A spontaneous point mutation i the aac(6′)-lb′ gene results in altered substrate specificity of aminoglycoside 6′-N-acetyltransferase of a Pseudomonas fluorescens strain

Lambert, T

doi:10.1016/0378-1097(94)90030-2

Cited by 28 publications

(43 citation statements)

References 9 publications

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“…The aac(6Ј)-Ib-encoded enzyme, a 6Ј-Nacetylaminoglycoside acetyltransferase type I [AAC(6Ј)-I], was characterized by one amino acid substitution (L119S). This substitution was responsible for the susceptibility of G and the resistance of T, N, and A (24).…”

Section: Discussionmentioning

confidence: 97%

Successive Emergence of Extended-Spectrum β-Lactamase-Producing and Carbapenemase-Producing Enterobacter aerogenes Isolates in a University Hospital

et al. 2008

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Sixty-two clinical isolates of Enterobacter aerogenes resistant to expanded-spectrum cephalosporins were collected between July 2003 and May 2005. Among these isolates, 23 (37.1%) were imipenem (IPM) susceptible, and 39 (62.9%) were IPM insusceptible, of which 89.7% (35/39) were resistant and 10.3% (4/39) were intermediate. Isolate genotypes were compared by pulsed-field gel electrophoresis. Of 62 isolates, 48 belonged to epidemic pulsotype A (77.4%). This pulsotype included 37.5% and 58.4% of ␤-lactam phenotypes b and a, respectively. Nine isolates (14.5%) belonged to pulsotype E, which included 22.3% and 77.7% of phenotypes b and a, respectively. The ␤-lactamases with pIs of 5.4, 6.5, 8.2, and 8.2 corresponded to extended-spectrum ␤-lactamases (ESBLs) TEM-20, TEM-24, SHV-5, and SHV-12, respectively. Of 39 IPM-insusceptible E. aerogenes isolates, 26 (66.6%) were determined to be metallo-␤-lactamase producers, by using a phenotypic method. Of these isolates, 24 harbored a bla IMP-1 gene encoding a protein with a pI of >9.5, and two carried the bla VIM-2 gene encoding a protein with a pI of 5.3, corresponding to ␤-lactamases IMP-1 and VIM-2, respectively. The remaining 13 (33.4%) isolates were negative for the bla IMP-1 and bla VIM-2 genes but showed an alteration of their outer membrane proteins (OMPs). Ten of these isolates produced the two possible OMPs (32 and 42 kDa), with IPM MICs between 8 and 32 g/ml, and three others produced only a 32-kDa OMP with IPM MICs >32 g/ml. This work demonstrates that, in addition to resistance to expanded-spectrum cephalosporins, IPM resistance can occur in ESBL-producing E. aerogenes isolates by carbapenemase production or by the loss of porin in the outer membrane.Enterobacter aerogenes has recently emerged as an important hospital pathogen (13). The prevalence of this bacterial species has increased considerably since the introduction of extendedspectrum cephalosporins into clinical practice (18). Various resistance mechanisms have been described in this species, such as extended-spectrum ␤-lactamases (ESBLs), plasmidmediated production (21), and hyperproduction of the Bush group 1 chromosomally mediated cephalosporinases (32). Although these enzymes are specifically characteristic of some Enterobacter spp., the appearance of similar plasmid-mediated ␤-lactamases in Klebsiella pneumoniae and Escherichia coli raises concerns about the spread of resistance (21, 32). For both ESBL-producing and AmpC-producing isolates, carbapenems are the only ␤-lactam agents active against both resistance mechanisms. Data from previous studies have shown that both ESBL and AmpC ␤-lactamase producers had reduced susceptibility to imipenem (IPM) due to either carbapenemhydrolyzing enzymes (carbapenemase) (30), decreased membrane permeability due to loss of porin in the outer membrane (14, 39), or active efflux (27).The IPM resistance of ESBL-producing E. aerogenes strains was observed for the first time at Amiens University Hospital (A.U.H.) in 2002. To determine whether the strains were epidem...

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Section: Discussionmentioning

confidence: 97%

Successive Emergence of Extended-Spectrum β-Lactamase-Producing and Carbapenemase-Producing Enterobacter aerogenes Isolates in a University Hospital

et al. 2008

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“…specificities of the respective enzymes (Hall, 1981 ;Doten & Mortlock, 1984;Buchholz et al, 1988 ;Srinivasan et al, 1993;Lambert et al, 1994). Finally, cryptic genes may be expressed (Hall et al, 1983) which often results in the constitutive formation of the encoded enzyme (Szymona & Doudoroff, 1960;Rode & Gifiorn, 1982.…”

Section: -H S C H N E I D E R a N D O T H E R Smentioning

confidence: 99%

Enzyme evolution in Rhodobacter sphaeroides: selection of a mutant expressing a new galactitol dehydrogenase and biochemical characterization of the enzyme

et al. 1995

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A gain of function mutant of Rhodobacter sphaeroides Si4, capable of growing on galactitol, was isolated from a chemostat culture. Continuous cultivation was performed for 54 d with a limiting concentration (1 mM) of the substrate D-glucitol and an excess (20 mM) of the non-metabolizable galactitol. The mutant strain, R. sphaeroides D, grew in galactitol minimal medium with a growth rate of 0-11 h-1 (t d = 6-3 h). In crude extracts of R. sphaeroides D, a specific galactitol dehydrogenase (GDH) activity of 380 mU mg-1 was found, while the wild-type strain exhibited GDH activities lower than 50 mU mg-1 when grown on different polyols. Unlike mannitol, sorbitol or ribitol dehydrogenase from the wild-type strain, the new GDH was expressed constitutively. To study whether it was a newly evolved enzyme or an improved side activity of one of the pre-existing polyol dehydrogenases, GDH was purified to apparent homogeneity by ammonium sulfate precipitation and chromatography on Phenyl-Sepharose, Q-Sepharose, Matrex Gel Red-A and Mono-Q. The relative molecular mass (M r) of the native GDH was 110000. SDS-PAGE resulted in one single band that represented a polypeptide with a M r of 28000, indicating that the native protein is a tetramer. The isoelectric point of GDH was determined to be pH 4-2. The enzyme was specific for NAD+ but catalysed the oxidation of different sugar alcohols as well as different diols and secondary alcohols. The apparent K m values were: galactitol, 240 mM; D-threitol, 85 mM; 1,2-hexandiol, 0-2 mM; NAD+, 12μM; L-erythrulose, 144 mM; acetoin, 62 mM; dihydroxyacetone, 48 mM; and NADH, 4μM. GDH activity was strictly dependent on the presence of divalent cations. The properties of GDH are different to any of the three polyol dehydrogenases from R. sphaeroides Si4. In addition, comparison of the N-terminal amino acid sequence of the isolated GDH with the N-terminal sequence of the other three polyol dehydrogenases clearly demonstrates that GDH is an additional enzyme, so far unrecognized in the wild-type strain.

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“…MexXY-OprA of PA7 is a 'hybrid' system in that the PA7-specific oprA gene that is absent from the other known P. (Mugnier et al, 1998). This amino acid change has been associated with a shift from amikacin to gentamicin resistance in vitro after site-directed mutagenesis (Rather et al., 1992) and in various clinical isolates (Lambert et al, 1994;Mugnier et al, 1998;Casin et al, 2003;Poole, 2005). A recombinant Escherichia coli strain carrying AAC(69)-Ib 9 is strongly resistant to tobramycin and gentamicin and moderately (or slightly) resistant to amikacin (Rather et al, 1992;Casin et al, 2003).…”

Section: Introductionmentioning

confidence: 99%

Primary mechanisms mediating aminoglycoside resistance in the multidrug-resistant Pseudomonas aeruginosa clinical isolate PA7

2012

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The multiresistant taxonomic outlier Pseudomonas aeruginosa PA7 possesses the conserved efflux genes, mexXY; however these are linked to a unique gene encoding an outer membrane channel, dubbed oprA, that is absent in most P. aeruginosa strains. Using genetic knockouts and single copy chromosomal complementation, we showed that aminoglycoside resistance in PA7 is mediated in part by the MexXY-OprA pump, and intriguingly that MexXY in this strain can utilize either the OprA or OprM outer membrane channel, linked to the mexAB efflux genes. We also identified a small portion of the oprA gene immediately downstream of the mexY gene in PAO1, suggesting that non-PA7 P. aeruginosa strains might have possessed, but lost, the intact mexXYoprA efflux pump locus. Consistent with this, most of a panel of serotype strains possessed the truncated oprA but the serotype O12 isolate had an intact mexXY-oprA locus, similar to PA7 and the related strain DSM 1128. We also showed that the mexZ repressor gene upstream of mexXYoprA in PA7 is mutated, leading to overexpression of mexXY-oprA, using sequencing, homologous replacement and real-time quantitative reverse transcriptase PCR. Finally we assessed the contribution of MexXY and aminoglycoside modifying enzymes AAC together to resistance in PA7 and the AAC(69)-Iae-mediated amikacin-resistant clinical isolate IMCJ2.S1, concluding that the effect of the modifying enzymes is enhanced by functional efflux, especially in the presence of divalent cations, to develop high-level aminoglycoside resistance in P. aeruginosa. INTRODUCTIONPseudomonas aeruginosa is a common nosocomial pathogen that causes a broad range of infections with a high mortality rate (Mahar et al., 2010; Iida et al., 2010;Lambert et al., 2011). A major factor in its prominence as a pathogen is, in part, its intrinsic resistance to a number of antibacterial agents (Poole et al., 1993;Hancock, 1998;Poole, 2002) and, particularly, the development of increased multidrug resistance in healthcare settings (Giamarellos-Bourboulis et al., 2006;Kirikae et al., 2008;Kallen et al., 2010;Keen et al., 2010). This organism readily acquires resistance via chromosomal mutations (e.g. overexpression of the efflux pump) and lateral gene transfer (e.g. acquisition of metallob-lactamase) (Lister et al., 2009;Poole, 2011). The emergence and spread of multidrug-, extensive drug-and pan-drug-resistant P. aeruginosa infections are very serious and of great concern, as few agents are effective against these organisms. In addition, antibiotic-resistant Gram-negative bacteria, including P. aeruginosa, increase the hospital costs and length of stay associated with healthcare-associated infections (Mauldin et al., 2010).Aminoglycosides such as amikacin, gentamicin and tobramycin are a vital component of antipseudomonal chemotherapy for a variety of infections, particularly pulmonary infections in cystic fibrosis (CF) patients (Poole, 2005(Poole, , 2011. Aminoglycoside resistance in P. aeruginosa has often arisen via acquired aminoglycoside-modifyi...

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A spontaneous point mutation i the aac(6′)-lb′ gene results in altered substrate specificity of aminoglycoside 6′-N-acetyltransferase of a Pseudomonas fluorescens strain

Cited by 28 publications

References 9 publications

Successive Emergence of Extended-Spectrum β-Lactamase-Producing and Carbapenemase-Producing Enterobacter aerogenes Isolates in a University Hospital

Successive Emergence of Extended-Spectrum β-Lactamase-Producing and Carbapenemase-Producing Enterobacter aerogenes Isolates in a University Hospital

Enzyme evolution in Rhodobacter sphaeroides: selection of a mutant expressing a new galactitol dehydrogenase and biochemical characterization of the enzyme

Primary mechanisms mediating aminoglycoside resistance in the multidrug-resistant Pseudomonas aeruginosa clinical isolate PA7

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