We describe the nonnatural antimicrobial peptide KKIRVRLSA (M33) and its capacity to neutralize LPS-induced cytokine release, preventing septic shock in animals infected with bacterial species of clinical interest. M33 showed strong resistance to proteolytic degradation when synthesized in tetrabranched form with 4 peptides linked by a lysine core, making it suitable for use in vivo. HPLC and mass spectrometry demonstrated its stability in serum beyond 24 h. M33 was found to be very selective for gram-negative bacteria. Minimal inhibitory concentration (MIC) ranged from 0.3 to 3 muM for multidrug resistant clinical isolates of several pathogenic species, including Pseudomonas aeruginosa, Klebsiella pneumoniae, and Acinetobacter baumannii. M33 neutralized LPS derived from P. aeruginosa and K. pneumoniae, and prevented TNF-alpha release from LPS-activated macrophages, with an EC(50) of 3.8e-8 M and 2.8e-7 M, respectively, as detected by sandwich ELISA. M33 activity was also tested in sepsis animal models. It averted septic shock symptoms due to Escherichia coli and P. aeruginosa in doses compatible with clinical use (5-25 mg/kg). These properties make tetrabranched M33 peptide a good candidate for the development of a new antibacterial drug.-Pini, A., Falciani, C., Mantengoli, E., Bindi, S., Brunetti, J., Iozzi, S., Rossolini, G. M., Bracci, L. A novel tetrabranched antimicrobial peptide that neutralizes bacterial lipopolysaccharide and prevents septic shock in vivo.