MOLECULAR CHARACTERIZATION BY PCR-RESTRICTION FRAGMENT LENGTH POLYMORPHISM OF TEM BETA-LACTAMASES

To rapidly characterise TEM-derived extended-spectrum beta-lactamases a fast and easy method using polymerase chain reaction-restriction fra gment length polymorphism was developed. This method was validated wit h ten reference TEM-type extended-spectrum beta-lactamases. The mutati ons involved in TEM-20 and TEM-21, which were previously reported only with biochemical analysis, were then characterised. TEM-20 differed f rom TEM-19 by a silent mutation at position 925 (A for G), and TEM-21 differed from TEM-3 and TEM-14 by a single mutation (G for A) in an un reported position 660, involving an amino acid substitution, arginine for histidine, at position 153. Moreover, a new extended-spectrum beta -lactamase conferring low resistance to ceftazidime (TEM-29), was desc ribed. TEM-29 derived from TEM-1, with an amino acid substitution, his -164. Finally, the combination of polymerase chain reaction-restrictio n fragment length polymorphism and plasmid analysis allowed us to inve stigate nosocomial outbreaks due to clinical isolates of multi-resista nt Klebsiella pneumoniae in three hospitals.