• hLF1-11
• rifampicin
• antimicrobial peptide
• Klebsiella pneumoniae

The worldwide spread of carbapenem-resistant Klebsiella pneumoniae strains has become a major source of concern for public health.
In light of the need to develop new drugs, cationic antimicrobial peptides have gained attention as possible therapeutic candidates. The antibacterial activity of the antimicrobial synthetic peptide representing the first cationic domain of human lactoferricin, further referred to as hLF1-11, was evaluated against sensitive and resistant K. pneumoniae strains. In addition, the antibacterial activity of hLF1-11 in combination with several antimicrobial drugs (rifampicin, clarithromycin, clindamycin, gentamycin and tigecycline) was evaluated against clinical isolates of carbapenemase-producing K. pneumoniae harboring different resistance genes. The results revealed that the hLF1-11 peptide exhibited antimicrobial activity in a dose dependent manner. This antibacterial activity was significantly more effective against a sensitive than against a resistant K. pneumoniae strain. In addition, the results revealed a synergistic effect between hLF1-11 and all the tested antimicrobial drugs against several multidrug (MDR) KPC K. pneumoniae strains.
Another important finding pertains to the mechanism of action of the synergistic effect induced by hLF1-11 and rifampicin. When these cells were exposed to rifampicin for 30 or 60 min, washed and then treated with a sub-inhibitory concentration of hLF1-11 up to 2 h, were significantly (P < 0.05) killed than untreated cells, while no antibacterial activity was observed when they were first incubated with hLF1-11 and then with rifampicin. CCCP, which is an uncoupler of oxidative phosphorylation, was used to dissipate the proton motive force (PMF). The effect of CCCP on the antibacterial activity of these compounds revealed that the antibacterial activity of hLF1-11 as well as the synergistic effect between hLF1-11 and rifampicin were significantly reduced by CCCP, while no effect was obtained on the antibacterial activity of rifampicin. In agreement, EtBr accumulation increased upon exposure to hLF1-11 dose dependently as well as upon exposure to the combination of hLF1-11 and rifampicin, while no increase in fluorescence intensity was induced by treatment with rifampicin. Moreover, hLF1-11 was able to induce cytoplasmic membrane depolarization, as measured by the anionic dye DiBAC4(3), which enters the depolarized cells. All together, these data suggest that the rifampicin molecules accumulated by Klebsiella pneumoniae during the 30 min preincubation were not taken away by washing. The subsequent addition of hLF1-11, by inducing a transient loss of membrane potential, could have facilitated the entrance and retention of rifampicin into the cytoplasm. In agreement, both cell membrane permeabilization, as well as membrane depolarization, increased upon exposure to hLF1-11, thus supporting our hypothesis.
In conclusion, hLF1-11 induces sensitization of KPC K. pneumoniae to rifampicin, an otherwise impermeable hydrophobic antibiotic, by facilitating access of rifampicin to and subsequent retention of rifampicin in the cytoplasm.