Evaluation of antibiotic susceptibilities of Ehrlichia canis, Ehrlichia chaffeensis, and Anaplasma phagocytophilum by real-time PCR
Evaluation of antibiotic susceptibilities of Ehrlichia canis, Ehrlichia chaffeensis, and Anaplasma phagocytophilum by real-time PCR
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DOI：
10.1128/aac.48.12.4822-4828.2004
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发表时间：
2004-12-01
影响因子：
4.9
通讯作者：
Raoult, D
中科院分区：
文献类型：
--
作者：
Branger, S;Rolain, JM;Raoult, D
采用实时荧光定量聚合酶链式反应测定抗生素对巨噬细胞无形体、查菲埃利希菌和犬埃立克体的最低抑菌浓度。建立了细菌的倍增时间：查菲埃希菌19h，吞噬艾美耳球虫26h，犬埃希氏菌28h。与使用Diff-Quick染色确定灵敏度的参考方法相比，我们的PCR方法非常灵敏和特异。我们证实多西环素和利福平对这些细菌有很高的活性，并发现对氟喹诺酮类药物有不同的敏感性；吞噬弧菌对氟喹诺酮类药物敏感，而犬埃希菌和查菲埃希菌只对部分敏感。β-内酰胺类化合物、复方新诺明、大环内酯类化合物和泰利霉素对上述三种微生物均无抗菌活性。甲氧西林的活性高于氯霉素。我们首次发现这三个物种的23S RNA基因存在大量的点突变，其中第754、2057、2058、2059和2611位(大肠杆菌编号)的突变是已知的，在其他细菌中对大环内酯类化合物具有耐药性。这些突变中的每一个在对这些药物的耐药性中的作用应该在未来进行调查。我们的研究证实了先前的报道，即定量聚合酶链式反应是确定抗生素敏感性的可靠方法；因此，它可能有助于筛选新药。
We determined MICs of antibiotics against Anaplasma phagocytophilum, Ehrlichia chaffeensis, and Ehrlichia canis by real-time quantitative PCR. The doubling times of the organisms were established: 19 h for E. chaffeensis, 26 h for A. phagocytophilum, and 28 h for E. canis. In comparison to the reference method for determining sensitivities, which uses Diff-Quick staining, our PCR assay was very sensitive and specific. We confirmed that doxycycline and rifampin are highly active against these bacteria and found variable susceptibilities to fluoroquinolones; A. phagocytophilum was susceptible, but E. canis and E. chaffieensis were only partly susceptible. P-Lactam compounds, cotrimoxazole, macrolide compounds, and telithromycin showed no activity against any of the three organisms. Thiamphenicol was found to be more active than chloramphenicol. For the first time, we showed that these three species have numerous point mutations in their 23S RNA genes, with those at positions 754, 2057, 2058, 2059, and 2611 (Escherichia coli numbering) known to confer resistance to macrolide compounds in other bacteria. The role of each of these mutations in resistance to these drugs should be investigated in the future. Our study confirms previous reports that quantitative PCR is a reliable method for determining antibiotic susceptibility; therefore, it might be useful for screening new drugs.