Antibiotic-potentiation activities of three animal species extracts, Bitis arietans, Helix aspersa, and Aristaeomorpha foliacea and mode of action against MDR Gram-negative bacteria phenotypes

Abstract

Background: In recent years, drug resistance to human pathogenic bacteria has been commonly reported from all over the world. As antimicrobial activities of most medicinal plants and antibiotics have been already explored, it is more important to make investigations on animal species mainly invertebrates which could constitute an efficient source of antimicrobial molecules. This work was aimed at contributing to the fight against microbial resistance through the study of antibacterial potential of three animal species (Helix aspersa, Bitis arietans, Aristaeomorpha foliacea) on several multidrug-resistant (MDR) Gram-negative strains overexpressing efflux pumps including Escherichia coli, Enterobacter aerogenes, Klebsiella pneumoniae, Pseudomonas aeruginosa. Methods: The microdilution technique was used to evaluate the antibacterial activities of the tested samples by determining their minimal inhibitory concentrations (MICs), as well as the effect of their combination with antibiotics. Studies on the mechanisms of action of the most active sample, dried Bitis arietans extract, was carried out using standard methods for evaluating the effects of this extract on bacterial H+-ATPases-mediated proton pumps and on bacterial growth kinetics. In this latter case, the optical density was read spectrophotometrically. Results: Zoochemical screening indicated the presence of protein constituents and alkaloids and the absence of other metabolites in all tested extracts. Dried B. arietans showed the best antibacterial activity by inhibiting the growth of 90% of studied bacterial strains with MICs ranging from 128 to 2048 μg/ml. Moreover, this extract presented a significant activity (100≤MIC≤512 µg/ml) against 35% of bacteria that are E. coli (ATCC8739, AG100ATet, MC4100), E. aerogenes EA27, K. pneumoniae ATCC11296, P. aeruginosa (PA01, PA124) and a moderate activity (512<MIC≤2048 µg/ml) against 55% of studied bacteria. It was followed by fresh B. arietans which inhibited the growth of 65% of bacteria with significant activity on three bacteria (E. coli ATCC8739, E. aerogenes ATCC13048 and K. pneumoniae ATCC11296. These two extracts showed bactericidal effects on many strains. The other extracts samples selectively exhibited an antibacterial activity against less than 40% of strains. All samples potentiated the activity of at least 56% of used antibiotics against at least 70% of studied bacterial strains. B. arietans extracts at MIC/2 and MIC/4 mostly improved the activities of more than 78% of antibiotics on at least 70% of bacteria with improvement activity factors (IAF) ranging from 2 – 128 suggesting that this animal contains bioactive compounds which could act as efflux pumps inhibitors. Bacterial growth kinetic study showed that when treated with dried B. arietans extract (the most active sample) at different concentrations MIC/2, MIC and 2xMIC, the growth of tested bacteria (E. coli ATCC8739) decreased respectively when the concentrations increased. Furthermore, this extract inhibited the H+-ATPase-mediated proton pumps of this bacterium increasing the pH values. Conclusion: Results obtained in the present work provide interesting data for the use of dried B. arietans extract and invertebrates in general in the traditional therapy for the treatment of bacterial infections involving multidrug-resistant phenotypes. Keywords: Gram-negative bacteria; multidrug resistance; efflux pumps; infectious diseases; animal species; secondary metabolites.

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