Xu Su, Fu Zhuyingjie, Zhou Ying, Liu Yang, Xu Xiaogang, Wang Minggui
Institute of Antibiotics, Huashan Hospital, Fudan UniversityShanghai, China.
Key Laboratory of Clinical Pharmacology of Antibiotics, National Health and Family Planning CommissionShanghai, China.
Front Microbiol. 2017 May 19;8:914. doi: 10.3389/fmicb.2017.00914. eCollection 2017.
With the increasing spread of methicillin-resistant worldwide, fosfomycin has begun to be used more often, either alone or in combination with other antibiotics, for treating methicillin-resistant infections, resulting in the emergence of fosfomycin-resistant strains. Fosfomycin resistance is reported to be mediated by fosfomycin-modifying enzymes (FosA, FosB, FosC, and FosX) and mutations of the target enzyme MurA or the membrane transporter proteins UhpT and GlpT. Our previous studies indicated that the genes might not the major fosfomycin resistance mechanism in , whereas mutations of and seemed to be more related to fosfomycin resistance. However, the precise role of these two genes in fosfomycin resistance remains unclear. The aim of the present study was to investigate the role of and in fosfomycin resistance. Homologous recombination was used to knockout the and genes in Newman. Gene complementation was generated by the plasmid pRB473 carrying these two genes. The fosfomycin minimal inhibitory concentration (MIC) of the strains was measured by the -test to observe the influence of gene deletion on antibiotic susceptibility. In addition, growth curves were constructed to determine whether the mutations have a significant influence on bacterial growth. Deletion of , and both of them led to increased fosfomycin MIC 0.5 μg/ml to 32 μg/ml, 4 μg/ml, and >1024 μg/ml, respectively. By complementing and into the deletion mutants, the fosfomycin MIC decreased from 32 to 0.5 μg/ml and from 4 to 0.25 μg/ml, respectively. Moreover, the transporter gene-deleted strains showed no obvious difference in growth curves compared to the parental strain. In summary, our study strongly suggests that mutations of and lead to fosfomycin resistance in , and that mutation may play a more important role. The high resistance and low biological fitness cost resulting from and deletion suggest that these strains might have an evolutionary advantage in a fosfomycin-rich clinical situation, which should be closely monitored.
随着耐甲氧西林金黄色葡萄球菌在全球范围内的日益传播，磷霉素已开始更频繁地单独或与其他抗生素联合用于治疗耐甲氧西林金黄色葡萄球菌感染，这导致了耐磷霉素菌株的出现。据报道，磷霉素耐药性是由磷霉素修饰酶（FosA、FosB、FosC和FosX）以及靶酶MurA或膜转运蛋白UhpT和GlpT的突变介导的。我们之前的研究表明，某些基因可能不是金黄色葡萄球菌中主要的磷霉素耐药机制，而某些基因和的突变似乎与磷霉素耐药性更相关。然而，这两个基因在金黄色葡萄球菌磷霉素耐药性中的确切作用仍不清楚。本研究的目的是调查和在金黄色葡萄球菌磷霉素耐药性中的作用。采用同源重组技术敲除金黄色葡萄球菌Newman中的和基因。通过携带这两个基因的质粒pRB473进行基因互补。通过肉汤稀释法测定菌株的磷霉素最小抑菌浓度（MIC），以观察基因缺失对抗生素敏感性的影响。此外，构建生长曲线以确定这些突变是否对细菌生长有显著影响。单独敲除和同时敲除两者导致磷霉素MIC分别从0.5μg/ml增加到32μg/ml、4μg/ml和>1024μg/ml。通过将和互补到缺失突变体中，磷霉素MIC分别从32μg/ml降至0.5μg/ml和从4μg/ml降至0.25μg/ml。此外，与亲本菌株相比，转运蛋白基因缺失的菌株在生长曲线上没有明显差异。总之，我们的研究强烈表明，和的突变导致金黄色葡萄球菌对磷霉素耐药，并且突变可能起更重要的作用。和缺失导致的高耐药性和低生物学适应性成本表明，这些菌株在富含磷霉素的临床环境中可能具有进化优势，应密切监测。
