Effects of CO2 and pH on inhibition of TEM-1 and other beta-lactamases by penicillanic acid sulfones

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Abstract
Incubation in 5% CO2 reduced the inhibition zones of piperacillin-tazobactam (75/10 micrograms) disks for Escherichia coli strains with TEM-1, TEM-2, and SHV-1 beta-lactamases. Similarly, MICs of piperacillin-tazobactam and other penicillin-sulfone combinations for TEM producers were up to 500-fold higher at pH 6.5 than at pH 8.0. This effect was greatest for organisms with high levels of enzyme activity. CO2 and mild acidity did not affect the susceptibility of beta-lactamase-negative strains to penicillin-sulfone combinations, and the effects of these conditions were variable for organisms with beta-lactamases other than TEM-1, TEM-2, and SHV-1. These last observations discounted acid-mediated inactivation of piperacillin or tazobactam. MICs of amoxicillin or piperacillin alone or with clavulanate for TEM and SHV producers were affected only less than or equal to 16-fold by 5% CO2 or acidity, indicating that the greater effects seen with the penicillin-sulfone combinations depended on the behavior of the sulfones and not on that of the penicillins. This pH effect was studied in detail for TEM-1 enzyme. Inhibition of this enzyme by sulfones but not clavulanate varied grossly with pH, with 50% inhibitory concentrations of tazobactam and sulbactam up to 300-fold higher at pH 6.5 than at 8.0. By contrast, the hydrolytic activity of TEM-1 enzyme for substrates and its level of production varied threefold or less between pH 6.5 and pH 8.0. Increased inhibition at pH 8.0 reflected sequestration of the enzyme into a secondary noncovalent complex rather than increased irreversible inactivation.