Antimicrobial activity, cytotoxicity and chemical analysis of lemongrass essential oil (Cymbopogon flexuosus) and pure citral.

Bacterial strains Five

A. baumannii strains from the University of the West of England, Bristol, UK, microbiology culture collection were used in this study. These were ATCC® BAA-1709™ (human isolate), ATCC® BAA-1710™ (human isolate), NCTC 12156 (ATCC 19606; type strain), ATCC 17978 (lung infection model; human isolate) and SM 37212, a clinical isolate obtained from the Pathology department at Southmead Hospital, Bristol, UK. The strains were maintained on brain heart infusion (BHI) agar (CM1136; Oxoid Ltd, Basingstoke, UK) and sub-cultured on a weekly basis. For inoculum preparation, single colonies were picked from a BHI agar plate into BHI broth (CM1135; Oxoid Ltd., Basingstoke, UK) and incubated overnight at 37 °C. EO and component The lemongrass EO ( C. flexuosus ) used in this study was donated by Amphora Aromatics, Bristol, UK, whilst citral (95 %; synonym—3,7-dimethyl-2,6-octadienal, geranial and neral mixture; CAS Number 5392–40-5) was purchased from Sigma-Aldrich, Dorset, UK. The EO and citral were stored in a cool dark place and the containers kept tightly closed in a dry and well-ventilated place according to the safety data information.

Susceptibility testing

The disk diffusion assay was used to determine antimicrobial susceptibility of the A. baumannii to a selection of antibiotics (Table 1 ) using the British Society for Antimicrobial Chemotherapy guidelines, version 13 ( 2014 ) and to whole lemongrass EO and citral. Using the agar overlay assay, bacterial lawns were prepared with the inoculum size adjusted to approximately 1.5 × 10 8 CFU/ml. Ten microliters of lemongrass EO and citral were deposited onto 6-mm filter paper discs before placing them on the surface of Iso-sensitest agar (Oxoid; Basingstoke, UK). The agar plates were then incubated at 37 °C for 24 h and the diameter of the zone of inhibition (ZOI) measured in millimetres using a Vernier calliper. Each experiment was performed in triplicate. The controls were bacterial cultures without treatment. Table 1 Inhibition zones (mm) of A. baumannii strains after 24 h exposure to selected antibiotics following BSAC guidelines (version 13, June 2014) Antibiotic Disc content (μg) NCTC 12156 (ATCC 19606) ATCC 17978 SM 37212 ATCC® BAA-1709™ ATCC® BAA-1710™ Ciprofloxacin 1 18.30 (R) 17.6 (R) 0.00 (R) 30.10 (S) 0.00 (R) Gentamicin 10 20.70 (S) 19.6 (R) 7.42 (R) 31.70 (S) 7.10 (R) Meropenem 10 26.50 (S) 27.7 (R) 21.00 (R) 36.80 (S) 25.70 (S) Piperacillin/Tazobactam 75/10 24.50 (S) 25.00 (S) 18.90 (R) 86.00 (S) 20.00 (I) Criteria for defining MDR, XDR and PDR in Acinetobacter spp. (Magiorakos et al. 2012 ); MDR: non-susceptible to ≥1 agent in ≥3 antimicrobial categories; XDR: non-susceptible to ≥1 agent in all but ≤2 categories; PDR: non-susceptible to all antimicrobial agents listed ( R ) resistant, ( S ) sensitive, ( I ) intermediate Minimum inhibitory concentration and minimum bactericidal concentration The method used in this study for determination of inhibitory and bactericidal activity of both lemongrass EO and citral was similar to that use in Adukwu et al. ( 2012 ) with similar concentration ranges 0.03, 0.06, 0.12, 0.5, 1, 2 and 4 % ( v / v ), and the only difference was the microplate reader. In this study, we used the Tecan Infinite 200 PRO, Reading, UK for optical density measurements. For minimum bactericidal concentration (MBC) determination, 10 μl was taken from each well (treated and untreated) after incubation and spot inoculated on BHI agar and incubated for 24 h at 37 °C. The concentration at which no growth was observed on subculture was determined as the MBC.