Fractional Maximal Effect Method for In Vitro Synergy between Amoxicillin and Ceftriaxone and between Vancomycin and Ceftriaxone against Enterococcus faecalis and Penicillin-Resistant Streptococcus pneumoniae

Abstract

In the present study we assessed the use of a new in vitro testing method and graphical representation of the results to investigate the potential effectiveness of combinations of amoxicillin (AMZ) plus ceftriaxone (CRO) and of CRO plus vancomycin (VAN) against strains of Streptococcus pneumoniae highly resistant to penicillin and cephalosporins (PRP strains). We used the fractional maximal effect (FME) method of time-kill curves to calculate adequate concentrations of the drugs to be tested rather than relying on arbitrary choices. The concentrations obtained, each of which corresponded to a fraction of the maximal effect, were tested alone and in combination with the bacterial strains in a broth medium. Synergy was defined as a ratio of observed effect/theoretical effect, called FME, of greater than 1, additivity was defined as an FME equal to 1, and antagonism was defined as an observed effect lower than the best effect of one of the antibiotics used alone. The area between antagonism and additivity is the indifference zone. The well-known synergy between amoxicillin and gentamicin against a reference strain of Enterococcus faecalis was confirmed, with a best FME equal to 1.07. Two strains of PRP, strains PRP-1 and PRP-2, were studied. The MICs for PRP-1 and PRP-2 were as follows: penicillin, 4 and 16 g/ml, respectively; AMZ, 2 and 8 g/ml, respectively, CRO, 1 and 4 g/ml, respectively; and VAN, 0.5 and 0.25 g/ml, respectively. For PRP-1 the best FME for the combination AMZ-CRO was 1.22 with drug concentrations of 1.68 mg/liter for AMZ and 0.17 mg/liter for CRO; the best FME for the combination VAN-CRO was 1.75 with VAN at 0.57 mg/liter and CRO at 0.17 mg/liter. For PRP-2 the best FME obtained for the combination AMZ-CRO was 1.05 with drug concentrations of 11.28 mg/liter for AMZ and 0.64 mg/liter for CRO; the best FME obtained for the combination VAN The emergence of strains of Streptococcus pneumoniae highly resistant to penicillin and cephalosporins (PRP strains) (5, 10) and, often, to other classes of antimicrobial agents (33) complicates empirical treatment of pneumococcal infections. Therefore, all potential modalities for the treatment of these infections must be considered (6). Ceftriaxone or vancomycin is often one of the drugs recommended for the treatment of severe infections due to PRP such as meningitis. However, a combination of amoxicillin and ceftriaxone could also be a candidate for the treatment of such infections.In this context, our purpose was, first, to investigate the in vitro interaction between amoxicillin and ceftriaxone and between vancomycin and ceftriaxone against PRP and, second, to identify the range of concentrations that are of clinical interest.The traditional methods used to test the in vitro interactions between drugs are not very satisfactory, with difficulties in the interpretation of the results (16). All these methods come from two original models: the Loewe additivity model (24), illustrated by isobolograms, and the Bliss independence model (4), illustrated by dose-effe...

Intracellular Activity of Antibiotics in a Model of Human THP-1 Macrophages Infected by a Staphylococcus aureus Small-Colony Variant Strain Isolated from a Cystic Fibrosis Patient: Study of Antibiotic Combinations

“…The ordinate shows the value of the observed FME for each antibiotic alone (open symbols) or for the combination [FME obs(AϩB) ; shown as closed symbols in A and B and as stars in C]. Values of FME obs(AϩB) that are Ͼ1 denote a synergistic effect; values equal to 1 indicate an additive effect; values of Ͻ1 but higher than FME of the antibiotics alone (FME obs A or FME obsB ) indicate an indifferent effect (a value of Ͻ1 and below FME obsA or FME obsB would indicate an antagonistic effect, which was not observed here) (10). For oritavancin, two ranges of concentrations were examined based on the observation of a bimodal concentration-effect relationship with two successive E max values of Ϫ0.43 log CFU at low (L) and Ϫ3.13 log CFU at high (H) concentrations (see Table 2 in reference 21).…”

“…To this effect, we used the FME method (10,16), focusing on the combinations between rifampin, oritavancin, and moxifloxacin Maximal effects used for calculating concentrations were a 1.72-log and 1.32-log CFU decrease for rifampin and moxifloxacin, respectively (21). As studies with oritavancin alone revealed a bimodal dose-effect relationship (21), we considered for this case two E max values (at 0.43-and 3.13-log reduction), corresponding to the two successive plateaus observed in these experiments.…”

“…In these graphs, a synergistic effect will yield an ordinate value of Ͼ1, an additive effect yields a value of 1, an indifferent effect yields a value Ͻ1 but higher than the values of the of FME obs of the corresponding antibiotics alone, and an antagonistic effect has a value of Ͻ1 and below the FME obs of the corresponding antibiotic alone (10).…”

“…No difference was seen between samples treated with charcoal or left untreated or between samples exposed to cell lysates challenged by antibiotics or to unchallenged lysates, ruling out that any carryover effect could take place under our experimental conditions. In a second instance, selected combinations were examined in detail using the FME approach (10,16). In the present study, all measurements were made after a 24-h incubation, and the decrease in the intracellular bacterial counts (E) was used as the end point.…”

“…In the present study, antibiotic combinations were first tested at two fixed concentrations selected to provide meaningful microbiological and pharmacodynamic information. The most promising combinations were then tested using the fractional maximal effect (FME) approach (10,16), a method that allows analysis of the effects of combinations when dose-effect relationships are not linear (21) and that uses calculated rather than arbitrarily chosen concentration ratios. The study demonstrates that the combination of rifampin and oritavancin allows nearly complete eradication of an intracellular SCV strain, and it calls for a systematic assessment of antibiotic combinations in difficult-to-treat infections caused by these variants of S. aureus.…”