Enzymatic modification of aminoglycoside antibiotics mediated by regioselective aminoglycoside N-acetyltransferases is the predominant cause of bacterial resistance to aminoglycosides. A recently ...discovered bifunctional aminoglycoside acetyltransferase (AAC(6′)-Ib variant, AAC(6′)-Ib-cr) has been shown to catalyze the acetylation of fluoroquinolones as well as aminoglycosides. We have expressed and purified AAC(6′)-Ib-wt and its bifunctional variant AAC(6′)-Ib-cr in Escherichia coli and characterized their kinetic and chemical mechanism. Initial velocity and dead-end inhibition studies support an ordered sequential mechanism for the enzyme(s). The three-dimensional structure of AAC(6′)-Ib-wt was determined in various complexes with donor and acceptor ligands to resolutions greater than 2.2 Å. Observation of the direct, and optimally positioned, interaction between the 6′-NH2 and Asp115 suggests that Asp115 acts as a general base to accept a proton in the reaction. The structure of AAC(6′)-Ib-wt permits the construction of a molecular model of the interactions of fluoroquinolones with the AAC(6′)-Ib-cr variant. The model suggests that a major contribution to the fluoroquinolone acetylation activity comes from the Asp179Tyr mutation, where Tyr179 makes π-stacking interactions with the quinolone ring facilitating quinolone binding. The model also suggests that fluoroquinolones and aminoglycosides have different binding modes. On the basis of kinetic properties, the pH dependence of the kinetic parameters, and structural information, we propose an acid/base-assisted reaction catalyzed by AAC(6′)-Ib-wt and the AAC(6′)-Ib-cr variant involving a ternary complex.
A Standard Numbering Scheme for Class C β-Lactamases Mack, Andrew R; Barnes, Melissa D; Taracila, Magdalena A ...
Antimicrobial agents and chemotherapy,
02/2020, Letnik:
64, Številka:
3
Journal Article, Web Resource
Recenzirano
Odprti dostop
Unlike for classes A and B, a standardized amino acid numbering scheme has not been proposed for the class C (AmpC) β-lactamases, which complicates communication in the field. Here, we propose a ...scheme developed through a collaborative approach that considers both sequence and structure, preserves traditional numbering of catalytically important residues (Ser
, Lys
, Tyr
, and Lys
), is adaptable to new variants or enzymes yet to be discovered and includes a variation for genetic and epidemiological applications.
Introduction: The importance of plasmid-mediated quinolone resistance (PMQR) in Enterobacterales and its high incidence has been emphasised many times. However, a clinical strain carrying more than ...two PMQR genes is rare. This study sequenced plasmid transconjugants from a donor strain carrying four different PMQR genes to establish their genetic locations.
Methods: An Escherichia coli clinical strain displayed remarkable quinolone resistance with a ciprofloxacin MIC of 1024 mg/L carrying four PMQR genes: qnrA1, qepA1, aac(6′)1b-cr and oqxAB. When outcrossed to Escherichia coli J53 AziR, different PMQR genes were transferred and the resulting strains 7C and 8C were chosen for further characterisation. Plasmids were extracted and sequenced by the Illumina and Oxford Nanopore Technologies platforms. S1 nuclease-PFGE was used to estimate the number and size of plasmids.
Results: The parental strain had three plasmid bands, as determined by PFGE. Transconjugant 8C obtained three plasmids: pMG336 (162 647 bp, F18:A-:B1:C4) carrying oqxAB; pMG335 carrying qepA1 (73 874 bp, F2:A-:B-); and pMG334 (59 724 bp, IncN (ST5)) with qnrA1 and aac(6′)1b-cr. Interestingly, strain 7C obtained plasmid pMG333 (134 435 bp), which was not present in the parental strain but was an IncN-IncF cointegrate of plasmids pMG334 and pMG335 linked via insertion sequence IS26.
Conclusion: This study described the complete nucleotide sequence of three plasmids carrying four PMQR genes in a single strain and the plasmid profile obtained after outcrosses. In addition, it described a cointegrate of two plasmids formed with flanking insertion sequences.
In a previous study, mutants with enhanced ciprofloxacin resistance (Cip
) were selected from
J53/pMG252 carrying
Strain J53 Cip
8-2 showed an increase in the copy number and transcription level of
...We sequenced the plasmids on Illumina and MinION platforms. Parental plasmid pMG252 and plasmid pMG252A from strain J53 Cip
8-2 were almost identical, except for the region containing
that in pMG252A contained 4 additional copies of the
Δ
-IS
region.
In a previous study, mutants with enhanced ciprofloxacin resistance (Cipr) were selected from Escherichia coli J53/pMG252 carrying qnrA1. Strain J53 Cipr 8-2 showed an increase in the copy number and ...transcription level of qnrA1. We sequenced the plasmids on Illumina and MinION platforms. Parental plasmid pMG252 and plasmid pMG252A from strain J53 Cipr 8-2 were almost identical, except for the region containing qnrA1 that in pMG252A contained 4 additional copies of the qnrA1-qacEΔ1-sul1-ISCR1 region.
Plasmid toxins CcdB and ParE are part of addiction systems promoting plasmid maintenance. Both target host DNA gyrase, as do quinolones and plasmid-determined Qnr proteins that protect gyrase from ...quinolone inhibition. We cloned qnrB4, qnrS1, ccdB, parE, and the antitoxin-encoding genes ccdA and parD on compatible plasmids and tested them in combination. CcdB and ParE had no specific effect on quinolone susceptibility or Qnr protection, and Qnr did not act as a CcdB or ParE antitoxin.
Plasmid-mediated quinolone resistance (PMQR) caused by qnr genes has been known for 15 years. Information about global distribution and prevalence of qnr genes is abundant, but clinical information ...concerning infections produced by these isolates and risk factors for their acquisition is limited.
Klebsiella pneumoniae blood isolates (n = 227) from a 1 year prospective cohort of patients in Taiwan were studied. MICs of quinolones were determined for all isolates, and multiplex PCR for the presence of PMQR genes and DNA gyrase mutations was applied to all 24 isolates with ciprofloxacin MICs ≥ 0.12 mg/L and a control group of 72 isolates with MICs ≤ 0.06 mg/L.
All qnr isolates were in the group with ciprofloxacin MICs ≥ 0.12 mg/L, constituting 9.4% of tested isolates and 3.9% (qnrB 2.6% and qnrS 1.3%) of total isolates. aac(6')-Ib-cr and qepA were not found. Risk factors for qnr included nosocomial infection, bedridden status, surgery within 3 months, non-K1/K2 serotypes and prior antimicrobial use. Ciprofloxacin MIC ≥ 0.12 mg/L was associated with prior quinolone use; in contrast, prior cephalosporin use was more closely linked to the presence of qnr. Fourteen-day mortality was similar in patients infected with qnr-positive versus qnr-negative isolates, but there was a trend for increased in-hospital mortality in patients infected with qnr-positive isolates.
In K. pneumoniae blood isolates collected at a hospital in Taiwan, the overall prevalence of qnr genes was 3.9%. Prior quinolone use was linked to increased ciprofloxacin MIC, but not with the prevalence of qnr, which was most strongly linked to exposure to other antimicrobials, especially cephalosporins.
QnrB1 is a plasmid-encoded pentapeptide repeat protein (PRP) that confers a moderate degree of resistance to fluoroquinolones. Its gene was cloned into an expression vector with an N-terminal ...polyhistidine tag, and the protein was purified by nickel affinity chromatography. The structure of QnrB1 was determined by a combination of trypsinolysis, surface mutagenesis, and single anomalous dispersion phasing. QnrB1 folds as a right-handed quadrilateral β-helix with a highly asymmetric dimeric structure typical of PRP-topoisomerase poison resistance factors. The threading of pentapeptides into the β-helical fold is interrupted by two noncanonical PRP sequences that produce outward projecting loops that interrupt the regularity of the PRP surface. Deletion of the larger upper loop eliminated the protective effect of QnrB1 on DNA gyrase toward inhibition by quinolones, whereas deletion of the smaller lower loop drastically reduced the protective effect. These loops are conserved among all plasmid-based Qnr variants (QnrA, QnrC, QnrD, and QnrS) and some chromosomally encoded Qnr varieties. A mechanism in which PRP-topoisomerase poison resistance factors bind to and disrupt the quinolone-DNA-gyrase interaction is proposed.
In order to study the interactions between Escherichia coli DNA gyrase and the gyrase interacting protein QnrB in vivo, we constructed a gyrB-gyrA fusion and validated its ability to correct the ...temperature-sensitive growth of gyrA and gyrB mutants. Like wild-type gyrA, the gyrB-gyrA fusion complemented a quinolone-resistant gyrA mutant to increase susceptibility. It functioned as an active type II topoisomerase, catalyzed negative supercoiling of DNA, was inhibited by quinolone, and was protected by QnrB.
Expression of the quinolone resistance gene
is increased by quinolones, but unlike induction of some other
genes, the bacterial SOS system is not involved and no
box is found upstream. Nonetheless, ...at least 205 bp of upstream sequence is required for induction to take place. An upstream sequence bound to beads trapped potential binding proteins from cell extracts that were identified by mass spectrometry as Dps, Fis, Ihf, Lrp, CysB, and YjhU. To further elucidate their role, a reporter plasmid linking the
upstream sequence to
was introduced into cells of the Keio collection with single-gene deletions and screened for
expression. Mutants in
and
had decreased
induction, while induction in a
mutant was increased and
,
,
,
, and other mutants showed no change. The essential upstream sequence contains potential binding sites for Ihf and DnaA. A
deletion could not be tested because it provides essential functions in cell replication; however, increased
expression decreased
induction while decreased
expression enhanced it, implying a role for DnaA as a repressor. In a mobility shift assay, purified IhfA, IhfB, and DnaA proteins (but not CysB) were shown to bind to the upstream segment. Induction decreased in a
quinolone-resistant mutant, indicating that GyrA also has a role. Thus, quinolones acting through proteins DnaA, GyrA, IhfA, and IhfB regulate expression of
.
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