Study of the Adhesion of Clinical Strains of Staphylococcus aureus on an Abiotic Surface Using the Biofilm Ring Test®

J. M. Liesse Iyamba, N. B. Takaisi-Kikuni, S. Dulanto, J. P. Dehaye

Laboratoire de Chimie biologique et médicale et de Microbiologie pharmaceutique, Faculté de Pharmacie, Université libre de Bruxelles, Brussels, Belgium.
Laboratoire de Microbiologie Expérimentale et Pharmaceutique, Faculté des Sciences Pharmaceu- tiques, Université de Kinshasa, Democratic Republic of Congo.

DOI: 10.4236/jbnb.2012.324057                

Abstract

Four methicillin-sensitive (MSSA) and 4 methicillin-resistant (MRSA) strains of Staphylococcus aureus were collected and isolated at the Laboratory of Bacteriology of the Provincial General Reference Hospital of Kinshasa in the Democratic Republic of Congo. The microbial adhesion to solvents (MATS) test showed that the MRSA strains had a less hydrophobic membrane than the MSSA strains. Using the Biofilm Ring Test^? (BFRT^?) to investigate on the adhesion of these bacterial strains to smooth surfaces, we observed that the MSSA strains adhered more rapidly than the MRSA strains. The biomass of the produced biofilm measured by the Crystal violet staining method (CVSM) was more important with MSSA than with MRSA strains. Ethylene glycol-bis(2-aminoethylether)-N,N,N',N'-tetraacetic acid (EGTA) inhibited the adhesion and the formation of a biofilm by MRSA strains; this inhibition was reversed by calcium, magnesium and manganese. The MRSA strains adhered less to silicon tubing and the adhesion was inhibited by EGTA in 2 of the 4 MRSA strains and none of the MSSA strains. In conclusion, the MSSA and MRSA strains adhered on an abiotic surface and formed a biofilm at distinct rates and with different sensitivities to ions. The results also confirm the utility as well as the limits of the BFRT^? to study the adhesion of bacteria on a surface.

Keywords

Biofilm Ring Test^®; Crystal Violet; Cell Surface; Hydrophobicity; Adhesion; Catheter Tube

Conflicts of Interest

The authors declare no conflicts of interest.

References

[1]	N. K. Archer, M. J. Mazaitis, J. W. Costerton, J. G. Leid, M. E. Powers and M. E. Shirtliff, “Staphylococcus aureus Biofilms: Properties, Regulation, and Roles in Human Disease,” Virulence, Vol. 2, No. 5, 2011, pp. 445-459. doi:10.4161/viru.2.5.17724
[2]	L. Hall-Stoodley, P. Stoodley, S. Kathju, N. Hoiby, C. Moser, J. W. Costerton, A. Moter and T. Bjarnsholt, “Towards Diagnostic Guidelines for Biofilm-Associated Infections,”. FEMS Immunology and Medical Microbiology, Vol. 65, No. 2, 2012, pp. 127-145. doi:10.1111/j.1574-695X. 2012.00968
[3]	D. G. Cvitkovitch, Y. H. Li and R. P. Ellen, “Quorum Sensing and Biofilm Formation in Streptococcal Infections,” Journal of Clinical Investigation, Vol. 112, No. 11, 2003, pp. 1626-1632. doi:10.1172/JCI200320430
[4]	M. Leone and L. R. Dillon, “Catheter Outcomes in Home Infusion,” Journal of Infusion Nursing, Vol. 31, No. 2, 2008, pp. 84-91. doi:10.1097/01.NAN.0000313655. 65410.4e
[5]	P. S. Stewart and J. W. Costerton, “Antibiotic Resistance of Bacteria in Biofilms,” The Lancet, Vol. 358, No. 9276, 2001, pp. 135-138. doi:10.1016/S0140-6736(01)05321-1
[6]	L. Hall-Stoodley and P. Stoodley, “Evolving Concepts in Biofilm Infections,” Cellular Microbiology, Vol. 11, No. 7, 2009, pp.1034-1043. doi:10.1111/j.1462-5822.2009. 01323
[7]	M. Otto, “Staphy-lococcal Biofilms,” Current Topics in Microbiology and Immunology, Vol. 322, 2008, pp. 207- 228. doi:10.1007/978-3-540-75418-3_10
[8]	C. von Eiff, B. Jansen, W. Kohnen and K. Becker, “Infections Associated with Medical Devices: Pathogenesis, Management and Prophylaxis,” Drugs, Vol. 65, No. 2, 2005, pp. 179-214. doi:10.2165/00003495-200565020-00003
[9]	L. D. Renner and D. B. Weibel, “Physicochemical Regulation of Biofilm Formation,” MRS Bulletin, Vol. 36, No. 5, 2011, pp. 347-355. doi:10.1557/mrs.2011.65
[10]	D. López, H. Vlamakis and R. Kolter, “Biofilms,” Cold Spring Harbor Perspectives in Biology, Vol. 2, No. 7, 2010, p. a000398. doi:10.1016/j.pnsc.2008.04.001
[11]	T. R. Garrett, M. Bhakoo and Z. Zhang, “Bacterial Adhesion and Biofilms on Surfaces,” Progress in Natural Sciencs, Vol. 18, No. 9, 2008, pp.1049-1056.
[12]	B. R. Boles and A. R. Horswill, “Staphylococcal Biofilm Disassembly,” Trends in Microbiology, Vol. 19, No. 9, 2011, pp. 449-455. doi:10.1016/j.tim.2011.06.004
[13]	E. Peeters, H. J. Nelis and T.Coenye “Comparison of Multiple Methods for Quantification of Biofilms Grown in Microtiter Plates,” Journal of Microbiological Methods, Vol. 72, No. 2, 2008, pp. 157-165. doi.10.1016/j.mimet. 2007.11.010
[14]	S. Stepanovic, D. Vu-kovic, I. Dakic, B. Savic and M. Svabic-Vlahovic, “A Modified Microtiter-Plate Test for Quantification of Staphylococcal Bio-film Formation,” Journal of Microbiological Methods, Vol. 40, No. 2, 2000, pp. 175–179.
[15]	P. Chavant, B. Gaillard-Martinie, R. Talon, M. Hébraud and T. Bernardi, “A New Device for Rapid Evaluation of Biofilm Formation Potential by Bacteria,” Journal of Microbiological Methods, Vol. 68, No. 3, 2007, pp. 605-612. doi:10.1016/j.mimet.2006.11.010
[16]	S. Sulaeman, G. Le Bihan, A. Rossero, M. Federighi, E. Dé and O. Tresse, “Comparison between the Biofilm Initiation of Campylobacter Jejuni and Campylobacter Coli Strains to an Inert Surface Using BioFilm Ring Test,” Journal of Applied Microbiology, Vol. 108, No. 4, 2010, pp. 1303-1312. doi :10.1111/j.1365-2672. 2009.04534l
[17]	C. Nagant, M. Tré-Hardy, M. Devleeschouwer and J. P. Dehaye, “Study of the Initial Phase of Biofilm Formation Using a Biofomic Approach,” Journal of Microbiological Methods, Vol. 82, No. 3, 2010, 243-248. doi:10.1016/j. mimet.2010.06.011
[18]	J. M. Liesse Iyamba, M. Seil, M. Devleeschouwer, N. B. Takaisi Kikuni and J. P. Dehaye, “Study of the Formation of a Biofilm by Clinical Strains of Staphylococcus aureus,” Biofouling, Vol. 27, No. 8, 2011, pp. 811-821. doi:10.1080/08927014.2011.604776
[19]	M.-N. Bellon-Fontaine, J. Rault and C. J. Van Oss, “Microbial Adhesion to Solvents: A Novel Method to Determine the Electron-Donor/Electron-Acceptor or Lewis Acid-Base Properties of Microbial Cells,” Colloids and Surfaces B: Biointerfaces, Vol. 71, No. 1-2, 1996, pp. 147-153.
[20]	N. Ozerdem Akpolat, S. Elci, S. Atmaca, H. Akbayin and K. Gül, “The Effects of Magnesium, Calcium and EDTA on Slime Production by Staphylococcus Epidermidis Strains,” Folia Microbiologica (Praha), Vol. 48, No. 5, 2003, pp. 649-653. doi:10.1007/BF02993473
[21]	D. E. Caldwell and J. R. Lawrence, “Study of Attached Cells in Continuous-Flow Slide Culture,” In: J. W. T. Wimpenny, Ed., Handbook of Laboratory Model Systems for Microbial Ecosystems, CRC Press, Boca Raton, 1988, pp. 117-138.
[22]	J. R. Lawrence, D. R. Korber, B. D. Hoyle, J. W. Costerton and D. E. Caldwell, “Optical Sectioning of Microbial Biofilms,” Journal of Bacteriology, Vol. 173, No. 20, 1991, pp. 6558-6567.
[23]	A. Escher and W. G. Characklis, “Modeling the Initial Events in Biofilm Accumulation,” In: W. G. Characklis and K. C. Marshall, Eds., Bio-films, John Wiley and Sons, New York, 1990, pp. 445-486.
[24]	H. W. Fowler, “Microbial Adhesion to Surfaces,” In: J. V. T. Wimpenny, Ed., Handbook of Laboratory Model Systems for Microbial Ecosystems, CRC Press, Boca Raton, 1988, pp. 139-153.
[25]	M. Vorachit, K. Lam, P. Jayanetra and J. W. Costerton, “Resistance of Pseudomonas Pseudomallei Growing as a Biofilm on Silastic Disks to Ceftazidime and Cotrimoxazole,” Antimicrobial Agents and Chemotherapy, Vol. 37, No. 9, 1993, pp. 2000-2002. doi:/10.1128/AAC.37.9.2000
[26]	R. Murga, J. M. Miller and R. M. Donlan, “Biofilm Formation by Gram-Negative Bacteria on Central Venous Catheter Connectors: Effect of Conditioning Films in a Laboratory Model,” Journal of Clinical Microbiology, Vol. 39, No. 6, 2001, pp. 2294-2297. doi:10.1128/JCM. 39.6.2294-2297.2001
[27]	C. Nagant, B. Pitts, N. Kamran, M. Vandenbranden, J. G. Bolscher, P. S. Stewart and J. P. Dehaye, “Identification of the Domains of the Human Antimicrobial Peptide LL-37 Active against the Biofilms Formed by Pseudomonas Aeruginosa Using a Library of Truncated Fragments,” Unpublished.
[28]	S. B. Surman, J. T. Walker, D. T. Goddard, L. H. G. Morton, C. W. Keevil, W. Weaver, A. Skinner, A. Hanson, D. Caldwell and J. Kurtz, “Comparison of Microscope Techniques Examination of Bio-films,” Journal of Microbiological Methods, Vol. 25, No. 1, 1996, pp. 57-70. doi:10.1016/0167-7012(95)00085-2
[29]	A. Sénéchal, S. D. Carrigan and M. Tabrizian, “Probing Surface Adhesion Forces of Enterococcus Faecalis to Medical-Grade Polymers Using Atomic Force Micro-scopy,” Langmuir, Vol. 20, No. 10, 2004, pp. 4172-4177. doi:10.1021/la035847y
[30]	S. L. Burnett, J. Chen and L. R. Beuchat, “Attachment of Escherichia Coli O157:H7 to the Surfaces and Internal Structures of Apples as Detected by Confocal Scanning Laser Microscopy,” Applied and Environmental Micro-biology, Vol. 66, No. 11, 2000, pp. 4679-4687. doi:/10.1128/AEM.66.11.4679-4687.2000
[31]	L. Kodjikian, C. Burillon, G. Lina, C. Roques, G. Pellon, J. Freney and F. N. Renaud, “Biofilm Formation on Intraocular Lenses by a Clinical Strain Encoding the Ica Locus: A Scanning Electron Microscopy Study,” Investigative Ophthalmology and Visual Science, Vol. 44, No. 10, 2003, pp. 4382-4387. doi:10.1167/iovs.03-0185
[32]	J. B. Xavier, D. C. White and J. S. Almeida, “Automated Biofilm Morphology Quantification from Confocal Laser Scanning Microscopy Imaging,” Water Science and Technology, Vol. 47, No. 5, 2003, pp. 31-37.
[33]	H. Anwar, J. L. Strap and J. W. Costerton, “Eradication of Biofilm Cells of Staphylococcus aureus with Tobramycin and Cephalexin,” Canadian Journal of Microbiology, Vol. 38, No. 7, 1992, pp. 618-625. doi: 10.1139/m92-102
[34]	N. Oulahal, A. Martial-Gros, M. Bonneau and L. J. Blum, “Combined Effect of Chelating Agents and Ultrasound on Biofilm Removal from Stainless Steel Surfaces. Application to Escherichia Coli Milk and Staphylococcus aureus Milk Biofilms,” Biofilms, Vol. 1, No. 1, 2004, pp. 65-73. doi:10.1017/S1479050504001140
[35]	G. D. Christensen, W. A. Simpson, J. J. Younger, L. M. Baddour, F. F. Barrett, D. M. Melton and E. H. Beachey, “Adherence of Coagulase-Negative Staphylococci to Plastic Tissue Culture Plates: A Quantitative Model for the Adherence of Staphylococci to Medical Devices,” Journal of Clinical Microbiology, Vol. 22, No. 6, 1985, pp. 996-1006.
[36]	E. O’Neill, C. Pozzi, P. Houston, D. Smyth, H. Humphreys, D. A. Robinson and J. P. O'Gara, “Association between Methicillin Susceptibility and Biofilm Regulation in Staphylococcus aureus Isolates from Device-Related Infections,” Journal of Clinical Microbiology, Vol. 45, No. 5, 2007, pp. 1379-1388. doi: 10.1128/JCM.02280-06
[37]	S. Badel, C. Laroche, C. Gardarin, T. Bernardi and P. Michaud, “New Method Showing the Influence of Matrix Components in Leuconostoc Mesenteroides Biofilm Formation,” Applied Biochemistry Biotechnology, Vol. 151, No. 2-3, 2008, pp. 364-370. doi:10.1007/ s12010-008-8199-y
[38]	S. Badel, F. Callet, C. Laroche, C. Gardarin, E. Petit, H. El Alaoui, T. Bernardi and P. Michaud, “A New Tool to Detect High Viscous Exopolymers from Microalgae,” Journal of Industrial Microbiology and Biotechnology Vol. 32, No. 2, 2011, pp. 319-326. doi:10.1007/ s10295-010-0775-9
[39]	G. L. Smith and D. J. Miller, “Potentiometric Measurements of Stoichiometric and Apparent Affinity Constants of EGTA for Protons and Divalent Ions Including Calcium,” Biochimica et Biophysica Acta, Vol. 839, No. 3, 1985, pp. 287-299. doi:10.1016/ 0304-4165(85)90011-X
[40]	M.-H. Lin, J.-C. Shu, H.-Y. Huang and Y.-C. Cheng, “Involvement of Iron in Biofilm Formation by Staphylococcus aureus,” PLOS One, Vol. 7, No. 3, 2012, p. e34388. doi:10.1371/journal.pone.0034388
[41]	D. G. Conrady, C. C. Brescia, K. Horii, A. A. Weiss, D. J. Hassett and A. B. Herr, “A Zinc-Dependent Adhesion Module is Responsible for Intercellular Adhesion in Staphylococcal Biofilms,” Proceedings of the National Academy of Sciences USA, Vol. 105, No. 49, 2008, pp. 19456-19461. doi:10.1073/pnas.0807717105
[42]	D. T. Gruszka, J. A. Wojdyla, R. J. Bingham, J. P. Turkenburg, I. W. Manfield, A. Steward, A. P. Leech, J. A. Geoghegan, T. J. Foster, J. Clarke and J. R. Potts, “Staphylococcal Biofilm-Forming Protein Has a Contiguous Rod-Like Structure,” Proceedings of the National Academy of Sciences USA, Vol. 109, No. 17, 2012, pp. E1011-E1018. doi:10.1073/pnas.1119456109
[43]	M. M. Amaral, L. R. Coelho, R. P. Flores, R. R. Souza, M. C. Silva-Carvalho, L. A. Teixeira, B. T. Ferreira-Carvalho and A. M. S. Figueiredo, “The Predominant Variant of the Brazilian Epidemic Clonal Complex of Methicillin-Resistant Staphylococcus aureus Has an Enhanced Ability to Produce Biofilm and to Adhere to and Invade Airway Epithelial Cells,” Journal of Infectious Diseases, Vol. 192, No. 5, 2005, pp. 801-810. doi:10.1086/432515
[44]	C. Pozzi, E. M. Waters, J. K. Rudkin, C. R. Schaeffer, A. J. Lohan, P. Tong, B. J. Loftus, G. B. Pier, P. D. Fey, R. C. Massey and J. P. O'Gara, “Methicillin-Resistance Alters the Biofilm Phenotype and Attenuates Virulence in Staphylococcus aureus Device-Associated Infections,” PLOS Pathogy, Vol. 8, No. 4, 2012, p. e1002626.
[45]	S. Aathithan, R. Dybowski and G. L. French, “Highly Epidemic Strains of Methicillin-Resistant Staphylococcus aureus not Distinguished by Capsule Formation, Protein a Content or Adherence to HEP-2 Cells,” European Journal of Clinical Microbiology & Infectious Diseases, Vol. 20, No. 1, 2001, pp. 27-32. doi:10.1007/PL00011233
[46]	H. Karauzum, T. Ferry, S. de Bentzmann, G. Lina, M. Bes, F. Vandenesch, M. Schmaler, B. Berger-Bachi, J. Etienne and R. Landmann, “Comparison of Adhesion and Virulence of Two Predominant Hospital-Acquired Methicillin-Resistant Staphylococcus aureus Clones and Clonal Methicillin-Susceptible Staphylococcus aureus Isolates,” Infection and Immunity, Vol. 76, No. 11, 2008, pp. 5133- 5138. doi:10.1128/IAI.01697-07.