Department of Microbiology and Immunology, Faculty of Pharmacy, Tanta University, Tanta, Egypt.
Department of Pharmacognosy, Faculty of Pharmacy, Delta University for Science and Technology, International Coastal Road, Gamasa, 11152, Egypt.
Microb Cell Fact. 2024 Aug 8;23(1):221. doi: 10.1186/s12934-024-02421-1.
β-lactam resistance is a significant global public health issue. Outbreaks of bacteria resistant to extended-spectrum β-lactams and carbapenems are serious health concerns that not only complicate medical care but also impact patient outcomes. The primary objective of this work was to express and purify two soluble recombinant representative serine β‑lactamases using Escherichia coli strain as an expression host and pET101/D as a cloning vector. Furthermore, a second objective was to evaluate the potential, innovative, and safe use of galloylquinic acid (GQA) from Copaifera lucens as a potential β-lactamase inhibitor.In the present study, bla and bla represented genes encoding for serine β-lactamases that were cloned from parent isolates of E. coli and K. pneumoniae, respectively, and expression as well as purification were performed. Moreover, susceptibility results demonstrated that recombinant cells became resistant to all test carbapenems (MICs; 64-128 µg/mL) and cephalosporins (MICs; 128-512 µg/mL). The MICs of the tested β-lactam antibiotics were determined in combination with 4 µg/mL of GQA, clavulanic acid, or tazobactam against E. coli strains expressing CTX-M-15 or KPC-2-β-lactamases. Interestingly, the combination with GQA resulted in an important reduction in the MIC values by 64-512-fold to the susceptible range with comparable results for other reference inhibitors. Additionally, the half-maximal inhibitory concentration of GQA was determined using nitrocefin as a β-lactamase substrate. Data showed that the test agent was similar to tazobactam as an efficient inhibitors of the test enzymes, recording smaller IC values (CTX-M-15; 17.51 for tazobactam, 28.16 µg/mL for GQA however, KPC-2; 20.91 for tazobactam, 24.76 µg/mL for GQA) compared to clavulanic acid. Our work introduces GQA as a novel non-β-lactam inhibitor, which interacts with the crucial residues involved in β-lactam recognition and hydrolysis by non-covalent interactions, complementing the enzyme's active site. GQA markedly enhanced the potency of β-lactams against carbapenemase and extended-spectrum β-lactamase-producing strains, reducing the MICs of β-lactams to the susceptible range. The β-lactamase inhibitory activity of GQA makes it a promising lead molecule for the development of more potent β-lactamase inhibitors.
β-内酰胺耐药性是一个重大的全球公共卫生问题。对扩展谱β-内酰胺类和碳青霉烯类抗生素耐药的细菌的爆发是严重的健康问题，不仅使医疗复杂化，还影响患者的治疗效果。本研究的主要目的是使用大肠杆菌菌株作为表达宿主和 pET101/D 作为克隆载体，表达和纯化两种可溶性重组代表性丝氨酸β-内酰胺酶。此外，第二个目标是评估来自 Copaifera lucens 的没食子酰奎宁酸（GQA）作为潜在的β-内酰胺酶抑制剂的潜在、创新和安全用途。
在本研究中，bla 和 bla 代表分别从大肠杆菌和肺炎克雷伯菌的亲本分离株中克隆的编码丝氨酸β-内酰胺酶的基因，并进行了表达和纯化。此外，药敏结果表明，重组细胞对所有测试的碳青霉烯类（MICs；64-128μg/mL）和头孢菌素类（MICs；128-512μg/mL）均产生耐药性。用 4μg/mL 的 GQA、克拉维酸或他唑巴坦与表达 CTX-M-15 或 KPC-2-β-内酰胺酶的大肠杆菌菌株一起测定测试β-内酰胺类抗生素的 MIC 值。有趣的是，与 GQA 联合使用可使 MIC 值降低 64-512 倍，达到敏感范围，其他参考抑制剂的结果相当。此外，还使用硝噻吩作为β-内酰胺酶底物测定了 GQA 的半最大抑制浓度。数据表明，该测试剂与他唑巴坦一样，是测试酶的有效抑制剂，记录的 IC 值较小（CTX-M-15；他唑巴坦为 17.51，GQA 为 28.16μg/mL；然而，KPC-2；他唑巴坦为 20.91，GQA 为 24.76μg/mL）与克拉维酸相比。
我们的工作介绍了 GQA 作为一种新型的非β-内酰胺抑制剂，它通过非共价相互作用与涉及β-内酰胺识别和水解的关键残基相互作用，补充了酶的活性位点。GQA 显著增强了碳青霉烯类和扩展谱β-内酰胺酶产生菌株对β-内酰胺类抗生素的效力，将β-内酰胺类抗生素的 MIC 值降低到敏感范围。GQA 的β-内酰胺酶抑制活性使其成为开发更有效的β-内酰胺酶抑制剂的有前途的先导分子。
