Prevalent extended-spectrum beta-lactamases in Argentina: structural insights into the interaction with oxyimino-cephalosporins and mechanism-based inhibitors

P. POWER; P. CHARLIER; M. RUGGIERO; B. GHIGLIONE; M. GALLENI; G. GUTKIND; E. SAUVAGE

Las Palmas de Gran Canaria

Congreso; 12th Beta-Lactamase Meeting; 2014

The occurrence of bacterial isolates recovered from clinical specimens displaying high resistance levels to beta-lactams has been kept almost invariable during the last years in Argentina. These resistance microorganisms produce almost exclusively CTX-M b-lactamases and, at a lower degree, PER-2, which is an infrequent enzyme in other parts of the world. Within the CTX-M b-lactamases family, diversification of their sequences led to the emergence of variants responsible for decreased susceptibility to ceftazidime, being the Asp240Gly mutation the most prevalent among the so called ceftazidimases. For PER-2, we recently observed that mutations at Arg220 (equivalent to Arg244 in IRT-TEMs) have an impact on the inhibition susceptibility of PER-2, as well as towards the substrate profile. Therefore, we solved the crystallographic structures of PER-2 (2.1 Å) and CTX-M-96 (1.2 Å), and we were able to analyze in detail the organization of the active site and to evaluate the possible role of key amino acid residues in the overall stabilization of the structure and also in the interaction with different b-lactams and mechanism-based inhibitors. Compared to other CTX-M beta-lactamases, CTX-M-96 presents some differences in the disposition of specific secondary structures: Thr51-Gln56, His141-Pro145, Pro226-Val230, and Pro252-Ala257; and residues whose side chains point towards opposite directions: Arg94, Glu158, Arg191, Asn192. In the absence of oxyimino-cephalosporins (OC), Asn132, Glu166, Pro167 and Asn170 seem to be shifted up to 0.7 Å away from the catalytic cleft, suggesting that the presence of antibiotic induces the approach of these residues towards the OC through hydrogen bonds. Structural differences do not seem to be conclusive to determine the ceftazidimase behavior, and additional data are still needed to explain the observed in vivo resistance to OC. In PER-2, B3 strand is shifted up to ca. 5 Å towards the catalytic cleft in comparison with other class A b-lactamases, creating an apparently more favorable environment for stabilization of molecules like the oxyimino-cephalosporins through hydrogen bonds. This strand seems to be also stabilized by hydrogen bonds between Thr237 and Arg220, and mutations in the latter could disrupt the network leading to the already observed differences in the hydrolytic/inhibition behavior towards several drugs, especially cefotaxime, ceftazidime, benzyl-penicillin and clavulanic acid. The structural evidences suggest that the hydrogen-bond network including Arg220-Thr237-Ser238-b-lactam is important for the proper activity and inhibition of the enzyme. Mutations in these positions seem to also affect the whole hydrolytic behavior.