Exposing a β-Lactamase “Twist”: the Mechanistic Basis for the High Level of Ceftazidime Resistance in the C69F Variant of the Burkholderia pseudomallei PenI β-Lactamase

Papp-Wallace, Krisztina M.; Becka, Scott A.; Taracila, Magdalena A.; Winkler, Marisa L.; Gatta, Julian A.; Rholl, Drew A.; Schweizer, Herbert P.

doi:10.1128/aac.02073-15

Cited by 25 publications

(19 citation statements)

References 42 publications

(63 reference statements)

Supporting

Mentioning

Contrasting

Order By: Relevance

“…The authors concluded that increased flexibility of the B3 ␤-strand that parallels the active site is correlated with higher activity against ceftazidime. Curiously, it was previously shown that in a B. pseudomallei C69F variant, CAZ interacted with D240 via a unique hydrogen bond formation not seen in wild-type PenA (65). The V261I substitution did not affect the susceptibility profile of Bp1651, which could be explained by the conservative amino acid change and location, which (61) and location of amino acid changes leading to antimicrobial resistance.…”

Section: Discussionmentioning

confidence: 92%

Antibiotic Resistance Markers in Burkholderia pseudomallei Strain Bp1651 Identified by Genome Sequence Analysis

Bugrysheva

Sue

Gee

et al. 2017

Antimicrob Agents Chemother

Self Cite

View full text Add to dashboard Cite

Burkholderia pseudomallei Bp1651 is resistant to several classes of antibiotics that are usually effective for treatment of melioidosis, including tetracyclines, sulfonamides, and ␤-lactams such as penicillins (amoxicillin-clavulanic acid), cephalosporins (ceftazidime), and carbapenems (imipenem and meropenem). We sequenced, assembled, and annotated the Bp1651 genome and analyzed the sequence using comparative genomic analyses with susceptible strains, keyword searches of the annotation, publicly available antimicrobial resistance prediction tools, and published reports. More than 100 genes in the Bp1651 sequence were identified as potentially contributing to antimicrobial resistance. Most notably, we identified three previously uncharacterized point mutations in penA, which codes for a class A ␤-lactamase and was previously implicated in resistance to ␤-lactam antibiotics. The mutations result in amino acid changes T147A, D240G, and V261I. When individually introduced into select agent-excluded B. pseudomallei strain Bp82, D240G was found to contribute to ceftazidime resistance and T147A contributed to amoxicillin-clavulanic acid and imipenem resistance. This study provides the first evidence that mutations in penA may alter susceptibility to carbapenems in B. pseudomallei. Another mutation of interest was a point mutation affecting the dihydrofolate reductase gene folA, which likely explains the trimethoprim resistance of this strain. Bp1651 was susceptible to aminoglycosides likely because of a frameshift in the amrB gene, the transporter subunit of the AmrAB-OprA efflux pump. These findings expand the role of penA to include resistance to carbapenems and may assist in the development of molecular diagnostics that predict antimicrobial resistance and provide guidance for treatment of melioidosis.

show abstract

Section: Discussionmentioning

confidence: 92%

Antibiotic Resistance Markers in Burkholderia pseudomallei Strain Bp1651 Identified by Genome Sequence Analysis

Bugrysheva

Sue

Gee

et al. 2017

Antimicrob Agents Chemother

Self Cite

View full text Add to dashboard Cite

show abstract

“…18 For the experiments, 10 μ M PenA was incubated with 10 μ M avibactam for set times (i.e., 5 min, 5 h, and 24 h) at room temperature in 10m MPBS (pH 7.4). Reactions were terminated by the addition of 0.1% formic acid and 1% acetonitrile.…”

Section: Methodsmentioning

confidence: 99%

Overcoming an Extremely Drug Resistant (XDR) Pathogen: Avibactam Restores Susceptibility to Ceftazidime for Burkholderia cepacia Complex Isolates from Cystic Fibrosis Patients

et al. 2017

Self Cite

View full text Add to dashboard Cite

Burkholderia multivorans is a significant health threat to persons with cystic fibrosis (CF). Infections are difficult to treat as this pathogen is inherently resistant to multiple antibiotics. Susceptibility testing of isolates obtained from CF respiratory cultures revealed that single agents selected from different antibiotic classes were unable to inhibit growth. However, all isolates were found to be susceptible to ceftazidime when combined with the novel non-β-lactam β-lactamase inhibitor, avibactam (all minimum inhibitor concentrations (MICs) were ≤8 mg/L of ceftazidime and 4 mg/L of avibactam). Furthermore, a major β-lactam resistance determinant expressed in B. multivorans, the class A carbapenemase, PenA was readily inhibited by avibactam with a high k2/K of (2 ± 1) × 106 μM−1 s−1 and a slow koff of (2 ± 1) × 10−3 s−1. Mass spectrometry revealed that avibactam formed a stable complex with PenA for up to 24 h and that avibactam recyclized off of PenA, re-forming the active compound. Crystallographic analysis of PenA–avibactam revealed several interactions that stabilized the acyl–enzyme complex. The deacylation water molecule possessed decreased nucleophilicity, preventing decarbamylation. In addition, the hydrogen-bonding interactions with Lys-73 were suggestive of a protonated state. Thus, Lys-73 was unlikely to abstract a proton from Ser-130 to initiate recyclization. Using Galleria mellonella larvae as a model for infection, ceftazidime–avibactam was shown to significantly (p < 0.001) improve survival of larvae infected with B. multivorans. To further support the translational impact, the ceftazidime–avibactam combination was evaluated using susceptibility testing against other strains of Burkholderia spp. that commonly infect individuals with CF, and 90% of the isolates were susceptible to the combination. In summary, ceftazidime–avibactam may serve as a preferred therapy for people that have CF and develop Burkholderia spp. infections and should be considered for clinical trials.

show abstract

“…To discern the nature of the intermediates of inactivation by AVI in the reaction pathway with the PER-2 ␤-lactamase, electrospray ionization mass spectrometry (ESI-MS) was performed using a Waters SynaptG2-Si quadrupole time of flight (QTOF) mass spectrometer equipped with a Waters Acquity H class Ultra Performance liquid chromatograph (UPLC) on an Acquity UPLC BEH C 18 column (1.7-m particle size; 2.1 by 100 mm), using standards and calibrations as previously described (28). For the experiments, the PER-2 ␤-lactamase was incubated with AVI for set times (i.e., 5 min and 24 h) at room temperature in 10 mM PBS, pH 7.4.…”

Section: Esi-msmentioning

confidence: 99%

Exploring the Landscape of Diazabicyclooctane (DBO) Inhibition: Avibactam Inactivation of PER-2 β-Lactamase

Ruggiero

Papp-Wallace

Taracila

et al. 2017

Antimicrob Agents Chemother

Self Cite

View full text Add to dashboard Cite

PER ␤-lactamases are an emerging family of extended-spectrum ␤-lactamases (ESBL) found in Gram-negative bacteria. PER ␤-lactamases are unique among class A enzymes as they possess an inverted omega (⍀) loop and extended B3 ␤-strand. These singular structural features are hypothesized to contribute to their hydrolytic profile against oxyimino-cephalosporins (e.g., cefotaxime and ceftazidime). Here, we tested the ability of avibactam (AVI), a novel non-␤-lactam ␤-lactamase inhibitor to inactivate PER-2. Interestingly, the PER-2 inhibition constants (i.e., k 2 /K ϭ 2 ϫ 10 3 Ϯ 0.1 ϫ 10 3 M Ϫ1 s Ϫ1 , where k 2 is the rate constant for acylation (carbamylation) and K is the equilibrium constant) that were obtained when AVI was tested were reminiscent of values observed testing the inhibition by AVI of class C and D ␤-lactamases (i.e., k 2 /K range of Ϸ10 3 M Ϫ1 s Ϫ1 ) and not class A ␤-lactamases (i.e., k 2 /K range, 10 4 to 10 5 M Ϫ1 s Ϫ1 ). Once AVI was bound, a stable complex with PER-2 was observed via mass spectrometry (e.g., 31,389 Ϯ 3 atomic mass units [amu] ¡ 31,604 Ϯ 3 amu for 24 h). Molecular modeling of PER-2 with AVI showed that the carbonyl of AVI was located in the oxyanion hole of the ␤-lactamase and that the sulfate of AVI formed interactions with the ␤-lactam carboxylate binding site of the PER-2 ␤-lactamase (R220 and T237). However, hydrophobic patches near the PER-2 active site (by Ser70 and B3-B4 ␤-strands) were observed and may affect the binding of necessary catalytic water molecules, thus slowing acylation (k 2 /K) of AVI onto PER-2. Similar electrostatics and hydrophobicity of the active site were also observed between OXA-48 and PER-2, while CTX-M-15 was more hydrophilic. To demonstrate the ability of AVI to overcome the enhanced cephalosporinase activity of PER-2 ␤-lactamase, we tested different ␤-lactam-AVI combinations. By lowering MICs to Յ2 mg/liter, the ceftaroline-AVI combination could represent a favorable therapeutic option against Enterobacteriaceae expressing bla PER-2 . Our studies define the inactivation of the PER-2 ESBL by AVI and suggest that the biophysical properties of the active site contribute to determining the efficiency of inactivation.

show abstract

Exposing a β-Lactamase “Twist”: the Mechanistic Basis for the High Level of Ceftazidime Resistance in the C69F Variant of the Burkholderia pseudomallei PenI β-Lactamase

Cited by 25 publications

References 42 publications

Antibiotic Resistance Markers in Burkholderia pseudomallei Strain Bp1651 Identified by Genome Sequence Analysis

Antibiotic Resistance Markers in Burkholderia pseudomallei Strain Bp1651 Identified by Genome Sequence Analysis

Overcoming an Extremely Drug Resistant (XDR) Pathogen: Avibactam Restores Susceptibility to Ceftazidime for Burkholderia cepacia Complex Isolates from Cystic Fibrosis Patients

Exploring the Landscape of Diazabicyclooctane (DBO) Inhibition: Avibactam Inactivation of PER-2 β-Lactamase

Contact Info

Product

Resources

About