Comparison of Single Doses of Amoxicillin or of Amoxicillin-Gentamicin for the Prevention of Endocarditis Caused by Streptococcus faecalis and by Viridans Streptococci

Recent recommendations for the prophylaxis of endocarditis in humans have advocated single doses or short courses of antibiotic combinations (β-lactam plus aminoglycoside) for susceptible patients in whom enterococcal bacteremia might develop or for patients at especially high risk of developing endocarditis (e.g., patients with prosthetic cardiac valves). We tested the prophylactic efficacy (in rats with catheter-induced aortic vegetations) of single doses of amoxicillin plus gentamicin against challenge with various streptococcal strains (two strains of Streptococcus faecalis, one of Streptococcus bovis, and three of viridans streptococci); we then compared this efficacy with that of single doses of amoxicillin alone. Successful prophylaxis against all six strains was achieved with single doses of both amoxicillin alone and amoxicillin plus gentamicin. This protection, however, was limited, for both regimens, to the lowest bacterial-inoculum size producing endocarditis in 90% of control rats and was not extended to higher inocula by using the combination of antibiotics. We concluded that a single dose of amoxicillin alone was protective against enterococcal and nonenterococcal endocarditis in the rat, but that its efficacy was limited and could not be improved by the simultaneous administration of gentamici

Excision of staphylococcal cassette chromosome mec in methicillin-resistant Staphylococcus aureus assessed by quantitative PCR.

BACKGROUND: Methicillin-resistance in staphylococci is conferred by the mecA gene, located on the genomic island Staphylococcal Cassette Chromosome mec (SCCmec). SCCmec mobility relies on the Ccr recombinases, which catalyze insertion and excision form the host's chromosome. Although being a crucial step in its horizontal transfer, little is known about the dynamics of SCCmec excision. RESULTS: A quantitative PCR-based method was used to measure the rate of SCCmec excision by amplifying the chromosome-chromosome junction and the circularized SCCmec resulting from excision. SCCmec excision rate was measured in methicillin-resistant Staphylococcus aureus (MRSA) strain N315 at various growth times in broth cultures. In the present experimental settings, excision of SCCmec occurred at a rate of approximately 2 × 10(-6) in MRSA N315. CONCLUSION: This work brings new insights in the poorly understood SCCmec excision process. The results presented herein suggest a model in which excision occurs during a limited period of time at the early stages of growth

Expression of SCCmec cassette chromosome recombinases in methicillin-resistant Staphylococcus aureus and Staphylococcus epidermidis

Objectives Methicillin resistance in staphylococci is mediated by the mecA gene, which is carried on the staphylococcal cassette chromosome mec (SCCmec). SCCmec is responsible for vertical and horizontal transfer of methicillin resistance. Horizontal transfer implies first SCCmec excision from the chromosome. Site-specific excision is catalysed by the Ccr recombinases, which are encoded by ccrAB genes located on the cassette. The aim of this study is to determine the promoter activity of ccrAB genes in individual cells of methicillin-resistant Staphylococcus aureus (N315, COL and MW2) and Staphylococcus epidermidis (RP62A). One mutant cured of its SCCmec (N315EX) was also used. Exposure to various stresses was included in the study. Methods For each strain, translational promoter-green fluorescent protein (gfp) fusions were used to assess the levels of ccr promoter activity in individual cells. Analyses were performed using epifluorescence microscopy and flow cytometry. Results ccr promoter activity was observed in only a small percentage of cell populations. This ‘bistable' phenotype was strain dependent (GFP was expressed in N315 and RP62A, but not in COL and MW2) and growth dependent (GFP-expressing cells decreased from approximately 3% to 1% between logarithmic and stationary growth phases). The ccr promoter of strain N315 displayed normal promoter activity when expressed in SCCmec-negative N315EX. Likewise, the ccr promoter of strain COL (which was inactive in COL) showed normal N315-like activity when transformed into N315 and N315EX. Conclusions SCCmec excision operates through bistability, favouring a small fraction of cells to ‘sacrifice' their genomic islands for transfer, while the rest of the population remains intact. Determinants responsible for the activity of the ccr promoter were not located on SCCmec, but were elsewhere on the genome. Thus, the staphylococcal chromosome plays a key role in determining SCCmec stability and transferabilit

Successful Single-Dose Amoxicillin Prophylaxis Against Experimental Streptococcal Endocarditis: Evidence for Two Mechanisms of Protection

Amoxicillin prophylaxis against experimental endocarditis due to one nontolerant and two tolerant strains of streptococci was studied in rats. Single-dose amoxicillin protected against the two tolerant strains in animals challenged with the 90% infective dose (ID90), but protection diminished with increasing inoculum sizes. Protection against the nontolerant strain was successful with inocula that were 100-and I,OOO-fold larger than the ID90. Close correlation existed between the speed of bacterial killing in vitro, the time of exposure to bactericidal levels in vivo, and the range of inocula against which prophylaxis was effective. Amoxicillin seemed to protect by at least two mechanisms. (1) When in vitro tests indicated adequate bacterial killing, protection was independent of the inoculum size and was probably conferred by bacterial killing. (2) When in vitro tests indicated bacterial inhibition but not. killing, protection was inoculum-dependent and was probably mediated by inhibition of bacterial adherenc

Experimental Bacterial Endocarditis After Dental Extractions in Rats with Periodontitis

The development of bacterial endocarditis was analyzed after dental extractions in rats with or without periodontal disease. Periodontal disease was produced in rats by tying silk ligatures around the two maxillary first molars and placing the animals on a high sucrose diet for 14 weeks. Sterile aortic valve vegetations were produced by means of a transaortic catheter, and 24 hr later the maxillary first molars were extracted. The animals were killed 72 hr after the extractions. In rats with periodontal disease, extractions resulted in a 48% (14 of 29) incidence of bacterial endocarditis, most cases of which were due to Streptococcus spp. (one was caused by Staphylococcus aureus). In contrast, when the teeth with a healthy periodontium were extracted, only 6% (one of 15) of the rats developed endocarditis. When catheters were placed in anim, tis with periodontal disease but no extractions were performed, no endocarditis occurre

Mechanisms of Successful Amoxicillin Prophylaxis of Experimental Endocarditis Due to Streptococcus intermedius

Prophylaxis with amoxicillin (40 mg/kg) was studied in rats with aortic valve vegetations. Bacteria on the valves were quantitated early (10 min to 6 hr) and late (three days) after intravenous challenge with tolerant Streptococcus intermedius. Amoxicillin reduced by 40% the number of bacteria per valve 10 min after intravenous challenge with 105 S. intermedius (P < .05) and by 74% the incidence of endocarditis three days thereafter (P < .0001). Bacterial multiplication started 2 hr after challenge in control rats, whereas bacteria disappeared in 6 hr in amoxicillin-treated rats. Intravenous penicillinase 30 min after challenge abolished successful amoxicillin prophylaxis, a result demonstrating the necessity of prolonged growth inhibition for protection. Growth inhibition for 18 hr (two subsequent amoxicillin doses) was necessary for protection after intravenous challenge with 105 S. intermedius. Thus, in the absence of bacterial killing, inhibition of valvular colonization by amoxicillin was not as important a mechanism of endocarditis prophylaxis as was prolonged inhibition of bacterial growth, which allowed adherent bacteria to be cleared from the valve

Role of Amoxicillin Serum Levels for Successful Prophylaxis of Experimental Endocarditis Due to Tolerant Streptococci

The importance of amoxicillin serum profiles for successful prophylaxis of experimental endocarditis in rats was assessed. Animals with catheter-induced vegetations were challenged intravenously with large inocula of Streptococcus sanguis and received one of the following amoxicillin dosages: single or multiple bolus injection of 40 mg/kg; 40 mg/kg administered as a continuous infusion over 12 h; or either 9 or 18 mg/kg administered over 12 or 24 h, respectively. The regimen producing a single transient high peak serum level failed to prevent experimental endocarditis; in contrast, a second injection 6 h after the first resulted in successful prophylaxis. Likewise, the three regimens of continuous, relatively low-dose regimens prevented infections. Thus, the most important parameter for successful prophylaxis was the duration of inhibitory concentration of the drug in the serum. The total dose of antibiotic, the peak serum levels, or the area-under-the-curve values were not predictive of successful prophylaxi

Antibiotic Treatment of Experimental Endocarditis Due to Methicillin-Resistant Staphylococcus epidermidis

The natural history and treatment of experimental endocarditis due to heterogeneous and homogeneous methicillin-resistant Staphylococcus epidermidis was investigated. Amoxicillin/clavulanate or vancomycin were administered for 3 days via a computerized pump to mimic human drug kinetics in animals. After challenge with the minimum inoculum producing 90% of infections (ID90) , bacteria in the vegetations grew logarithmically for 16 h. Then, bacterial densities stabilized (at ∼108 cfu/g) and growth rates sharply declined. Both regimens cured ⩾60% of endocarditis (due to heterogeneous or homogeneous bacteria) when started 12-16 h after infection, although the bacterial densities in the vegetations had increased by 20 times in between. In contrast, treatment started after 24 h failed in most animals, while bacterial densities had not increased any more. Thus, while both regimens were equivalent, the therapeutic outcome was best predicted by growth rates in the vegetations, not by bacterial densities. These observations highlight the importance of phenotypic tolerance developing in viv