Unidad de Gestión Clínica de Enfermedades Infecciosas, Microbiología y Medicina Preventiva, Hospital Universitario Virgen Macarena, Seville, Spain.
Departamento de Microbiología, Universidad de Sevilla, Seville, Spain.
Antimicrob Agents Chemother. 2021 Feb 17;65(3). doi: 10.1128/AAC.01911-20.
The objectives of this study were to characterize the role of the , , and genes in fosfomycin resistance in and evaluate the use of sodium phosphonoformate (PPF) in combination with fosfomycin. Seven clinical isolates of and the reference strain (ATCC 700721) were used, and their genomes were sequenced. Δ, Δ, and Δ mutants were constructed from two isolates and ATCC 700721. Fosfomycin susceptibility testing was done by the gradient strip method. Synergy between fosfomycin and PPF was studied by checkerboard assay and analyzed using SynergyFinder. Spontaneous fosfomycin mutant frequencies at 64 and 512 mg/liter, activity using growth curves with fosfomycin gradient concentrations (0 to 256mg/liter), and time-kill assays at 64 and 307 mg/liter were evaluated with and without PPF (0.623 mM). The MICs of fosfomycin against the clinical isolates ranged from 16 to ≥1,024 mg/liter. The addition of 0.623 mM PPF reduced fosfomycin MIC between 2- and 8-fold. Deletion of led to a 32-fold decrease. Synergistic activities were observed with the combination of fosfomycin and PPF (most synergistic area at 0.623 mM). The lowest fosfomycin-resistant mutant frequencies were found in Δ mutants, with decreases in frequency from 1.69 × 10 to 1.60 × 10 for 64 mg/liter of fosfomycin. In the final growth monitoring and time-kill assays, fosfomycin showed a bactericidal effect only with the deletion of and not with the addition of PPF. We conclude that gene inactivation leads to a decrease in fosfomycin resistance in The pharmacological approach using PPF did not achieve enough activity, and the effect decreased with the presence of fosfomycin-resistant mutations.
本研究的目的是描述 和 基因在磷霉素耐药中的作用，并评估膦甲酸钠（PPF）与磷霉素联合使用的效果。我们使用了 7 株临床分离的 和参考菌株（ATCC 700721），并对其基因组进行了测序。我们从两株分离株和 ATCC 700721 中构建了 Δ 、Δ 、和 Δ 突变体。采用梯度条法进行磷霉素药敏试验。通过棋盘试验研究磷霉素和 PPF 之间的协同作用，并使用 SynergyFinder 进行分析。在有无 PPF（0.623mM）的情况下，通过生长曲线中磷霉素浓度梯度（0 至 256mg/L）和时间杀伤试验（64 和 307mg/L）评估了 64 和 512mg/L 时磷霉素自发突变频率和 活性。临床分离株对磷霉素的 MIC 值范围为 16 至≥1024mg/L。添加 0.623mM PPF 使磷霉素 MIC 值降低了 2 至 8 倍。缺失 导致 32 倍的下降。磷霉素和 PPF 联合使用具有协同作用（最协同区域在 0.623mM）。在 Δ 突变体中发现最低的磷霉素耐药突变频率，64mg/L 磷霉素时，突变频率从 1.69×10降至 1.60×10。在最终的生长监测和时间杀伤试验中，只有缺失 时，磷霉素才表现出杀菌作用，而添加 PPF 则没有。我们的结论是， 基因失活导致 中磷霉素耐药性降低。使用 PPF 的药理学方法没有达到足够的活性，并且随着耐药突变的存在，效果降低。
