Evaluation of the antimicrobial activities of Napoleona imperialis leaf extracts against multi-drug resistant bacterial isolates from diabetic foot ulcer

Background: The increased resistance of Gram-negative bacteria, particularly those that produce extended beta-lactamase, are limiting the efficacy of antimicrobial drugs in treating infected diabetic foot ulcers. This study evaluates antimicrobial activities of Napoleona imperialis leaf extracts on bacterial isolates of diabetic foot ulcers as a way of developing novel antimicrobials that will be effective in treating infections caused by multi-drug-resistant (MDR) bacterial isolates. Methodology: Fresh leaves of N. imperialis were collected and identified by a plant taxonomist at the Department of Plant Biology and Biotechnology, Nnamdi Azikiwe University, Awka, Nigeria. The leaves were washed and air- dried under shade and pulverized into fine powder using local milling machine. The pulverized plant was extracted using methanol, hexane, ethyl-acetate, and water. The test organisms used were MDR bacteria isolated from fresh clinical samples collected from patients with diabetic ulcer. The samples were processed using conventional cultures, biochemical identification and molecular detection by PCR methods, and antimicrobial susceptibility testing was done by the disc diffusion technique. The phytochemical compositions of the extract were assessed using standard methods. Antibacterial activity of the extracts was performed at a concentration of 400mg/ml of the extracts using agar well diffusion method. The minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MIC) of the extracts were determined using serial (doubling) dilution technique. The time-kill assay of the plant extract was also evaluated. Results: The MDR isolates recovered from the diabetic ulcer were Escherichia coli, Klebsiella pneumoniae, Streptococcus pneumoniae, Pseudomonas aeruginosa and Staphylococcus aureus. The plant extract yield showed that aqueous fraction of the leaf extract gave higher yield of 37.3%, followed by ethyl acetate fraction of the leaf extract at 26.8%, hexane fraction of the leaf extract at 22.3%, and the least was crude methanol leaf extract at 13.4%. The phytochemical analysis showed that the leaf extracts contained phenols, flavonoid, tannins, glycosides, alkaloids, saponin, terpenoids and triterpenes. The methanol extract produced highest mean inhibition zone diameter of 19.7±00mm against E. coli, 19.3±1.2mm against P. aeruginosa, 19.3±1.2mm against S. pneumoniae, 18.7±1.2mm against S. aureus and 17.7±1.5mm against K. pneumoniae. The bacteriostatic (MIC) activities of this methanol extract at different concentrations ranged from 3.125 to 25.00mg/ml, and bactericidal (MBC) activities ranged from 6.25 to 50.00mg/ml. The time- kill assay of the crude methanol leaf extract at 1xMIC, 2xMIC and 3x MIC showed a decrease in the number of viable cells count of the initial inoculum within 2-8 hours of incubation, indicating high activity. Conclusion: The methanol leaf extracts of N. imperialis could be used as a complementary source of antimicrobials to the conventional antibiotic in the treatment of wound infections caused by MDR bacteria due to the contents of essential secondary metabolites in the plant extract and the antibacterial activities observed.