Andreasen Minna Rud, Rick Tim, Alexandersen Nicolai Riff, Hansen Katrine Hartung, Pedersen Martin Schou, Warweitzky Jakob K, Botelho Carolina Mastella, Häussler Susanne, Jelsbak Lotte, Schønning Kristian
Department of Science and Environment, Roskilde University, Roskilde, Denmark.
Department of Clinical Microbiology, Copenhagen University Hospital-Amager and Hvidovre, Copenhagen, Denmark.
J Antimicrob Chemother. 2024 Apr 2;79(4):810-814. doi: 10.1093/jac/dkae033.
An Escherichia coli isolate, WGS1363, showed resistance to piperacillin/tazobactam but susceptibility to cephalosporins and contained a previously unrecognized β-lactamase, CTX-M-255, as the only acquired β-lactamase. CTX-M-255 was identical to CTX-M-27 except for a G239S substitution. Here, we characterize the hydrolytic spectrum of CTX-M-255 and a previously reported β-lactamase, CTX-M-178, also containing a G239S substitution and compare it to their respective parental enzymes, CTX-M-27 and CTX-M-15.
All β-lactamase genes were expressed in E. coli TOP10 and MICs to representative β-lactam-antibiotics were determined. Furthermore, blaCTX-M-15, blaCTX-M-27, blaCTX-M-178 and blaCTX-M-255 with C-terminal His-tag fusions were affinity purified for enzyme kinetic assays determining Michaelis-Menten kinetic parameters against representative β-lactam-antibiotics and IC50s of clavulanate, sulbactam, tazobactam and avibactam.
TOP10-transformants expressing blaCTX-M-178 and blaCTX-M-255 showed resistance to penicillin/β-lactamase combinations and susceptibility to cephalothin and cefotaxime in contrast to transformants expressing blaCTX-M-15 and blaCTX-M-27. Determination of enzyme kinetic parameters showed that CTX-M-178 and CTX-M-255 both lacked hydrolytic activity against cephalosporins and showed impaired hydrolytic efficiency against penicillin antibiotics compared to their parental enzymes. Both enzymes appeared more active against piperacillin compared to benzylpenicillin and ampicillin. Compared to their parental enzymes, IC50s of β-lactamase-inhibitors were increased more than 1000-fold for CTX-M-178 and CTX-M-255.
CTX-M-178 and CTX-M-255, both containing a G239S substitution, conferred resistance to piperacillin/tazobactam and may be characterized as inhibitor-resistant CTX-M β-lactamases. Inhibitor resistance was accompanied by loss of activity against cephalosporins and monobactams. These findings add to the necessary knowledge base for predicting antibiotic susceptibility from genotypic data.
大肠杆菌分离株WGS1363对哌拉西林/他唑巴坦耐药，但对头孢菌素敏感，且含有一种此前未被识别的β-内酰胺酶CTX-M-255，为唯一获得性β-内酰胺酶。CTX-M-255与CTX-M-27相同，只是存在G239S替换。在此，我们对CTX-M-255和此前报道的同样含有G239S替换的β-内酰胺酶CTX-M-178的水解谱进行了表征，并将其与其各自的亲本酶CTX-M-27和CTX-M-15进行比较。
所有β-内酰胺酶基因均在大肠杆菌TOP10中表达，并测定对代表性β-内酰胺抗生素的最低抑菌浓度（MIC）。此外，对带有C端His标签融合的blaCTX-M-15、blaCTX-M-27、blaCTX-M-178和blaCTX-M-255进行亲和纯化，用于酶动力学测定，以确定针对代表性β-内酰胺抗生素的米氏动力学参数以及克拉维酸、舒巴坦、他唑巴坦和阿维巴坦的半数抑制浓度（IC50）。
与表达blaCTX-M-15和blaCTX-M-27的转化体相比，表达blaCTX-M-178和blaCTX-M-255的TOP10转化体对青霉素/β-内酰胺酶组合耐药，而对头孢噻吩和头孢噻肟敏感。酶动力学参数测定表明，与它们的亲本酶相比，CTX-M-178和CTX-M-255均缺乏对头孢菌素的水解活性，且对青霉素类抗生素的水解效率受损。与苄青霉素和氨苄青霉素相比，这两种酶对哌拉西林的活性似乎更高。与它们的亲本酶相比，CTX-M-178和CTX-M-255的β-内酰胺酶抑制剂的IC50增加了1000倍以上。
均含有G239S替换的CTX-M-178和CTX-M-255赋予了对哌拉西林/他唑巴坦的耐药性，可被表征为耐抑制剂的CTX-Mβ-内酰胺酶。抑制剂耐药性伴随着对头孢菌素和单环β-内酰胺类药物活性的丧失。这些发现为从基因型数据预测抗生素敏感性增添了必要的知识库。
