Loss of penicillin tolerance by inactivating the carbon catabolite repression determinant CcpA in Streptococcus gordonii.
OBJECTIVE
Antibiotic tolerance is a phenomenon allowing bacteria to withstand drug-induced killing. Here, we studied a penicillin-tolerant mutant of Streptococcus gordonii (Tol1), 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 Tol1. 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 Tol1 strains. In killing assays, the WT lost 3.5 +/- 0.6 and 5.3 +/- 0.6 log(10) cfu/mL and Tol1 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 Tol1 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 Tol1 (4.1 log(10) cfu/g) and Tol1DeltaccpA (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 Tol1 strains, other factors are probably involved in tolerance.
Bizzini
A
A
Faculty of Biology and Medicine, Department of Fundamental Microbiology, Biophore Building, University of Lausanne, 1015 Lausanne, Switzerland.
Entenza
J M
JM
Moreillon
P
P
D000818:Animals; D000900:Anti-Bacterial Agents / Q000627:therapeutic use; D001426:Bacterial Proteins / Q000235:genetics*; D001483:Base Sequence; D015139:Blotting, Southern; D002244:Carbon / Q000378:metabolism*; D017931:DNA Primers; D004269:DNA, Bacterial / Q000235:genetics; D004268:DNA-Binding Proteins / Q000235:genetics*; D004697:Endocarditis, Bacterial / Q000188:drug therapy / Q000382:microbiology; D005260:Female; D005798:Genes, Bacterial / Q000235:genetics*; D008826:Microbial Sensitivity Tests; D008969:Molecular Sequence Data; D010403:Penicillin Resistance / Q000235:genetics*; D010406:Penicillins / Q000627:therapeutic use; D010641:Phenotype; D010957:Plasmids / Q000235:genetics; D051381:Rats; D017208:Rats, Wistar; D012097:Repressor Proteins / Q000235:genetics*; D013290:Streptococcal Infections / Q000188:drug therapy / Q000382:microbiology; D013291:Streptococcus / Q000235:genetics* / Q000254:growth & development; D014169:Transformation, Bacterial
D000900:Anti-Bacterial Agents; D001426:Bacterial Proteins; D017931:DNA Primers; D004269:DNA, Bacterial; D004268:DNA-Binding Proteins; D010406:Penicillins; D012097:Repressor Proteins; C069286:catabolite control proteins, bacteria; D002244:Carbon