A Methylazanediyl Bisacetamide Derivative Sensitizes Staphylococcus aureus Persisters to a Combination of Gentamicin And Daptomycin.

Infections caused by Staphylococcus aureus, notably methicillin-resistant S. aureus (MRSA), pose treatment challenges due to its ability to tolerate antibiotics and develop antibiotic resistance. The former, a mechanism independent of genetic changes, allows bacteria to withstand antibiotics by altering metabolic processes. Here, a potent methylazanediyl bisacetamide derivative, MB6, is described, which selectively targets MRSA membranes over mammalian membranes without observable resistance development. Although MB6 is effective against growing MRSA cells, its antimicrobial activity against MRSA persisters is limited. Nevertheless, MB6 significantly potentiates the bactericidal activity of gentamicin against MRSA persisters by facilitating gentamicin uptake. In addition, MB6 in combination with daptomycin exhibits enhanced anti-persister activity through mutual reinforcement of their membrane-disrupting activities. Crucially, the "triple" combination of MB6, gentamicin, and daptomycin exhibits a marked enhancement in the killing of MRSA persisters compared to individual components or any double combinations. These findings underscore the potential of MB6 to function as a potent and selective membrane-active antimicrobial adjuvant to enhance the efficacy of existing antibiotics against persister cells. The molecular mechanisms of MB6 elucidated in this study provide valuable insights for designing anti-persister adjuvants and for developing new antimicrobial combination strategies to overcome the current limitations of antibiotic treatments.

Arctic Psychrotolerant Pseudomonas sp. B14-6 Exhibits Temperature-Dependent Susceptibility to Aminoglycosides.

Bacteria can evade antibiotics by acquiring resistance genes, as well as switching to a non-growing dormant state without accompanying genetic modification. Bacteria in this quiescent state are called persisters, and this non-inheritable ability to withstand multiple antibiotics is referred to as antibiotic tolerance. Although all bacteria are considered to be able to form antibiotic-tolerant persisters, the antibiotic tolerance of extremophilic bacteria is poorly understood. Previously, we identified the psychrotolerant bacterium Pseudomonas sp. B14-6 from the glacier foreland of Midtre Lovénbreen in High Arctic Svalbard. Herein, we investigated the resistance and tolerance of Pseudomonas sp. B14-6 against aminoglycosides at various temperatures. This bacterium was resistant to streptomycin and susceptible to apramycin, gentamicin, kanamycin, and tobramycin. The two putative aminoglycoside phosphotransferase genes aph1 and aph2 were the most likely contributors to streptomycin resistance. Notably, unlike the mesophilic Pseudomonas aeruginosa PA14, this cold-adapted bacterium demonstrated reduced susceptibility to all tested aminoglycosides in a temperature-dependent manner. Pseudomonas sp. B14-6 at a lower temperature formed the persister cells that shows tolerance to the 100-fold minimum inhibitory concentration (MIC) of gentamicin, as well as the partially tolerant cells that withstand 25-fold MIC gentamicin. The temperature-dependent gentamicin tolerance appears to result from reduced metabolic activity. Lastly, the partially tolerant Pseudomonas sp. B14-6 cells could slowly proliferate under the bactericidal concentrations of aminoglycosides. Our results demonstrate that Pseudomonas sp. B14-6 has a characteristic ability to form cells with a range of tolerance, which appears to be inversely proportional to its growth rate.

Antimicrobial activity of the membrane-active compound nTZDpa is enhanced at low pH.

The opportunistic human pathogen Staphylococcus aureus can evade antibiotics by acquiring antibiotic resistance genes or by entering into a non-growing dormant state. Moreover, the particular circumstances of a specific infection site, such as acidity or anaerobicity, often weaken antibiotic potency. Decreased bacterial susceptibility combined with diminished antibiotic potency is responsible for high failure rates when treating S. aureus infections. Here, we report that the membrane-active antimicrobial agent nTZDpa does not only exhibit enhanced antibiotic activity against multidrug-resistant Gram-positive pathogens in acidic pH, but also retains antimicrobial potency under anaerobic conditions. This agent completely eradicated highly antibiotic-tolerant cells and biofilms formed by methicillin-resistant S. aureus at pH 5.5 at concentrations at which it was not potent at pH 7.4. Furthermore, nTZDpa was more potent at synergistically potentiating gentamicin killing against antibiotic-tolerant MRSA cells at low pH than at high pH. All-atom molecular dynamics simulations combined with membrane-permeabilization assays revealed that the neutral form of nTZDpa, which contains carboxylic acid, is more effective than the deprotonated form at penetrating the bacterial membrane and plays an essential role in membrane activity. An acidic pH increases the proportion of the neutrally charged nTZDpa, which results in antimicrobial enhancement. Our results provide key insights into rational design of pH-sensitive membrane-active antimicrobials and antibiotic adjuvants that are effective in an infection environment. These findings demonstrate that nTZDpa is a promising lead compound for developing new therapeutics against hard-to-cure infections caused by drug-resistant and -tolerant S. aureus.

Characterization of Phenethyl Cinnamamide Compounds from Hemp Seed and Determination of Their Melanogenesis Inhibitory Activity.

Hyperpigmentation is induced by the overactivation of tyrosinase, which is a rate-limiting enzyme in melanogenesis. The defatted extract of hemp (Cannabis sativa L.) seed is known to have inhibitory effects on melanogenesis; however, effective compounds in the extract have not been identified yet. In this study, three phenethyl cinnamamides present in hemp seed extract were prepared by purification and chemical synthesis and were assessed for their inhibitory effect on melanogenesis in B16F10 melanoma cells. A comparison of the anti-melanogenesis and anti-tyrosinase activity of hemp seed solvent fractions revealed that the ethyl acetate fraction possessed the greatest potential for suppressing melanogenesis in melanoma cells by decreasing tyrosinase activity. We tentatively identified 26 compounds in the ethyl acetate fraction by comparing spectroscopic data with the literature. Three phenethyl cinnamamides such as N-trans-caffeoyltyramine, N-trans-coumaroyltyramine, and N-trans-feruloyltyramine present abundantly in the ethyl acetate fraction were prepared and their anti-melanogenesis and anti-tyrosinase activities in melanoma cells were evaluated. We found that N-trans-caffeoyltyramine and N-trans-feruloyltyramine inhibited alpha melanocyte stimulating hormone (α-MSH)-induced melanogenesis without cytotoxicity, while N-trans-coumaroyltyramine inhibited melanogenesis with cytotoxicity. IC50 values of N-trans-caffeoyltyramine, N-trans-feruloyltyramine, and N-trans-coumaroyltyramine for inhibition of α-MSH-mediated tyrosinase activation were 0.8, 20.2, and 6.3 µM, respectively. Overall, N-trans-caffeoyltyramine possessed the strongest anti-melanogenesis activity among the three phenethyl cinnamamides evaluated. The inhibitory effect of N-trans-caffeoyltyramine was verified by determining the melanin content and tyrosinase activity in melanoma after treating the cells with synthetic compounds. Thus, N-trans-caffeoyltyramine isolated from hemp seed extract could be useful in cosmetics as a skin-whitening agent.

Anti-Melanogenic Properties of Velutin and Its Analogs.

Velutin, one of the flavones contained in natural plants, has various beneficial activities, such as skin whitening, as well as anti-inflammatory, anti-allergic, antioxidant, and antimicrobial activities. However, the relationship between the structure of velutin and its anti-melanogenesis activity is not yet investigated. In this study, we obtained 12 velutin derivatives substituted at C5, C7, C3', and C4' of the flavone backbone with hydrogen, hydroxyl, and methoxy functionalities by chemical synthesis, to perform SAR analysis of velutin structural analogues. The SAR study revealed that the substitution of functional groups at C5, C7, C3', and C4' of the flavone backbone affects biological activities related to melanin synthesis. The coexistence of hydroxyl and methoxy at the C5 and C7 position is essential for inhibiting tyrosinase activity. However, 1,2-diol compounds substituted at C3' and C4' of flavone backbone induce apoptosis of melanoma cells. Further, substitution at C3' and C4' with methoxy or hydrogen is essential for inhibiting melanogenesis. Thus, this study would be helpful for the development of natural-derived functional materials to regulate melanin synthesis.