Collection of botanical samples
The leaves of S. nutans were collected from plants growing in a natural population in nearby the locality of Toconce (22°15′11.16″S, 68°5′44.68″W, at 3788 m.a.s.l.) in April of 2014 at II Región of Antofagasta, Chile. The specimen was authenticated by Dr. Roberto Rodríguez, Departamento de Botánica, Facultad de Ciencias Naturales y Oceanográficas, Universidad de Concepción, Concepción, Chile. A sample was kept for the herbarium collection, under register CONC 139.929.
Isolation of essential oil (EO)
The essential oils were obtained from 300 g of ground fresh plants by hydrodistillation during 3 h in a Clevenger-type apparatus, and was collected from the aqueous phase using ethyl ether extraction. The oil was sealed in a glass vial and refrigerated at 4 °C to avoid its decomposition by light or heat. The yield of oil was calculated in terms of the obtained oil mass and plant mass used.
Gas chromatography: mass spectrometry
The determination and quantification of the composition of the essential oil was performed by gas chromatography coupled with mass spectrometry (GC–MS) using an Agilent 5973N GC/MS system, with a fused silica capillary column (30 m × 0.25 mm coated with DB-5, film thickness 0.25 μm), the flow rate was 1 mL/min with helium carrier gas at 7 psi, injector port at a temperature of 250 °C and a split ratio of 1:30 and oven temperature programmed from 60 to 280 °C at 2 °C per min, injection of 2 μL of sample (10 % n-hexane).
The mass spectrometer conditions were: electron impact mode (EI) 70 eV, ion source at 230 °C, quadrupole analyzer 150 °C, a scanning range of m/z = 35–450. An HP MSD Enhanced ChemStation Software module controlled all parameter. Identification of components were made by comparing retention indices (RI) according to the Wiley Library and NIST 2000 database.
Getting the pathogenic strain
The pathogenic strain Vibrio cholerae tor1 was obtained from the strain collection of the Laboratorio Mesocosmos Marino, Centro de Bioinnovación de Antofagasta, Facultad de Ciencias del Mar y Recursos Biológicos, Universidad de Antofagasta. The strain was kept in a cryo-pearl collection and in culture plates with Trypticase soy agar, TSA (Oxoid Ltd, Basingstoke, Hampshire, England) under axenic conditions at 20 ± 1 °C.
Antibiogram of essential oil from S. nutans against V. cholera
Antimicrobial activity testing was carried out using a disc-diffusion method. Petri dishes were prepared using Mueller–Hinton agar (38 g/L with addition of 2 % NaCl). A 100 μL aliquot of the pathogenic bacteria was used to inoculate a Trypticase soy culture broth (TSB), incubating at 20 °C during 12 h. Sterile filter paper discs of 6 mm in diameter were impregnated directly with 10 μL of the oil (which correspond to 9.28 mg of essential oils S. nutans). A 100 μL sample was removed from the 12-h culture and it was streaked on the surface of the solid medium using a sterile loop. The disc with the product was then placed on the surface seeded with the pathogen. The control antibiotics used were Chloramphenicol (CL) (30 μg/disc), Streptomycin (S) (10 μg/disc), Sulfamethoxazol/Trimetoprim (SXT) (25 μg/disc), and Cefotaxime (CTX) (30 μg/disc). Each treatment was performed in triplicate. The plates were incubated at 37 °C during 24 h. A halo >5 mm around the filter caused by the absence of bacterial growth was considered as inhibition.
Identification of the inhibiting concentration of S. nutans
The minimum inhibitory concentration (MIC) was determined according to the micro-dilution in broth method described by (Rejiniemon et al. 2014; Wiegand et al. 2008), with the following modifications: (a) sterile 96-well plates were used, (12 mm × 8 mm. Costar©, 96 Well Cell Culture Cluster, Corning Incorporated); (B) seawater, previously filtered to 0.2 μm and kept at 4 °C, was used as the test medium; (C) the essential oil concentration range studied was 0.05–12.8 mg/mL, obtained from three stock solutions in ethanol: Stock Solution A—50 mg/mL (0.075 g of sample in 1.5 mL of ethanol); Stock Solution B—12.8 mg/mL (89.6 μL of Stock Solution A were diluted in 260 μL of solvent); Stock Solution C—0.8 mg/mL: (31.25 μL of Stock Solution B were diluted in 469 μL of solvent. Then the desired concentrations were placed in each well considering a final volume of 200 μL.
The bacterial inoculum was added at a concentration of 1 × 108 CFU/mL, obtained from an 18-h culture in Trypticase soy broth. Each plate was incubated at 37 °C for 24 h. The controls were seawater (medium in which the bacteria were diluted and inoculated in each well), and ethanol (solvent used to dilute the essential oil), with one control for each sample concentration. The resulting turbidity was observed and MIC value was determined to be where growth was no longer visible by assessment of turbidity by optical density reading at 620 nm with a Tecan© Sunrise 96 well Microplate Readers. All essential oil concentrations and controls used were analyzed in triplicate.
Bacterial growth in the presence of S. nutans essential oil
Aliquots of 10, 30 and 60 μL of S. nutans essential oil were added directly to test tubes, getting concentrations of 1, 3 and 6 mg/mL. Bacteria without essential oil (CT), and bacteria plus Chloramphenicol (CT CL) in concentration of 1 mg/mL were used as positive control. From an 18-h culture of the bacteria in TSB, an initial concentration of 1 × 103 CFU/mL was inoculated. The treatments and control were made in triplicate. Bacterial growth inhibition was evaluated at 48 h of culture by counting viable bacteria in TSA plates that were incubated at 37 °C during 48 h and the bacterial count was expressed in colony forming units per milliliter (CFU/mL).
Statistical analysis
The results were reported as mean ± standard error. One way ANOVA and Tukey post Hoc multiple comparison test were used to analyze data with the sofware GraphPad Prism, version 5.0 for Mac OS X. The level of significance was set at P < 0.05.
