The Anti-biofilm Effects Of Linolenic Acid With Tobramycin On Pseudomonas Aeruginosa Through Quorum Sensing System

Background: The world of microorganisms has provided both harmful and helpful effects on the human race.The beneficial effects originate from the symbiotic existence between humans and microbiota but several human illnesses have turned friendly microbes into opportunistic pathogens leading to the eruption of numerous microbial related diseases.Pseudomonas aeruginosa,a ubiquitous Gram negative opportunistic pathogen is capable of instigating severe nosocomial diseases in humans and currently,tobramycin is utilized to treat P.aeruginosa related lung infections.The bacterium is capable of forming biofilms as one of its defensive mechanisms from hostile environment.This biofilm formation is an active process that is dependent highly on environmental signals that sensitizes a cell to undergo stages of cycle growth.Moreover,the biofilm structure is fortified by the production of extracellular polymeric substances which are composed of polysaccharides,proteins,lipids and extracellular DNA.These substances contribute to resistance in providing a physiological barrier to antimicrobial diffusion.The process of biofilm formation is regulated by an array of genes that seem to be governed by the system of quorum sensing(QS).P.aeruginosa uses three interconnected QS and virulence factor production is regulated through these systems.All these features have allowed Pseudomonas aeruginosa to cause life threatening infections demanding clinicians to extensively use antibiotics to manage its infections resulting into emerging multiple drug resistant strains.As a result currently utilized antibiotics including tobramycin have become resistant.Numerous efforts through creation and utilization of antimicrobial agents have been made to treat and prevent these infections but these efforts have further been hampered by the emergence of antimicrobial resistance.Despite extensive studies on drug discovery to alleviate microbial drug resistance,the toxicity and tolerance issues of certain compounds and the continued microbial evolution has forced researchers to focus on screening various phytochemicals and dietary compounds for antimicrobial potential.Fatty acids are an essential component for human nutrition and play very vital roles in human growth.The utilization of fatty acids in pharmaceutical industries such as carrier of active substances or drug vehicles for example liposome formulation,and the presence of a plethora of information that support the effectiveness of omega-3fatty acids in lipid abnormities,diabetes,cancer,and reports of anti-inflammatory and immunomodulatory activities,stimulate interest for employing or assessing them in microbial associated infections which may provide more benefits than harm to human health.In bacteria,free fatty acids primarily target bacterial cell membrane in various ways resulting into bacteriostatic or bactericidal effects that may be seen with growth inhibition or cell apoptosis.Linoleic acid and linolenic acid,amongst these free fatty acids are essential fatty acids which cannot be synthesized by the human body but are acquired through nutrition and dietary supplements.Linolenic acid(LNA)together with its omega-3 derivatives possess antimicrobial actions on various microorganisms.We hypothesized that LNA may as well possess anti-biofilm effects on P.aeruginosa and improve the effectiveness of tobramycin.Methodology: To prove this hypothesis,we evaluated the biofilm formation using crystal violet stain for detecting the differences in biofilm biomass thicknesses.As the thicker the biomass the more stain is absorbed,and the lighter the stain in comparison with the control was considered as the drug inhibitory effect.Since the stain does not differentiate between attached live and dead cells in biofilm biomass,we employed the biofilm metabolic activity assay to understand the effects of LNA and/or tobramycin using alarmablue cell viability staining.Furthermore,the fluorescence microscopic analysis was used to visualize the biofilm biomass changes.Because biofilm formation is controlled by QS systems and since through literature,QS inhibitors have been shown to disrupt biofilm formation suggesting an antimicrobial target for novel agents,we examined the gene expression levels of selected QS,biofilm and virulence related genes using quantitative real time PCR.On the other hand,P.aeruginosa produces various virulence factors that causes tissue damage in the host.QS-related virulence factor such as swarming motility,pyocyanin production,Las A staphylolytic activity and azocasein protease production were analyzed in the absence/presence of LNA alone or in combination with tobramycin.This was to understand whether LNA or in combination with tobramycin would attenuate production of these virulence phenotypic features thereby reducing the virulence and pathogenesis of P.aeruginosa associated infections.Results: This study examined the effect of LNA and tobramycin on five clinical stains,five environmental strains and one standard stain(ATCC 27853)of P.aeruginosa.The median minimum inhibitory concentration(MIC)of LNA and tobramycin on these strains was 1.56 mg/ml and 0.3125 mg/ml,respectively.Amongst the strains used,a clinical strain identified as C2 in this study was very resistant to the sub-MIC of tobramycin(0.078 mg/ml),and hence was used to understand the effect of sub-MIC doses of LNA without much influence from tobramycin.LNA alone and in combination with tobramycin managed to disrupt biofilm formation and reduced the metabolic activities of biofilm cells.Further inhibitory effects was noticed in motility assay where all the doses used prevented swarming motility compared with the untreated control.LNA reduced pyocyanin production and azocasein activity in a dose dependent fashion while tobramycin had no significant effects.Similarly,Las A staphylolytic activity was reduced by high sub-MIC doses of LNA alone and in combination with tobramycin.However,the gene expression analysis favored the combination of LNA and tobramycin which downregulated most of the QS,biofilm and virulence factor related genes.This was also supported by the drug interaction analysis which showed additive and synergism effects.Conclusion: LNA possesses anti-biofilm effects on P.aeruginosa and can be considered as a potential agent for adjunctive therapy.A combined therapy can decrease tobramycin concentration and reduce the adverse effects of aminoglycosides which could be valuable for the therapy of P.aeruginosa infection in clinical medicine.