Bifunctional Enzyme AAC Research Articles

Aminoglycoside resistance profile and structural architecture of the aminoglycoside acetyltransferase AAC(6')-Im.

Aminoglycoside 6’-acetyltransferase-Im (AAC(6’)-Im) is the closest monofunctional homolog of the AAC(6’)-Ie acetyltransferase of the bifunctional enzyme AAC(6’)-Ie/APH(2”)-Ia. The AAC(6’)-Im acetyltransferase confers 4- to 64-fold higher MICs to 4,6-disubstituted aminoglycosides and the 4,5-disubstituted aminoglycoside neomycin than AAC(6’)-Ie, yet unlike AAC(6’)-Ie, the AAC(6’)-Im enzyme does not confer resistance to the atypical aminoglycoside fortimicin. The structure of the kanamycin A complex of AAC(6’)-Im shows that the substrate binds in a shallow positively-charged pocket, with the N6’ amino group positioned appropriately for an efficient nucleophilic attack on an acetyl-CoA cofactor. The AAC(6’)-Ie enzyme binds kanamycin A in a sufficiently different manner to position the N6’ group less efficiently, thereby reducing the activity of this enzyme towards the 4,6-disubstituted aminoglycosides. Conversely, docking studies with fortimicin in both acetyltransferases suggest that the atypical aminoglycoside might bind less productively in AAC(6’)-Im, thus explaining the lack of resistance to this molecule.

Structure of the bifunctional aminoglycoside-resistance enzyme AAC(6')-Ie-APH(2'')-Ia revealed by crystallographic and small-angle X-ray scattering analysis.

Broad-spectrum resistance to aminoglycoside antibiotics in clinically important Gram-positive staphylococcal and enterococcal pathogens is primarily conferred by the bifunctional enzyme AAC(6')-Ie-APH(2'')-Ia. This enzyme possesses an N-terminal coenzyme A-dependent acetyltransferase domain [AAC(6')-Ie] and a C-terminal GTP-dependent phosphotransferase domain [APH(2'')-Ia], and together they produce resistance to almost all known aminoglycosides in clinical use. Despite considerable effort over the last two or more decades, structural details of AAC(6')-Ie-APH(2'')-Ia have remained elusive. In a recent breakthrough, the structure of the isolated C-terminal APH(2'')-Ia enzyme was determined as the binary Mg2GDP complex. Here, the high-resolution structure of the N-terminal AAC(6')-Ie enzyme is reported as a ternary kanamycin/coenzyme A abortive complex. The structure of the full-length bifunctional enzyme has subsequently been elucidated based upon small-angle X-ray scattering data using the two crystallographic models. The AAC(6')-Ie enzyme is joined to APH(2'')-Ia by a short, predominantly rigid linker at the N-terminal end of a long α-helix. This α-helix is in turn intrinsically associated with the N-terminus of APH(2'')-Ia. This structural arrangement supports earlier observations that the presence of the intact α-helix is essential to the activity of both functionalities of the full-length AAC(6')-Ie-APH(2'')-Ia enzyme.

Editorial Commentary: Enterococcus faecalisInfective Endocarditis: Is It Time to Abandon Aminoglycosides?

Keywords. Enterococcus faecalis; infective endocarditis; high-level resistance; aminoglycosides.Since the first description of enterococ-cal infective endocarditis (IE) in 1899and the subsequent availability of antibi-otics for the treatment of this life-threat-ening infection, clinicians have facedimportant challenges in the manage-ment of this disease [1, 2]. Enterococciexhibit intrinsic antibiotic resistance (eg,to cephalosporins, clindamycin), are lesssusceptible to various antibiotics (eg, β-lactams) that are active against strepto-cocci and staphylococci, and are oftentolerant to compounds (penicillin) thatnormallyhaveabactericidaleffectagainstother susceptible bacteria. Indeed, thelack of bactericidal activity of penicillinagainst enterococci was recognized whenfailure rates using penicillin mono-therapy for the treatment of IE causedby enterococci were found to be higherthan when caused by staphylococci andstreptococci [3]. The lack of efficacy ofpenicillin for many cases of enterococcalIE sparked interest in possible alternativetherapies. Reports of clinical cure in pa-tients with enterococcal IE failing peni-cillin monotherapy when streptomycinwas added to penicillin resulted inseminal experiments in the 1950s thatshowed that the combination of penicil-lin plus an aminoglycoside was bacterici-dal in vitro [4, 5]. This combination wasused clinically with success and im-proved cure rates for enterococcal IE [6].However, after this regimen became thestandard of care, enterococcal strains ex-hibiting high-level resistance (HLR) tostreptomycin (mainly due to ribosomalmutations) and, eventually, gentamicin(due to acquisition of the bifunctionalenzyme AAC [6′]-Ie-APH[2′′]-Ia) weredescribed. More recently, the widespreaddissemination of antimicrobial resistancedeterminants in enterococci has furthercomplicated the clinical picture with theemergence and dissemination of strainsof Enterococcus faecium with HLR toampicillin, indicative of the hospital-associated clade of multidrug-resistantE. faecium causing nosocomial infec-tions worldwide [7, 8]. Modern-day clin-icalisolatesbelongingtothisgeneticcladeoften exhibit resistance to vancomycin(harboring the vanA gene cluster) andHLR to aminoglycosides, in addition toHLR to ampicillin (minimum inhibitoryconcentration >64 µg/mL), reducing an-tibiotic choices even further [9,10].Despitethehigh frequencyof ampicillin-resistant E. faecium, ampicillin-resistantE. faecalis is strikingly uncommon. Thepressing issue in the latter species is theincreasing frequency of HLR to all amino-glycosides, as the presence of HLR toaminoglycosides abolishes the synergis-tic, bactericidal effect of the penicillin-aminoglycoside combination againstenterococci. Additionally, toxicity of theaminoglycosidesisalimitingfactorfortheir use, even against E. faecalis isolateslacking HLR to aminoglycosides. Indeed,renal injury and ototoxicity are the 2 mostfeared complications of prolonged amino-glycoside therapy. An article entitled “Deafor Dead?” published in 1959 dramaticallyillustrates, albeit with neomycin, the poten-tial risks of using these drugs for E. faecalisIE [11]. Furthermore, the safety of amino-glycosides may be even more of an issuetoday, as patients who develop enterococcalIE in the modern-day era tend to be older,with a higher number of comorbidities andlikelyreceivingmorepotentiallynephrotox-ic agents than in previous decades, increas-ing the risk of aminoglycoside-related renaldysfunction.Theabove-mentionedlimitationsintheuseofaminoglycosideshavepromptedthestudy of alternative therapeutic strategies

Aranorosin circumvents arbekacin-resistance in MRSA by inhibiting the bifunctional enzyme AAC(6′)/APH(2″)

Aranorosin circumvents arbekacin-resistance in MRSA by inhibiting the bifunctional enzyme AAC(6′)/APH(2″)

Effects of Altering Aminoglycoside Structures on Bacterial Resistance Enzyme Activities

Aminoglycoside-modifying enzymes (AMEs) constitute the most prevalent mechanism of resistance to aminoglycosides by bacteria. We show that aminoglycosides can be doubly modified by the sequential actions of AMEs, with the activity of the second AME in most cases unaffected, decreased, or completely abolished. We demonstrate that the bifunctional enzyme AAC(3)-Ib/AAC(6')-Ib' can diacetylate gentamicin. Since single acetylation does not always inactivate the parent drugs completely, two modifications likely provide more-robust inactivation in vivo.

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

Escherichia coli SCH92111602 expresses an aminoglycoside resistance profile similar to that conferred by the aac(6')-Ie-aph(2")-Ia gene found in gram-positive cocci and was found to contain the aminoglycoside resistance genes aph(2")-Ib and aac(6')-Im (only 44 nucleotides apart). aph(2")-Ib had been reported previously in Enterococcus faecium SF11770. aac(6')-Im had not been detected previously in enterococci and was found to be present also 44 nucleotides downstream from aph(2")-Ib in E. faecium SF11770. aph(2")-Ib and aac(6')-Im are separate open reading frames, each with its own putative ribosome binding site, whereas aac(6')-Ie-aph(2")-Ia appears to be a fusion of two genes with just one start and one stop codon. The deduced AAC(6')-Im protein exhibits 56% identity and 80% similarity to the AAC(6')-Ie domain of the bifunctional enzyme AAC(6')-APH(2"). Our results document the existence of a member of the aph(2") family of genes in gram-negative bacteria and provide evidence suggesting the horizontal transfer of aph(2")-Ib and aac(6')-Im as a unit between gram-positive and gram-negative bacteria.

Functional characterization of a multiple-antibiotic resistant plasmid from clinical isolates of methicillin-resistant Staphylococcus aureus

During surveillance done as part of the investigation of nosocominal infections due to methicillin-resistant Staphylococcus aureus (MRSA), we have been aware of the close relationship between the presence of certain plasmids and the characteristic patterns of antibiotic resistance. The hypothesis that a mechanism for the rapid and widespread dissemination of resistance to multiple aminoglycosides in clinical isolates of MRSA at our university hospital was the result of transfer of a single plasmid among these strains was examined. About 45% of the total isolates of MRSA (91/200 isolates) carried a 35.5 kb plasmid (designated pCL4) and these isolates always showed resistance to gentamicin, tobramycin, kanamycin, amikacin, astromicin, and arbekacin. Mating experiments between a susceptible strain and resistant MRSA isolates carrying pCL4 showed high frequency transfer of the plasmid. The aminoglycoside resistance patterns of all the transconjugants obtained corresponded well with those of parental strains. However, the plasmid could not necessarily be detected in the transconjugants, whereas the transformants obtained by means of electroporation usually possessed the plasmid. The plasmid-encoded aminoglycoside-resistance determinant, which has been identified as the gene aacA/aphD that encodes the bifunctional enzyme AAC (6')/APH(2"), either in the transconjugants and transformants could be transposed to their chromosomes in the absence of whole-plasmid integration resulting from a recombination event. Southern hybridization analysis using an aacA/aphD specific probe demonstrated that there are multiple sites of the insertions indistinguishable among the chromosomes of plasmid-free transconjugants and transformants. These results indicate that rapid dissemination of multiple-aminoglycoside-resistance in nosocominal strains of MRSA resulted from transfer of a conjugative plasmid and has been facilitated by translocations of the resistance gene to the chromosome.

Aminoglycoside-modifying enzymes in high-level streptomycin and gentamicin resistant Enterococcus spp. in Spain

Aminoglycoside resistance was evaluated in 690 enterococcus strains isolated from different clinical sources originating from patients at the University Clinic Hospital of Zaragoza (Spain). The enterococci obtained from clinically significant samples (blood, urine, or exudates) showed more high-level resistance to gentamicin and streptomycin (65 and 42%, respectively) than those isolated from faecal samples (49 and 23%, respectively). Aminoglycoside-modifying enzymes (AME) from 119 of these high-level gentamicin and streptomycin resistant enterococcus strains were studied. The most frequent AMEs found were APH(3′) and AAC(6′)-APH(2″). More than one enzyme was detected in 71% of the strains (four different enzymes in 5% of the strains). Three Enterococcus faecalis strains had ANT(4′)(4″) enzymatic activity. Different enzymatic expressions of the bifunctional enzyme AAC(6′)-APH(2″) were demonstrated in strains in which the complete aac(6′)-aph(2″ ) gene was detected by PCR and hybridization: (i) AAC(6′)+APH(2″) activity; (ii) AAC(6′) only; (iii) APH(2″) only; and (iv) no activity of AAC(6′) or APH(2″).

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

Enterococcus casseliflavus UC73 is a clinical blood isolate with high-level resistance to gentamicin. DNA preparations from UC73 failed to hybridize with intragenic probes for aac(6')-Ie-aph(2'')-Ia and aph(2'')-Ic. A 4-kb fragment from UC73 was cloned and found to confer resistance to gentamicin in Escherichia coli DH5alpha transformants. Nucleotide sequence analysis revealed the presence of a 906-bp open reading frame whose deduced amino acid sequence had a region with homology to the aminoglycoside-modifying enzyme APH(2'')-Ic and to the C-terminal domain of the bifunctional enzyme AAC(6')-APH(2''). The gene is designated aph(2'')-Id, and its observed phosphotransferase activity is designated APH(2'')-Id. A PCR-generated intragenic probe hybridized to the genomic DNA from 17 of 118 enterococcal clinical isolates (108 with high-level gentamicin resistance) from five hospitals. All 17 were vancomycin-resistant Enterococcus faecium isolates, and pulsed-field typing revealed three distinct clones. The combination of ampicillin plus either amikacin or neomycin exhibited synergistic killing against E. casseliflavus UC73. Screening and interpretation of high-level aminoglycoside resistance in enterococci may need to be modified to include detection of APH(2'')-Id.

A novel gentamicin resistance gene in Enterococcus.

Enterococcus gallinarum SF9117 is a veterinary isolate for which the MIC of gentamicin is 256 micrograms/ml. Time-kill studies with a combination of ampicillin plus gentamicin failed to show synergism against SF9117. A probe representing aac(6')-aph(2") did not hybridize to DNA from SF9117. A 3.2-kb fragment from plasmid pYN134 of SF9117 was cloned and conferred resistance to gentamicin in Escherichia coli DH5 alpha. Nucleotide sequence analysis revealed the presence of a 918-bp open reading frame whose deduced amino acid sequence had a region with homology to the C-terminal domain of the bifunctional enzyme AAC(6')-APH(2"). The gene is designated aph(2")-Ic, and its observed phosphotransferase activity is provisionally designated APH(2")-Ic. An intragenic probe hybridized to the genomic DNA from an Enterococcus faecium isolate from the peritoneal fluid of one patient and to the plasmid DNA of an Enterococcus faecalis isolate from the blood of another patient. An enterococcal isolate containing this novel resistance gene might not be readily detected in clinical laboratories that use gentamicin at 500 or 2,000 micrograms/ml for screening for high-level resistance.