The role of RND-type efflux pumps in multidrug-resistant mutants of Klebsiella pneumoniae

Rui Ting Ni,Tomofusa Tsuchiya,Teruo Kuroda,Ryoko Kitagawa,Takanori Kishino,Minako Mizusawa,Wakano Ogawa,Futoshi Matsubara,Motoyasu Onishi

doi:10.1038/s41598-020-67820-x

Rui Ting Ni, Tomofusa Tsuchiya + Show 7 more

Open Access

https://doi.org/10.1038/s41598-020-67820-x

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Abstract

The emergence of multidrug-resistant Klebsiella pneumoniae is a worldwide problem. K. pneumoniae possesses numerous resistant genes in its genome. We isolated mutants resistant to various antimicrobials in vitro and investigated the importance of intrinsic genes in acquired resistance. The isolation frequency of the mutants was 10−7–10−9. Of the multidrug-resistant mutants, hyper-multidrug-resistant mutants (EB256-1, EB256-2, Nov1-8, Nov2-2, and OX128) were identified, and accelerated efflux activity of ethidium from the inside to the outside of the cells was observed in these mutants. Therefore, we hypothesized that the multidrug efflux pump, especially RND-type efflux pump, would be related to changes of the phenotype. We cloned all RND-type multidrug efflux pumps from the K. pneumoniae genome and characterized them. KexEF and KexC were powerful multidrug efflux pumps, in addition to AcrAB, KexD, OqxAB, and EefABC, which were reported previously. It was revealed that the expression of eefA was increased in EB256-1 and EB256-2: the expression of oqxA was increased in OX128; the expression of kexF was increased in Nov2-2. It was found that a region of 1,485 bp upstream of kexF, was deleted in the genome of Nov2-2. K. pneumoniae possesses more potent RND-multidrug efflux systems than E. coli. However, we revealed that most of them did not contribute to the drug resistance of our strain at basic levels of expression. On the other hand, it was also noted that the overexpression of these pumps could lead to multidrug resistance based on exposure to antimicrobial chemicals. We conclude that these pumps may have a role to maintain the intrinsic resistance of K. pneumoniae when they are overexpressed. The antimicrobial chemicals selected many resistant mutants at the same minimum inhibitory concentration (MIC) or a concentration slightly higher than the MIC. These results support the importance of using antibiotics at appropriate concentrations at clinical sites.

Highlights

The emergence of multidrug-resistant Klebsiella pneumoniae is a worldwide problem
They are a typical antibiotic from each category of aminoglycosides, cephems, β-lactams, and quinolones, respectively
32 mutants were isolated from kanamycin-containing plates; 32 mutants were isolated from cefotaxime-containing plates; 34 mutants were isolated from oxacillincontaining plates; 49 mutants were isolated from norfloxacin-containing plates; 505 mutants were isolated from novobiocin-containing plates; 76 mutants were isolated from ethidium Br-containing plates (Table 1)

Summary

Introduction

The emergence of multidrug-resistant Klebsiella pneumoniae is a worldwide problem. K. pneumoniae possesses numerous resistant genes in its genome. In K. pneumoniae, there are reports of clinical isolates overexpressing an RND-type efflux Minimum inhibitory concentrations (MICs) of antimicrobial chemicals were measured in a part of the mutants, based on resistance to several chemicals on plates.

Results

Conclusion