PH826噬菌体与抗生素联用有效抑制多重耐药铜绿假单胞菌生物被膜的形成

Combination of phage PH826 and antibiotics inhibits biofilm formation of multidrug-resistant Pseudomonas aeruginosa

摘要:

【目的】铜绿假单胞菌（Pseudomonas aeruginosa）是一种重要的革兰氏阴性病原体，可以加重囊性纤维化患者的肺部感染，最终会导致患者的死亡。然而由于多重耐药（multi-drug resistant，MDR）和泛耐药（pan-drug resistant，PDR）铜绿假单胞菌菌株的出现，使其防控变得更为严峻。【方法】从养殖场污水中分离能有效裂解多重耐药铜绿假单胞菌的噬菌体，研究其形态特征、生物学特性、宿主谱范围、基因组特征和体外抑菌能力等，并采用噬菌体和抗生素联用的方法进行生物被膜的抑制试验。【结果】透射电子显微镜的形态分析和基因组分析结合表明，该噬菌体属于Nankokuvirus病毒属。生物学特性试验表明，PH826具有广泛的温度稳定性（4-60℃）和pH稳定性（3.0-11.0）。宿主谱测试显示，PH826可以裂解13株铜绿假单胞菌（包括人源和动物源），体外抑菌试验显示，PH826在感染复数（multiplicity of infection，MOI）分别为10、1、0.1时对铜绿假单胞菌均有强烈的裂解作用。根据基因组分析，PH826噬菌体的基因组大小为87 956 bp，G+C含量为54.70%，编码165个开放阅读框（open reading frames，ORFs），并预测携带有裂解酶。值得注意的是，在24 h和48 h内，PH826和环丙沙星联用在1×、2×、4×最小抑菌浓度（minimum inhibitory concentration，MIC）的条件下都减少了80%以上的生物被膜的形成；PH826和美罗培南联用在24 h和48 h内，在2×MIC和4×MIC的条件下也减少了80%以上的生物被膜的形成。【结论】PH826可能是一种潜在的消毒剂和治疗剂，可以单独使用或与抗生素联合使用，用于预防和治疗致病性铜绿假单胞菌。

Abstract:

[Objective] Pseudomonas aeruginosa is a major Gram-negative pathogen that can exacerbate lung infections in the patients with cystic fibrosis, which can ultimately lead to death. The emerging multi-drug resistant and pan-drug resistant strains pose challenges to the prevention and control of P. aeruginosa infection. [Methods] A phage capable of lysing multi-drug resistant P. aeruginosa was isolated from farm sewage. The morphological characteristics, biological characteristics, host range, genomic features, and in vitro inhibitory ability of the isolate were studied. The biofilm inhibition test was conducted with both the phage and antibiotics. [Results] The phage PH826 was identified to be a member of the genus Nankokuvirus and it can keep stability within a wide temperature range from 4℃ to 60℃ and pH range (pH 3.0-11.0). The host range test showed that PH826 could lyse 13 P. aeruginosa strains from human and animals. The antibacterial test in vitro showed that PH826 was strongly lytic against P. aeruginosa at multiplicity of infection (MOI) of 10, 1, and 0.1. The genome of PH826 was 87 956 bp in length, with the G+C content of 54.70%, encoding 165 open reading frames (ORFs) including an endolysin gene. The combination of PH826 at 1×MIC (minimum inhibitory concentration, MIC), 2×MIC, or 4×MIC and ciprofloxacin reduced biofilm formation by more than 80% at the time points of 24 h and 48 h. The combination of PH826 at 2×MIC or 4×MIC and meropenem reduced biofilm formation by more than 80% at the time points of 24 h and 48 h. [Conclusion] PH826 may be a potential disinfectant and therapeutic agent that can be used alone or in combination with antibiotics for the prevention and treatment of pathogenic P. aeruginosa.