Therapeutic effect of phages on extensively drug-resistant Acinetobacter baumannii-induced sepsis in mice
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摘要: 目的 研究噬菌体对泛耐药鲍氏不动杆菌所致脓毒症小鼠的治疗效果。 方法 (1)将60只BALB/c小鼠,按随机数字表法分为空白对照组、脓毒症对照组、抗生素治疗组、噬菌体治疗组、噬菌体对照组,每组12只。空白对照组小鼠腹腔(注射部位下同)注射生理盐水1 mL;脓毒症对照组、抗生素治疗组、噬菌体治疗组小鼠注射5×107 CFU/mL泛耐药鲍氏不动杆菌(菌株分离自笔者单位收治的严重烧伤患者血液)1 mL建立脓毒症模型,2 h后,脓毒症对照组、抗生素治疗组、噬菌体治疗组小鼠分别注射1 mL生理盐水、1 mg/mL亚胺培南/西司他丁、1×108噬斑形成单位(PFU)/mL根据前述鲍氏不动杆菌筛选的噬菌体(下同);噬菌体对照组小鼠注射1×108 PFU/mL噬菌体1 mL。各组存活小鼠连续注射7 d,每天观察存活情况并统计1周存活比。(2)另取60只BALB/c小鼠,同实验(1)分组处理,各组存活小鼠连续注射5 d。实验第2、4、6天,每组选3只小鼠(取材时存活小鼠不足3只组,取所有存活小鼠),每只鼠取血检测白细胞计数,各组各时相点样本数为3。实验第2天取血小鼠,另取血行细菌培养;另取小鼠肺、肝、肾、脾组织,匀浆、稀释后加入LB固体培养基中行细菌培养,行菌落计数后计算细菌含量。对数据行Wilcoxon秩和检验、单因素方差分析、LSD检验及Kruskal-Wallis秩和检验。 结果 (1)实验第7天,空白对照组、脓毒症对照组、抗生素治疗组、噬菌体治疗组、噬菌体对照组小鼠存活数分别为12、0、8、10、12只。与脓毒症对照组比较,其余4组小鼠存活比明显升高(
Z 值为55.635~106.593,P 值均小于0.05);噬菌体治疗组小鼠存活比稍高于抗生素治疗组,差异无统计学意义(Z =2.797,P >0.05)。(2)实验第2天,空白对照组、噬菌体治疗组、噬菌体对照组小鼠白细胞计数相近,分别为(5.60±0.94)×109/L、(5.16±0.36)×109/L、(5.26±1.89)×109/L,3组均明显低于脓毒症对照组的(8.64±0.64)×109/L(P <0.05或P <0.01),且后2组明显低于抗生素治疗组的(7.80±1.76)×109/L(P 值均小于0.05)。实验第4天,抗生素治疗组、噬菌体治疗组、噬菌体对照组小鼠白细胞计数相近,明显低于空白对照组(P <0.05或P <0.01),前述4组白细胞计数均明显低于脓毒症对照组(P 值均小于0.01)。实验第6天,空白对照组、抗生素治疗组、噬菌体治疗组、噬菌体对照组小鼠白细胞计数总体比较,差异无统计学意义(χ 2=4.128,P >0.05)。实验第2天,空白对照组、脓毒症对照组、抗生素治疗组、噬菌体治疗组、噬菌体对照组分别有0、12、7、2、0只小鼠血细菌培养结果为阳性。空白对照组、噬菌体治疗组、噬菌体对照组小鼠血细菌培养阳性比显著低于脓毒症对照组(χ 2值为-30.000~30.000,P 值均小于0.01)。抗生素治疗组小鼠血细菌培养阳性比显著高于空白对照组和噬菌体对照组(χ 2值分别为17.500、-17.500,P 值均小于0.05)。实验第2天,除噬菌体治疗组小鼠肾组织外,空白对照组、噬菌体治疗组、噬菌体对照组小鼠各脏器组织细菌含量均显著低于脓毒症对照组(χ 2值为-9.000~9.000,P 值均小于0.01);抗生素治疗组小鼠肾组织细菌含量显著高于空白对照组和噬菌体对照组(χ 2值分别为-7.500、7.500,P 值均小于0.05)。 结论 噬菌体治疗泛耐药鲍氏不动杆菌所致脓毒症小鼠,能明显提高其存活比,控制其炎症反应,有效清除肺、肝、脾、肾中细菌。Abstract: Objective To study the therapeutic effect of phages on extensively drug-resistantAcinetobacter baumannii -induced sepsis in mice. Methods (1) Sixty BALB/c mice were divided into blank control group, sepsis control group, antibiotics treatment group, phage treatment group, and phage control group according to the random number table, with 12 mice in each group. Mice in blank control group were intraperitoneally (the same injection position below) injected with 1 mL normal saline. Mice in sepsis control group, antibiotics treatment group, and phage treatment group were injected with 1 mL extensively drug-resistantAcinetobacter baumannii (the strain was isolated from the blood of a severely burned patient hospitalized in our unit) in the concentration of 5×107 colony-forming unit/mL to reproduce sepsis model. Two hours later, mice in sepsis control group, antibiotics treatment group, and phage treatment group were injected with 1 mL saline, 1 mg/mL imipenem/cilastatin, and 1×108 plaque-forming unit (PFU)/mL phages screened based on above-mentionedAcinetobacter baumannii (the same phages below) respectively. Mice in phage control group were injected with 1 mL phages in the titer of 1×108 PFU/mL. The injection was performed continuously for 7 days in each living mouse, and the survival situation of mice was observed each day to calculate the survival ratio in one week. (2) Another 60 BALB/c mice were grouped and treated as in experiment (1), and the injection was performed continuously for 5 days in each living mouse. On experiment day 2, 4, and 6, 3 mice from each group were selected (if the number of survived mouse in any group was less than 3 at sample collecting, all the survived mice were selected), and blood was drawn to determine white blood cell count (WBC, with 3 samples at each time point in each group). On experiment day 2, blood was drawn from the mice that had their blood taken earlier for bacterial culture, and lung, liver, kidney, and spleen tissue was collected from the same mice. The tissue samples were added to the LB solid medium after being homogenized and diluted for bacterial culture. The content of bacteria was calculated after the bacterial colony number was counted. Data were processed Wilcoxon rank sum test, one-way analysis of variance, LSD test and Kruskal-Wallis rank sum test. Results (1) On experiment day 7, there were 12, 8, 10, and 12 mice survived in blank control group, antibiotics treatment group, phage treatment group, and phage control group respectively, while no mouse survived in sepsis control group. Compared with that in sepsis control group, the survival ratio of mice was significantly higher in the other four groups (withZ values from 55.635 to 106.593,P values below 0.05). The survival ratio of mice in phage treatment group was slightly higher than that in antibiotics treatment group, without statistically significant difference (Z =2.797,P >0.05). (2) On experiment day 2, WBC data of mice in blank control group, phage treatment group, and phage control group were close[respectively (5.60±0.94)×109/L, (5.16±0.36)×109/L, and (5.26±1.89)×109/L], all significantly lower than the datum in sepsis control group[(8.64±0.64)×109/L,P <0.05 orP <0.01], and the WBC data in the latter two groups were significantly lower than the datum in antibiotics treatment group[(7.80±1.76)×109/L, withP values below 0.05]. On experiment day 4, WBC data of mice in antibiotics treatment group, phage treatment group, and phage control group were close, all significantly lower than the datum in blank control group (P <0.05 orP <0.01), and WBC data in the above-mentioned four groups were all lower than the datum in sepsis control group (withP values below 0.01). On experiment day 6, there was no statistically significant difference in WBC among blank control group, antibiotics treatment group, phage treatment group, and phage control group (χ 2=4.128,P >0.05). On experiment day 2, respectively 12, 7, and 2 mice were detected as blood bacterial culture-positive in sepsis control group, antibiotics treatment group, and phage treatment group, while no positive result was detected in the other two groups. Positive ratios of blood bacterial culture of mice in blank control group, phage treatment group, phage control group were significantly lower than the ratio in sepsis control group (withχ 2 values from -30.000 to 30.000,P values below 0.01). Positive ratio of blood bacterial culture of mice in antibiotics treatment group was significantly higher than that in blank control group or phage control group (withχ 2 values respectively 17.500 and -17.500,P values below 0.05). On experiment day 2, except for the kidney tissue of mice in phage treatment group, the bacteria load in each viscus of mice in blank control group, phage treatment group, and phage control group was significantly lower than that in sepsis control group (withχ 2 values from -9.000 to 9.000,P values below 0.01). The bacteria load in kidney of mice in antibiotics treatment group was significantly higher than that in blank control group or phage control group (withχ 2 values respectively -7.500 and 7.500,P values below 0.05). Conclusions Phages can significantly improve survival ratio, control inflammation response, and effectively clean bacteria in lung, liver, spleen, and kidney in treating extensively drug-resistantAcinetobacter baumannii -induced sepsis in mice.-
Key words:
- Burns /
- Acinetobacter baumannii /
- Bacteriophages /
- Sepsis /
- Extensively drug-resistant
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