Vol. 57, No. 3

APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Mar. 1991, p. 863-866

0099-2240/91/030863-04$02.00/0 Copyright ©) 1991, American Society for Microbiology

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In Vitro Measurement of the Adherence of Staphylococcus epidermidis to Plastic by Using Cellular Urease as a Marker W. MICHAEL DUNNE,

JR.,1"2*

EILEEN M. BURD2 Departments of Pathology1 and Pediatrics,2 Children's Hospital of Wisconsin, Medical College of Wisconsin, AND

Milwaukee, Wisconsin 53201 Received 27 September 1990/Accepted 6 December 1990

A rapid and sensitive in vitro assay was developed to quantitatively assess the adherence of Staphylococcus epidermidis to a hydrophobic plastic surface. The assay is based upon the detection of cell-associated urease activity as a marker of bacteria remaining adherent to the polystyrene microwells of flat-bottomed, 96-well tissue culture plates. Using ATCC 35984, a slime-producing strain of S. epidermidis, the assay could detect as few as 3 x 103 bacteria and was linear to 3.5 x 107 bacteria. The adherence of both slime-positive and slime-negative coagulase-negative staphylococci could be evaluated by using this method. This assay could be used to examine factors which influence the adherence of individual S. epidermidis strains to hydrophobic surfaces and to develop agents or coating materials which suppress the adherence of coagulase-negative staphylococci to biomedical implants.

Coagulase-negative staphylococci (CONS), especially Staphylococcus epidermidis, frequently infect biomedical implants and transcutaneous devices such as intravascular and peritoneal catheters (16, 21), cerebrospinal fluid shunts (1), and prosthetic cardiac valves (11). The pathogenesis of biomedical implant infections caused by S. epidermidis can be separated into at least two distinct phases: (i) primary bacterial adherence to the surface of the device and (ii) the production (by certain strains) of a stable biofilm matrix over the surface of the device. Primary adherence is initiated through hydrophobic and/or ligand-specific interactions between the exterior surface of the cell wall and the surface of the device (9, 19, 22). Biofilm production is an attribute of strains of S. epidermidis which elaborate an extracellular polysaccharide or slime (3). In vivo, the slime polysaccharide complexes with host factors to form an extracellular matrix which anchors bacteria firmly to the surfaces of biomedical implants (14) and protects adherent organisms from host defenses (8, 10). Sterilization of a biomedical implant infected by S. epidermidis without surgical revision or removal is difficult, especially if the offending strain produces a slime polysaccharide (6, 23). Recent efforts aimed at reducing the incidence of biomedical implant infections have relied upon improvements in aseptic handling techniques and site care (12, 18), reduction of bacterial pathogen reservoirs by using prophylactic antimicrobial agents (13), and modifications in catheter design and materials that decrease the likelihood of bacterial contamination (17, 20). Future strategies may include the development of agents or antimicrobial coatings which inhibit or suppress the primary adherence of S. epidermidis to the surface of biomedical implants, thus circumventing the production of a biofilm layer by slime-producing strains. For the purpose of examining factors which affect the adherence of S. epidermidis to hydrophobic surfaces, we have developed *

a simple and rapid in vitro assay of adherence which is independent of bacterial growth and slime production. The assay is based upon the quantitative measurement of the cell-associated urease activity of S. epidermidis as an estimate of the number of bacteria remaining adherent to the microwells of plastic tissue culture plates. This method can also be used for screening the efficacy of antiadhesive

agents.

ATCC 35984 (Se+), a slime-producing strain of S. epidermidis previously described by Christensen et al. (4), was used for the development of the adherence assay. Additional strains of CONS were used for some experiments and included three previously characterized laboratory isolates of slime-positive S. epidermidis, designated Se+1, Se+4, and Se+10 (6, 7), and ATCC 35982, a slime-negative strain of Staphylococcus hominis (Sh-). A slime-negative phase variant of Se+1 (Se-1) was obtained by selecting several colonial morphotypes of the parent strain on Memphis agar as previously described (2) and screening for slime production according to the method of Christensen et al. (4). All strains of CONS used in this study were urease positive, and species were determined by using Staph-Ident (Analytab Products, Plainview, N.Y.). Se+ was used initially in the development of the assay. Cultures were grown overnight in 200 ml of tryptic soy broth (Difco Laboratories, Detroit, Mich.) at 37°C with constant shaking. Cells were harvested by centrifugation at 3,000 x g for 10 min, washed twice in phosphate-buffered saline, pH 7.0, and adjusted to contain approximately 4 x 107, 2 x 108, 4 x 108, 6.5 x 108, or 1 x 109 CFU/ml with phosphate-buffered saline on the basis of the A6,0. The bacterial densities of each resulting inoculum were estimated by limiting serial dilution and colony counts. To perform the assay, eight microwells (one column) of a sterile, 96-well flat-bottomed tissue culture plate (GIBCO, Grand Island, N.Y.) were inoculated with 100 ,ul of each inoculum and the plate was incubated for 1 h at 370C. Preliminary studies had shown that extending the incubation period beyond 1 h did not generate a significant increase in

Corresponding author. 863

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In vitro measurement of the adherence of Staphylococcus epidermidis to plastic by using cellular urease as a marker.

A rapid and sensitive in vitro assay was developed to quantitatively assess the adherence of Staphylococcus epidermidis to a hydrophobic plastic surfa...
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