Carbapenem resistance in a clinical isolate of Citrobacter freundii

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Abstract
Carbapenem resistance was studied in two sets of Citrobacter freundii strains: (i) strain CFr950, resistant to imipenem (MIC, 16 microg/ml) and isolated in vivo during imipenem therapy, and strain CFr950-Rev, the spontaneous, imipenem-susceptible revertant of CFr950 selected in vitro, and (ii) strains CFr801 and CFr802, two imipenem-resistant mutants selected in vitro from the susceptible clinical isolate CFr800. In all strains, whether they were imipenem-susceptible or -resistant strains, production of the cephalosporinase was derepressed and their Km values for cephaloridine were in the range of 128 to 199 microM. No carbapenemase activity was detected in vitro. The role of cephalosporinase overproduction in the resistance was demonstrated after introduction of the ampD gene which decreased the level of production of cephalosporinase at least 250-fold and resulted in an 8- to 64-fold decrease in the MICs of the carbapenems. The role of reduced permeability in the resistance was suggested by the absence, in CFr950 and CFr802, of two outer 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 confirmed 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 that the association of reduced, porin-mediated permeability with high-level cephalosporinase production, observed previously in other gram-negative bacteria, may also confer carbapenem resistance on C. freundii.

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