Identification of bla(IMP-22) in Pseudomonas spp. in urban wastewater and nosocomial environments: biochemical characterization of a new IMP metallo-enzyme variant and its genetic location.
OBJECTIVE
The aim of the study was the biochemical characterization of a new variant of the metallo-beta-lactamase, IMP-22. Moreover, the genetic environment of the bla(IMP-22) gene was investigated in Pseudomonas fluorescens and Pseudomonas aeruginosa collected from urban wastewater and a teaching hospital in L'Aquila, Italy.


METHODS
Molecular characterization of genetic elements was carried out by PCR and DNA sequencing methods. The new enzyme was purified from recombinant Escherichia coli BL21(DE)Rosetta/pBC-SK/IMP-22. Steady-state kinetic parameters (K(m) and V(max)) were determined for a large pattern of substrates.


RESULTS
A new IMP metallo-beta-lactamase gene was found in a class 1 integron and in one case, in a plasmid of Pseudomonas spp. The bla(IMP-22) encodes for a pre-protein of 246 amino acids and the N-terminus of the mature beta-lactamase (NH(2)-PDLK) was also determined. The molecular mass and pI were 24 930 Da and 6.2, respectively. On the basis of the kinetic parameters calculated (K(m) and V(max)), IMP-22 was found to hydrolyse narrow- and extended-spectrum beta-lactams. Enzyme activity was found to be inhibited by metal chelators such as EDTA, 1,10-o-phenathroline and dipicolinic acid with an IC(50) of 800, 750 and 300 microM, respectively.


CONCLUSIONS
The finding of the bla(IMP-22) gene in P. fluorescens environmental strains and P. aeruginosa clinical isolate suggests the ongoing spread of bla(MBL) genes in several bacterial species and in different environments.
Pellegrini
Cristina
C
Dipartimento di Scienze e Tecnologie Biomediche, University of L'Aquila, Italy.
Mercuri
Paola Sandra
PS
Celenza
Giuseppe
G
Galleni
Moreno
M
Segatore
Bernardetta
B
Sacchetti
Elisa
E
Volpe
Roberto
R
Amicosante
Gianfranco
G
Perilli
Mariagrazia
M
D000595:Amino Acid Sequence; D000900:Anti-Bacterial Agents / Q000378:metabolism*; D001483:Base Sequence; D003428:Cross Infection / Q000382:microbiology; D004269:DNA, Bacterial / Q000737:chemistry / Q000235:genetics; D004791:Enzyme Inhibitors / Q000494:pharmacology; D023061:Gene Order; D006801:Humans; D020128:Inhibitory Concentration 50; D007526:Isoelectric Point; D007558:Italy; D007700:Kinetics; D008969:Molecular Sequence Data; D008970:Molecular Weight; D016133:Polymerase Chain Reaction; D011552:Pseudomonas Infections / Q000382:microbiology*; D011550:Pseudomonas aeruginosa / Q000201:enzymology* / Q000302:isolation & purification; D011551:Pseudomonas fluorescens / Q000201:enzymology* / Q000302:isolation & purification; D017422:Sequence Analysis, DNA; D014871:Water Microbiology; D001618:beta-Lactamases / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D047090:beta-Lactams / Q000378:metabolism*
D000900:Anti-Bacterial Agents; D004269:DNA, Bacterial; D004791:Enzyme Inhibitors; D047090:beta-Lactams; D001618:beta-Lactamases