Formation of Proteus mirabilis Persister in Chicken under Gentamicin Sulphate Treatment
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摘要: 为了解奇异变形杆菌持留菌形成特征和防控家禽养殖中细菌性疾病，以鸡源致病菌奇异变形杆菌PM2658为研究对象，通过二倍稀释法测得抗生素对鸡源奇异变形杆菌PM2658的最小抑菌浓度（Minimum inhibitory of concentration，MIC），用斑点培养（spot-plate）法分析PM2658在不同抗生素和营养条件下持留菌形成特征。结果显示，硫酸庆大霉素、头孢噻肟钠、氧氟沙星对PM2658的MIC值分别为1.56、0.78和3.13 μg·mL-1。在16×MIC和100×MIC硫酸庆大霉素处理下，鸡源奇异变形杆菌PM2658都可以形成持留菌，形成率分别为8.04×10-4和1.03×10-6。16×MIC硫酸庆大霉素-4×MIC头孢噻肟钠、16×MIC硫酸庆大霉素-4×MIC氧氟沙星、4×MIC头孢噻肟钠-4×MIC氧氟沙星、16×MIC硫酸庆大霉素-4×MIC头孢噻肟钠-4×MIC氧氟沙星不同抗生素组合处理下，鸡源奇异变形杆菌PM2658的持留菌形成规律与单独硫酸庆大霉素处理的持留菌形成规律一致，也表明鸡源奇异变形杆菌PM2658持留菌具有多重抗生素耐受性。在16×MIC硫酸庆大霉素处理下，与LB和1/2 LB培养条件相比，1/5 LB培养条件下鸡源奇异变形杆菌持留菌形成率最高，比前两者分别提高了17.67倍和5.05倍，说明鸡源奇异变形杆菌持留菌的形成还与营养环境密切相关。持留菌遗传学特性检测结果显示，鸡源奇异变形杆菌PM2658持留菌的遗传学特性并没有发生改变。鸡源奇异变形杆菌确实可以形成持留菌，其形成特征取决于特定的抗生素条件。Abstract: For understanding the biological characteristics of persister formation of Proteus mirabilis, and control and prevention of bacterial diseases in poultry, Proteus mirabilis PM2658 from chicken was used as materials. Minimum inhibitory of concentrations (MIC) of antibiotics to Proteus mirabilis PM2658 were determined by two-fold dilution method. Under different antibiotic treatments and nutritional conditions, the characteristics of persister formation of Proteus mirabilis were determined by spot-plate protocol. Results showed that MIC of gentamicin sulphate (Gen), cefotaxime sodium (Cef) and ofloxacin (Ofx) to Proteus mirabilis PM2658 were 1.56, 0.78 and 3.13 μg·mL-1, respectively. Treated by 16×MIC and 100×MIC of Gen, stationary phase of Proteus mirabilis PM2658 could form persister, and the percentages of persister formation were 8.04×10-4 and 1.03×10-6, respectively. Moreover, the regularity of peresister formation under antibiotic combinations of 16×MIC Gen-4×MIC Cef, 16×MIC Gen-4×MIC Ofx, 4×MIC Cef-4×MIC Ofx, 16×MIC Gen-4×MIC Cef-4×MIC Ofx was the same as that of single Gen treatment, indicating that persister of Proteus mirabilis PM2658 had multiple drug tolerance. The rate of formation of Proteus mirabilis PM2658 persister in 1/5 LB was higher than those in LB and 1/2 LB under 16×MIC Gen treatment, which separately increased by 17.67 times and 5.05 times, which indicated that the formation of Proteus mirabils PM2658 persister was related to nutrition of environment. The heritage of Proteus mirabilis PM2658 persister did not change, which was further determined after every different treatments mentioned above. Taken together, Proteus mirabilis from chicken do form persister, and characteristics of persister formation depends on specific antibiotic.
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Key words:
- Proteus mirabilis /
- drug tolerance /
- persister /
- gentamicin sulphate
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图 1 不同Gen浓度下鸡源奇异变形杆菌PM2658持留菌形成情况
Figure 1. Formation of P. mirabilis PM2658 persisters under various concentrations of Gen
图 2 鸡源奇异变形杆菌PM2658持留菌遗传学特性检测结果
注：A为未经抗生素处理PM2658和经16×MIC和100×MIC Gen处理4 h后持留菌在不含抗生素培养基上的生长情况；B为经过16×MIC和100×MIC Gen处理4 h后持留菌在含有相应浓度Gen培养基上的生长情况。
Figure 2. Growth of P. mirabilis PM2658 persisters on culture media containing different amount of Gen
图 3 鸡源奇异变形杆菌持留菌多重抗生素耐受性
注：A为持留菌对Gen-Cef耐受性；B为持留菌对Gen-Ofx耐受性；C为持留菌对Gen-Cef-Ofx耐受性。
Figure 3. Multi-drug tolerance of P. mirabilis PM2658 persisters
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