ECL-1, a novel plasmid-mediated class A b-lactamase with carbenicillinase characteristics from Escherichia coli
Abstract number: 1733_359
Papagiannitsis C., Loli A., Tzouvelekis L.S., Tzelepi E., Arlet G., Miriagou V.
Objectives: Characterisation of a novel class A b-lactamase (ECL-1) encoded by an 80-kb self-transferable plasmid from Escherichia coli.
Methods:E. coli EC-3521r was isolated in 2002 from a urine sample of a patient treated in a general hospital. MICs of b-lactam antibiotics were determined by agar dilution. Isoelectric points of the produced b-lactamases were determined by IEF of cell extracts. Hydrolysis rates of b-lactams and inhibitory activity of clavulanic acid, tazobactam, and sulbactam, were determined by UV spectrophotometry. Bla-genes were identified by PCR. Mating experiments were performed in mixed broth cultures. Bla gene encoding fragments were cloned into the pBCSK(+) vector and the nucleotide sequences of inserts were determined.
Results: By PCR screening, the E. coli EC-3521r isolate was found to be positive for blaACC and blaTEM. Analysis of the b-lactamase content by IEF, indicated production of three main b-lactamase species with isoelectric points (pIs) of 7.8 corresponding to ACC-1, 5.4 (TEM-1) and 5.8. Identities of the former two enzymes were confirmed by sequencing of PCR products. Beta-lactam resistance was transferred by an 80-kb plasmid (pR3521) that encoded all three b-lactamases.
A cloned fragment of pR3521 encoded production of the b-lactamase with a pI of 5.8 and mediated resistance to penicillins but not cephalosporins. The respective recombinant plasmid carried a 3,833-bp Sau3A fragment containing an ORF of 867-bp homologous to the orf1 observed in ACC-1-encoding plasmids. This orf did not exhibit significant homology with any known sequence. However, the deduced polypeptide (288aa) possessed the typical motifs of a class A b-lactamase and was designated blaECL-1. ECL-1 exhibited 51% amino acid sequence identity with the chromosomal carbenicillinases of the RTG. The enzyme was effective against ampicillin and carbenicillin while the relative hydrolysis rates of oxacillin, cephalothin and cephaloridine were low. Tazobactam was the most potent inhibitor of ECL-1 followed by clavulanic acid and sulbactam.
Conclusion: The ECL-1 is a novel class A b-lactamase of unknown origin, that is functionally and phylogenetically related to the RTG subgroup of the CARB b-lactamases.
|Session name:||European Society of Clinical Microbiology and Infectious Diseases|
|Location:||ICC, Munich, Germany|
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