A Shift in Activity from Antimicrobial to Anticancer Agents.

The potential anticancer effect of fluoroquinolone antibiotics has been recently unveiled and related to their ability to interfere with DNA topoisomerase II. We herein envisioned the design and synthesis of novel Ciprofloxacin and Norfloxacin nitric oxide (NO) photo-donor hybrids to explore the potential synergistic antitumor effect exerted by the fluoroquinolone scaffold and NO eventually produced upon light irradiation. Anticancer activity, evaluated on a panel of tumor cell lines, showed encouraging results with IC50 values in the low micromolar range. Some compounds displayed intense antiproliferative activity on triple-negative and doxorubicin-resistant breast cancer cell lines, paving the way for their potential use to treat aggressive, refractory and multidrug-resistant breast cancer. No significant additive effect was observed on PC3 and DU145 cells following NO release. Conversely, antimicrobial photodynamic experiments on both Gram-negative and Gram-positive microorganisms displayed a significant killing rate in Staphylococcus aureus, accounting for their potential effectiveness as selective antimicrobial photosensitizers.

3D Reconstruction of HvRNASET2 Molecule to Understand Its Antibacterial Role.

Recent studies performed on the invertebrate model Hirudo verbana (medicinal leech) suggest that the T2 ribonucleic enzyme HvRNASET2 modulates the leech's innate immune response, promoting microbial agglutination and supporting phagocytic cells recruitment in challenged tissues. Indeed, following injection of both lipoteichoic acid (LTA) and Staphylococcus aureus in the leech body wall, HvRNASET2 is expressed by leech type I granulocytes and induces bacterial aggregation to aid macrophage phagocytosis. Here, we investigate the HvRNASET2 antimicrobial role, in particular assessing the effects on the Gram-negative bacteria Escherichia coli. For this purpose, starting from the three-dimensional molecule reconstruction and in silico analyses, the antibacterial activity was evaluated both in vitro and in vivo. The changes induced in treated bacteria, such as agglutination and alteration in wall integrity, were observed by means of light, transmission and scanning electron microscopy. Moreover, immunogold, AMPs (antimicrobial peptides) and lipopolysaccharide (LPS) binding assays were carried out to evaluate HvRNASET2 interaction with the microbial envelopes and the ensuing ability to affect microbial viability. Finally, in vivo experiments confirmed that HvRNASET2 promotes a more rapid phagocytosis of bacterial aggregates by macrophages, representing a novel molecule for counteracting pathogen infections and developing alternative solutions to improve human health.

Antimicrobial Role of RNASET2 Protein During Innate Immune Response in the Medicinal Leech Hirudo verbana.

The innate immune response represents a first-line defense against pathogen infection that has been widely conserved throughout evolution. Using the invertebrate Hirudo verbana (Annelida, Hirudinea) as an experimental model, we show here that the RNASET2 ribonuclease is directly involved in the immune response against Gram-positive bacteria. Injection of lipoteichoic acid (LTA), a key component of Gram-positive bacteria cell wall, into the leech body wall induced a massive migration of granulocytes and macrophages expressing TLR2 (the key receptor involved in the response to Gram-positive bacteria) toward the challenged/inoculated area. We hypothesized that the endogenous leech RNASET2 protein (HvRNASET2) might be involved in the antimicrobial response, as already described for other vertebrate ribonucleases, such as RNase3 and RNase7. In support of our hypothesis, HvRNASET2 was mainly localized in the granules of granulocytes, and its release in the extracellular matrix triggered the recruitment of macrophages toward the area stimulated with LTA. The activity of HvRNASET2 was also evaluated on Staphylococcus aureus living cells by means of light, transmission, and scanning electron microscopy analysis. HvRNASET2 injection triggered the formation of S. aureus clumps following a direct interaction with the bacterial cell wall, as demonstrated by immunogold assay. Taken together, our data support the notion that, during the early phase of leech immune response, granulocyte-released HvRNASET2 triggers bacterial clumps formation and, at the same time, actively recruits phagocytic macrophages in order to elicit a rapid and effective eradication of the infecting microorganisms from inoculated area.

Cationic diarylporphyrins: In vitro versatile anticancer and antibacterial photosensitizers.

The visible light combined with photosensitizers (PSs) is exploited in both antitumoral and antimicrobial fields inducing a photo-oxidative stress within the target cells. Among the different PSs, porphyrins belong to the family of the most promising compounds to be used in clinical photodynamic applications. Although in the last years many porphyrins have been synthesised and tested, only a few reports concern the in vitro effects of the 5,15-diarylporphyrins. In this work, the activity of four 5,15-diarylporphyrins (compounds 7-10), bearing alkoxy-linked pyridinium appendixes, have been tested on cancer cell lines and against bacterial cultures. Among the synthetized PSs, compounds 7 and 9 are not symmetrically substituted porphyrins showing one cationic charge tethered at the end of one 4C or 8C carbon chains, respectively. On the other hand, compounds 8 and 10 are symmetrically substituted and show two chains of C4 and C8 carbons featuring a cationic charge at the end of both chains. The dicationic 8 and 10 were more hydrophilic than monocationic 7 and 9, outlining that the presence of two pyridinium salts have a higher impact on the solubility in the aqueous phase than the lipophilic effect exerted by the length of the alkyl chains. Furthermore, these four PSs showed a similar rate of photobleaching, irrespective of the length and number of chains and the number of positive charges. Among the eukaryotic cell lines, the SKOV3 cells were particularly sensitive to the photodynamic activity of all the tested diarylporphyrins, while the HCT116 cells were found more sensitive to PSs bearing C4 chain (7 and 8), regardless the number of cationic charges. The photo-induced killing effect of these porphyrins was also tested against two different bacterial cultures. As expected, the Gram positive Bacillus subtilis was more sensitive than the Gram negative Escherichia coli, and the dicationic porphyrin 8, bearing two C4 chains, was the most efficient on both microorganisms. In conclusion, the new compound 8 seems to be an optimal candidate to deepen as versatile anticancer and antibacterial photosensitizer.

Catalase A is involved in the response to photooxidative stress in Pseudomonas aeruginosa.

BACKGROUND: Pseudomonas aeruginosa is the etiological agent of systemic and skin infections that are often difficult to treat. Photodynamic therapy (PDT) and, more recently, phototherapy (PT), are emerging among antimicrobial treatments to be combined with antibiotics. Visible light, either alone or combined with a photosensitizer (PS), elicits photooxidative stress that induces microbial death. The response of bacteria to phototherapy seems to involve the antioxidant machinery. This study relies on the effects of detoxifying catalase A (KatA) in response to PDT and PT-induced photooxidative stress. METHODS: The photo- and photodynamic inactivation experiments have been targeted at P. aeruginosa PAO1 and its isogenic derivative katA- mutant. The microorganisms were irradiated by a wide-spectrum halogen-tungsten lamp or light-emitting diodes (LEDs). Two photosensitizers, Tetrakis-(1-methyl-4-pyridyl)-21H, 23porphine, tetra-p-tosylate (TMPyP) porphyrin and Toluidine Blue O (TBO), were applied as part of the photodynamic approach. RESULTS: P. aeruginosa katA- mutant was more sensitive than wild-type strain PAO1 to wide-spectrum light and blue LED (464 nm) treatments. The complementation of KatA, in katA- mutant, restored the light response of wild-type PAO1. Upon TBO treatment and irradiation by visible light (halogen lamp or LED), the sensitivity of katA- mutant was significant higher (p = 0.028 and p = 0.045, respectively) than that of the PAO1 strain. CONCLUSIONS: This study provides the first description of KatA in the response to photooxidative stress induced by photo- and photodynamic therapy.