Staphylococcus epidermidis remains the predominant pathogen in prosthetic-device infections. Ventricular assist devices, a recently developed form of therapy for end-stage congestive heart failure, have had considerable success. However, infections, most often caused by Staphylococcus epidermidis, have limited their long-term use. The transcutaneous driveline entry site acts as a potential portal of entry for bacteria, allowing development of either localized or systemic infections. A novel in vitro binding assay using explanted drivelines obtained from patients undergoing transplantation and a heterologous lactococcal system of surface protein expression were used to identify S. epidermidis surface components involved in the pathogenesis of driveline infections. Of the four components tested, SdrF, SdrG, PIA, and GehD, SdrF was identified as the primary ligand. SdrF adherence was mediated via its B domain attaching to host collagen deposited on the surface of the driveline. Antibodies directed against SdrF reduced adherence of S. epidermidis to the drivelines. SdrF was also found to adhere with high affinity to Dacron, the hydrophobic polymeric outer surface of drivelines. Solid phase binding assays showed that SdrF was also able to adhere to other hydrophobic artificial materials such as polystyrene. A murine model of infection was developed and used to test the role of SdrF during in vivo driveline infection. SdrF alone was able to mediate bacterial adherence to implanted drivelines. Anti-SdrF antibodies reduced S. epidermidis colonization of implanted drivelines. SdrF appears to play a key role in the initiation of ventricular assist device driveline infections caused by S. epidermidis. This pluripotential adherence capacity provides a potential pathway to infection with SdrF-positive commensal staphylococci first adhering to the external Dacron-coated driveline at the transcutaneous entry site, then spreading along the collagen-coated internal portion of the driveline to establish a localized infection. This capacity may also have relevance for other prosthetic device–related infections.

Objective Infections, especially those involving drivelines, are among the most serious complications that follow ventricular assist device implantation. Staphylococci are the most common causes of these infections. Once driveline infections are established, they may remain localized or progress as an ascending infection to cause metastatic seeding of other tissue sites. While elaboration of biofilm appears to be critical in prosthetic device infections, its role as a facilitator of staphylococcal infection and migration along the driveline and other prosthetic devices is unclear. Methods A mouse model of driveline infection was used to investigate staphylococcal migration along the driveline. A biofilm producing strain of Staphylococcus epidermidis, and a Staphylococcus aureus strain and its ica negative (biofilm deficient) isogenic mutant were used in these studies. Bacterial density on the driveline and the underlying tissue was measured over time. Scanning electron microscopy was used to examine the morphology of S. epidermidis biofilm formation as the infection progressed. Results The biofilm-deficient S. aureus mutant was less effective at infecting and migrating along the driveline than the wild type strain over time. However the ica mutation had no effect on the ability of the strain to infect underlying tissue. S. aureus exhibited more rapid migration than S. epidermidis. Scanning electron microscopy revealed the deposition of host matrix on the Dacron™ material following implantation. This was followed by elaboration of a bacterial biofilm that correlated with more rapid migration along the driveline. Conclusion Biofilm formation is a critical virulence determinant that facilitates the progression of drivelines infections.

Phages have recently been implicated as important in biofilm development, although the mechanisms whereby phages impact biofilms remain unclear. One defective lambdoid phage carried by Escherichia coli K-12 is DLP12. Among the genes found in DLP12 are essD, ybcS and rzpD/rzoD, which are homologues of the Lambda phage genes encoding cell-lysis proteins (S, R and Rz/Rz 1 ). The role that these DLP12 lysis genes play in biofilm formation was examined in deletion mutants of E. coli PHL628, a curli-overproducing, biofilm-forming K-12 derivative. Strains lacking essD, ybcS and rzpD/rzoD were unable to form wild-type biofilms. While all mutants were compromised in attachment to abiotic surfaces and aggregated less well than the wild-type, the effect of the essD knockout on biofilm formation was less dramatic than that of deleting ybcS or rzpD/rzoD. These results were consistent with electron micrographs of the mutants, which showed a decreased number of curli fibres on cell surfaces. Also consistent with this finding, we observed that expression from the promoter of csgB, which encodes the curli subunits, was downregulated in the mutants. As curli production is transcriptionally downregulated in response to cell wall stress, we challenged the mutants with SDS and found them to be more sensitive to the detergent than the wild-type. We also examined the release of 14 C-labelled peptidoglycan from the mutants and found that they did not lose labelled peptidoglycan to the same extent as the wild-type. Given that curli production is known to be suppressed by N-acetylglucosamine 6-phosphate (NAG-6P), a metabolite produced during peptidoglycan recycling, we deleted nagK, the N-acetylglucosamine kinase gene, from the lysis mutants and found that this restored curli production. This suggested that deletion of the lysis genes affected cell wall status, which was transduced to the curli operon by NAG-6P via an as yet unknown mechanism. These observations provide evidence that the S, R and Rz/Rz 1 gene homologues encoded by DLP12 are not merely genetic junk, but rather play an important, though undefined, role in cell wall maintenance.

The biofilm-specific gene expression of Escherichia coli PHL628 was compared with that from exponentially growing planktonic cells using macroarray technology. In duplicate experiments, both biofilm and planktonic cells were grown in separate continually stirred tank reactors at 57% of the maximal planktonic growth rate. When transcriptional results from planktonic cultures were compared with that of biofilm grown cells, c. 4.5% of the genome showed a significant change in expression. The results presented here point to an extremely heterogeneous biofilm wherein specific gene induction was consistent with the response of biofilm cells to gradients in electron acceptors, nutrients, carbon source and a variety of stresses. A mutant in one of the genes, gspM (pshM), that was induced in biofilms was constructed and was shown to be compromised for its ability to form mature biofilms. This analysis provides additional insight into the genes induced during biofilm development, the gradients they respond to, the contribution of one gene to biofilm development, and a comparison of this with other transcriptional profiles from E. coli biofilms.

Staphylococcus epidermidis infections are common complications of prosthetic device implantation. SdrF, a surface protein, appears to play a critical role in the initial colonization step by adhering to type I collagen and Dacron™. The role of ionic interactions in S. epidermidis adherence to prosthetic material was examined. SdrF was cloned and expressed in Lactococcus lactis. The effect of pH, cation concentration and detergents on adherence to different types of plastic surfaces was assessed by crystal violet staining and bacterial cell counting. SdrF, in contrast with controls and other S. epidermidis surface proteins, bound to hydrophobic materials such as polystyrene. Binding was an ionic interaction and was affected by surface charge of the plastic, pH and cation concentration. Adherence of the SdrF construct was increased to positively charged plastics and was reduced by increasing concentrations of Ca2+ and Na+. Binding was optimal at pH 7.4. Kinetic studies demonstrated that the SdrF B domain, as well as one of the B subdomains was sufficient to mediate binding. The SdrF construct also bound more avidly to Goretex™ than the lacotococcal control. SdrF is a multifunctional protein that contributes to prosthetic devices infections by ionic, as well as specific receptor-ligand interactions.