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Detection of the high-level aminoglycoside resistance gene aph(2")-Ib in Enterococcus faecium.

ABSTRACT: A new high-level gentamicin resistance gene, designated aph(2")-Ib, was cloned from Enterococcus faecium SF11770. The deduced amino acid sequence of the 897-bp open reading frame of aph(2")-Ib shares homology with the aminoglycoside-modifying enzymes AAC(6')-APH(2"), APH(2")-Ic, and APH(2")-Id. The observed phosphotransferase activity is designated APH(2")-Ib.

SUBMITTER: Kao SJ

PROVIDER: S-EPMC90169 | biostudies-literature | 2000 Oct

REPOSITORIES: biostudies-literature

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Detection of the high-level aminoglycoside resistance gene aph(2")-Ib in Enterococcus faecium.

Kao S J SJ You I I Clewell D B DB Donabedian S M SM Zervos M J MJ Petrin J J Shaw K J KJ Chow J W JW

Antimicrobial agents and chemotherapy 20001001 10

A new high-level gentamicin resistance gene, designated aph(2")-Ib, was cloned from Enterococcus faecium SF11770. The deduced amino acid sequence of the 897-bp open reading frame of aph(2")-Ib shares homology with the aminoglycoside-modifying enzymes AAC(6')-APH(2"), APH(2")-Ic, and APH(2")-Id. The observed phosphotransferase activity is designated APH(2")-Ib. ...[more]

PMID: 10991878

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Aminoglycoside resistance genes aph(2")-Ib and aac(6')-Im detected together in strains of both Escherichia coli and Enterococcus faecium.

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A new high-level gentamicin resistance gene, aph(2'')-Id, in Enterococcus spp.

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High Prevalence and Predominance of the aph(2?)-If Gene Conferring Aminoglycoside Resistance in Campylobacter.

Project description:Campylobacter is a major foodborne pathogen, and previous studies revealed that Campylobacter isolates from food-producing animals are increasingly resistant to gentamicin in China. The molecular epidemiology and genetic mechanisms responsible for gentamicin resistance in China have not been well understood. In this study, 607 Campylobacter isolates of chicken and swine origins collected in 2014 were analyzed, revealing that 15.6% (25/160) of the Campylobacter jejuni isolates and 79.9% (357/447) of the Campylobacter coli isolates were resistant to gentamicin. PCR detection of the gentamicin resistance genes indicated that aph(2?)-If was more prevalent than the previously identified aacA/aphD gene and has become the dominant gentamicin resistance determinant in Campylobacter Transformation and whole-genome sequencing as well as long-range PCR discovered that aph(2?)-If was located on a chromosomal segment inserted between two conserved genes, Cj0299 and panB Cloning of aph(2?)-If into gentamicin-susceptible C. jejuni NCTC 11168 confirmed its function in conferring high-level resistance to gentamicin and kanamycin. Molecular typing by pulsed-field gel electrophoresis suggested that both regional expansion of a particular clone and horizontal transmission were involved in the dissemination of the aph(2?)-If gene in Campylobacter To our knowledge, this is the first report describing the high prevalence of a chromosomally encoded aph(2?)-If gene in Campylobacter The high prevalence and predominance of this gene might be driven by the use of aminoglycoside antibiotics in food animal production in China and potentially compromise the usefulness of gentamicin as a therapeutic agent for Campylobacter-associated systemic infection.

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The crystal structures of substrate and nucleotide complexes of Enterococcus faecium aminoglycoside-2''-phosphotransferase-IIa [APH(2'')-IIa] provide insights into substrate selectivity in the APH(2'') subfamily.

Project description:Aminoglycoside-2''-phosphotransferase-IIa [APH(2'')-IIa] is one of a number of homologous bacterial enzymes responsible for the deactivation of the aminoglycoside family of antibiotics and is thus a major component in bacterial resistance to these compounds. APH(2'')-IIa produces resistance to several clinically important aminoglycosides (including kanamycin and gentamicin) in both gram-positive and gram-negative bacteria, most notably in Enterococcus species. We have determined the structures of two complexes of APH(2'')-IIa, the binary gentamicin complex and a ternary complex containing adenosine-5'-(beta,gamma-methylene)triphosphate (AMPPCP) and streptomycin. This is the first crystal structure of a member of the APH(2'') family of aminoglycoside phosphotransferases. The structure of the gentamicin-APH(2'')-IIa complex was solved by multiwavelength anomalous diffraction methods from a single selenomethionine-substituted crystal and was refined to a crystallographic R factor of 0.210 (R(free), 0.271) at a resolution of 2.5 A. The structure of the AMPPCP-streptomycin complex was solved by molecular replacement using the gentamicin-APH(2'')-IIa complex as the starting model. The enzyme has a two-domain structure with the substrate binding site located in a cleft in the C-terminal domain. Gentamicin binding is facilitated by a number of conserved acidic residues lining the binding cleft, with the A and B rings of the substrate forming the majority of the interactions. The inhibitor streptomycin, although binding in the same pocket as gentamicin, is orientated such that no potential phosphorylation sites are adjacent to the catalytic aspartate residue. The binding of gentamicin and streptomycin provides structural insights into the substrate selectivity of the APH(2'') subfamily of aminoglycoside phosphotransferases, specifically, the selectivity between the 4,6-disubstituted and the 4,5-disubstituted aminoglycosides.

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High level aminoglycoside resistance and distribution of aminoglycoside resistant genes among clinical isolates of Enterococcus species in Chennai, India.

Project description:Enterococci are nosocomial pathogen with multiple-drug resistance by intrinsic and extrinsic mechanisms. Aminoglycosides along with cell wall inhibitors are given clinically for treating enterococcal infections. 178 enterococcal isolates were analyzed in this study. E. faecalis is identified to be the predominant Enterococcus species, along with E. faecium, E. avium, E. hirae, E. durans, E. dispar and E. gallinarum. High level aminoglycoside resistance (HLAR) by MIC for gentamicin (GM), streptomycin (SM) and both (GM + SM) antibiotics was found to be 42.7%, 29.8%, and 21.9%, respectively. Detection of aminoglycoside modifying enzyme encoding genes (AME) in enterococci was identified by multiplex PCR for aac(6')-Ie-aph(2'')-Ia; aph(2'')-Ib; aph(2'')-Ic; aph(2'')-Id and aph(3')-IIIa genes. 38.2% isolates carried aac(6')-Ie-aph(2'')-Ia gene and 40.4% isolates carried aph(3')-IIIa gene. aph(2'')-Ib; aph(2'')-Ic; aph(2'')-Id were not detected among our study isolates. aac(6')-Ie-aph(2'')-Ia and aph(3')-IIIa genes were also observed in HLAR E. durans, E. avium, E. hirae, and E. gallinarum isolates. This indicates that high level aminoglycoside resistance genes are widely disseminated among isolates of enterococci from Chennai.

| S-EPMC3932257 | biostudies-literature

Modification of penicillin-binding protein 5 associated with high-level ampicillin resistance in Enterococcus faecium.

Project description:High-level ampicillin resistance in Enterococcus faecium has been shown to be associated with the synthesis of a modified penicillin-binding protein 5 (PBP 5) which had apparently lost its penicillin-binding capability (R. Fontana, M. Aldegheri, M. Ligozzi, H. Lopez, A. Sucari, and G. Satta. Antimicrob. Agents Chemother. 38:1980-1983, 1994). The pbp5 gene of the highly resistant strain E. faecium 9439 was cloned and sequenced. The deduced amino acid sequence showed 77 and 54% homologies with the PBPs 5 of Enterococcus hirae and Enterococcus faecalis, respectively. A gene fragment coding for the C-terminal part of PBP 5 containing the penicillin-binding domain was also cloned from several E. faecium strains with different levels of ampicillin resistance. Sequence comparison revealed a few point mutations, some of which resulted in amino acid substitutions between SDN and KTG motifs in PBPs 5 of highly resistant strains. One of these converted a polar residue (the T residue at position 562 or 574) of PBP 5 produced by susceptible and moderately resistant strains into a nonpolar one (A or I). This alteration could be responsible for the altered phenotype of PBP 5 in highly resistant strains.

| S-EPMC163115 | biostudies-other

vanM, a new glycopeptide resistance gene cluster found in Enterococcus faecium.

Project description:Since glycopeptide-resistant enterococci (GRE) were reported in 1988, they have appeared in hospitals worldwide. Seven van gene cluster types (vanA, vanB, vanC, vanD, vanE, vanG, and vanL) are currently known. We investigated a clinical strain of Enterococcus faecium Efm-HS0661 that was isolated in 2006 from an inpatient with intra-abdominal infection in Shanghai. It was resistant to most antimicrobials, including vancomycin (MIC, >256 ?g/ml) and teicoplanin (MIC, 96 ?g/ml). Glycopeptide resistance could be transferred to E. faecium BM4105RF by conjugation. The donor and its transconjugant were negative by PCR for the known van genes. By cloning and primer walk sequencing, we discovered a novel van gene cluster, designated vanM. The vanM ligase gene was 1,032-bp in length and encoded a 343-amino-acid protein that shared 79.9, 70.8, 66.3, and 78.8% amino acid identity with VanA, VanB, VanD, and VanF, respectively. Although the vanM DNA sequence was closest to vanA, the organization of the vanM gene cluster was most similar to that of vanD. Upstream from the vanM cluster was an IS1216-like element, which may play a role in the dissemination of this resistance determinant. Liquid chromatography-mass spectrometry analysis of peptidoglycan precursors extracted from the VanM-type strain Efm-HS0661 treated with vancomycin or teicoplanin revealed a modified precursor (UDP-N-acetylmuramic acid [MurNAc]-tetrapeptide-D-Lac), indicating that VanM, like VanA, confers glycopeptide resistance by the inducible synthesis of precursor ending in D-Ala-D-Lac.

| S-EPMC2976141 | biostudies-literature

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