Genetic and Biochemical Characterization of the Chromosomal Class A β-Lactamases of Raoultella (formerly Klebsiella ) planticola and Raoultella ornithinolytica

Abstract

ABSTRACT Enterobacterial strains of Raoultella spp. display a penicillinase-related β-lactam resistance pattern suggesting the presence of a chromosomal bla gene. From whole-cell DNA of Raoultella planticola strain ATCC 33531 T and Raoultella ornithinolytica strain ATCC 31898 T , bla genes were cloned and expressed into Escherichia coli . Each gene encoded an Ambler class A β-lactamase, named PLA-1 and ORN-1 for R. planticola and R. ornithinolytica , respectively. These β-lactamases (291 amino acids), with the same pI value of 7.8, had a shared amino acid identity of 94%, 37 to 47% identity with the majority of the chromosome-encoded class A β-lactamases previously described for Enterobacteriaceae , and 66 to 69% identity with the two β-lactamases LEN-1 and SHV-1 from Klebsiella pneumoniae . However, the highest identity percentage (69 to 71%) was found with the plasmid-mediated β-lactamase TEM-1. PLA-1, which displayed very strong hydrolytic activity against penicillins, also displayed significant hydrolytic activity against cefepime and, to a lesser extent, against cefotaxime and aztreonam, but there was no hydrolytic activity against ceftazidime. Such a substrate profile suggests that the Raoultella β-lactamases PLA-1 and ORN-1 should be classified into the group 2be of the β-lactamase classification of K. Bush, G. A. Jacoby, and A. A. Medeiros (Antimicrob. Agents Chemother. 39: 1211-1233, 1995). The highly homologous regions upstream of the bla PLA-1A and bla ORN-1A genes comprised a nucleotide sequence identical to the −35 region and another one very close to the −10 region of the bla LEN-1 gene. From now on, as the bla gene sequences of the most frequent Raoultella and Klebsiella species are available, the bla gene amplification method can be used to differentiate these species from each other, which the biochemical tests currently carried out in the clinical laboratory are unable to do.