Relationship of cell surface hydrophobicity with biofilm formation and growth rate: A study on Pseudomonas aeruginosa, Staphylococcus aureus, and Escherichia coli

Document Type: Original Article


1 Microbiology Analytical Centre, PCSIR Laboratories Complex Karachi, Pakistan

2 Department of Microbiology, University of Karachi, Pakistan

3 Department of Microbiology, Federal Urdu University of Arts, Science, and Technology, Pakistan

4 Department of Pathobiology, Bahauddin Zakariya University, Multan, Pakistan


Objective(s): This study was designed to determine the relationship of Pseudomonas aeruginosa, Staphylococcus aureus, and Escherichia coli isolates in multispecies biofilms and their individual phenotypic characters in biofilm consortia.
Materials and Methods:  The subject isolates were recovered from different food samples and identified on the basis of growth on differential and selective media.  Tube methods, Congo-red agar method, and scanning electron microscopy (SEM) were used to study biofilms phenotypes. The hydrophobicity of the strains was evaluated by the adhesion to apolar solvent.
Results: The results showed that E. coli dominated the pre-biofilm stage. It has been observed that E. coli adopted biofilm life much before S. aureus and P. aeruginosa. However, after adopting biofilm lifestyle, slowly and gradually, P. aeruginosa dominated the consortia and dispersed other stakeholders. The subject isolates of P. aeruginosa produce cis-2-decanoic acid to disperse or inhibit S. aureus and E. coli biofilms. Gas-chromatography and mass spectrometry results showed that cis-2-decanoic was higher in the co-culture condition and increased at late log-phase or at stationary phase. Although majority of S. aureus were unable to compete with P. aeruginosa, however, a minor population competed, survived, and persisted in biofilm consortia as small colony variants. The survivors showed higher expression of sigB and sarA genes. P. aeruginosa showed comparatively higher hydrophobic surface properties.
Conclusion: Comparative analysis showed that cell surface hydrophobicity, growth rate, and small colony variants (SCVs) are correlated in biofilm consortia of the P. aeruginosa, S. aureus, and E. coli.


Main Subjects

1. Berlanga M, Guerrero R. Living together in biofilms: the microbial cell factory and its biotechnological implications. Microb Cell Fact 2016; 15:165-175.
2. von Bodman SB, Willey JM, Diggle SP. Cell-cell communication in bacteria: united we stand. J Bacteriol 2008; 190:4377-4391.
3. Flemming H, Wingender J, Szewzyk U, Steinberg P, Rice SA, Kjelleberg S. Biofilms: an emergent form of bacterial life. Nature Rev Microbiol 2016; 14:563-575.
4. Giaouris E, Heir E, Desvaux M, Hébraud M, Møretrø T, Langsrud S, et al. Intra- and inter-species interactions within biofilms of important foodborne bacterial pathogens. Front Microbiol 2015; 6:841-866.
5. Limoli D, Jones C, Wozniak D. Bacterial extracellular polysaccharides in biofilm formation and function. Microbiol Spectr 2015; 3:1-30.
6. Li HY, Tian X. Quorum sensing and bacterial social interactions in biofilms. Sensors 2012; 12:2519-2538.
7. Atkinson S, Williams P. Quorum sensing and social networking in the microbial world. J R Soc Interface 2009; 6:959-978.
8. Donlan RM. Biofilms: microbial life on surfaces. Emerg Infect Dis 2002; 8:881-890.
9. Shank EA, Kolter R. New developments in microbial interspecies signalling. Curr Opin Microbiol 2009; 12:205-214.
10. Filkins LM, Graber JA, Olson DG, Dolben EL, Lynd LR, Bhuju S, et al. Co culture of Staphylococcus aureus with Pseudomonas aeruginosa drives S. aureus towards fermentative metabolism and reduced viability in a cystic fibrosis model. J Bacteriol 2015; 197:2252-2264.
11. Hotterbeekx A, Singh SK, Goossens H, Kumar SM. In vivo and in vitro interactions between Pseudomonas aeruginosa and Staphylococcus spp. Front Cell Infect Microbiol 2017; 7:106-118.
12. Orazi G,  O’Toole GA. Pseudomonas aeruginosa alters Staphylococcus aureus sensitivity to vancomycin in a biofilm model of cystic fibrosis infection. MBio 2017; 4:e00873-17.
13. Korgaonkar A, Trivedi U, Rumbaugh KP, Whiteley M. Community surveillance enhances Pseudomonas aeruginosa virulence during polymicrobial infection. Proc Natl Acad Sci           USA 2013; 110:1059-1064.
14. Culotti A, Packman AI. Pseudomonas aeruginosa promotes Escherichia coli biofilm formation in nutrient-limited medium. PLoS One 2014; 9:e107186.
15. Kim HS, Park HD. Ginger extract inhibits biofilm formation by Pseudomonas aeruginosa PA14. PLoS One 2013; 8:e76106.
16. Kaiser TD, Pereira EM, Dos Santos KR, Maciel EL, Schuenck RP, Nunes AP.  Modification of the congo red agar method to detect biofilm production by Staphylococcus epidermidis. Diagn Microbiol Infect Dis 2013; 75:235-239.
17. Reichhardt C, Jacobson AN, Maher MC, Uang J,  McCrate OA, Eckart M, Cegelski L. Congo red interactions with curli-producing E. coli and native curli amyloid fibres. PLoS One 2015; 10:e0140388.
18. 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.
19. 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.
20. Allegrucci M, Sauer K. Characterization of colony morphology variants isolated from Streptococcus pneumonia biofilms. J Bacteriol 2007; 189:2030-2038.
21. Keren I, Kaldalu N, Spoering A, Wang Y, Lewis K. Persister cells and tolerance to antimicrobials. FEMS Microbiol Lett 2004; 230:13-18.
22. Bayston R, Ashraf W, Smith T. Triclosan resistance in methicillin-resistant Staphylococcus aureus expressed as small colony variants: a novel mode of evasion of susceptibility to antiseptics. J Antimicrob Chemother 2007; 59:848-853.
23. 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.
24. 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; 49:14-22.
25. Liu YZ, Yuan K, Ji CR. New method of extraction on the chemical components of Chinese medicinal plants extracting method by smashing of plant tissues (EMS). Henan Sc 1993; 11:2652-2681.
26. Mirani ZA, Jamil N. Role of extracellular fatty acids in vancomycin-induced biofilm formation by vancomycin-resistant Staphylococcus aureus. Pak J Pharm Sci 2013; 26:383-389.
27. Kilic A, Guclu AU, Senses Z, Bedir O, Aydogan H, Basustaoglu AC. Staphylococcal cassette chromosome mec(SCCmec) characterization and panton-valentine leukocidin gene occurrence for methicillin-resistant Staphylococcus aureus in Turkey, from 2003 to 2006.  Antonie van Leeuwenhoek 2008; 94:607-614.
28. Zhang K, McClure JA, Elsayed S, Louie T, Conly JM. Novel multiplex PCR assay for characterization and concomitant subtyping of staphylococcal cassette chromosome mec types I to V in methicillin-resistant Staphylococcus aureus. J Clin Microbiol 2005; 43:5026-5033.
29. Tuchscherr L, Bischoff M, Lattar SM, Llana MN, Pfortner H, Niemann S, et al. Sigma factor sigB is crucial to mediate Staphylococcus aureus adaptation during chronic infections. PLoS Pathog 2015; 11:e1004870.
30. Iqbal Z, Seleem MN, Hussain HI, Huang L, Hao H, Yuan Z. Comparative virulence studies and transcriptome analysis of Staphylococcus aureus strains isolated from animals. Sci Rep 2016; 6:35442-35453.
31. Nuryastuti T, van der Mei HC, Busscher HJ, Iravati S, Aman AT, Krom BP. Effect of cinnamon oil on icaA expression and biofilm formation by Staphylococcus epidermidis. Appl Environ Microbiol 2009; 75:6850-6855.
32. 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 Clin Microbiol 2004; 42:2074-2079.
33. Naravaneni R, Jamil K. Rapid detection of food-borne pathogens by using molecular techniques. J Med Microbiol 2005; 54:51-54.
34. Oliveira MN, Martinez-Garcia E, Xavier J, Durham WM, Kolter R, Kim W, et al. Biofilm formation as a response to ecological competition. PLoS Biol 2015; 13:e1002191.
35. Cornforth DM, Foster KR. Antibiotics and the art of bacterial war. Proc Natl Acad Sci U S A 2015; 112:10827-10828.
36. Romero D, Traxler MF, Lopez D, Kolter R. Antibiotics as signal molecules. Chem Rev 2011; 111:5492-5505.
37. Davey ME, O’Toole GA. Microbial biofilms: from ecology to molecular genetics. Microbiol Mol Biol Rev 2000; 64:847-867.
38. Vega NM, Gore J. Collective antibiotic resistance: mechanisms and implications. Curr Opin Microbiol 2014; 21C:28-34.
39. Beloin C, Renard S, Ghigo JM, Lebeaux D. Novel approaches to combat bacterial biofilms. Curr Opin Pharmacol 2014; 18C:61-68.
40. Kochkodan V, Tsarenko S, Potapchenko N, Kosinova V, Goncharuk V. Adhesion of microorganisms to polymer membranes: a photobactericidal effect of surface treatment with TiO2. Desalination 2008; 220:380-385.
41. Giaouris E, Chapot-Chartier MP, Briandet R. Surface physicochemical analysis of natural Lactococcus lactis strains reveals the existence of hydrophobic and low charged strains with altered adhesive properties. Int J Food Microbiol 2009; 131:2-9.
42. Krasowska A, Sigler K. How microorganisms use hydrophobicity and what does this mean for human needs?. Front Cell Infect Microbiol 2014; 4:112.
43. Mirani ZA, Naz S, Khan F, Aziz M, Khan MN, Khan SI. Antibacterial fatty acids destabilize hydrophobic and multicellular aggregates of biofilm in S. aureus. J Antibiot 2017; 70:115-121.
44. Kahl BC, Becker K, Löffler B. Clinical significance and pathogenesis of staphylococcal small colony variants in persistent infections. Clin Microbiol Rev 2016; 2:401-427.  
45. Mirani ZA, Aziz M, Khan SI. Small colony variants have a major role in stability and persistence of Staphylococcus aureus biofilms. J Antibiot 2015; 68:98-105.
46. Valle J, Toledo-Arana A, Berasain C, Ghigo JM, Amorena B, Penades JR, et al. SarA and not sigmaB is essential for biofilm development by Staphylococcus aureus. Mol Microbiol 2003; 48:1075-1087.
47. Tuchscherr L, Loffler B. Staphylococcus aureus dynamically adapts global regulators and virulence factor expression in the course from acute to chronic infection. Curr Genet 2016; 62:15-17.
48. Häussler S. Biofilm formation by the small colony variant phenotype of Pseudomonas aeruginosa. Environ Microbiol 2004; 4:546-551.
49. Chu W, Zere TR, Weber MM, Wood TK, Whiteley M, Hidalgo-Romano B, et al. Indole production promotes Escherichia coli mixed-culture growth with Pseudomonas aeruginosa by inhibiting quorum signalling. Appl Environ Microbiol 2012; 78:411-419.
50. Sadowska B, Walencka E, Wieckowska-Szakiel M, Różalska B. Bacteria competing with the adhesion and biofilm formation by Staphylococcus aureus. Folia Microbiol (Praha) 2010; 55:497-501.
51. Hoffman LR, Deziel E, D’Argenio DA, Lepine F, Emerson J, McNamara S, et al. Selection for Staphylococcus aureus small-colony variants due to growth in the presence of Pseudomonas aeruginosa. Proc Natl Acad Sci U S A 2006; 103:19890-19895.
52. Estrela AB, Abraham WR. Combining biofilm-controlling compounds and antibiotics as a promising new way to control biofilm infections. Pharmaceuticals 2010; 3:1374-1393.
53. Davies DG, Marques CN. A fatty acid messenger is responsible for inducing dispersion in microbial biofilms. J Bacteriol 2009; 191:1393-1403.
54. Kim S, Yoon Y, Choi KH. Pseudomonas aeruginosa DesB promotes Staphylococcus aureus growth inhibition in coculture by controlling the synthesis of HAQs. PLoS One 2015; 10:1-16.
55. 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.