Synergism between  -Lactam Antibiotics: A Test of Theoretical Predictions Made with Staphylococcus Aureus

Abstract

S Y NERGI s M between p-lactam antibiotics against Gram-negative bacteria is a well-recognised phenomenon (Hamilton-Miller, Smith and Knox, 1964;Sabath and Abraham, 1964;Sutherland and Batchelor, 1964), but little attention has been paid in this respect to Gram-positive bacteria. This may be because the effect is difficult to demonstrate because of the generally unfavourable kinetic characteristics of p-lactamases from Gram-positive organisms, and there seems to be little practical application for it. Kinetic considerations of a model of this type of synergism produced a means whereby the extent of synergism could be predicted (Hamilton-Miller, 1971a), and it is the purpose of the present paper to test practically the validity of the earlier theoretical analysis. In order to do this, it was decided to use a system involving synergism between cephalothin and various penicillins against Staphylococcus aureus; use of this system was suggested by the earlier findings (Hamilton-Miller, 1966 and1970) that cephalothin acted as a competitive inhibitor of staphylococcal /3-lactamase under both rigidly controlled (enzyme assay) and physiological (broth culture) conditions. METHODS AND MATERIALSThe model. The naturally occurring lag phase in a culture of p-lactamase-producing organisms will be increased in the presence of a hydrolysable penicillin the initial concentration of which (SO) is too high to allow growth to occur. The extent of this increase VmaX. = maximum rate of hydrolysis of penicillin by penicillinase; and S, = concentration of penicillin at which the organisms can start to grow. The parameter S, represents the intrinsic resistance of the strain (see Hamilton-Miller, 1965), and will be referred to hereafter as the maximum non-inhibitory concentration (MNIC).The lag phase of the culture will be further prolonged if a competitive inhibitor of penicillinase is present, as the rate of penicillin destruction will be thereby decreased. This prolongation of lag phase (At) can be expressed (Hamilton-Miller, 1971a) by where i = concentration of competitive inhibitor and Kt = dissociation constant of penicillinase-inhibitor complex.

beta-Lactamases in laboratory and clinical resistance

“…The extracellular ␤-lactamases of gram-positive species serve to reduce the external drug concentration (S 0 ), with growth commencing only once this falls below a toxic threshold (S ϩ ) that reflects PBP sensitivity. The period for this to be achieved (t) can be predicted, with reasonable accuracy (79,82), from the integrated Michaelis-Menten equation:…”

β-Lactamases: quantity and resistance

“…The classic case is Staphylococcus uureus: as more 0-lactamase-positive staphylococci are added to an M I C test, they can destroy a greater amount of substrate in a given time (see equation 2) and so the MIC is raised. Benzylpenicifin is a very good substrate MIC data from Borifiglio and Livermore [28]; kinetic data from Hamilton-Miller [3] and Hamilton-Miller and Ramsay [4]. MIC, minirnuin inhibitory concentration; n, number of organisms; V,,,,,,, niaxirnuni rate of antibiotic hydrolysis; KM, Michaelis constant (substrate concentration that allows the enzyme to function at 50% of for staphylococcal penicillinase [34] and its MICs for p-lactamase producers increase almost 10-fold as the inoculum is raised from lo4 to lo6 (Table 2), whereas there is no analogous increase in MICs for p-lactamasenegative isolates [28].…”

“…Then, and only then, does bacterial growth begin. The period (t) required to achieve this reduction in drug concentration is predictable with good accuracy [3,4] from an integrated form of the Michaelis-Menten equation:…”

Use of the Biophotometer in the Evaluation of Antibacterial Compounds

“…As we have shown before [4] valuable information can be obtained by observing what happens when various concentrations of an antibiotic act upon single bacterial strains, producing 'families' of growth curves. A mathematical description of each curve can be made by evaluating the param eters described above.…”