M.L. Gómez-Lus

Effect of clavulanic acid and/or polymorphonuclear neutrophils on amoxicillin bactericidal activity againstStreptococcus pneumoniae

The effects of polymorphonuclear neutrophils (PMNs) and/or clavulanic acid on the bactericidal activity of amoxicillin (at human serum achievable concentrations) against a serotype 3 penicillin-susceptibleStreptococcus pneumoniae strain [minimal inhibitory concentration /minimal bactericidal concentration (MIC/MBC) values of penicillin, amoxicillin, and amoxicillin/clavulanic acid (2:1)=0.01/0.01 μg/ml] and a serotype 9 penicillin-resistant strain [MIC/MBC of penicillin, amoxicillin, and amoxicillin/clavulanic acid (2:1)=1/2 μg/ml] were studied. Against the penicillin-resistant strain, subinhibitory concentrations of amoxicillin reduced the growth rate; this effect was increased by the addition of clavulanic acid. A reduction of the penicillin-resistant initial inocula (3 × 106 cfu/ml) at subinhibitory concentrations was obtained only with amoxicillin plus clavulanic acid and PMNs. At suprainhibitory concentrations, both clavulanic acid and PMNs increased the bactericidal activity of amoxicillin, as evidenced by an increased reduction in the penicillin-resistant initial inocula. The combined effect of these antibiotics and immune defenses may help explain the maintenance of their clinical efficacy in respiratory tract infections, despite the increase in the incidence of penicillin-resistant pneumococci.

Influence of the MBC/MIC ratio on the antibacterial activity of vancomycin versus linezolid against methicillin-resistant Staphylococcus aureus isolates in a pharmacodynamic model simulating serum and soft tissue interstitial fluid concentrations reported in diabetic patients

OBJECTIVES To explore serum and tissue pharmacodynamics of linezolid versus vancomycin against methicillin-resistant Staphylococcus aureus (MRSA) clinical isolates with different MBC/MIC ratios. METHODS Five strains (vancomycin MIC/MBCs, mg/L) were used: TOL-1 (2/≥64), TOL-2 (1/16), LT-1 and LT-2 (1/8) and NT (1/2). The linezolid MIC/MBC for all strains was 2/≥64 mg/L. A two-compartment dynamic computerized device was used (inocula 10(7) cfu/mL). Free concentrations obtained in serum and interstitial fluid with twice-daily regimens of 1 g of vancomycin or 600 mg of linezolid were simulated over 48 h. ABBCs (differences between control growth curves and killing curves of bacteria exposed to antibiotics; log10 cfu × h/mL) and log10 reductions in initial inocula were calculated. RESULTS In serum simulations, vancomycin (AUC0-24/MIC = 251.8 for TOL-1 and 503.6 for the remaining strains) was bacteriostatic against strains with MBC/MIC ≥8, but bactericidal against NT. In interstitial fluid simulations (AUC0-24/MIC = 54.6 for TOL-1 and 109.2 for the remaining strains), initial inocula grew in all cases. Linezolid, both in serum (AUC0-24/MIC = 87.0) and in interstitial fluid (AUC0-24/MIC = 130.6) simulations, reduced initial inocula ≥2.2 log10 for all strains (apart from LT-1 in serum simulations that showed a bacteriostatic profile). ABBCs were similar in serum and interstitial fluid with linezolid, but significantly lower in interstitial fluid simulations with vancomycin. CONCLUSIONS From the pharmacodynamic perspective (serum concentrations), vancomycin tolerance should include MBC/MIC ≥8 since strains exhibiting this ratio showed bacteriostatic profiles similar to those obtained with isolates with MBC/MIC ratios of 16 or 32. Insufficient concentrations of vancomycin at the simulated infected site were linked to bacteriological failure. Free concentrations of linezolid at the infection site pharmacodynamically covered MRSA.

Effect of polymorphonuclear neutrophils on serum bactericidal activity againstStreptococcus pneumoniae after amoxicillin administration

The effect of phagocytic killing on serum bactericidal activity againstStreptococcus pneumoniae was investigated 0, 1.5, 8 and 12 h after a single 875 mg oral dose of amoxicillin in healthy adults. Killing curves were determined with polymorphonuclear neutrophils (PMN), serum or PMN plus serum. Global killing (i. e. intracellular and extracellular killing) over 3 h of incubation was expressed as the area under the killing curve (AUKC; log cfu × h/ml). Amoxicillin did not affect the activity of PMN alone. For serum alone, the AUKC of post-administration samples (with supra-inhibitory amoxicillin concentrations) was significantly lower than in baseline samples. For serum plus PMN, significant bactericidal activity of serum was still found in samples after antibiotic concentrations had reached sub-inhibitory levels.

Opsonophagocytosis versus complement bactericidal killing as effectors following Neisseria meningitidis group C vaccination

Abstract.Background: Opsonophagocytosis and complement-mediated Neisseria meningitidis killing after vaccination were investigated. Methods: Twelve seronegative healthy volunteers received one dose of polysaccharide A/C vaccine and were followed for 3 years. Ex vivo serum killing rates with polymorphonuclear cells (PMN) and/or complement were performed at 0, 1.5, 6, 12, 18, 24, 30 and 36 months. Results: High mean total and median bactericidal antibodies were detected over time in all subjects. Considerable reduction of the initial inoculum was obtained only in the presence of complement, with or without PMN (with significant differences compared to curves without complement) a long time after vaccination. Conclusion: PMN did not increase post-vaccination bacterial killing, suggesting that antibody complement-mediated killing, and not opsonophagocytosis, is the main immune effector of the vaccine protection against N. meningitidis.

Evaluation of two in vitro pharmacodynamic simulation models: microfiltration versus centrifugation–filtration

Pharmacodynamic in vitro models that simulate serum antimicrobial concentrations provide more information about the activity of an antibiotic than MICs or traditional time-kill methods. The aim of this study was to compare two pharmacodynamic simulation models using ATCC strains of five different species and five antibiotics. In the first model (Centriprep-10 system), a filtration-centrifugation process was used to eliminate the antibiotic; in the second model (microfiltration system) no centrifugation was necessary. The antibiotic concentrations tested were similar to those in serum after normal doses of cefuroxime, clarithromycin, ciprofloxacin, gentamicin and cefotaxime. No significant differences were observed in the killing rates between the models except in the case of Haemophilus influenzae and cefotaxime. The new microfiltration model had the following advantages: lack of the carry-over effect, the absence of centrifugation that could damage bacteria and the possibility of increasing the number of incubation periods to give a better fit of the kinetic profile of man.

Evolving architectural patterns in microbial colonies development.

Semithin sections of colonies of three ATCC strains (Staphylococcus aureus, Escherichia coli, and Candida albicans) showed that their internal structure had specific patterns that evolved over the time. These patterns generally were defined by the presence of different layers composed of microorganisms with variable population densities and dead cells. The observed structures in this study could be explained as a particular form of biofilm with an air‐semisolid interface. Microsc. Res. Tech. 2010.

Increase of in vitro amoxycillin bactericidal activity by clavulanic acid against Neisseria meningitis using time-kill curves

Carriage of Neisseria meningitidis in the nasopharynx in healthy humans has been recognized since the last century. A primary factor determining the effectiveness of a drug as a meningococcal prophylactic agent is the ability to achieve bactericidal levels in saliva [1]. The time-kill method is a reliable method for evaluation of antibacterial activity, as it provides information on changes in colony counts over time and correlates better with in vivo activity [2]. In this study, killing curves investigating the bactericidal activity over 24 h of amoxycillin, clavulanic acid and amoxycillin/clavulanate (2:1) at 0.25, 0.5, 1, 4 and 10×MIC were performed against the N. meningitidis strain C11. This strain is currently used to prepare the licensed polysaccharide vaccine and is recommended by the CDC for measurement of bactericidal antibodies. The MIC of amoxycillin, clavulanic acid and amoxycillin/clavulanate (2:1) were determined by microdilution using cation-adjusted Mueller–Hinton broth and a final inoculum of 5×10 cfu/ml after incubation at 37 °C in 5% CO2 for 24 h. The modal value of three determinations was considered. Bacteria in the logarithmic phase of growth were obtained by diluting growth from a chocolate agar overnight culture in Mueller–Hinton broth supplemented with Ca and Mg and placing it in a shaking water bath until reaching the spectrophotometrically measured absorbance corresponding to 10 cfu/ml. A control without antibiotic was used. Experimental tubes were incubated at 37 °C in a shaking water bath and agitated at 110 oscillations per minute during the assay. Aliquots of 20 l taken after 1, 2, 4, 6, 8 and 24 h from the experimental tubes were plated onto a Mueller–Hinton supplemented agar for colony counting. Experiments were carried out in triplicate. Bactericidal activity was defined as the capability to reduce the initial inocula using the formula: