scholarly journals Characterization of VIM-2, a Carbapenem-Hydrolyzing Metallo-β-Lactamase and Its Plasmid- and Integron-Borne Gene from a Pseudomonas aeruginosa Clinical Isolate in France

2000 ◽  
Vol 44 (4) ◽  
pp. 891-897 ◽  
Author(s):  
Laurent Poirel ◽  
Thierry Naas ◽  
Delphine Nicolas ◽  
Louis Collet ◽  
Samuel Bellais ◽  
...  
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Amino Acid ◽  
Variable Region ◽  
Gene Cassette ◽  
Amino Acid Identity ◽  
Resistance Pattern ◽  
Relative Molecular Mass ◽  
Class 1 Integron ◽  
E Coli

ABSTRACT Pseudomonas aeruginosa COL-1 was identified in a blood culture of a 39-year-old-woman treated with imipenem in Marseilles, France, in 1996. This strain was resistant to β-lactams, including ureidopenicillins, ticarcillin-clavulanic acid, cefepime, ceftazidime, imipenem, and meropenem, but remained susceptible to the monobactam aztreonam. The carbapenem-hydrolyzing β-lactamase gene of P. aeruginosa COL-1 was cloned, sequenced, and expressed in Escherichia coli DH10B. The deduced 266-amino-acid protein was an Ambler class B β-lactamase, with amino acid identities of 32% with B-II from Bacillus cereus; 31% with IMP-1 from several gram-negative rods in Japan, includingP. aeruginosa; 27% with CcrA from Bacteroides fragilis; 24% with BlaB from Chryseobacterium meningosepticum; 24% with IND-1 from Chryseobacterium indologenes; 21% with CphA-1 from Aeromonas hydrophila; and 11% with L-1 from Stenotrophomonas maltophilia. It was most closely related to VIM-1 β-lactamase recently reported from Italian P. aeruginosa clinical isolates (90% amino acid identity). Purified VIM-2 β-lactamase had a pI of 5.6, a relative molecular mass of 29.7 kDa, and a broad substrate hydrolysis range, including penicillins, cephalosporins, cephamycins, oxacephamycins, and carbapenems, but not monobactams. As a metallo-β-lactamase, its activity was zinc dependent and inhibited by EDTA (50% inhibitory concentration, 50 μM). VIM-2 conferred a resistance pattern to β-lactams in E. coli DH10B that paralleled its in vitro hydrolytic properties, except for susceptibility to ureidopenicillins, carbapenems, and cefepime.bla VIM-2 was located on a ca. 45-kb plasmid that in addition conferred resistance to sulfamides and that was not self-transmissible either from P. aeruginosa to E. coli or from E. coli to E. coli. bla VIM-2 was the only gene cassette located within the variable region of a novel class 1 integron, In56, that was weakly related to the bla VIM-1-containing integron. VIM-2 is the second carbapenem-hydrolyzing metalloenzyme characterized from a P. aeruginosa isolate outside Japan.

scholarly journals OXA-28, an Extended-Spectrum Variant of OXA-10 β-Lactamase from Pseudomonas aeruginosa and Its Plasmid- and Integron-Located Gene

2001 ◽  
Vol 45 (2) ◽  
pp. 447-453 ◽  
Author(s):  
Laurent Poirel ◽  
Delphine Girlich ◽  
Thierry Naas ◽  
Patrice Nordmann
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Amino Acid ◽  
Variable Region ◽  
Gene Cassette ◽  
Cloning And Expression ◽  
Marginal Effect ◽  
Class 1 Integron ◽  
E Coli ◽  
Class 1 ◽  
Lactamase Gene

ABSTRACT Pseudomonas aeruginosa ED-1, isolated from a pulmonary brush of a patient hospitalized in a suburb of Paris, France, was resistant to ceftazidime and of intermediate susceptibility to ureidopenicillins and to cefotaxime. Cloning and expression of the β-lactamase gene content of this isolate in Escherichia coli DH10B identified a novel OXA-10 variant, OXA-28, with a pI value of 8.1 and a molecular mass of 29 kDa. It differed from OXA-10 by 10 amino acid changes and from OXA-13 and OXA-19 by 2 amino acid changes, including a glycine instead of tryptophan at position 164, which is likely involved in its resistance to ceftazidime. Like OXA-11, -14, -16, and -19 and as opposed to OXA-17, OXA-28 predominantly compromised ceftazidime and had only marginal effect on the MICs of aztreonam and cefotaxime in P. aeruginosa. Once expressed in E. coli, OXA-28 raised the MIC of ceftazidime to a much higher level than those of amoxicillin, cephalothin, and cefotaxime (128, 16, 8, and 4 μg/ml, respectively). OXA-28 β-lactamase had a broad spectrum of activity, including ceftazidime. Its activity was partially antagonized by clavulanic acid (50% inhibitory concentration, 10 μM) and NaCl addition. The oxa28 gene cassette was inserted in the variable region of a class 1 integron, In57, immediately downstream of an amino 6′-N-acetyltransferase gene cassette,aac(6′)Ib. The structures of the integrons carrying eitheroxa28, oxa13, or oxa19 gene cassettes were almost identical, suggesting that they may have derived from a common ancestor as a result of the common European origin of theP. aeruginosa isolates. In57 was located on a self-transferable plasmid of ca. 150 kb that was transferred fromP. aeruginosa to P. aeruginosa.

scholarly journals qnrVC-Like Gene Located in a Novel Complex Class 1 Integron Harboring the ISCR1 Element in an Aeromonas punctata Strain from an Aquatic Environment in Shandong Province, China

2010 ◽  
Vol 54 (8) ◽  
pp. 3471-3474 ◽  
Author(s):  
Ruirui Xia ◽  
Xianhu Guo ◽  
Yuzhen Zhang ◽  
Hai Xu
Keyword(s):  
Amino Acid ◽  
Gene Cassette ◽  
Multidrug Resistant ◽  
Amino Acid Identity ◽  
Complex Class ◽  
Class 1 Integron ◽  
Class 1 ◽  
Gyrase A ◽  
Ciprofloxacin Resistance ◽  
Novel Complex

ABSTRACT A qnrVC-like gene, qnrVC4, was found in a novel complex class 1 integron gene cassette array following the ISCR1 element and bla PER-1 in a multidrug-resistant strain of the aquatic bacterium Aeromonas punctata. The deduced QnrVC4 protein sequence shares 45% to 81% amino acid identity with quinolone resistance determinants QnrB6, QnrA1, QnrS1, QnrC, QnrVC1, and QnrVC3. A Ser-83 to Ile amino acid substitution in gyrase A may be mainly responsible for ciprofloxacin resistance in this strain.

scholarly journals Cloning and Characterization of blaVIM, a New Integron-Borne Metallo-β-Lactamase Gene from a Pseudomonas aeruginosa Clinical Isolate

1999 ◽  
Vol 43 (7) ◽  
pp. 1584-1590 ◽  
Author(s):  
Laura Lauretti ◽  
Maria Letizia Riccio ◽  
Annarita Mazzariol ◽  
Giuseppe Cornaglia ◽  
Gianfranco Amicosante ◽  
...  
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Clinical Isolate ◽  
Genomic Library ◽  
Plasmid Vector ◽  
Gene Cassette ◽  
University Hospital ◽  
Class 1 Integron ◽  
E Coli ◽  
Class B ◽  
Cloned Gene

ABSTRACT Production of a metallo-β-lactamase activity was detected in a carbapenem-resistant Pseudomonas aeruginosa clinical isolate (isolate VR-143/97) from an Italian inpatient at the Verona University Hospital (northern Italy). The metallo-β-lactamase determinant was isolated from a genomic library of VR-143/97, constructed in an Escherichia coli plasmid vector, by screening for clones with reduced susceptibility to imipenem. Sequencing of the cloned gene revealed that it encoded a new class B β-lactamase that was named VIM-1. At the sequence level VIM-1 was rather divergent from the other class B enzymes (16.4 to 38.7% identity), overall being more similar to members of subclass B1 including the β-lactamase II of Bacillus cereus (Bc-II), the Bacteroides fragilis CcrA, the Chryseobacterium meningosepticum BlaB, and the cassette-encoded IMP-1 enzymes. Among these, VIM-1 showed the highest degree of similarity to Bc-II. Similarly to bla IMP,bla VIM was also found to be carried on a gene cassette inserted into a class 1 integron. Thebla VIM-containing integron was located on the chromosome of P. aeruginosa VR-143/97, and the metallo-β-lactamase-encoding determinant was not transferable toE. coli by conjugation. Expression of the integron-bornebla VIM gene in E. coli resulted in a significant decrease in susceptibility to a broad array of β-lactams (ampicillin, carbenicillin, piperacillin, mezlocillin, cefotaxime, cefoxitin, ceftazidime, cefoperazone, cefepime, and carbapenems), revealing a very broad substrate specificity of the VIM-1 enzyme.

scholarly journals Characterization of Class 1 Integrons from Pseudomonas aeruginosa That Contain the blaVIM-2Carbapenem-Hydrolyzing β-Lactamase Gene and of Two Novel Aminoglycoside Resistance Gene Cassettes

2001 ◽  
Vol 45 (2) ◽  
pp. 546-552 ◽  
Author(s):  
Laurent Poirel ◽  
Thierry Lambert ◽  
Salih Türkoglü ◽  
Esthel Ronco ◽  
Jean-Louis Gaillard ◽  
...  
Keyword(s):  
Amino Acids ◽  
Pseudomonas Aeruginosa ◽  
Amino Acid ◽  
Resistance Gene ◽  
Gene Cassette ◽  
Amino Acid Sequences ◽  
Aminoglycoside Resistance ◽  
Class 1 Integron ◽  
Gene Cassettes ◽  
Class 1

ABSTRACT Two clonally unrelated Pseudomonas aeruginosa clinical strains, RON-1 and RON-2, were isolated in 1997 and 1998 from patients hospitalized in a suburb of Paris, France. Both isolates expressed the class B carbapenem-hydrolyzing β-lactamase VIM-2 previously identified in Marseilles in the French Riviera. In both isolates, thebla VIM-2 cassette was part of a class 1 integron that also encoded aminoglycoside-modifying enzymes. In one case, two novel aminoglycoside resistance gene cassettes,aacA29a and aacA29b, were located at the 5′ and 3′ end of the bla VIM-2 gene cassette, respectively. The aacA29a and aacA29b gene cassettes were fused upstream with a 101-bp part of the 5′ end of theqacE cassette. The deduced amino acid sequence AAC(6′)-29a protein shared 96% identity with AAC(6′)-29b but only 34% identity with the aacA7-encoded AAC(6′)-I1, the closest relative of the AAC(6′)-I family enzymes. These aminoglycoside acetyltransferases had amino acid sequences much shorter (131 amino acids) than the other AAC(6′)-I enzymes (144 to 153 amino acids). They conferred resistance to amikacin, isepamicin, kanamycin, and tobramycin but not to gentamicin, netilmicin, and sisomicin.

scholarly journals Oxacillinase-Mediated Resistance to Cefepime and Susceptibility to Ceftazidime in Pseudomonas aeruginosa

2001 ◽  
Vol 45 (6) ◽  
pp. 1615-1620 ◽  
Author(s):  
Daniel Aubert ◽  
Laurent Poirel ◽  
Jacqueline Chevalier ◽  
Sophie Leotard ◽  
Jean-Marie Pages ◽  
...  
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Amino Acid ◽  
Clinical Isolate ◽  
Amino Acid Identity ◽  
Class 1 Integron ◽  
Acid Identity ◽  
Resistance Phenotype ◽  
Class D ◽  
Class 1 ◽  
Efflux Mechanism

ABSTRACT Pseudomonas aeruginosa clinical isolate SOF-1 was resistant to cefepime and susceptible to ceftazidime. This resistance phenotype was explained by the expression of OXA-31, which shared 98% amino acid identity with a class D β-lactamase, OXA-1. Theoxa-31 gene was located on a ca. 300-kb nonconjugative plasmid and on a class 1 integron. No additional efflux mechanism for cefepime was detected in P. aeruginosa SOF-1. Resistance to cefepime and susceptibility to ceftazidime in P. aeruginosawere conferred by OXA-1 as well.

scholarly journals Multidrug-Resistant Pseudomonas aeruginosa Strain That Caused an Outbreak in a Neurosurgery Ward and Its aac(6′)-Iae Gene Cassette Encoding a Novel Aminoglycoside Acetyltransferase

2005 ◽  
Vol 49 (9) ◽  
pp. 3734-3742 ◽  
Author(s):  
Jun-ichiro Sekiguchi ◽  
Tsukasa Asagi ◽  
Tohru Miyoshi-Akiyama ◽  
Tomoko Fujino ◽  
Intetsu Kobayashi ◽  
...  
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Amino Acid ◽  
Resistance Gene ◽  
Gene Cassette ◽  
Multidrug Resistant ◽  
Aminoglycoside Resistance ◽  
Amino Acid Sequence Level ◽  
Class 1 Integron ◽  
Class 1 ◽  
Tract Infections

ABSTRACT We characterized multidrug-resistant Pseudomonas aeruginosa strains isolated from patients involved in an outbreak of catheter-associated urinary tract infections that occurred in a neurosurgery ward of a hospital in Sendai, Japan. Pulsed-field gel electrophoresis of SpeI-, XbaI-, or HpaI-digested genomic DNAs from the isolates revealed that clonal expansion of a P. aeruginosa strain designated IMCJ2.S1 had occurred in the ward. This strain possessed broad-spectrum resistance to aminoglycosides, β-lactams, fluoroquinolones, tetracyclines, sulfonamides, and chlorhexidine. Strain IMCJ2.S1 showed a level of resistance to some kinds of disinfectants similar to that of a control strain of P. aeruginosa, ATCC 27853. IMCJ2.S1 contained a novel class 1 integron, In113, in the chromosome but not on a plasmid. In113 contains an array of three gene cassettes of bla IMP-1, a novel aminoglycoside resistance gene, and the aadA1 gene. The aminoglycoside resistance gene, designated aac(6′)-Iae, encoded a 183-amino-acid protein that shared 57.1% identity with AAC(6′)-Iq. Recombinant AAC(6′)-Iae protein showed aminoglycoside 6′-N-acetyltransferase activity by thin-layer chromatography. Escherichia coli expressing exogenous aac(6′)-Iae showed resistance to amikacin, dibekacin, isepamicin, kanamycin, netilmicin, sisomicin, and tobramycin but not to arbekacin, gentamicins, or streptomycin. Alterations of gyrA and parC at the amino acid sequence level were detected in IMCJ2.S1, suggesting that such mutations confer the resistance to fluoroquinolones observed for this strain. These results indicate that P. aeruginosa IMCJ2.S1 has developed multidrug resistance by acquiring resistance determinants, including a novel member of the aac(6′)-I family and mutations in drug resistance genes.

scholarly journals Integron-Located oxa-32 Gene Cassette Encoding an Extended-Spectrum Variant of OXA-2 β-Lactamase from Pseudomonas aeruginosa

2002 ◽  
Vol 46 (2) ◽  
pp. 566-569 ◽  
Author(s):  
Laurent Poirel ◽  
Patrick Gerome ◽  
Christophe De Champs ◽  
Jean Stephanazzi ◽  
Thierry Naas ◽  
...  
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Amino Acid ◽  
Clinical Isolate ◽  
Amino Acid Substitution ◽  
Gene Cassette ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
Class 1 Integron ◽  
Class D ◽  
Class 1

ABSTRACT Pseudomonas aeruginosa clinical isolate CY-1, which was resistant to ceftazidime, harbored a conjugative ca. 250-kb plasmid that contained a class 1 integron with two gene cassettes encoding OXA-32, an OXA-2- type β-lactamase, and the aminoglycoside acetyltransferase AAC(6′)Ib9. OXA-32 differed from OXA-2 by an Leu169Ile amino acid substitution (class D numbering). Site-directed mutagenesis established that Ile169 is responsible for resistance to ceftazidime but not to cefotaxime.

scholarly journals Molecular and Biochemical Heterogeneity of Class B Carbapenem-Hydrolyzing β-Lactamases in Chryseobacterium meningosepticum

2000 ◽  
Vol 44 (7) ◽  
pp. 1878-1886 ◽  
Author(s):  
Samuel Bellais ◽  
Daniel Aubert ◽  
Thierry Naas ◽  
Patrice Nordmann
Keyword(s):  
Amino Acid ◽  
Broad Spectrum ◽  
Bacterial Species ◽  
Catalytic Efficiency ◽  
Amino Acid Identity ◽  
Relative Molecular Mass ◽  
Acid Identity ◽  
E Coli ◽  
Class B ◽  
Conserved Amino Acids

ABSTRACT Although the carbapenem-hydrolyzing β-lactamase (CHβL) BlaB-1 is known to be in Chryseobacterium meningosepticum NCTC 10585, a second CHβL gene, bla GOB-1, was cloned from another C. meningosepticum clinical isolate (PINT). The G+C content of bla GOB-1 (36%) indicated the likely chromosomal origin of this gene. Its expression inEscherichia coli DH10B yields a mature CHβL with a pI of 8.7 and a relative molecular mass of 28.2 kDa. In E. coli, GOB-1 conferred resistance to narrow-spectrum cephalosporins and reduced susceptibility to ureidopenicillins, broad-spectrum cephalosporins, and carbapenems. GOB-1 had a broad-spectrum hydrolysis profile including penicillins and cephalosporins (but not aztreonam). The catalytic efficiency for meropenem was higher than for imipenem. GOB-1 had low amino acid identity with the class B CHβLs, sharing 18% with the closest, L-1 from Stenotrophomonas maltophilia, and only 11% with BlaB-1. Most of the conserved amino acids that may be involved in the active site of CHβLs (His-101, Asp-103, His-162, and His-225) were identified in GOB-1. Sequence heterogeneity was found for GOB-1-like and BlaB-1-like β-lactamases, having 90 to 100% and 86 to 100% amino acid identity, respectively, among 10 unrelated C. meningosepticumisolates. Each isolate had a GOB-1-like and a BlaB-1-like gene. The same combination of GOB-1-like and BlaB-1-like β-lactamases was not found in two different isolates. C. meningosepticum is a bacterial species with two types of unrelated chromosome-borne class B CHβLs that can be expressed in E. coli and, thus, may represent a clinical threat if spread in gram-negative aerobes.

scholarly journals Biochemical-Genetic Characterization and Regulation of Expression of an ACC-1-Like Chromosome-Borne Cephalosporinase fromHafnia alvei

2000 ◽  
Vol 44 (6) ◽  
pp. 1470-1478 ◽  
Author(s):  
Delphine Girlich ◽  
Thierry Naas ◽  
Samuel Bellais ◽  
Laurent Poirel ◽  
Amal Karim ◽  
...  
Keyword(s):  
Amino Acid ◽  
Resistant Mutant ◽  
Amino Acid Identity ◽  
Relative Molecular Mass ◽  
Regulation Of Expression ◽  
Acid Identity ◽  
E Coli ◽  
Naturally Occurring ◽  
Its Gene ◽  
Biochemical Genetic

ABSTRACT A naturally occurring AmpC β-lactamase (cephalosporinase) gene was cloned from the Hafnia alvei 1 clinical isolate and expressed in Escherichia coli. The deduced AmpC β-lactamase (ACC-2) had a pI of 8 and a relative molecular mass of 37 kDa and showed 50 and 47% amino acid identity with the chromosome-encoded AmpCs from Serratia marcescens andProvidentia stuartii, respectively. It had 94% amino acid identity with the recently described plasmid-borne cephalosporinase ACC-1 from Klebsiella pneumoniae, suggesting the chromosomal origin of ACC-1. The hydrolysis constants (k cat and Km ) showed that ACC-2 was a peculiar cephalosporinase, since it significantly hydrolyzed cefpirome. Once its gene was cloned and expressed inE. coli (pDEL-1), ACC-2 conferred resistance to ceftazidime and cefotaxime but also an uncommon reduced susceptibility to cefpirome. A divergently transcribed ampR gene with an overlapping promoter compared with ampC(bla ACC-2) was identified in H. alvei 1, encoding an AmpR protein that shared 64% amino acid identity with the closest AmpR protein from P. stuartii. β-Lactamase induction experiments showed that the ampCgene was repressed in the absence of ampR and was activated when cefoxitin or imipenem was added as an inducer. From H. alvei 1 cultures that expressed an inducible-cephalosporinase phenotype, several ceftazidime- and cefpirome-cross-resistant H. alvei 1 mutants were obtained upon selection on cefpirome- or ceftazidime-containing plates, and H. alvei 1 DER, a ceftazidime-resistant mutant, stably overproduced cephalosporinase. Transformation of H. alvei 1 DER or E. coliJRG582 (ampDE mutant) harboring ampC andampR from H. alvei 1 with a recombinant plasmid containing ampD from E. coli resulted in a decrease in the MIC of β-lactam and recovery of an inducible phenotype for H. alvei 1 DER. Thus, AmpR and AmpD proteins may regulate biosynthesis of the H. alvei cephalosporinase similarly to other enterobacterial cephalosporinases.

Cloning and Characterization of a Carotenoid Cleavage Dioxygenase from Artemisia Annua L

2011 ◽  
Vol 108 ◽  
pp. 274-281
Author(s):  
Shuo Qian Liu ◽  
Na Tian ◽  
Zhong Hua Liu ◽  
Jia Nan Huang ◽  
Juan Li
Keyword(s):  
Amino Acid ◽  
Artemisia Annua ◽  
Amino Acid Identity ◽  
Carotenoid Cleavage Dioxygenase ◽  
E Coli ◽  
Acid Protein ◽  
Rapid Amplification ◽  
First Time

In order to discover the formation mechanism of carotenoid derived aroma, which has been wildly used on protection of crop against insect attacks, the full-length cDNA of an Artemisia annua carotenoid cleavage dioxygenase (AaCCD1) was cloned by rapid amplification of cDNA ends. The function of AaCCD1 was characterized by expression of AaCCD1 in a strain of E. coli accumulating carotenoids and enzyme assay in vitro. The completed open read frame of AaCCD1 was 1629 bp and it encoded a 542-amino acid protein with a 77% amino acid identity to Arabidopsis thaliana CCD1, a predicted molecular mass of 61.04 kDa and a pI of 5.8. AaCCD1 efficiently cleaves carotenoids and regulate the formation of terpenoid compounds. This is the first time to report the cloning and identification of carotenoid cleavage dioxygenase from Atemisia annua, which will play a great role on understanding the regulation of volatile compounds.

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