Avibactam activity against extended-spectrum AmpC -lactamases

Sir, The effectiveness of β-lactams against Gram-negative bacilli has declined over time due to the emergence and spread of β-lactamases. Avibactam, which is currently in development in clinical trials in combination with the β-lactams ceftazidime, ceftaroline fosamil and aztreonam, is a potent inhibitor of class A, class C and some class D β-lactamases.1 Paired with ceftazidime, it is effective against a wide range of β-lactamase-producing Gram-negative strains and superior to clavulanate- or tazobactam-based combinations.1,2 Recently, Livermore et al.3 showed that structural modifications in the AmpC β-lactamase of Enterobacter cloacae could alter the susceptibility to avibactam. These AmpC variants obtained in vitro had either an amino acid deletion in the Ω loop or an Asn-346→Ile replacement in helix H-11 of the mature enzyme. Interestingly, the Asn-346→Ile substitution had already been shown to extend the hydrolysis spectrum of the plasmid-borne CMY-10 β-lactamase.4 Moreover, this replacement occurred in the vicinity of the R2 binding site, which is a hotspot for mutations accounting for an extended hydrolysis spectrum.5 Indeed, extended-spectrum AmpC β-lactamases, which constitute an emerging mechanism of resistance that compromises the clinical activity of extended-spectrum cephalosporins, including cefepime,5 and carbapenems when combined with outer membrane impermeability,6 derive mainly from wild-type cephalosporinases by structural alterations in the R2 binding site that accommodates the R2 lateral side chain of β-lactams. Taken together, these findings suggest that acquired resistance to avibactam and extension of the hydrolysis spectrum of class C β-lactamases might result from the same structural changes. It prompted us to investigate the inhibitory activity of avibactam against extended-spectrum AmpC β-lactamases containing structural changes in the R2 binding site. Six isogenic Escherichia coli recombinant clones—E. coli TOP10 pEC14, E. coli TOP10 pEC16, E. coli TOP10 pEC17, E. coli TOP10 pEC18, E. coli TOP10 pMEV and E. coli TOP10 pBER—that have been characterized in previous work6–8 were tested in this study. They expressed extended-spectrum AmpC enzymes with representative structural alterations in the R2 binding site, such as the Val-298→Leu and His-296→Pro replacements,7 the Ser-287→Asn and Ser-287→Cys substitutions,7 the Ser-282 duplication6 and the tandem duplication of the alanine residues at positions 294 and 295.8 All these modifications occurred in the R2 loop7 or the H-9 and H-10 helices,6–8 which are secondary structures surrounding the R2 binding site.5 The MICs of ceftazidime, cefepime and imipenem were determined using an agar dilution technique on Mueller–Hinton agar (Sanofi Diagnostics Pasteur, Paris, France) plates and Mueller–Hinton agar supplemented with 4 mg/L avibactam with an inoculum of 104 cfu per spot, and were interpreted according to the guidelines of the CLSI.9 Results are shown in Table 1. Avibactam restored the susceptibility to ceftazidime and cefepime of all recombinant clones, thus confirming its potency against these variant enzymes. These results are in agreement with the crystallographic study carried out by Lahiri et al.,10 which showed the weak interaction between avibactam and the R2 binding site of the AmpC β-lactamase of Pseudomonas aeruginosa. According to that study, the carboxamide and sulphate groups of avibactam, and the carbonyl oxygen of the acylated compound, did not interact with the R2 binding site of the AmpC enzyme. By contrast, the hydrophobic side chain of the Ala-293 residue, which is located in the R2 loop, interacted with the piperidine ring of the inhibitor.10 MICs of ceftazidime, cefepime and imipenem alone and in combination with avibactam for extended-spectrum AmpC-producing E. coli recombinant clones MICs of ceftazidime, cefepime and imipenem alone and in combination with avibactam for extended-spectrum AmpC-producing E. coli recombinant clones In conclusion, the present study demonstrates that avibactam, which is a novel covalent non-β-lactam β-lactamase inhibitor, remains active against extended-spectrum AmpC β-lactamases, suggesting that this molecule could be used in clinical practice with limited risk of selecting AmpC variants. This study was supported by internal funding. None to declare. This study was presented in part at the Thirty-third Interdisciplinary Meeting of Anti-Infectious Chemotherapy, Paris, France, 2013 (Abstract 334). We thank AstraZeneca Pharmaceuticals (Waltham, MA, USA) for providing avibactam. N. P.-O. has a predoctoral fellowship from Instituto de Salud Carlos III, Ministry of Economy and Competitivity of Spain.