The pathogenesis of gonococcal urethritis in men: confocal and immunoelectron microscopic analysis of urethral exudates from men infected with Neisseria gonorrhoeae

Confocal and immunoelectron microscopic analysis of urethral exudates from 12 men with gonococcal urethritis showed that Neisseria gonorrhoeae can invade urethral epithelial cells. Studies with acridine orange stain demonstrated that the majority of organisms within urethral epithelial cells were viable at the time of fixation. Three-dimensional modeling of an infected epithelial cell using image analysis of 21 digitized confocal sections stained with YOYO-1 and DiIC 18(3) revealed that gonococcal invasion of these cells occurred in a polar fashion, most likely at the epithelial luminal surface. Serial immunoelectron micrographs showed evidence of membrane fusion with pedestal formation between the gonococcus and the epithelial cell, gonococci within vacuoles, and occasional gonococci free in the cytoplasm. Immunoelectron microscopy studies showed ruptured vacuoles at the cell surface releasing organisms. These studies demonstrate that urethral epithelial cells are invaded by gonococci during the course of infection in males.     

Physical limitations on Salmonella typhi entry into cultured human intestinal epithelial cells

Kinetic studies of Salmonella typhi invasion of INT407 cells at different multiplicities of infection (MOIs) have revealed a strict physical limitation on S. typhi entry at MOIs of >/=40. Staining of infected monolayers to distinguish intracellular from extracellular bacteria revealed that all monolayer cells are susceptible to infection and that internalized bacteria are typically contained in one to three separate clusters per cell during the first 60 min. Scanning and transmission electron microscopic analyses of time course-infected monolayers showed that at early times postinfection, bacteria bind to shortened, coalesced microvilli in one to three focal aggregate structures per host cell surface. As reported previously for S. typhimurium, focal aggregates progress to conical membrane ruffles that appear to engulf one or a few centrally contained S. typhi cells by a macropinocytic process, which enhanced the entry of simultaneously added Escherichia coli HB101 about 30-fold. Additionally, kinetic studies showed that at an MOI of approximately 400, maximal S. typhi entry is virtually completed within 30 to 35 min. Monolayers pretreated with S. typhi for 30 min to saturate the entry process were severely reduced in the ability to internalize subsequently added kanamycin-resistant strains of S. typhi or S. typhimurium, but E. coli HB101(pRI203) expressing the cloned Yersinia inv gene was not reduced in entry. In invasion inhibition assays, anti-beta1 integrin antibodies markedly reduced E. coli HB101(pRI203) invasion efficiency but did not reduce S. typhi entry. Collectively, these data provide direct physical and visual evidence which indicates that S. typhi organisms are internalized at a limited number (i.e., two to four) of sites on host cells. S. typhi and S. typhimurium likely share INT407 cell entry receptors which do not appear to be members of the beta1 integrin superfamily.     

Gentamicin-resistant menadione and hemin auxotrophic Staphylococcus aureus persist within cultured endothelial cells

Staphylococcus aureus menadione and hemin auxotrophs, generated by in vitro gentamicin selection, demonstrated reduced hemolytic activity and enhanced intracellular survival within cultured bovine aortic endothelial cells relative to their hemolytic parent. Supplementation of the auxotrophs with exogenous menadione or hemin resulted in rapid growth, increased hemolytic activity, and reduced intracellular persistence to the level found for the hemolytic clinical parent. Aminoglycoside selection of staphylococcal menadione and hemin auxotrophs and subsequent persistence of these variants in the intracellular milieu may adapt S. aureus for evasion of host defenses and resistance to antimicrobial therapy.     

Apoptosis of human intestinal epithelial cells after bacterial invasion

Epithelial cells that line the human intestinal mucosa are the initial site of host invasion by bacterial pathogens. The studies herein define apoptosis as a new category of intestinal epithelial cell response to bacterial infection. Human colon epithelial cells are shown to undergo apoptosis following infection with invasive enteric pathogens, such as Salmonella or enteroinvasive Escherichia coli. In contrast to the rapid onset of apoptosis seen after bacterial infection of mouse monocyte-macrophage cell lines, the commitment of human intestinal epithelial cell lines to undergo apoptosis is delayed for at least 6 h after bacterial infection, requires bacterial entry and replication, and the ensuing phenotypic expression of apoptosis is delayed for 12-18 h after bacterial entry. TNF-alpha and nitric oxide, which are produced as components of the intestinal epithelial cell proinflammatory program in the early period after bacterial invasion, play an important role in the later induction and regulation of the epithelial cell apoptotic program. Apoptosis in response to bacterial infection may function to delete infected and damaged epithelial cells and restore epithelial cell growth regulation and epithelial integrity that are altered during the course of enteric infection. The delay in onset of epithelial cell apoptosis after bacterial infection may be important both to the host and the invading pathogen since it provides sufficient time for epithelial cells to generate signals important for the activation of mucosal inflammation and concurrently allows invading bacteria time to adapt to the intracellular environment before invading deeper mucosal layers.     

Receptor-mediated recognition and uptake of iron from human transferrin by Staphylococcus aureus and Staphylococcus epidermidis

Staphylococcus aureus and Staphylococcus epidermidis both recognize and bind the human iron-transporting glycoprotein, transferrin, via a 42-kDa cell surface protein receptor. In an iron-deficient medium, staphylococcal growth can be promoted by the addition of human diferric transferrin but not human apotransferrin. To determine whether the staphylococcal transferrin receptor is involved in the removal of iron from transferrin, we employed 6 M urea-polyacrylamide gel electrophoresis, which separates human transferrin into four forms (diferric, monoferric N-lobe, and monoferric C-lobe transferrin and apotransferrin). S. aureus and S. epidermidis but not Staphylococcus saprophyticus (which lacks the transferrin receptor) converted diferric human transferrin into its apotransferrin form within 30 min. During conversion, iron was removed sequentially from the N lobe and then from the C lobe. Metabolic poisons such as sodium azide and nigericin inhibited the release of iron from human transferrin, indicating that it is an energy-requiring process. To demonstrate that this process is receptor rather than siderophore mediated, we incubated (i) washed staphylococcal cells and (ii) the staphylococcal siderophore, staphyloferrin A, with porcine transferrin, a transferrin species which does not bind to the staphylococcal receptor. While staphyloferrin A removed iron from both human and porcine transferrins, neither S. aureus nor S. epidermidis cells could promote the release of iron from porcine transferrin. In competition binding assays, both native and recombinant N-lobe fragments of human transferrin as well as a naturally occurring human transferrin variant with a mutation in the C-lobe blocked binding of 125I-labelled transferrin. Furthermore, the staphylococci removed iron efficiently from the iron-loaded N-lobe fragment of human transferrin. These data demonstrate that the staphylococci efficiently remove iron from transferrin via a receptor-mediated process and provide evidence to suggest that there is a primary receptor recognition site on the N-lobe of human transferrin.     

Effects of calcitonin on the secretion of pancreatic juice induced by dopamine, secretin and pancreozymin

Non-lactating, multiparous ewes were immunized either by subcutaneous infection with live Staphylococcus aureus (seventeen ewes) or by intramuscular injections of a killed S. aureus-oil adjuvant vaccine (seventeen ewes). Polymorphs which were subsequently collected from the mammary glands of the animals were used in in vitro phagocytosis assays against Pseudomonas sp. or S. aureus. There was no difference between polymorphs from the two groups of ewes in their ability to phagocytose Pseudomonas organisms. Polymorphs from the infected ewes showed significant phagocytic superiority over cells from ewes given the killed vaccine when S. aureus was the target organism. This phagocytic superiority could be abrogated by removal of cytophilic immunoglobulin from polymorphs and restored by replacement of cytophilic immunoglobulin. It was shown by staining polymorphs with FITC-conjugated anti-immunoglobulin sera that cytophilic immunoglobulin on the surface of polymorphs belonged to the IgG2 class of immunoglobulins. When 'neutral' polymorphs (from non-immunized ewes) were coated with IgG2 purified from the sera of infected ewes, they exhibited enhanced phagocytosis of staphylococci compared with 'neutral' polymorphs carying IgG2 from the sera of ewes given the killed vaccine.     

Penicillin G-induced microbicidal activity of endothelial cells cultured on gelfoam blocks

A body of evidence has surfaced documenting the ability of endothelial cells cultured on monolayers to phagocytose but not kill bacteria. Several years ago, a new three-dimensional endothelial cell culturing model was developed, which simulated the morphology of the endothelium in small vessels and capillaries. Given that endothelial cells may be derived from the same pluripotent stem cells as macrophages, the question of whether endothelial cells might phagocytose and kill bacteria was explored. Endothelial cells grown on Gelfoam blocks exhibited bactericidal activity towards Staphylococcus aureus, reaching maximal killing of > 90% after 2 h. Evidence documents the involvement of bacterial adherence to the plasma membrane of the endothelial cell. This is followed by phagocytosis of S. aureus, leading to intracellular killing. Penicillin G, included in the endothelial cell growth medium, was found to be a critical factor in the bactericidal activity demonstrated by Gelfoam blocks laden with endothelial cells.     

Isolation of endothelial cells from human umbilical cords and development of low-cost culture medium

A one-day point prevalence of infection analysis was undertaken in 1417 intensive care units (ICUs) (10,038 patients) in 17 western European countries. The prevalence of ICU-acquired infection was 20.6% (2064 patients), representing almost half the cases of infection. Pneumonia was the most commonly reported infection (46.9%), followed by infection of the lower respiratory tract (17.8%), urinary tract (17.6%), and blood (13.0%). Staphylococcus aureus was the most frequently isolated organism (30.1%), followed by Pseudomonas aeruginosa (28.7%), coagulase-negative staphylococci (19.1%), yeasts (17.1%), and enterococci (11.7%). As a group, the Enterobacteriaceae were the most commonly isolated organisms (34.4%). The study also revealed that resistance to antimicrobial agents is common among Staphylococcus aureus, Pseudomonas aeruginosa, and coagulase-negative staphylococci.     

Decreased susceptibility to antibiotic killing of a stable small colony variant of Staphylococcus aureus in fluid phase and on fibronectin-coated surfaces

The frequency of small colony variants of staphylococci associated with persistent, antibiotic resistant and relapsing infections is probably underestimated. These variants demonstrate decreased metabolism, leading to slow growth, increased resistance to cell-wall-active antibiotics, and decreased uptake of aminoglycoside antibiotics. This altered phenotype arises from defects in menadione and haemin biosynthesis resulting in impaired electron transport and decreased ATP concentrations. The recent acquisition of a stable small colony variant (SCV strain JB1), generated from strain 6850 of Staphylococcus aureus, allowed us to study the susceptibilities to antibiotic killing of parent and variant strains. Because differences in susceptibilities have been found between unattached and surface-adherent organisms, we tested both strains in solid and fluid-phase assays. Suspensions of SCV strain JB1 exposed to 8 x MIC of either oxacillin, vancomycin or fleroxacin, exhibited lower reductions in viable counts than the parent strain 6850, especially when high bacterial concentrations (1-2 x 10(7) cfu/mL) of either strain were tested. Susceptibility to antibiotic killing of bacteria attached to fibronectin-coated coverslips was markedly influenced by their growing or nongrowing state on the surface. In the latter condition, surface-bound SCV organisms were highly resistant to the bactericidal action of oxacillin or vancomycin in contrast to the parental strain which was normally eliminated by each antimicrobial agent. In conclusion, the decreased susceptibility of the stable SCV strain of S. aureus to bactericidal concentrations of antibiotics may help to explain the persistence of such organisms in chronic infections.     

Entry and trafficking of granzyme B in target cells during granzyme B-perforin-mediated apoptosis

In the widely accepted model of granule-mediated killing by cytotoxic lymphocytes, granzyme B entry into the target cell is facilitated by the pore forming molecule, perforin. Using indirect immunofluorescence and also direct visualization of fluorescein isothiocyanate (FITC)-conjugated granzyme B, we demonstrate internalization in the absence of perforin. Induction of the lytic pathway, however, required a second signal that was provided by perforin or adenovirus (Ad2). The combination of agents also resulted in a dramatic relocalization of the granzyme. Microinjection of granzyme B directly into the cytoplasm of target cells resulted in apoptosis without the necessity of a second stimulus. This suggested that the key event is the presence of granzyme B in the cytoplasm, and that when the enzyme is internalized by a target cell, it trafficks to an intracellular compartment and accumulates until release is stimulated by the addition of perforin. We found that the proteinase passed through rab5-positive vesicles and then accumulated within a novel compartment. On the basis of these results, we propose a new model for granzyme-perforin-induced target cell lysis in which granzyme B is subjected to trafficking events in the target cell that control and contribute to cell death.     

Extracellular and bacterial factors influencing staphylococcal phagocytosis and killing by human polymorphonuclear leukocytes

Extracellular and bacterial factors that influence the phagocytosis and killing of staphylococci by human polymorphonuclear leukocytes have been studied. Staphylococcus epidermidis strains were, in general, more rapidly phagocytized than were S. aureus strains. However, two strains of S. epidermidis had a very slow rate of ingestion. Although the rate of phagocytosis of S. aureus Wood 46 was greater than that of S. aureus 502A, the Wood 46 strain was more difficult to kill. Serum was essential for phagocytosis of both S. aureus and S. epidermidis. The opsonic titer of pooled serum was similar for S. aureus and S. epidermidis. In normal pooled serum, heat-labile factors were more important for effective phagocytosis than they were in immune serum. Although a saturation point for ingestion was reached, the percentage of ingested bacteria that remained alive within the leukocyte remained relatively fixed. Heat-killed and live staphylococci were igested in a similar fashion. The rate of phagocytosis was greatly reduced at 41 degrees C.     

Intramuscular clindamycin for therapy of infective endocarditis. Report of 23 cases and review of the literature

Twenty-three patients with infective endocarditis received intramuscular clindamycin (Cleocin) for treatment. Thirteen had acute Staphylococcus (S.) aureus endocarditis but none had involvement of the aortic valve. Eleven of these 13 infections were heroin-related and involved the tricuspid valve.Twenty-one patients were successfully treated. Two patients with heroin-related S. aureus infection failed to respond to intramuscularly administered clindamycin, but responded to retreatment with methicillin. There have been 34 reported cases of endocarditis treated with clindamycin. Although 80 percent of all cases due to staphylococci responded favorably, almost all were heroin-related tricuspid valve infections. In addition 91 percent of cases due to aerobic streptococci responded but, surpisingly, treatment failed in three of four cases of anaerobic endocarditis. Although clindamycin can be useful in streptococcal endocarditis and in some cases of heroin-related S. aureus tricuspid endocarditis, caution should be exercised in its use. It is "less" bactericidal than the penicillins or cephalosporins, and organisms have become resistant during treatment. Furthermore, patients with anaerobic endocarditis have not responded well, and data are not available to recommend administration of clindamycin for acute S. aureus infections engrafted on the aortic or mitral valve.     

Staphylococci express a receptor for human transferrin: identification of a 42-kilodalton cell wall transferrin-binding protein

Staphylococcus aureus and the coagulase-negative staphylococci are commonly responsible for peritonitis in renal patients undergoing continuous ambulatory peritoneal dialysis. To simulate growth conditions in vivo, staphylococci isolated from peritoneal infections were cultured in used human peritoneal dialysate (HPD). Immunoblotting experiments using cell wall preparations from these staphylococci revealed the presence of the host iron-binding glycoprotein transferrin bound to S. aureus, S. epidermidis, S. capitis, S. haemolyticus, and S. hominis but not to S. warneri or S. saprophyticus. Similar results were obtained by incubating broth-grown staphylococci with human transferrin, although, in contrast to S. aureus, the coagulase-negative staphylococci bound more transferrin after growth in iron-restricted broth. To determine whether the staphylococci express a saturable specific receptor for human transferrin, the interaction of human 125I-transferrin with the staphylococci was examined. Both S. aureus and S. epidermidis bound the radiolabelled iron-saturated ligand in a time- and concentration-dependent manner. From competition binding assays, the affinity (Kd) and number of receptors were estimated for S. epidermidis (Kd, 0.27 microM; 4,200 receptors per cell) and S. aureus (Kd, 0.28 microM; 4,200 receptors per cell). S. epidermidis but not S. aureus receptor activity was partially iron regulated. Human apotransferrin and iron-saturated transferrin and rabbit and rat transferrins competed equally well for the staphylococcal receptor. Bovine and porcine transferrins and ovotransferrin as well as human and bovine lactoferrins were much less effective at competing with human transferrin. Treatment of whole staphylococci with protease abolished transferrin binding, indicating the involvement of cell surface protein. Western blots (immunoblots) of cell wall preparations probed with human transferrin revealed the presence of a 42-kDa transferrin-binding protein common to both S. aureus and S. epidermidis. On Western strip blots, the binding of human transferrin to this protein was blocked by labelled human transferrin but not by albumin, immunoglobulin G, or bovine transferrin or ovotransferrin. To assess the conservation of the 42-kDa transferrin-binding protein, cell wall proteins of S. epidermidis, S. haemolyticus, S. capitis, S. hominis, S. warneri, and S. saprophyticus were Western blotted and probed with human transferrin. Only S. warneri and S. saprophyticus lacked the 42-kDa wall protein, consistent with their inability to bind transferrin. These data show that the staphylococci express a specific receptor for human transferrin based at least in part on a common 42-kDa cell wall protein.     

Characteristics of the staphylococci isolated from the udder of cows with mastitis

A total of 175 strains of Staphylococcus aureus and 67 strains of Staphylococcus epidermidis were studied, isolated from 486 samples of milk secretion taken aceptically from the individual quarters of the udder of cows affected with subclinical and purulent (clinical) mastitis. The staphylococci were referred to as the causative agent of mastitis in case they were the only microflora in the seedings of the investigated material. Tests were applied as given in Fig. 1 to characterize the strains. It was found that mastitis in cows could be due to both plasma coagulating staphylococci (Staphylococcus aureus) and coagulase-negative Staphylococcus epidermidis organisms. The two Staphylococcus species were isolated from cows with clinical and subclinical mastitis. The division between pathogenic and nonpathogenic Staphylococcus strains by the plasma-coagulating symptom proved impossible, and this made it necessary to use other tests for pathogenicity. It became evident that the thing Staph. aureus and Staph. epidermidis had in common when isolated from cows with mastitis was the production of a gold-like pigment and delta hemolysin. Similarly to Staph. aureus isolated animals, the bovine Staph. epidermidis organisms did not possess fibrinolysin and rarely produced hemolysin. The isolated organisms belonging to the coagulase-positive staphylococci corresponded by their basic properties to Staphylococcus aureus var. bovis as described in the literature. The cultures of Staphylococcus epidermidis isolated under similar conditions showed in a considerable per cent of the cases somewhat different behaviour.     

Induction of apoptotic cell death in human endothelial cells treated with snake venom: implication of intracellular reactive oxygen species and protective effects of glutathione and superoxide dismutases

Human vascular endothelial cells play a pivotal role in atherosclerotic changes but are resistant to apoptotic inducers such as Fas ligand and it has been difficult to induce apoptosis. We developed an experimental model for the apoptosis in the endothelial cells by using snake venom treatment. Snake venom was found to generate intracellular reactive oxygen species (ROS) in the endothelial cells, which leads to apoptosis as judged by electron microscopy as well as by DNA cleavage. Buthionine sulfoximine (BSO) and diethyldithiocarbamate (DDC) accelerated the apoptosis, indicating intracellular glutathione and superoxide levels play a critical role. Pretreatment with tumor necrosis factor (TNF) or phorbol ester (TPA), which increases the Mn-SOD level, prevented the apoptosis. These data suggest that intracellular ROS enhances apoptosis whereas several anti-oxidants are protective in human endothelial cells. The induction of apoptosis by ROS of endothelial cells may be related to initiation of atherosclerotic changes.     

Inhibition of thymidine uptake by staphylococci, a new method for the investigation of phagocytosis

A radioassay employing (3H) thymidine, to measure inhibition of growth of Staphylococcus aures by human phagocytes is presented. The principle of this new method is that viable and dividing staphylococci take up thymidine more rapidly than white cells, so that whereas in control cultures containing staphylococci alone high counts per minute (cpm) are obtained within 90 min of incubation in test cultures both leucocytes and plasma from 25 normal subjects reduced the cpm, following ingestion and killing of staphylococci.     

Clearance of Shigella flexneri infection occurs through a nitric oxide-independent mechanism

Nitric oxide (NO) generated by gamma interferon (IFN-gamma) activation of macrophages mediates the killing of many intracellular pathogens. IFN-gamma is essential to innate resistance to Shigella flexneri infection. We demonstrate that NO is produced following S. flexneri infection both in mice and in activated cells in vitro and that while it is able to kill S. flexneri in a cell-free system, it is not required for clearance of S. flexneri in either infected mice or in activated cells in vitro.     

In vitro and in vivo efficacy of a rifampin-loaded silicone catheter for the prevention of CSF shunt infections

Infection of cerebrospinal fluid (CSF) shunts is one of the major complications associated with their use and is usually managed by shunt removal, temporary insertion of an external drainage and implantation of a new shunt system. We have evaluated the efficacy of a rifampin-loaded silicone ventricular catheter to prevent bacterial colonization and infection in vitro and in an animal model. On the basis of an incorporation process a rifampin-loaded catheter was developed which is capable of releasing rifampin in bacteriocidal concentrations for 60 days and more. In a stationary bacterial adherence assay using S. epidermidis as test strain, the colonization resistance of the device was demonstrated. To assess the capability of the catheter to prevent CSF shunt infections, a rabbit model was developed which allowed the establishment of a reliable and reproducible CSF infection by implantation of silicone catheters into the ventricle and inoculating S. epidermidis (minimal dose 10(6) cfu) or S. aureus (minimal dose 10(3) cfu). Rifampin-loaded catheters (12 animals inoculated with S. epidermidis, 8 animals inoculated with S. aureus) were compared with non-loaded (14 animals inoculated with S. epidermidis, 19 animals inoculated with S. aureus) control catheters, and infection was documented by clinical, microbiological and histological methods. In contrast to the control group, none of the animals with rifampin-loaded catheters showed clinical signs of infection. Furthermore, in none of the materials obtained after sacrifice of the animals (catheter, brain tissue, CSF, blood) could the infecting bacteria be cultured, whereas in materials from animals with the unloaded catheter the infecting strains could always be cultured from the catheter and from surrounding brain tissue. The histological examination of catheter-adjacent tissue supported these findings. We conclude that a rifampin-loaded silicone ventricular catheter is capable of completely preventing bacterial colonization and infection by staphylococci as the main causative organisms in CSF shunt infections and should be further evaluated in clinical trials.     

Endothelial cell dysfunction in response to intracellular overexpression of amyloid precursor protein

Previous reports have shown that exposure of vascular endothelial and smooth muscle cells to exogenous amyloid beta (Abeta) peptide results in cell damage and toxicity via oxidative injury. In this study we demonstrate that overexpression of the amyloid precursor protein (APP) is toxic to bovine aortic endothelial cells but not to bovine aortic smooth muscle cells. Intracellular coexpression of the free radical scavenger proteins metallothionein or MnSOD abolished the toxic effect of APP overexpression in endothelial cells. Our results demonstrate that endothelial cells are specifically susceptible to intracellular overexpression of APP and free radical generation is the likely mechanism of cell damage due to APP overexpression.