scholarly journals Genetic and Biochemical Characterization of OXA-63, a New Class D β-Lactamase from Brachyspira pilosicoli BM4442

2008 ◽  
Vol 52 (4) ◽  
pp. 1264-1268 ◽  
Author(s):  
Djalal Meziane-Cherif ◽  
Thierry Lambert ◽  
Marine Dupêchez ◽  
Patrice Courvalin ◽  
Marc Galimand
Keyword(s):  
Escherichia Coli ◽  
Clavulanic Acid ◽  
Inhibitory Concentration ◽  
Biochemical Characterization ◽  
Fusobacterium Nucleatum ◽  
Cloning And Sequencing ◽  
Brachyspira Pilosicoli ◽  
New Class ◽  
Class D

ABSTRACT Brachyspira pilosicoli BM4442, isolated from the feces of a patient with diarrhea at the Hospital Saint-Michel in Paris, was resistant to oxacillin (MIC > 256 μg/ml) but remained susceptible to cephalosporins and to the combination of amoxicillin and clavulanic acid. Cloning and sequencing of the corresponding resistance determinant revealed a coding sequence of 807 bp encoding a new class D β-lactamase named OXA-63. The bla OXA-63 gene was chromosomally located and not part of a transposon or of an integron. OXA-63 shared 54% identity with FUS-1 (OXA-85), an oxacillinase from Fusobacterium nucleatum subsp. polymorphum, and 25 to 44% identity with other class D β-lactamases (DBLs) and contained all the conserved structural motifs of DBLs. Escherichia coli carrying the bla OXA-63 gene exhibited resistance to benzylpenicillin and amoxicillin but remained susceptible to amoxicillin in combination with clavulanic acid. Mature OXA-63 consisted of a 31.5-kDa polypeptide and appeared to be dimeric. Kinetic analysis revealed that OXA-63 exhibited a narrow substrate profile, hydrolyzing oxacillin, benzylpenicillin, and ampicillin with catalytic efficiencies of 980, 250, and 150 mM−1 s−1, respectively. The enzyme did not apparently interact with oxyimino-cephalosporins, imipenem, or aztreonam. Unlike FUS-1 and other DBLs, OXA-63 was strongly inhibited by clavulanic acid (50% inhibitory concentration [IC50] of 2 μM) and tazobactam (IC50 of 0.16 μM) and exhibited low susceptibility to NaCl (IC50 of >2 M). OXA-63 is the first DBL described for the anaerobic spirochete B. pilosicoli.

scholarly journals Genetic and Biochemical Characterization of FUS-1 (OXA-85), a Narrow-Spectrum Class D β-Lactamase from Fusobacterium nucleatum subsp. polymorphum

2006 ◽  
Vol 50 (8) ◽  
pp. 2673-2679 ◽  
Author(s):  
Christine Voha ◽  
Jean-Denis Docquier ◽  
Gian Maria Rossolini ◽  
Thierry Fosse
Keyword(s):  
Biochemical Characterization ◽  
Fusobacterium Nucleatum ◽  
Cloning And Sequencing ◽  
Gene Encoding ◽  
Brachyspira Pilosicoli ◽  
Reading Frame ◽  
New Class ◽  
Class D ◽  
Lactamase Gene

ABSTRACT Previous studies have reported β-lactamase-mediated penicillin resistance in Fusobacterium nucleatum, but no β-lactamase gene has yet been identified in this species. An F. nucleatum subsp. polymorphum strain resistant to penicillin and amoxicillin was isolated from a human periodontitis sample. DNA cloning and sequencing revealed a 765-bp open reading frame encoding a new class D β-lactamase named FUS-1 (OXA-85). A recombinant Escherichia coli strain carrying the bla FUS-1 gene exhibited resistance to amoxicillin with a moderate decrease in the MICs with clavulanic acid. The bla FUS-1 gene was found in two additional clonally unrelated F. nucleatum subsp. polymorphum isolates. It was located on the chromosome in a peculiar genetic environment where a gene encoding a putative transposase-like protein is found, suggesting a possible acquisition of this class D β-lactamase gene. The FUS-1 enzyme showed the closest ancestral relationship with OXA-63 from Brachyspira pilosicoli (53% identity) and with putative chromosomal β-lactamases of Campylobacter spp. (40 to 42% identity). FUS-1 presents all of the conserved structural motifs of class D β-lactamases. Kinetic analysis revealed that FUS-1 exhibits a narrow substrate profile, efficiently hydrolyzing benzylpenicillin and oxacillin. FUS-1 was poorly inactivated by clavulanate and NaCl. FUS-1 is the first example of a class D β-lactamase produced by a gram-negative, anaerobic, rod-shaped bacterium to be characterized.

scholarly journals Genetic and Biochemical Characterization of CAD-1, a Chromosomally Encoded New Class A Penicillinase from Carnobacterium divergens

2007 ◽  
Vol 52 (2) ◽  
pp. 551-556 ◽  
Author(s):  
Djalal Meziane-Cherif ◽  
Dominique Decré ◽  
E. Arne Høiby ◽  
Patrice Courvalin ◽  
Bruno Périchon
Keyword(s):  
Clavulanic Acid ◽  
Mobile Genetic Element ◽  
Inhibitory Concentration ◽  
Biochemical Characterization ◽  
Cloning And Sequencing ◽  
New Class ◽  
Class A ◽  
Amoxicillin Clavulanic Acid ◽  
Carnobacterium Divergens

ABSTRACT Carnobacterium divergens clinical isolates BM4489 and BM4490 were resistant to penicillins but remained susceptible to combinations of amoxicillin-clavulanic acid and piperacillin-tazobactam. Cloning and sequencing of the responsible determinant from BM4489 revealed a coding sequence of 912 bp encoding a class A β-lactamase named CAD-1. The bla CAD-1 gene was assigned to a chromosomal location in the two strains that had distinct pulsed-field gel electrophoresis patterns. CAD-1 shared 53% and 42% identity with β-lactamases from Bacillus cereus and Staphylococcus aureus, respectively. Alignment of CAD-1 with other class A β-lactamases indicated the presence of 25 out of the 26 isofunctional amino acids in class A β-lactamases. Escherichia coli harboring bla CAD-1 exhibited resistance to penams (benzylpenicillin and amoxicillin) and remained susceptible to amoxicillin in combination with clavulanic acid. Mature CAD-1 consisted of a 34.4-kDa polypeptide. Kinetic analysis indicated that CAD-1 exhibited a narrow substrate profile, hydrolyzing benzylpenicillin, ampicillin, and piperacillin with catalytic efficiencies of 6,600, 3,200, and 2,900 mM−1 s−1, respectively. The enzyme did not interact with oxyiminocephalosporins, imipenem, or aztreonam. CAD-1 was inhibited by tazobactam (50% inhibitory concentration [IC50] = 0.27 μM), clavulanic acid (IC50 = 4.7 μM), and sulbactam (IC50 = 43.5 μM). The bla CAD-1 gene is likely to have been acquired by BM4489 and BM4490 as part of a mobile genetic element, since it was not found in the susceptible type strain CIP 101029 and was adjacent to a gene for a resolvase.

scholarly journals Biochemical Characterization of SFC-1, a Class A Carbapenem-Hydrolyzing β-Lactamase

2007 ◽  
Vol 51 (12) ◽  
pp. 4512-4514 ◽  
Author(s):  
Fátima Fonseca ◽  
Ana Cristina Sarmento ◽  
Isabel Henriques ◽  
Bart Samyn ◽  
Jozef van Beeumen ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Sodium Dodecyl Sulfate ◽  
Molecular Mass ◽  
Clavulanic Acid ◽  
Gel Electrophoresis ◽  
Biochemical Characterization ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Class A

ABSTRACT The carbapenem-hydrolyzing β-lactamase SFC-1 from Serratia fonticola UTAD54 was overexpressed in Escherichia coli, purified, and characterized. The enzyme exhibited an apparent molecular mass of 30.5 kDa, determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. SFC-1 hydrolyzes penicillins, cephalosporins, aztreonam, and carbapenems and is inhibited by clavulanic acid, sulbactam, and tazobactam.

scholarly journals Genetic and Biochemical Characterization of a Chromosome-Encoded Carbapenem-Hydrolyzing Ambler Class D β-Lactamase from Shewanella algae

2004 ◽  
Vol 48 (5) ◽  
pp. 1670-1675 ◽  
Author(s):  
Claire Héritier ◽  
Laurent Poirel ◽  
Patrice Nordmann
Keyword(s):  
Escherichia Coli ◽  
Clinical Isolate ◽  
Biochemical Characterization ◽  
Resistance Phenotype ◽  
Narrow Spectrum ◽  
Class D ◽  
Shewanella Algae ◽  
Lactamase Gene

ABSTRACT A chromosome-encoded β-lactamase gene from Shewanella algae clinical isolate KB-1 was cloned and expressed in Escherichia coli. It encoded the Ambler class D enzyme OXA-55, sharing less than 55% identity with any other oxacillinases. Although conferring a narrow-spectrum β-lactam resistance phenotype, OXA-55 had carbapenem-hydrolyzing activity that mirrored the reduced susceptibility to imipenem observed in S. algae KB-1. Very similar oxacillinases were found in other S. algae isolates.

Purification and biochemical characterization of a recombinant Anopheles gambiae tryptophan 2,3-dioxygenase expressed in Escherichia coli

2008 ◽  
Vol 38 (9) ◽  
pp. 871-876 ◽  
Author(s):  
Alessandra Paglino ◽  
Fabrizio Lombardo ◽  
Bruno Arcà ◽  
Menico Rizzi ◽  
Franca Rossi
Keyword(s):  
Escherichia Coli ◽  
Anopheles Gambiae ◽  
Biochemical Characterization

scholarly journals Molecular and Biochemical Characterization of the xlnD-Encoded 3-Hydroxybenzoate 6-Hydroxylase Involved in the Degradation of 2,5-Xylenol via the Gentisate Pathway in Pseudomonas alcaligenes NCIMB 9867

2005 ◽  
Vol 187 (22) ◽  
pp. 7696-7702 ◽  
Author(s):  
Xiaoli Gao ◽  
Chew Ling Tan ◽  
Chew Chieng Yeo ◽  
Chit Laa Poh
Keyword(s):  
Escherichia Coli ◽  
Fusion Protein ◽  
Molecular Mass ◽  
Electron Donor ◽  
Biochemical Characterization ◽  
Aromatic Substrates ◽  
Gentisate Pathway ◽  
A Subunit ◽  
Pseudomonas Alcaligenes

ABSTRACT The xlnD gene from Pseudomonas alcaligenes NCIMB 9867 (strain P25X) was shown to encode 3-hydroxybenzoate 6-hydroxylase I, the enzyme that catalyzes the NADH-dependent conversion of 3-hydroxybenzoate to gentisate. Active recombinant XlnD was purified as a hexahistidine fusion protein from Escherichia coli, had an estimated molecular mass of 130 kDa, and is probably a trimeric protein with a subunit mass of 43 kDa. This is in contrast to the monomeric nature of the few 3-hydroxybenzoate 6-hydroxylases that have been characterized thus far. Like other 3-hydroxybenzoate 6-hydroxylases, XlnD could utilize either NADH or NADPH as the electron donor. P25X harbors a second 3-hydroxybenzoate 6-hydroxylase II that was strictly inducible by specific aromatic substrates. However, the degradation of 2,5-xylenol and 3,5-xylenol in strain P25X was found to be dependent on the xlnD-encoded 6-hydroxylase I and not the second, strictly inducible 6-hydroxylase II.

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