Pseudomonas aeruginosa is a ubiquitous organism and opportunistic pathogen that can cause persistent infections due to its peculiar antibiotic resistance mechanisms and to its ability to adhere and form biofilm. The interest in the development of new approaches for the prevention and treatment of biofilm formation has recently increased. The aim of this study was to seek new non-biocidal agents able to inhibit biofilm formation, in order to counteract virulence rather than bacterial growth and avoid the selection of escape mutants. Herein, different essential oils extracted from Mediterranean plants were analyzed for their activity against P. aeruginosa. Results show that they were able to destabilize biofilm at very low concentration without impairing bacterial viability. Since the action is not related to a bacteriostatic/bactericidal activity on P. aeruginosa, the biofilm change of growth in presence of the essential oils was possibly due to a modulation of the phenotype. To this aim, application of machine learning algorithms led to the development of quantitative activity–composition relationships classification models that allowed to direct point out those essential oil chemical components more involved in the inhibition of biofilm production. The action of selected essential oils on sessile phenotype make them particularly interesting for possible applications such as prevention of bacterial contamination in the community and in healthcare environments in order to prevent human infections. We assayed 89 samples of different essential oils as P. aeruginosa anti-biofilm. Many samples inhibited P. aeruginosa biofilm at concentrations as low as 48.8 µg/mL. Classification of the models was developed through machine learning algorithms.

Biofilm resistance to antimicrobials is a complex phenomenon, driven not only by genetic mutation induced resistance, but also by means of increased microbial cell density that supports horizontal gene transfer across cells. The prevention of biofilm formation and the treatment of existing biofilms is currently a difficult challenge; therefore, the discovery of new multi-targeted or combinatorial therapies is growing. The development of anti-biofilm agents is considered of major interest and represents a key strategy as non-biocidal molecules are highly valuable to avoid the rapid appearance of escape mutants. Among bacteria, staphylococci are predominant causes of biofilm-associated infections. Staphylococci, especially Staphylococcus aureus (S. aureus) is an extraordinarily versatile pathogen that can survive in hostile environmental conditions, colonize mucous membranes and skin, and can cause severe, non-purulent, toxin-mediated diseases or invasive pyogenic infections in humans. Staphylococcus epidermidis (S. epidermidis) has also emerged as an important opportunistic pathogen in infections associated with medical devices (such as urinary and intravascular catheters, orthopaedic implants, etc.), causing approximately from 30% to 43% of joint prosthesis infections. The scientific community is continuously looking for new agents endowed of anti-biofilm capabilities to fight S. aureus and S epidermidis infections. Interestingly, several reports indicated in vitro efficacy of non-biocidal essential oils (EOs) as promising treatment to reduce bacterial biofilm production and prevent the inducing of drug resistance. In this report were analyzed 89 EOs with the objective of investigating their ability to modulate bacterial biofilm production of different S. aureus and S. epidermidis strains. Results showed the assayed EOs to modulated the biofilm production with unpredictable results for each strain. In particular, many EOs acted mainly as biofilm inhibitors in the case of S. epidermidis strains, while for S. aureus strains, EOs induced either no effect or stimulate biofilm production. In order to elucidate the obtained experimental results, machine learning (ML) algorithms were applied to the EOs’ chemical compositions and the determined associated anti-biofilm potencies. Statistically robust ML models were developed, and their analysis in term of feature importance and partial dependence plots led to indicating those chemical components mainly responsible for biofilm production, inhibition or stimulation for each studied strain, respectively.

BackgroundGlioblastoma (GBM) is the most lethal and aggressive malignant primary brain tumor in adults. After surgical resection of the tumor, the patient typically should be subjected to chemotherapy (temozolomide, TMZ) and concomitant radiotherapy. Since the TMZ treatment does not lead to complete remission and often develops resistance, the identification of efficacious therapeutics is strongly to pursue. Among the epigenetic players, the H3K27 methyltransferase (MT) EZH2 (enhancer of zeste homologue 2) has been found overexpressed or mutated in several human cancers including gliomas, and its overexpression is associated with poor outcome in GBM. Two EZH2 inhibitors (EZH2i), UNC1999 and GSK343, suppressed GBM growth in vitro and in vivo indicating that EZH2i can be potential drugs against GBM.ResultsTwo new EZH2i, MC4040 and MC4041, were designed, prepared, and tested by us to determine their effects in primary GBM cell cultures. MC4040 and MC4041 displayed single-digit micromolar inhibition of EZH2, 10-fold less potency against EZH1, and no activity towards other MTs. In primary GBM cells as well as in U-87 GBM cells, the two compounds reduced H3K27me3 levels, and dose- and time-dependently impaired GBM cell viability without inducing apoptosis and arresting the cell cycle in the G0/G1 phase, with increased p21 and p27 levels. In combination with TMZ, MC4040 and MC4041 displayed stronger, but not additive, effects on cell viability. The potent clinical candidate as EZH2i tazemetostat, alone or in combination with TMZ, exhibited a similar potency of inhibition of GBM cell growth when compared to MC4040 and MC4041. At the molecular level, MC4040 and MC4041 reduced the VEGFR1/VEGF expression, reversed the epithelial-mesenchymal transition (EMT), and hampered cell migration and invasion attenuating the cancer malignant phenotype. Treatment of GBM cells with MC4040 and MC4041 also impaired the GBM pro-inflammatory phenotype, with a significant decrease of TGF-β, TNF-α, and IL-6, joined to an increase of the anti-inflammatory cytokine IL-10.ConclusionsThe two novel EZH2i MC4040 and MC4041 impaired primary GBM cell viability, showing even stronger effects in combination with TMZ. They also weakened the aggressive malignant phenotype by reducing angiogenesis, EMT, cell migration/invasion and inflammation, thus they may be considered potential candidates against GBM also for combination therapies.

A comprehensive study on essential oil samples of Foeniculum vulgare Miller from Tarquinia (Italy) is reported. A 24-h systematic steam distillation was performed on different harvested samples applying different extraction times. The GC-MS analysis of the residue outcome showed o-cymene, α-phellandrene, α-pinene and estragole as the major constituents. The predominance and continued presence of o-cymene makes this fennel oil a rather unique chemotype. An evident correlation between the antifungal activity and phenological stage is demonstrated. The most active fractions were particularly rich in estragole, as well as a significant amount of fenchone that possibly exerts some additive effect in the expression of overall antifungal potency. Pre-fruiting material produced oil particularly rich in o-cymene. With reference to the duration of the extraction, the maximum amount of oil was released within the first 3 h, whereas the reproductive phase material needed at least 6 h for the extraction.

Bacterial biofilm plays a pivotal role in chronic Staphylococcus aureus (S. aureus) infection and its inhibition may represent an important strategy to develop novel therapeutic agents. The scientific community is continuously searching for natural and “green alternatives” to chemotherapeutic drugs, including essential oils (EOs), assuming the latter not able to select resistant strains, likely due to their multicomponent nature and, hence, multitarget action. Here it is reported the biofilm production modulation exerted by 61 EOs, also investigated for their antibacterial activity on S. aureus strains, including reference and cystic fibrosis patients’ isolated strains. The EOs biofilm modulation was assessed by Christensen method on five S. aureus strains. Chemical composition, investigated by GC/MS analysis, of the tested EOs allowed a correlation between biofilm modulation potency and putative active components by means of machine learning algorithms application. Some EOs inhibited biofilm growth at 1.00% concentration, although lower concentrations revealed different biological profile. Experimental data led to select antibiofilm EOs based on their ability to inhibit S. aureus biofilm growth, which were characterized for their ability to alter the biofilm organization by means of SEM studies.