Time-resolved analysis of Staphylococcus aureus invading the endothelial barrier.

Staphylococcus aureus is a leading cause of infections world-wide. Once this pathogen has reached the bloodstream, it can invade different parts of the human body by crossing the endothelial barrier. Infected endothelial cells may be lysed by bacterial products, but the bacteria may also persist intracellularly, where they are difficult to eradicate with antibiotics and cause relapses of infection. Our present study was aimed at investigating the fate of methicillin resistant S. aureus (MRSA) isolates of the USA300 lineage with different epidemiological origin inside endothelial cells. To this end, we established two in vitro infection models based on primary human umbilical vein endothelial cells (HUVEC), which mimic conditions of the endothelium when infection occurs. For comparison, the laboratory strain S. aureus HG001 was used. As shown by flow cytometry and fluorescence- or electron microscopy, differentiation of HUVEC into a cell barrier with cell-cell junctions sets limits to the rates of bacterial internalization, the numbers of internalized bacteria, the percentage of infected cells, and long-term intracellular bacterial survival. Clear strain-specific differences were observed with the HG001 strain infecting the highest numbers of HUVEC and displaying the longest intracellular persistence, whereas the MRSA strains reproduced faster intracellularly. Nonetheless, all internalized bacteria remained confined in membrane-enclosed LAMP-1-positive lysosomal or vacuolar compartments. Once internalized, the bacteria had a higher propensity to persist within the differentiated endothelial cell barrier, probably because internalization of lower numbers of bacteria was less toxic. Altogether, our findings imply that intact endothelial barriers are more likely to sustain persistent intracellular infection.

Neonatal liver abscesses due to Candida infection in a preterm infant, secondary to malpositioned umbilical lines--a rare entity.

Neonatal liver abscess is an uncommon seen condition in neonatology and it holds a very high neonatal mortality because of difficulty in diagnosis and treatment. Till today, only few instances are reported that too are mainly in preterm. Its diagnosis requires a high index of suspicion. Fungal hepatic abscess is very rare and in medical literature very few case reports are there in the medical literature. Here, we report a case of Candida albicans liver abscess in a preterm neonate, secondary to malpositioned umbilical lines that presented with respiratory difficulty and other clinical features of sepsis that was managed medically and discharged successfully.

Staphylococcus aureus host cell invasion and virulence in sepsis is facilitated by the multiple repeats within FnBPA.

Entry of Staphylococcus aureus into the bloodstream can lead to metastatic abscess formation and infective endocarditis. Crucial to the development of both these conditions is the interaction of S. aureus with endothelial cells. In vivo and in vitro studies have shown that the staphylococcal invasin FnBPA triggers bacterial invasion of endothelial cells via a process that involves fibronectin (Fn) bridging to alpha(5)beta(1) integrins. The Fn-binding region of FnBPA usually contains 11 non-identical repeats (FnBRs) with differing affinities for Fn, which facilitate the binding of multiple Fn molecules and may promote integrin clustering. We thus hypothesized that multiple repeats are necessary to trigger the invasion of endothelial cells by S. aureus. To test this we constructed variants of fnbA containing various combinations of FnBRs. In vitro assays revealed that endothelial cell invasion can be facilitated by a single high-affinity, but not low-affinity FnBR. Studies using a nisin-inducible system that controlled surface expression of FnBPA revealed that variants encoding fewer FnBRs required higher levels of surface expression to mediate invasion. High expression levels of FnBPA bearing a single low affinity FnBR bound Fn but did not invade, suggesting that FnBPA affinity for Fn is crucial for triggering internalization. In addition, multiple FnBRs increased the speed of internalization, as did higher expression levels of FnBPA, without altering the uptake mechanism. The relevance of these findings to pathogenesis was demonstrated using a murine sepsis model, which showed that multiple FnBRs were required for virulence. In conclusion, multiple FnBRs within FnBPA facilitate efficient Fn adhesion, trigger rapid bacterial uptake and are required for pathogenesis.

Putative outer membrane proteins of Leptospira interrogans stimulate human umbilical vein endothelial cells (HUVECS) and express during infection.

Cell adhesion molecules (CAMs) are surface receptors present in eukaryotic cells that mediate cell-cell or cell-extracellular matrix interactions. Vascular endothelium stimulation in vitro that lead to the upregulation of CAMs was reported for the pathogenic spirochaetes, including rLIC10365 of Leptospira interrogans. In this study, we report the cloning of LIC10507, LIC10508, LIC10509 genes of L. interrogans using Escherichia coli as a host system. The rational for selecting these sequences is due to their location in L. interrogans serovar Copenhageni genome that has a potential involvement in pathogenesis. The genes encode for predicted lipoproteins with no assigned functions. The purified recombinant proteins were capable to promote the upregulation of intercellular adhesion molecule 1 (ICAM-1) and E-selectin on monolayers of human umbilical vein endothelial cells (HUVECS). In addition, the coding sequences are expressed in the renal tubules of animal during bacterial experimental infection. The proteins are probably located at the outer membrane of the bacteria since they are detected in detergent-phase of L. interrogans Triton X-114 extract. Altogether our data suggest a possible involvement of these proteins during bacterial infection and provide new insights into the role of this region in the pathogenesis of Leptospira.

Tissue culture assays used to analyze invasion by Staphylococcus aureus.

Many successful pathogens have developed the ability to adhere to and invade animal tissues. Recent experimental evidence suggests that S. aureus, generally perceived as an extracellular pathogen, can also invade and, in some cases, multiply within host cells. As a proxy to infections in animal hosts, the study of S. aureus interactions with tissue culture cells has become an important research tool in many aspects of bacterial pathogenesis. In this unit, we describe two cell culture models, including bovine mammary epithelial cells and human umbilical vein endothelial cells, that investigators have used to study the interactions of S. aureus with host cells.

Listeria monocytogenes induced Rac1-dependent signal transduction in endothelial cells.

Infection of endothelial cells by Listeria monocytogenes is an essential step in the pathogenesis of listeriosis. Small GTPases of the Rho family act as molecular switches in signal transduction. We tested the hypothesis that Rho GTPases contribute to the regulation of cytokine expression following L. monocytogenes infection. L. monocytogenes induced release of distinct CC and CXC, as well as Th1 and Th2 cytokines and growth factors by endothelial cells and activated RhoA and Rac1. Inhibition of Rac1 by inhibitor Nsc23766 reduced cytokine expression, and slightly yet significantly the uptake of bacteria. Blocking of Rho proteins by Clostridium difficile toxin B-10463 (TcdB) reduced Listeria-dependent cytokine expression, whereas activating Rho proteins by Escherichia coli CNF1 increased it. We analyzed regulation of IL-8 expression in more detail: Listeria-induced IL-8 release was reduced by inhibition of RhoA, Rac1 and Cdc42 (TcdB) or Rac1 while blocking of RhoA/B/C by Clostridium limosum C3 fusion toxin (C3FT) or Rho kinase by Y27632 reduced cytokine expression only slightly. Activation of RhoA, Rac1 and Cdc42 (CNF1), but not of RhoA alone (CNF(Y)), enhanced Listeria-dependent IL-8 release significantly. Furthermore, inhibition of RhoA, Rac1 and Cdc42 (TcdB) and Rac1 (Nsc23766), but not of RhoA (C3FT) reduced Listeria-related recruitment of NF-kappaB/p65 and RNA polymerase II to the il8 promoter, as well as acetylation of histone H4 and Ser10/Lys14-phosphorylation/acetylation of histone H3 at the il8 gene promoter in HUVEC. In conclusion, Rac1 contributed to L. monocytogenes-induced cytokine expression by human endothelial cells.

Streptococcus pneumoniae R6x induced p38 MAPK and JNK-mediated caspase-dependent apoptosis in human endothelial cells.

Streptococcus pneumoniae is the major pathogen of community-acquired pneumonia and a common cause of otitis, meningitis and sepsis. During pneumococci infection accompanied with bacterial invasion and hematogenous spreading, the endothelium is directly targeted by pneumococci and their virulence factors. Therefore, we tested the hypothesis that pneumococci induced endothelial apoptosis. Unencapsulated R6x pneumococci strongly induced apoptosis of human endothelial cells both from lung microvasculature and umbilical vein, whereas an encapsulated strain D39 mainly led to necrotic cell death. Deletion of the gene coding for pneumolysin reduced pneumococci-induced apoptosis in HUVEC. Furthermore, N-acetyl-L-cysteine, an antioxidant thiol, significantly reduced apoptosis caused by R6x, and LDH release induced by D39, pointing to a role for reactive oxygen species in the pathogenesis. Apoptotic cells showed increased cleavage and activity of caspases 6 and 9 but only late activation of caspase 3. Programmed cell death could be strongly reduced by pan-caspase inhibitor zVAD. Reduced levels of Bcl2 and cytosolic increase of apoptosis-inducing factor in pneumococci-infected cells implicated involvement of mitochondrial death pathways. Caspase activation and apoptosis were abolished by cAMP elevation. Moreover, p38 mitogen-activated protein kinase and c-Jun NH(2)-terminal kinase were activated in pneumococci-infected cells and inhibitors of both kinases strongly reduced pneumococci-induced caspase activation and apoptosis. Hence, kinase- and caspase-dependence of pneumococci-induced endothelial apoptosis may bear relevance to novel therapeutic approaches to pneumococci-related disease.

Intestinal bacterial overgrowth induces the production of biologically active intestinal lymph.

OBJECTIVE: We have previously documented that gut-derived lymph from rats subjected to trauma plus hemorrhagic shock (T/HS) is injurious to vascular endothelial cells and activates neutrophils (PMNs), two key events in postshock organ injury. Because T/HS leads to gut injury, intestinal bacterial overgrowth, and the loss of gut barrier function, the relative role of gut injury as opposed to intestinal bacterial overgrowth per se in the pathogenesis of biologically active intestinal lymph is unclear. We therefore studied whether mesenteric lymph can injure endothelial cells and/or active PMNs in an intestinal bacterial overgrowth model where there is no gut injury (monoassociation). METHODS: Bacterial overgrowth was established in male rats by treating the animals with 4 days of oral antibiotics followed by administration of a nonpathogenic, streptomycin-resistant strain of Escherichia coli C25. Mesenteric lymph was then collected from rats with normal flora and from E. coli C25 monoassociated rats. Its effects were tested on human umbilical vein endothelial cells (HUVECs) and human PMNs. As an additional control, lymph was collected from antibiotic-decontaminated rats that received antibiotics but were not colonized with E. coli C25. RESULTS: As compared with medium, normal flora intestinal lymph, antibiotic-decontaminated lymph, or portal plasma from the monoassociated rats, mesenteric lymph from the monoassociated rats killed HUVECs and increased the permeability of a HUVEC monolayer. In contrast to the effects on HUVECs, lymph from the monoassociated rats did not increase PMN CD11b expression or prime PMNs for an augmented respiratory burst, as compared with lymph from the rats with normal flora or from antibiotic-decontaminated rats. The effects of lymph from the monoassociated rats was not caused by bacteria, because these lymph samples were sterile. CONCLUSION: These results indicate that disruption of the normal intestinal microflora resulting in bacterial overgrowth with enteric bacilli may participate in the production of mesenteric lymph that is injurious to endothelial cells in shock, but this mechanism does not appear to be significantly involved in the activation of PMNs.

Characterization of YopT effects on Rho GTPases in Yersinia enterocolitica-infected cells.

Pathogenic yersiniae employ a type III secretion system for translocating up to six effector proteins (Yersinia outer proteins (Yops)) into eukaryotic target cells. YopT is a cysteine protease that was shown to remove the C-terminal isoprenoid group of RhoA, Rac, and CDC42Hs. Here we characterized the cell biological and biochemical activities of YopT in cells infected with pathogenic Yersinia enterocolitica. Bacterially injected YopT located to cell membranes from which it released RhoA but not Rac or CDC42Hs. In the infected cells RhoA was dissociated from guanine nucleotide dissociation inhibitor-1 (GDI-1) and accumulated as a monomeric protein in the cytosol, whereas Rac and CDC42Hs remained GDI-bound. Direct transfer of isoprenylated RhoA to YopT and RhoA modification could be reconstituted in vitro by guanosine 5'-3-O-(thio)triphosphate loading of a recombinant RhoA.GDI-1 complex. Finally, in macrophages infected with a Yersinia strain selectively translocating YopT podosomal adhesion structures required for chemotaxis as well as phagocytic cups mediating uptake of yersiniae were disrupted. These findings indicate that bacterially translocated YopT acts on membrane-bound and GDI-complexed RhoA but not Rac or CDC42, and this is sufficient for disruption of macrophage immune functions.

The REP2 repeats of the genome of Neisseria meningitidis are associated with genes coordinately regulated during bacterial cell interaction.

Interaction with host cells is essential in meningococcal pathogenesis especially at the blood-brain barrier. This step is likely to involve a common regulatory pathway allowing coordinate regulation of genes necessary for the interaction with endothelial cells. The analysis of the genomic sequence of Neisseria meningitidis Z2491 revealed the presence of many repeats. One of these, designated REP2, contains a -24/-12 type promoter and a ribosome binding site 5 to 13 bp before an ATG. In addition most of these REP2 sequences are located immediately upstream of an ORF. Among these REP2-associated genes are pilC1 and crgA, described as being involved in steps essential for the interaction of N. meningitidis with host cells. Furthermore, the REP2 sequences located upstream of pilC1 and crgA correspond to the previously identified promoters known to be induced during the initial localized adhesion of N. meningitidis with human cells. This characteristic led us to hypothesize that at least some of the REP2-associated genes were upregulated under the same circumstances as pilC1 and crgA. Quantitative PCR in real time demonstrated that the expression of 14 out of 16 REP2-associated genes were upregulated during the initial localized adhesion of N. meningitidis. Taken together, these data suggest that these repeats control a set of genes necessary for the efficient interaction of this pathogen with host cells. Subsequent mutational analysis was performed to address the role of these genes during meningococcus-cell interaction.

Stimulation of adhesion molecule expression by Helicobacter pylori and increased neutrophil adhesion to human umbilical vein endothelial cells.

Helicobacter pylori upregulates endothelial adhesion molecules but the pattern is unclear. Human umbilical vein endothelial cells (HUVEC) were exposed to control medium or H. pylori 60190. Binding of monoclonal antibodies against P-selectin, E-selectin, vascular adhesion molecule-1 (VCAM-1) and intercellular adhesion molecule-1 (ICAM-1) was determined using enzyme-linked immunosorbent assay. Binding of polymorphonuclear leukocytes to HUVEC was determined on cells exposed as above. After 6 h exposure to H. pylori, there were 30%, 124%, 167% and 100% increases in P-selectin, E-selectin, VCAM-1 and ICAM-1 levels and a 400% increase in polymorphonuclear leukocyte adhesion in HUVEC exposed to H. pylori. Effects of incubation for other intervals between 0 and 18 h are also described. H. pylori exerts some of its effects on gastric mucosa via gastric vasculature. This study gives insight into the pattern of H. pylori-associated endothelial adhesion molecule upregulation.

Mechanisms of Staphylococcus aureus induced apoptosis of human endothelial cells.

Staphylococcus aureus plays an important role in sepsis, pneumonia and wound infections. Here, we demonstrate that infection with several S. aureus strains results in apoptosis of human endothelial cells. S. aureus induced an activation of cellular caspases, the acid sphingomyelinase, a release of cytochrome c and a stimulation of Jun NH2-terminal kinase (JNK). The significance of these findings is indicated by a prevention of S. aureus triggered apoptosis of human cells deficient for ASM or upon genetic or pharmacological inhibition of JNK or caspases, respectively.

Formation of stress fibres in human endothelial cells infected with Bartonella bacilliformis is associated with altered morphology, impaired migration and defects in cell morphogenesis.

Bartonella bacilliformis, a Gram-negative, flagellated bacterium, infects human erythrocytes (haematic phase) and endothelial cells (tissue phase), resulting in a biphasic disease. In the tissue phase of disease (verruga peruana), infection leads to infection of endothelial cells and a pronounced proliferation of these cells, resulting in characteristic skin eruptions of papules and nodules. We have studied the properties of endothelial cells infected in vitro. Extensive cytoskeletal remodelling of endothelial cells occurred after infection in vitro with B. bacilliformis. The cells became spindle shaped and contained arrays of actin stress fibres orientated parallel to the long axis of the cell. Cell-cell contacts were disrupted, along with the distribution of the plasma membrane marker protein, PECAM-1, which participates in cell-cell junctions. The prominent stress fibres terminated in an increased number of focal contacts, which were studied using immunofluorescent staining for paxillin, a cytoplasmic protein that localizes in the focal adhesions. These morphological changes are consistent with activation of intracellular Rho by B. bacilliformis. Formation of stress fibres and the increased number of focal adhesions could be prevented by preincubation of the endothelial cells with C3 exoenzyme, which inactivates intracellular Rho by ADP ribosylation. Endothelial cell motility was greatly diminished in infected cells and the cells did not respond effectively to a stimulus that would evoke motility. In addition, infection of endothelial cells interfered with their ability to form networks of capillary tubes when suspended within three-dimensional collagen matrices. If the properties of infected endothelial cells in vivo are similar, the infected cells will probably not participate effectively in angiogenesis.

Human endothelial cells are activated by interferon-gamma plus tumour necrosis factor-alpha to kill intracellular Pseudomonas aeruginosa.

Proinflammatory cytokines have been shown to activate endothelial cells. To investigate the effect of cytokines on the interaction of human umbilical vein endothelial cells (HUVEC) with Pseudomonas aeruginosa, cells were treated with interferon-gamma (IFN-gamma) plus tumour necrosis factor-alpha (TNF-alpha) for 24 hr and exposed to P. aeruginosa suspension for 1 hr. Light microscopy showed that activated cells internalized significantly more bacteria than control cells. To ascertain the effect of cytokines on the microbicidal activity of HUVEC, the concentrations of viable intracellular (IC) bacteria in control and activated cells were determined, at 1 and 5 hr postinfection, by the gentamicin exclusion assay. In control cells, no significant decrease in the concentration of bacteria was detected 5 hr postinfection. In contrast, in activated cells the concentration of viable bacteria at 5 hr was significantly lower. Concentrations of superoxide and hydrogen peroxide detected in supernatants of activated cells were significantly higher than in control cell supernatants. HUVEC anti-P. aeruginosa activity was insensitive to the antioxidants superoxide dismutase, dimethylthiourea and allopurinol as well as to the L-arginine analogues aminoguanidine and NG-monomethyl-L-arginine (L-NMMA), but was significantly inhibited by catalase. Our results indicate that HUVEC can be activated by IFN-gamma plus TNF-alpha to kill IC P. aeruginosa and suggest a role for reactive oxygen radicals, notably hydrogen peroxide, in HUVEC antibacterial activity.

Altered gene expression in Staphylococcus aureus upon interaction with human endothelial cells.

Staphylococcus aureus is isolated from a substantial number of patients with infective endocarditis who are not known to have predisposing heart abnormalities. It has been suggested that the infection is initiated by the direct binding of S. aureus to human vascular endothelium. To determine the mutual response of the endothelial cells and the bacteria, we studied the interaction between S. aureus and human vascular endothelium. Scanning electron microscopic analyses showed that binding of S. aureus to human umbilical vein endothelial cells (HUVEC) mainly occurred via thread-like protrusions extending from the cell surface. Bound bacteria appeared to be internalized via retraction of the protrusions into newly formed invaginations of the endothelial cell surface. The growth phase of S. aureus had a major impact on the interaction with HUVEC. Logarithmically growing bacteria showed increased binding to, and were more readily internalized by, HUVEC compared to stationary-phase bacteria. To assess the bacterial response to the cellular environment, an expression library of S. aureus was used to identify genes whose expression was induced after 4 h of exposure to HUVEC. The identified genes could be divided into different categories based on the functions of the encoded proteins (transport, catabolism, biosynthesis, and DNA repair). Further analyses of five of the S. aureus transposon clones showed that HUVEC as well as human serum are stimuli for triggering gene expression in S. aureus.

Laparoscopically assisted resection of umbilical structures in foals.

A technique for laparoscopically assisted resection of umbilical structures in foals was developed. Eleven foals ranging from 8 to 42 days old underwent this procedure. Results of bacteriologic culture of umbilical structures were positive in 7 foals. Mean duration of anesthesia was 99 minutes, of which the initial 20 to 25 minutes were typically devoted to positioning and preparation of the foal for surgery. Major complications did not develop in any foal. Minor complications, such as slippage of the endoscopic ligating clip or laceration of the bladder during dissection, were dealt with successfully during the procedure. Potential benefits of use of laparoscopy (i.e., decreased postoperative morbidity, smaller incisions, and increased intraoperative access to structures) must be carefully weighed against the risks of increased duration of anesthesia. However, anesthetic-related complications were not observed in any foal.

NF-kappa B-dependent inhibition of apoptosis is essential for host cellsurvival during Rickettsia rickettsii infection.

The possibility that bacteria may have evolved strategies to overcome host cell apoptosis was explored by using Rickettsia rickettsii, an obligate intracellular Gram-negative bacteria that is the etiologic agent of Rocky Mountain spotted fever. The vascular endothelial cell, the primary target cell during in vivo infection, exhibits no evidence of apoptosis during natural infection and is maintained for a sufficient time to allow replication and cell-to-cell spread prior to eventual death due to necrotic damage. Prior work in our laboratory demonstrated that R. rickettsii infection activates the transcription factor NF-kappa B and alters expression of several genes under its control. However, when R. rickettsii-induced activation of NF-kappa B was inhibited, apoptosis of infected but not uninfected endothelial cells rapidly ensued. In addition, human embryonic fibroblasts stably transfected with a superrepressor mutant inhibitory subunit Ikappa B that rendered NF-kappa B inactivatable also underwent apoptosis when infected, whereas infected wild-type human embryonic fibroblasts survived. R. rickettsii, therefore, appeared to inhibit host cell apoptosis via a mechanism dependent on NF-kappa B activation. Apoptotic nuclear changes correlated with presence of intracellular organisms and thus this previously unrecognized proapoptotic signal, masked by concomitant NF-kappa B activation, likely required intracellular infection. Our studies demonstrate that a bacterial organism can exert an antiapoptotic effect, thus modulating the host cell's apoptotic response to its own advantage by potentially allowing the host cell to remain as a site of infection.

Infection of human vascular endothelial cells with Staphylococcus aureus induces hyperadhesiveness for human monocytes and granulocytes.

The consequences of internalization of Staphylococcus aureus by HUVEC with respect to their adhesiveness for human monocytes and granulocytes were investigated. Viable and UV-killed, but not heat-killed, S. aureus were internalized by HUVEC, which required participation of the endothelial cytoskeleton. S. aureus-infected HUVEC displayed increased surface expression of CD106 (VCAM-1), CD54 (ICAM-1), and MHC I molecules. Expression of CD62P (P-selectin), CD62E (E-selectin), CD31 (PECAM-1), and CD102 (ICAM-2) was not affected. Concomitantly, these HUVEC expressed a time- and inoculum size-dependent hyperadhesiveness for monocytes and granulocytes. Monocyte adhesion reached maximal levels (approximately 60% adhesion) 23 h after the initial 1 h period of infection of HUVEC with about 50 bacteria per single HUVEC. To induce maximal (approximately 20%) adhesion of granulocytes, five times higher concentrations of HUVEC-infecting bacteria were required. Using the appropriate mAb, granulocyte adhesion to S. aureus-infected HUVEC was shown to be entirely mediated by the beta2 (CD11/CD18) integrins. Monocyte adhesion to these HUVEC was largely (approximately 70%) dependent on both CD11a/CD18 (LFA-1) and CD49d/CD29 (VLA-4). This demonstrates that infection of HUVEC with S. aureus potentiates CD11/CD18-mediated granulocyte adhesion and shifts the mechanism of monocyte adhesion from being completely CD11/CD18 dependent to one that also utilizes the VLA-4/VCAM-1 dependent pathway. Together, these findings indicate that in response to internalization of S. aureus, vascular endothelial cells may initiate recruitment of monocytes and granulocytes, which may be an important initial event in the pathogenesis of endovascular diseases.

A one-stage marsupialization procedure for management of infected umbilical vein remnants in calves and foals.

Five Holstein calves and two foals with omphalophlebitis were treated by surgical marsupialization of the umbilical vein remnant because complete resection of the infected tract was not possible. The infected umbilical stalk was resected, and the umbilical vein remnant was marsupialized in a one-stage procedure by suturing it into the abdominal wall lateral to the abdominal incision. Antimicrobial drugs were administered, and the marsupialized tract was irrigated until closure by second intention healing. Cellulitis associated with the marsupialization site occurred in two calves but resolved with antimicrobial therapy. Owners reported that, 9 to 60 months after surgery, there were no complications associated with the procedure.

Antibiotic penetration of and bactericidal activity within endothelial cells.

It has been observed that the number of cases of infective endocarditis arising in patients who have no previous identifiable cardiac abnormalities is increasing, suggesting that direct bacterial interactions with endothelium may occur. Furthermore, the prolonged natural history of endocarditis, need for lengthy therapy, and frequency of relapse suggest that intracellular bacteria that may be protected from antimicrobial action and host responses exist. Using high-performance liquid chromatography, we investigated the penetration of seven antibiotics used to treat Staphylococcus aureus infections into cultured human umbilical vein endothelial cells and the effect of these antibiotics on the intracellular killing of two strains of the organism. In general, endothelial cell penetration of lipophilic drugs, such as minocycline, ciprofloxacin, and rifampin, exceeded that of hydrophilic drugs, represented by nafcillin, cefazolin, cefuroxime, and vancomycin. Bacterial killing paralleled the intracellular penetration of all the antibiotics except rifampin, which concentrated well inside cells but had poor killing activity. However, when combined with the other antibiotics, rifampin potentiated their killing activity against intracellular S. aureus.