scholarly journals Detection of a Streptomycin/Spectinomycin Adenylyltransferase Gene (aadA) in Enterococcus faecalis

1999 ◽  
Vol 43 (1) ◽  
pp. 157-160 ◽  
Author(s):  
Nancye C. Clark ◽  
Ørjan Olsvik ◽  
Jana M. Swenson ◽  
Carol A. Spiegel ◽  
Fred C. Tenover
Keyword(s):  
Enterococcus Faecalis ◽  
Pcr Primers ◽  
Binding Assays ◽  
Aminoglycoside Resistance ◽  
Cycle Sequencing ◽  
E Coli ◽  
Genes Encoding ◽  
Gram Negative Organisms ◽  
High Level ◽  
Rhodamine Dye

Genes encoding streptomycin/spectinomycin adenylyltransferases [ANT(3")(9)] have been reported to exist in gram-negative organisms and Staphylococcus aureus. During a study of high-level aminoglycoside resistance in enterococci, we encountered an isolate ofEnterococcus faecalis that was streptomycin resistant but did not appear to contain the 6′-adenylyltransferase gene (aadE) when examined by PCR with specific primers. Phosphocellulose paper binding assays indicated the presence of an ANT(3")(9) enzyme. Streptomycin and spectinomycin MICs of 4,000 and 8,000 μg/ml, respectively, were observed for the isolate. PCR primers corresponding to a highly conserved region of the aadA gene were used to amplify a specific 284-bp product. The product hybridized with a digoxigenin-labeled PCR product from E. coliC600(pHP45Ω) known to contain the aadA gene. TheaadA gene was transferred via filter matings from theE. faecalis donor to E. faecalisJH2-2. PCR primers designed for analysis of integrons were used to amplify a 1-kb product containing the aadA gene, which was cloned into the vector pCRII and transformed into Escherichia coli DH5-α competent cells. d-Rhodamine dye terminator cycle sequencing was used to determine the gene sequence, which was compared to previously reported sequences of aadAgenes. We found the aadA gene in E. faecalis to be identical to the aadA genes reported by Sundström et al. for E. coli plasmid R6-5 (L. Sundström, P. Rådström, G. Swedberg, and O. Sköld, Mol. Gen. Genet. 213:191–201, 1988), by Fling et al. for theaadA within transposon Tn7 (M. E. Fling, J. Kopf, and C. Richards, Nucleic Acids Res. 13:7095–7106, 1985), and by Hollingshead and Vapnek for E. coliR538-1 (S. Hollingshead and D. Vapnek, Plasmid 13:17–30, 1985). Previous reports of the presence of the aadA gene in enterococci appear to be erroneous and probably describe anaadE gene, since the isolates were reported to be susceptible to spectinomycin.

scholarly journals Distribution of aminoglycoside resistance genes in recent clinical isolates of Enterococcus faecalis, Enterococcus faecium and Enterococcus avium

2001 ◽  
Vol 126 (2) ◽  
pp. 197-204 ◽  
Author(s):  
N. KOBAYASHI ◽  
MD. MAHBUB ALAM ◽  
Y. NISHIMOTO ◽  
S. URASAWA ◽  
N. UEHARA ◽  
...  
Keyword(s):  
Enterococcus Faecalis ◽  
Enterococcus Faecium ◽  
Clinical Isolates ◽  
University Hospital ◽  
Aminoglycoside Resistance ◽  
Genes Encoding ◽  
Enterococcal Species ◽  
Aminoglycoside Resistance Genes ◽  
Major Factors ◽  
High Level

Aminoglycoside modifying enzymes (AMEs) are major factors which confer aminoglycoside resistance on bacteria. Distribution of genes encoding seven AMEs was investigated by multiplex PCR for 279 recent clinical isolates of enterococci derived from a university hospital in Japan. The aac(6′)-aph(2″), which is related to high level gentamicin resistance, was detected at higher frequency in Enterococcus faecalis (42·5 %) than in Enterococcus faecium (4·3 %). Almost half of E. faecalis and E. faecium isolates possessed ant(6)-Ia and aph(3′)-IIIa. The profile of AME gene(s) detected most frequently in individual strains of E. faecalis was aac(6′)-aph(2″)+ant(6)-Ia+aph(3′)-IIIa, and isolates with this profile showed high level resistance to both gentamicin and streptomycin. In contrast, AME gene profiles of aac(6′)-Ii+ant(6)-Ia+aph(3′)-IIIa, followed by aac(6′)-Ii alone, were predominant in E. faecium. Only one AME gene profile of ant(6)-Ia+aph(3′)-IIIa was found in Enterococcus avium. The ant(4′)-Ia and ant(9)-Ia, which have been known to be distributed mostly among Staphylococcus aureus strains, were detected in a few enterococcal strains. An AME gene aph(2″)-Ic was not detected in any isolates of the three enterococcal species. These findings indicated a variety of distribution profiles of AME genes among enterococci in our study site.

scholarly journals Factors influencing determination of high-level aminoglycoside resistance in Enterococcus faecalis.

1991 ◽  
Vol 29 (9) ◽  
pp. 1934-1939 ◽  
Author(s):  
D F Sahm ◽  
S Boonlayangoor ◽  
P C Iwen ◽  
J L Baade ◽  
G L Woods
Keyword(s):  
Enterococcus Faecalis ◽  
Aminoglycoside Resistance ◽  
Factors Influencing ◽  
High Level

Evaluation of Modified MicroScan Screening Tests for High-Level Aminoglycoside Resistance in Enterococcus faecalis

1993 ◽  
Vol 99 (3) ◽  
pp. 286-288 ◽  
Author(s):  
Frederick S. Nolte ◽  
Janis M. Williams ◽  
Kathyrn L. Maher ◽  
Everly Metchock
Keyword(s):  
Enterococcus Faecalis ◽  
Screening Tests ◽  
Aminoglycoside Resistance ◽  
High Level

scholarly journals Enterococcus faecalis Endocarditis and Outpatient Treatment: A Systematic Review of Current Alternatives

Antibiotics ◽  
2020 ◽  
Vol 9 (10) ◽  
pp. 657
Author(s):  
Laura Herrera-Hidalgo ◽  
Arístides de Alarcón ◽  
Luis E. López-Cortes ◽  
Rafael Luque-Márquez ◽  
Luis F. López-Cortes ◽  
...  
Keyword(s):  
Enterococcus Faecalis ◽  
Outpatient Care ◽  
Oral Therapy ◽  
Outpatient Setting ◽  
Aminoglycoside Resistance ◽  
Design Data ◽  
Treatment Regimens ◽  
Continuation Treatment ◽  
High Level ◽  
Treatment Alternatives

The selection of the best alternative for Enterococcus faecalis infective endocarditis (IE) continuation treatment in the outpatient setting is still challenging. Three databases were searched, reporting antibiotic therapies against E. faecalis IE in or suitable for the outpatient setting. Articles the results of which were identified by species and treatment regimen were included. The quality of the studies was assessed accordingly with the study design. Data were extracted and synthesized narratively. In total, 18 studies were included. The treatment regimens reported were classified regarding the main antibiotic used as regimen, based on Aminoglycosides, dual β-lactam, teicoplanin, daptomycin or dalbavancin or oral therapy. The regimens based on aminoglycosides and dual β-lactam combinations are the treatment alternatives which gather more evidence regarding their efficacy. Dual β-lactam is the preferred option for high level aminoglycoside resistance strains, and for to its reduced nephrotoxicity, while its adaptation to the outpatient setting has been poorly documented. Less evidence supports the remaining alternatives, but many of them have been successfully adapted to outpatient care. Teicoplanin and dalbavancin as well as oral therapy seem promising. Our work provides an extensive examination of the potential alternatives to E. faecalis IE useful for outpatient care. However, the insufficient evidence hampers the attempt to give a general recommendation.

scholarly journals Fitness Cost and Interference of Arm/Rmt Aminoglycoside Resistance with the RsmF Housekeeping Methyltransferases

2012 ◽  
Vol 56 (5) ◽  
pp. 2335-2341 ◽  
Author(s):  
Belen Gutierrez ◽  
Jose A. Escudero ◽  
Alvaro San Millan ◽  
Laura Hidalgo ◽  
Laura Carrilero ◽  
...  
Keyword(s):  
16S Rrna ◽  
Pathogenic Bacteria ◽  
Binding Pocket ◽  
Maldi Mass Spectrometry ◽  
Fitness Cost ◽  
Resistance Pattern ◽  
Aminoglycoside Resistance ◽  
Content Type ◽  
E Coli ◽  
High Level

ABSTRACTArm/Rmt methyltransferases have emerged recently in pathogenic bacteria as enzymes that confer high-level resistance to 4,6-disubstituted aminoglycosides through methylation of the G1405 residue in the 16S rRNA (like ArmA and RmtA to -E). In prokaryotes, nucleotide methylations are the most common type of rRNA modification, and they are introduced posttranscriptionally by a variety of site-specific housekeeping enzymes to optimize ribosomal function. Here we show that while the aminoglycoside resistance methyltransferase RmtC methylates G1405, it impedes methylation of the housekeeping methyltransferase RsmF at position C1407, a nucleotide that, like G1405, forms part of the aminoglycoside binding pocket of the 16S rRNA. To understand the origin and consequences of this phenomenon, we constructed a series of in-frame knockout and knock-in mutants ofEscherichia coli, corresponding to the genotypesrsmF+, ΔrsmF,rsmF+rmtC+, and ΔrsmF rmtC+. When analyzed for the antimicrobial resistance pattern, the ΔrsmFbacteria had a decreased susceptibility to aminoglycosides, including 4,6- and 4,5-deoxystreptamine aminoglycosides, showing that the housekeeping methylation at C1407 is involved in intrinsic aminoglycoside susceptibility inE. coli. Competition experiments between the isogenicE. colistrains showed that, contrary to expectation, acquisition ofrmtCdoes not entail a fitness cost for the bacterium. Finally, matrix-assisted laser desorption ionization (MALDI) mass spectrometry allowed us to determine that RmtC methylates the G1405 residue not only in presence but also in the absence of aminoglycoside antibiotics. Thus, the coupling between housekeeping and acquired methyltransferases subverts the methylation architecture of the 16S rRNA but elicits Arm/Rmt methyltransferases to be selected and retained, posing an important threat to the usefulness of aminoglycosides worldwide.

scholarly journals Glutamate Decarboxylase Genes as a Prescreening Marker for Detection of Pathogenic Escherichia coliGroups

2001 ◽  
Vol 67 (7) ◽  
pp. 3110-3114 ◽  
Author(s):  
Michael A. Grant ◽  
Stephen D. Weagant ◽  
Peter Feng
Keyword(s):  
Shiga Toxin ◽  
Glutamate Decarboxylase ◽  
Pcr Primers ◽  
Enteric Bacteria ◽  
Food Samples ◽  
E Coli ◽  
Virulence Markers ◽  
Multiplex Pcr Assay ◽  
Lactose Fermentation ◽  
Genes Encoding

ABSTRACT The enzyme glutamate decarboxylase (GAD) is prevalent inEscherichia coli but few strains in the various pathogenicE. coli groups have been tested for GAD. Using PCR primers that amplify a 670-bp segment from the gadA andgadB genes encoding GAD, we examined the distribution of the gadAB genes among enteric bacteria. Analysis of 173 pathogenic E. coli strains, including 125 enterohemorrhagicE. coli isolates of the O157:H7 serotype and its phenotypic variants and 48 isolates of enteropathogenic E. coli, enterotoxigenic E. coli, enteroinvasive E. coli, and other Shiga toxin-producing E. coli (STEC) serotypes, showed that gadAB genes were present in all these strains. Among the 22 non-E. coli isolates tested, only the 6 Shigella spp. carried gadAB. Analysis of naturally contaminated water and food samples using agadAB-specific DNA probe that was labeled with digoxigenin showed that a gadAB-based assay is as reliable as standard methods that enumerate E. coli organisms on the basis of lactose fermentation. The presence of few E. coli cells initially seeded into produce rinsates could be detected by PCR togadA/B genes after overnight enrichment. A multiplex PCR assay using the gadAB primers in combination with primers to Shiga toxin (Stx) genes stx 1 andstx 2 was effective in detecting STEC from the enrichment medium after seeding produce rinsate samples with as few as 2 CFU. The gadAB primers may be multiplexed with primers to other trait virulence markers to specifically identify other pathogenicE. coli groups.

scholarly journals Characterization of Dihydrofolate Reductase Genes from Trimethoprim-Susceptible and Trimethoprim-Resistant Strains ofEnterococcus faecalis

1999 ◽  
Vol 43 (1) ◽  
pp. 141-147 ◽  
Author(s):  
Teresa M. Coque ◽  
Kavindra V. Singh ◽  
George M. Weinstock ◽  
Barbara E. Murray
Keyword(s):  
Amino Acids ◽  
Nucleotide Sequence ◽  
E Coli ◽  
Integrative Vector ◽  
Dihydrofolate Reductases ◽  
Multiple Copies ◽  
Gram Negative Organisms ◽  
High Level

Enterococci are usually susceptible in vitro to trimethoprim; however, high-level resistance (HLR) (MICs, >1,024 μg/ml) has been reported. We studied Enterococcus faecalis DEL, for which the trimethoprim MIC was >1,024 μg/ml. No transfer of resistance was achieved by broth or filter matings. Two different genes that conferred trimethoprim resistance when they were cloned in Escherichia coli (MICs, 128 and >1,024 μg/ml) were studied. One gene that coded for a polypeptide of 165 amino acids (MIC, 128 μg/ml forE. coli) was identical to dfr homologs that we cloned from a trimethoprim-susceptible E. faecalis strain, and it is presumed to be the intrinsic E. faecalis dfr gene (which causes resistance in E. coli when cloned in multiple copies); this gene was designated dfrE. The nucleotide sequence 5′ to this dfr gene showed similarity to thymidylate synthetase genes, suggesting that the dfr andthy genes from E. faecalis are located in tandem. The E. faecalis gene that conferred HLR to trimethoprim in E. coli, designated dfrF, codes for a predicted polypeptide of 165 amino acids with 38 to 64% similarity with other dihydrofolate reductases from gram-positive and gram-negative organisms. The nucleotide sequence 5′ to dfrFdid not show similarity to the thy sequences. A DNA probe for dfrF hybridized under high-stringency conditions only to colony lysates of enterococci for which the trimethoprim MIC was >1,024 μg/ml; there was no hybridization to plasmid DNA from the strain of origin. To confirm that this gene causes trimethoprim resistance in enterococci, we cloned it into the integrative vector pAT113 and electroporated it into RH110 (E. faecalisOG1RF::Tn916ΔEm) (trimethoprim MIC, 0.5 μg/ml), which resulted in RH110 derivatives for which the trimethoprim MIC was >1,024 μg/ml. These results indicate thatdfrF is an acquired but probably chromosomally located gene which is responsible for in vitro HLR to trimethoprim in E. faecalis.

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