CRISPR-Cas1对鲍曼不动杆菌耐药性与毒力的抑制作用

CRISPR-Cas1 attenuates antibiotic resistance and virulence of Acinetobacter baumannii

摘要:

鲍曼不动杆菌的耐药性和毒力与日俱增。研究表明，鲍曼不动杆菌中CRISPR-Cas系统的存在可以降低细菌的耐药性并减弱细菌毒力。然而，CRISPR-Cas系统中各组分对鲍曼不动杆菌的具体影响尚不明晰。目的 探究CRISPR-Cas系统的组分在鲍曼不动杆菌中对细菌耐药性和毒力的调控作用。方法 通过构建cas1基因鲍曼不动杆菌过表达株，并与之对应检测相应过表达株与原始菌株的细菌生长情况、血清抗性、细菌耐药性、生物膜形成能力、细菌感染小鼠模型小鼠的存活率等变化。结果 使用纸片扩散法对临床常用的22种抗生素的耐药性进行检测，结果显示，过表达株AB26::cas1对6种抗生素的耐药性由耐药变为敏感。使用结晶紫染色法检测细菌生物膜形成能力，AB26::cas1株生物膜形成能力较AB26株降低。腹腔注射感染小鼠模型显示，相较于AB26株体内感染组小鼠，AB26::cas1株体内感染组小鼠未发生死亡。荧光定量PCR检测结果显示，大部分耐药/毒力基因mRNA的表达量下降。人血清与灭活血清孵育后进行存活性检测，野生株与过表达株的血清抗性差异无统计学意义。结论 来源于AB43株的cas1基因对鲍曼不动杆菌的耐药性及生物膜形成能力具有抑制作用，同时细菌对小鼠的致病性也有抑制效果。

Abstract:

The antibiotic resistance and virulence of Acinetobacter baumannii are increasingly serious. Studies have shown that the CRISPR-Cas system in A. baumannii attenuates the antibiotic resistance and virulence, while the effects of CRISPR-Cas system components on A. baumannii remain unclear.Objective Investigation of the role of components of the CRISPR-Cas system in the modulation of bacterial drug resistance and virulence in A. baumannii.Methods The impact of cas1 overexpression on bacterial growth, serum resistance, bacterial drug resistance, biofilm formation ability, and survival rate of mice in a mouse model of bacterial infection was investigated by constructing cas1 overexpression strains and corresponding with them to detect the aforementioned changes.Results The resistance of the bacterial strain AB26::cas1 to 22 commonly used antibiotics was determined using the paper diffusion method. Of these antibiotics, six were found to have an effect on the strain, causing it to change from resistant to sensitive. The capacity of the bacterial strain AB26::cas1 to form biofilms was evaluated through the use of crystal violet staining, which revealed a reduction in biofilm formation ability when compared to the AB26 strain. The intraperitoneal injection of the infected mice model demonstrated that no mortality occurred in the AB26::cas1 strain in vivo infected group in comparison to the AB26 strain in vivo infected group. The fluorescence quantitative PCR assay demonstrated a reduction in mRNA expression of the majority of resistance and toxicity genes. The survival assay, which involved incubating normal human serum with inactivated serum, revealed no statistically significant difference in serum resistance between the wild strains and the overexpression strains.Conclusion The cas1 gene, derived from strain AB43, was observed to exert an inhibitory effect on the drug resistance and biofilm-forming ability of A. baumannii, as well as the pathogenicity of the bacteria in mice.