ABSTRACT: Objectives: The aim of the study was to characterize (-)-α-Pinene, which is one of the chemical constituents of Alpinia katsumadai seed essential oil responsible for its resistance modulatory activity in Campylobacter jejuni. Methods: Broth microdilution method was used to evaluate the antimicrobial and resistance modulatory potential of (-)-α-Pinene and ethidium bromide accumulation assay to determine its efflux-inhibitory activity in subinhibitory concentration. The target efflux system was identified using knock-out mutants in several efflux related genes. Furthermore, the influence of subinhibitory concentration of (-)-α-Pinene on C. jejuni NCTC 11168 was investigated using microarray technology in order to elucidate the adaptive mechanism of bacteria to treatment with this phytochemical. Knock-out mutants of key adaptation genes were constructed and their role in adaptation to several stress factors, including (-)-α-Pinene, different osmolites and pH, was investigated using Biolog phenotypical microarrays and CFU counts. Results: (-)-α-Pinene was confirmed as highly efficient Campylobacter jejuni resistance modulator, due to its efflux inhibitory activity, which was significantly higher compared to reference inhibitors CCCP and reserpine. The CmeABC along with newly characterized CmeI (Cj1687) was confirmed as its main target efflux system. The transcriptional analysis indicated that the heat shock regulators HspR and HrcA are the main transcription regulators involved in adaptation to (-)-α-Pinene treatment. Conclusions: (-)-α-Pinene is a novel CmeABC and CmeI efflux inhibitor, which evokes the heat shock response in Campylobacter jejuni. Influence of (-)-α-Pinene on gene expression in C. jejuni NCTC 11168 was determined by expressional analysis and qRT-PCR. Exponential phase culture was adjusted to OD600=0.2 in MHB using spectrophotometer (Smart Spec, Bio-Rad, Hercules, CA, USA). Five ml of culture was treated with 62.5 mg/L of (-)-alpha-pinene dissolved in DMSO. Only DMSO (0.048 %) was added to untreated samples. Cultures were treated for 2 h, incubated microaerobically shaking (160 rpm) at 42°C. Experiments were carried out in 4 biological replicates. RNA Protect Bacteria reagent (Qiagen, Maryland, USA) was added to the culture and total RNA was isolated using RNeasy mini kit (Qiagen) and treated with Ambion® Turbo DNA-freeTM kit (Invitrogen, USA). Microarrays with 4751 probes targeting 1756 genes specific for Campylobacter jejuni subsp. jejuni NCTC 11168 (Mycroarray, Biodiscovery-LLC, MI, USA) were used for gene expression analysis. The cDNA was synthesized with random hexamers, SuperScriptTM III Reverse Transcriptase and amynoallyl dUTP (all supplied by Invitrogen, USA) and labeled with monofunctional NHS-ester dye Amersham C3 or Cy5 (GE Healthcare, Buckinghamshire, UK). Concentration of cDNA and labeling efficiency was determined spectrophotometrically with NanoDrop 1000. Four biological replicates were hybridized to four microarrays according to manufacturerâs protocol, incubated for 24 hours at 42â°C and scanned at 532-nm (Cy3) and 635-nm (Cy5) wavelengths using GenePix 4100A (Molecular Devices, Sunnyvale, CA) following the manufacturer's protocol. Fluorescence intensities of each spot were extracted using GenePix Pro 7.0 (Molecular Devices).
