Mechanisms and fitness costs of tigecycline resistance in Escherichia coli
Mechanisms and fitness costs of tigecycline resistance in Escherichia coli
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DOI：
10.1093/jac/dkt263
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发表时间：
2013-12-01
影响因子：
5.2
通讯作者：
Andersson, Dan I.
中科院分区：
文献类型：
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作者：
Linkevicius, Marius;Sandegren, Linus;Andersson, Dan I.
目的：体外逐步筛选替加环素耐药大肠埃希菌突变株，测定突变率，鉴定耐药机制，测定对其他抗生素的耐药水平和交叉耐药，评估替加环素耐药机制的适应度成本，并调查临床分离株中是否存在相同的体外鉴定靶基因突变。在补充有替加环素的琼脂平板上选择对替加环素敏感性降低的自发突变体。通过进行MIC测定和使用Luria-Delbruck波动试验测定突变率来评价耐药性水平和交叉耐药性。通过测量指数生长速率、滞后期和总产量来估计突变体适合度。Illumina全基因组测序用于鉴定增加替加环素MICs的突变。结果：对替加环素敏感性降低的自发突变体以每代每个细胞10（-8）至10（-6）的相似速率被选择;然而，未达到临床MIC折点。替加环素的耐药水平较低，一些突变株对疏水性药物（氯霉素、红霉素和新生霉素）的MIC升高，或对SOS反应诱导剂（环丙沙星和呋喃妥因）的MIC降低。在外排调控网络基因（lon、acrR和马尔R）或脂多糖核心生物合成途径基因（lpcA、rfaE、rfaD、rfaC和rfaF）中鉴定出突变。在临床分离株中发现了相同靶基因的突变。结论：替加环素选择具有相对高突变率的低水平耐药突变，其中大多数具有相当大的适应性成本。需要进一步的体内实验来评估这些突变如何影响细菌的毒力和建立成功感染的能力。
Objectives: To stepwise select tigecycline-resistant Escherichia coli mutants in vitro, determine the mutation rates, identify the resistance mechanisms, determine the resistance level and cross-resistance to other antibiotic classes, evaluate the fitness costs of tigecycline resistance mechanisms and investigate if the same in vitro-identified target genes were mutated in clinical isolates.Methods: Spontaneous mutants with reduced susceptibility to tigecycline were selected on agar plates supplemented with tigecycline. Resistance levels and cross-resistance were evaluated by performing MIC assays and determining mutation rates using Luria-Delbruck fluctuation tests. Mutant fitness was estimated by measuring exponential growth rates, lag phase and total yield. Illumina whole-genome sequencing was used to identify mutations increasing MICs of tigecycline.Results: Spontaneous mutants with reduced susceptibility to tigecycline were selected at a rate of similar to 10(-8) to 10(-6) per cell per generation; however, the clinical MIC breakpoint was not reached. The resistance level of tigecycline was low and some of the mutants had elevated MICs of hydrophobic drugs (chloramphenicol, erythromycin and novobiocin) or decreased MICs of SOS response inducers (ciprofloxacin and nitrofurantoin). Mutations were identified in efflux regulatory network genes (lon, acrR and marR) or lipopolysaccharide core biosynthesis pathway genes (lpcA, rfaE, rfaD, rfaC and rfaF). Mutations in the same target genes were found in clinical isolates.Conclusions: Tigecycline selects for low-level resistance mutations with relatively high mutation rates and the majority of them come with a substantial fitness cost. Further in vivo experiments are needed to evaluate how these mutations affect bacterial virulence and ability to establish a successful infection.