Loss of penicillin tolerance by inactivating the carbon catabolite repression determinant CcpA in Streptococcus gordonii

Objectives: Antibiotic tolerance is a phenomenon allowing bacteria to withstand drug-induced killing. Here, we studied a penicillin-tolerant mutant of Streptococcus gordonil (Toll), which was shown to be deregulated in the expression of the arginine deiminase operon (arc). arc was not directly responsible for tolerance, but is controlled by the global regulator CcpA. Therefore, we sought whether CcpA might be implicated in tolerance. Methods: The ccpA gene was characterized and subsequently inactivated by PCR ligation mutagenesis in both the susceptible wild-type (WT) and Toll. The minimal inhibitory concentration and time-kill curves for the strains were determined and the outcome of penicillin treatment in experimental endocarditis assessed. Results: ccpA sequence and expression were similar between the WT and Toll strains. In killing assays, the WT lost 3.5 +/- 0.6 and 5.3 +/- 0.6 log(10) cfu/mL and Toll lost 0.4 +/- 0.2 and 1.4 +/- 0.9 log(10) cfu/mL after 24 and 48 h of penicillin exposure, respectively. Deletion of ccpA almost totally restored Toll kill susceptibility (loss of 2.5 +/- 0.7 and 4.9 +/- 0.7 log(10) cfu/mL at the same endpoints). In experimental endocarditis, penicillin treatment induced a significant reduction in vegetation bacterial densities between Toll (4.1 log(10) cfu/g) and Toll Delta ccpA (2.4 log(10) cfu/g). Restitution of ccpA re-established the tolerant phenotype both in vitro and in vivo. Conclusions: CcpA, a global regulator of the carbon catabolite repression system, is implicated in penicillin tolerance both in vitro and in vivo. This links antibiotic survival to bacterial sugar metabolism. However, since ccpA sequence and expression were similar between the WT and Toll strains, other factors are probably involved in tolerance.