Type 8 capsular polysaccharide (CP8) is widely prevalent among clinical isolates of Staphylococcus aureus, but the role that the capsule plays in the pathogenesis of staphylococcal infections is unclear. This study was performed to identify growth conditions that would optimize the production of CP8 and to determine whether enhanced CP8 expression would influence staphylococcal virulence. S. aureus Becker grown in a chemically defined broth medium with < 1 microM ferric nitrate produced up to eightfold more CP8 per milligram of biomass than did bacteria cultivated in the same medium containing 20 microM ferric nitrate. The bacteria produced > 350-fold more cell-associated CP8 per milligram of biomass when grown on the surface of Columbia agar than when grown in Columbia broth. Most of the CP8 produced by broth-grown cells was secreted into the culture medium. S. aureus cultivated on the surface of nitrocellulose membranes floating on Columbia broth produced levels of CP8 similar to those produced by cells grown on Columbia agar. Similarly, bacteria harvested from endocardial vegetations of rabbits infected with S. aureus produced high levels of CP8. These results indicate that staphylococci grown on surfaces, both in vitro and in vivo, produce larger quantities of cell-associated CP8 than those grown in liquid cultures. However, no differences were observed in the 50% lethal dose for mice of strain Becker grown on solid medium (high levels of capsule expression) or in liquid medium (low levels of capsule expression).

Clinical isolates of coagulase-negative staphylococci were analyzed for elaboration of the capsular polysaccharide/adhesion (PS/A) and extracellular biofilm or slime. Of the 151 analyzed, 103 (68%) produced PS/A and 69 (46%) made extracellular slime; 87% of the slime-producing isolates made PS/A. Among isolates from all clinical infections examined except peritonitis, PS/A-positive isolates bound significantly (P < .001) more colony-forming units after 15 min to 1.5-cm segments of silicone-elastomer catheter than did PS/A-negative isolates. Slime-positive isolates were not more adherent than slime-negative isolates, because 42% of the PS/A-positive isolates were slime-negative. Thus, PS/A expression is common among clinical isolates of coagulase-negative staphylococci, accounting for most slime-positive and a proportion of slime-negative isolates.

We used transposon (Tn) mutagenesis to study the role of capsular polysaccharide/adhesin (PS/A) and slime in adherence of Staphylococcus epidermidis to catheters. pLTV1, containing Tn917-LTV1, was transformed into S. epidermidis M187 by protoplast fusion with S. aureus RN4220(pLTV1), creating M187(pLTV1). Tn mutants were isolated following growth at 42 degrees C; mutants deficient in PS/A and slime production were selected. PS/A- and slime-deficient Tn mutants had a 10-fold decrease in vitro in the initial phase of adherence to catheters, comparable to levels of strains that do not produce PS/A. Introduction of Tn917-LTV1-interrupted DNA from PS/A-deficient mutant M187sn3 into the parental strain via transformation of protoplasts yielded recipients with inserts identical to those of the Tn mutant that were PS/A and slime deficient. Chromosomal DNA flanking the Tn in mutant M187sn3 was cloned into Escherichia coli. The cloned DNA was found to hybridize to approximately 5-kb EcoRI fragments from the parental strain and from control Tn mutants that express parental levels of PS/A and to either approximately 9- or approximately 14-kb EcoRI fragments from other highly adherent, PS/A-producing strains. Mapping studies demonstrated that in the eight PS/A-deficient mutants that have been isolated, the Tn insertions all occur within a region of approximately 11.6 kb that is defined by three EcoRI sites. These results support previous findings indicating that in S. epidermidis PS/A is involved with in vitro adherence to plastic biomaterials and elaboration of PS/A is closely associated with slime production.

Background. Staphylococcus epidermidis is the principal pathogen in prosthetic valve endocarditis. The

We studied the effects of in vitro and in vivo coating of catheters with human blood proteins on binding of coagulase-negative staphylococci. Coating resulted in no enhancement of binding. Catheters coated in vitro bound fewer organisms than uncoated catheters. Host proteins do not enhance adherence of coagulase-negative staphylococci to biomaterials.Coagulase-negative staphylococci (CoNS) have become major nosocomial pathogens because of their capacity to colonize plastic biomaterials used in catheters and prosthetic devices (7). Several mechanisms have been proposed to explain this adherence to biomaterials, but the problem is far from solved (6,10,18,20). Recent studies suggest that host proteins coated onto biomaterials promote adherence of CoNS (10, 21).We have proposed that the capsular polysaccharide/adhesin (PS/A) mediates the initial adherence of CoNS to plastic biomaterials. We noted that strains of CoNS which produced PS/A and slime (PS/A' slime') (20) adhered in high numbers to native, uncoated plastics, such as silicon elastomer. PS/Aslime-strains adhere to plastics in much smaller numbers. In the present study, we examined the effect of precoating catheters in vitro with human blood, serum, and purified blood proteins on adherence of both PS/A' slime' and PS/Aslimestrains of CoNS. We also studied adherence of CoNS to catheters coated in vivo with blood proteins after insertion into volunteers or hospitalized patients.CoNS were isolated from patients with clear-cut clinical diagnoses of catheter-related sepsis, dialysis-related peritonitis, endocarditis, or cerebrospinal fluid shunt infection and were identified as Staphylococcus epidermidis by a Sceptor System (Staphylococcus MIC/ID Panel; Becton Dickinson, Towson, Md.). Strains were categorized as PS/A' or PS/Aby enzyme-linked immunosorbent assay inhibition using polyclonal antibody specific for the PS/A antigen purified from S. epidermidis RP62A (ATCC 35984) (20). PS/A' strains showed a mean (+ standard deviation) of 75% + 12% inhibition of binding of PS/A-specific polyclonal rabbit antibody, while PS/Astrains showed a mean of 3.1% + 7% inhibition. Slime production was quantitated by measuring the optical density at 490 nm of the safranin-stained biofilms produced by inoculation of 105 to 106 CFU of CoNS per ml followed by overnight growth at 37°C in tryptic soy broth (TSB) in flat-bottomed 96-well tissue culture plates (Costar, Cambridge, Mass.). PS/A' strains produced a mean optical density of 1.25 ± 0.4, while PS/Astrains produced a mean optical density of 0.34 ± 0.08. Staphylococcus aureus Reynolds, Becker, Wood 46, and Cowan 1 were provided by Jean Lee of Harvard Medical School and were also used in the studies. * Corresponding author.To measure adherence to catheter tubing, CoNS were grown at 37°C overnight in static TSB cultures containing 25 ,uCi of Na [1-'4C]acetate (Du Pont, NEN Research Products, Boston, Mass.). The bacteria were sedimented by centrifugation and suspended in TSB. On the basis of previous reports (10, 21) that a protein ...