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Front. Microbiol. | doi: 10.3389/fmicb.2019.00337

Essential oil, extracts, and sesquiterpenes obtained from the heartwood of Pilgerodendron uviferum act as potential inhibitors of the Staphylococcus aureus NorA multidrug efflux pump

 Javier Espinoza1, 2*,  Alejandro Urzúa3*, Loreto Sanhueza4, Mariana Walter5, Paola Fincheira1, 2 and  Marcela Wilkens6
  • 1Department of Chemical Sciences and Natural Resources, University of La Frontera, Chile
  • 2Centro de Excelencia en Investigación Biotecnológica Aplicada al Medio Ambiente, Universidad de La Frontera, Chile
  • 3Universidad de Santiago de Chile, Chile
  • 4Universidad Mayor, Chile
  • 5Departamento de Química de los Materiales, Facultad de Química y Biología, Universidad de Santiago de Chile, Chile
  • 6Center for Biomedical and Applied Research, Faculty of Medical Sciences, University of Santiago, Chile, Chile

Staphylococcus aureus is a serious human pathogen that is highly adaptive to environmental conditions and rapidly develops antibiotic resistance. The use of efflux pumps to reduce antibiotic concentrations at the intracellular level is one of the main mechanisms by which bacteria develop antibiotic resistance. The management of efflux pumps, specifically NorA, which is expressed by S. aureus strains, is a valuable strategy for restoring susceptibility in strains resistant to antibacterial agents. In recent years, many studies have focused on searching for natural substances that can reverse efflux pump-mediated resistance in S. aureus. Extracts and compounds obtained from plants can be efficient efflux pump inhibitors (EPIs) and represent a potentially patient-friendly strategy for controlling S. aureus. In the present study, we evaluated the ability of essential oils, petroleum ether extracts, dichloromethane extract (DCME) and six compounds isolated from the heartwood of Pilgerodendron uviferum (Cupressaceae) and two synthetic derivatives to inhibit efflux in NorA pumps in the following three S. aureus strains: K2378, which overexpressed the norA gene (norA++), K1902 (norA-deleted, △norA) and the parental strain, NCTC 8325-4. Efflux activity was evaluated using a fluorometric method that measured the accumulation of the universal efflux pump substrate ethidium bromide (EtBr). Only DCME and the compounds 15-copaenol and epi-cubenol inhibited EtBr efflux by K2378. Even the lowest concentration of 15-copaenol exhibited a stronger inhibitory effect than carbonyl cyanide m-chlorophenyl hydrazone on EtBr efflux by K2378. 15-copaenal only showed inhibition of EtBr efflux in K2378 cells at 125 μg/mL, but not superior to the control inhibitor and 15-copaenyl acetate exerted no intrinsic EPI activity against K2378. Fractional inhibitory concentration index (FICI) values obtained in the checkerboard assays, indicated that all combinations between DCME, epi-cubenol and 15-copaenol, and tested antibiotics showed a synergistic effect in wild type, norA++ and △norA strains. Moreover, those were not toxic for the HeLa cell line at concentrations in which the synergistic effect and inhibitory activity of efflux pumps was determined. Other extracts and compounds obtained from P. uviferum did not display EtBr efflux-inhibiting activity against the evaluated S. aureus strains.

Keywords: antibiotic resistance, efflux pump, NORA, efflux pumps inhibitors, Sesquiterpenes, 15-copaenol.

Received: 30 Oct 2018; Accepted: 08 Feb 2019.

Edited by:

Ren-You Gan, School of Agriculture and Biology, Shanghai Jiao Tong University, China

Reviewed by:

Jianhua Wang, Gene Engineering Laboratory, Feed Research Institute, Chinese Academy of Agricultural Sciences, China
Tommaso Felicetti, Dipartimento Di Scienze Farmaceutiche, Università Degli Studi Di Perugia, Italy  

Copyright: © 2019 Espinoza, Urzúa, Sanhueza, Walter, Fincheira and Wilkens. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

* Correspondence:
Dr. Javier Espinoza, Department of Chemical Sciences and Natural Resources, University of La Frontera, Temuco, Chile, javier.espinoza@ufrontera.cl
Dr. Alejandro Urzúa, Universidad de Santiago de Chile, Santiago, 9170022, Metropolitan Region, Chile, alejandro.urzua@usach.cl