Effect of selected terpenoids on antibiotic potentiation and eradication of Staphylococcus aureus biofilms – a structure activity relationship study

Research on the discovery of new drugs to treat bacterial biofilm infections has been a priority for biomedical and scientific communities. Actually, the sessile mode of growth accounts for more than 80% of human bacterial infections and displays increased resistance to antibiotics. To date, there are no drugs with total efficacy against biofilms and the treatment failure is a recurrent clinical situation. This lack of alternatives boosted up again the exploration of the pharmacological properties of health-promoting agents from natural origin. In this connection, the potential of phytotherapeutic agents for the treatment/prevention of complex infectious diseases, such as those that involve biofilm formation has been intensified[1]. In view of that, the main aim of the present study was to evaluate the activity of four phytochemicals bellowing to terpenoids class [cis-6-nonen-1-ol (CIS), citronellic acid (CITACID), citronellol (CITRO) and 3-7-dimethyl-1-octanol (3,7DOC)] against Staphylococcus aureus, both in planktonic and sessile state. Firstly, the minimum inhibitory and bactericidal concentrations (MIC and MBC) were determined by the broth microdilution method and culturability on plate count agar, respectively. Then, the potential of each terpenoid as resistance modifying agent was assessed by the disc diffusion method, using antibiotics from different classes. Besides, its potential to eradicate pre-formed S. aureus biofilms (24-h old) was performed using a microtiter plate assay and characterized in terms of biofilm mass removal (crystal violet staining), metabolic activity reduction (alamar blue staining) and culturability (colony forming units - CFU - counts). Considering that the selected terpenoids are chemical structurally related, i.e. present a similar backbone and differ only on the functional groups location, a structure activity relationship (SAR) analysis was also established. Both CITO and 3,7DOC presented the lowest MIC value (200 μg/mL) followed by CIS (400 μg/mL) and CITACID (1000 μg/mL). The MBC was found to be 1000 µg/mL with CIS, 2000 µg/mL with CITACID and > 2000 µg/mL (the maximum concentration tested) with CITO/3,7DOC. Apparently, the hydrophobicity of the molecules appear to affect positively its inhibitory proprieties – molecules with higher hydrophobicity presented lower MIC values. Moreover, it seems that the hydroxyl and methyl functional groups play the major influence on the antimicrobial properties. Indeed, CITO and 3,7DOC presented the higher hydrophobicity values and both had hydroxyl and methyl functional groups, possessing the lowest MIC value. Independently of the terpenoid tested, all combinations (terpenoid-antibiotic) resulted in a potentiation effect. Regarding biofilm eradication, although no biomass removal was observed, metabolic activity reductions from 25% (CIS at 5×MIC) up to 44% (CITACID at 10×MIC) and total loss of culturability (CITACID at 10×MIC; 6-log CFU/cm² reduction) was found. These effects were found to be dose dependent. Overall, the results obtained suggest that all the tested terpenoids might be interesting antibiotic adjuvants and emphasize the use of CITACID for biofilm cells inactivation. The results obtained are promising since the terpenoids studied are natural occurring flavoring ingredients, generally recognized as safe by the FDA, which are usually applied as food additives for human consumption.

References: [1] A.Borges, A.Abreu, C.Dias, M.Saavedra, F.Borges, M.Simões, Molecules 21(2016)877.