Novel Anti-Biofilm Agents from Unexploited Soil and Marine Microorganisms

Abstract

Background: Biofilm formation, especially on indwelling medical devices, is highly problematic and represents a crucial problem in patient care. Biofilms are considered a protective environment for the bacterial communities, making those biofilm-forming microorganisms 500-5000 times more resistant to conventional antibiotics. Therefore, therapeutic interventions are needed not only to inhibit biofilm formation, but also to try and eradicate pre-formed biofilms. This gave rise to plenty of studies that resorted to plant-derived, soil-derived and marine-derived extracts as agents to combat these infections. Hence, we aimed at investigating the activity of unexploited Lebanese soil and marine microorganisms in inhibiting and/or eradicating biofilms of Pseudomonas aeruginosa and Acinetobacter baumannii, two potent biofilm-forming pathogens. Materials and Methods: Water and soil samples were collected from four regions in Lebanon, Tabarja (Tbj), Beit Meri (BM), Mazrat Meshref (MM) and Zekrit (ZK). From the collected samples, secondary metabolites were extracted in 14 different production media to be further tested. First, they were tested for their antibacterial activity on a panel of ESKAPE pathogens, and then for their anti-biofilm activity using biofilm formation assays. The active extracts were then subjected to bio-guided fractionation and physiological, phenotypic and biochemical characterization. Finally, pure compounds were obtained after several rounds of purification and those were again tested for their ability to inhibit or eradicate Pseudomonas aeruginosa Pan14 and Acinetobacter baumannii DSM biofilms. Results: The extracts generated by the isolates in the 14 different production media demonstrated minimal or no antibacterial activity on any of the pathogens, so we proceeded to test these bacterial extracts for their anti-biofilm activity. Crude extracts derived from the medium C of both BM9 and TBJ13 significantly reduced the biofilm formation in Pseudomonas aeruginosa Pan14 o 24.1% and 11.8%, respectively. On the other hand, crude extracts derived from medium C of ZK31 reduced the biofilm formation in Acinetobacter baumannii DSM to -3.4%. Therefore, pH and NaCl tolerance, API and Gram staining were performed for the three extracts to properly characterize them before proceeding with 16S sequencing for Tbj13 and BM9. Medium C of the three isolates was then produced on a larger scale and subjected to liquid-liquid partitioning. The fractions obtained were further tested showing anti-biofilm activity in Tbj13 C Hexane, BM9 C Chloroform + Hexane and BM9 C Ethyl acetate against Pseudomonas aeruginosa Pan14 and ZK31 C chloroform against Acinetobacter baumannii DSM. Extracts showing the highest activity were purified using column chromatography, thin layer chromatography and high-performance liquid chromatography. All of this led us to obtain three pure compounds: Tbj13 C Hexane, BM9 C (C + H) G a and BM9 C (C + H) G b which significantly inhibit Pseudomonas aeruginosa Pan14 biofilm formation with a P value < 0.0001. Conclusion: Targeted anti-biofilm therapy is crucial to effectively reduce biofilm- associated infections. This highlights the importance of this study and more importantly, of the natural products as a source of potentially active soil and marine- derived bioactive compounds. Due to the scarcity of anti-biofilm drugs and to the fact that most of the discovered agents have not succeeded until now, we aim to discover and develop anti-biofilm agents with proper bioavailability, pharmacokinetic properties, and most importantly to provide safe, non-toxic administration to infected individuals.