Paola Di Fermo

This paper aimed to verify how a new protocol, recently proposed for treating chronic wounds due to its excellent antimicrobial properties, affects human dermal fibroblasts (NHDFs). Single and combined action of light-emitting diodes (LED), complex magnetic fields (CMFs), and methylglyoxal (MGO) on cell viability and activity of NHDFs were investigated. Our first objective was to exclude any toxicity of this combined treatment on these cells. NHDFs were exposed to LED light for 17 min, CMFs for 22 min, MGO, MGO + LED, and MGO + CMFs, and then were assessed for cell viability, morphology, cytoskeletal integrity, collagen type I production, and migration capacity. Results of combined treatments were compared with those of single treatments and unexposed controls. NHDFs exposed to both single and combined treatments maintained viability, morphology, and cytoskeletal integrity, showing no signs of cytotoxicity. MGO at low concentrations was non-toxic and, when combined with other technologies, could confer beneficial effects on cell adhesion. LED stimulated collagen type I synthesis, and the production increased in samples subjected to the combined action of MGO + LED. CMFs notably accelerated fibroblasts’ migration in scratch assays, and when combined with MGO, they further enhanced this effect. The effects of MGO + LED and MGO + CMFs were probably due to cellular uptake and receptor sensitivity. The tested protocols were not only non-toxic but also promoted beneficial effects on the vitality and activity of dermal fibroblasts, confirming their potential in treating chronic wounds.

Chronic wound infections associated with resistant polymicrobial biofilms are often refractory to conventional therapies with sustained healing time. This study evaluated the efficacy of non-antibiotic treatments including Methylglyoxal—MGO—Light-Emitting Diode—LED—and Complex Magnetic Fields—CMFs—alone/combined against the biofilms of two polymicrobial mixes (MIX 1, MIX 2) containing S. aureus, P. aeruginosa and C. albicans using the Lubbock chronic wound biofilm model. At 24 h after treatment, the effects were evaluated by (i) CFU/mg reduction, (ii) Confocal Laser Scanning Microscopy—CLSM—and (iii) Scanning Electron Microscopy—SEM. All treatments significantly reduced biofilms in terms of CFU/mg in both mixes versus the controls, 24 h after treatment. MGO showed remarkable activity, especially against P. aeruginosa. In MIX 1, LED/MGO + LED was highly effective against C. albicans. The combinations MGO + LED/MGO + CMFs enhanced the antibiofilm activity compared to each single treatment against S. aureus and P. aeruginosa, in both MIX1/MIX2. CLSM and SEM analysis showed biofilm disaggregation and reduction in cell viability with combined treatments, and Candida hyphal inhibition after CMFs application. In conclusion, the results demonstrate that MGO, alone or combined with LED or CMFs, shows high efficacy against resistant biofilms in the LCWB model 24 h after treatment, and encourage further studies on potential non-antibiotic and eco-friendly strategies as future alternative therapeutic approaches for chronic wound infections.

Background: antimicrobial resistance represents a critical issue leading to delayed wound healing; hence, it is necessary to develop novel strategies to address this phenomenon. Objectives: this study aimed to explore the antimicrobial/anti-virulence action of Methylglyoxal-MGO alone or combined with novel technologies such as Light-Emitting Diodes-LED and Complex Magnetic Fields-CMFs against resistant clinical strains isolated from chronic wounds. Methods: characterized planktonic Staphylococcus aureus, Pseudomonas aeruginosa, and Candida albicans isolates were used. Antimicrobial activity was evaluated by measuring optical density, Colony Forming Units-CFU, and synergy between MGO/LED or CMFs. Cellular membrane permeability by propidium iodide fluorescence and fluidity by Laurdan generalized polarization measurements were performed. P. aeruginosa motility was tested using the soft agar method. A docking study was performed to evaluate the possible interaction between MGO and urease in P. aeruginosa. Results: single/combined treatments showed significant antimicrobial activity. Major CFU reduction was detected after CMFs/MGO+CMFs application on C. albicans. Treatments exhibited significant changes in membrane permeability and fluidity. The treatments decreased P. aeruginosa motility with a major reduction after LED application. Docking analysis showed that MGO could bind with P. aeruginosa urease leading to defective folding and functional alterations. Conclusions: the results suggest that these treatments could represent promising and green therapeutic solutions against resistant isolates from chronic wounds.

The microorganisms living around and inside the human host in an extraordinary way for their morphologies often remind, microscopically we find every day foodstuffs that in our tables. Background: Many food products “are similar” to bacteria, a sort of “convergent evolution” between the microscopic and the macroscopic. Here below, we present some “similarities“between bacteria and food suggesting “New Recipes” of the Abruzzo Region folk tradition in Central Italy, which handed down from father to son constituted the history of our land. We believe that the history of a people is the true identity of man and passion for their roots should never be circumvented. Passion that binds us to our traditions, the same that binds us to our work, microbiology, making it natural for us during lunch hour, in the laboratory, to speak of the particular similarities of bacteria found with the foods we consume daily. Methods: We compared to same macroscopic food products to the following bacteria: Helicobacter pylori, H. heilmannii, Neisseria meningitidis, Staphylococcus aureus, Leptospira interrogans, and Pseudomonas aeruginosa. Results: In particular, we identified for each microorganism “similarities“with food and proposed New Recipes inspired for their “assonance” to microbial morphology and behavior and hence, suggest the following: “the Conversion”, “the Unidentical Fusilli Twins”, “the Dangerous Peaches”, “the Greek Grape Cluster Cake”, “the Black Cuttlefish Spirals sauce” and “the Biofilm Green Zucchini”. Conclusions: Therefore, arriving at a “special” approach that wants to link the microscopic and macroscopic worlds underlining that each shape is not casual and that everything is connected.

Helicobacter pylori colonizes human gastric mucosa, overcoming stressful conditions and entering in a dormant state. This study evaluated: (i) H. pylori’s physiological changes from active to viable-but-non-culturable (VBNC) and persister (AP) states, establishing times/conditions; (ii) the ability of vitamin C to interfere with dormancy generation/resuscitation. A dormant state was induced in clinical MDR H. pylori 10A/13 by: nutrient starvation (for VBNC generation), incubating in an unenriched medium (Brucella broth) or saline solution (SS), and (for AP generation) treatment with 10xMIC amoxicillin (AMX). The samples were monitored after 24, 48, and 72 h, 8–14 days by OD₆₀₀, CFUs/mL, Live/Dead staining, and an MTT viability test. Afterwards, vitamin C was added to the H. pylori suspension before/after the generation of dormant states, and monitoring took place at 24, 48, and 72 h. The VBNC state was generated after 8 days in SS, and the AP state in AMX for 48 h. Vitamin C reduced its entry into a VBNC state. In AP cells, Vitamin C delayed entry, decreasing viable coccal cells and increasing bacillary/U-shaped bacteria. Vitamin C increased resuscitation (60%) in the VBNC state and reduced the aggregates of the AP state. Vitamin C reduced the incidence of dormant states, promoting the resuscitation rate. Pretreatment with Vitamin C could favor the selection of microbial vegetative forms that are more susceptible to H. pylori therapeutical schemes.

Chronic wounds have harmful effects on both patients and healthcare systems. Wound chronicity is attributed to an impaired healing process due to several host and local factors that affect healing pathways. The resulting ulcers contain a wide variety of microorganisms that are mostly resistant to antimicrobials and possess the ability to form mono/poly-microbial biofilms. The search for new, effective and safe compounds to handle chronic wounds has come a long way throughout the history of medicine, which has included several studies and trials of conventional treatments. Treatments focus on fighting the microbial colonization that develops in the wound by multidrug resistant pathogens. The development of molecular medicine, especially in antibacterial agents, needs an in vitro model similar to the in vivo chronic wound environment to evaluate the efficacy of antimicrobial agents. The Lubbock chronic wound biofilm (LCWB) model is an in vitro model developed to mimic the pathogen colonization and the biofilm formation of a real chronic wound, and it is suitable to screen the antibacterial activity of innovative compounds. In this review, we focused on the characteristics of chronic wound biofilms and the contribution of the LCWB model both to the study of wound poly-microbial biofilms and as a model for novel treatment strategies.

Innovative non-antibiotic compounds such as graphene oxide (GO) and light-emitting diodes (LEDs) may represent a valid strategy for managing chronic wound infections related to resistant pathogens. This study aimed to evaluate 630 nm LED and 880 nm LED ability to enhance the GO antimicrobial activity against Staphylococcus aureus- and Pseudomonas aeruginosa-resistant strains in a dual-species biofilm in the Lubbock chronic wound biofilm (LCWB) model. The effect of a 630 nm LED, alone or plus 5-aminolevulinic acid (ALAD)-mediated photodynamic therapy (PDT) (ALAD-PDT), or an 880 nm LED on the GO (50 mg/l) action was evaluated by determining the CFU/mg reductions, live/dead analysis, scanning electron microscope observation, and reactive oxygen species assay. Among the LCWBs, the best effect was obtained with GO irradiated with ALAD-PDT, with percentages of CFU/mg reduction up to 78.96% ± 0.21 and 95.17% ± 2.56 for S. aureus and P. aeruginosa, respectively. The microscope images showed a reduction in the cell number and viability when treated with GO + ALAD-PDT. In addition, increased ROS production was detected. No differences were recorded when GO was irradiated with an 880 nm LED versus GO alone. The obtained results suggest that treatment with GO irradiated with ALAD-PDT represents a valid, sustainable strategy to counteract the polymicrobial colonization of chronic wounds.

Chronic wound infections represent an important health problem due to the reduced response to antimicrobial treatment of the pathogens organized in structured biofilms. This study investigated the effects of the previously described antifungal peptide L18R against three representative wound pathogens: Staphylococcus aureus, Pseudomonas aeruginosa, and Candida albicans. The antimicrobial activity of L18R was evaluated (i) against single planktonic microbial populations; (ii) on single, dual, and triadic species of biofilms in both the early stage and mature stage; and (iii) in the polymicrobial Lubbock chronic wound biofilm (LCWB) model, mimicking spatial microbial colonization. This study used the evaluation of CFUs, biofilm biomass detection, and confocal and scanning electron microscopy analysis. L18R showed a significant antimicrobial activity against planktonic microorganisms and was able to differentially reduce the biomass of monomicrobial biofilms. No reduction of biomass was observed against the polymicrobial biofilm. In mature LCWB, L18R caused a moderate reduction in total CFU number, with a variable effect on the different microorganisms. Microscopy images confirmed a predominant presence of P.aeruginosa and a lower percentage of C. albicans cells. These findings suggest a modulating action of L18R and recommend further studies on its potential role in chronic wound management in association with conventional antibiotics or alternative treatments.

Background: An unbalanced skin microbiota due to an increase in pathogenic vs. commensal bacteria can be efficiently tackled by using prebiotics. The aim of this work was to identify novel prebiotic combinations by exerting species-specific action between S. aureus and S. epidermidis strains. Methods: First, the antimicrobial/antibiofilm effect of Xylitol-XYL and Galacto-OligoSaccharides–GOS combined with each other at different concentrations (1, 2.5, 5%) against S. aureus and S. epidermidis clinical strains was evaluated in time. Second, the most species-specific concentration was used to combine XYL with Fructo-OligoSaccharides–FOS, IsoMalto-Oligosaccharides–IMO, ArabinoGaLactan–LAG, inulin, dextran. Experiments were performed by OD₆₀₀ detection, biomass quantification and LIVE/DEAD staining. Results: 1% XYL + 1% GOS showed the best species-specific action with an immediate antibacterial/antibiofilm action against S. aureus strains (up to 34.54% ± 5.35/64.68% ± 4.77) without a relevant effect on S. epidermidis. Among the other prebiotic formulations, 1% XYL plus 1% FOS (up to 49.17% ± 21.46/37.59% ± 6.34) or 1% IMO (up to 41.28% ± 4.88/36.70% ± 10.03) or 1% LAG (up to 38.21% ± 5.31/83.06% ± 5.11) showed antimicrobial/antibiofilm effects similar to 1% XYL+1% GOS. For all tested formulations, a prevalent bacteriostatic effect in the planktonic phase and a general reduction of S. aureus biofilm formation without loss of viability were recorded. Conclusion: The combinations of 1% XYL with 1% GOS or 1% FOS or 1% IMO or 1% LAG may help to control the balance of skin microbiota, representing good candidates for topic formulations.

The drug-resistance phenomenon in Helicobacter pylori underlines the need of novel strategies to improve the eradication rate including alternative treatments combining antibiotic and non-antibiotic compounds with synergistic action. In this study, the antibacterial (MIC/MBC) and anti-virulence effects (biofilm reduction and swarming motility inhibition) of resveratrol-RSV and new synthetized RSV-phenol derivatives, with a higher bioavailability, alone and combined with levofloxacin-LVX were evaluated against resistant H. pylori clinical strains. The experiments were confirmed in vivo using the Galleria mellonella model. Among the studied RSV derivatives, RSV-3 and RSV-4 possessed higher antibacterial activity with respect to RSV (MICs from 6.25 to 200 µg/mL and from 3.12 to 200 µg/mL, respectively). RSV, RSV-3, and RSV-4 were able to synergize with LVX restoring its effect in two out of seven clinical resistant strains tested for the study. RSV, RSV-3, and RSV-4, alone and with LVX at sub-MIC and sub-synergistic concentrations, significantly reduced the biofilm formation. Moreover, RSV-3 and RSV-4 reduced the H. pylori swarming motility on soft agar. RSV, RSV-3, and RSV-4 were non-toxic for G. mellonella larvae and displayed a protective effect against H. pylori infection. Overall, RSV–phenol derivatives should be considered interesting candidates for innovative therapeutic schemes to tackle the H. pylori antibiotic resistance.