title: Antibiotic resistance modulation and modes of action of (-)-α-pinene in Campylobacter jejuni.
abstract: The aim of the study was to investigate the mode of action of (-)-α-pinene in terms of its modulation of antibiotic resistance in Campylobacter jejuni. Broth microdilution and ethidium bromide accumulation assays were used to evaluate the (-)-α-pinene antimicrobial activity, modulation of antimicrobial resistance, and inhibition of antimicrobial efflux. The target antimicrobial efflux systems were identified using an insertion mutagenesis approach, and C. jejuni adaptation to (-)-α-pinene was evaluated using DNA microarrays. Knock-out mutants of the key up-regulated transcriptional regulators hspR and hrcA were constructed to investigate their roles in C. jejuni adaptation to several stress factors, including osmolytes, and pH, using Biolog phenotypical microarrays. Our data demonstrate that (-)-α-pinene efficiently modulates antibiotic resistance in C. jejuni by decreasing the minimum inhibitory concentrations of ciprofloxacin, erythromycin and triclosan by up to 512-fold. Furthermore, (-)-α-pinene promotes increased expression of cmeABC and another putative antimicrobial efflux gene, Cj1687. The ethidium bromide accumulation was greater in the wild-type strain than in the antimicrobial efflux mutant strains, which indicates that these antimicrobial efflux systems are a target of action of (-)-α-pinene. Additionally, (-)-α-pinene decreases membrane integrity, which suggests that enhanced microbial influx is a secondary mode of action of (-)-α-pinene. Transcriptomic analysis indicated that (-)-α-pinene disrupts multiple metabolic pathways, and particularly those involved in heat-shock responses. Thus, (-)-α-pinene has significant activity in the modulation of antibiotic resistance in C. jejuni, which appears to be mediated by multiple mechanisms that include inhibition of microbial efflux, decreased membrane integrity, and metabolic disruption. These data warrant further studies on (-)-α-pinene to develop its use in the control of antibiotic resistance in Campylobacter.
authors.affiliation: Department of Food Science and Technology, Biotechnical Faculty, University of Ljubljana, Ljubljana, Slovenia.
authors.forename: Jasna
authors.initials: J
authors.lastname: Kovač
authors.affiliation: Department of Food Science and Technology, Biotechnical Faculty, University of Ljubljana, Ljubljana, Slovenia.
authors.forename: Katarina
authors.initials: K
authors.lastname: Šimunović
authors.affiliation: Department of Veterinary Microbiology and Preventive Medicine, College of Veterinary Medicine, Iowa State University, Ames, United States of America.
authors.forename: Zuowei
authors.initials: Z
authors.lastname: Wu
authors.affiliation: Department of Food Science and Technology, Biotechnical Faculty, University of Ljubljana, Ljubljana, Slovenia.
authors.forename: Anja
authors.initials: A
authors.lastname: Klančnik
authors.affiliation: Department of Pharmacognosy, Institute of Pharmaceutical Sciences, University of Graz, Graz, Austria.
authors.forename: Franz
authors.initials: F
authors.lastname: Bucar
authors.affiliation: Department of Veterinary Microbiology and Preventive Medicine, College of Veterinary Medicine, Iowa State University, Ames, United States of America.
authors.forename: Qijing
authors.initials: Q
authors.lastname: Zhang
authors.affiliation: Department of Food Science and Technology, Biotechnical Faculty, University of Ljubljana, Ljubljana, Slovenia.
authors.forename: Sonja Smole
authors.initials: SS
authors.lastname: Možina
mesh_terms: D000900:Anti-Bacterial Agents / Q000494:pharmacology*; D000081008:Bicyclic Monoterpenes; D016123:Campylobacter jejuni / Q000187:drug effects* / Q000502:physiology; D002258:Carbonyl Cyanide m-Chlorophenyl Hydrazone / Q000494:pharmacology; D002463:Cell Membrane Permeability / Q000187:drug effects; D002939:Ciprofloxacin / Q000494:pharmacology; D024901:Drug Resistance, Multiple, Bacterial; D004917:Erythromycin / Q000494:pharmacology; D053858:Metabolic Networks and Pathways / Q000187:drug effects; D008826:Microbial Sensitivity Tests; D039821:Monoterpenes / Q000494:pharmacology*; D016254:Mutagenesis, Insertional; D012110:Reserpine / Q000494:pharmacology; D059467:Transcriptome / Q000187:drug effects
chemical_list: D000900:Anti-Bacterial Agents; D000081008:Bicyclic Monoterpenes; D039821:Monoterpenes; D002258:Carbonyl Cyanide m-Chlorophenyl Hydrazone; D002939:Ciprofloxacin; D004917:Erythromycin; D012110:Reserpine; C005451:alpha-pinene
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