Antibacterial activity and mode of action of acetone crude leaf extracts of underinvestigated Syzygium and Eugenia (Myrtaceae) species on multidrug resistant porcine diarrhoeagenic Escherichia coli

Abstract

BACKGROUND : Diarrhoea, a global economically important disease burden affecting swine and, especially piglets, is commonly caused by infection with entero-toxigenic E. coli (ETEC). Adherence of ETEC to porcine intestinal epithelial cells following infection, is necessary for its pathogenesis. While antimicrobials are commonly given as therapy or as feed additives for prophylaxis against microbial infections, the concern over increased levels of antimicrobial resistance necessitate the search for safe and effective alternatives in livestock feed. Attention is shifting to natural products including plants as suitable alternatives to antimicrobials. The activity of acetone crude leaf extracts of nine under-explored South African endemic plants from the Myrtaceae family with good antimicrobial activity were tested against pathogenic E. coli of porcine origin using a microplate serial dilution method. Bioautography, also with p-iodonitrotetrazolium violet as growth indicator was used to view the number of bioactive compounds in each extract. In vitro toxicity of extracts was determined against Caco-2 cells using the 3-(4,5-dimethythiazolyl-2)-2,5-diphenyltetrazolium bromide reduction assay. The antimicrobial susceptibility of E. coli isolates was tested on a panel of antimicrobials using the Kirby-Bauer agar diffusion method while the anti-adherence mechanism was evaluated using a Caco-2 cell enterocyte anti-adhesion model. RESULTS : The MIC of the extracts ranged from 0.07–0.14 mg/mL with S. legatii having the best mean MIC (0.05 mg/ mL). Bioautography revealed at least two active bands in each plant extract. The 50% lethal concentration (LC50) values ranged between 0.03–0.66 mg/mL. Eugenia zeyheri least cytotoxic (LC50 = 0.66 mg/ml) while E. natalitia had the highest cytotoxicity (LC50 = 0.03 mg/mL). All the bacteria were completely resistant to doxycycline and colistin sulphate and many of the plant extracts significantly reduced adhesion of E. coli to Caco-2 cells. CONCLUSIONS : The extracts of the plants had good antibacterial activity as well as a protective role on intestinal epithelial cells against enterotoxigenic E. coli bacterial adhesion. This supports the potential use of these species in limiting infection causes by E. coli. Some of these plants or extracts may be useful as phytogenic feed additives but it has to be investigated by animal feed trials.