Cabassi Clotilde Silvia, Sala Andrea, Santospirito Davide, Alborali Giovanni Loris, Carretto Edoardo, Ghibaudo Giovanni, Taddei Simone
Department of Veterinary Science, University of Parma, Via del Taglio 10, 43126, Parma, Italy.
Istituto Zooprofilattico Sperimentale della Lombardia e dell'Emilia Romagna, Via Bianchi 7/9, 25124, Brescia, Italy.
Ann Clin Microbiol Antimicrob. 2017 Mar 23;16(1):17. doi: 10.1186/s12941-017-0193-1.
Antimicrobial resistance is a growing threat to public health. Pseudomonas aeruginosa is a relevant pathogen causing human and animal infections, frequently displaying high levels of resistance to commonly used antimicrobials. The increasing difficulty to develop new effective antibiotics have discouraged investment in this area and only a few new antibiotics are currently under development. An approach to overcome antibiotic resistance could be based on antimicrobial peptides since they offer advantages over currently used microbicides.
The antimicrobial activity of the synthetic peptide AMP2041 was evaluated against 49 P. aeruginosa clinical strains with high levels of antimicrobial resistance, isolated from humans (n = 19) and animals (n = 30). In vitro activity was evaluated by a microdilution assay for lethal dose 90% (LD), while the activity over time was performed by time-kill assay with 12.5 µg/ml of AMP2014. Evidences for a direct membrane damage were investigated on P. aeruginosa ATCC 27853 reference strain, on animal isolate PA-VET 38 and on human isolate PA-H 24 by propidium iodide and on P. aeruginosa ATCC 27853 by scanning electron microscopy.
AMP2041 showed a dose-dependent activity, with a mean (SEM) LD of 1.69 and 3.3 µg/ml for animal and human strains, respectively. AMP2041 showed microbicidal activity on P. aeruginosa isolates from a patient with cystic fibrosis (CF) and resistance increased from first infection isolate (LD = 0.3 μg/ml) to the mucoid phenotype (LD = 10.4 μg/ml). The time-kill assay showed a time-dependent bactericidal effect of AMP2041 and LD was reached within 20 min for all the strains. The stain-dead assay showed an increasing of membrane permeabilization and SEM analysis revealed holes, dents and bursts throughout bacterial cell wall after 30 min of incubation with AMP2041.
The obtained results assessed for the first time the good antimicrobial activity of AMP2041 on P. aeruginosa strains of human origin, including those deriving from a CF patient. We confirmed the excellent antimicrobial activity of AMP2041 on P. aeruginosa strains derived from dog otitis. We also assessed that AMP2041 antimicrobial activity is linked to changes of the P. aeruginosa cell wall morphology and to the increasing of membrane permeability.
抗生素耐药性对公众健康构成日益严重的威胁。铜绿假单胞菌是一种引起人类和动物感染的重要病原体，对常用抗菌药物常常表现出高度耐药性。开发新型有效抗生素的难度不断增加，这使得该领域的投资减少，目前仅有少数新型抗生素正在研发中。克服抗生素耐药性的一种方法可能基于抗菌肽，因为它们相对于目前使用的杀菌剂具有优势。
评估了合成肽AMP2041对49株高度耐药的铜绿假单胞菌临床菌株的抗菌活性，这些菌株分别从人类（n = 19）和动物（n = 30）中分离得到。通过微量稀释法测定90%致死剂量（LD）来评估体外活性，同时用12.5μg/ml的AMP2014通过时间杀菌试验来测定随时间的活性。通过碘化丙啶对铜绿假单胞菌ATCC 27853参考菌株、动物分离株PA-VET 38和人类分离株PA-H 24研究直接膜损伤的证据，并通过扫描电子显微镜对铜绿假单胞菌ATCC 27853进行研究。
AMP2041表现出剂量依赖性活性，动物菌株和人类菌株的平均（标准误）LD分别为1.69μg/ml和3.3μg/ml。AMP2041对一名囊性纤维化（CF）患者分离出的铜绿假单胞菌菌株具有杀菌活性，耐药性从首次感染分离株（LD = 0.3μg/ml）增加到黏液样表型（LD = 10.4μg/ml）。时间杀菌试验显示AMP2041具有时间依赖性杀菌作用，所有菌株在20分钟内达到LD。染色死亡试验显示膜通透性增加，扫描电子显微镜分析显示与AMP2041孵育30分钟后细菌细胞壁出现孔洞、凹陷和破裂。
所获得的结果首次评估了AMP2041对源自人类的铜绿假单胞菌菌株，包括源自CF患者的菌株具有良好的抗菌活性。我们证实了AMP2041对源自犬中耳炎的铜绿假单胞菌菌株具有优异的抗菌活性。我们还评估了AMP2041的抗菌活性与铜绿假单胞菌细胞壁形态的变化以及膜通透性的增加有关。
