铜绿假单胞菌对碳青霉烯类抗生素耐药相关基因的筛选

为了在基因组水平筛选获得铜绿假单胞菌PAO1对碳青霉烯类抗生素耐药相关基因,本研究通过构建PAO1转座突变体文库、筛选对亚胺培南、美罗培南和比亚培南耐药及敏感突变株;通过随机PCR、核苷酸测序及序列比对的手段,确定了突变体中转座子的插入位点及其破坏的基因,获得了48个PAO1中对碳青霉烯类抗生素耐药和敏感相关的基因,其中27个基因突变后表现为耐药性增强,21个基因突变后表现为药物敏感性增强.本实验筛选获得的48个基因中有5个与Alvarez-Ortega和Dotsch等人筛选的基因重复.通过对PA0011,PA0667及PA3901进行基因敲除及遗传互补,进一步确证这3个基因均与PAO1对碳青霉烯类的耐药性相关.本次筛选发现了38个新的与碳青霉烯类耐药相关的基因,其中包括13个class4类基因.对这些新基因的进一步研究,不仅有利于全面了解铜绿假单胞菌的耐药机制及其调控网络,而且有利于药物作用靶点的发现,为有效治疗铜绿假单胞菌的感染提供新思路.

Determination of Genes Involved in the Resistance of Pseudomonas aeruginosa to Carbapenems

To investigate genes involved in the resistance of P. aeruginosa to carbapenems, a transposon mutation library containing more than 30000 clones was constructed and screened with three kinds of carbapenems, imipenem, meropenem and biapenem, for mutants with increased or reduced susceptibility to these antimicrobials. The insertion sites of the transposon were identified by arbitrary PCR and subsequent DNA sequencing. Forty-eight mutants with altered susceptibility to these antimicrobials compared with the wild type PAO1 were identified, 27 mutants demonstrated a reduced susceptibility phenotype and 21 mutants with an increased susceptibility. We identified five overlapping genes between our study and the study of Alvarez-Ortega and Dotsch et al. PA0011, PA0667 and PA3901 knockout mutant and complementary strain were constructed. That the antibiotics susceptibility analysis of these knockout mutants and complementary stains coincide with the mutagenesis mutants and wild type PAO1 respectively demonstrated that the three genes play a role in the carbapenems resistance of PAO1. In this study, 38 novel and unknown genetic determinants involved in susceptibility to carbapenems in PAO1 are found, including 13 class4 genes. Knowledge of novel resistance genes may contribute to understanding the mechanism of carbapenems resistance and discovering new drugs＇ target in P. aeruginosa.