Substitutions at Position 105 in SHV Family β-Lactamases Decrease Catalytic Efficiency and Cause Inhibitor Resistance

Abstract

Ambler position 105 in class A ␤-lactamases is implicated in resistance to clavulanic acid, although no clinical isolates with mutations at this site have been reported. We hypothesized that Y105 is important in resistance to clavulanic acid because changes in positioning of the inhibitor for ring oxygen protonation could occur. In addition, resistance to bicyclic 6-methylidene penems, which are interesting structural probes that inhibit all classes of serine ␤-lactamases with nanomolar affinity, might emerge with substitutions at position 105, especially with nonaromatic substitutions. All 19 variants of SHV-1 with variations at position 105 were prepared. Antimicrobial susceptibility testing showed that Escherichia coli DH10B expressing Y105 variants retained activity against ampicillin, except for the Y105L variant, which was susceptible to all ␤-lactams, similar to the case for the host control strain. Several variants had elevated MICs to ampicillin-clavulanate. However, all the variants remained susceptible to piperacillin in combination with a penem inhibitor (MIC, <2/4 mg/liter). The Y105E, -F, -M, and -R variants demonstrated reduced catalytic efficiency toward ampicillin compared to the wild-type (WT) enzyme, which was caused by increased K m . Clavulanic acid and penem K i values were also increased for some of the variants, especially Y105E. Mutagenesis at position 105 in SHV yields mutants resistant to clavulanate with reduced catalytic efficiency for ampicillin and nitrocefin, similar to the case for the class A carbapenemase KPC-2. Our modeling analyses suggest that resistance is due to oxyanion hole distortion. Susceptibility to a penem inhibitor is retained although affinity is decreased, especially for the Y105E variant. Residue 105 is important to consider when designing new inhibitors.

The intrinsic resistome of bacterial pathogens

“…The commercially available β-lactamase inhibitors (BLIs), clavulanic acid, sulbactam and tazobactam, are only effective against class A β-lactamases. However, their combinations with β-lactam antibiotics are still effective, despite the emergence of resistance, either because of gene-dosage effect (Martinez et al, 1987, 1989; Reguera et al, 1991), either because of mutations that make β-lactamase resilient to inhibition (Blazquez et al, 1993; Canica et al, 1998; Salverda et al, 2010; Frase et al, 2011; Li et al, 2012). …”

N152G, -S, and -T Substitutions in CMY-2 β-Lactamase Increase Catalytic Efficiency for Cefoxitin and Inactivation Rates for Tazobactam

“…After discarding the supernatant, the pellet was resuspended in 80 l of 5ϫ sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) loading dye and boiled at 100°C for 10 min. Immunoblotting was performed with the samples as described previously (20). The membrane was probed with rabbit polyclonal anti-CMY-2 antibody at 1.0 g/ml in blocking buffer, and bands were detected with protein G-horseradish peroxidase conjugate (Bio-Rad).…”

“…CMY-2 and N152G, -S, and -T CMY-2 ␤-lactamases were expressed in E. coli AS226-51 cells (Invitrogen), derived from a strain that does not produce a chromosomal AmpC ␤-lactamase. As described previously, crude extracts were purified to homogeneity by preparative isoelectric focusing and gel filtration or cation exchange chromatography using a GE Superdex 75 10/300 column with 1ϫ phosphatebuffered saline (pH 7.4) or GE HiTrap Q HP columns with 50 mM HEPES (pH 7.0) and elution buffer consisting of 50 mM HEPES and 1 M NaCl (pH 7.0) (20). Protein purity was assessed with SDS-PAGE and was Ͼ95%.…”

“…K I (the inhibitor concentration required for half-maximal inactivation) and k inact were determined by using timed inactivation, i.e., mixing fixed amounts of enzyme (4 nM) and variable concentrations of inhibitor and, at various time points (0, 15, 30, 60, 120, 300, and 600 s), assaying an aliquot of the mixture with NCF as the substrate (20,25). The timed-inactivation plots were fitted with a singleexponential decay to obtain the apparent inhibition constant (k app ) for each inhibitor concentration.…”

Rapid Detection of β-Lactamase-Hydrolyzing Extended-Spectrum Cephalosporins in Enterobacteriaceae by Use of the New Chromogenic βLacta Test

“…Variable expression of ESBLs was described several years ago, particularly with SHV variants, affecting the hydrolysis of cephalosporins and MIC values (18). This could also eventually affect the hydrolysis of HMRZ-86, a fact that has been shown with nitrocefin and in vitro variants obtained by mutagenesis of bla SHV-1 (19). On the other hand, results for carbapenemase producers (n ϭ 67) were mainly positive (92.5% [62/67 isolates]), including results for producers of OXA-48, an enzyme with minor hydrolytic activity against extended-spectrum cephalosporins (20).…”