Impact of High-Level Daptomycin Resistance in the Streptococcus mitis Group on Virulence and Survivability during Daptomycin Treatment in Experimental Infective Endocarditis.

Among the viridans group streptococci, the Streptococcus mitis group is the most common cause of infective endocarditis. These bacteria have a propensity to be ß-lactam resistant, as well as to rapidly develop high-level and durable resistance to daptomycin (DAP). We compared a parental, daptomycin-susceptible (DAPs) S. mitis/S. oralis strain and its daptomycin-resistant (DAPr) variant in a model of experimental endocarditis in terms of (i) their relative fitness in multiple target organs in this model (vegetations, kidneys, spleen) when animals were challenged individually and in a coinfection strategy and (ii) their survivability during therapy with daptomycin-gentamicin (an in vitro combination synergistic against the parental strain). The DAPr variant was initially isolated from the cardiac vegetations of animals with experimental endocarditis caused by the parental DAPs strain following treatment with daptomycin. The parental strain and the DAPr variant were comparably virulent when animals were individually challenged. In contrast, in the coinfection model without daptomycin therapy, at both the 106- and 107-CFU/ml challenge inocula, the parental strain outcompeted the DAPr variant in all target organs, especially the kidneys and spleen. When the animals in the coinfection model of endocarditis were treated with DAP-gentamicin, the DAPs strain was completely eliminated, while the DAPr variant persisted in all target tissues. These data underscore that the acquisition of DAPr in S. mitis/S. oralis does come at an intrinsic fitness cost, although this resistance phenotype is completely protective against therapy with a potentially synergistic DAP regimen.

Diminished virulence of a sar-/agr- mutant of Staphylococcus aureus in the rabbit model of endocarditis.

Microbial pathogenicity in Staphylococcus aureus is a complex process involving a number of virulence genes that are regulated by global regulatory systems including sar and agr. To evaluate the roles of these two loci in virulence, we constructed sar-/agr- mutants of strains RN6390 and RN450 and compared their phenotypic profiles to the corresponding single sar- and agr- mutants and parents. The secretion of all hemolysins was absent in the sar-/agr- mutants while residual beta-hemolysin activity remained in single agr- mutants. The fibronectin binding capacity was significantly diminished in both single sar- mutants and double mutants when compared with parents while the reduction in fibrinogen binding capacity in the double mutants was modest. In the rabbit endocarditis model, there was a significant decrease in both infectivity rates and intravegetation bacterial densities with the double mutant as compared to the parent (RN6390) at 10(3)-10(6) CFU inocula despite comparable levels of early bacteremia among various challenge groups. Notably, fewer bacteria in the double mutant group adhered to valvular vegetations at 30 min after challenge (10(6) CFU) than the parent group. These studies suggest that both the sar and agr loci are involved in initial valvular adherence, intravegetation persistence and multiplication of S. aureus in endocarditis.

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.

Antimicrobial peptides versus invasive infections.

It has been estimated that there are more microorganisms within and upon the human body than there are human cells. By necessity, every accessible niche must be defended by innate mechanisms to prevent invasive infection, and ideally that precludes the need for robust inflammatory responses. Yet the potential for pathogens to transcend the integument actively or passively and access the bloodstream emphasizes the need for rapid and potent antimicrobial defense mechanisms within the vascular compartment. Antimicrobial peptides from leukocytes have long been contemplated as being integral to defense against these infections. Recently, platelets are increasingly recognized for their likely multiple roles in antimicrobial host defense. Platelets and leukocytes share many structural and functional archetypes. Once activated, both cell types respond in specific ways that emphasize key roles for their antimicrobial peptides in host defense efficacy: (a) targeted accumulation at sites of tissue injury or infection; (b) direct interaction with pathogens; and (c) deployment of intracellular (leukocyte phagosomes) or extracellular (platelet secretion) antimicrobial peptides. Antimicrobial peptides from these cells exert rapid, potent, and direct antimicrobial effects against organisms that commonly access the bloodstream. Experimental models in vitro and in vivo show that antimicrobial peptides from these cells significantly contribute to prevent or limit infection. Moreover, certain platelet antimicrobial proteins are multifunctional kinocidins (microbicidal chemokines) that recruit leukocytes to sites of infection, and potentiate the antimicrobial mechanisms of these cells. In turn, pathogens pre-decorated by kinocidins may be more efficiently phagocytosed and killed by leukocytes and their antimicrobial peptide arsenal. Hence, multiple and relevant interactions between platelets and leukocytes have immunologic functions yet to be fully understood. A clearer definition of these interactions, and the antimicrobial peptide effectors contributing to these functions, will significantly advance our understanding of antimicrobial host defense against invasive infection. In addition, this knowledge may accelerate development of novel anti-infective agents and strategies against pathogens that have become refractory to conventional antimicrobials.

Acetylsalicylic acid reduces vegetation bacterial density, hematogenous bacterial dissemination, and frequency of embolic events in experimental Staphylococcus aureus endocarditis through antiplatelet and antibacterial effects.

BACKGROUND: Platelets are integral to cardiac vegetations that evolve in infectious endocarditis. It has been postulated that the antiplatelet aggregation effect of aspirin (ASA) might diminish vegetation evolution and embolic rates. METHODS AND RESULTS: Rabbits with Staphylococcus aureus endocarditis were given either no ASA (controls) or ASA at 4, 8, or 12 mg. kg-1. d-1 IV for 3 days beginning 1 day after infection. Vegetation weights and serial echocardiographic vegetation size, vegetation and kidney bacterial densities, and extent of renal embolization were evaluated. In addition, the effect of ASA on early S aureus adherence to sterile vegetations was assessed. In vitro, bacterial adherence to platelets, fibrin matrices, or fibrin-platelet matrices was quantified with either platelets exposed to ASA or S aureus preexposed to salicylic acid (SAL). ASA at 8 mg. kg-1. d-1 (but not at 4 or 12 mg. kg-1. d-1) was associated with substantial decreases in vegetation weight (P<0.05), echocardiographic vegetation growth (P<0.001), vegetation (P<0.05) and renal bacterial densities and renal embolic lesions (P<0.05) versus controls. Diminished aggregation resulted when platelets were preexposed to ASA or when S aureus was preexposed to SAL (P<0.05). S aureus adherence to sterile vegetations (P<0.05) or to platelets in suspension (P<0.05), fibrin matrices (P<0.05), or fibrin-platelet matrices (P<0.05) was significantly reduced when bacteria were preexposed to SAL. CONCLUSIONS: ASA reduces several principal indicators of severity and metastatic events in experimental S aureus endocarditis. These benefits involve ASA effects on both the platelet and the microbe.

Staphylococcal bacteremia and endocarditis: state of the art.

The major clinical difficulties in bacteremic Staphylococcus aureus infections involve the following: (1) differentiation of infective endocarditis (IE) from non-IE staphylococcemias and (2) questions concerning optimum chemotherapy of deep-seated staphylococcemic infections. In this article, an algorithmic approach to the differentiation of S aureus IE from non-IE staphylococcemias will be developed, particularly in regard to specific clinical, echocardiographic, radioisotopic, and serological "markers" for recognizing patients as at "high risk" for underlying S aureus IE. Also, data will be presented relating to the controversies of monotherapy v combination chemotherapy in S aureus IE as well as in bacteremic S aureus infections caused by antibiotic-tolerant S aureus strains.

Pulmonary aspergilloma. Diagnostic and therapeutic considerations.

Pulmonary aspergillomas usually arise from colonization and proliferation of Aspergillus in preexisting parenchymal cavities. The most common symptom in this disorder is hemoptysis, which may be massive and life-threatening. Although positive sputum cultures for Aspergillus are present in more than half of patients with aspergilloma, this is neither a sensitive nor specific diagnostic marker. Virtually all patients with this syndrome have serum precipitating antibodies to Aspergillus antigens, and this serves as a useful confirmatory test in patients with suspected aspergilloma. The routine chest roentgenograph and standard tomography remain the most important diagnostic procedures. The computed tomograph of the chest may be helpful in certain cases. Routine surgical resection of aspergillomas is not recommended but should be reserved for patients with recurrent, severe hemoptysis who can tolerate thoracotomy. Parenteral antifungal therapy has not been effective in this disease; however, selected patients may be candidates for intracavitary antifungal therapy.

Chronic progressive coccidioidal pneumonitis. Report of six cases with clinical, roentgenographic, serologic, and therapeutic features.

Chronic progressive coccidioidal pneumonitis (CPCP) is an uncommon sequela of acute pulmonary coccidiodomycosis. Six recent patients with CPCP are described, most of whom were previously healthy. The clinical presentation was indolent, resulting in long diagnostic delays. Serial chest roentgenograms showed progressive pulmonary infiltration and sputum cultures were persistently positive for Coccidioides immitis. Serum complement fixation (CF) antibody titers were high, with five of six patients having titers greater than or equal to 1:16. No patients had evidence of extrapulmonary coccidioidal spread at time of diagnosis of CPCP, although hematogenous dissemination occurred later in one patient. Five patients received amphotericin B intravenously (greater than or equal to 30 mg/kg total), resulting in rapid clinical and mycologic cure, decline in CF titers, and roentgenographic improvement or stabilization. However, two of these five patients suffered permanent physiologic impairment. One patient refused therapy and remains clinically symptomatic, with chronic positivity of sputum cultures for C immitis and high CF titers.

Non-group D streptococcal meningitis misidentified as enterococcal meningitis. Diagnostic and therapeutic implications of misdiagnosis by screening microbiology.

Two patients had nonhemolytic Gram-positive coccal meningitis. Both pathogens were initially misidentified as a group D enterococcus by growth in "selective" media, which led to the use of inappropriate and potentially toxic systemic and intrathecal aminoglycosides. Careful evaluation of the antibiotic sensitivity data and additional microbiological studies allowed correct identification of the organism. The important diagnostic and therapeutic considerations in differentiating true enterococcal infections, especially meningitis, from those caused by other alpha-hemolytic or nonhemolytic streptococci are emphasized. A simple laboratory schema for rapid recognition of such pathogens is reviewed.

Staphylococcus aureus bacteremia. Clinical, serologic, and echocardiographic findings in patients with and without endocarditis.

Seventy-two adult patients with Staphylococcus aureus bacteremia were prospectively studied clinically, serologically, and echocardiographically. Multivariate analysis identified four parameters that significantly predicted endocarditis in staphylococcemic patients at time of initial evaluation: absence of a primary site of infection; community acquisition of infection; metastatic sequelae; and valvular vegetations detected by echocardiography. Echocardiography was most predictive of endocarditis in patients with community-acquired S aureus bacteremia from an obvious primary focus. In 11 (69%) of 16 patients with endocarditis and vegetations on two-dimensional echocardiography, this technique also revealed other important findings, including ventricular dilatation, and/or underlying valvular lesions. In 18% of patients with S aureus bacteremia without stigmata of endocarditis, echocardiography provided information that led to a diagnosis of endocarditis and a subsequent change in therapy. Our findings support the routine use of two-dimensional echocardiography in all cases of community-acquired S aureus bacteremia to identify occult endocarditis in patients without classic stigmata of disease, and to provide important prognostic data in clinically apparent endocarditis.

Solid-phase radioimmunoassay for IgG antibodies to Staphylococcus epidermidis. Use in serious coagulase-negative staphylococcal infections.

A radioimmunoassay (RIA) for human IgG antibodies to Staphylococcus epidermidis was compared with an agar-gel-diffusion assay in patients with a variety of infections. The RIA was sensitive and reproducible and discriminated between endocarditis and uncomplicated bacteremias due to coagulase-negative staphylococci. Anti-S epidermidis antibodies by RIA were elevated in 16 (89%) of 18 patients with coagulase-negative staphylococcal endocarditis but in none of 28 patients with uncomplicated bacteremia (n = 18) or with blood culture contaminated with these organisms (n = 10). Cross-reacting IgG antibodies to S epidermidis antigens were also detected by RIA in 13 (76%) of 17 patients with Staphylococcus aureus endocarditis but in none of 17 patients with nonvalvular S aureus bacteremias and in none of 25 patients with endocarditis or bacteremia caused by other pathogens. Agar-gel-diffusion assay was less sensitive than RIA for detecting coagulase-negative staphylococcal endocarditis, being positive in nine (50%) of 18 such patients. This RIA may be useful in distinguishing patients with endocarditis from those with nonvalvular staphylococcemias or blood culture contamination.

Group D enterococcal meningitis. Clinical and therapeutic considerations with report of three cases and review of the literature.

Three patients with meningitis due to the Lancefield group D enterococci are described and the pertinent literature is reviewed. Anatomic central nervous system (CNS) defects, prior neurologic or neurosurgical interventions, group D enterococcal endocarditis, and urinary tract infection appear to be important predisposing factors. Of note is the frequent lack of cellular response in the spinal fluid to enterococci. The mortality of this infection is high (33%) and is probably dictated as much by the underlying disorder as the infection itself. The therapeutic importance of careful separation of group D streptococcal isolates into enterococci and nonenterococci, especially in instances of CNS infections, is emphasized. We discuss the appropriate antimicrobial therapy for enterococcal meningitis.

Sternoarticualr pyoarthrosis due to gram-negative bacilli. Report of eight cases.

Of eight patients with Gram-negative bacillary sternoarticular pyoarthrosis, seven were long-term intravenous heroin abusers. Clinical onset was insidious and a long delay (one month or more) in seeking hospitalization was usually noted. Anterior chest discomfort and painful, restricted homolateral shoulder motion were the chief complaints. Fever and monoarticular arthritis were universally present, Open synovial biopsy examination was frequently required for etiologic diagnosis. Pseudomonas aeruginosa was the most common pathogen isolated. Roentgenographic evidence of associated osteomyelitis was usually seen, but tomography was often necessary to delineate this lesion. Intraoperatively, associated osteomyelitis of the clavicular head and/or sternum was present in all eight cases and a perisynovial and/or retrosternal abscess was found in five patients. Early surgical exploration and prolonged antimicrobial therapy yielded excellent results.

Circulating immune complexes in prosthetic valve endocarditis.

Using the Raji cell radioimmunoassay, we have determined circulating immune complex (CIC) levels in 36 patients with prosthetic valves during 38 episodes of fever. Fever resulted from prosthetic valve endocarditis (PVE) in 27 instances and from other causes in 11 instances. Peak initial CIC levels higher than 100 micrograms/mL occurred more frequently in the group with PVE, while peak initial CIC values less than 30 micrograms/mL were more frequent in the control group. Circulating immune complex levels fell substantially with completion of antibiotic therapy in 28 (78%) of the patients with PVE. Late CIC elevations were associated with drug-related rashes and replacement of persistently infected prostheses. Our data suggest that the predictive value of measurement of CIC levels in patients with fever and prosthetic valves is in excluding PVE in patients with CIC levels persisting below 30 micrograms/mL.

Infective endocarditis following human-to-human enterococcal transmission: a complication of intravenous narcotic abuse.

Two heroin addicts, husband and wife, who shared injection paraphernalia extensively, developed enterococcal endocarditis within six weeks of one another. The etiologic organisms were of the same subspecies and had identical antibiotic susceptibilities and biochemical profiles. The clinical, epidemiologic, and bacteriologic data strongly suggest human-to-human transmission of the pathogen.

Candida meningitis. Report of seven cases and review of the english literature.

Seven patients with Candida meningitis are reported. These 7, plus 21 previously cited cases, were reviewed. This infection arose by two distinct mechanisms: hematogenous dissemination and direct inoculation. Recent antibiotic therapy, corticosteroid administration and severe underlying diseases were important predisposing factors. Fever, meningismus, elevated CSF pressures and localizing neurologic signs were commonly noted. Organisms were seen on gram-stain of CSF in only 43% of cases. Mortality rate in patients receiving inadequate or no antifungal therapy was high (greater than 90%), while those patients given appropriate antifungal drugs, especially intravenous amphotericin B, had a significantly lower mortality rate (38%). Several factors associated with poor prognosis were delineated in this study: diagnostic interval after symptomatic onset longer than two weeks, CSF glucose levels below 35 mg/100 ml and presence of intracranial hypertension and focal neurologic deficits.

Serious infections due to group G streptoccocci. Report of 15 cases with in vitro-in vivo correlations.

Serious infections due to group G streptococci have been infrequently reported. Fifteen such cases are described. Endovascular infection, particularly endocarditis, and septic arthritis were the most common clinical syndromes observed. Despite exquisite in vitro sensitivity of group G streptococci to penicillin G, the in vivo clinical response was disappointing in six of nine patients with either endocarditis or septic arthritis. The group G streptococcal isolates from the patients in this study were uniformly sensitive to the inhibitory and killing action of penicillin G, ampicillin, cefotaxime, cephalothin, cefoxitin, and vancomycin. In contrast, clindamycin, erythromycin, and chloramphenicol had relatively poor bactericidal activity against these strains, including several "tolerant" strains. Timed-kill studies with penicillin G revealed impaired killing of group G streptococci at in vitro conditions of high inocula and stationary growth phases. This may partially explain the poor clinical responses in cases of group G streptococcal endocarditis.

Lactobacillemia--report of nine cases. Important clinical and therapeutic considerations.

Serious infections due to lactobacilli have been rarely cited. We report our findings in nine recent patients with lactobacillemia. In the combined literature and current experience, endocarditis and sepsis from localized suppuration were the most common clinical syndromes, most frequently arising from prior oropharyngeal infections. Lactobacillus endocarditis showed a predilection for left-sided cardiac involvement (100 per cent) and systemic arterial embolization (55 per cent). The nine clinical isolates were tested for minimal inhibitory and bactericidal concentrations (MICs and MBCs) against five drugs with broad gram-positive spectrums; of note, these organisms demonstrated a high incidence of both unachievable MBCs (64 per cent) and widely disparate (greater than 100 fold) MIC:MBC ratios (38 per cent). This is in accord with observations in Lactobacillus endocarditis of poor in vivo clinical response despite "appropriate" regimens and achievable MICs of the organisms. Bactericidal synergistic studies on two endocarditis isolates indicated that the penicillins plus aminoglycosides may be potentially useful in the treatment of deep-seated Lactobacillus infections when single antimicrobials fail to achieve a cure.

Use of circulating immune complex levels in the serodifferentiation of endocarditic and nonendocarditic septicemias.

Distinguishing endocarditic from nonendocarditic septicemias is prognostically and therapeutically important. One hundred two patients with both valvular and nonvalvular sepsis were studied for the presence and quantitation of circulating immune complexes. Ninety per cent of the patients with infective endocarditis versus 50 per cent of septic patients without infective endocarditis had circulating immune complex levels (p less than 0.005). Mean circulating immune complex levels in patients with infective endocarditis were significantly higher than in those without infective endocarditis, 106 +/- 18.58 microgram/ml versus 31 +/- 7.4 microgram/ml (p less than 0.005). Only three of 52 patients without infective endocarditis had circulating immune complex levels greater than 100 microgram/ml, as opposed to 16 of 50 patients with infective endocarditis (p less than 0.005). Similarly, one of 52 patients without infective endocarditis has circulating immune complex levels greater than 200 microgram/ml, as opposed to eight of 50 patients with infective endocarditis (p less than 0.05). In 92 per cent of the patients without infective endocarditis and 76 per cent of those with infective endocarditis peak circulating immune complex levels developed within 14 days after their entry into the study, often on the initial sampling. In febrile, septicemic patients with clinical symdromes nonclassic for endocarditis, measurements of serial circulating immune complex levels may be of adjunctive diagnosis importance. If circulating immune complex levels are undetectable, endocarditis would appear less likely; alternatively, levels above 100 to 200 microgram/ml would suggest a valvular rather than nonvalvular septic focus.

Evaluation of new clinical criteria for the diagnosis of infective endocarditis.

PURPOSE: The clinical diagnosis of infective endocarditis (IE) can be difficult. A new diagnostic schema for IE (the Duke criteria) has been proposed, utilizing clinical, microbiologic, and echocardiographic data. We evaluated the Duke criteria in a cohort of prospectively enrolled patients suspected of having IE and compared the diagnostic efficiency of these criteria with the previously published criteria of von Reyn. PATIENTS: Sixty-three febrile patients with suspected IE at a non-referral, municipal hospital were evaluated. All patients had the following parameters defined: the presence and nature of underlying heart disease; recent abuse of intravenous drugs; peripheral stigmata of IE; blood culture results; findings on two-dimensional transthoracic and transesophageal echocardiography (TTE, TEE); and the results of open heart surgery. RESULTS: Twelve of 63 patients underwent open heart surgery, at which time IE was pathologically confirmed in 10 patients and excluded in 2 patients. All 10 patients with pathologically confirmed IE were classified as "clinically definite" by Duke criteria, whereas 5 of 10 were rejected by von Reyn criteria (p < 0.05). Among the remaining 51 patients suspected of IE and evaluated by both von Reyn and Duke clinical criteria, significantly more cases were classified as "definite" IE by Duke criteria than by von Reyn criteria (p < 10(-5)). Similarly, significantly fewer cases were rejected as IE by the Duke criteria as compared with the von Reyn criteria (p < 10(-6). Duke criteria were also significantly better at diagnosing IE than von Reyn criteria in the following clinical settings: suspected right-sided IE (p < 0.01); suspected left-sided IE (p = 0.014); suspected culture-negative IE (p < 10(-2); and IE complicating Staphylococcus aureus or viridans streptococcal bacteremias (p < 10(-5); p < 0.05, respectively). Among 30 cases defined as clinically definite by the Duke criteria, the presence of blood culture positivity and echocardiographically defined vegetations was important in this classification of 77% and 57% of cases, respectively. Among the 17 patients in the clinically definite category with vegetative endocarditis observed by echocardiography, 7 (41%) had vegetations defined only by TEE. CONCLUSION: The Duke criteria are superior to the von Reyn criteria for the clinical diagnosis of IE, predominantly reflecting use of two-dimensional echocardiographic demonstration of valvular vegetations in the Duke schema.