Original Article

Different effects of sub-minimum inhibitory concentrations of gentamicin on the expression of genes involved in alginate production and biofilm formation of Pseudomonas aeruginosa

Abstract

Background and Objectives: Antibiotics at sub-minimum inhibitory concentrations (sub-MIC) may alter bacterial virulence factors. The objective of this study was to investigate the effect of gentamicin at sub-MIC concentrations on the expression of genes involved in alginate production and biofilm formation of Pseudomonas aeruginosa.
Materials and Methods: The broth microdilution method was used to determine the MIC of gentamicin for three P. aeruginosa clinical isolates (P1-P3) and standard strains (PAO1 and 8821M). Alginate production and biofilm formation of the bacteria in the presence and absence of sub-MIC concentrations of gentamicin were measured using microtiter plate and carbazole assay, respectively. The real-time PCR method was used to determine the effect of gentamicin at sub-MIC concentrations on the expression level of genes involved in biofilm formation (pelA and pslA) and alginate production (algD and algU).
Results: Gentamicin at sub-MIC concentrations significantly reduced alginate production, biofilm formation, and the expression of alginate and biofilm-encoding genes in clinical isolate P1. This inhibitory effect was also observed on the alginate production of 8821M strain and biofilm formation of PAO1strain. In clinical isolates, P2 and P3, alginate production, biofilm formation, and the expression of alginate and biofilm-encoding genes were significantly increased in exposure to sub-MIC concentrations of gentamicin.
Conclusion: This study showed that different phenotypic changes in clinical isolates and standard strains of P. aeruginosa in exposure to sub-MIC concentrations of gentamicin are associated with changes in the expression of virulence genes. Further researches are required to understand the mechanisms involved in regulating the expression of virulence genes after exposure to sub-MIC concentrations of antibiotics.

1. Cho H, Huang X, Lan Piao Y, Eun Kim D, Yeon Lee S, Jeong Yoon E, et al. Molecular modeling and redesign of alginate lyase from Pseudomonas aeruginosa for accelerating CRPA biofilm degradation. Proteins 2016; 84: 1875-1887.
2. Farjah A, Owlia P, Siadat SD, Mousavi SF, Shafiee Ardestani M, Khorsand Mohammad pour H. Immunological evaluation of an alginate‐based conjugate as a vaccine candidate against Pseudomonas aeruginosa. APMIS 2015; 123: 175-183.
3. Wu DQ, Cheng H, Duan Q, Huang W. Sodium houttuyfonate inhibits biofilm formation and alginate biosynthesis‑associated gene expression in a clinical strain of Pseudomonas aeruginosa in vitro. Exp Ther Med 2015; 10: 753-758.
4. Lau CH, Hughes D, Poole K. MexY-promoted aminoglycoside resistance in Pseudomonas aeruginosa: involvement of a putative proximal binding pocket in aminoglycoside recognition. mBio 2014; 5(2): e01068.
5. Shen L, Shi Y, Zhang D, Wei J, Surette MG, Duan K. Modulation of secreted virulence factor genes by subinhibitory concentrations of antibiotics in Pseudomonas aeruginosa. J Microbiol 2008; 46: 441-447.
6. A Al-Kafaween M, Mohd Hilmi AB, A Nagi Al-Jamal H, A Elsahoryi N, Jaffar N, Khairi Zahri M. Pseudomonas aeruginosa and Streptococcus Pyogenes exposed to Malaysian Trigona honey in vitro demonstrated downregulation of virulence factor. Iran J Biotechnol 2020; 18(4): e2542.
7. Wilton M, Charron-Mazenod L, Moore R, Lewenza S. Extracellular DNA acidifies biofilms and induces aminoglycoside resistance in Pseudomonas aeruginosa. Antimicrob b Agents Chemother 2015; 60: 544-553.
8. Skariyachan S, Sridhar VS, Packirisamy S, Kumargowda ST, Challapilli SB. Recent perspectives on the molecular basis of biofilm formation by Pseudomonas aeruginosa and approaches for treatment and biofilm dispersal. Folia Microbiol (Praha) 2018; 63: 413-432.
9. Otani S, Hiramatsu K, Hashinaga K, Komiya K, Umeki K, Kishi K, et al. Sub-minimum inhibitory concentrations of ceftazidime inhibit Pseudomonas aeruginosa biofilm formation. J Infect Chemother 2018; 24: 428-433.
10. Ghafoor A, Hay ID, Rehm BH. Role of exopolysaccharides in Pseudomonas aeruginosa biofilm formation and architecture. Appl Environ Microbiol 2011; 77: 5238-5246.
11. Pournajaf A, Razavi S, Irajian G, Ardebili A, Erfani Y, Solgi S, et al. Integron types, antimicrobial resistance genes, virulence gene profile, alginate production and biofilm formation in Iranian cystic fibrosis Pseudomonas aeruginosa isolates. Infez Med 2018; 26: 226-236.
12. Hay ID, Gatland K, Campisano A, Jordens JZ, Rehm BH. Impact of alginate overproduction on attachment and biofilm architecture of a supermucoid Pseudomonas aeruginosa strain. Appl Environ Microbiol 2009; 75: 6022-6025.
13. Powell LC, Pritchard MF, Ferguson EL, Powell KA, Patel SU, Rye PD, et al. Targeted disruption of the extracellular polymeric network of Pseudomonas aeruginosa biofilms by alginate oligosaccharides. NPJ Biofilms Microbiomes 2018; 4: 13.
14. Withers TR, Yin Y, Yu HD. Identification of novel genes associated with alginate production in Pseudomonas aeruginosa using mini-himar1 mariner transposon-mediated mutagenesis. J Vis Exp 2014; (85): 51346.
15. Sautter R, Ramos D, Schneper L, Ciofu O, Wassermann T, Koh CL, et al. A complex multilevel attack on Pseudomonas aeruginosa algT/U expression and algT/U activity results in the loss of alginate production. Gene 2012; 498: 242-253.
16. Jones CJ, Wozniak DJ. Psl produced by mucoid Pseudomonas aeruginosa contributes to the establishment of biofilms and immune evasion. mBio 2017; 8(3): e00864-17.
17. Hoffman LR, D'Argenio DA, MacCoss MJ, Zhang Z, Jones RA, Miller SI. Aminoglycoside antibiotics induce bacterial biofilm formation. Nature 2005; 436: 1171-1175.
18. Sato Y, Unno Y, Ubagai T, Ono Y. Sub-minimum inhibitory concentrations of colistin and polymyxin B promote Acinetobacter baumannii biofilm formation. PLoS One 2018; 13(3): e0194556.
19. Fonseca AP, Extremina C, Fonseca AF, Sousa JC. Effect of subinhibitory concentration of piperacillin/tazobactam on Pseudomonas aeruginosa. J Med Microbiol G12004; 53(Pt 9): 903-910.
20. Bruchmann J, Kirchen S, Schwartz T. Sub-inhibitory concentrations of antibiotics and wastewater influencing biofilm formation and gene expression of multi-resistant Pseudomonas aeruginosa wastewater isolates. Environ Sci Pollut Res Int 2013; 20: 3539-3549.
21. Clinical and laboratory standard institute (CLSI). Performance standards for antimicrobial susceptibility testing, 27th edition ( M100-S30). Wayne, PA: CLSI; 2020.
22. Müsken M, Di Fiore S, Römling U, Häussler S. A 96-well-plate–based optical method for the quantitative and qualitative evaluation of Pseudomonas aeruginosa biofilm formation and its application to susceptibility testing. Nat Protoc 2010; 5: 1460-1469.
23. Davarzani F, Saidi N, Besharati S, Saderi H, Rasooli I, Owlia P. Evaluation of antibiotic resistance pattern, alginate and biofilm production in clinical isolates of Pseudomonas aeruginosa. Iran J Public Health 2021; 50: 341-349.
24. Al-Kafaween MA, Al-Jamal HAN, Hilmi ABM, Elsahoryi NA, Jaffar N, Zahri MK. Antibacterial properties of selected Malaysian Tualang honey against Pseudomonas aeruginosa and Streptococcus pyogenes. Iran J Microbiol 2020; 12: 565-576.
25. Ghadaksaz A, Imani Fooladi AA, Mahmoodzadeh Hosseini H, Amin M. The prevalence of some Pseudomonas virulence genes related to biofilm formation and alginate production among clinical isolates. J Appl Biomed 2015; 13: 61-68.
26. Livak KJ, Schmittgen TD. Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) method. Methods 2001; 25: 402-408.
27. Mousavi Nadoshan S, Owlia P, Moein Najafabadi L, Rasooli I, Saderi H, Salari MH. Effects of sub-inhibitory concentrations of essential oils of Mentha spicata and Cumminum cyminum on virulence factors of Pseudomonas aeruginosa. J Med Plants 2010; 9: 124-130.
28. Subrt N, Mesak LR, Davies J. Modulation of virulence gene expression by cell wall active antibiotics in Staphylococcus aureus. J Antimicrob Chemother 2011; 66: 979-984.
29. Bahari S, Zeighami H, Mirshahabi H, Roudashti S, Haghi F. Inhibition of Pseudomonas aeruginosa quorum sensing by subinhibitory concentrations of curcumin with gentamicin and azithromycin. J Glob Antimicrob Resist 2017; 10: 21-28.
30. Gupta P, Chhibber S, Harjai K. Subinhibitory concentration of ciprofloxacin targets quorum sensing system of Pseudomonas aeruginosa causing inhibition of biofilm formation & reduction of virulence. Indian J Med Res 2016; 143: 643-651.
31. Bagge N, Schuster M, Hentzer M, Ciofu O, Givskov M, Greenberg EP, et al. Pseudomonas aeruginosa biofilms exposed to imipenem exhibit changes in global gene expression and beta-lactamase and alginate production. Antimicrob Agents Chemother 2004; 48: 1175-1187.
32. Navidifar T, Amin M, Rashno M. Effects of sub-inhibitory concentrations of meropenem and tigecycline on the expression of genes regulating pili, efflux pumps and virulence factors involved in biofilm formation by Acinetobacter baumannii. Infect Drug Resist 2019; 12: 1099-1111.
Files
IssueVol 13 No 6 (2021) QRcode
SectionOriginal Article(s)
DOI https://doi.org/10.18502/ijm.v13i6.8085
Keywords
Alginate; Biofilm; Gene expression; Gentamicin; Pseudomonas aeruginosa

Rights and permissions
Creative Commons License This work is licensed under a Creative Commons Attribution-NonCommercial 4.0 International License.
How to Cite
1.
Davarzani F, Yousefpour Z, Saidi N, Owlia P. Different effects of sub-minimum inhibitory concentrations of gentamicin on the expression of genes involved in alginate production and biofilm formation of Pseudomonas aeruginosa. Iran J Microbiol. 2021;13(6):808-816.