Tania Blanco-Martín, Lucía González-Pinto, Pablo Aja-Macaya, Salud Rodríguez-Pallares, Lucía Sánchez-Peña, Eva Gato, María Del Carmen Fernández-López, Michelle Outeda-García, Arianna Rodríguez-Coello, Rosa Pedraza-Merino, Isaac Alonso-García, Juan Carlos Vázquez-Ucha, Luis Martínez-Martínez, Jorge Arca-Suárez, Alejandro Beceiro, Germán Bou
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We analyzed imipenem and imipenem/relebactam single-step mutant frequencies, resistance development trajectories and differentially selected resistance mechanisms using two representative <i>K. pneumoniae</i> isolates that had developed ceftazidime/avibactam resistance during therapy (ST512/KPC-31 and ST258/KPC-35). Mutant frequencies and mutant prevention concentrations were measured in Mueller-Hinton agar plates containing incremental concentrations of imipenem or imipenem/relebactam. Resistance dynamics were determined after incubation for 7 days in 10 mL MH tubes containing incremental concentrations of each antibiotic or combination, up to 64 times their baseline MIC. Two colonies per strain from each experiment were characterized by antimicrobial susceptibility testing and whole genome sequencing. The impact of KPC variants identified in resistant mutants on β-lactam resistance was investigated by cloning experiments. Imipenem/relebactam suppressed the emergence of resistant mutants at lower concentrations than imipenem, slowed down resistance development for both strains, and the resulting mutants yielded lower MICs of carbapenems and carbapenem/β-lactamase inhibitors than those selected with imipenem alone. Characterization of resistant mutants revealed that imipenem resistance was mainly caused by inactivation of OmpK36 and mutations in the KPC β-lactamase. Imipenem/relebactam-resistant mutants also maintained OmpK36 alterations, but mutations in KPC were much less frequent compared with those selected with imipenem alone. Genetic and biochemical characterization of the KPC derivatives identified in the resistant mutants confirmed their role in carbapenem resistance. Our data positions imipenem/relebactam as an attractive therapeutic option for combating ceftazidime/avibactam-resistant KPC-producing <i>K. pneumoniae</i> infections.</p>","PeriodicalId":8152,"journal":{"name":"Antimicrobial Agents and Chemotherapy","volume":" ","pages":"e0112024"},"PeriodicalIF":4.1000,"publicationDate":"2024-12-05","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11619344/pdf/","citationCount":"0","resultStr":"{\"title\":\"Mutant prevention concentrations, <i>in vitro</i> resistance evolution dynamics, and mechanisms of resistance to imipenem and imipenem/relebactam in carbapenem-susceptible <i>Klebsiella pneumoniae</i> isolates showing ceftazidime/avibactam resistance.\",\"authors\":\"Tania Blanco-Martín, Lucía González-Pinto, Pablo Aja-Macaya, Salud Rodríguez-Pallares, Lucía Sánchez-Peña, Eva Gato, María Del Carmen Fernández-López, Michelle Outeda-García, Arianna Rodríguez-Coello, Rosa Pedraza-Merino, Isaac Alonso-García, Juan Carlos Vázquez-Ucha, Luis Martínez-Martínez, Jorge Arca-Suárez, Alejandro Beceiro, Germán Bou\",\"doi\":\"10.1128/aac.01120-24\",\"DOIUrl\":null,\"url\":null,\"abstract\":\"<p><p><i>Klebsiella pneumoniae</i> carbapenemase (KPC) variants selected during ceftazidime/avibactam treatment usually develop susceptibility to carbapenems and carbapenem/β-lactamase inhibitors, such as imipenem and imipenem/relebactam. We analyzed imipenem and imipenem/relebactam single-step mutant frequencies, resistance development trajectories and differentially selected resistance mechanisms using two representative <i>K. pneumoniae</i> isolates that had developed ceftazidime/avibactam resistance during therapy (ST512/KPC-31 and ST258/KPC-35). Mutant frequencies and mutant prevention concentrations were measured in Mueller-Hinton agar plates containing incremental concentrations of imipenem or imipenem/relebactam. Resistance dynamics were determined after incubation for 7 days in 10 mL MH tubes containing incremental concentrations of each antibiotic or combination, up to 64 times their baseline MIC. Two colonies per strain from each experiment were characterized by antimicrobial susceptibility testing and whole genome sequencing. The impact of KPC variants identified in resistant mutants on β-lactam resistance was investigated by cloning experiments. 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引用次数: 0
摘要
肺炎克雷伯菌碳青霉烯酶(KPC)变异株在头孢他啶/阿维菌素治疗过程中被筛选出来,通常会对碳青霉烯类和碳青霉烯/β-内酰胺酶抑制剂(如亚胺培南和亚胺培南/雷巴坦)产生敏感性。我们利用两个在治疗过程中对头孢他啶/阿维菌素产生耐药性的代表性肺炎克雷伯菌分离株(ST512/KPC-31 和 ST258/KPC-35),分析了亚胺培南和亚胺培南/雷巴坦单步突变频率、耐药性发展轨迹和不同选择的耐药性机制。在含有递增浓度亚胺培南或亚胺培南/雷巴坦的穆勒-欣顿琼脂平板中测量了突变频率和突变预防浓度。在含有递增浓度亚胺培南或亚胺培南/瑞雷巴坦的 10 mL MH 管中培养 7 天后,测定耐药性动态,最高可达其基线 MIC 的 64 倍。通过抗菌药物敏感性测试和全基因组测序,对每个实验中每个菌株的两个菌落进行了鉴定。通过克隆实验研究了在耐药突变体中发现的 KPC 变体对 β-内酰胺耐药性的影响。与亚胺培南相比,亚胺培南/雷巴坦能以更低的浓度抑制耐药突变体的出现,减缓两种菌株的耐药性发展,而且与单独使用亚胺培南相比,所产生的突变体对碳青霉烯类和碳青霉烯类/β-内酰胺酶抑制剂的 MIC 值更低。耐药突变体的特征显示,亚胺培南耐药主要是由 OmpK36 失活和 KPC β-内酰胺酶突变引起的。亚胺培南/雷巴坦耐药突变体也保持了 OmpK36 的改变,但与仅用亚胺培南筛选出的突变体相比,KPC 突变的频率要低得多。耐药突变体中发现的 KPC 衍生物的遗传和生化特征证实了它们在碳青霉烯类耐药性中的作用。我们的数据表明,亚胺培南/雷巴坦是应对头孢他啶/阿维菌素耐药的 KPC 产肺炎克菌感染的一种有吸引力的治疗选择。
Mutant prevention concentrations, in vitro resistance evolution dynamics, and mechanisms of resistance to imipenem and imipenem/relebactam in carbapenem-susceptible Klebsiella pneumoniae isolates showing ceftazidime/avibactam resistance.
Klebsiella pneumoniae carbapenemase (KPC) variants selected during ceftazidime/avibactam treatment usually develop susceptibility to carbapenems and carbapenem/β-lactamase inhibitors, such as imipenem and imipenem/relebactam. We analyzed imipenem and imipenem/relebactam single-step mutant frequencies, resistance development trajectories and differentially selected resistance mechanisms using two representative K. pneumoniae isolates that had developed ceftazidime/avibactam resistance during therapy (ST512/KPC-31 and ST258/KPC-35). Mutant frequencies and mutant prevention concentrations were measured in Mueller-Hinton agar plates containing incremental concentrations of imipenem or imipenem/relebactam. Resistance dynamics were determined after incubation for 7 days in 10 mL MH tubes containing incremental concentrations of each antibiotic or combination, up to 64 times their baseline MIC. Two colonies per strain from each experiment were characterized by antimicrobial susceptibility testing and whole genome sequencing. The impact of KPC variants identified in resistant mutants on β-lactam resistance was investigated by cloning experiments. Imipenem/relebactam suppressed the emergence of resistant mutants at lower concentrations than imipenem, slowed down resistance development for both strains, and the resulting mutants yielded lower MICs of carbapenems and carbapenem/β-lactamase inhibitors than those selected with imipenem alone. Characterization of resistant mutants revealed that imipenem resistance was mainly caused by inactivation of OmpK36 and mutations in the KPC β-lactamase. Imipenem/relebactam-resistant mutants also maintained OmpK36 alterations, but mutations in KPC were much less frequent compared with those selected with imipenem alone. Genetic and biochemical characterization of the KPC derivatives identified in the resistant mutants confirmed their role in carbapenem resistance. Our data positions imipenem/relebactam as an attractive therapeutic option for combating ceftazidime/avibactam-resistant KPC-producing K. pneumoniae infections.
期刊介绍:
Antimicrobial Agents and Chemotherapy (AAC) features interdisciplinary studies that build our understanding of the underlying mechanisms and therapeutic applications of antimicrobial and antiparasitic agents and chemotherapy.