INVESTIGATIONS ON THE EFFECTS OF EFFLUX PUMP INHIBITORS ON ANTIBIOTIC RESISTANCE, BIOFILM FORMATION AND LIPID BIOSYNTHESIS IN MYCOBACTERIUM ABSCESSUS

Mycobacterium abscessus (Mab) is a non-tuberculous mycobacterium that is highly resistant to many antibiotics. Mab causes pulmonary infections in immunocompromised individuals. The presence of efflux pumps to pump out antibiotics and its ability to form biofilms makes Mab a virulent pathogen. Studies have been done on Mab antibiotic tolerance but there are still a lot of gaps in knowledge about the effects of efflux pump inhibitors (EPIs) on antibiotic resistance and lipid biosynthesis in this bacterium during biofilm formation. In this study, we investigated the effects of the EPIs chlorpromazine (CPZ), 1-(1-naphthylmethyl)-piperazine (NMP), thioridazine (TRZ), Phenylalanine-arginine β-naphthylamide (PBN) and plumbagin (PLU) on antibiotic resistance, efflux, biofilm formation and lipid biosynthesis associated with log-phase growth and biofilm formation in Mab. We used the resazurin assay to determine the minimum inhibitory concentration (MIC) of the EPIs. We investigated the effects of the EPIs during biofilm-forming growth conditions on the MICs of antibiotics such as clarithromycin, amikacin, cefoxitin, ciprofloxacin which are the frontline antibiotics used to treat non-tuberculous mycobacterial infections. We also assessed the effects of the EPIs on the accumulation and efflux activities of the Mab cells through ethidium bromide (EtBr) assay. Furthermore, we evaluated the effects of the EPIs at sub-MIC concentrations on Mab biofilm formation under normoxic and hypoxic conditions. We utilized metabolic radiolabeling methods using 14C-palmitic acid and 14C- acetic acid which are precursors of lipid biosynthesis and analyzed lipids by silica-thin layer chromatography and autoradiography. We observed that Mab cells developed higher tolerance to the EPIs in a biofilm-stimulating medium. Furthermore, a decrease in the MICs of antibiotics was observed in the presence of the EPIs. Also, in the presence of the EPIs, there was less efflux activity within the Mab cells. In addition, EPIs inhibited biofilm formation significantly. We also noticed that NMP and PBN inhibited 14C-palmitic acid and 14C- acetic acid incorporation into polar lipids such as glycopeptidolipids, trehalose monomycolate, phosphatidylethanolamine, phosphatidylglycerol/cardiolipin, phosphatidylinositol mannosides at specific tested conditions. Our findings suggest that the EPIs inhibited the activities of the efflux pumps associated with the efflux of the antibiotics and lipid biosynthesis involved in biofilm formation. In conclusion, the results from this study gives insights on possible therapeutic opportunities.