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The rapidly evolving crisis of antibiotic resistance among microorganisms has contributed to the rise of patient morbidity and mortality from nosocomial and community-acquired infections. Therefore, innovative antimicrobial technology targeting mechanisms to which the bacteria are unlikely to evolve resistance is urgently needed. We have previously described a nitric oxide-releasing nanoparticle (NO-np) with efficacy against methicillin-resistant Staphylococcus aureus (MRSA) and Acinetobacter baumannii in vitro and in murine wound and abscess models. Although the prior findings suggest that the NO-np can be a useful therapeutic for skin and soft tissue infections, the antimicrobial spectrum of NO-np has yet to be fully elucidated. In the current study, we investigated the efficacy of a NO-np against several Grampositive and -negative clinical isolates. We found that the NO-np were uniformly active against all of the Streptococcus pyogenes, Enterococcus faecalis, Klebsiella pneumoniae, Escherichia coli and Pseudomonas aeruginosa clinical isolates examined, including strains that were both sensitive and resistant to commonly used antibiotics. We concluded that the NO-np have the potential to serve as a novel broad spectrum antimicrobial agent.