Adaptation of clinical isolates of Klebsiella pneumoniae to the combination of niclosamide with the efflux pump inhibitor phenyl-arginine-β-naphthylamide (PaβN): co-resistance to antimicrobials

[Objectives] To search for new means of combatting carbapenemase-producing strains of Klebsiella pneumoniae by repurposing the anti-helminth drug niclosamide as an antimicrobial agent and combining it with the efflux pump inhibitor (EPI) phenyl-arginine-β-naphthylamide (PaβN).
[Methods] Niclosamide and PaβN MICs were determined for six clinical K. pneumoniae isolates harbouring different carbapenemases by broth microdilution and chequerboard assays. Time–kill curves in the presence of each drug alone and in combination were conducted. The viability of bacterial cells in the presence of repetitive exposures at 8 h to the treatment at the same concentration of niclosamide and/or PaβN (adapted isolates) was determined. The acrAB-tolC genes and their regulators were sequenced and quantitative RT–PCR was performed to assess whether the acrA gene was overexpressed in adapted isolates compared with non-adapted isolates. Finally, the MICs of several antimicrobials were determined for the adapted isolates.
[Results] Niclosamide and PaβN had synergistic effects on the six isolates in vitro, but adaptation appeared when the treatment was applied to the medium every 8 h, with an increase of 6- to 12-fold in the MIC of PaβN. Sequencing revealed different mutations in the regulators of the tripartite AcrAB-TolC efflux pump (ramR and acrR) that may be responsible for the overexpression of the efflux pump and the adaptation to this combination. Co-resistance to different antimicrobials confirmed the overexpression of the AcrAB-TolC efflux pump.
[Conclusions] Despite the synergistic effect that preliminary in vitro stages may suggest, the combinations of drugs and EPI may generate adapted phenotypes associated with antimicrobial resistance that must be taken into consideration.
This study was funded by grant PI19/00878 awarded to M. Tomás within the State Plan for R+D+I 2013-2016 (National Plan for Scientific Research, Technological Development and Innovation 2008-2011) and co-financed by the ISCIII-Deputy General Directorate for Evaluation and Promotion of Research - European Regional Development Fund ‘A way of Making Europe’ and Instituto de Salud Carlos III FEDER, Spanish Network for Research in Infectious Diseases (REIPI, RD16/0016/0001, RD16/0016/0006 and RD16/CIII/0004/0002) and by the Study Group on Mechanisms of Action and Resistance to Antimicrobials, GEMARA (SEIMC, http://www.seimc.org/). M. Tomás was financially supported by the Miguel Servet Research Programme (SERGAS and ISCIII). I. Bleriot was financially supported by the pFIS programme (ISCIII, FI20/00302). O. Pacios, L. Fernández-García and M. López were financially supported by the grants IN606A-2020/035, IN606B-2021/013 and IN606B-2018/008, respectively (GAIN, Xunta de Galicia).