Effect of azithromycin and phenylalanine-arginine beta-naphthylamide on quorum sensing and virulence factors in clinical isolates of Pseudomonas aeruginosa
,
Abstract
Background and Objectives: Pseudomonas aeruginosa is a problematic opportunistic pathogen causing several types of nosocomial infections with a high resistance rate to antibiotics. Production of many virulence factors in P. aeruginosa is regulated by quorum sensing (QS), a cell-to-cell communication mechanism. In this study, we aimed to assess and compare the inhibitory effect of azithromycin (AZM) and EPI- PAβN (efflux pump inhibitor- Phenylalanine-Arginine Beta-Naphthylamide) on QS system and QS-dependent virulence factors in P. aeruginosa clinical isolates.
Materials and Methods: A total of 50 P. aeruginosa isolates were obtained from different types of clinical specimens. Isolates were investigated for detection of QS system molecules by AHL cross-feeding bioassay and QS-dependent virulence factors; this was also confirmed by detection of QS genes (lasR, lasI, rhlR, and rhlI) using PCR assay. The inhibitory effect of sub-MIC AZM and EPI PAβN on these virulence factors was assessed.
Results: All the P. aeruginosa, producing QS signals C4HSL, failed to produce C4HSL in the presence of sub-MIC AZM, In the presence of EPI PAβN (20 µg/ml) only 14 isolates were affected, there was a significant reduction in QS-dependent virulence factors production (protease, biofilm, rhamnolipid and pyocyanin) in the presence of either 20 µg/ml EPI or sub-MIC of AZM with the inhibitory effect of AZM was more observed than PAβN.
Conclusion: Anti-QS agents like AZM and EPI (PAβN) are useful therapeutic options for P. aeruginosa due to its inhibitory effect on QS-dependent virulence factors production without selective pressure on bacteria growth, so resistance to these agents is less likely to develop.
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2. Lee J, Zhang L. The hierarchy quorum sensing network in Pseudomonas aeruginosa. Protein Cell 2015;6:26-41.
3. Williams P, Cámara M. Quorum sensing and environmental adaptation in Pseudomonas aeruginosa: a tale of regulatory networks and multifunctional signal molecules. Curr Opin Microbiol 2009;12:182-191.
4. Schuster M, Greenberg EP. A network of networks: quorum-sensing gene regulation in Pseudomonas aeruginosa. Int J Med Microbiol 2006;296:73-81.
5. Rutherford ST, Bassler BL. Bacterial quorum sensing: its role in virulence and possibilities for its control. Cold Spring Harb Perspect Med 2012;2(11): a012427.
6. González-Valdez A, Servín-González L, Juárez K, Hernandez-Aligio A, Soberón-Chávez G. The effect of specific rhlA-las-box mutations on DNA binding and gene activation by Pseudomonas aeruginosa quorum-sensing transcriptional regulators RhlR and LasR. FEMS Microbiol Lett 2014;356:217-225.
7. Nelson LK, D'Amours GH, Sproule-Willoughby KM, Morck DW, Ceri H. Pseudomonas aeruginosa las and rhl quorum-sensing systems are important for infection and inflammation in a rat prostatitis model. Microbiology (Reading) 2009;155:2612-2619.
8. Meletis G, Bagkeri M (2013). Pseudomonas aeruginosa: Multi-Drug-Resistance Development and Treatment Options. In: Infection Control; Basak, S. Ed.; IntechOpen, pp.33-56.
9. Tomás M, Doumith M, Warner M, Turton J F, Beceiro A, Bou G, et al. Efflux pumps, OprD porin, AmpC beta-lactamase, and multiresistance in Pseudomonas aeruginosa isolates from cystic fibrosis patients. Antimicrob Agents Chemother 2010;54:2219-2224.
10. Pang Z, Raudonis R, Glick BR, Lin TJ, Cheng Z. Antibiotic resistance in Pseudomonas aeruginosa: mechanisms and alternative therapeutic strategies. Biotechnol Adv 2019;37:177-192.
11. Bassetti M, Vena A, Croxatto A, Righi E, Guery B. How to manage Pseudomonas aeruginosa infections. Drugs Context 2018;7:212527.
12. Giamarellou H. Treatment options for multidrug-resistant bacteria. Expert Rev Anti Infect Ther 2006;4:601-618.
13. Jakobsen TH, Bjarnsholt T, Jensen PØ, Givskov M, Høiby N. Targeting quorum sensing in Pseudomonas aeruginosa biofilms: current and emerging inhibitors. Future Microbiol 2013;8:901-921.
14. Morohoshi T, Kato M, Fukamachi K, Kato N, Ikeda T. N-acylhomoserine lactone regulates violacein production in Chromobacterium violaceum type strain ATCC 12472. FEMS Microbiol Lett 2008;279:124-130.
15. Lutz L, Pereira DC, Paiva RM, Zavascki AP, Barth AL. Macrolides decrease the minimal inhibitory concentration of anti-pseudomonal agents against Pseudomonas aeruginosa from cystic fibrosis patients in biofilm. BMC Microbiol 2012;12:196.
16. van Delden C, Köhler T, Brunner-Ferber F, François B, Carlet J, Pechère JC. Azithromycin to prevent Pseudomonas aeruginosa ventilator-associated pneumonia by inhibition of quorum sensing: a randomized controlled trial. Intensive Care Med 2012;38:1118-1125.
17. Askoura M, Mottawea W, Abujamel T, Taher I. Efflux pump inhibitors (EPIs) as new antimicrobial agents against Pseudomonas aeruginosa. Libyan J Med 2011;6: 10.3402/ljm.v6i0.5870.
18. Hirakata Y, Kondo A, Hoshino K, Yano H, Arai K, Hirotani A, et al. Efflux pump inhibitors reduce the invasiveness of Pseudomonas aeruginosa. Int J Antimicrob Agents 2009;34:343-346.
19. Baldelli V, D'Angelo F, Pavoncello V, Fiscarelli EV, Visca P, Rampioni G, et al. Identification of FDA-approved antivirulence drugs targeting the Pseudomonas aeruginosa quorum sensing effector protein PqsE. Virulence 2020;11:652-668.
20. Santos AF, Cayô R, Schandert L, Gales AC. Evaluation of MALDI-TOF MS in the microbiology laboratory. J Bras Patol Med Lab 2013;49:191-197.
21. Clinical and Laboratory Standards Institute (CLSI). Performance Standards for Antimicrobial Susceptibility Testing, 30th edition (M100-S30). Wayne, PA: CLSI; 2020.
22. Karatuna O, Yagci A. Analysis of quorum sensing-dependent virulence factor production and its relationship with antimicrobial susceptibility in Pseudomonas aeruginosa respiratory isolates. Clin Microbiol Infect 2010;16:1770-1775.
23. Essar DW, Eberly L, Hadero A, Crawford IP. Identification and characterization of genes for a second anthranilate synthase in Pseudomonas aeruginosa: interchangeability of the two anthranilate synthases and evolutionary implications. J Bacteriol 1990; 172: 884-900.
24. Vijayaraghavan P, Vincent SGP. A simple method for the detection of protease activity on agar plate using bromocresolgreen dye. J Biochem Tech 2013; 4:628-630.
25. Bodour AA, Miller-Maier RM. Application of a modified drop-collapse technique for surfactant quantitation and screening of biosurfactant-producing microorganisms. J Microbiol Methods 1998;32:273-280.
26. Skindersoe ME, Alhede M, Phipps R, Yang L, Jensen PO, Rasmussen TB, et al. Effects of antibiotics on quorum sensing in Pseudomonas aeruginosa. Antimicrob Agents Chemother 2008;52:3648-3663.
27. Hassan A, Usman J, Kaleem F, Omair M, Khalid A, Iqbal M. Evaluation of different detection methods of biofilm formation in the clinical isolates. Braz J Infect Dis 2011;15:305-311.
28. Ahmed OB, Asghar AH, Elhassan MM. Comparison of three DNA extraction methods for polymerase chain reaction (PCR) analysis of bacterial genomic DNA. Afr J Microbiol Res 2014;8:598-602.
29. Schaber JA, Carty NL, McDonald NA, Graham ED, Cheluvappa R, Griswold JA, et al. Analysis of quorum sensing-deficient clinical isolates of Pseudomonas aeruginosa. J Med Microbiol 2004;53:841-853.
30. Beceiro A, Tomás M, Bou G. Antimicrobial resistance and virulence: a successful or deleterious association in the bacterial world? Clin Microbiol Rev 2013;26:185-230.
31. Fleitas Martínez O, Cardoso MH, Ribeiro SM, Franco OL. Recent advances in anti-virulence therapeutic strategies with a focus on dismantling bacterial membrane microdomains, toxin neutralization, quorum-sensing interference and biofilm inhibition. Front Cell Infect Microbiol 2019;9:74.
32. Wang H, Tu F, Gui Z, Lu X, Chu W. Antibiotic resistance profiles and quorum sensing-dependent virulence factors in clinical isolates of Pseudomonas aeruginosa. Indian J Microbiol 2013;53:163-167.
33. Senturk S, Ulusoy S, Bosgelmez-Tinaz G, Yagci A. Quorum sensing and virulence of Pseudomonas aeruginosa during urinary tract infections. J Infect Dev Ctries 2012;6:501-507.
34. Pearson JP, Feldman M, Iglewski BH, Prince A. Pseudomonas aeruginosa cell-to-cell signaling is required for virulence in a model of acute pulmonary infection. Infect Immun 2000;68:4331-4334.
35. Zhu H, Bandara R, Conibear TC, Thuruthyil SJ, Rice SA, Kjelleberg S, et al. Pseudomonas aeruginosa with lasI quorum-sensing deficiency during corneal infection. Invest Ophthalmol Vis Sci 2004;45:1897-1903.
36. Boşgelmez-Tinaz G, Ulusoy S. Characterization of N-butanoyl-L-homoserine lactone (C4-HSL) deficient clinical isolates of Pseudomonas aeruginosa. Microb Pathog 2008;44:13-19.
37. Micek ST, Wunderink RG, Kollef MH, Chen C, Rello J, Chastre J, et al. An international multicenter retrospective study of Pseudomonas aeruginosa nosocomial pneumonia: impact of multidrug resistance. Crit Care 2015;19:219.
38. Neville N, Jia Z. Approaches to the structure-based design of antivirulence drugs: therapeutics for the post-antibiotic era. Molecules 2019;24:378.
39. Rasko DA, Sperandio V. Anti-virulence strategies to combat bacteria-mediated disease. Nat Rev Drug Discov 2010;9:117-128.
40. Imperi F, Leoni L, Visca P. Antivirulence activity of azithromycin in Pseudomonas aeruginosa. Front Microbiol 2014;5:178.
41. G Gillis RJ, Iglewski BH. Azithromycin retards Pseudomonas aeruginosa biofilm formation. J Clin Microbiol 2004;42:5842-5845.
42. Hoffmann N, Lee B, Hentzer M, Rasmussen TB, Song Z, Johansen HK, et al. Azithromycin blocks quorum sensing and alginate polymer formation and increases the sensitivity to serum and stationary-growth-phase killing of Pseudomonas aeruginosa and attenuates chronic P. aeruginosa lung infection in Cftr(-/-) mice. Antimicrob Agents Chemother 2007;51:3677-3687.
43. Sofer D, Gilboa-Garber N, Belz A, Garber NC. ‘Subinhibitory’erythromycin represses production of Pseudomonas aeruginosa lectins, autoinducer and virulence factors. Chemotherapy 1999;45:335-341.
44. Wagner T, Soong G, Sokol S, Saiman L, Prince A. Effects of azithromycin on clinical isolates of Pseudomonas aeruginosa from cystic fibrosis patients. Chest 2005;128:912-919.
45. Hirakata Y, Srikumar R, Poole K, Gotoh N, Suematsu T, Kohno S, et al. Multidrug efflux systems play an important role in the invasiveness of Pseudomonas aeruginosa. J Exp Med 2002;196:109-118.
46. Join-Lambert OF, Michéa-Hamzehpour M, Köhler T, Chau F, Faurisson F, Dautrey S, et al. Differential selection of multidrug efflux mutants by trovafloxacin and ciprofloxacin in an experimental model of Pseudomonas aeruginosa acute pneumonia in rats. Antimicrob Agents Chemother 2001;45(2):571-576.
47. Mahamoud A, Chevalier J, Alibert-Franco S, Kern WV, Pagès JM. Antibiotic efflux pumps in Gram-negative bacteria: the inhibitor response strategy. J Antimicrob Chemother 2007;59:1223-1229.
48. Renau TE, Léger R, Filonova L, Flamme E M, Wang M, Yen R, et al. Conformationally-restricted analogues of efflux pump inhibitors that potentiate the activity of levofloxacin in Pseudomonas aeruginosa. Bioorg Med Chem Lett 2003;13:2755-2758.
49. Alcalde-Rico M, Hernando-Amado S, Blanco P, Martínez JL. Multidrug efflux pumps at the crossroad between antibiotic resistance and bacterial virulence. Front Microbiol 2016;7:1483.
2. Lee J, Zhang L. The hierarchy quorum sensing network in Pseudomonas aeruginosa. Protein Cell 2015;6:26-41.
3. Williams P, Cámara M. Quorum sensing and environmental adaptation in Pseudomonas aeruginosa: a tale of regulatory networks and multifunctional signal molecules. Curr Opin Microbiol 2009;12:182-191.
4. Schuster M, Greenberg EP. A network of networks: quorum-sensing gene regulation in Pseudomonas aeruginosa. Int J Med Microbiol 2006;296:73-81.
5. Rutherford ST, Bassler BL. Bacterial quorum sensing: its role in virulence and possibilities for its control. Cold Spring Harb Perspect Med 2012;2(11): a012427.
6. González-Valdez A, Servín-González L, Juárez K, Hernandez-Aligio A, Soberón-Chávez G. The effect of specific rhlA-las-box mutations on DNA binding and gene activation by Pseudomonas aeruginosa quorum-sensing transcriptional regulators RhlR and LasR. FEMS Microbiol Lett 2014;356:217-225.
7. Nelson LK, D'Amours GH, Sproule-Willoughby KM, Morck DW, Ceri H. Pseudomonas aeruginosa las and rhl quorum-sensing systems are important for infection and inflammation in a rat prostatitis model. Microbiology (Reading) 2009;155:2612-2619.
8. Meletis G, Bagkeri M (2013). Pseudomonas aeruginosa: Multi-Drug-Resistance Development and Treatment Options. In: Infection Control; Basak, S. Ed.; IntechOpen, pp.33-56.
9. Tomás M, Doumith M, Warner M, Turton J F, Beceiro A, Bou G, et al. Efflux pumps, OprD porin, AmpC beta-lactamase, and multiresistance in Pseudomonas aeruginosa isolates from cystic fibrosis patients. Antimicrob Agents Chemother 2010;54:2219-2224.
10. Pang Z, Raudonis R, Glick BR, Lin TJ, Cheng Z. Antibiotic resistance in Pseudomonas aeruginosa: mechanisms and alternative therapeutic strategies. Biotechnol Adv 2019;37:177-192.
11. Bassetti M, Vena A, Croxatto A, Righi E, Guery B. How to manage Pseudomonas aeruginosa infections. Drugs Context 2018;7:212527.
12. Giamarellou H. Treatment options for multidrug-resistant bacteria. Expert Rev Anti Infect Ther 2006;4:601-618.
13. Jakobsen TH, Bjarnsholt T, Jensen PØ, Givskov M, Høiby N. Targeting quorum sensing in Pseudomonas aeruginosa biofilms: current and emerging inhibitors. Future Microbiol 2013;8:901-921.
14. Morohoshi T, Kato M, Fukamachi K, Kato N, Ikeda T. N-acylhomoserine lactone regulates violacein production in Chromobacterium violaceum type strain ATCC 12472. FEMS Microbiol Lett 2008;279:124-130.
15. Lutz L, Pereira DC, Paiva RM, Zavascki AP, Barth AL. Macrolides decrease the minimal inhibitory concentration of anti-pseudomonal agents against Pseudomonas aeruginosa from cystic fibrosis patients in biofilm. BMC Microbiol 2012;12:196.
16. van Delden C, Köhler T, Brunner-Ferber F, François B, Carlet J, Pechère JC. Azithromycin to prevent Pseudomonas aeruginosa ventilator-associated pneumonia by inhibition of quorum sensing: a randomized controlled trial. Intensive Care Med 2012;38:1118-1125.
17. Askoura M, Mottawea W, Abujamel T, Taher I. Efflux pump inhibitors (EPIs) as new antimicrobial agents against Pseudomonas aeruginosa. Libyan J Med 2011;6: 10.3402/ljm.v6i0.5870.
18. Hirakata Y, Kondo A, Hoshino K, Yano H, Arai K, Hirotani A, et al. Efflux pump inhibitors reduce the invasiveness of Pseudomonas aeruginosa. Int J Antimicrob Agents 2009;34:343-346.
19. Baldelli V, D'Angelo F, Pavoncello V, Fiscarelli EV, Visca P, Rampioni G, et al. Identification of FDA-approved antivirulence drugs targeting the Pseudomonas aeruginosa quorum sensing effector protein PqsE. Virulence 2020;11:652-668.
20. Santos AF, Cayô R, Schandert L, Gales AC. Evaluation of MALDI-TOF MS in the microbiology laboratory. J Bras Patol Med Lab 2013;49:191-197.
21. Clinical and Laboratory Standards Institute (CLSI). Performance Standards for Antimicrobial Susceptibility Testing, 30th edition (M100-S30). Wayne, PA: CLSI; 2020.
22. Karatuna O, Yagci A. Analysis of quorum sensing-dependent virulence factor production and its relationship with antimicrobial susceptibility in Pseudomonas aeruginosa respiratory isolates. Clin Microbiol Infect 2010;16:1770-1775.
23. Essar DW, Eberly L, Hadero A, Crawford IP. Identification and characterization of genes for a second anthranilate synthase in Pseudomonas aeruginosa: interchangeability of the two anthranilate synthases and evolutionary implications. J Bacteriol 1990; 172: 884-900.
24. Vijayaraghavan P, Vincent SGP. A simple method for the detection of protease activity on agar plate using bromocresolgreen dye. J Biochem Tech 2013; 4:628-630.
25. Bodour AA, Miller-Maier RM. Application of a modified drop-collapse technique for surfactant quantitation and screening of biosurfactant-producing microorganisms. J Microbiol Methods 1998;32:273-280.
26. Skindersoe ME, Alhede M, Phipps R, Yang L, Jensen PO, Rasmussen TB, et al. Effects of antibiotics on quorum sensing in Pseudomonas aeruginosa. Antimicrob Agents Chemother 2008;52:3648-3663.
27. Hassan A, Usman J, Kaleem F, Omair M, Khalid A, Iqbal M. Evaluation of different detection methods of biofilm formation in the clinical isolates. Braz J Infect Dis 2011;15:305-311.
28. Ahmed OB, Asghar AH, Elhassan MM. Comparison of three DNA extraction methods for polymerase chain reaction (PCR) analysis of bacterial genomic DNA. Afr J Microbiol Res 2014;8:598-602.
29. Schaber JA, Carty NL, McDonald NA, Graham ED, Cheluvappa R, Griswold JA, et al. Analysis of quorum sensing-deficient clinical isolates of Pseudomonas aeruginosa. J Med Microbiol 2004;53:841-853.
30. Beceiro A, Tomás M, Bou G. Antimicrobial resistance and virulence: a successful or deleterious association in the bacterial world? Clin Microbiol Rev 2013;26:185-230.
31. Fleitas Martínez O, Cardoso MH, Ribeiro SM, Franco OL. Recent advances in anti-virulence therapeutic strategies with a focus on dismantling bacterial membrane microdomains, toxin neutralization, quorum-sensing interference and biofilm inhibition. Front Cell Infect Microbiol 2019;9:74.
32. Wang H, Tu F, Gui Z, Lu X, Chu W. Antibiotic resistance profiles and quorum sensing-dependent virulence factors in clinical isolates of Pseudomonas aeruginosa. Indian J Microbiol 2013;53:163-167.
33. Senturk S, Ulusoy S, Bosgelmez-Tinaz G, Yagci A. Quorum sensing and virulence of Pseudomonas aeruginosa during urinary tract infections. J Infect Dev Ctries 2012;6:501-507.
34. Pearson JP, Feldman M, Iglewski BH, Prince A. Pseudomonas aeruginosa cell-to-cell signaling is required for virulence in a model of acute pulmonary infection. Infect Immun 2000;68:4331-4334.
35. Zhu H, Bandara R, Conibear TC, Thuruthyil SJ, Rice SA, Kjelleberg S, et al. Pseudomonas aeruginosa with lasI quorum-sensing deficiency during corneal infection. Invest Ophthalmol Vis Sci 2004;45:1897-1903.
36. Boşgelmez-Tinaz G, Ulusoy S. Characterization of N-butanoyl-L-homoserine lactone (C4-HSL) deficient clinical isolates of Pseudomonas aeruginosa. Microb Pathog 2008;44:13-19.
37. Micek ST, Wunderink RG, Kollef MH, Chen C, Rello J, Chastre J, et al. An international multicenter retrospective study of Pseudomonas aeruginosa nosocomial pneumonia: impact of multidrug resistance. Crit Care 2015;19:219.
38. Neville N, Jia Z. Approaches to the structure-based design of antivirulence drugs: therapeutics for the post-antibiotic era. Molecules 2019;24:378.
39. Rasko DA, Sperandio V. Anti-virulence strategies to combat bacteria-mediated disease. Nat Rev Drug Discov 2010;9:117-128.
40. Imperi F, Leoni L, Visca P. Antivirulence activity of azithromycin in Pseudomonas aeruginosa. Front Microbiol 2014;5:178.
41. G Gillis RJ, Iglewski BH. Azithromycin retards Pseudomonas aeruginosa biofilm formation. J Clin Microbiol 2004;42:5842-5845.
42. Hoffmann N, Lee B, Hentzer M, Rasmussen TB, Song Z, Johansen HK, et al. Azithromycin blocks quorum sensing and alginate polymer formation and increases the sensitivity to serum and stationary-growth-phase killing of Pseudomonas aeruginosa and attenuates chronic P. aeruginosa lung infection in Cftr(-/-) mice. Antimicrob Agents Chemother 2007;51:3677-3687.
43. Sofer D, Gilboa-Garber N, Belz A, Garber NC. ‘Subinhibitory’erythromycin represses production of Pseudomonas aeruginosa lectins, autoinducer and virulence factors. Chemotherapy 1999;45:335-341.
44. Wagner T, Soong G, Sokol S, Saiman L, Prince A. Effects of azithromycin on clinical isolates of Pseudomonas aeruginosa from cystic fibrosis patients. Chest 2005;128:912-919.
45. Hirakata Y, Srikumar R, Poole K, Gotoh N, Suematsu T, Kohno S, et al. Multidrug efflux systems play an important role in the invasiveness of Pseudomonas aeruginosa. J Exp Med 2002;196:109-118.
46. Join-Lambert OF, Michéa-Hamzehpour M, Köhler T, Chau F, Faurisson F, Dautrey S, et al. Differential selection of multidrug efflux mutants by trovafloxacin and ciprofloxacin in an experimental model of Pseudomonas aeruginosa acute pneumonia in rats. Antimicrob Agents Chemother 2001;45(2):571-576.
47. Mahamoud A, Chevalier J, Alibert-Franco S, Kern WV, Pagès JM. Antibiotic efflux pumps in Gram-negative bacteria: the inhibitor response strategy. J Antimicrob Chemother 2007;59:1223-1229.
48. Renau TE, Léger R, Filonova L, Flamme E M, Wang M, Yen R, et al. Conformationally-restricted analogues of efflux pump inhibitors that potentiate the activity of levofloxacin in Pseudomonas aeruginosa. Bioorg Med Chem Lett 2003;13:2755-2758.
49. Alcalde-Rico M, Hernando-Amado S, Blanco P, Martínez JL. Multidrug efflux pumps at the crossroad between antibiotic resistance and bacterial virulence. Front Microbiol 2016;7:1483.
| Files | ||
| Issue | Vol 13 No 1 (2021) | |
| Section | Original Article(s) | |
| DOI | https://doi.org/10.18502/ijm.v13i1.5491 | |
| Keywords | ||
| Pseudomonas aeruginosa; Virulence factors; Quorum sensing; Azithromycin; Phenylalanine-arginine β-naphthylamide | ||
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How to Cite
1.
Elsheredy A, El-Soudany I, Elsherbini E, Metwally D, Ghazal A. Effect of azithromycin and phenylalanine-arginine beta-naphthylamide on quorum sensing and virulence factors in clinical isolates of Pseudomonas aeruginosa. Iran J Microbiol. 2021;13(1):37-49.
