NEW SYSTEM BASED ON SITE-DIRECTED MUTAGENESIS FOR HIGHLY ACCURATE COMPARISON OF RESISTANCE LEVELS CONFERRED BY SHV BETA-LACTAMASES

We developed a system based on site-directed mutagenesis that allows a precise comparison of SHV enzymes under isogenic conditions, In addit ion, the influences of two different, naturally occurring promoters we re examined for each SHV derivative. The system comprised two separate ly cloned DNA fragments, each the size of 3.6 kb, Both fragments encod ed an SHV gene originating from clinical isolates but with different p romoters. The structural genes were made identical by site-directed mu tagenesis. Other mutations were then introduced into both fragments by means of site-directed mutagenesis, resulting in the SHV derivatives SHV-1, SHV-2, SHV-2a, SHV-3, and SHV-5, The amino acid exchange of glu tamic acid at position 235 for lysine in SHV-5 resulted in the highest resistance levels, SHV-3, differing from SHV-2 by the exchange of arg inine at position 201 for leucine and previously described as indistin guishable from SHV-2, was shown to cause slightly higher resistance to ceftazidime and lower resistance to ceftriaxone, cefotaxime, and cefe pime than SHV-2. The point mutation in SHV-2a, with the leucine-to-glu tamine replacement at the unusual position 31, previously considered a lmost insignificant, proved to increase resistance to ceftazidime but reduced the MICs of all other cephalosporins tested when compared with those for SHV-2, For all clones harboring SHV derivatives, resistance was increased by a stronger promoter, in some cases masking the effec t of the point mutation itself and demonstrating the importance of reg ulatory mechanisms of resistance.