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Biomedical subjects

A S Bayer

Publications and source records attributed to A S Bayer.

At least 37 records · Page 2Linked to original sources
PubMed1999

Comparative efficacies of liposomal amikacin (MiKasome) plus oxacillin versus conventional amikacin plus oxacillin in experimental endocarditis induced by Staphylococcus aureus: microbiological and echocardiographic analyses.

Optimal treatment strategies for serious infections caused by Staphylococcus aureus have not been fully characterized. The combination of a beta-lactam plus an aminoglycoside can act synergistically against S. aureus in vitro and in vivo. MiKasome, a new liposome-encapsulated formulation of conventional amikacin, significantly prolongs serum half-life (t1/2) and increases the area under the concentration-time curve (AUC) compared to free amikacin. Microbiologic efficacy and left ventricular function, as assessed by echocardiography, were compared in animals administered either oxacillin alone or oxacillin in combination with conventional amikacin or MiKasome in a rabbit model of experimental endocarditis due to S. aureus. In vitro, oxacillin, combined with either free amikacin or MiKasome, prevented the bacterial regrowth observed with aminoglycosides alone at 24 h of incubation. Rabbits with S. aureus endocarditis were treated with either oxacillin alone (50 mg/kg, given intramuscularly three times daily), oxacillin plus daily amikacin (27 mg/kg, given intravenously twice daily), or oxacillin plus intermittent MiKasome (160 mg/kg, given intravenously, a single dose on days 1 and 4). The oxacillin-alone dosage represents a subtherapeutic regimen against the infecting strain in the endocarditis model (L. Hirano and A. S. Bayer, Antimicrob. Agents Chemother. 35:685-690, 1991), thus allowing recognition of any enhanced bactericidal effects between oxacillin and either aminoglycoside formulation. Treatment was administered for either 3 or 6 days, and animals were sacrificed after each of these time points or at 5 days after a 6-day treatment course (to evaluate for posttherapy relapse). Left ventricular function was analyzed by utilizing serial transthoracic echocardiography during treatment and posttherapy by measurement of left ventricular fractional shortening. At all sacrifice times, both combination regimens significantly reduced S. aureus vegetation counts versus control counts (P < 0.05). In contrast, oxacillin alone did not significantly reduce S. aureus vegetation counts after 3 days of therapy. Furthermore, at this time point, the two combinations were significantly more effective than oxacillin alone (P < 0.05). All three regimens were effective in significantly decreasing bacterial counts in the myocardium during and after therapy compared to controls (P < 0.05). In kidney and spleen abscesses, all regimens significantly reduced bacterial counts during therapy (P < 0.0001); however, only the combination regimens prevented bacteriologic relapse in these organs posttherapy. By echocardiographic analysis, both combination regimens yielded a significant physiological benefit by maintaining normal left ventricular function during treatment and posttherapy compared with oxacillin alone (P < 0.001). These results suggest that the use of intermittent MiKasome (similar to daily conventional amikacin) enhances the in vivo bactericidal effects of oxacillin in a severe S. aureus infection model and preserves selected physiological functions in target end organs.

Amikacin↗
PubMed1999

Impact of the high-affinity proline permease gene (putP) on the virulence of Staphylococcus aureus in experimental endocarditis.

Staphylococcus aureus causes a wide variety of invasive human infections. However, delineation of the genes which are essential for the in vivo survival of this pathogen has not been accomplished to date. Using signature tag mutagenesis techniques and large mutant pool screens, previous investigators identified several major gene classes as candidate essential gene loci for in vivo survival; these include genes for amino acid transporters, oligopeptide transporters, and lantibiotic synthesis (W. R. Schwan, S. N. Coulter, E. Y. W. Ng, M. H. Langhorne, H. D. Ritchie, L. L. Brody, S. Westbrock-Wadman, A. S. Bayer, K. R. Folger, and C. K. Stover, Infect. Immun. 66:567-572, 1998). In this study, we directly compared the virulence of four such isogenic signature tag mutants with that of the parental strain (RN6390) by using a prototypical model of invasive S. aureus infection, experimental endocarditis (IE). The oligonucleotide signature tag (OST) mutant with insertional inactivation of the gene (putP) which encodes the high-affinity transporter for proline uptake exhibited significantly reduced virulence in the IE model across three challenge inocula (10(4) to 10(6) CFU) in terms of achievable intravegetation densities (P, <0.05). The negative impact of putP inactivation on in vivo survival in the IE model was confirmed by simultaneous challenge with the original putP mutant and the parental strain as well as by challenge with a putP mutant in which this genetic inactivation was transduced into a distinct parental strain (S6C). In contrast, inactivation of loci encoding an oligopeptide transporter, a purine repressor, and lantibiotic biosynthesis had no substantial impact on the capacity of OST mutants to survive within IE vegetations. Thus, genes encoding the uptake of essential amino acids may well represent novel targets for new drug development. These data also confirm the utility of the OST technique as an important screening methodology for identifying candidate genes as requisite loci for the in vivo survival of S. aureus.

Amino Acid Transport Systems, Neutral↗
PubMed1999

Hyperproduction of alpha-hemolysin in a sigB mutant is associated with elevated SarA expression in Staphylococcus aureus.

To evaluate the role of SigB in modulating the expression of virulence determinants in Staphylococcus aureus, we constructed a sigB mutant of RN6390, a prototypic S. aureus strain. The mutation in the sigB gene was confirmed by the absence of the SigB protein in the mutant on an immunoblot as well as the failure of the mutant to activate sigmaB-dependent promoters (e.g., the sarC promoter) of S. aureus. Phenotypic analysis indicated that both alpha-hemolysin level and fibrinogen-binding capacity were up-regulated in the mutant strain compared with the parental strain. The increase in fibrinogen-binding capacity correlated with enhanced expression of clumping factor and coagulase on immunoblots. The effect of the sigB mutation on the enhanced expression of the alpha-hemolysin gene (hla) was primarily transcriptional. Upon complementation with a plasmid containing the sigB gene, hla expression returned to near parental levels in the mutant. Detailed immunoblot analysis as well as a competitive enzyme-linked immunosorbent assay of the cell extract of the sigB mutant with anti-SarA monoclonal antibody 1D1 revealed that the expression of SarA was higher in the mutant than in the parental control. Despite an elevated SarA level, the transcription of RNAII and RNAIII of the agr locus remained unaltered in the sigB mutant. Because of a lack of perturbation in agr, we hypothesize that inactivation of sigB leads to increased expression of SarA which, in turn, modulates target genes via an agr-independent but SarA-dependent pathway.

Animals↗
PubMed1999

Membrane permeabilization by thrombin-induced platelet microbicidal protein 1 is modulated by transmembrane voltage polarity and magnitude.

Thrombin-induced platelet microbicidal protein 1 (tPMP-1) is a small, cationic peptide generated from rabbit platelets when they are exposed to thrombin in vitro. It has potent microbicidal activity against a broad spectrum of bacterial and fungal pathogens, including Staphylococcus aureus. Previous in vitro studies involving whole staphylococcal cells and planar lipid bilayers (as artificial bacterial membrane models) suggested that membrane permeabilization by tPMP-1 is voltage dependent (S.-P. Koo, M. R. Yeaman, and A. S. Bayer, Infect. Immun. 64:3758-3764, 1996; M. R. Yeaman, A. S. Bayer, S. P. Koo, W. Foss, and P. M. Sullam, J. Clin. Investig. 101:178-187, 1998). Thus, the aims of the present study were to specifically characterize the electrophysiological events associated with membrane permeabilization by tPMP-1 by using artificial planar lipid bilayer membranes. We assessed the influence of transmembrane voltage polarity and magnitude on the initiation and modulation of tPMP-1 membrane permeabilization at various concentrations of tPMP-1 (range, 1 to 100 ng/ml) added to the cis side of the membranes. The incidence of membrane permeabilization induced by tPMP-1 at all of the concentrations tested was more frequent at -90 mV than at +90 mV. It is noteworthy that membrane permeabilization due to 1-ng/ml tPMP-1 was successfully initiated at -90 mV but not at +90 mV. Further, the mean onset times of induction of tPMP-1 activity were comparable under the various conditions. Modulation of ongoing membrane permeabilization was dependent on voltage and tPMP-1 concentration. Membrane permeabilization at a low tPMP-1 concentration (1 ng/ml) was directly correlated with trans-negative voltages, while a higher tPMP-1 concentration (100 ng/ml) induced conductance which was more dependent on trans-positive voltages. Collectively, these data indicate that the mechanism of tPMP-1 microbicidal activity at the bacterial cytoplasmic membrane may involve distinct induction and propagation stages of membrane permeabilization which, in turn, are modulated by transmembrane potential, as well as peptide concentration.

Animals↗
PubMed1998

Platelet microbicidal proteins and neutrophil defensin disrupt the Staphylococcus aureus cytoplasmic membrane by distinct mechanisms of action.

Platelet microbicidal proteins (PMPs) are hypothesized to exert microbicidal effects via cytoplasmic membrane disruption. Transmission electron microscopy demonstrated a temporal association between PMP exposure, damage of the Staphylococcus aureus cytoplasmic membrane ultrastructure, and subsequent cell death. To investigate the mechanisms of action of PMPs leading to membrane damage, we used flow cytometry to compare the effects of two distinct PMPs (thrombin-induced PMP-1 [tPMP-1] or PMP-2) with human neutrophil defensin-1 (hNP-1) on transmembrane potential (Deltapsi), membrane permeabilization, and killing of S. aureus. Related strains 6850 (Deltapsi -150 mV) and JB-1 (Deltapsi -100 mV; a respiration-deficient menadione auxotroph of 6850) were used to assess the influence of Deltapsi on peptide microbicidal effects. Propidium iodide (PI) uptake was used to detect membrane permeabilization, retention of 3,3'-dipentyloxacarbocyanine (DiOC5) was used to monitor membrane depolarization (Deltapsi), and quantitative culture or acridine orange accumulation was used to measure viability. PMP-2 rapidly depolarized and permeabilized strain 6850, with the extent of permeabilization inversely related to pH. tPMP-1 failed to depolarize strain 6850, but did permeabilize this strain in a manner directly related to pH. Depolarization, permeabilization, and killing of strain JB-1 due to PMPs were significantly less than in strain 6850. Growth in menadione reconstituted Deltapsi of JB-1 to a level equivalent to 6850, and was associated with greater depolarization due to PMP-2, but not tPMP-1. Reconstitution of Deltapsi also enhanced permeabilization and killing of JB-1 due to tPMP-1 or PMP-2. Both PMP-2 and tPMP-1 caused significant reductions in viability of strain 6850. In contrast to tPMP-1 or PMP-2, defensin hNP-1 depolarized, permeabilized, and killed both strains 6850 and JB-1 equally, and in a manner directly related to pH. Collectively, these data indicate that membrane dysfunction and cell death due to tPMP-1, PMP-2, or hNP-1 likely involve different mechanisms. These findings may also reveal new insights into the microbicidal activities versus mammalian cell toxicities of antimicrobial peptides.

Animals↗
PubMed1998

Staphylococcus aureus genetic loci impacting growth and survival in multiple infection environments.

The Gram-positive bacterium Staphylococcus aureus infects diverse tissues and causes a wide spectrum of diseases, suggesting that it possesses a repertoire of distinct molecular mechanisms promoting bacterial survival in disparate in vivo environments. Signature-tag transposon mutagenesis screening of a 1520-member library identified numerous S. aureus genetic loci affecting growth and survival in four complementary animal infection models including mouse abscess, bacteraemia and wound and rabbit endocarditis. Of a total of 237 in vivo attenuated mutants identified by the murine models, less than 10% showed attenuation in all three models, emphasizing the advantage of screening in diverse disease environments. The largest gene class identified by these analyses encoded peptide and amino acid transporters, some of which were important for S. aureus survival in all animal infection models tested. The identification of staphylococcal loci affecting growth, persistence and virulence in multiple tissue environments provides insight into the complexities of human infection and on the molecular mechanisms that could be targeted by new antibacterial therapies.

ATP-Binding Cassette Transporters↗
PubMed1998

In vitro resistance to thrombin-induced platelet microbicidal protein among clinical bacteremic isolates of Staphylococcus aureus correlates with an endovascular infectious source.

Platelet microbicidal proteins (PMPs), small cationic peptides released at sites of endovascular damage, kill common bloodstream pathogens in vitro. Our group previously showed that in vitro resistance of clinical staphylococcal and viridans group streptococcal bacteremic strains to PMPs correlated with the diagnosis of infective endocarditis (IE) (Wu et al., Antimicrob. Agents Chemother. 38:729-732, 1994). However, that study was limited by (i) the small number of Staphylococcus aureus isolates from IE patients, (ii) the retrospective nature of the case definitions, and (iii) the diverse geographic sources of strains. The present study evaluated the in vitro PMP susceptibility phenotype of a large number of staphylococcemic isolates (n = 60), collected at a single medical center and categorized by defined and validated clinical criteria. A significantly higher proportion of staphylococcemic strains from patients with IE was PMP resistant in vitro than the proportion of strains from patients with soft tissue sepsis (83% and 33%, respectively; P < 0.01). Moreover, the levels of PMP resistance (mean percent survival of strains after 2-h exposure to PMP in vitro) were significantly higher for isolates from patients with IE and with vascular catheter sepsis than for strains from patients with abscess sepsis (P < 0.005 and P < 0.01, respectively). These data further support the concept that bloodstream pathogens that exhibit innate or acquired PMP resistance have a survival advantage with respect to either the induction or progression of endovascular infections.

Anti-Bacterial Agents↗
PubMed1998

Efficacy of trovafloxacin, a new quinolone antibiotic, in experimental staphylococcal endocarditis due to oxacillin-resistant strains.

Therapeutic options for severe infections caused by strains of oxacillin-resistant Staphylococcus aureus (ORSA) and coagulase-negative staphylococci (ORSE) are very limited. With the increasing resistance of such strains to aminoglycosides, rifampin, and currently available quinolone agents, as well as the recent documentation of increasing resistance of ORSA to vancomycin (VANCO), new treatment alternatives are imperative. The in vivo efficacy of trovafloxacin (TROVA), a new quinolone agent with excellent antistaphylococcal activity in vitro, against experimental endocarditis (IE) due to beta-lactamase-producing ORSA and ORSE strains (ORSA and ORSE IE) was evaluated. TROVA (25 mg/kg of body weight intravenously [i.v.] twice daily [b.i.d]) was compared to VANCO (20 mg/kg i.v. b.i.d.) and two regimens of ampicillin-sulbactam (AMP-SUL; 200 mg/kg intramuscularly [i.m.] three times a day [t.i.d.] and 20 mg/kg i.m. b.i.d.), with all agents given for 3 or 6 days. AMP-SUL was included as a comparative treatment regimen because of its proven efficacy against experimental ORSA and ORSE IE. For both ORSA and ORSE IE, TROVA, AMP-SUL, and VANCO each reduced staphylococcal densities in vegetations compared to untreated controls (P < 0.01). For ORSA IE, TROVA was the most rapidly bactericidal agent--although not to a statistically significant degree--correlating with its superior bactericidal effect in vitro compared to those of VANCO and AMP-SUL.

Animals↗
PubMed1998

Selective activation of sar promoters with the use of green fluorescent protein transcriptional fusions as the detection system in the rabbit endocarditis model.

The global regulatory locus sar is composed of three overlapping transcripts initiated from a triple-promoter system (designated P1, P3, and P2). To explore if the individual sar promoters are differentially expressed in vitro and in vivo, we constructed a shuttle plasmid (pALC1434) containing a promoterless gfpUV gene (a gfp derivative [Clontech]) preceded by a polylinker region. Recombinant shuttle vectors containing individual sar promoters upstream of the gfpUV reporter gene were then introduced into Staphylococcus aureus RN6390. Northern and immunoblot analysis revealed that P1 is stronger than the P2 and P3 promoters in vitro. Additionally, the levels of the gfpUV transcript driven by individual sar promoters also correlated with the growth cycle dependency of these promoters in liquid cultures, thus suggesting the utility of pALC1434 as a vehicle for reporter fusion. Using the rabbit endocarditis model, we examined the expression of these three GFPUV fusions in vivo by fluorescence microscopy of infected cardiac vegetations 24 h after initial intravenous challenge. Similar to the in vitro findings, P1 was activated both in the center and on the surface of the vegetations. In contrast, the P3 promoter was silent both in vivo and in vitro as determined by fluorescence microscopy. Remarkably, P2 was silent in vitro but became highly activated in vivo. In particular, the sar P2 promoter was activated on the surface of the vegetation but not in the center of the lesion. These data imply that in vivo promoter activation of sar differed from that observed in vitro. Moreover, the individual sar promoters may be differentially expressed in different areas within the same anatomic niche, presumably reflecting the microbial physiological response to distinct host microenvironments. As the sar locus controls the synthesis of both extracellular and cell wall virulence determinants, these promoter-gfpUV constructs should be useful to characterize many aspects of S. aureus gene regulation in vivo.

Animals↗
PubMed1998

Identification and characterization of the PutP proline permease that contributes to in vivo survival of Staphylococcus aureus in animal models.

Staphylococcus aureus is an important pathogen of humans and other animals, causing bacteremia, abscesses, endocarditis, and other infectious syndromes. A signature-tagged mutagenesis (STM) system was adapted for use in studying the genes required for in vivo survival of S. aureus. An STM library was ultimately created in S. aureus RN6390, with Tn917 being used to create the transposon mutations. Pools of S. aureus RN6390 mutants were screened in mouse abscess, bacteremia, and wound infection models for growth attenuation after in vivo passage. One of the mutants that was identified displayed marked attenuation following large-pool screening in all three animal models, which was confirmed in bacteremia and endocarditis models of infection with a smaller pool of mutants. Sequence analysis of the entire open reading frame showed a 99% identity to the high-affinity proline permease (putP) gene characterized in another strain of S. aureus. In wound and murine abscess infection models, the putP mutant was approximately 10-fold more attenuated than was wild-type strain RN6390. Another S. aureus strain transduced with the putP mutation also displayed an attenuated phenotype after passage in the wound model. A [3H]proline uptake assay showed that less proline was specifically transported into the putP mutant than into strain RN6390. The reduced viability of the bacteria possessing the mutation in the S. aureus high-affinity proline permease suggests that proline scavenging by the bacteria is important for in vivo growth and proliferation and that analogs of proline may serve as potential antistaphylococcal therapeutic agents.

Amino Acid Transport Systems, Neutral↗
PubMed1998

In vitro resistance to thrombin-induced platelet microbicidal protein is associated with enhanced progression and hematogenous dissemination in experimental Staphylococcus aureus infective endocarditis.

We examined the influence of thrombin-induced platelet microbicidal protein 1 (tPMP-1) on the progression and hematogenous dissemination of experimental endocarditis caused by isogenic Staphylococcus aureus strains differing in tPMP susceptibility (tPMPs) or resistance (tPMPr) in vitro. Following simultaneous challenge of animals with both strains, significantly higher tPMPr bacterial densities were present in vegetations (P < 0.0001), kidneys (P < 0. 0001), and spleens (P < 0.0001) compared with those for the tPMPs strain. These data indicate that tPMP-1 limits the intravegetation proliferation and hematogenous dissemination of a tPMPs strain in experimental endocarditis, while the tPMPr phenotype confers a selective advantage associated with the enhanced progression of this infection.

Animals↗
PubMed1997

Adaptive resistance of Pseudomonas aeruginosa induced by aminoglycosides and killing kinetics in a rabbit endocarditis model.

Adaptive resistance following the first exposure to aminoglycosides is a recently described in vitro phenomenon in Pseudomonas aeruginosa and other aerobic gram-negative bacilli. We investigated the in vivo relevance of adaptive resistance in P. aeruginosa following a single dose of amikacin in the experimental rabbit endocarditis model. Rabbits with P. aeruginosa endocarditis received either no therapy (control) or a single intravenous (i.v.) dose of amikacin (80 mg/kg of body weight) at 24 h postinfection, after which they were sacrificed at 5, 8, 12, 16, or 24 h postdose. Excised aortic vegetations were subsequently exposed ex vivo to amikacin at 2.5, 5, 10 or 20 times the MIC for 90 min. In vivo adaptive resistance was identified when amikacin-induced pseudomonal killing within excised aortic vegetations was less in animals receiving single-dose amikacin in vivo than in vegetations from control animals not receiving amikacin in vivo. Maximal adaptive resistance occurred between 8 and 16 h after the in vivo amikacin dose, with complete refractoriness to ex vivo killing by amikacin seen at 12 h postdose. By 24 h postdose, bacteria within excised vegetations had partially recovered their initial amikacin susceptibility. In a parallel treatment study, we demonstrated that amikacin given once daily (but not twice daily) at a total dose of 80 mg/kg i.v. for 1-day treatment significantly reduced pseudomonal densities within aortic vegetations versus those in untreated controls. When therapy was continued for 3 days with the same total daily dose (80 mg/kg/day), amikacin given once or twice daily significantly reduced intravegetation pseudomonal densities versus those in controls. However, amikacin given once daily was still more effective than the twice-daily regimen. These data confirm the induction of aminoglycoside adaptive resistance in vivo and further support the advantages of once-daily aminoglycoside dosing regimens in the treatment of serious pseudomonal infections.

Amikacin↗
PubMed1997

Combination therapy with amphotericin B and fluconazole against invasive candidiasis in neutropenic-mouse and infective-endocarditis rabbit models.

Although there are an increasing number of new antifungal agents available, the morbidity and mortality due to invasive mycoses remain high. The high rates of polyene toxicities and the development of azole resistance have raised the issue of using antifungal agents of these classes in combination, despite theoretical concerns regarding antagonism between such agents. This study was designed to evaluate the in vivo efficacy of combined therapy with amphotericin B and fluconazole against Candida albicans. Two distinct animal models were used in this study: a neutropenic-mouse model of hematogenously disseminated candidiasis and the infective-endocarditis rabbit model. Treatment efficacy was assessed by determining reductions in mortality as well as decreases in tissue fungal densities. In the neutropenic-mouse model, amphotericin B, as well as combination therapy, significantly prolonged survival compared to untreated controls (P < 10(-5) and P = 0.001, respectively). The fungal densities in the kidneys of neutropenic mice were significantly reduced with either amphotericin B monotherapy or amphotericin B-fluconazole combined therapy compared to those of controls (P < 10(-6)). Fluconazole monotherapy also reduced fungal densities in the kidneys; however, this decrease was not statistically significant (P = 0.17). In contrast, treatment with either fluconazole alone or combined with amphotericin B (but not amphotericin B monotherapy) significantly decreased fungal densities in the brain (P = 0.025). In the rabbit endocarditis model, amphotericin B monotherapy or combined therapy significantly decreased fungal densities in cardiac vegetations (P < 0.01 versus the controls). Although no significant antagonism was seen when fluconazole was given in combination with amphotericin B, combination therapy did not augment the antifungal activity of amphotericin B.

Amphotericin B↗
PubMed1997

Hyperproduction of alpha-toxin by Staphylococcus aureus results in paradoxically reduced virulence in experimental endocarditis: a host defense role for platelet microbicidal proteins.

Staphylococcal alpha-toxin targets several cell types which are important components of cardiac vegetations in endocarditis, including platelets, erythrocytes, and endothelial cells. We evaluated the in vivo role of Staphylococcus aureus alpha-toxin in experimental endocarditis by using isogenic strains differing in the capacity to produce functional alpha-toxin, including 8325-4 (wild-type strain), DU-1090 (a mutant strain with allelic replacement of the alpha-toxin gene [hla]), DU1090(pH35L) (a mutant strain producing a target cell-binding but nonlytic toxin), DU1090(pDU1212) (a variant of DU1090 carrying the cloned hla gene on a multicopy plasmid), and DU1090(pCL84::hla) (a variant of DU1090 with a single copy of the hla gene cloned into the chromosomal lipase locus). In vitro, wild-type alpha-toxin (from parental strain 8325-4) extensively lysed both erythrocytes and platelets. In contrast, mutant alpha-toxin [from strain DU1090(pH35L)] lysed neither cell type. Following exposure to the wild-type alpha-toxin, platelet lysates were found to contain microbicidal activity against Bacillus subtilis (but not against Micrococcus luteus), as well as against the parental and alpha-toxin variant S. aureus strains noted above. Furthermore, lysate microbicidal activity was heat stable, neutralized by polyanionic filters or compounds, and recoverable from anionic filter membranes by hypertonic saline elution. These characteristics are consistent with those of cationic platelet microbicidal proteins (PMPs). Reverse-phase high-pressure liquid chromatography and polyacrylamide gel electrophoresis confirmed the presence of three distinct PMPs (1, 2, and 3) in platelet lysates. In experimental endocarditis, the two variant staphylococcal strains producing either minimal alpha-toxin or nonlytic alpha-toxin in vitro [strains DU1090 and DU1090(pH35L), respectively] exhibited significantly lower virulence in vivo than the parental strain (decreased intravegetation staphylococcal densities). Paradoxically, the two variant staphylococcal strains producing alpha-toxin at supraparental levels in vitro [strains DU1090(p1212) and DU1090(pCL84::hla)] also exhibited significantly decreased induction rates and intravegetation staphylococcal densities in experimental endocarditis versus the parental strain. The reduced in vivo virulence of the latter variant staphylococcal strains could not be explained by differences in bacteremic clearance or initial adherence to sterile vegetations (compared to the parental strain). These findings suggest that the reduced virulence exhibited by the variant staphylococcal strains in this model was related to pathogenetic events subsequent to bacterial adherence to the damaged endocardium. Excess intravegetation secretion of alpha-toxin, leading to increased PMP release (secondary to either increased platelet secretion or lysis), may well explain the reduced virulence observed in experimental endocarditis.

Animals↗
PubMed1997

The cytoplasmic membrane is a primary target for the staphylocidal action of thrombin-induced platelet microbicidal protein.

Thrombin-induced platelet microbicidal protein (tPMP-1) is a small, cationic peptide released from rabbit platelets exposed to thrombin in vitro. tPMP-1 is microbicidal against a broad spectrum of bloodstream pathogens, including Staphylococcus aureus. Preliminary evidence suggests that tPMP-1 targets and disrupts the staphylococcal cytoplasmic membrane. However, it is not clear if the cytoplasmic membrane is a direct or indirect target of tPMP-1. Therefore, we assessed the in vitro activity of tPMP-1 versus protoplasts prepared from logarithmic-phase (LOG) or stationary-phase (STAT) cells of the genetically related S. aureus strains 19S and 19R (tPMP-1 susceptible and resistant, respectively). Protoplasts exposed to tPMP-1 (2 microg/ml) for 2 h at 37 degrees C were monitored for lysis (decrease in optical density at 420 nm) and ultrastructural alterations (by transmission electron microscopy [TEM]). Exposure to tPMP-1 resulted in substantial lysis of LOG but not STAT protoplasts of 19S, coinciding with protoplast membrane disruption observed by TEM. Thus, it appears that tPMP-1-induced membrane damage is influenced by the bacterial growth phase but is independent of the staphylococcal cell wall. In contrast to 19S, neither LOG nor STAT protoplasts of 19R were lysed by tPMP-1. tPMP-1-induced membrane damage was further characterized with anionic planar lipid bilayers subjected to various trans-negative voltages. tPMP-1 increased conductance across bilayers at -90 mV but not at -30 mV. Once initiated, a reduction in voltage from -90 to -30 mV diminished conductance magnitude but did not eliminate tPMP-1-mediated membrane permeabilization. Therefore, tPMP-1 appears to directly target the staphylococcal cytoplasmic membrane as a primary event in its mechanism of action. Specifically, tPMP-1 likely leads to staphylococcal death, at least in part by permeabilizing the bacterial membrane in a voltage-dependent manner.

Animals↗
PubMed1997

Purification and in vitro activities of rabbit platelet microbicidal proteins.

Recent in vitro studies have demonstrated that rabbit platelets release a small, cationic antimicrobial protein in response to thrombin stimulation under physiological conditions (M. R. Yeaman, S. M. Puentes, D. C. Norman, and A. S. Bayer, Infect. Immun. 60:1202-1209, 1992). This observation prompted our present investigation, focused on determining the array of antimicrobial proteins contained within rabbit platelets and their in vitro activity against common bloodstream pathogens. A group of small (6.0- to 9.0-kDa), cationic proteins with in vitro antimicrobial activity was purified from whole and thrombin-stimulated rabbit platelets by gel filtration and reversed-phase high-performance liquid chromatography. Purified proteins in micromolar concentrations (10 to 40 microg/ml) exerted in vitro microbiostatic and/or microbicidal activities against Staphylococcus aureus, Escherichia coli, and Candida albicans in a dose-dependent manner. The antimicrobial activities of proteins purified from rabbit platelet acid extracts were generally inversely related to pH, with maximal activity observed at pH 5.5. In contrast, the predominant protein isolated from thrombin-stimulated rabbit platelets, though biochemically and microbiologically similar to proteins extracted by acid, exhibited antimicrobial activities which were modestly enhanced at pH 7.2 compared with pH 5.5. Amino acid compositional analyses in combination with molecular mass determinations suggest that the majority of these proteins are distinct molecules not derived from a single common precursor. Collectively, these data indicate that rabbit platelets contain proteins which exert potent in vitro antimicrobial activity against bacterial and fungal pathogens which commonly invade the bloodstream. Moreover, several of these proteins were released from platelets stimulated with thrombin under physiological conditions and exerted potent antimicrobial activities in physiological pH ranges. These observations support the hypothesis that platelets serve an important role in host defense against infection, via localized release of antimicrobial proteins in response to stimuli associated with tissue injury or microbial colonization.

Amino Acids↗
PubMed1997

Phenotypic resistance to thrombin-induced platelet microbicidal protein in vitro is correlated with enhanced virulence in experimental endocarditis due to Staphylococcus aureus.

Thrombin-induced platelet microbicidal protein (tPMP) is secreted by rabbit platelets following thrombin stimulation, and it kills common endovascular pathogens in vitro, including Staphylococcus aureus. Therefore, pathogens which exhibit tPMP resistance in vitro possess a potential survival advantage in vivo at sites of endovascular damage. We generated an isogenic S. aureus strain pair, differing in tPMP susceptibility, by transposon (Tn551) mutagenesis of a tPMP-susceptible (tPMPs) parental strain (ISP479) to derive a stably tPMP-resistant (tPMPr) strain, ISP479R. ISP479 and ISP479R were equivalent in vitro in the following phenotypes: biotyping, antiobiograms, platelet adherence and aggregation, growth kinetics, cell wall-associated protein A expression, and fibrinogen binding. Genotypic comparisons of chromosomal DNA of strains ISP479 and ISP479R following restriction endonuclease digestion revealed indistinguishable pulsed-field gel electrophoretic patterns. The genotype exhibited by strain ISP479R was linked to the tPMP-resistant phenotype, as it was transducible into the initially tPMP-susceptible parental strain, ISP479. Southern hybridization verified the presence of a single copy of Tn551 in the same chromosomal restriction site of both ISP479R and tPMPr transductants of ISP479. The correlation of in vitro tPMP susceptibility phenotypes with the ability to induce experimental endocarditis (a prototypical endovascular infection) was evaluated. Despite equivalent rates of endocarditis induction, animals infected with strain ISP479R achieved significantly higher vegetation bacterial densities over a 7-day post-challenge period than did animals infected with strain ISP479. These data suggest that tPMPr microbial strains have a selective advantage in experimental staphylococcal endocarditis. Furthermore, the major impact of tPMP resistance upon endocarditis pathogenesis appears to involve a postvalvular adherence event(s), most probably by facilitating bacterial proliferation within vegetations.

Animals↗