Role of the cephalosporinase gene in the resistance of the clinically isolated cephem-resistant Escherichia coli.

A small number of highly cephem-resistant strains was found in extensive susceptibility testing of clinical isolates of Escherichia coli to the new cephalosporin derivatives. The cephem-resistance of these clinical isolates appeared to be due to the increased cephalosporinase activities. To clarify the mechanism of the resistance, we cloned the cephalosporinase genes from two typical cephem-resistant clinical isolates as well as from an E. coli K-12 strain. The following two lines of evidence indicated that the cephem-resistance resulted from hyper production of the cephalosporinase due to the up-mutation of the regulatory sequence of the cephalosporinase gene. 1) Reciprocal exchange of the regulatory sequence including a short segment of N-terminal coding sequence and the rest of the coding sequence between the cephalosporinase genes from E. coli K-12 and the cephem-resistant clinical isolate showed that the higher cephalosporinase activity was accompanied by the regulatory sequence of the cephalosporinase gene from the clinical isolate. 2) The promoter activities of the cephalosporinase genes were determined by cloning the regulatory sequences into a promoter analysis vector. The promoter activities of the cephalosporinase genes from the clinical isolates were 23-33-fold higher than that of the cephalosporinase gene from E. coli K-12.