Section of Microbiology, Department of Biomedical and Biotechnological Sciences (BIOMETEC), Microbiologia Medica Molecolare e Antibiotico Resistenza (MMARLab), University of Catania, Catania, Italy.
Front Cell Infect Microbiol. 2022 Jan 20;11:816657. doi: 10.3389/fcimb.2021.816657. eCollection 2021.
Penicillin-resistance among clinical isolates has been recently associated with overexpression or aminoacidic substitutions in low-affinity PBP4. Ceftobiprole (BPR), a new-generation cephalosporin, is a therapeutic option against Here, we present evidence that 4 gene sequence alterations may influence the expression level of the gene and ceftobiprole binding to PBP4 in clinical isolates showing remarkable MDR-phenotypes, and how this could interfere with BPR antibacterial and bactericidal activity. Seven strains from bloodstream infections were analyzed for their antibiotic and β-lactam resistance. BPR bactericidal activity was assessed by time-kill analysis; 4 genes were sequenced and 4 relative expression levels of transcription were performed by RT-qPCR. Five penicillin-resistant ampicillin-susceptible (PRAS) isolates were detected, 4 of which were also BPR non-susceptible (BPR-NS). In the time-kill experiments, BPR exposure resulted in a potent bactericidal activity (3-5 log reduction) at the different concentrations tested. 4 gene sequence analysis revealed some mutations that may account for the changes in PBP4 affinity and MIC increase in the 4 BPR-NS strains (MICs 4-16 mg/L): the deletion of an adenine (A) in the promoter region in all PRAS/BPR-NS strains; 12 different amino acid substitutions, 7 of which were next to the PBP catalytic-sites. The most significant were: T418A, located 6 amino acids (aa) upstream of the catalytic-serine included in the STFK I; L475Q, 7 aa upstream of the SDN II; V606A and the novel Y605H, 13/14 aa upstream of the KTGT III. Taken together, our data showed that elevated BPR MICs were attributable to increased transcription of 4 - associated with a single upstream adenine deletion and PBP4 alterations in the catalytic-site - which might interfere with the formation of the BPR/PBP4 complex. 4 molecular alterations may account for the changes in PBP4 affinity and MIC increase, without affecting BPR activity. Indeed, our dynamic analysis by time-kill assays showed that BPR exerted a bactericidal activity against clinical isolates, despite their MDR phenotypes.
青霉素耐药性最近与低亲和力 PBP4 的过度表达或氨基酸取代有关。头孢托罗（BPR）是一种新型头孢菌素，是治疗具有显著多药耐药表型的临床分离株的治疗选择。在这里，我们提供了证据表明，4 种基因序列改变可能影响基因的表达水平和头孢托罗与 PBP4 的结合，在表现出显著多药耐药表型的临床分离株中，以及这如何干扰 BPR 的抗菌和杀菌活性。分析了 7 株来自血流感染的菌株，以评估其抗生素和β-内酰胺耐药性。通过时间杀伤分析评估 BPR 的杀菌活性；对 4 个基因进行测序，并通过 RT-qPCR 进行 4 个相对转录表达水平的检测。检测到 5 株青霉素耐药性但氨苄西林敏感性（PRAS）分离株，其中 4 株也是头孢托罗非敏感性（BPR-NS）。在时间杀伤实验中，BPR 暴露在不同测试浓度下导致强大的杀菌活性（3-5 对数减少）。4 种基因序列分析显示，一些突变可能导致 4 株 BPR-NS 菌株中 PBP4 亲和力的变化和 MIC 增加（MICs 4-16mg/L）：所有 PRAS/BPR-NS 菌株启动子区域的腺嘌呤（A）缺失；12 种不同的氨基酸取代，其中 7 种位于 PBP 催化部位附近。最显著的是：T418A，位于包含 STFK I 的催化丝氨酸上游 6 个氨基酸（aa）；L475Q，SDN II 上游 7 aa；V606A 和新的 Y605H，KTGT III 上游 13/14 aa。总之，我们的数据表明，BPR MIC 升高归因于 4 的转录增加 - 与单个上游腺嘌呤缺失和催化部位的 PBP4 改变相关 - 这可能干扰 BPR/PBP4 复合物的形成。4 种分子改变可能导致 PBP4 亲和力的改变和 MIC 的增加，而不影响 BPR 的活性。事实上，我们通过时间杀伤试验进行的动态分析表明，尽管临床分离株具有多药耐药表型，但 BPR 仍对其具有杀菌活性。
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