Low-level resistance of Staphylococcus aureus to thrombin-induced platelet microbicidal protein 1 in vitro associated with qacA gene carriage is independent of multidrug efflux pump activity

Thrombin-induced platelet microbial protein 1 (tPMP-1), a cationic antimicrobial polypeptide released from thrombin-stimulated rabbit platelets, targets the Staphylococcus aureus cytoplasmic membrane to initiate its microbicidal effects. In vitro resistance to tPMP-1 correlates with survival advantages in vivo. In S. aureus, the plasmid-carried qacA gene encodes a multidrug transporter, conferring resistance to organic cations (e.g., ethidium [Et]) via proton motive force (PMF)-energized export. We previously showed that qacA also confers a tPMP-1-resistant (tPMP-1r) phenotype in vitro. The current study evaluated whether (i) transporters encoded by the qacB and qacC multidrug resistance genes also confer tPMP-1r and (ii) tPMP-1r mediated by qacA is dependent on efflux pump activity. In contrast to tPMP-1r qacA-bearing strains, the parental strain and its isogenic qacB- and qacC-containing strains were tPMP-1 susceptible (tPMP-1s). Efflux pump inhibition by cyanide m-chlorophenylhydrazone abrogated Etr, but not tPMP-1r, in the qacA-bearing strain. In synergy assays, exposure of the qacA-bearing strain to tPMP-1 did not affect the susceptibility of Et (ruling out Et-tPMP-1 cotransport). The following cytoplasmic membrane parameters did not differ significantly between the qacA-bearing and parental strains: contents of the major phospholipids; asymmetric distributions of the positively charged species, lysyl-phosphotidylglycerol; fatty acid composition; and relative surface charge. Of note, the qacA-bearing strain exhibited greater membrane fluidity than that of the parental, qacB-, or qacC-bearing strain. In conclusion, among these families of efflux pumps, only the multidrug transporter encoded by qacA conferred a tPMP-1r phenotype. These data suggest that qacA-encoded tPMP-1r results from the impact of a specific transporter upon membrane structure or function unrelated to PMF-dependent peptide efflux.     

Platelet antistaphylococcal responses occur through P2X1 and P2Y12 receptor-induced activation and kinocidin release

Platelets (PLTs) act in antimicrobial host defense by releasing PLT microbicidal proteins (PMPs) or PLT kinocidins (PKs). Receptors mediating staphylocidal efficacy and PMP or PK release versus isogenic PMP-susceptible (ISP479C) and -resistant (ISP479R) Staphylococcus aureus strains were examined in vitro. Isolated PLTs were incubated with ISP479C or ISP479R (PLT/S. aureus ratio range, 1:1 to 10,000:1) in the presence or absence of a panel of PLT inhibitors, including P2X and P2Y receptor antagonists of increasingly narrow specificity, and PLT adhesion receptors (CD41, CD42b, and CD62P). PLT-to-S. aureus exposure ratios of ≥10:1 yielded significant reductions in the viability of both strains. Results from reversed-phase high-performance liquid chromatography indicated that staphylocidal PLT releasates contained PMPs and PKs. At ratios below 10:1, the PLT antistaphylococcal efficacy relative to the intrinsic S. aureus PMP-susceptible or -resistant phenotype diminished. Apyrase (an agent of ADP degradation), suramin (a general P2 receptor antagonist), pyridoxal 5′-phosphonucleotide derivative (a specific P2X₁ antagonist), and cangrelor (a specific P2Y₁₂ antagonist) mitigated the PLT staphylocidal response against both strains, correlating with reduced levels of PMP and PK release. Specific inhibition occurred in the presence and absence of homologous plasma. The antagonism of the thromboxane A₂, cyclooxygenase-1/cyclooxygenase-2, or phospholipase C pathway or the hindrance of surface adhesion receptors failed to impede PLT anti-S. aureus responses. These results suggest a multifactorial PLT anti-S. aureus response mechanism involving (i) a PLT-to-S. aureus ratio sufficient for activation; (ii) the ensuing degranulation of PMPs, PKs, ADP, and/or ATP; (iii) the activation of P2X₁/P2Y₁₂ receptors on adjacent PLTs; and (iv) the recursive amplification of PMP and PK release from these PLTs.     

Diagnosis of culture-negative endocarditis: the role of the Duke criteria and the impact of transesophageal echocardiography.

BACKGROUND: The Duke criteria have been shown to be more sensitive than the von Reyn criteria in the diagnosis of culture-positive endocarditis but to date have not been fully validated for culture-negative endocarditis (CNE). The aim of this study was (1) to compare the diagnostic accuracy of the Duke criteria versus clinical judgment and the von Reyn criteria in CNE and (2) to assess the diagnostic impact of transesophageal echocardiography (TEE) on the Duke criteria in CNE. METHODS: The study group consisted of 49 patients with suspected CNE in whom the presence (n = 32) or absence (n = 17) of endocarditis was confirmed by surgery, autopsy, or both. All patients underwent transthoracic echocardiography (TTE) and TEE. They were classified into a Duke category initially with TTE data only, and then the Duke categories were reevaluated with the additional TEE data. RESULTS: The Duke criteria demonstrated a significantly higher sensitivity (72%) than the von Reyn criteria (28%; P =.0008) and a higher specificity (100%) than clinical judgment (76%; P =.02). No major differences were noted between sensitivities of the Duke criteria and clinical judgement. TEE significantly augmented the capacity to diagnose CNE by Duke criteria versus TTE (P <.05). CONCLUSIONS: The Duke criteria are of high diagnostic validity for the conduction of clinical studies on CNE. They have the potential to affect clinical decision-making, based on the higher specificity versus clinical judgment. TEE appears to be crucial for the diagnosis of CNE when the Duke criteria are applied. The diagnostic differentiation between CNE, sclerotic valve degeneration, and nonbacterial thrombotic endocarditis remains a challenge.     

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.     

Kulturnegative Endokarditis: Ätiologie, Diagnostik, Management und Therapie

BACKGROUND: Culture-negative endocarditis is a diagnostic challenge with variable clinical presentation and protean manifestations. ETIOLOGY AND DIAGNOSIS: The two main causes why endocarditis may be culture-negative are 1. antibiotic treatment prior to obtaining blood cultures, and 2. the presence of fastidious microorganisms with limited or no capacity to grow in routine blood cultures (Table 1). If initial blood cultures remain negative for 48-72 hours, these cultures should be incubated for at least an additional 2-4 weeks. Moreover, subcultures should be plated onto chocolate agar and incubated in an atmosphere of increased CO2 environment to facilitate recovery of fastidious bacteria. Additional techniques for identification of a causative organism include serologic tests and DNA/RNA-based molecular techniques. If the patient is clinically stable, the clinician can wait until culture results from initial samples are known before deciding upon either administering an empiric antibiotic therapy or obtaining further blood cultures. Certain predisposing patient characteristics or epidemiologic exposures may be associated with particular causative microorganisms in culture-negative endocarditis. In the absence of positive blood cultures echocardiography is a crucial tool in the diagnosis and management of culture-negative endocarditis which provides the basis for the visualization of endocarditis-associated cardiac lesions. In this context, transesophageal echocardiography is associated with a significantly higher sensitivity in the detection of vegetations and perivalvular complications and is, therefore, considered the diagnostic imaging method of choice in the diagnosis of culture-negative endocarditis. The Duke criteria have been shown to have a high accuracy in the diagnosis of culture-negative endocarditis. In this context global clinical judgment demonstrated a comparable sensitivity but a lower specificity. Main differential diagnoses include diseases which can mimic the clinical endocarditis syndrome as well as the echocardiographic pattern of culture-negative endocarditis, especially 1. nonbacterial thrombotic endocarditis and 2. valvular sclerosis in the presence of systemic infection (Table 2). TREATMENT: The selection of a particular antibiotic regimen in a suspected case of culture-negative endocarditis depends on demographics (e.g., age or geographic area), epidemiologic history (e.g., animal exposures, drug-use history, alcohol abuse, homelessness) and clinical characteristics which may be suggestive of an etiologic organism.     

Has Transesophageal Echocardiography Changed the Approach to Patients with Suspected or Known Infective Endocarditis?

Infective endocarditis is still a great clinical challenge. Its diagnosis is difficult to establish, and mortality has remained around 30%. Early diagnosis and optimal treatment are crucial fo prognosis improvement. Echocardiography plays an indispensable role in the management of this disease, especially with the recently introduced approach, transesophageal echocardiography (TEE). TEE can overcome the limitations of transthoracic echocardiography (TTE) and is superior to TTE in almost every way in providing earlier and more information for the diagnosis and treatment of infective endocarditis. TEE detects valve vegetations with much higher sensitivity and specificity than TTE. It can demonstrate smaller vegetations in the early stage of the disease and vegetations on atypical locations (e.g., mitral valve annulus), and provides detailed characterization of vegetations (e.g., location, size, mobility, and changes during treatment). Such information is of great prognostic value and may help in selecting proper treatment. TEE is more sensitive for detecting complications, such as mitral valve perforation, abscess, and subaortic complications, which respond poorly to medicine and for which timely surgery may be the best treatment. For those with prosthetic valve endocarditis, TEE is especially useful because TTE is greatly limited by the acoustic shadow of prostheses. Both positive and negative results of TEE examination are valuable for confirming or excluding infective endocarditis. TEE also plays a unique role in intraoperative monitoring and can assess surgical results before the chest is closed. TEE has become an invaluable tool for the diagnosis and management of patients with suspected or known infective endocarditis.     

Nonredundant Roles of Interleukin-17A (IL-17A) and IL-22 in Murine Host Defense against Cutaneous and Hematogenous Infection Due to Methicillin-Resistant Staphylococcus aureus

Staphylococcus aureus is the leading cause of skin and skin structure infections (SSSI) in humans. Moreover, the high frequency of recurring SSSI due to S. aureus, particularly methicillin-resistant S. aureus (MRSA) strains, suggests that infection induces suboptimal anamnestic defenses. The present study addresses the hypothesis that interleukin-17A (IL-17A) and IL-22 play distinct roles in immunity to cutaneous and invasive MRSA infection in a mouse model of SSSI. Mice were treated with specific neutralizing antibodies against IL-17A and/or IL-22 and infected with MRSA, after which the severity of infection and host immune response were determined. Neutralization of either IL-17A or IL-22 reduced T cell and neutrophil infiltration and host defense peptide elaboration in lesions. These events corresponded with increased abscess severity, MRSA viability, and CFU density in skin. Interestingly, combined inhibition of IL-17A and IL-22 did not worsen abscesses but did increase gamma interferon (IFN-γ) expression at these sites. The inhibition of IL-22 led to a reduction in IL-17A expression, but not vice versa. These results suggest that the expression of IL-17A is at least partially dependent on IL-22 in this model. Inhibition of IL-17A but not IL-22 led to hematogenous dissemination to kidneys, which correlated with decreased T cell infiltration in renal tissue. Collectively, these findings indicate that IL-17A and IL-22 have complementary but nonredundant roles in host defense against cutaneous versus hematogenous infection. These insights may support targeted immune enhancement or other novel approaches to address the challenge of MRSA infection.     

Comparison of drug-eluting stents with bare metal stents in unselected patients with acute myocardial infarction.

OBJECTIVES: The aim of this study was to compare the procedural characteristics and outcomes of patients with acute myocardial infarction treated with drug-eluting stents (DES) vs. bare metal stents (BMS). BACKGROUND: DES have been shown to reduce the incidence of restenosis and target vessel revascularization (TVR) in clinical randomized studies when compared with BMS in patients undergoing elective percutaneous intervention. Limited data are available with the use of DES in patients with acute ST-segment elevation myocardial infarction. METHODS: Two hundred and sixty-one consecutive patients who presented with myocardial infarction between 7/2001 and 8/2005 were studied. The procedural characteristics, 30-day and 12-month outcomes of 131 patients treated with DES were compared with 130 patients treated with BMS. RESULTS: At 12-months follow-up DES therapy was associated with a substantial decrease in major adverse cardiovascular events (MACE) (HR 0.33; P =0.002), TVR (HR 0.19; P =0.002), and recurrent myocardial infarction (HR 0.23; P =0.051) vs. BMS therapy. Coronary interventions utilizing DES were characterized by a marked increase in the number of stent per target vessel (DES: 1.9 +/- 0.9 vs. BMS: 1.38 +/- 0.6, P < 0.0001), treatment of bifurcation (DES: 21% vs. BMS: 5%, P =0.0004), and multivessel intervention (DES: 22% vs. BMS: 8%, P =0.003). CONCLUSION: The routine use of DES in acute myocardial infarction is associated with reduced rates of MACE at 12 months vs BMS, despite a higher rate of complex procedures in the DES treated patients. In addition to its anti-restenosis effect, the improved outcome of patients treated with DES may be linked to a more complete revascularization in association with prolonged clopidogrel therapy.