CARBAPENEM RESISTANCE IN A CLINICAL ISOLATE OF CITROBACTER-FREUNDII

Carbapenem resistance was studied in two sets of Citrobacter freundii strains: (i) strain CFr950, resistant to imipenem (MIC, 16 mu g/ml) an d isolated in vivo during imipenem therapy, and strain CFr950-Rev, the spontaneous, imipenem-susceptible revertant of CFr950 selected in vit ro, and (ii) strains CFr801 and CFr802, two imipenem-resistant mutants selected in vitro from the susceptible clinical isolate CFr800. In al l strains, whether they were imipenem-susceptible or -resistant strain s, production of the cephalosporinase was derepressed and their K-m va lues for cephaloridine were in the range of 128 to 199 mu M. No carbap enemase activity was detected in vitro. The role of cephalosporinase o verproduction in the resistance was demonstrated after introduction of the ampD gene which decreased the level of production of cephalospori nase at least 250-fold and resulted in an 8- to 64-fold decrease in th e MICs of the carbapenems. The role of reduced permeability in the res istance was suggested by the absence, in CFr950 and CFr802, of two out er membrane proteins (the 42- and 40-kDa putative porins whose levels were considerably decreased in CFr801) and the reappearance of the 42- kDa protein in imipenem-susceptible strain CFr950-Rev. This role was c onfirmed after introduction of the ompF gene of Escherichia coli into the CFr strains, which resulted in 8- to 16-fold decreases In the MICs of carbapenems for CFr802 and CFr950. We infer from these results tha t the association of reduced, porin-mediated permeability with high-le vel cephalosporinase production, observed previously in other gram-neg ative bacteria, may also confer carbapenem resistance on C. freundii.