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Resistance to aminoglycoside antibiotics is a serious problem, typically arising from inactivating enzymes, reduced uptake, or increased efflux in the important pathogens for which they are used as treatment. Conjugating aminoglycosides to proline-rich antimicrobial peptides (PrAMPs), which also target ribosomes and have a distinct bacterial uptake mechanism, might mutually benefit their individual activities. To this aim we have developed a strategy for noninvasively modifying tobramycin to link it to a Cys residue and through this covalently link it to a Cys-modified PrAMP by formation of a disulfide bond. Reduction of this bridge in the bacterial cytosol should release the individual antimicrobial moieties. We found that the conjugation of tobramycin to the well-characterized N-terminal PrAMP fragment Bac7(1-35) resulted in a potent antimicrobial capable of inactivating not only tobramycin-resistant bacterial strains but also those less susceptible to the PrAMP. To a certain extent, this activity also extends to the shorter and otherwise poorly active fragment Bac7(1-15). Although the mechanism that allows the conjugate to act when its individual components do not is as yet unclear, results are very promising and suggest this may be a way of resensitizing pathogens that have developed resistance to the antibiotic.

Electrospun polycaprolactone (PCL) membranes have been widely explored in the literature as a solution for several applications in tissue engineering and regenerative medicine. PCL hydrophobicity and its lack of bioactivity drastically limit its use in the medical field. To overcome these drawbacks, many promising strategies have been developed and proposed in the literature. In order to increase the bioactivity of electrospun PCL membranes designed for guided bone and tissue regeneration purposes, in the present work, the membranes were functionalized with a coating of bioactive lactose-modified chitosan (CTL). Since CTL can be used for the synthesis and stabilization of silver nanoparticles, a coating of this compound was employed here to provide antibacterial properties to the membranes. Scanning electron microscopy imaging revealed that the electrospinning process adopted here allowed us to obtain membranes with homogeneous fibers and without defects. Also, PCL membranes retained their mechanical properties after several weeks of aging in simulated body fluid, representing a valid support for cell growth and tissue development. CTL adsorption on membranes was investigated by fluorescence microscopy using fluorescein-labeled CTL, resulting in a homogeneous and slow release over time. Inductively coupled plasma-mass spectrometry was used to analyze the release of silver, which was shown to be stably bonded to the CTL coating and to be slowly released over time. The CTL coating improved MG63 osteoblast adhesion and proliferation on membranes. On the other hand, the presence of silver nanoparticles discouraged biofilm formation by Pseudomonas aeruginosa and Staphylococcus aureus without being cytotoxic. Overall, the stability and the biological and antibacterial properties make these membranes a valid and versatile material for applications in guided tissue regeneration and in other biomedical fields like wound healing.

Proline-rich antimicrobial peptides (PrAMPs) are promising lead compounds for developing new antimicrobials; however, their narrow spectrum of action is limiting. PrAMPs kill bacteria binding to their ribosomes and inhibiting protein synthesis. In this study, 133 derivatives of the PrAMP Bac7(1-16) were synthesized to identify the crucial residues for ribosome inactivation and antimicrobial activity. Then, five new Bac7(1-16) derivatives were conceived and characterized by antibacterial and membrane permeabilization assays, X-ray crystallography, and molecular dynamics simulations. Some derivatives displayed broad spectrum activity, encompassing Escherichia coli , Klebsiella pneumoniae , Acinetobacter baumanii , Pseudomonas aeruginosa , and Staphylococcus aureus . Two peptides out of five acquired a weak membrane-perturbing activity while maintaining the ability to inhibit protein synthesis. These derivatives became independent of the SbmA transporter, commonly used by native PrAMPs, suggesting that they obtained a novel route to enter bacterial cells. PrAMP-derived compounds could become new-generation antimicrobials to combat antibiotic-resistant pathogens.