Klebsiella pneumoniae (K. pneumoniae) exhibits the ability to form biofilms as a means of adapting to its adverse surroundings. K. pneumoniae in this biofilm state demonstrates remarkable resistance, evades immune system attacks, and poses challenges for complete eradication, thereby complicating clinical anti-infection efforts. Moreover, the precise mechanisms governing biofilm formation and disruption remain elusive. Recent studies have discovered that fingolimod (FLD) exhibits biofilm properties against Gram-positive bacteria. Therefore, the antibiofilm properties of FLD were evaluated against multidrug-resistant (MDR) K. pneumoniae in this study. The antibiofilm activity of FLD against K. pneumoniae was assessed utilizing the Alamar Blue assay along with confocal laser scanning microscopy (CLSM), scanning electron microscopy (SEM), and crystal violet (CV) staining. The results showed that FLD effectively reduced biofilm formation, exopolysaccharide (EPS), motility, and bacterial abundance within K. pneumoniae biofilms without impeding its growth and metabolic activity. Furthermore, the inhibitory impact of FLD on the production of autoinducer-2 (AI-2) signaling molecules was identified, thereby demonstrating its notable anti-quorum sensing (QS) properties. The results of qRT-PCR analysis demonstrated that FLD significantly decreased the expression of genes associated with the efflux pump gene (AcrB, kexD, ketM, kdeA, and kpnE), outer membrane (OM) porin proteins (OmpK35, OmpK36), the quorum-sensing (QS) system (luxS), lipopolysaccharide (LPS) production (wzm), and EPS production (pgaA). Simultaneously, FLD exhibited evident antibacterial synergism, leading to an increased survival rate of G. mellonella infected with MDR K. pneumoniae. These findings suggested that FLD has substantial antibiofilm properties and synergistic antibacterial potential for colistin in treating K. pneumoniae infections.

The objectives of the study were to design, synthesize, and evaluate the antibacterial activity of a series of novel aminoguanidine-indole derivatives. Thirty-seven new compounds were effectively synthesized through nucleophilic substitution reaction and guanidinylation reaction. Chemical structures of all the desired compounds were identified by NMR and HR-MS spectroscopy. Most of the synthesized compounds showed significant antibacterial activity against ESKAPE pathogens and clinical resistant Klebsiella pneumoniae (K. pneumoniae) isolates. K. pneumoniae is an important opportunistic pathogen that often threatens the health of immunocompromised people such as the elderly, children, and ICU patients. The most active compound 4P showed rapid bactericidal activity against resistant K. pneumoniae 2108 with MIC and MBC values that were 4 and 8 µg/mL, respectively. The hemolytic activity of 4P was low, with an HC₅₀ value of 123.6 µg/mL. Compound 4P induced the depolarization of the bacterial membrane and disrupted bacterial membrane integrity and was not prone to antibiotic resistance. The dihydrofolate reductase (DHFR) activity was also notably inhibited by 4P in vitro. Molecular docking revealed that the aminoguanidine moiety and indole structure of 4P played an important role in binding to the target site of the K. pneumoniae dihydrofolate reductase (DHFR) receptor. In the mouse pneumonia model caused by K. pneumoniae, 4P improved the survival rate of mice, reduced bacterial loads, and alleviated tissues’ pathological injuries at a dosage of 4 mg/kg. Therefore, compound 4P may be a promising lead compound or drug candidate for antibacterial purposes against K. pneumoniae.