Unidad intercentros de Enfermedades Infecciosas, Microbiología y Medicina Preventiva, Hospital Universitario Virgen Macarena, Seville, Spain; Instituto de Biomedicina de Sevilla IBIS, Hospital Universitario Virgen del Rocío/CSIC/Universidad de Sevilla, Seville, Spain.
Instituto de Biomedicina de Sevilla IBIS, Hospital Universitario Virgen del Rocío/CSIC/Universidad de Sevilla, Seville, Spain; Departmento de Microbiología, Universidad de Sevilla, Seville, Spain; Red Española de Investigación en Patología Infecciosa (REIPI), Instituto de Salud Carlos III, Madrid, Spain.
Clin Microbiol Infect. 2017 May;23(5):325-331. doi: 10.1016/j.cmi.2016.12.022. Epub 2017 Jan 3.
Fosfomycin is re-evaluated as a treatment of multidrug-resistant Enterobacteriaceae infections. However, MIC differences have been described among the different susceptibility testing. The aim was to study the role of the different inoculum size used in agar dilution with respect to broth microdilution, according to CLSI, in the fosfomycin MIC discrepancies.
Fosfomycin MICs were determined using agar dilution (reference) and broth microdilution in 220 Escherichia coli (n=81) and Klebsiella pneumoniae (n=139) clinical isolates. Fosfomycin mutant frequencies were determined in 21 E. coli (MIC=1mg/L) and 21 K. pneumoniae (MIC=16mg/L). The emergence of resistant subpopulations of five E. coli strains (MIC=1mg/L) was monitored over the time by microdilution assay using 0, 4 and 8 mg/L of fosfomycin, and eight different inocula (5×10-3.91×10 CFU/well, 1 : 2 dilutions).
For E. coli, 86.4% of categorical agreement (CA), 9.1% very major errors (VME), 3.3% major errors (ME) and 9.9% minor errors (mE) were found. For K. pneumoniae, CA was 51.1%, VME 15.7%, ME 28.4% and mE 25.2%. Essential agreement (±1-log) was observed in 55.45%. By microdilution, 35.9% of the MICs showed discrepancies of ≥2 dilutions. Initial inoculum used was 5.63 times higher in the microdilution method, in range with CLSI methodology for both techniques. Fosfomycin mutant frequencies were 6.05×10 (4×MIC) to 5.59×10 (256×MIC) for E. coli, and 1.49×10 (4×MIC) to 1.58×10 (16×MIC) for K. pneumoniae. Resistant subpopulations arose mainly after 8 h of incubation with inocula >3.13×10 CFU/well.
The higher inoculum used in the microdilution method enriched the initial inoculum with resistant subpopulations and could partially explain the fosfomycin MIC discrepancies with respect to the agar dilution method.
磷霉素作为治疗多重耐药肠杆菌科感染的药物正在重新评估。然而，不同的药敏试验方法已经描述了 MIC 差异。本研究旨在根据 CLSI 标准，研究琼脂稀释法和肉汤微量稀释法中不同接种量对磷霉素 MIC 差异的影响。
采用琼脂稀释法（参考方法）和肉汤微量稀释法测定 220 株大肠埃希菌（n=81）和肺炎克雷伯菌（n=139）临床分离株的磷霉素 MIC。测定 21 株大肠埃希菌（MIC=1mg/L）和 21 株肺炎克雷伯菌（MIC=16mg/L）的磷霉素突变频率。采用微量稀释法，用 0、4 和 8mg/L 的磷霉素，8 个不同的接种量（5×10-3.91×10 CFU/孔，1：2 稀释），监测 5 株大肠埃希菌（MIC=1mg/L）的耐药亚群在时间上的出现情况。
对于大肠埃希菌，分类符合率（CA）为 86.4%，非常大的主要错误（VME）为 9.1%，主要错误（ME）为 3.3%，次要错误（mE）为 9.9%。对于肺炎克雷伯菌，CA 为 51.1%，VME 为 15.7%，ME 为 28.4%，mE 为 25.2%。±1 对数的基本符合率（±1-log）为 55.45%。通过微量稀释法，35.9%的 MIC 差异≥2 个稀释度。微量稀释法中初始接种量比 CLSI 方法高 5.63 倍，两种方法均在范围内。大肠埃希菌的磷霉素突变频率为 6.05×10（4×MIC）至 5.59×10（256×MIC），肺炎克雷伯菌的磷霉素突变频率为 1.49×10（4×MIC）至 1.58×10（16×MIC）。在接种量>3.13×10 CFU/孔的情况下孵育 8 小时后，主要出现耐药亚群。
微量稀释法中使用的较高接种量使初始接种物中富集了耐药亚群，这可能部分解释了与琼脂稀释法相比，磷霉素 MIC 差异的原因。
