Squaramide Homoserine Lactone Derivatives and their Abilities to Modulate Pseudomonas aeruginosa Biofilm Formation

Abstract

Biofilm formation is a bacterial group behavior that is responsible for chronic and persistent infections. In addition, the biofilm mode of growth can decrease the susceptibility of bacteria to antibiotics, leading to antibiotic resistance. The gram-negative opportunistic bacterial pathogen Pseudomonas aeruginosa communicates using autoinducers. One of the key autoinducers is N-acyl-l-homoserine lactones (AHL). Herein, we reported the design and synthesis of squaramide homoserine lactone derivatives and their abilities to inhibit Pseudomonas aeruginosa biofilm formation. Subtle changes in chemical structure resulted in the inversion of biofilm formation activity. Among the AHL mimics tested, the squaramide homoserine lactone with an oligoethylene glycol chain 4H exhibited the most potent anti-biofilm activity, with an IC₅₀ of 0.28 mM. In addition, these AHL derivatives did not affect bacterial survival. Hence, biofilm formation could be suppressed without direct pressure on bacterial growth, preventing the rapid development of bacterial resistance.

Key words

biofilm inhibitors - Pseudomonas aeruginosa - quorum sensing - N-acyl-l-homoserine lactone - squaramide

Publication History

Received: 03 March 2025

Accepted after revision: 19 May 2025

Article published online:
08 July 2025

• References and Notes


Current address:
• 1a Molecular Biology of Dengue and Flaviviruses Research Team, Medical Molecular Biotechnology Research Group and
• 1b Medical Biotechnology Research Unit, National Center for Genetic Engineering and Biotechnology (BIOTEC), National Science and Technology Development Agency (NSTDA), Pathum Thani 12120, Thailand.
• 2 Hall-Stoodley L, Costerton J, Stoodley P. Nat. Rev. Microbiol. 2004; 2: 95
• 3 Davies D. Nat. Rev. Drug Discovery 2003; 2: 114
• 4 Costerton JW, Cheng KJ, Geesey GG, Ladd TI, Nickel JC, Dasgupta M, Marrie TJ. Annu. Rev. Microbiol. 1987; 41: 435
• 5 Stewart PS, Costerton JW. Lancet 2001; 358: 135
• 6a Worthington RJ, Richards JJ, Melander C. Org. Biomol. Chem. 2012; 10: 7457
• 6b Mah T.-FC, O’Toole GA. Trends Microbiol. 2001; 9: 34
• 7 Ceri H, Olson ME, Stremick C, Read RR, Morck D, Buret A. J. Clin. Microbiol. 1999; 37: 1771
• 8 Olson ME, Ceri H, Morck DW, Buret AG, Read RR. Can. J. Vet. Res. 2002; 66: 86
• 9 Yassien M, Khardori N, Ahmedy A, Toama M. Antimicrob. Agents Chemother. 1995; 39: 2262
• 10 Williams I, Venables WA, Lloyd D, Paul F, Critchley I. Microbiology 1997; 143: 2407
• 11 Geske GD, O’Neill JC, Blackwell HE. Chem. Soc. Rev. 2008; 37: 1432
• 12 Tuon FF, Dantas LR, Suss PH, Tasca Ribeiro VS. Pathogens 2022; 11: 300
• 13 Geske GD, Wezeman RJ, Siegel AP, Blackwell HE. J. Am. Chem. Soc. 2005; 127: 12762
• 14 Smith RS, Harris SG, Phipps R, Iglewski B. J. Bacteriol. 2002; 184: 1132
• 15 Schaefer AL, Hanzelka BL, Eberhard A, Greenberg EP. J. Bacteriol. 1996; 178: 2897
• 16 Frezza M, Castang S, Esteplane J, Soulère L, Deshayes C, Chantegrel B, Nasser W, Queneau Y, Reverchon S, Doutheau A. Bioorg. Med. Chem. 2006; 14: 4781
• 17 Chemical syntheses: All commercially available materials were purchased from commercial suppliers and were used without additional purification, unless otherwise noted. The syntheses of AHL derivatives are described in the Supporting Information.
• 18 Biological evaluation: Luria Broth (LB), Tryptic soy broth (TSB), and Muller–Hinton broth (MHB) were purchased from BD (Sparks, MD, USA). Bacto agar was from LabM (Quest Park, UK). P. aeruginosa ATCC 27853 was kindly given by the Department of Microbiology, Faculty of Science, Mahidol University. Disk diffusion: The disk diffusion susceptibility testing was performed based on the standard protocol suggested by the Clinical and Laboratory Standard Institute (CLSI).¹⁹ P. aeruginosa ATCC 27853 was sub-cultured in LB and cultures were then incubated in a shaking incubator at 37 °C. An overnight culture (100 μL) with an inoculum density of 1 × 10⁶ CFU/mL was spread on the surface of Muller–Hinton agar (MHA) plates by using a sterile swab. Whatman filter paper disks (6 mm) were gently placed on the inoculated surface and the test compound (30 µg) in DMSO was subsequently added onto each small filter paper disks. The resulting plates were incubated at 37 °C for 18–20 hours in an ambient air incubator. After incubation, the diameters of the inhibitory zones were measured using sliding calipers. The experiments were performed in triplicate and the average zone with standard error is reported. Biofilm inhibition assay: A quantitative biofilm assay using crystal violet staining solution was performed based on a previously reported procedure.²⁰ In brief, P. aeruginosa was grown statically for 18 hours at 37 °C in LB. An overnight culture was diluted to 2 × 10⁷ CFU/mL in LB. A diluted culture (100 μL) was transferred into a sterilized polystyrene 96-well microtiter plate. The inoculum was incubated in the presence and in the absence of our synthetic compounds for 24 hours in a 37 °C incubator. Planktonic bacteria were removed and each well was washed with distilled water. The biofilm was stained with 0.1% crystal violet solution for 30 minutes at room temperature. Crystal violet solution was extracted from the biofilm using 33% acetic acid solution. The amount of biofilm formed was determined by measuring the optical density (OD) at a wavelength of 595 nm.
• 19 Performance Standards for Antimicrobial Susceptibility Testing, 32nd ed., CLSI Supplement M100. Clinical and Laboratory Standards Institute; Wayne: 2022
• 20 Merritt JH, Kadouri DE, O’Toole GA. Curr. Protoc. Microbiol. 2005; 00 (01) 1B.1.1-1B.1.17
• 21 Vannini A, Volpari C, Gargioli C, Muraglia E, Cortese R, De Francesco R, Neddermann P, Di Marco S. EMBO J. 2002; 21: 4393
• 22 Ishida T, Ikeda T, Takiguchi N, Kuroda A, Ohtake H, Kato J. Appl. Environ. Microbiol. 2007; 73: 3183
• 23 Tang D, Lin Y, Yao H, Liu Y, Xi Y, Li M, Mao A. Appl. Microbiol. Biotechnol. 2024; 108: 418