Abstract
As with many other bacterial species, the most commonly used method to assess staphylococcal biofilm formation in vitro is the microtiter plate assay. This assay is particularly useful for comparison of multiple strains including large-scale screens of mutant libraries. When such screens are applied to the coagulase-negative staphylococci in general, and Staphylococcus epidermidis in particular, they are relatively straightforward by comparison with microtiter plate assays used to assess biofilm formation in other bacterial species. However, in the case of clinical isolates of Staphylococcus aureus, including methicillin-resistant S. aureus, we have found it necessary to employ specific modifications including precoating of the wells of the microtiter plate with plasma proteins and supplementation of the medium with both salt and glucose. In this chapter, we describe the microtiter plate assay in the specific context of clinical isolates of S. aureus and the use of these modifications. A second in vitro method, which also is generally dependent on coating with plasma proteins and supplementation of the growth medium, is the use of flow cells. In this method, bacteria are allowed to attach to a surface and then monitored with respect to their ability to remain attached to the substrate and differentiate into mature biofilms under the constant pressure of fluid shear force. Although flow cells are not applicable to large-scale screens, we have found that they provide a more reproducible and accurate assessment of the capacity of S. aureus clinical isolates to form a biofilm. They also provide a means of analyzing structural differences in biofilm architecture and isolating bacteria and/or spent media for analysis of physiological and metabolic changes associated with the adaptive response to growth in a biofilm. While a primary focus of this chapter is on the use of in vitro assays to assess biofilm formation in clinical isolates of S. aureus, it is important to emphasize two additional considerations. First, it has become increasingly evident that biofilm formation in S. epiderimidis and S. aureus is not equivalent. Additionally, to date, most studies with S. aureus have been done with a very limited number of strains, almost all of which are derived from the NCTC strain designated 8325, and we have found that these strains are not representative of the most relevant clinical isolates. As with the specific elements of our flow cell system, we have written this chapter to reflect our focus on clinical isolates of S. aureus and the specific methods that we have found most reliable in that context. Second, as is often the case, in vitro methods do not necessarily reflect events that occur in vivo. Several in vivo methods to assess biofilm formation have been described, and these generally fall into one of two categories. The first focuses directly on staphylococcal diseases that are generally thought to include a biofilm component (e.g., endocarditis, osteomyelitis, septic arthritis). A discussion of these models is also beyond the scope of this chapter, but examples are easily found in the staphylococcal literature. The second approach uses some form of implanted device in an attempt to focus more directly on implant-associated biofilms. We use a model in which a small piece of Teflon catheter is implanted subcutaneously in mice and used as a substrate for colonization. We have the advantage of using bioluminescent derivatives of S. aureus clinical isolates and the IVISĀ® imaging system. However, because this system is not generally available, we restrict technical comments in this chapter to our use of an implanted catheter model evaluated by direct microbiological analysis of explanted catheters (2).
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References
Lewis, K. (2001) Riddle of biofilm resistance. Antimicrob. Agents Chemother. 45, 999ā1007.
Keren, I., Kaldalu, N., Spoering, A., Wang, Y., and Lewis, K. (2004) Persister cells and tolerance to antimicrobials. FEMS Microbiol. Lett. 230, 13ā18.
Christensen, G. D., Simpson, W. A., Bisno, A. L., and Beachey, E. H. (1983) Experimental foreign body infections in mice challenged with slime-producing Staphylococcus epidermidis. Infect. Immun. 40, 407ā410.
Rupp, M. E., Ulphani, J. S., Fey, P. D., and Mack, D. (1999) Characterization of Staphylococcus epidermidis polysaccharide intercellular adhesin/hemagglutinin in the pathogenesis of intravascular catheter-associated infection in a rat model. Infect. Immun. 67, 2656ā2659.
Kadurugamuwa, J. L., Sin, L., Albert, E., et al. (2003) Direct continuous method for monitoring biofilm infection in a mouse model. Infect. Immun. 71, 882ā890.
Patti, J. M., Allen, B. L., McGavin, M. J., and Hook, M. (1994) MSCRAMM-mediated adherence of microorganisms to host tissues. Annu. Rev. Microbiol. 48, 585ā617.
Sillanpaa, J., Xu, Y., Nallapareddy, S. R., Murray, B. E., and Hook, M. (2004) A family of putative MSCRAMMs from Enterococcus faecalis. Microbiology 150, 2069ā2078.
Beenken, K. E., Blevins, J. S., and Smeltzer, M. S. (2003) Mutation of sarA in Staphylococcus aureus limits biofilm formation. Infect. Immun. 71, 4206ā4211.
Cafiso, V., Bertuccio, T., Santagati, M., et al. (2004) Presence of the ica operon in clinical isolates of Staphylococcus epidermidis and its role in biofilm production. Clin. Microbiol. Infect. 10, 1081ā1088.
Fitzpatrick, F., Humphreys, H., and OāGara, J. P. (2005) The genetics of staphylococcal biofilm formation-will a greater understanding of pathogenesis lead to better management of device-related infection? Clin. Microbiol. Infect. 11, 967ā973.
Cramton, S. E., Gerke, C., Schnell, N. F., Nichols, W. W., and Gotz, F. (1999) The intracellular adhesin (ica) locus is present in Staphylococcus aureus and is required for biofilm formation. Infect. Immun. 67, 5427ā5433.
Beenken, K. E., Dunman, P. M., McAleese, F., et al. (2004) Global gene expression in Staphylococcus aureus biofilms. J. Bacteriol. 186, 4665ā4684.
Fitzpatrick, F., Humpreys, H., and OāGara, J. P. (2005) Evidence for icaADBC-independent biofilm development mechanism in methicillin-resistant Staphylococcus aureus clinical isolates. J. Clin. Microbiol. 43, 1973ā1976.
Fluckiger, U., Ulrich, M., Steinhuber, A., et al. (2005) Biofilm formation, icaADBC transcription, and polysaccharide intercellular adhesin synthesis by staphylococci in a device-related infection model. Infect. Immun. 73, 1811ā1819.
McKenney, D., Pouliot, K. L., Wang, Y., et al. (1999) Broadly protective vaccine for Staphylococcus aureus based on an in vivo-expressed antigen. Science 284, 1523ā1527.
Yao, Y., Sturdevandt, D. E., and Otto, M. (2005) Genomewide analysis of gene expression in Staphylococcus epidermidis biofilms: insights into the pathophysiology of S. epidermidis biofilms and the role of phenol-soluble modulins in formation of biofilms. J. Infect. Dis. 191, 289ā298.
Yarwood, J. M., Bartels, D. J., Volper, E. M., and Greenberg, E. P. (2004) Quorum sensing in Staphylococcus aureus biofilms. J. Bacteriol. 186, 1838ā1850.
Vuong, C., Gerke, C., Somerville, G. A., Fischer, E. R., and Otto, M. (2003) Quorum-sensing control of biofilm factors in Staphylococcus epidermidis. J. Infect. Dis. 188, 706ā718.
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Cassat, J.E., Lee, C.Y., Smeltzer, M.S. (2007). Investigation of Biofilm Formation in Clinical Isolates of Staphylococcus aureus . In: Ji, Y. (eds) Methicillin-Resistant Staphylococcus aureus (MRSA) Protocols. Methods in Molecular Biology, vol 391. Humana Press. https://doi.org/10.1007/978-1-59745-468-1_10
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DOI: https://doi.org/10.1007/978-1-59745-468-1_10
Publisher Name: Humana Press
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