From bugs to drugs: novel anti-microbial agents from Lebanese soil and marine dwelling microorganisms

Abstract

Antimicrobial resistance (AMR) is the phenomenon by which microorganisms become non-susceptible to their originally designed drugs. AMR became a serious public health issue, as multidrug-resistant microorganisms are claiming millions of lives globally, and are expected to become, by 2050, the leading cause of death worldwide. Indeed, community-acquired ESKAPE pathogens and their nosocomial infections are threatening millions of individuals. This risk is further increasing as these pathogens are resisting all clinically available antibiotics. Under another infectious facet, parasitic infections such as toxoplasmosis and leishmaniasis still affect millions of individuals around the globe and cause life-changing disabilities. While there is still a lack of a golden standard treatment for toxoplasmosis, cutaneous leishmaniasis causative species acquired resistance to current first-line therapies. Recently, there has been a void in the development of novel antimicrobial agents, further aggravating the serious issue of AMR. Therefore, the discovery of novel classes of antimicrobials with unexploited targets and to which resistance has not been acquired yet is fundamental. Natural products produced by environmental microbes are the basis of the majority of clinically utilized antimicrobial agents. We sought to revisit this source to identify new antimicrobials of understudied microorganisms in unexploited niches such as Lebanese soil and seawater. We successfully isolated previously uncultured bacterial strains inhabiting these environments and characterized them at the morphological, biochemical, and genomic levels. We then triggered their biosynthetic pathways by fermentation in fourteen different production media. Thereafter, we screened the in vitro bioactivities of the isolated crude extracts against the planktonic cells of ESKAPE pathogens and the biofilms of P. aeruginosa. We also assessed their activity against the intra-macrophagic Toxoplasma gondii tachyzoites and amastigotes of a clinical Leishmania tropica isolate. ZK3 RA3 remarkably inhibited the growth of Methicillin-resistant S. aureus (MRSA), while BM9 C and TBJ13 C significantly inhibited the formation of the biofilms of a Pseudomonas aeruginosa clinical isolate (PAN 14). Furthermore, HAS1 VEG inhibited the intracellular replication of T. gondii tachyzoites, whereas HAS1 INA and KR24 V6 significantly decreased the intramacrophage replication of L. tropica amastigotes. This potent bioactivity prompted us to produce upscaled fermentations using the bacteria and media that generated the aforementioned crude extracts. Subsequently, the bioactive compounds were isolated through bio-guided fractionation, and the purified molecules proved to be chemically novel and extremely potent. Antibacterial agents ZK3-F2 and ZK3-F6’ purified from the culture of ZK3 in media RA3 showcased remarkable activity against Methicillin-resistant Staphylococcus aureus with MICs of 0.0625 and 0.25 μg/mL respectively. This activity is more effective than the clinically utilized Vancomycin antibiotic. Similar results were obtained against Vancomycin-resistant Enterococcus feacium. Whereas for BM9 C and TBJ13 C, all obtained liquid-liquid partition fractions were able to significantly inhibit PAN 14 biofilm formation and eradicate its preformed biofilms. In the case of T. gondii, both the Ethyl Acetate and Water fractions obtained from HAS1 VEG, by liquid-liquid partition significantly inhibited the replication of T. gondii tachyzoites with a decrease in SAG-1 expression to 37%. For L. tropica, HAS1- F1 and HAS1-F2, compounds isolated from a culture of HAS1 in media INA significantly inhibited at the relatively low concentration of 1 μg/mL the intracellular Leishmania tropica amastigote replication (47 and 38% decrease in the kinetoplast marker respectively). These compounds also showed no cytotoxicity against human peripheral blood mononuclear cells. Altogether, our data shows that Lebanese environmental bacteria are an invaluable source of novel, potent, and safe antimicrobials worthy of further investigation.