Role of Phenotypic Switching in Stability and Persistence of Pseudomonas aeruginosa Biofilms

Objectives: Objective: This study was designed to explore the role of different phenotypes of P. aeruginosa in the development, stability and persistence of biofilm. Methods: A total of seventeen (17) waterborne biofilm producing strains of P. aeruginosa were studied. These isolates were identified on the basis of typical phenotypic characters and the tube method was used for the study of biofilms. Population and phenotypic variance were studied by the drop plate method. The hydrophobicity of strains was evaluated by the bacterial adhesion to apolar solvent test. Results: Study showed that the subject isolates of P. aeruginosa adopted a biofilm life style after 36 h of incubation at 35 °C. After 24 h the adhesion started, but it was reversible and easily dispersed by simple washing. However, after 36 h the irreversible adhesion was noticed. The biofilm consortia harbor three different phenotypes: i. wild types, showed typical P. aeruginosa characters on Cetrimide agar; ii. Slow growers, showed poor pigmentation and take >36 h for colony development, and iii. Small colony variants (SCVs) are metabolically inactive and producing pinpointed non pigmented colonies. Comparative analysis showed that these phenotypes i.e. SCVs were highly hydrophobic and persistent in biofilm consortia due to the production of excessive amounts of exopolysaccharides. Conclusions: This study showed that phenotypic heterogeneity is a characteristic feature of P. aeruginosa biofilms and all of these phenotypes have a major role in stability and persistence of biofilm consortia. J Microbiol Infect Dis 2020; 10(1):10-17.

___

  • 1. Sousa AM, Pereira MO. Pseudomonas aeruginosa Diversification during Infection Development in Cystic Fibrosis Lungs-A Review. Pathogens (Basel, Switzerland) 2014; 3(3):680–703. 2. Sadikot RT, Blackwell TS, Christman JW, Prince AS. Pathogen-host interactions in Pseudomonas aeruginosa pneumonia. Am J Respir Crit Care Med 2005; 171: 1209–1223. 3. Algburi A, Comito N, Kashtanov D, Dicks LM, Chikindas ML. Control of biofilm formation: Antibiotics and beyond. Appl Environ Microbiol 2017; 83. doi:10.1128/AEM.02508-16. 4. Alguri A, Comito N, Kashtanov D, Dicks LM, Chikindas ML. Control of Biofilm formation: Antibiotics and beyond. Appl Environ Microbio Mo lBiol Rev 2014: 78:510-543. 5. Machado I, Graça J, Lopes H, Lopes S, Pereira MO. Antimicrobial pressure of ciprofloxacin and gentamicin on biofilm development by an endoscope-isolated Pseudomonas aeruginosa. ISRN Biotechnol 2012; 2013: 178646. 6. Hall CW, Mah TF. Molecular mechanisms of biofilm-based antibiotic resistance and tolerance in pathogenic bacteria. FEMS Microbiol Rev 2017; 41: 276–301. doi: 10.1093/femsre/fux010. 7. Moradali MF, Ghods S, Rehm BHA. Pseudomonas aeruginosa lifestyle: A paradigm for adaptation, survival, and persistence. Front. Cell. Infect. Microbiol 2017; 7-39. 8. Sauer K, and Camper AK. Characterization of phenotypic changes in Pseudomonas putida in response to surface-associated growth. J Bacteriol 2001; 183: 6579–6589. 9. Malone JG. Role of small colony variants in persistence of Pseudomonas aeruginosa infections in cystic fibrosis lungs. Infect Drug Resist 2015; 8: 237-247. 10. Mirani ZA, Jamil N. Effect of sub-lethal doses of vancomycin and oxacillin on biofilm formation by vancomycin intermediate resistant Staphylococcus aureus. J Basic Microbiol 2011; 51: 191–195. 11. O'Toole GA, Kolter R. Initiation of biofilm formation in Pseudomonas fluorescens WCS365 proceeds via multiple, convergent signalling pathways: a genetic analysis. Mol Microbiol 1998; 28: 449-461. 12. Allegrucci M, Sauer K. Characterization of colony morphology variants isolated from Streptococcus pneumonia biofilms. J Bacteriol 2007; 189: 2030–2038. 13. Keren I, Kaldalu N, Spoering A, Wang Y, Lewis K. Persister cells and tolerance to antimicrobials. FEMS Microbiol Lett 2004; 230:13-18. 14. Chen CY, Nace GW, Irwin PL. A 6×6 drop plate method for simultaneous colony counting and MPN enumeration of Campylobacter jejuni, Listeria monocytogenes, and Escherichia coli. J Microbiol Meth 2003; 55: 475–479. 15. Kouidhi B, Zmantar T, Hentati H, Bakhrouf A. Cell surface hydrophobicity, biofilm formation, adhesives properties and molecular detection of adhesins genes in Staphylococcus aureus associated to dental caries. Microb Pathog 2010; 49 (1-2):14–22. 16. Qin Z, Yang Y, Qu D, Molin S, Tolker-Nielsen T. Pseudomonas aeruginosa extracellular products inhibit staphylococcal growth, and disrupt established biofilms produced by Staphylococcus epidermidis. Microbiol 2009; 155: 2148–2156. 17. Spilker T, Coenye T, Vandamme P, LiPuma JJ. PCR-based assay for differentiation of Pseudomonas aeruginosa from other Pseudomonas species recovered from cystic fibrosis patients. J ClinMicrobiol 2004; 42: 2074–2079. 18. Ma L, Conover M, Lu H, Parsek MR, Bayles K, Wozniak DJ. Assembly and development of the Pseudomonas aeruginosa biofilm matrix. PLoS Pathog 2009; 5:e1000354. 19. Basta DW, Bergkessel M, Newman DK. Identification of fitness determinants during energy-limited growth arrest in Pseudomonas aeruginosa. mBio 2017; 8:e01170-17. 20. Häussler S, Ziegler I, Löttel A, et al. Highly adherent small-colony variants of Pseudomonas aeruginosa in cystic fibrosis lung infection. J Med Microbiol 2003; 52:295-301. 21. Onyango LA, Hugh Dunstan R, Roberts TK, Macdonald MM, Gottfries J. Phenotypic variants of staphylococci and their underlying population distributions following exposure to stress. PLoS One 2013; 8:e77614. 22. Petráčková D, Buriánková K, Tesařová E, et al. Surface hydrophobicity and roughness influences the morphology and biochemistry of streptomycetes during attached growth and differentiation. FEMS Microbiol Lett 2013; 342, 147–156. 23. Kaiser D. Bacterial swarming: a re-examination of cell-movement patterns. Curr Biol 2007; 17: R561–R570. 24. Johns BE, PurdyKJ, Tucker NP, Maddocks SE. Phenotypic and genotypic characteristics of small colony variants and their role in chronic infection. Microbiol Insights 2015; 8: 15–23. 25. Sandt C, Smith-Palmer T, Comeau J, Pink D. Quantification of water and biomass in small colony variant PAO1 biofilms by confocal Raman microspectroscopy. Appl Microbiol Biotechnol 2009; 83 (6): 1171–1182. 26. Williamson KS, Richards LA, Perez-Osorio AC, et al. Heterogeneity in Pseudomonas aeruginosa biofilms includes expression of ribosome hibernation factors in the antibiotic-tolerant subpopulation and hypoxia-induced stress response in the metabolically active population. J Bacteriol 2012; 194: 2062–2073. 27. Valentini M, Gonzalez D, Mavridou DA, Filloux A. Lifestyle transitions and adaptive pathogenesis of Pseudomonas aeruginosa. Curr Opin Microbiol 2017; 18:41:15-20. 28. Singh S, Singh SK, Chowdhury I, Singh R. Understanding the Mechanism of Bacterial Biofilms Resistance to Antimicrobial Agents. The open microbiology journal. 2017; 11:53–62. 29. Wentland EJ, Stewart PS, Huang CT, McFeters GA. Spatial variations in growth rate within Klebsiella pneumoniae colonies and biofilm. Biotechnol Prog 1996; 12(3):316–321. 30. Boles BR, Thoendel M, Singh PK. Self-generated diversity produces “insurance effects” in biofilms communities. Proc Natl Acad Sci USA 2004; 101: 16630–16635.