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Persister cells are growth-arrested subpopulations that can survive possible fatal environments and revert to wild types after stress removal. Clinically, persistent pathogens play a key role in antibiotic therapy failure, as well as chronic, recurrent, and antibiotic-resilient infections. In general, molecular and physiological research on persister cells formation and compounds against persister cells are much desired. In this study, we firstly demonstrated that the spore forming Gram-positive model organism Bacillus subtilis can be used to generate persister cells during exposure to antimicrobial compounds. Interestingly, instead of exhibiting a unified antibiotic tolerance profile, different number of persister cells and spores were quantified in various stress conditions. qPCR results also indicated that differential stress responses are related to persister formation in various environmental conditions. We propose, for the first time to the best of our knowledge, an effective method to isolate B. subtilis persister cells from a population using fluorescence-activated cell sorting (FACS), which makes analyzing persister populations feasible. Finally, we show that alpha-helical cationic antimicrobial peptides SAAP-148 and TC-19, derived from human cathelicidin LL-37 and human thrombocidin-1, respectively, have high efficiency against both B. subtilis vegetative cells and persisters, causing membrane permeability and fluidity alteration. In addition, we confirm that in contrast to persister cells, dormant B. subtilis spores are not susceptible to the antimicrobial peptides.

Antimicrobial peptides are considered promising candidates for the development of novel antimicrobial agents to combat infections by multi-drug-resistant (MDR) bacteria. Here, we describe the identification and characterization of the synthetic peptide TC19, derived from the human thrombocidin-1-derived peptide L3. Biophysical experiments into the interaction between TC19 and mimics of human and bacterial plasma membranes demonstrated that the peptide is highly selective for bacterial membranes. In agreement, TC19 combined low cytotoxicity towards human fibroblasts with efficient and rapid killing in human plasma of MDR strains of several bacterial species of the ESKAPE panel. In addition, TC19 induced minor resistance in vitro, neutralized pro-inflammatory activity of bacterial cell envelope components while displaying slight chemotactic activity for human neutrophils. Importantly, topical application of TC19-containing hypromellose gel significantly reduced numbers of viable methicillin-resistant Staphylococcus aureus (MRSA) and MDR Acinetobacter baumannii in a superficial wound infection in mice. Together, TC19 is an attractive candidate for further development as a novel agent against (MDR) bacterial skin wound infections., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020. Published by Elsevier B.V.)

Antibiotic resistance and biofilm formation are the main reasons for failure in treatment of bacterial infections. This study aimed to identify synergistic combinations of conventional antibiotics and novel synthetic antimicrobial and antibiofilm peptides (SAAPs) inspired by the structures of the natural human cationic peptides LL-37 and thrombocidin-1 (TC-1). The LL-37-inspired lead peptide SAAP-148 was combined with antibiotics of different classes against Staphylococcus aureus, and showed synergy with teicoplanin. Synergy with teicoplanin was also observed with LL-37, the LL-37-inspired SAAP-276 and the TC-1-inspired TC84. Interestingly, no synergy was observed against Staphylococcus epidermidis. Furthermore, teicoplanin combined with SAAP-148 or SAAP-276 showed strong interaction against S. aureus biofilms. The dltABCD operon and the mprF gene in S. aureus conferred resistance to LL-37, but SAAP-148 proved to be indifferently potent against wild-type, ΔdltA and ΔmprF S. aureus strains. When used alone, relatively high concentrations of both LL-37 and teicoplanin (30-120 µM and 4-32 mg/L, respectively) were required to kill S. aureus. Resistance to LL-37 in S. aureus was overcome by combined use of teicoplanin and LL-37. Thus, teicoplanin potentiates peptide LL-37, enhancing the efficacy of the innate defence, and combining the novel peptides with teicoplanin offers potential for enhanced efficacy of treatment of S. aureus infections, including biofilms., (Copyright © 2018 Elsevier B.V. and International Society of Chemotherapy. All rights reserved.)

Background: Amphipathic cationic antimicrobial peptides (AMPs) TC19 and TC84, derived from the major AMPs of human blood platelets, thrombocidins, and Bactericidal Peptide 2 (BP2), a synthetic designer peptide showed to perturb the membrane of Bacillus subtilis. We aimed to determine the means by which the three AMPs cause membrane perturbation in vivo using B. subtilis and to evaluate whether the membrane alterations are dependent on the phospholipid composition of the membrane., Methods: Physiological analysis was employed using Alexa Fluor 488 labelled TC84, various fluorescence dyes, fluorescent microscopy techniques and structured illumination microscopy., Results: TC19, TC84 and BP2 created extensive fluidity domains in the membrane that are permeable, thus facilitating the entering of the peptides and the leakage of the cytosol. The direct interaction of the peptides with the bilayer create the fluid domains. The changes caused in the packing of the phospholipids lead to the delocalization of membrane bound proteins, thus contributing to the cell's destruction. The changes made to the membrane appeared to be not dependent on the composition of the phospholipid bilayer., Conclusions: The distortion caused to the fluidity of the membrane by the AMPs is sufficient to facilitate the entering of the peptides and leakage of the cytosol., General Significance: Here we show in vivo that cationic AMPs cause "membrane leaks" at the site of membrane insertion by altering the organization and fluidity of the membrane. Our findings thus contribute to the understanding of the membrane perturbation characteristic of cationic AMPs., (Copyright © 2018 The Author(s). Published by Elsevier B.V. All rights reserved.)

Background: Three amphipathic cationic antimicrobial peptides (AMPs) were characterized by determining their effect on Gram-positive bacteria using Bacillus subtilis strain 168 as a model organism. These peptides were TC19 and TC84, derivatives of thrombocidin-1 (TC-1), the major AMPs of human blood platelets, and Bactericidal Peptide 2 (BP2), a synthetic designer peptide based on human bactericidal permeability increasing protein (BPI)., Methods: To elucidate the possible mode of action of the AMPs we performed a transcriptomic analysis using microarrays. Physiological analyses were performed using transmission electron microscopy (TEM), fluorescence microscopy and various B. subtilis mutants that produce essential membrane bound proteins fused to green fluorescent protein (GFP)., Results: The transcriptome analysis showed that the AMPs induced a cell envelope stress response (cell membrane and cell wall). The cell membrane stress response was confirmed with the physiological observations that TC19, TC84 and BP2 perturb the membrane of B. subtilis. Using B. subtilis mutants, we established that the cell wall stress response is due to the delocalization of essential membrane bound proteins involved in cell wall synthesis. Other essential membrane proteins, involved in cell membrane synthesis and metabolism, were also delocalized due to alterations caused by the AMPs., Conclusions: We showed that peptides TC19, TC84 and BP2 perturb the membrane causing essential proteins to delocalize, thus preventing the possible repair of the cell envelope after the initial interference with the membrane., General Significance: These AMPs show potential for eventual clinical application against Gram-positive bacterial cells and merit further application-oriented investigation., (Copyright © 2018 The Author(s). Published by Elsevier B.V. All rights reserved.)

Development of novel antimicrobial agents is a top priority in the fight against multidrug-resistant (MDR) and persistent bacteria. We developed a panel of synthetic antimicrobial and antibiofilm peptides (SAAPs) with enhanced antimicrobial activities compared to the parent peptide, human antimicrobial peptide LL-37. Our lead peptide SAAP-148 was more efficient in killing bacteria under physiological conditions in vitro than many known preclinical- and clinical-phase antimicrobial peptides. SAAP-148 killed MDR pathogens without inducing resistance, prevented biofilm formation, and eliminated established biofilms and persister cells. A single 4-hour treatment with hypromellose ointment containing SAAP-148 completely eradicated acute and established, biofilm-associated infections with methicillin-resistant Staphylococcus aureus and MDR Acinetobacter baumannii from wounded ex vivo human skin and murine skin in vivo. Together, these data demonstrate that SAAP-148 is a promising drug candidate in the battle against antibiotic-resistant bacteria that pose a great threat to human health., (Copyright © 2018 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.)