Background Although traditional antibiotic therapy provided an effective approach to combat pathogenic bacteria, the long-term and widespread use of antibiotic results in the evolution of multidrug-resistant bacteria. Recent progress in nanotechnology offers an alternative opportunity to discover and develop novel antibacterial agents. Methods A total of 51 K. pneumoniae strains were collected from several specimens of hospitalized patients and identified by two parallel methods (biochemical tests and Vitek-2 system). The antibiotic sensitivity of isolates was evaluated by disk diffusion antibiogram and Vitek-2 system. The biofilms formation ability of antibiotic-resistant strains was examined by microtiter plate and tube methods based on crystal violet staining. The molecular technique was used to determine key genes responsible for biofilms formation of clinical isolates. The antibacterial and antibiofilm activities of Ag NPs, Ni NPs, Al2O3 NPs singly (NPs) and in combination (cNPs) were investigated against selected strains using standard methods. Moreover, the cytotoxicity of NPs was evaluated on mouse neural crest-derived (Neuro-2A) cell line. Results The results of bacterial studies revealed that more than 80% of the isolates were resistant to commonly used antibiotics and about 95% of them were able to form biofilms. Moreover, the presence of fimA and mrkA genes were determined in all biofilm-producing strains. The results of antibacterial and antibiofilm activities of NPs and cNPs demonstrated the lower MIC and MBEC values for Al2O3 NPs singly as well as for Ag/Ni cNPs and Ag/Al2O3 cNPs in combination, respectively. Overall, the inhibitory effects of cNPs were superior to NPs against all strains. Furthermore, the results of the checkboard assays showed that Ag NPs act synergistically with two other NPs against multidrug-resistant Klebsiella pneumonia (MDR-K. pneumoniae) isolates. The in vitro cytotoxicity assay revealed no significant toxicity of NPs against Neuro-2A cells. Conclusion In the present study, the combination of Ag NPs, Ni NPs, and Al2O3 NPs were used against MDR-K. pneumoniae strains and antibacterial and antibiofilm activities were observed for Ag/Ni cNPs and Ag/Al2O3 cNPs.

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