## 2 Materials and Methods  
### Essential Oils, Working Solutions and Bacterial Strains  
The essential oils originated from the same manufacturer (doTERRA International LLC, Pleasant Grove, United States), obtained by distillation, and were purchased from a local retailer. Through the precise traceability system provided by the manufacturer of the oils used in the present study (each EO bottle bears the inception of a lot number that is fully traceable), every HPLC analysis is publicly available (Supplementary Materials). The essential oils used in this study originated from crops cultured in different countries: Lemongrass--harvested in India; Clove- harvested in Madagascar; Melaleuca--harvested in Kenya and Australia; Thyme--harvested in Germany; Oregon--harvested in Turkey. The purity of the extracted oils is certified by the Certified Pure Therapeutic Grade (CPTG) certification.  
Aqueous extracts of each essential oil (AqEO) were prepared. For this, equal amounts of each EO (oregano, thyme, lemongrass, melaleuca and clove) and sterile bi-distilled water (4 mL of each) were gently mixed overnight in 15 mL centrifuge tubes using an orbital mixer. The aqueous phase was recovered and further used as canal irrigant. One ml of the aqueous extract of each essential oil (AqEO) was also sent to the University of Bari for high-performance liquid chromatographic (HPLC) analysis. HPLC analysis was carried out on a chromatograph LC20 Chromatography Enclosure (Thermo Fisher Scientific Inc., Monza, Milan), with a PDA-100 Photodiode Array Detector and a Fluorescent detector Ultimate 3000. Anhydrous powders (sodium acetate, sodium ascorbate, boric acid, ascorbic acid) and all the chemical reagents were purchased, if not specifically reported, from Sigma-Aldrich (Milan, Italy). Distilled water, 96% ethanol, dimethyl-sulfoxide (DMSO), acetonitrile, 99.8% glacial acetic acid, 100% methanol and all solvents used for HPLC and optical density readings were of analytical grade or HPLC grade from Fisher Scientific (Thermo Fisher Scientific Inc., Monza, Italy). Reversed phase columns Hypersep C-18 supplied by Thermo Scientific were used to carry out a solid phase extraction (SPE). HPLC analysis was carried out using a PolarAdvantage II (4.6 \(\times\) 150 mm; 3 \(\upmu\)m) C18 column directly connected to an Adsorbosphere C18 pre-column. The injection volume of analysis samples was of 100 \(\upmu\)L. Elution was carried out at a flow rate of 0.80 mL/min for 40 min using, as mobile phase, a mixture of water and acetonitrile (A = acetonitrile, B = distillated water) characterized by the following gradient: 20% A in B for 16 min, from 16 to 30 min the mixture gradually changes to 70% A, remaining stable for 5 min, then returning to 20% A until the end of the analysis. Different chromatographic runs were carried out on different biological matrices (AqEO). analytes were monitored with a diode array detector and CHROMELEON v.6.8 software connected to the chromatographing system was used for the acquisition and processing of data.  
A solution of 5.25% sodium hypochlorite (NaOCl--commercial name Chloraxid, Cerkamed, Poland) was used as an antimicrobial agent (irrigant with documented antimicrobial properties) (27) and was considered a positive standard to whom the antimicrobial effects of EO could be reported to. Sterile saline solution was used for negative standard (irrigant without antimicrobial properties).  
_Enterococcus faecalis_ ATCC29212 (Oxoid Ltd., Basingstoke, United Kingdom) was revitalized from \(-80\)\({}^{\circ}\)C glycerol stocks on blood agar. After checking the purity of the culture, a bacterial inoculum of was prepared from in sterile saline solution, at a concentration of 1 McFarland units.