Carprofen-induced depletion of proton motive force reverses TetK-mediated doxycycline resistance in methicillin-resistant Staphylococcus pseudintermedius

Andrew Hesketh,Cristian De Gobba,Bimal Jana,Luca Guardabassi,Rikke Prejh Brochmann,Zofia Magnowska,Rene Lametsch

doi:10.1038/s41598-019-54091-4

Abstract

We previously showed that doxycycline (DOX) and carprofen (CPF), a veterinary non-steroidal anti-inflammatory drug, have synergistic antimicrobial activity against methicillin-resistant Staphylococcus pseudintermedius (MRSP) carrying the tetracycline resistance determinant TetK. To elucidate the molecular mechanism of this synergy, we investigated the effects of the two drugs, individually and in combination, using a comprehensive approach including RNA sequencing, two-dimensional differential in-gel electrophoresis, macromolecule biosynthesis assays and fluorescence spectroscopy. Exposure of TetK-positive MRSP to CPF alone resulted in upregulation of pathways that generate ATP and NADH, and promote the proton gradient. We showed that CPF is a proton carrier that dissipates the electrochemical potential of the membrane. In the presence of both CPF and DOX, the energy compensation strategy was attenuated by downregulation of all the processes involved, such as citric acid cycle, oxidative phosphorylation and ATP-providing arginine deiminase pathway. Furthermore, protein biosynthesis inhibition increased from 20% under DOX exposure alone to 75% upon simultaneous exposure to CPF. We conclude that synergistic interaction of the drugs restores DOX susceptibility in MRSP by compromising proton-motive-force-dependent TetK-mediated efflux of the antibiotic. MRSP is unable to counterbalance CPF-mediated PMF depletion by cellular metabolic adaptations, resulting in intracellular accumulation of DOX and inhibition of protein biosynthesis.

Highlights

Staphylococci are common colonizers of the skin and mucosae in both humans and animals, and cause a wide range of opportunistic infections such as urinary tract, skin and soft-tissue infections, otitis, endocarditis, osteomyelitis and sepsis
We showed that co-exposure to CPF restored susceptibility to DOX in DOX-resistant methicillin-resistant Staphylococcus pseudintermedius (MRSP) ST71 containing tetK, a tetracycline resistance gene encoding a drug-specific efflux pump[6]
No synergy was observed for other MRSP strains where DOX resistance was mediated by other mechanisms

Summary

Introduction

Staphylococci are common colonizers of the skin and mucosae in both humans and animals, and cause a wide range of opportunistic infections such as urinary tract, skin and soft-tissue infections, otitis, endocarditis, osteomyelitis and sepsis. In a previous study[4], we identified a strong synergy between doxycycline (DOX), a tetracycline antibiotic that inhibits bacterial protein synthesis by binding to the 30 S ribosomal subunit, and carprofen (CPF), a Transcriptome 30 min Transcriptome 90 min Proteome 90 min Regulations: Up Down Sum Up Down Sum Up Down Sum. DOX-CPF Synergistic interactions veterinary non-steroidal anti-inflammatory drug (NSAID) that inhibits cyclooxygenase activity in eukaryotic cells. We showed that co-exposure to CPF restored susceptibility to DOX in DOX-resistant MRSP ST71 containing tetK, a tetracycline resistance gene encoding a drug-specific efflux pump[6]. The effects of the two drugs, individually and in combination, were investigated in DOX-resistant MRSP ST71 using a comprehensive systems biology approach consisting of RNA sequencing (RNA-seq), two-dimensional differential in-gel electrophoresis (2D DIGE), macromolecule biosynthesis assays and fluorescence spectroscopy

Objectives

Methods

Results

Conclusion