Characterization of a novel extended-spectrum beta-lactamase from Pseudomonas aeruginosa.
A clinical isolate of Pseudomonas aeruginosa RNL-1 showed resistance to extended-spectrum cephalosporins which was inhibited by clavulanic acid. Although this strain contained three plasmids ca. 80, 20, and 4 kb long, the resistance could not be transferred by mating-out assays with P. aeruginosa or Escherichia coli. Cloning of a 2.1-kb Sau3A fragment from P. aeruginosa RNL-1 into plasmid pACYC184 produced pPZ1, a recombinant plasmid that encodes a beta-lactamase. This beta-lactamase (PER-1) had a relative molecular mass of 29 kDa and a pI of 5.4 and was biosynthesized by P. aeruginosa RNL-1 along with a likely cephalosporinase with a pI of 8.7. PER-1 showed a broad substrate profile by hydrolyzing benzylpenicillin, amoxicillin, ticarcillin cephalothin, cefoperazone, cefuroxime, HR 221, ceftriaxone, ceftazidime, and (moderately) aztreonam but not oxacillin, imipenem, or cephamycins. Vmax values for extended-spectrum cephalosporins were uncommonly high, and the affinity of the enzyme for most compounds was relatively low (i.e., high Km). PER-1 activity was inhibited by clavulanic acid, sulbactam, imipenem, and cephamycins but not by EDTA. A 1.1-kb SnaBI fragment from pPZ1 failed to hybridize with plasmids that encode TEM-, SHV-, OXA-, or CARB/PSE-type beta-lactamase or with the ampC gene of P. aeruginosa. However, the same probe appeared to hybridize with chromosomal but not plasmid DNA from P. aeruginosa RNL-1. This study reports the properties of a novel extended-spectrum beta-lactamase in P. aeruginosa which may not be derived by point mutations from previously known enzymes of this species.
Laboratoire de Microbiologie, Hôpital Raymond Poincaré, Faculté de Médecine Paris-Ouest, Garches, France.
P
P
Nordmann
E
E
Ronco
T
T
Naas
C
C
Duport
Y
Y
Michel-Briand
R
R
Labia
D003001:Cloning, Molecular; D004269:DNA, Bacterial / Q000302:isolation & purification; D004352:Drug Resistance, Microbial; D005798:Genes, Bacterial; D007525:Isoelectric Focusing; D008970:Molecular Weight; D010957:Plasmids; D011552:Pseudomonas Infections / Q000382:microbiology; D011550:Pseudomonas aeruginosa / Q000201:enzymology* / Q000235:genetics; D012689:Sequence Homology, Nucleic Acid; D013045:Species Specificity; D001618:beta-Lactamases / Q000737:chemistry* / Q000235:genetics
D004269:DNA, Bacterial; D001618:beta-Lactamases