Novel anti-biofilm natural products from microorganisms isolated from Lebanon

Abstract

Background: Antimicrobial resistance (AMR) is rising at an alarming pace causing a major crisis threatening public health globally. Among various mechanisms of drug resistance, bacterial biofilms, which are layers of microbial cells attached to a surface and buried firmly in an exopolysaccharide matrix, is considered a predominant one. Biofilm associated bacteria are less sensitive to antibiotics than the planktonic cells. Indeed, resistance to gentamicin and ceftazidime in Escherichia coli, tazobactam, and colistin in Klebsiella pneumonia, and ciprofloxacin in Pseudomonas aeruginosa was related to biofilm formation. Since it is becoming increasingly clear that biofilm formation is implicated in the spread of multidrug-resistance, and conventional antibiotics are being inadequate at eradicating biofilm-forming pathogens, it is highly urgent to discover novel molecules that exhibit strong antibiofilm activity and can be combined with antibacterial agents. Fortunately, soil and marine microorganisms are potential natural sources for the isolation of natural compounds with effective biological activities. However, scarce material is found in the literature, and little research is being done about screening for antibiofilm compounds. This study aims to isolate novel antibiofilm compounds produced by environmental bacteria from several regions in Lebanon.

Methods: Marine and soil samples were collected from three different areas in Lebanon. Serial dilutions were done on the samples for Actinomycetes isolation. Subsequently, 30μl of each dilution were plated on ISP3 and soil agar. After purification, pure colonies were stored in 50% glycerol at -80C. Then, these selected environmental bacteria were cultivated in 14 different culture media in order to produce secondary metabolites. The latter was then extracted via acetone/methanol and then tested for their anti-biofilm activity against Pseudomonas aeruginosa (PAN14), Staphylococcus aureus (N315), Acinetobacter baumannii (DSM 30008 & T36). Screening of the crude extracts was done in microtiter plates via two assays. The first checks extracts’ potency to inhibit biofilm occurrence, whereas the other evaluates its capacity to inhibit biofilm formation after the film is established.

Results: According to our findings, crude extracts derived from the marine sample TBJ13C and soil sample BM9C demonstrated a strong anti-biofilm activity against P. Aeruginosa (PAN14). Both extracts with their hexane and ethyl acetate fractions, respectively, showed highly effective inhibition of biofilm growth, as well as eradication of preformed ones in PAN14. Further, crude extracts produced by MM3, MM7, and MM9 also exhibited significant anti-biofilm activity in preventing biofilm formation, primarily in PAN14, as some had the ability to do so in two other models, namely, Staphylococcus aureus MRSA and Acinetobacter baumannii (T36). Conclusion: Therefore, this process steps forward from a screening hit to phenotypic and biochemical characterization, anti-biofilm compound isolation, molecular characterization, and structure elucidation.