Faecalis Endocarditis Research Articles

Influence of gentamicin dosing interval on the efficacy of penicillin- containing regimens in experimental Enterococcus faecalis endocarditis

The influence of the gentamicin dosing regimen was studied in experimental Enterococcus faecalis endocarditis. After inoculation, animals received penicillin, or penicillin plus once-daily gentamicin, or penicillin plus thrice-daily gentamicin, or no treatment. After the treatment period, bacterial densities within the vegetations (mean +/- SEM) were 6.06 +/- 0.30, 5.42 +/- 0.29, 4.98 +/- 0.10 and 9.97 +/- 0.16 log cfu/g for the four groups. All regimens produced significant reductions in bacterial density when compared with controls; penicillin plus thrice-daily gentamicin resulted in a significant difference from penicillin alone. Although once-daily regimens have proved effective in trials involving other organisms, such regimens do not appear to be so optimal for the treatment of enterococcal endocarditis.

Aggregation and binding substances enhance pathogenicity in a rabbit model of Enterococcus faecalis endocarditis.

We investigated the importance of Aggregation Substance (AS) and Binding Substance (BS) in a rabbit model of Enterococcus faecalis endocarditis. Aggregation substance (AS), a pheromone inducible, surface protein of E. faecalis, promotes mating aggregate formation during bacterial conjugation and is thought to be a virulence factor in enterococcal infections by promoting binding to a variety of eukaryotic cell surfaces2. AS is not produced constitutively, but may be induced by eukaryotic factors in serum. AS contains the amino acid motifs Arg-Gly-Asp-Ser and Arg-Gly-Asp-Val, found in fibronectin and other proteins which mediate adhesion to eukaryotic cell surfaces2. Binding Substance (BS) is the site recognized by AS and is also necessary for mating aggregate formation. Lipotechoic acid (LTA) inhibits aggregate formation and may function as enterococcal BS2. Because of the increasing frequency with which enterococci are identified as nosocomial pathogens and because of the increasing incidence of vancomycin and other antibiotic resistance among these organisms, greater attention is focusing on the pathogenesis of enterococcal infections. AS and BS are thought to play an important role in enterococcal infections.

Efficacy of vancomycin and teicoplanin alone and in combination with streptomycin in experimental, low-level vancomycin-resistant, VanB-type Enterococcus faecalis endocarditis.

The efficacy of vancomycin (VM) and teicoplanin (TE), alone and in combination with streptomycin (SM), against enterococci that express low-level VanB-type VM resistance was investigated in experimental endocarditis using isogenic strains of Enterococcus faecalis susceptible to glycopeptides and aminoglycosides or inducibly resistant to low levels of VM (MIC = 16 micrograms/ml). VM was significantly less active against the resistant strain than against the susceptible strain, establishing that low-level VanB-type VM resistance can influence therapeutic efficacy. By contrast, TE had equally good activity against both strains. VM or TE combined with SM was synergistic and bactericidal against the resistant strain in vitro. While both combinations were efficient in reducing bacterial density in vivo, TE plus SM was significantly superior to VM plus SM if valve sterilization was considered. These data suggest that despite the presence of low-level VanB-type resistance, combination therapy with a glycopeptide and SM (and presumably other aminoglycosides to which there is not high-level resistance) will nevertheless provide effective bactericidal activity.

Plasmid-associated hemolysin and aggregation substance production contribute to virulence in experimental enterococcal endocarditis

A rabbit endocarditis model was utilized to evaluate the virulence conferred by the conjugative plasmid pAD1 with the following strains: Enterococcus faecalis plasmid-free FA2-2 and FA2-2 containing plasmids pAD1 (hemolysin and aggregation substance positive), pAM9058 (insertional inactivation of hemolysin), and pAM944 or pAM947 (insertional inactivation of aggregation substance). All isolates were similar in ability to produce endocarditis. Mean vegetation weight was greater in animals inoculated with strains that produced aggregation substance (P < 0.01). Mortality was significantly increased in animals given FA2-2 containing pAD1 compared with those given all other strains (P < 0.01). These results suggest that the combination of hemolysin and aggregation substance is associated with increased mortality and that vegetation weight is associated with production of aggregation substance in experimental E. faecalis endocarditis.

Isolation and sequence determination of an immunodominant antigen from Enterococcus faecalis

Immunoblotting serum from patients with endocarditis due to Enterococcus faecalis has previously identified three antigenic bands of 112, 88–90 and 45–47 kDa which were demonstrated as being endocarditis specific. We have isolated a partial DNA clone for the highly immunogenic 88–90 kDa antigen of E. faecalis. The lambda ft11 libarary was screened with pooled sera from patients with E. faecalis endocarditis, and a positive clone for the 88–90 kda band identified by affinity-purified antibody probes. The continuous open reading frame of 1932 nucleotide encoded for a predicted polypeptide of 644 amino acids with a molecular weight of 72541 daltons. Homology sequence analysis indicated the presence of a putative internal repeat bearing consensus nucleotide binding motifs. We believe this is the first reported gram-positive bacterial ATP-binding protein that contains two ATP-binding domains within a single protein.

Importance of the aminoglycoside dosing regimen in the penicillin-netilmicin combination for treatment of Enterococcus faecalis-induced experimental endocarditis

The penicillin-aminoglycoside combination is recommended for the treatment of systemic enterococcal infections. However, the optimal dosing regimen of the aminoglycoside remains to be elucidated. We evaluated the efficacy of penicillin, alone or in combination with various dosing regimens of netilmicin, for the treatment of experimental left-sided Enterococcus faecalis endocarditis in rabbits. Animals were injected intramuscularly for 4 days with penicillin alone or in combination with netilmicin in one of the following regimens: netilmicin at a low dose (2 mg/kg of body weight every 8 h), netilmicin at a high dose (4 mg/kg every 8 h), or netilmicin at a single daily high dose (12 mg/kg every 24 h). MICs and MBCs were 3.1 and 6.2 micrograms/ml and 8 and 8 micrograms/ml for penicillin and netilmicin, respectively. A netilmicin concentration of 4 micrograms/ml was the lowest concentration that achieved synergism with penicillin, as shown by the kill-curve method. Mean peak levels of netilmicin in serum were 5.6 (netilmicin at 2 mg/kg), 9.8 (netilmicin at 4 mg/kg), and 20.6 (netilmicin at 12 mg/kg) micrograms/ml. Mean penicillin levels in serum were constantly above the MIC. Penicillin plus netilmicin at a high dose given three times daily was more effective (P less than 0.05) than any other regimen in reducing bacterial titers in vegetations and was the only treatment that induced a significant bactericidal activity in rabbit serum during the trough. We concluded that divided doses of aminoglycoside are more effective than the same total dose given once daily in combination with penicillin. Our data suggest that prolonged levels of aminoglycoside in serum might be important to exhibit the greatest in vivo efficacy of the combination against E. faecalis. They also indicate that use of a reduced total daily dose of aminoglycoside or an increase in the interval between each dose might reduce the efficacy of therapy in animals with this type of infection.

Serological response in Enterococcus faecalis endocarditis determined by enzyme-linked immunosorbent assay : Shorrock PJ, Lambert PA, Aitchison EJ, et al: J Clin Microbiol 28:195–200, 1990

Serological response in Enterococcus faecalis endocarditis determined by enzyme-linked immunosorbent assay : Shorrock PJ, Lambert PA, Aitchison EJ, et al: J Clin Microbiol 28:195–200, 1990

Serological response in Enterococcus faecalis endocarditis determined by enzyme-linked immunosorbent assay

Enterococcus (Streptococcus) faecalis expresses three species-specific surface protein antigens of molecular weights 73,000, 40,000, and 37,000. On Western blotting (immunoblotting), they were detected strongly by immunoglobulin G (IgG) in sera from patients with E. faecalis endocarditis, but not in sera from patients with other E. faecalis infections or with endocarditis due to other streptococci. We developed an enzyme-linked immunosorbent assay system to measure IgG, IgM, and IgA levels to these antigens and evaluated its potential as a serodiagnostic test for E. faecalis endocarditis. The test correctly diagnosed E. faecalis endocarditis in 15 of 16 cases. Of 10 cases of endocarditis due to other streptococci and 10 E. faecalis infections other than endocarditis, 9 and 8, respectively, gave negative results. The test should prove particularly useful in culture-negative cases, for which choice of appropriate antibiotic therapy for E. faecalis endocarditis is vital.

Diagnosing endocarditis with the cloned 112 kDa antigen of Enterococcus faecalis

A new indirect ELISA is presented for the diagnosis of enterococcal endocarditis. It is based on the cloning of Enterococcus faecalis DNA into λ gt11. The library was screened by antisera from three cases of E. faecalis endocarditis. Three positive clones were found, all of which cross-reacted with the 112 kDa antigen of E. faecalis. One of these was taken and lysogenised into E. coli Y1089 to produce a fusion protein of 125 kDa. This IPTG dependent protein was purified by affinity chromatography and used in an indirect ELISA. The test differentiated between five cases of enterococcal endocarditis (IgG ELISA optical density > 0.8) and patients with endocarditis due to staphylococci (IgG ELISA optical density < 0.243) and other streptococci (IgG ELISA optical density < 0.656). Patients with a simple E. faecalis septicaemia had a maximum IgG ELISA optical density of 0.636. The only major cross-reaction occurred in patients with Streptococcus bovis endocarditis.

Role of immunoblotting in the diagnosis of culture negative and enterococcal endocarditis.

Serum samples from patients with endocarditis and septicaemia due to Enterococcus faecalis, Enterococcus faecium, Streptococcus bovis, and Streptococcus sanguis were immunoblotted against antigenic extracts from all four species. In E faecalis endocarditis there was a strong IgM response to E faecalis antigenic bands of 112, 88-90, and 45-47 Kd and a strong IgG response to 88-90 and 45-47 Kd bands. In E faecium endocarditis there was a pronounced IgG response to an E faecium band of 82-90 Kd. For S bovis endocarditis, there was a strong IgG response to several components of S bovis including bands of 66, 58, 52 and 4 Kd. For S sanguis, there was a strong IgG response to bands of 80-82, 76, 60 and 45 Kd. These patterns of antibody production were absent in patients with uncomplicated septicaemia and in controls. The delineation of these patterns enabled confirmation of the final diagnosis in seven patients initially suspected of having culture negative endocarditis.

Comparison of single and multiple doses of prophylactic antibiotics in experimental streptococcal endocarditis.

Single-doses or short-term administration of beta-lactam antibiotics alone or combined with aminoglucoside antibiotics have failed to consistently prevent experimental streptococcal endocarditis induced by high inocula of bacteria poorly susceptible to killing by these antibiotics. The optimal duration of administration of antibiotics for successful prophylaxis under these circumstances has not been established. We therefore tested, in rats with catheter-induced sterile aortic vegetations, the duration of administration of antibiotic necessary to prevent endocarditis induced by bacterial inocula 100 to 10,000 times the 90% infective dose of two tolerant viridans-group streptococci and two Streptococcus faecalis strains. Multiple-dose regimens of amoxicillin alone or of amoxicillin combined with gentamicin were studied. Against the two viridans group streptococci, successful prophylaxis was achieved with multiple doses of amoxicillin alone given over 24 to 48 hr and by the combination of amoxicillin and gentamicin given for 6 to 24 hr. Against the two S. faecalis strains, multiple-dose regimens with amoxicillin alone failed, but the combination of amoxicillin and gentamicin was successful when administered for 48 to 72 hr. Thus, after challenge with high bacterial inocula, repeated doses of a beta-lactam antibiotic alone were sufficient to prevent viridans streptococcal endocarditis, but multiple doses of a bactericidal combination (beta-lactam plus aminoglucoside), as necessary for the treatment of established endocarditis, were a prerequisite for successful prophylaxis of S. faecalis endocarditis.

Serodiagnosis of Streptococcus faecalis endocarditis by immunoblotting of surface protein antigens.

We describe a method for the serodiagnosis of Streptococcus faecalis in infective endocarditis which could be of value in culture-negative cases. Serum-grown cells of S. faecalis produced three major characteristic protein antigens (73,000, 40,000, and 37,000 molecular weight) which were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis of solubilized whole cells. After electrophoretic transfer to a nitrocellulose membrane, these antigens were visualized by probing with serum from patients with endocarditis caused by S. faecalis. Serum from patients with endocarditis caused by other organisms did not react with the S. faecalis-specific antigens. This procedure should facilitate positive early diagnosis of S. faecalis endocarditis or establish its absence in culture-negative cases.

Influence of the developmental state of valvular lesions on the antimicrobial activity of cefotaxime in experimental enterococcal infections

Cefotaxime has little antimicrobial activity in vitro against most strains of enterococci, as measured by conventional MICs and MBCs. However, the MICs of cefotaxime against many enterococci are markedly reduced by the addition of serum to the test medium. To assess the relevance of this observation in vivo, we examined the efficacy of cefotaxime in experimental Streptococcus faecalis endocarditis. Since response to antimicrobial agents may vary with the degree of vegetation development, therapeutic efficacy was assessed both in rabbits with newly formed vegetations and in rabbits with well-developed endocardial lesions. Peak serum levels of cefotaxime (50.1 +/- 20.0 micrograms/ml) exceeded the MIC in medium supplemented with serum (4 micrograms/ml), but not in Mueller-Hinton broth alone (greater than 64 micrograms/ml). After 4 days of therapy, animals with newly formed lesions (therapy initiated 1 h after infection, transvalvular catheters removed) had lower mean vegetation bacterial titers than did untreated controls. Among animals with mature vegetations (therapy initiated 12 h after infection, catheters indwelling), the rate of mortality was significantly reduced by cefotaxime therapy. However, no difference in vegetation titers was observed. Thus, cefotaxime demonstrated antienterococcal activity within newly formed vegetations, but did not inhibit bacterial proliferation within well-established vegetations.

Therapeutic efficacy of teicoplanin in experimental enterococcal endocarditis.

The antimicrobial activities of teicoplanin and ampicillin, alone and in combination with gentamicin, were compared in experimental Streptococcus faecalis endocarditis. Bacterial titers in vegetations of rabbits treated with teicoplanin were significantly lower than those of untreated controls (P less than 0.01) and were equivalent to titers in ampicillin-treated animals. Gentamicin increased the activities of both drugs to a comparable degree.

EXPERIMENTAL ENDOCARDITIS IN RABBITS

A previously described model of experimental Streptococcus faecalis endocarditis in rabbits without an indwelling catheter during the infectious processes was used to study the effect of long‐term treatment with antibiotics. Groups of animals infected with six different strains were treated for four weeks and the following parameters were determined: survival rate, bacterial concentration in blood and vegetations, signs at autopsy indicating congestive heart failure. Before the therapeutic experiments, the tolerance of the rabbit to long‐term exposure of the drugs penicillin and streptomycin was considered in a group of non‐infected animals. Two out of 20 rabbits died with enteritis during the penicillin exposure, and a general weight reduction was observed. Streptomycin was apparently completely harmless. There was no therapeutic effect of streptomycin on S. faecalis endocarditis due to strains all designated resistant to streptomycin by MIC, except in rabbits infected with a strain, which showed partial susceptibility to the drug by IC50. Regardless of the therapeutic effect, evidence was obtained for rapid development of increased resistance of the infecting strains towards streptomycin. After long‐term treatment with penicillin in either low or high dose some of the animals survived and the valves were sterilized in 37% of the animals after low‐dose and in 3996 after high‐dose. It was observed that congestive heart failure occurred with the greatest frequency and intenstity after infection with proteolytic strains.

EXPERIMENTAL ENDOCARDITIS IN RABBITS

The purpose of this study was to investigate the efficacy of long‐term penicillin‐gentamicin treatment on experimental S. faecalis endocarditis due to selected strains; one strain was homogeneously resistant to 8000 μg/ml streptomycin (IC50 and MIC &gt; 8000 μg ml), the other strain heterogeneously resistant to 8000 μg/ml streptomycin (IC50: 3300 μg/ml, MIC: &gt; 8000 μg/ml. Both strains showed low‐grade resistance to gentamicin (MIC 10.5 and 25 μg/ml, respectively). The results showed that there was significant effect of the treatment in rabbits with endocarditis due to both strains, as measured by lowered mortality and by high bacteriologic cure rate. Despite effective antibiotic treatment, a high incidence (57%) of congestive heart failure was noted in rabbits late in the treatment period or after termination of antibiotic treatment, probably due to healing processes on the aortic valves. In human cases of S. faecalis endocarditis the antibiotic treatment should be either penicillin plus streptomycin or penicillin plus gentamicin. However, in cases where the infecting strain is homogeneously resistant to 8000 μg/ml streptomycin, the treatment with penicillin plus gentamicin is clearly the drug combination of choice.

EXPERIMENTAL ENDOCARDITIS IN RABBITS

The synergistic action of penicillin and streptomycin was investigated on animals with experimental endocarditis, using the previously described model and four different strains of Streptococcus faecalis. Two strains represented a moderate and two strains a high level of resistance to streptomycin. The purpose was to determine the effect of the combined penicillin and streptomycin treatment, since previous in vitro investigations showed that strains highly resistant to streptomycin were also resistant to combination of the drugs. Antibiotic treatment of the animals was carried out for 28 days, followed by a period of four weeks observation. The treatment resulted in a demonstrable effect against infection caused by the least streptomycin-resistant strain, but was completely ineffective in rabbits infected with the strain homogeneously resistant to 8000 micrograms/ml streptomycin. The synergistic effect of penicillin and streptomycin towards strains within a range of streptomycin resistance of 2000-8000 micrograms/ml could be predicted by the IC50 test, but not by MIC or the in vitro killing curve test for synergism. Besides the therapeutic results, this report also considers the following features: pathoanatomic and physiologic processes related to the number of viable bacteria in the endocardial vegetations; the influence of the proteolytic capacity of infecting strains; the development of congestive heart failure.

Identification of bacterial antigens in circulating immune complexes of infective endocarditis.

The presence of circulating immune complexes (IC) in patients with infective endocarditis has been well documented but the contributions of host and bacterial components to these IC have not been defined. To study this question, IC were isolated from serum of a patient with Streptococcus faecalis endocarditis by differential polyethylene glycol precipitation and competitive binding to staphylococcal protein A. A rabbit antiserum raised against the purified IC had reactivity by crossed immunoelectrophoresis primarily with an antigen derived from the cytoplasm of the infective organism. The antigen was a protein with a 12,000-dalton molecular mass. In situ radiolabeling of the IC bound to the protein A demonstrated a component of the same molecular mass as the bacterial antigen recognized by the antiserum. The patient serum had multiple antibody specificities reactive with bacterial antigens, including the antigen recognized by the rabbit anti-IC antiserum. These techniques for isolation and characterization of circulating IC may have value in the study of IC diseases in which the inciting antigens are not known.

EXPERIMENTAL ENDOCARDITIS IN RABBITS

Insertion of a polyethylene catheter in the left side of the heart was used to induce sterile endocarditis in 34 rabbits. Bacterial endocarditis was established by injection of approximately 10(8) Streptococcus faecalis into the blood stream simultaneous with the removal of the catheter which had been in place for 3 days. The course of the bacterial endocarditis was examined by autopsy of rabbits sacrificed at regular intervals after the infection. The results showed that the presence of the catheter was not essential for the induction or maintenance of the infection. Growth of the bacteria took place in the preformed vegetations in the aorta, on the aortic valves and in the left ventricle. However, increases in the size of the vegetations, a high density of bacteria in the vegetations and secondary spreading were found only on the aortic valves. The extracardial manifestations of left-sided S. faecalis endocarditis included constant bacteriaemia, a high frequency of septic kidney infarcts and enlargement of the spleen. This form of experimentally provoked bacterial endocarditis in rabbits provides a good imitation of human subacute endocarditis, and would thus seem to be suitable for further study of the pathophysiology of endocarditis and evaluation of the effect of treatment with antibiotics.

Streptoccocus faecalis endocarditis: an emerging problem?

The clinical features of 16 patients with Streptococcus faecalis endocarditis are reviewed and the currently favoured antimicrobial regimens for such infections are detailed.