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Biomedical subjects

Patrick M Schlievert

Publications and source records attributed to Patrick M Schlievert.

At least 19 recordsLinked to original sources
PubMed2007

Repression of Staphylococcus aureus SrrAB using inducible antisense srrA alters growth and virulence factor transcript levels.

Our laboratory has previously identified the staphylococcal respiratory response (SrrAB), a Staphylococcus aureus two-component system that acts in the global regulation of virulence factors. In strain RN4220, SrrAB downregulated production of agr RNAIII, protein A, and TSST-1, particularly under low-oxygen conditions. Work by another group showed that SrrAB regulates energy metabolism genes and indicated that SrrAB may regulate energy transduction in response to changes in oxygen availability. In this study we investigate the role of SrrAB in regulating RNAIII, tst, spa, icaR, and icaA in a clinical isolate of S. aureus, MN8. We employ an inducible antisense vector, pYJY4, in order to repress transcription of srrAB. Transcript levels were assessed by reverse transcription followed by quantitative PCR. Repression of srrAB in rich media under aerobic growth conditions shows that SrrAB is required for expression of tst, spa, and icaR transcripts at wild-type levels. Comparisons made between rich media under aerobic conditions vs low-oxygen conditions show that srrAB transcript levels are not altered by oxygen alone. Previous studies performed on strain RN4220 under low-oxygen conditions indicate that SrrAB represses tst and spa transcript when the amount of oxygen is limited. We propose that, under aerobic conditions, SrrAB enhances the levels of tst, spa, and icaR, while under low-oxygen conditions, SrrAB decreases the levels of these three transcripts.

Bacterial Proteins↗
PubMed2006

Glycerol monolaurate inhibits the effects of Gram-positive select agents on eukaryotic cells.

Many exotoxins of Gram-positive bacteria, such as superantigens [staphylococcal enterotoxins, toxic shock syndrome toxin-1 (TSST-1), and streptococcal pyrogenic exotoxins] and anthrax toxin are bioterrorism agents that cause diseases by immunostimulation or cytotoxicity. Glycerol monolaurate (GML), a fatty acid monoester found naturally in humans, has been reported to prevent synthesis of Gram-positive bacterial exotoxins. This study explored the ability of GML to inhibit the effects of exotoxins on mammalian cells and prevent rabbit lethality from TSS. GML (>or=10 microg/mL) inhibited superantigen (5 microg/mL) immunoproliferation, as determined by inhibition of (3)H-thymidine incorporation into DNA of human peripheral blood mononuclear cells (1 x 10(6) cells/mL) as well as phospholipase Cgamma1, suggesting inhibition of signal transduction. The compound (20 microg/mL) prevented superantigen (100 microg/mL) induced cytokine secretion by human vaginal epithelial cells (HVECs) as measured by ELISA. GML (250 microg) inhibited rabbit lethality as a result of TSST-1 administered vaginally. GML (10 microg/mL) inhibited HVEC and macrophage cytotoxicity by anthrax toxin, prevented erythrocyte lysis by purified hemolysins (staphylococcal alpha and beta) and culture fluids containing streptococcal and Bacillus anthracis hemolysins, and was nontoxic to mammalian cells (up to 100 microg/mL) and rabbits (250 microg). GML stabilized mammalian cell membranes, because erythrocyte lysis was reduced in the presence of hypotonic aqueous solutions (0-0.05 M saline) or staphylococcal alpha- and beta-hemolysins when erythrocytes were pretreated with GML. GML may be useful in the management of Gram-positive exotoxin illnesses; its action appears to be membrane stabilization with inhibition of signal transduction.

Animals↗
PubMed2006

Staphylococcal alpha-toxin causes increased tracheal epithelial permeability.

Staphylococcus aureus is an important cause of pulmonary infections. The role of S. aureus alpha-toxin as a virulence factor is unclear. We hypothesized that airway epithelium is a target of S. aureus alpha-toxin and that exposure of airway epithelium to alpha-toxin results in damage to the airway epithelium. To examine the hypothesis that alpha-toxin is capable of independently producing airway epithelium damage as measured by permeability and morphometry, an isolated whole mouse trachea test apparatus was developed. In vitro epithelial permeability (P) was calculated and digital micrographs were analyzed morphometrically. Purified S. aureus alpha-toxin produced a significant increase in tracheal epithelial P (P < 0.05). Morphometric analysis revealed the ratio of adherent tracheal epithelium attached to the basement membrane divided by the total length of the basement membrane decreased in a dose-dependent manner with 1 microg/ml alpha-toxin and 10 microg/ml alpha-toxin (P < 0.05). We developed a novel isolated whole mouse trachea test apparatus for the measurement of tracheal epithelium damage. Increased P and separation of the tracheal epithelium from the basement membrane occurred after S. aureus alpha-toxin exposure. We conclude that mammalian airway epithelium is a target of S. aureus alpha-toxin.

Animals↗
PubMed2006

An early favorable outcome of streptococcal toxic shock syndrome may require a combination of antimicrobial and intravenous gamma globulin therapy together with activated protein C.

Streptococcal toxic shock syndrome (STSS) associated with a group A beta hemolytic streptococcal infection was described 18 y ago. Since then, although the pathophysiology of the syndrome has been clarified, mortality can be as high as 80%. A middle-aged female developed STSS associated with a group A streptococcal pneumonia. Laboratory studies confirmed respiratory and renal failure as well as disseminated intravascular coagulation with a striking reduction in endogenous procoagulants. The patient, probably due to her HLA DRB1*14 haplotype was unable to generate anti-streptococcal antibodies. She was treated with appropriate antimicrobial therapy together with intravenous gamma globulin and drotrecogin or activated protein C. Her response to this combined therapy was accompanied by a rapid resolution of the multiorgan failure and correction of the accompanying disseminated intravascular coagulation. This rapid response to treatment supports the hypohesis that several host factors including the immune response and loss of procoagulants determine the development and severity of the toxic shock syndromes. Further studies with this combined approach appear warranted.

Anti-Infective Agents↗
PubMed2005

In vivo assessment of human vaginal oxygen and carbon dioxide levels during and post menses.

Previous in vitro and in vivo animal studies showed that O(2) and CO(2) concentrations can affect virulence of pathogenic bacteria such as Staphylococcus aureus. The objective of this work was to measure O(2) and CO(2) levels in the vaginal environment during tampon wear using newly available sensor technology. Measurements by two vaginal sensors showed a decrease in vaginal O(2) levels after tampon insertion. These decreases were independent of the type of tampons used and the time of measurement (mid-cycle or during menstruation). These results are not in agreement with a previous study that concluded that oxygenation of the vaginal environment during tampon use occurred via delivery of a bolus of O(2) during the insertion process. Our measurements of gas levels in menses showed the presence of both O(2) and CO(2) in menses. The tampons inserted into the vagina contained O(2) and CO(2) levels consistent with atmospheric conditions. Over time during tampon use, levels of O(2) in the tampon decreased and levels of CO(2) increased. Tampon absorbent capacity, menses loading, and wear time influenced the kinetics of these changes. Colonization with S. aureus had no effect on the gas profiles during menstruation. Taken collectively, these findings have important implications on the current understanding of gaseous changes in the vaginal environment during menstruation and the potential role(s) they may play in affecting bacterial virulence factor production.

Adult↗
PubMed2005

Purpura fulminans due to Staphylococcus aureus.

BACKGROUND: Purpura fulminans is an acute illness commonly associated with meningococcemia or invasive streptococcal disease, and it is typically characterized by disseminated intravascular coagulation (DIC) and purpuric skin lesions. In this article, we report the first 5 cases (to our knowledge) of purpura fulminans directly associated with Staphylococcus aureus strains that produce high levels of the superantigens toxic shock syndrome toxin-1 (TSST-1), staphylococcal enterotoxin serotype B (SEB), or staphylococcal enterotoxin serotype C (SEC). METHODS: Cases were identified in the Minneapolis-St. Paul, Minnesota, metropolitan area during 2000-2004. S. aureus infection was diagnosed on the basis of culture results, and susceptibility to methicillin was determined. The ability of the isolated organisms to produce TSST-1, SEB, SEC, and Panton-Valentine leukocidin (PVL) was determined. TSST-1, SEB, and SEC levels were also quantified after in vitro growth of the organisms. RESULTS: In 3 of the 5 cases, the infecting S. aureus strain was isolated from the blood cultures. In 2 of the 5 cases, the infecting S. aureus strain was isolated only from the respiratory tract, indicating that purpura fulminans and toxic shock syndrome resulted from exotoxin and/or other host factors, rather than septicemia. One of these latter 2 patients also had necrotizing pneumonia, and the isolated S. aureus was a methicillin-resistant strain that produced both SEC and PVL. Only 2 of the 5 patients survived, and 1 of the survivors received activated protein C. CONCLUSIONS: Staphylococcal purpura fulminans may be a newly emerging illness associated with superantigen production. Medical practitioners should be aware of this illness.

Adult↗
PubMed2005

Glycerol monolaurate inhibits virulence factor production in Bacillus anthracis.

Anthrax, caused by Bacillus anthracis, has been brought to the public's attention because of the 2001 bioterrorism attacks. However, anthrax is a disease that poses agricultural threats in the United States as well as human populations in Europe, China, Africa, and Australia. Glycerol monolaurate (GML) is a compound that has been shown to inhibit exotoxin production by Staphylococcus aureus and other gram-positive bacteria. Here, we study the effects of GML on growth and toxin production in B. anthracis. The Sterne strain of B. anthracis was grown to post-exponential phase with 0-, 10-, 15-, or 20-microg/ml concentrations of GML and then assayed quantitatively for protective antigen (PA) and lethal factor (LF). After 8 h, GML at concentrations greater than 20 microg/ml was bacteriostatic to growth of the organism. However, a 10-microg/ml concentration of GML was not growth inhibitory, but amounts of PA and LF made were greatly reduced. This effect was not global for all proteins when total secreted protein from culture fluids was examined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Through quantitative reverse transcription-PCR assays, this toxin-inhibitory effect was shown to occur at the transcriptional level, since amounts of mRNA for pagA (PA), lef (LF), and cya (edema factor) were reduced. Surprisingly, mRNA levels of atxA, a regulator of exotoxin gene expression, rose in the presence of GML. These data will be useful in developing therapeutic tools to treat anthrax disease, whether in animals or humans. These results also suggest that mechanisms of virulence regulation exist independent of atxA.

Antigens, Bacterial↗
PubMed2005

The innate immune system is activated by stimulation of vaginal epithelial cells with Staphylococcus aureus and toxic shock syndrome toxin 1.

Despite knowledge of the effects of toxic shock syndrome (TSS) toxin 1 (TSST-1) on the adaptive immune system, little is known about stimulation of the innate immune system, particularly epithelial cells. This study investigated the interactions of TSS Staphylococcus aureus and TSST-1 with human vaginal epithelial cells (HVECs) and porcine mucosal surfaces. When cocultured with HVECs for 6 h, TSS S. aureus MN8 proliferated, formed aggregates on the HVEC surfaces, and produced exotoxins. Receptor binding studies showed that 35S-TSST-1 bound to 5 x 10(4) receptors per HVEC, with saturation at 15 min. Affymetrix Human GeneChip U133A microarray analysis determined S. aureus MNSM (100 bacteria/HVEC) caused at least twofold up- or down-regulation of 410 HVEC genes by 6 h; these data were also confirmed with S. aureus MN8. TSST-1 (100 microg/ml) caused up- or down-regulation of 2,386 HVEC genes by 6 h. In response to S. aureus, the HVEC genes most up-regulated compared to those in controls were those coding for chemokines or cytokines--MIP-3alpha, 478-fold; GRO-alpha, 26-fold; GRO-beta, 14-fold; and GRO-gamma, 30-fold--suggesting activation of innate immunity. TSST-1 also caused up-regulation of chemokine/cytokine genes. Chemokine/cytokine gene up-regulation was confirmed by enzyme-linked immunosorbent assays measuring the corresponding proteins induced by S. aureus and TSST-1. S. aureus MN8, when incubated with porcine vaginal tissue, increased the flux of 35S-TSST-1 across the mucosal surface. This was accompanied by influx of lymphocytes into the upper layers of the tissue. These data suggest innate immune system activation through epithelial cells, reflected in chemokine/cytokine production and influx of lymphocytes, may cause changes in vaginal mucosa permeability, facilitating TSST-1 penetration.

Animals↗
PubMed2004

Virulence regulation in Staphylococcus aureus: the need for in vivo analysis of virulence factor regulation.

Staphylococcus aureus is a pathogenic microorganism that is responsible for a wide variety of clinical infections. These infections can be relatively mild, but serious, life-threatening infections may result from the expression of staphylococcal virulence factors that are coordinated by virulence regulators. Much work has been done to characterize the actions of staphylococcal virulence regulators in broth culture. Recently, several laboratories showed that transcriptional analyses of virulence regulators in in vivo animal models or in human infection did not correlate with transcriptional analyses accomplished in vitro. In describing the differences between in vitro and in vivo transcription of staphylococcal virulence regulators, we hope to encourage investigators to study virulence regulators using infection models whenever possible.

Animals↗
PubMed2004

Intravitreally injected human immunoglobulin attenuates the effects of Staphylococcus aureus culture supernatant in a rabbit model of toxin-mediated endophthalmitis.

OBJECTIVE: To determine whether human immunoglobulin attenuates the toxic effects of Staphylococcus aureus culture supernatant in a rabbit model of endophthalmitis. METHODS: Immunoglobulin binding to products of S aureus strain RN4220 was tested by Western blot analysis using known toxins (beta-hemolysin and toxic shock syndrome toxin-1) and a concentrated culture supernatant containing S aureus exotoxins (pooled toxin). To induce endophthalmitis, pooled toxin was injected into the rabbit vitreous. For immunoglobulin treatment, immunoglobulin and pooled toxin were either mixed and injected simultaneously or immunoglobulin was injected immediately after or 6 hours after pooled toxin injection. Severity of endophthalmitis was graded according to a 9-day course with clinical examination (slitlamp biomicroscopy or indirect ophthalmoscopy) and evaluation of histologic sections. RESULTS: The toxic effects of pooled toxin were markedly reduced when immunoglobulin was mixed with pooled toxin and injected simultaneously. Delayed injection of immunoglobulin diminished its ability to reduce toxicity. Clinical and histologic signs of toxicity were partially attenuated when immunoglobulin was injected immediately after pooled toxin, but only minimal clinically detectable reductions in toxicity were observed when immunoglobulin injection was delayed for 6 hours. CONCLUSION: Pooled human immunoglobulin can attenuate the toxic intravitreal effects of a concentrated culture supernatant containing S aureus exotoxins. Clinical Relevance Immunoglobulin may represent a novel adjuvant in the treatment of bacterial endophthalmitis. To optimize the potential therapeutic benefit, maximizing the mixture of immunoglobulin with bacterial products and early intervention are likely to be important.

Animals↗
PubMed2004

Temperature regulates bacterial protein production: possible role in rosacea.

Facial skin temperature is higher for patients with rosacea. Papules and pustules might arise because bacteria behave differently at these warmer temperatures. We sought to: (1) compare bacteria from facial skin of patients with rosacea with that of control subjects; and (2) grow these bacteria at 30 degrees C and 37 degrees C to compare growth curves and secreted proteins. Bacteria isolated from pustules/skin surfaces of patients with rosacea and skin surfaces of control subjects were identified and cultured at 37 degrees C and 30 degrees C. Secreted proteins were separated by electrophoresis. We found that Staphylococcus epidermidis isolated from patients with rosacea was consistently beta-hemolytic, whereas that from control subjects were nonhemolytic. Bacteria from patients with rosacea grew at the same rate and to the same stationary phase whether cultured at 37 degrees C or 30 degrees C. Isolates from patients with rosacea secreted more proteins, and generally more of each protein at 37 degrees C compared with 30 degrees C. In conclusion, bacteria isolated from patients with rosacea secrete different proteins and different amounts of protein at different temperatures.

Bacterial Proteins↗
PubMed2004

An amino-terminal domain of Enterococcus faecalis aggregation substance is required for aggregation, bacterial internalization by epithelial cells and binding to lipoteichoic acid.

Aggregation substance (AS), a plasmid-encoded surface protein of Enterococcus faecalis, plays important roles in virulence and antibiotic resistance transfer. Previous studies have suggested that AS-mediated aggregation of enterococcal cells could involve the binding of this protein to cell wall lipoteichoic acid (LTA). Here, a method to purify an undegraded form of Asc10, the AS of the plasmid pCF10, is described. Using this purified protein, direct binding of Asc10 to purified E. faecalis LTA was demonstrated. Equivalent binding of Asc10 to LTA purified from INY3000, an E. faecalis strain that is incapable of aggregation, was also observed. Surprisingly, mutations in a previously identified aggregation domain from amino acids 473 to 683 that abolished aggregation had no effect on LTA binding. In frame deletion analysis of Asc10 was used to identify a second aggregation domain located in the N-terminus of the protein from amino acids 156 to 358. A purified Asc10 mutant protein lacking this domain showed reduced LTA binding, while a purified N-terminal fragment from amino acids 44-331 had high LTA binding. Like the previously described aggregation domain, the newly identified Asc10((156-358)) aggregation domain was also required for efficient internalization of E. faecalis into HT-29 enterocytes. Thus, Asc10 possess two distinct domains required for aggregation and eukaryotic cell internalization: an N-terminal domain that promotes binding to LTA and a second domain located near the middle of the protein.

Bacterial Adhesion↗
PubMed2004

Characterization of virulence factor regulation by SrrAB, a two-component system in Staphylococcus aureus.

Workers in our laboratory have previously identified the staphylococcal respiratory response AB (SrrAB), a Staphylococcus aureus two-component system that acts in the global regulation of virulence factors. This system down-regulates production of agr RNAIII, protein A, and toxic shock syndrome toxin 1 (TSST-1), particularly under low-oxygen conditions. In this study we investigated the localization and membrane orientation of SrrA and SrrB, transcription of the srrAB operon, the DNA-binding properties of SrrA, and the effect of SrrAB expression on S. aureus virulence. We found that SrrA is localized to the S. aureus cytoplasm, while SrrB is localized to the membrane and is properly oriented to function as a histidine kinase. srrAB has one transcriptional start site which results in either an srrA transcript or a full-length srrAB transcript; srrB must be cotranscribed with srrA. Gel shift assays of the agr P2, agr P3, protein A (spa), TSST-1 (tst), and srr promoters revealed SrrA binding at each of these promoters. Analysis of SrrAB-overexpressing strains by using the rabbit model of bacterial endocarditis demonstrated that overexpression of SrrAB decreased the virulence of the organisms compared to the virulence of isogenic strains that do not overexpress SrrAB. We concluded that SrrAB is properly localized and oriented to function as a two-component system. Overexpression of SrrAB, which represses agr RNAIII, TSST-1, and protein A in vitro, decreases virulence in the rabbit endocarditis model. Repression of these virulence factors is likely due to a direct interaction between SrrA and the agr, tst, and spa promoters.

Animals↗
PubMed2004

A possible association of recurrent streptococcal infections and acute onset of obsessive-compulsive disorder.

Rheumatic fever is an immunologically mediated disease that follows infection by group A beta-hemolytic Streptococcus (GABHS). In rheumatic fever, antibodies generated against GABHS cross-react with the heart, joints, skin, and other sites, inducing an inflammatory, multisystem disease. Brain tissue-specific antibodies have been demonstrated in a subset of children with Sydenham chorea (a component of the Jones criteria for the diagnosis of rheumatic fever), and most Sydenham chorea patients manifest obsessive-compulsive symptoms very similar to those in traditional obsessive-compulsive disorder. The parallels drawn from the paradigm of Sydenham's chorea to Pediatric Autoimmune Neuropsychiatric Disorders Associated with Streptococcal Infections (PANDAS) is an area of active controversy. Newly emerging information on the role of GABHS superantigens in the pathogenesis of rheumatic fever is of particular interest. In this article, we review the microbial characteristics of GABHS and the subsequent immune responses to GABHS as a possible etiology of PANDAS.

Antibodies, Monoclonal↗
PubMed2003

Hospital transmission of community-acquired methicillin-resistant Staphylococcus aureus among postpartum women.

Infections caused by community-acquired methicillin-resistant Staphylococcus aureus (CA-MRSA) are being increasingly observed in patients who lack traditional risk factors. We described 8 postpartum women who developed skin and soft-tissue infections caused by MRSA at a mean time of 23 days (range, 4-73 days) after delivery. Infections included 4 cases of mastitis (3 of which progressed to breast abscess), a postoperative wound infection, cellulitis, and pustulosis. The outbreak strains were compared with the prototype CA-MRSA strain MW2 and found to be indistinguishable by pulsed-field gel electrophoresis. All were spa type 131, all contained the staphylococcal chromosomal cassette mec type IV, and all expressed Panton-Valentine leukocidin and staphylococcal enterotoxins C and H. The route of transmission was not discovered: the results of surveillance cultures of samples obtained from employees of the hospital, the hospital environment, and newborns were negative for the outbreak strain. We report that MW2, which was previously limited to the midwestern United States, has spread to the northeastern United States and has become a health care-associated pathogen.

Anti-Bacterial Agents↗
PubMed2003

Effects of total body irradiation and cyclosporin a on the lethality of toxic shock syndrome toxin-1 in a rabbit model of toxic shock syndrome.

Toxic shock syndrome (TSS) may be mediated by superantigen-activated T cells, a theory we tested in rabbits, which are more susceptible to the lethal effects of superantigens, such as TSS toxin-1 (TSST-1), than are mice. Rabbits exposed to 10 cGy of total body irradiation exhibited T cell deficiency, with profound depletion of splenic lymphocytes and circulating CD4(+) lymphocytes, as well as an inability to manifest delayed-type hypersensitivity. Nevertheless, these rabbits remained completely susceptible to TSST-1, indicating that TSS can occur in the setting of marked immunosuppression.

Animals↗