Efflux Pump Acrab Confers Decreased Susceptibility To Piperacillin-Tazobactam And Ceftolozane-Tazobactam In Tigecycline-Non-Susceptible Klebsiella Pneumoniae

Introduction: Drug efflux pumps are critical for resistance in Gram-negative organisms, but there are limited data on the role they play in decreased susceptibility to beta-lactam/beta-lactamase inhibitor combinations. In this study, we aimed to investigate the impact of efflux pump AcrAB on piperacillin-tazobactam (TZP) and ceftolozane-tazobactam (C/T) susceptibility in tigecycline-non-susceptible Klebsiella pneumoniae (TNSKP) strains.Methods: A tigecycline gradient was used to obtain various TNSKP strains, and in conjunction with the gradient derived strains, a TNSKP clinical strain (TNSKP24) was also included. Minimum inhibitory concentrations (MICs) of antibiotics were determined by the broth microdilution method, and whole-genome sequencing (WGS) was carried out to analyze genomic changes. PCR and sequencing were performed to confirm mutations in ramR, acrR, and the intergenic region of ramR-romA, and qRT-PCR was applied to evaluate levels of gene expression. In-frame acrB knockout and complementation were performed in 3 TNSKP strains.Results: Two derivatives of K. pneumoniae K2606 (K2606-4 and K2606-16) and TNSKP24 overexpressed efflux pump AcrAB were obtained for further study. The MICs of TZP and C/T exhibited a 4-to 8-fold increase in K2606-4 and K2606-16, respectively, when compared with K2606 (TZP, 2/4 mu g/mL; C/T, 0.25/4 mu g/mL). Deletion of acrB decreased the MICs of TZP and C/T by 4-to 16-fold in TNSKP24, K2606-4, and K2606-16, respectively, and complementation of acrB increased the MICs of these agents. MICs of clavulanate, sulbactam, and avibactam in the presence of beta-lactam compounds did not change after acrB deletion and subsequent introduction of complementation mutants.Conclusion: This study highlights that decreased susceptibility to TZP and C/T could be caused by the multidrug efflux pump AcrAB in TNSKP strains.