Carlos Arrecubieta

A Postinfluenza Model of Staphylococcus aureus Pneumonia

BACKGROUND Postinfluenza Staphylococcus aureus pneumonias are increasingly recognized as a major form of life-threatening infections. METHODS A mouse model of postinfluenza S. aureus pneumonia was developed. Mice were intranasally infected with bacteria alone or bacteria plus virus. Infection was assessed by mouse survival, lung histopathology, bacterial density in the lungs, and cellular response to infection. RESULTS Mice infected with both influenza virus and S. aureus showed higher mortality, greater lung parenchymal damage, and greater bacterial density at metastatic tissue sites than mice infected with only S. aureus. At 4 h, more polymorphonuclear leukocytes and fewer CD11c(+) cells were found in lung samples from mice infected with virus and bacteria than in those from mice infected with bacteria. alpha-Hemolysin and protein A were maximally expressed 4 h after infection, and Panton-Valentine leukocidin was maximally expressed 72 h after infection, with higher levels of alpha-hemolysin expression in mice infected with bacteria alone. Interferon gamma expression was higher in tissue collected from mice infected with virus plus bacteria than in those from bacteria-infected mice. CONCLUSIONS The results from this model demonstrate diverse effects caused by antecedent influenza virus infection, which have a profound influence on the morbidity and mortality associated with S. aureus pneumonia.

SdrF, a Staphylococcus epidermidis Surface Protein, Binds Type I Collagen

Staphylococcus epidermidis is the leading cause of device-related infections. These infections require an initial colonization step in which S. epidermidis adheres to the implanted material. This process is usually mediated by specific bacterial surface proteins and host factors coating the foreign device. Some of these surface proteins belong to the serine-aspartate repeat (Sdr) family, which includes adhesins from Staphyloccus aureus and S. epidermidis. Using a heterologous expression system in Lactococcus lactis to overcome possible staphylococcal adherence redundancy we observed that one of these Sdr proteins, SdrF, mediates binding to type I collagen when present on the lactococcal cell surface. We used lactococcal recombinant strains, a protein-protein interaction assay and Western ligand blot analysis to demonstrate that this process occurs via the B domain of SdrF and both the α1 and α2 chains of type I collagen. It was also found that a single B domain repeat of S. epidermidis 9491 retains the capacity to bind to type I collagen. We demonstrated that the putative ligand binding N-terminal A domain does not bind to collagen which suggests that SdrF might be a multiligand adhesin. Antibodies directed against the B domain significantly reduce in vitro adherence of S. epidermidis to immobilized collagen.

Vaccination with clumping factor A and fibronectin binding protein A to prevent Staphylococcus aureus infection of an aortic patch in mice.

Staphylococcus aureus is a leading cause of ventricular assist device-related infections. This study evaluated the protective effect against S. aureus infection of active and passive immunization that targeted 3 proteins involved in bacterial attachment to a murine intra-aortic polyurethane patch. Active immunization of mice with a combination of the A domains of clumping factor A (ClfA), fibronectin-binding protein A (FnBPA) and fibronectin-binding protein B or passive immunization with monoclonal antibodies against ClfA and FnBPA resulted in a higher level of protection than that obtained by vaccination with either protein or antibody alone. The combination of antibodies or protein antigens appears to provide enhanced protection against prosthetic-device infection.

SdrF, a Staphylococcus epidermidis surface protein, contributes to the initiation of ventricular assist device driveline-related infections.

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.

The Role of Staphylococcus aureus Adhesins in the Pathogenesis of Ventricular Assist Device–Related Infections

Ventricular assist devices (VADs) are an important form of therapy for end-stage congestive heart failure. However, infection of the VAD, which is often caused by Staphylococcus aureus, poses a major threat to survival. Using a novel in vitro binding assay with VAD membranes and a heterologous lactococcal system of expression, we identify 3 S. aureus proteins--clumping factor A (ClfA) and fibronectin binding proteins A and B (FnBPA and FnBPB) as the main factors involved in adherence to VAD polyurethane membranes. Adherence is greatly diminished by long implantation times, reflecting a change in topological features of the VAD membrane, and is primarily mediated by the FnBPA domains in the staphylococcal proteins. We also compare the adherence of S. aureus mutant strains and show that other staphylococcal components appear to be involved in adherence to VAD membranes. Finally, we demonstrate that ClfA, FnBPA, and FnBPB mediate bacterial infection of implanted murine intra-aortic polyurethane patches.

Role of biofilm in Staphylococcus aureus and Staphylococcus epidermidis ventricular assist device driveline 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 can remain localized or progress as an ascending infection to cause metastatic seeding of other tissue sites. Although 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 murine 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 intercellular adhesion gene cluster (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 after implantation. This was followed by elaboration of a bacterial biofilm that correlated with more rapid migration along the driveline. CONCLUSIONS Biofilm formation is a critical virulence determinant that facilitates the progression of drivelines infections.

Development of a murine ventricular assist device transcutaneous drive-line model.

Mechanical circulatory support devices (MCSD) are a major form of therapy for congestive heart failure. However, MCSD have been associated with a high incidence of infections. The most common of these infections involves the transcutaneous driveline 1–4. Here, we describe a murine driveline model developed to study MCSD-related infections.