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 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 FRI-1, a Carbapenem-Hydrolyzing Class A β-Lactamase from Enterobacter cloacae

2015 ◽  
Vol 59 (12) ◽  
pp. 7420-7425 ◽  
Author(s):  
Laurent Dortet ◽  
Laurent Poirel ◽  
Samia Abbas ◽  
Saoussen Oueslati ◽  
Patrice Nordmann
Keyword(s):  
Biochemical Characterization ◽  
Enterobacter Cloacae ◽  
Conjugative Plasmid ◽  
Lower Sensitivity ◽  
Gene Encoding ◽  
Content Type ◽  
Class A ◽  
Encoding Gene ◽  
Lactamase Gene

ABSTRACTAnEnterobacter cloacaeisolate was recovered from a rectal swab from a patient hospitalized in France with previous travel to Switzerland. It was resistant to penicillins, narrow- and broad-spectrum cephalosporins, aztreonam, and carbapenems but remained susceptible to expanded-spectrum cephalosporins. Whereas PCR-based identification of the most common carbapenemase genes failed, the biochemical Carba NP test II identified an Ambler class A carbapenemase. Cloning experiments followed by sequencing identified a gene encoding a totally novel class A carbapenemase, FRI-1, sharing 51 to 55% amino acid sequence identity with the closest carbapenemase sequences. However, it shared conserved residues as a source of carbapenemase activity. Purified β-lactamase FRI-1 hydrolyzed penicillins, aztreonam, and carbapenems but spared expanded-spectrum cephalosporins. The 50% inhibitory concentrations (IC50s) of clavulanic acid and tazobactam were 10-fold higher than those found forKlebsiella pneumoniaecarbapenemase (KPC), IMI, and SME, leading to lower sensitivity of FRI-1 activity to β-lactamase inhibitors. TheblaFRI-1gene was located on a ca. 110-kb untypeable, transferable, and non-self-conjugative plasmid. A putative LysR family regulator-encoding gene at the 5′ end of the β-lactamase gene was identified, leading to inducible expression of theblaFRI-1gene.

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 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.

scholarly journals Cloning, Nucleotide Sequence, and Expression of the Gene Encoding the Bacteriocin Boticin B from Clostridium botulinum Strain 213B

2000 ◽  
Vol 66 (12) ◽  
pp. 5480-5483 ◽  
Author(s):  
Sean S. Dineen ◽  
Marite Bradshaw ◽  
Eric A. Johnson
Keyword(s):  
Amino Acids ◽  
Nucleotide Sequence ◽  
Clostridium Botulinum ◽  
Shuttle Vector ◽  
Open Reading Frame ◽  
Gene Encoding ◽  
Reading Frame ◽  
Heat Stable ◽  
Group I

ABSTRACT Boticin B is a heat-stable bacteriocin produced byClostridium botulinum strain 213B that has inhibitory activity against various strains of C. botulinum and related clostridia. The gene encoding the bacteriocin was localized to a 3.0-kb HindIII fragment of an 18.8-kb plasmid, cloned, and sequenced. DNA sequencing revealed the boticin B structural gene,btcB, to be an open reading frame encoding 50 amino acids. A C. botulinum strain 62A transconjugant containing theHindIII fragment inserted into a clostridial shuttle vector expressed boticin B, although at much lower levels than those observed in C. botulinum 213B. To our knowledge, this is the first demonstration and characterization of a bacteriocin from toxigenic group I C. botulinum.

scholarly journals Physical and Biochemical Characterization of the qacA Gene Encoding Antiseptic and Disinfectant Resistance in Staphylococcus aureus

Microbiology ◽  
1989 ◽  
Vol 135 (1) ◽  
pp. 1-10 ◽  
Author(s):  
J. M. TENNENT ◽  
B. R. LYON ◽  
M. MIDGLEY ◽  
G. JONES ◽  
A. S. PUREWAL ◽  
...  
Keyword(s):  
Staphylococcus Aureus ◽  
Biochemical Characterization ◽  
Gene Encoding ◽  
Disinfectant Resistance

Sequence of a Rhodococcus gene cluster encoding the subunits of ethanolamine ammonia-lyase and an APC-like permease

1994 ◽  
Vol 40 (5) ◽  
pp. 403-407 ◽  
Author(s):  
René De Mot ◽  
Istvan Nagy ◽  
Geert Schoofs ◽  
Jos Vanderleyden
Keyword(s):  
Open Reading Frame ◽  
Genomic Fragment ◽  
Positive Bacterium ◽  
Gram Positive ◽  
Gene Encoding ◽  
Reading Frame ◽  
Transporter Family ◽  
Rhodococcus Species ◽  
Rhodococcus Sp

Sequence analysis of a 5173-bp genomic fragment from the nocardioform actinomycete Rhodococcus sp. strain NI86/21 revealed the presence of two genes, eutB and eutC, encoding the putative homologues of the large and small subunits of the ethanolamine ammonia-lyase, respectively, from Salmonella typhimurium. This is the first report of the characterization of these genes in a Gram-positive species. Immediately upstream of eutB, a gene encoding a putative permease of the APC (amino acids, polyamines, choline) transporter family was located. At present, no other Gram-positive members of this permease family are known. The translational coupling of these eut genes suggests an operon-like organization of the ethanolamine genes in Rhodococcus species. A truncated open reading frame downstream of eutC contained an N-terminal motif characteristic of membrane-anchored lipoproteins.Key words: nocardioform actinomycete, cobalamin, APC transporter, membrane-anchored lipoprotein, Gram-positive bacterium.

scholarly journals Genetic Context and Biochemical Characterization of the IMP-18 Metallo-β-Lactamase Identified in aPseudomonas aeruginosaIsolate from the United States

2010 ◽  
Vol 55 (1) ◽  
pp. 140-145 ◽  
Author(s):  
Luisa Borgianni ◽  
Silvia Prandi ◽  
Laurie Salden ◽  
Gisela Santella ◽  
Nancy D. Hanson ◽  
...  
Keyword(s):  
United States ◽  
Biochemical Characterization ◽  
Gene Cassette ◽  
The United States ◽  
Turnover Rates ◽  
Reading Frame ◽  
Class 1 ◽  
Original Array ◽  
Genetic Context

ABSTRACTThe production of metallo-β-lactamase (MBL) is an important mechanism of resistance to β-lactam antibiotics, including carbapenems. Despite the discovery and emergence of many acquired metallo-β-lactamases, IMP-type determinants (now counting at least 27 variants) remain the most prevalent in some geographical areas. In Asian countries, and notably Japan, IMP-1 and its closely related variants are most widespread. Some other variants have been detected in other countries and show either an endemic (e.g., IMP-13 in Italy) or sporadic (e.g., IMP-12 in Italy or IMP-18 in the United States) occurrence. The IMP-18-producingPseudomonas aeruginosastrain PS 297 from the southwestern United States carried at least two class 1 integrons. One was identical to In51, while the other, named In133and carrying theblaIMP-18gene cassette in the third position, showed an original array of five gene cassettes, includingaacA7,qacF,aadA1, and an unknown open reading frame (ORF). Interestingly. In133differed significantly from In96, theblaIMP-18-carrying integron identified in aP. aeruginosaisolate from Mexico. The meropenem and ertapenem MIC values were much lower forEscherichia colistrains producing IMP-18 (0.06 and 0.12 μg/ml, respectively) than for strains producing IMP-1 (2 μg/ml for each). Kinetic data obtained with the purified enzyme revealed lower turnover rates of IMP-18 than of other IMP-type enzymes with most substrates.

Genetic and biochemical characterization of an exopolygalacturonase and a pectate lyase fromYersinia enterocolitica

1999 ◽  
Vol 45 (5) ◽  
pp. 396-403 ◽  
Author(s):  
Ching-Hsing Liao ◽  
Larry Revear ◽  
Arland Hotchkiss ◽  
Brett Savary
Keyword(s):  
High Performance ◽  
Biochemical Characterization ◽  
Amperometric Detection ◽  
Pectate Lyase ◽  
Anion Exchange Chromatography ◽  
Exchange Chromatography ◽  
Human Pathogen ◽  
Gene Encoding ◽  
Reading Frame ◽  
Chromatography Analysis

Yersinia enterocolitica, an invasive foodborne human pathogen, degrades polypectate by producing two depolymerizing enzymes, pectate lyase (PL) and polygalacturonase (PG). The gene encoding the PG activity, designated pehY, was located in a 3-kb genomic fragment of Y. enterocolitica ATCC 49397. The complete nucleotide sequence of this 3-kb fragment was determined and an open reading frame consisting of 1803 bp was predicted to encode a PG protein with an estimated Mrof 66 kDa and pI of 6.3. The amino acid sequence of prePG showed 59 and 43% identity to that of the exopolygalacturonase (exoPG) of Erwinia chrysanthemi and Ralstonia solanacearum, respectively. The Y. enterocolitica PG overproduced in Escherichia coli was purified to near homogeneity using perfusion cation exchange chromatography. Analysis of the PG depolymerization products by high performance anion-exchange chromatography and pulsed amperometric detection (HPAEC-PAD) revealed the exolytic nature of this enzyme. The Y. enterocolitica PL overproduced in E. coli was also partially purified and the Mrand pI were estimated to be 55 kDa and 5.2, respectively. HPAEC-PAD analysis of the PL depolymerization products indicated the endolytic nature of this enzyme. Southern hybridization analyses revealed that pehY and pel genes of Y. enterocolitica are possibly encoded in the chromosome rather than in the plasmid. Purified exopolygalacturonase (over 10 activity units) was unable to macerate plant tissues.Key words: pectinase activities, human pathogen, HPLC analysis, pehY gene.

scholarly journals Genetic and Biochemical Characterization of CGB-1, an Ambler Class B Carbapenem-Hydrolyzing β-Lactamase from Chryseobacterium gleum

2002 ◽  
Vol 46 (9) ◽  
pp. 2791-2796 ◽  
Author(s):  
Samuel Bellais ◽  
Thierry Naas ◽  
Patrice Nordmann
Keyword(s):  
Reference Strain ◽  
Biochemical Characterization ◽  
Amino Acid Identity ◽  
Relative Molecular Mass ◽  
Class A ◽  
Molecular Subclass ◽  
Class B ◽  
Lactamase Gene ◽  
Chryseobacterium Gleum

ABSTRACT Chryseobacterium gleum (previously included in the Flavobacterium IIb species) is a gram-negative aerobe that is a source of nosocomial infections. An Ambler class B β-lactamase gene was cloned and expressed in Escherichia coli from reference strain C. gleum CIP 103039 that had reduced susceptibility to expanded-spectrum cephalosporins and carbapenems. The purified β-lactamase, CGB-1, with a pI value of 8.6 and a determined relative molecular mass of ca. 26 kDa, hydrolyzed penicillins; narrow- and expanded-spectrum cephalosporins; and carbapenems. CGB-1 was a novel member of the molecular subclass B1 of metallo-enzymes. It had 83 and 42% amino acid identity with IND-1 from Chryseobacterium indologenes and BlaB from C. meningosepticum, respectively. Thus, in addition to the previously characterized clavulanic acid-inhibited extended-spectrum β-lactamase CGA-1 of Ambler class A, C. gleum produces a very likely chromosome-borne class B β-lactamase.

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