Rima Mariam, Emeraud Cecile, Bonnin Rémy A, Gonzalez Camille, Dortet Laurent, Iorga Bogdan I, Oueslati Saoussen, Naas Thierry
Team ReSIST, INSERM U1184, School of Medicine Université Paris-Saclay, LabEx LERMIT, and Joint Research Unit EERA 'Evolution and Ecology of Resistance to Antibiotics', Institut Pasteur-APHP-University Paris Saclay, Le Kremlin-Bicêtre, France.
Bacteriology-Hygiene Unit, Assistance Publique/Hôpitaux de Paris, Bicêtre Hospital, Le Kremlin-Bicêtre, France.
J Antimicrob Chemother. 2021 Jul 15;76(8):2024-2028. doi: 10.1093/jac/dkab142.
OXA-48-producing Enterobacterales have widely disseminated globally with an increasing number of variants identified. Among them, OXA-244 is increasingly reported, despite detection difficulties.
To determine the steady-state kinetic parameters of OXA-244.
The blaOXA-244 gene was amplified, cloned into plasmids p-TOPO and pET41b+, and transformed into Escherichia coli TOP10 for MIC determination and E. coli BL21 DE3 for purification. Steady-state kinetic parameters and IC50s of clavulanic acid, tazobactam and NaCl were determined using purified OXA-244. Molecular modelling was also performed.
A reduction in MICs of temocillin and carbapenems was observed in E. coli expressing OXA-244 as compared with OXA-48. The kinetic parameters revealed a reduced carbapenemase activity of OXA-244 as compared with OXA-48, especially for imipenem, which was 10-fold lower. Similarly, catalytic efficiency (kcat/Km) was reduced by 4-fold and 20-fold for ampicillin and temocillin, respectively. Kinetic parameters for cephalosporins were, however, similar. Molecular modelling studies evidenced the key role of R214 in OXA-48, establishing salt bridges with D159 and with the carboxylate group of the R1 substituent of temocillin. These interactions are not possible with G214 in OXA-244, explaining the reduced affinity of temocillin for this enzyme. The R214G mutation in OXA-244 is also likely to induce changes in the active site's water network that would explain the decrease in the hydrolysis rate of carbapenems.
Our data confirm that the R214G mutation (present in OXA-244) results in reduced carbapenem- and temocillin-hydrolysing activity, confirming the crucial role of residue 214 in the hydrolysis of these substrates by OXA-48-like β-lactamases.
产OXA-48型肠杆菌科细菌已在全球广泛传播，且鉴定出的变体数量不断增加。其中，OXA-244的报道日益增多，尽管其检测存在困难。
确定OXA-244的稳态动力学参数。
扩增blaOXA-244基因，克隆到质粒p-TOPO和pET41b+中，转化到大肠杆菌TOP10中进行最低抑菌浓度（MIC）测定，转化到大肠杆菌BL21 DE3中进行纯化。使用纯化的OXA-244测定克拉维酸、他唑巴坦和氯化钠的稳态动力学参数及半数抑制浓度（IC50）。还进行了分子建模。
与表达OXA-48的大肠杆菌相比，表达OXA-244的大肠杆菌中替莫西林和碳青霉烯类药物的MIC降低。动力学参数显示，与OXA-48相比，OXA-244的碳青霉烯酶活性降低，尤其是对亚胺培南，其活性低10倍。同样，氨苄西林和替莫西林的催化效率（kcat/Km）分别降低了4倍和20倍。然而，头孢菌素的动力学参数相似。分子建模研究证明了R214在OXA-48中的关键作用，它与D159以及替莫西林R1取代基的羧基形成盐桥。在OXA-244中，G214无法形成这些相互作用，这解释了替莫西林对该酶的亲和力降低。OXA-244中的R214G突变也可能导致活性位点水网络的变化，这可以解释碳青霉烯类药物水解速率的降低。
我们的数据证实，R214G突变（存在于OXA-244中）导致碳青霉烯类药物和替莫西林水解活性降低，证实了214位残基在OXA-48样β-内酰胺酶水解这些底物中的关键作用。
