The increasing prevalence of antimicrobial-resistant (AMR) bacteria along with the limited development of antimicrobials warrant investigating novel antimicrobial modalities. Emerging inorganic engineered nanomaterials (ENMs), most notably silver nanoparticles (AgNPs), have demonstrated superior antimicrobial properties. However, AgNPs, particularly those of small size, could exert overt toxicity to mammalian cells. This study investigated whether combining AgNPs and conventional antimicrobials would produce a synergistic response and determined the optimal and safe minimum inhibitory concentration (MIC) range against several wild-type Gram-positive and -negative strains and three different clinical isolates of AMR Klebsiella pneumoniae. Furthermore, the cytotoxicity of the synergistic combinations was assessed in a human hepatocyte model. The results showed that the AgNPs (15–25 nm) were effective against Gram-negative bacteria (MIC of 16–128 µg/mL) but not Gram-positive strains (MIC of 256 µg/mL). Both wild-type and AMR K. pneumoniae had similar MIC values following exposure to AgNPs. Importantly, co-exposure to combinations of AgNPs and antimicrobial agents, including kanamycin, colistin, rifampicin, and vancomycin, displayed synergy against both wild-type and AMR K. pneumoniae isolates (except for vancomycin against AMR strain I). Notably, the tested combinations demonstrated no to minimal toxicity against hepatocytes. Altogether, this study indicates the potential of combining AgNPs with conventional antimicrobials to overcome AMR bacteria.

Antibiotic resistance, mainly caused by methicillin-resistant Staphylococcus aureus (MRSA), is a globally growing concern and a significant health issue in regard to infectious diseases. This issue will require a novel and comprehensive strategy to overcome the resistance to MRSA. Therefore, the current study advances TMPK-targeted drug discovery by screening a vast chemical space using virtual screening with detailed dynamics and binding energy evaluations. In contrast to the previous efforts, our study focuses on the pharmacological exploitation of TMPK's conserved and druggable active site in MRSA, which lacks homologs in humans and remains understated in antimicrobial pipelines. We screened traditional Chinese medicine and African natural compound databases, as well as the InterBioScreen, dark chemical matter, and Maybridge databases, using a three-step virtual screening strategy. From these databases, we identified 16 compounds as the most potent based on their docking and redocking scores and interaction profiles with the key residues. Of these, the top eight hits were re-validated using the induced-fit docking method, which confirmed the validity of these hits' interaction and inhibitory potential against TMPK. Thereafter, using molecular simulations, 500 ns each, we determined that these compounds exhibited stable dynamic profiles, minimal residual fluctuation, and well-folded conformations during the simulation. Furthermore, using the binding free energy calculations, including both the MMGBSA and MMPBSA methods, the top hits were ranked based on the strongest binding energy, which provides compelling evidence for the stability, specificity, and high binding affinity of these compounds towards TMPK. PCA (Principal Component Analysis) and FEL (Free Energy Landscape) analyses revealed the conformational behavior of these complexes. Furthermore, ADMET analysis confirmed the safety of these molecules. This study identified potential therapeutic hits using computational and theoretical approaches; however, experimental validation through in vitro enzymatic assays and MRSA culture-based inhibition studies is essential to confirm their therapeutic potencies.

This standard operating procedure (SOP) describes the standardized laboratory procedure for performing the MIC experiment.