The development of phages for therapy has been hampered by concerns over the potential for immune response, rapid toxin release

by the lytic action of phages, and difficulty of dose determination in clinical situations [5]. Phages multiply logarithmically in infected bacterial cells, and the release of progeny phage occurs by lysis of the infected cell at the end of the infection cycle, which involves the holin-endolysin system [6, 7]. Holins create a lesion in the cytoplasmic membrane through which endolysins gain access to the murein layer Histone Methyltransferase inhibitor [7]. Endolysins are peptidoglycan hydrolases that degrade the bacterial cell wall, leading to cell lysis and release of progeny phages [8]. An undesirable side effect of this phenomenon from a therapeutic perspective is the development of immunogenic reactions due to large uncontrolled amounts of phages in circulation [9]. Such concerns must be addressed before phage therapy can be widely accepted [5, 10]. This work features engineered bacteriophages that are incapable of lysing bacterial cells because they lack endolysin enzymatic activity. We previously produced, as a model, a recombinant lysis-deficient version of T4 bacteriophage that infects Escherichia coli [11, 12]. Phages have also been engineered to be non- replicating or to possess additional desirable

properties [13–15]. In an experimental E. coli infection model, the improved survival rate of rats treated Staurosporine in vitro with lysis-deficient T4LyD phage was attributed to lower endotoxin release [16]. We wished to generate an endolysin-deficient phage against a gram-positive bacterium, and chose S. aureus

because of Urocanase its clinical relevance. S. aureus is a major pathogen responsible for a variety of diseases ranging from minor skin infections to life-threatening conditions such as sepsis. This pathogen is often resistant to all β-lactam antibiotics; vancomycin-resistant strains may become untreatable [17–19]. This organism is the most common cause of nosocomial infections, and nasal carriage is implicated as a risk factor [20]. In the United States alone, invasive methicillin-resistant S. aureus (MRSA) infections occur in approximately 94,000 people each year, causing nearly 19,000 deaths [21]. Understandably, the progressive multidrug resistance of bacteria has motivated the re-evaluation of phages as therapy for diverse bacterial infections [22]. We report here that the recombinant endolysin-deficient S. aureus phage P954 kills cells without causing cell lysis and forms plaques on a host that expresses a plasmid-encoded heterologous endolysin, enabling its large-scale production. The recombinant phage P954 was evaluated for in vivo efficacy in an experimental mouse model and found to protect mice from fatal S. aureus infection.