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Blood infections caused by gram-negative bacteria are one of the major challenges facing modern medicine, despite treatment with the available conventional antibiotics (22). Mortality rates in the range of 20 to 80% for septicemia caused by gram-negative bacteria have been reported. Antibiotic treatment is often administered when the disease reaches an advanced stage, usually when symptoms appear, by which point insufficient time remains for the antibiotic to kill the pathogen before the onset of irreversible tissue damage. Moreover, in many cases the antibiotic is given before sensitivity tests have been performed to identify an effective treatment. The emergence of bacterial strains resistant to conventional antibiotics, the lack of a rapid means of diagnosing the infection, and the generally unknown antibiotic sensitivity pattern of the infecting bacteria are probably among the major causes of inefficient therapy and high mortality rates (26). Most of these blood infections are caused by opportunistic pathogens that are not usually capable of initiating bacteremia in otherwise healthy individuals. Host defense mechanisms of the innate, nonclonal immune system serve as the principal pathway for effective elimination of pathogens (11). Potent components of innate immunity are the macrophages and polymorphonuclear leukocytes (PMNs) that mediate the early clearance of bacteria by the phagocytic process (1, 23). Agents that enhance phagocytosis may promote clearance of the pathogen and termination of the infectious process. In some cases the phagocytotic process is mediated by adhesins expressed on the bacterial surface, in a process termed nonopsonic phagocytosis (18). However, the interaction of many bacteria with phagocytic cells is greatly facilitated by opsonins, which act as a bridge between the surfaces of these two types of cells (5, 8). The development of agents that can function as opsonins may provide a useful new approach to terminating the infectious process by enhancing bacterial attachment to phagocytic cells, followed by ingestion and digestion of the pathogen. For an agent to function as an opsonin, it must contain a moiety that recognizes a specific target molecule on the bacterial surface and another that recognizes specific receptors on phagocytic cells. Moreover, its toxicity must be relatively low, and most importantly, there should be little likelihood for the emergence of strains resistant to its action. In the present study, we describe the synthesis of antimicrobial peptide conjugates that act both as opsonins to enhance destruction by phagocytic cells and as agents that permeabilize the bacterial membrane to enhance eradication by hydrophobic antibiotics and other antimicrobial agents. The peptides are derived from polymyxin B (PMB) or polymyxin E (PME) covalently linked to a short chemotactic peptide. Early studies have established that although polymyxin-based antibiotics are relatively toxic, they can be rendered 10 to 15 times less toxic by cleaving the lipid moiety from the molecule (7, 10). These polymyxin-based peptides lack direct bactericidal activity but retain their ability to bind to lipopolysaccharides (LPSs) on the bacterial surface and to permeabilize the outer membrane (OM) to hydrophobic antibiotics that cannot otherwise penetrate the bacteria, as well as to other bactericidal agents, such as the complement (33). Since resistance to the parent polymyxins is rare (25), it is expected that resistance to these polymyxin-derived peptides will be rare as well. Indeed, we did not find strains resistant to the permeabilizing activity of a polymyxin B (PMB) nonapeptide (PMBN) among 59 PMB-sensitive strains tested (21). Moreover, administration of the nonapeptide in combination with erythromycin protected mice against a lethal dose of an erythromycin-resistant strain of Klebsiella pneumoniae (19). More recently, we showed that the polymyxin B nonapeptide binds to LPS, which is consistent with previous studies showing that the parent PMB and PME molecules bind to lipid A of LPS on bacterial surfaces (16, 25, 31). Encouraged by these results, we reasoned that conjugation of PMBN or PMEN to a moiety that recognizes specific receptors on phagocytic cells may confer opsonic activity on the molecule. We chose to use formyl methionine-leucine-phenylalanine (fMLF), a chemotactic peptide, as our conjugant because it has been studied extensively for its property of binding to specific receptors on phagocytic cells (17). It will be shown that conjugates consisting of polymyxin peptides covalently linked to fMLF enhance the bactericidal activities of conventional antibiotics, act as opsonins in promoting the phagocytic destruction of bacteria in vitro, and protect mice from lethal infections and bacteremia in vivo.