Antimicrobial Peptide Cec4 Eradicates Multidrug-Resistant Acinetobacter baumannii in vitro and in vivo.

Introduction: Acinetobacter baumannii has become a major difficulty in the treatment of bacteria-associated infection. The previously reported antimicrobial peptide Cec4 exhibited good and stable activity against A. baumannii in vitro, but the mechanisms and effects in vivo are elusive. Methods: The effects of Cec4 on bacterial membrane permeability, membrane potential and bacterial reactive oxygen species were measured. The cell membrane localization of antimicrobial peptides was studied by fluorescence labelling. The ability of bacteria to develop resistance to antimicrobial peptides was studied by continuous induction, and transcriptome difference was analysed. The in vivo toxicity of Cec4 against nematodes and mice was studied, and the in vivo therapeutic potential of Cec4 against A. baumannii was assessed. Results: Cec4 effectively cleared multidrug-resistant A. baumannii by altering bacterial cell membrane permeability, changing bacterial cell membrane polarity, and increasing bacterial intracellular reactive oxygen species. Cec4 affected the expression of the secretion system, outer membrane, and efflux pump genes of A. baumannii. In addition, the bacteria did not acquire stable drug-resistant ability. Cec4 at 1.024 mg/mL did not affect the proliferation of HeLa and HepG2 cells, and Cec4 at 45 mg/kg had little effect on the mortality of Caenorhabditis elegans, even the liver and kidney tissues of mouse. Most importantly, Cec4 could effectively improve the survival rates and reduce the bacterial load of various tissues in the mouse model of infection. Conclusion: In conclusion, Cec4 can damage the cell membrane of bacteria, and the bacteria is not easy to produce resistance to Cec4. Besides, Cec4 has good potential for the treatment of multidrug-resistant A. baumannii infections.

Cec4-Derived Peptide Inhibits Planktonic and Biofilm-Associated Methicillin Resistant Staphylococcus epidermidis.

Staphylococcus epidermidis is part of the normal microbiota that colonizes the skin and mucosal surfaces of human beings. Previous studies suggested that S. epidermidis possessed low virulence, but recent studies confirmed that it can acquire high virulence from Staphylococcus aureus and with the increasing detection of methicillin-resistant S. epidermidis. It has become a major pathogen of graft-associated and hospital-acquired infections. In previous studies, we modified the antimicrobial peptide Cec4 (41 amino acids) and obtained the derived peptide C9 (16 amino acids) showing better antimicrobial activity against S. epidermidis with an MIC value of 8 µg/mL. The peptide has rapid bactericidal activity without detectable high-level resistance, showing certain inhibition and eradication ability on S. epidermidis biofilms. The damage of cell membrane structures by C9 was observed by scanning emission microscopy (SEM) and transmission electron microscopy (TEM). In addition, C9 altered the S. epidermidis cell membrane permeability, depolarization levels, fluidity, and reactive oxygen species (ROS) accumulation and possessed the ability to bind genomic DNA. Analysis of the transcriptional profiles of C9-treated cells revealed changes in genes involved in cell wall and ribosome biosynthesis, membrane protein transport, oxidative stress, and DNA transcription regulation. At the same time, the median lethal dose of C9 in mice was more than 128 mg/kg, and the intraperitoneal administration of 64 mg/kg was less toxic to the liver and kidneys of mice. Furthermore, C9 also showed a certain therapeutic effect on the mouse bacteremia model. In conclusion, C9 may be a candidate drug against S. epidermidis, which has the potential to be further developed as an antibacterial therapeutic agent. IMPORTANCE S. epidermidis is one of the most important pathogens of graft-related infection and hospital-acquired infection. The growing problem of antibiotic resistance, as well as the emergence of bacterial pathogenicity, highlights the need for antimicrobials with new modes of action. Antimicrobial peptides have been extensively studied over the past 30 years as ideal alternatives to antibiotics, and we report here that the derived peptide C9 is characterized by rapid bactericidal and antibiofilm activity, avoiding the development of resistance by acting on multiple nonspecific targets of the cell membrane or cell components. In addition, it has therapeutic potential against S. epidermidis infection in vivo. This study provides a rationale for the further development and application of C9 as an effective candidate antibiotic.

Antimicrobial Peptide Cec4 Eradicates the Bacteria of Clinical Carbapenem-Resistant Acinetobacter baumannii Biofilm.

The drug resistance rate of Acinetobacter baumannii increases year on year, and the drugs available for the treatment of carbapenem-resistant A. baumannii (CRAB) infection are extremely limited. A. baumannii, which forms biofilms, protects itself by secreting substrates such as exopolysaccharides, allowing it to survive under adverse conditions and increasing drug resistance. Antimicrobial peptides are small molecular peptides with broad-spectrum antibacterial activity and immunomodulatory function. Previous studies have shown that the antimicrobial peptide Cec4 has a strong effect on A. baumannii, but the antibacterial and biofilm inhibition of this antimicrobial peptide on clinical carbapenem resistance A. baumannii is not thoroughly understood. In this study, it was indicated that most of the 200 strains of CRAB were susceptible to Cec4 with a MIC of 4 µg/ml. Cec4 has a strong inhibitory and eradication effect on the CRAB biofilm; the minimum biofilm inhibition concentration (MBIC) was 64-128 µg/ml, and the minimum biofilm eradication concentration (MBEC) was 256-512 µg/ml. It was observed that Cec4 disrupted the structure of the biofilm using scanning electron microscopy (SEM) and confocal laser scanning microscopy (CLSM). A comparative transcriptome analysis of the effects of the antimicrobial peptide Cec4 on CRAB biofilm, identified 185 differentially expressed genes, including membrane proteins, bacterial resistance genes, and pilus-related genes. The results show that multiple metabolic pathways, two-component regulation systems, quorum sensing, and antibiotic synthesis-related pathways in A. baumannii biofilms were affected after Cec4 treatment. In conclusion, Cec4 may represent a new choice for the prevention and treatment of clinical infections, and may also provide a theoretical basis for the development of antimicrobial peptide drugs.