Martinez Melina M B, Corleto Merlina, Weschenfeller Melanie, Urrea Montes Santiago, Salomón Camila N, Gonzalez Natalia, Garavaglia Matías, Faccone Diego, Maffía Paulo C
Laboratorio de Aplicaciones Biotecnológicas y Microbiología (LAByM), Secretaría de Investigación, Universidad Nacional de Hurlingham (UNAHUR), Hurlingham 1688, Buenos Aires, Argentina.
Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET), Ciudad Autónoma de Buenos Aires 1425, Argentina.
Biomolecules. 2025 Feb 27;15(3):339. doi: 10.3390/biom15030339.
The antimicrobial peptide P6.2 was previously de novo designed as an alpha helix cationic amphipathic molecule. In previous work, we have shown that this peptide displayed significant antimicrobial activity against both Gram-positive () and Gram-negative () bacteria. However, while P6.2 lacked biofilm-inhibiting properties against the strain PA01, it displayed anti-inflammatory effects in a murine acute lung infection model challenged with this pathogen. In this work, the peptide P6.2 antimicrobial activity and its possible synergy with meropenem were evaluated both in vitro and in vivo using a infection model against a carbapenem-resistant KPC-producing clinical isolate of . Firstly, the cytotoxic effect of the peptide on A549 and RAW264.7 cell lines was assayed, showing no cytotoxicity at 64 µg/mL and below. Then, the MIC (minimal inhibitory concentration) and bactericidal effect against the carbapenemase-producing strain M13513 strain were determined. P6.2 showed a MIC between 32 and 64 µg/mL, and a rapid bactericidal activity against this strain (less than 45 min). The peptide stability at different temperatures and in bovine serum at 37 °C was also analyzed, showing good stability and almost no degradation after 15 min of incubation at 100 °C or 24 h at 37 °C in serum, respectively. The antibiofilm activity was also evaluated, and although the peptide did not show biofilm inhibitory activity, it did demonstrate biofilm disruptive activity, together with bactericidal activity inside the pre-formed biofilm. The possible synergistic effect with the carbapenem meropenem was then analyzed in vitro by killing kinetics, revealing a synergistic interaction between P6.2 and the antibiotic against this strain. Finally, P6.2 was evaluated in vivo in the larvae infection model. Interestingly, in P6.2 alone did not completely clear the infection caused by M13513. However, when combined with meropenem, P6.2 demonstrated a synergistic effect, leading to increased survival rates in infected larvae. The results presented here highlight the potential that this peptide displays when used in combination with carbapenems against a clinically relevant KPC-producing .
抗菌肽P6.2先前被从头设计为一种α螺旋阳离子两亲性分子。在先前的工作中，我们已经表明，这种肽对革兰氏阳性菌（）和革兰氏阴性菌（）均表现出显著的抗菌活性。然而，虽然P6.2对PA01菌株缺乏生物膜抑制特性，但它在受到该病原体攻击的小鼠急性肺部感染模型中显示出抗炎作用。在这项工作中，使用针对产碳青霉烯酶KPC的临床分离株的感染模型，在体外和体内评估了肽P6.2的抗菌活性及其与美罗培南可能的协同作用。首先，测定了该肽对A549和RAW264.7细胞系的细胞毒性作用，结果表明在64μg/mL及以下浓度时无细胞毒性。然后，测定了对产碳青霉烯酶菌株M13513的MIC（最低抑菌浓度）和杀菌效果。P6.2的MIC在32至64μg/mL之间，并且对该菌株具有快速杀菌活性（少于45分钟）。还分析了该肽在不同温度下以及在37℃牛血清中的稳定性，结果表明分别在100℃孵育15分钟或在37℃血清中孵育24小时后具有良好的稳定性且几乎没有降解。还评估了其抗生物膜活性，尽管该肽未显示出生物膜抑制活性，但它确实表现出生物膜破坏活性，以及在预先形成的生物膜内的杀菌活性。然后通过杀菌动力学在体外分析了与碳青霉烯类美罗培南可能的协同作用，结果表明P6.2与该抗生素对该菌株存在协同相互作用。最后，在幼虫感染模型中对P6.2进行了体内评估。有趣的是，单独使用P6.2时不能完全清除由M13513引起的感染。然而，当与美罗培南联合使用时，P6.2表现出协同作用，导致感染幼虫的存活率提高。此处呈现的结果突出了该肽与碳青霉烯类联合使用时针对临床上相关的产KPC菌株所显示出的潜力。
