Efflux Pump AcrAB Confers Decreased Susceptibility to Piperacillin–Tazobactam and Ceftolozane–Tazobactam in Tigecycline-Non-Susceptible Klebsiella pneumoniae

Abstract

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 β-lactam/β-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 μg/mL; C/T, 0.25/4 μg/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 β-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.

Keywords:

• drug efflux pump
• Enterobacteriaceae
• multidrug resistance
• β-lactam/β-lactamase inhibitor combinations

Acknowledgments

We thank Yohei Doi for his critical review of the manuscript. This study was supported by the funding from the National Natural Science Foundation of China (NO. 81703567 to J. Li, NO. 81603166 to Z. Sheng, and NO. 81473250 and 81773785 to M. Wang), the Shanghai Three-Year Plan of the Key Subjects Construction in Public Health-Infectious Diseases and Pathogenic Microorganism (NO. 15GWZK0102 to Q. Xie), Shanghai Municipal Key Clinical Specialty (Infectious disease, YW20190002 to Q. Xie), and partially funded by the National Science and Technology Key Project on Major Infectious Diseases (NO. 2017ZX10302201-004-005 to Z. Sheng).

Abbreviations

TZP, piperacillin–tazobactam; C/T, ceftolozane–tazobactam; TNSKP, tigecycline-non-susceptible Klebsiella pneumoniae; MIC, minimum inhibitory concentration; WGS, whole-genome sequencing; CRKP, carbapenem-resistant Klebsiella pneumoniae; BL/BLIs, β-lactam/β-lactamase inhibitor combinations; ESBL, extended-spectrum-β-lactamase; RND, resistance-nodulation-division; CLSI, Clinical and Laboratory Standards Institute; MH, Muller-Hinton; PAβN, Phe-Arg-β-naphthylamide; SMRT, Single Molecule Real Time; qRT-PCR, quantitative real-time PCR; TSKP, tigecycline-susceptible K. pneumoniae clinical isolate; LB, Luria Bertani.

Data Sharing Statement

The complete genome sequence of K2606 is available at US National Center for Biotechnology Information (NCBI) website under the following GenBank accession numbers: CP047633 for chromosome, CP047634 for plasmid A, CP047635 for plasmid B, CP047636 for plasmid C, CP047637 for plasmid D, and CP047638 for plasmid E.

Supplemental material is available online in Figure S1.

Disclosure

The authors report no conflicts of interest in this work.