Sun Yue, Han Renru, Ding Li, Yang Yang, Guo Yan, Wu Shi, Hu Fupin, Yin Dandan
Institute of Antibiotics, Huashan Hospital, Fudan University, Shanghai, People's Republic of China.
Key Laboratory of Clinical Pharmacology of Antibiotics, Ministry of Health, Shanghai, People's Republic of China.
Infect Drug Resist. 2021 Dec 31;14:5725-5733. doi: 10.2147/IDR.S340662. eCollection 2021.
OXA-10-type class D β-lactamases have shown their evolutionary potential of enhancing carbapenem resistance. This study aimed to elucidate the role of OXA-10 variants in clinical isolated multidrug resistant (MDR) and characterize the first appearance of OXA-677 in China.
Six -positive strains were screened by PCR from 41 strains, which were resistant to both carbapenems and ceftazidime-avibactam, collected across China in 2018. The minimum inhibitory concentrations (MIC) were determined with the broth microdilution method. The resistance-associated genes and genetic environment were investigated by whole-genome sequencing (WGS). The function and mechanism of OXA-677 β-lactamase were identified by molecular cloning and protein structure modeling.
All the -positive were MDR strains. They also had outer membrane porin defects and produced β-lactam resistance gene fluoroquinolone-resistant gene , aminoglycoside-resistance gene and , fosfomycin-resistance gene , sulfamethoxazole-resistance gene , and chloramphenicol-resistance gene . All variants were located in a Tn1403-related transposon, containing - -, and - gene cassette arrays, respectively. Notably, the producer showed a high MIC level of meropenem (MIC>64 mg/L). Compared to , was found a G-to-T transversion at position 350, leading to a phenylalanine-for-valine substitution in position 117, which is closer to leucine155 in the omega loop of the active site. MIC of meropenem for DH5α with the recombinant plasmid pHSG398 carrying was elevated by 8 times.
We speculate that the OXA-10-like enzymes and the decrease of membrane permeability confer carbapenem resistance, and the V117 substitution in OXA-677 might lead to a higher resistance level of meropenem.
OXA-10型D类β-内酰胺酶已显示出增强对碳青霉烯类耐药性的进化潜力。本研究旨在阐明OXA-10变体在临床分离的多重耐药（MDR）菌株中的作用，并鉴定中国首次出现的OXA-677。
从2018年在中国各地收集的41株对碳青霉烯类和头孢他啶-阿维巴坦均耐药的菌株中，通过PCR筛选出6株阳性菌株。采用肉汤微量稀释法测定最低抑菌浓度（MIC）。通过全基因组测序（WGS）研究耐药相关基因和基因环境。通过分子克隆和蛋白质结构建模鉴定OXA-677β-内酰胺酶的功能和机制。
所有阳性菌株均为MDR菌株。它们还存在外膜孔蛋白缺陷，并产生β-内酰胺耐药基因、氟喹诺酮耐药基因、氨基糖苷耐药基因和、磷霉素耐药基因、磺胺甲恶唑耐药基因和氯霉素耐药基因。所有OXA-10变体均位于与Tn1403相关的转座子中，分别包含、和基因盒阵列。值得注意的是，产OXA-677的菌株对美罗培南显示出高MIC水平（MIC>64mg/L）。与OXA-10相比，OXA-677在第350位发生了G到T的颠换，导致第117位的缬氨酸被苯丙氨酸取代，这一位置更靠近活性位点ω环中的亮氨酸155。携带OXA-677的重组质粒pHSG398转化的DH5α对美罗培南的MIC升高了8倍。
我们推测，OXA-10样酶和膜通透性降低赋予了对碳青霉烯类的耐药性，OXA-677中的V117取代可能导致对美罗培南更高的耐药水平。
