23Pseudomonas aeruginosa is one of the leading causes of opportunistic and 24 hospital-acquired infections worldwide. The infection with P. aeruginosa is frequently 25 linked with clinical treatment difficulties given drug resistance and abuse of 26 antibiotics. Ibuprofen, a widely used non-steroidal anti-inflammatory drug, has been 27 previously reported to exert antimicrobial activity, although the specific mechanism of 28 its action requires additional investigation. Given the regulation effects on quorum 29 sensing (QS), we hypothesized that inhibition of P. aeruginosa with ibuprofen is 30 linked with the QS systems. First, we assessed the action of ibuprofen in P. 31 aeruginosa by measuring CFU. The antimicrobial activity of ibuprofen was evaluated 32 2 by crystal violent staining and acridine orange staining at various drug concentrations 33 (0, 50, 75, and 100 μg/mL). Moreover, the effect of ibuprofen on different QS 34 virulence factors, such as pyocyanin, elastase, protease, and rhamnolipids, was 35 assessed revealing a concentration-dependent decrease (P<0.05). The effect of 36 ibuprofen was confirmed by liquid chromatography/mass spectrometry analysis of 37 3-oxo-C 12 -HSL and C 4 -HSL production. In addition, qRT-PCR results identified 38 significant suppression of Las and Rhl gene expression after 18 hours of treatment 39 with ibuprofen (P<0.05), with the most significant suppression observed at the 40 concentration of 75 μg/mL. Functional complementation with exogenous 41 3-oxo-C 12 -HSL and C 4 -HSL suggested that C 4 -HSL can recover the production of 42 virulence factors and biofilm formation in P. aeruginosa. Molecular docking of 43 ibuprofen with QS-associated proteins revealed high binding affinity. In summary, the 44 results suggest that ibuprofen is a candidate drug for the treatment of clinical 45 infections with P. aeruginosa. 46 Keywords: P. aeruginosa, ibuprofen, antimicrobial activity, quorum sensing, N-acyl 47 homoserine lactones 48 49 Introduction 50 Pseudomonas aeruginosa, the most common Gram-negative non-fermentative 51 bacteria, is one of the leading causes of opportunistic and hospital-acquired infections 52 worldwide(1). The infection can cause serious complications in patients with burns, 53 cystic fibrosis, or in immunocompromised patients with respiratory infections, sepsis, 54 osteomyelitis, endocarditis, and urinary tract infections (UTIs) (2). The infection with 55 P. aeruginosa has been liked with difficulties in clinical treatment because of 56 significant resistance of the bacterium to antibiotics (3), which is further aggravated 57 by the worldwide abuse of antibiotics. Specifically, every year approximately 700,000 58 people die from antibiotic resistance worldwide, which can likely increase to 10 59 million by 2050 if no action is taken to reduce drug resistance or to develop new 60 antibiotics. In 2017, the World Health Organization (WHO) published a list of 61 drug-resistant bacteria threating the lives of people which needs require for which 62 3 new antibio...
