In vitro antimicrobial activities of phyllanthus amarus extract, antibiotics and their combination against leptospira interrogans

P. amarus had been traditionally used in treating ailments including infectious diseases. Previous researches proved its antibacterial properties towards groups of microorganisms including Leptospira spp., but scanted. In vitro antimicrobial activity of P. amarus extracts (aqueous and methanol) and antibiotics were studied against Leptospira interrogans serovars Australis, Batavie, Canicola and Javanica. Minimal inhibitory concentrations (MIC) of aqueous and methanol extracts were determined using Ellinghausen-McCullough-Johnson-Harris (EMJH) broth microdilution method in 96-well plates, by determining the absorbance values using ELISA tray reader at 420 nm and viewed under dark field microscope after incubation of 7 days , at 30 °C and 40 rpm. Minimal bactericidal concentrations (MBC) were determined by sub-culturing Leptospira inoculum from MIC test onto EMJH solid media for two to three weeks. Synergistic testing of P. amarus extracts in combination with antibiotics were determined using checkerboard assays in the form of fractional inhibitory concentration index (FICi) values with their interpretations. The combinations with the lowest FICI values are preceded with scanning electron microscopic analysis (SEM) based on their MIC values; P. amarus extract and antibiotic against the Leptospira culture, both in singly and in combination after 18 hours of treatment at 30 °C and 40 rpm. Both aqueous and methanol extracts showed antimicrobial activity towards all tested L. interrogans serovars. MICs and MBCs ranges of aqueous extract were 100 to 400 μg/ml, and methanol extract ranging from 400 to 800 μg/ml. All tested antibiotics; ceftriaxone, doxycycline and penicillin G, exhibited antimicrobial activities with MICs and MBCs values ranging from 0.05 to 0.78 μg/ml and 0.05 to 0.78 μg/ml, 0.39 to 3.13 μg/ml and 12.5 to 25.0 μg/ml, and <0.01 to 0.78 μg/ml and