Enhancing the activity of ß-lactamase inhibitory protein-II with cell-penetrating peptide against KPC-2-carrying Klebsiella pneumoniae.

Carbapenem-resistant Enterobacterales (CRE) is considered a paramount threat due to its rapid spread and high mortality rate. Klebsiella pneumoniae carbapenemases (KPCs), specifically KPC-2, are prevalent enzymes responsible for carbapenem resistance in many countries. While combinations of antibiotics are commonly used, they must be tailored to match the remaining susceptibility of the infecting strains. Therefore, there is a need to develop the ß-lactamase inhibitor to effectively address this issue. ß-lactamase inhibitor protein (BLIP) and its variants, BLIP-I and BLIP-II, have demonstrated the ability to inhibit class A ß-lactamases. In particular, BLIP-II shows strong binding to the KPC-2 carbapenemase, making it a potential candidate for inhibition. To improve the intracellular penetration of BLIP-II, a cell-penetrating peptide (CPP) was employed. In this study, a KRK-rich peptide was introduced at either the N-terminal or C-terminal region of tBLIP-II, excluding the signal sequence of the BLIP-II protein. tBLIP-II, tBLIP-II-CPP, and CPP-BLIP-II were successfully expressed, and the chimeric proteins retained inhibitory activity compared to tBLIP-II alone. It is apparent that homology modeling demonstrated neither the poly-histidine tag nor the CPP interfered with the essential interaction residues of tBLIP-II. Interestingly, BLIP-II-CPP exhibited the highest inhibitory activity, reducing the minimal inhibitory concentration (MIC) of meropenem by 8 folds. Moreover, the combination of tBLIP-CPP with meropenem significantly decreased the viable bacterial cell count compared to the combination of tBLIP-II with meropenem or meropenem alone. These findings suggest that tBLIP-CPP is a promising candidate for restoring carbapenem susceptibility against CRE and provides a valuable therapeutic option for infections caused by CRE.

Synergistic effect of two antimicrobial peptides, BP203 and MAP-0403 J-2 with conventional antibiotics against colistin-resistant Escherichia coli and Klebsiella pneumoniae clinical isolates.

Drug-resistant Enterobacterales infections are a great health concern due to the lack of effective treatments. Consequently, finding novel antimicrobials or combining therapies becomes a crucial approach in addressing this problem. BP203 and MAP-0403 J-2, novel antimicrobial peptides, have exhibited effectiveness against Gram-negative bacteria. In this study, we assessed the in vitro antibacterial activity of BP203 and MAP-0403 J-2, along with their synergistic interaction with conventional antibiotics including colistin, rifampicin, chloramphenicol, ceftazidime, meropenem, and ciprofloxacin against colistin-resistant Escherichia coli and Klebsiella pneumoniae clinical isolates. The minimal inhibitory concentrations (MIC) of BP203 and MAP-0403 J-2 against tested E. coli isolates were 2-16 and 8-32 µg/mL, respectively. However, for the majority of K. pneumoniae isolates, the MIC of BP203 and MAP-0403 J-2 were >128 µg/mL. Notably, our results demonstrated a synergistic effect when combining BP203 with rifampicin, meropenem, or chloramphenicol, primarily observed in most K. pneumoniae isolates. In contrast, no synergism was evident between BP203 and colistin, chloramphenicol, ceftazidime, rifampicin, or ciprofloxacin when tested against all E. coli isolates. Furthermore, synergistic effects between MAP-0403 J-2 and rifampicin, ceftazidime or colistin were observed against the majority of E. coli isolates. Similarly, the combined effect of MAP-0403 J-2 with rifampicin or chloramphenicol was synergistic in the majority of K. pneumoniae isolates. Importantly, these peptides displayed the stability at high temperatures, across a wide range of pH values, in specific serum concentrations and under physiological salt conditions. Both peptides also showed no significant hemolysis and cytotoxicity against mammalian cells. Our findings suggested that BP203 and MAP-0403 J-2 are promising candidates against colistin-resistant E. coli. Meanwhile, the synergism of these peptides and certain antibiotics could be of great therapeutic value as antimicrobial drugs against infections caused by colistin-resistant E. coli and K. pneumoniae.

Synergistic effect and antibiofilm activity of the antimicrobial peptide K11 with conventional antibiotics against multidrug-resistant and extensively drug-resistant Klebsiella pneumoniae.

Introduction: Infections caused by drug-resistant Klebsiella pneumoniae are now a serious problem for public health, associated with high morbidity and mortality due to limited treatment options. Therefore, new antibacterial agents or a combination of agents as the first line of treatment are urgently needed. K11 is a novel antimicrobial peptide (AMP) that has demonstrated in vitro antimicrobial activity against several types of bacteria. Additionally, K11 has previously shown no hemolytic activity. Herein, the antibacterial activity, the synergistic action of K11 in combination with different conventional antibiotics and the antibiofilm activity of K11 against multidrug-resistant (MDR) and extensively drug-resistant (XDR) K. pneumoniae were investigated. Meanwhile, the stability and ability to induce the bacterial resistance of K11 were also tested. Methods: Fifteen clinical isolates of MDR/XDR K. pneumoniae were used in this study. The minimum inhibitory concentration (MIC) of K11 against these isolates was determined by the broth microdilution method. In vitro synergy between K11 and antibiotics was evaluated using the checkerboard methodology. The antibiofilm activity of K11 against K. pneumoniae strong biofilm producers were explored by the crystal violet staining. The stability in different environments and resistance induction of K11 were evaluated by MIC determination. Results: The MIC values of K11 against MDR/XDR K. pneumoniae isolates were 8-512 µg/mL. Intriguingly, the synergistic effects were clearly observed for K11 in combination with chloramphenicol, meropenem, rifampicin, or ceftazidime, whereas no synergy was observed when K11 was combined with colistin. Besides, K11 effectively prevented biofilm formation against K. pneumoniae strong biofilm producers in a concentration-dependent manner starting at 0.25×MIC and exerted an enhancing effect when administered in combination with meropenem, chloramphenicol, or rifampicin. Additionally, K11 demonstrated high thermal and wide pH stability along with good stability in serum and physiological salts. Significantly, K. pneumoniae showed no induction of resistance even after prolonged exposure to a sub-inhibitory concentration of K11. Conclusion: These findings indicate that K11 is a promising candidate with potent antibacterial and antibiofilm activities without inducing resistance and acts synergistically with conventional antibiotics against drug-resistant K. pneumoniae.