应用噬菌体喷雾消毒剂防控细菌性奶牛乳腺炎

Phage spray disinfectant to prevent and control cow mastitis

目的为探究噬菌体消毒制剂防控奶牛乳腺炎的潜力,本研究在奶牛场环境样本中分离鉴定了2株肺炎克雷伯菌噬菌体SWPA1和SWPA2,与2株大肠埃希菌噬菌体共同制备噬菌体JPW鸡尾酒喷雾消毒剂,通过JPW与过氧乙酸(PAA)联用进行奶牛场喷雾效果评估。方法本研究在上海某牛场乳腺炎患病奶牛乳汁及乳尖表面开展致病菌分离及耐药分析;以乳汁中分离的肺炎克雷伯菌做指示菌,进行牛场污水中噬菌体分离;通过透射电镜观察噬菌体形态;并进行裂菌谱、最佳感染复数(MOI)、一步生长曲线、对pH、紫外线和温度耐受性的测定,分析噬菌体的生物学特性;制备噬菌体鸡尾酒消毒制剂JPW,与PAA进行联合喷雾消毒,检测噬菌体喷雾消毒剂对牛场环境中细菌的消杀效果。结果本研究在乳腺炎奶牛乳汁及乳尖表面分离到大肠埃希菌5株,肺炎克雷伯菌10株;耐药分析结果显示分离的肺炎克雷伯菌呈现多重耐药性;随后在污水中分离到2个肺炎克雷伯菌噬菌体,命名为SWPA1和SWPA2;电镜下观察SWPA1为肌尾噬菌体、SWPA2为短尾噬菌体;2株噬菌体对分离的肺炎克雷伯菌的裂解谱覆盖率达到70%;体外抗菌试验发现,噬菌体SWPA1和SWPA2的最佳感染复数均为0.1,最适pH均为7,在25~45℃范围内保持接近100%存活率;制备了肺炎克雷伯菌和大肠埃希菌噬菌体鸡尾酒消毒制剂JPW;消毒结果显示,与PAA单独消毒效果相比,JPW与PAA联用能够显著降低奶牛体表及牛场环境中的细菌数。结论噬菌体喷雾消毒剂JPW与PAA组合消毒模式能够有效降低养殖场环境及乳房表面的细菌数,提高消毒效果,提示噬菌体鸡尾酒喷雾消毒制剂具有防控养殖场奶牛乳腺炎的潜力。

Objective In order to explore the potential of phage disinfectants for the prevention and control of dairy cow mastitis,two strains of Klebsiella pneumoniae phages SWPA1 and SWPA2 were isolated and identified in dairy farm environmental samples,and a spray disinfectant was prepared by previously reported Escherichia coli phages and Klebsiella pneumoniae phages SWPA1 and SWPA2(named JPW).The effect of disinfection in dairy farms was evaluated by JPW in combination with peracetic acid(PAA).Methods Pathogenic bacteria were isolated and analyzed on the milk and nipple tip surfaces of cows suffering from mastitis on a cattle farm in Shanghai.K.pneumoniae,isolated from milk,was used as an indicator bacterium to isolate phages from cattle farm sewage.Phage morphology could be visualized using transmission electron microscopy.The rhizobacterial profile,optimal multiplicity of infection(MOI),phage one-step growth curve,and determination of pH,ultraviolet,and temperature tolerance are utilized to assess the biological characteristics of phages.Bacteriophage cocktail spray disinfectant preparation JPW was prepared and combined with PAA for spray disinfection to detect the sanitizing effect in the cattle farm environment.Results In this study,5 strains of Escherichia coli and 10 strains of Klebsiella pneumoniae were isolated from the milk and nipple tip surfaces of mastitis cows.The results of the drug resistance analysis showed that the isolated K.pneumoniae exhibited multidrug resistance.Subsequently,two K.pneumoniae phages were isolated in sewage and named SWPA1 and SWPA2;SWPA1 and SWPA2 belong to Myoviridae and Podoviridae recognized by electron microscopy,respectively.The lysis spectrum of two phages against Klebsiella pneumoniae isolates reached 70%.The in vitro antibacterial test found that the optimal multiplicity of infection of both of the two phages was 0.1,and the optimal pH was 7,maintaining a survival rate close to 100%in the range of 25~45℃.The results showed that the combination of JPW and PAA could significantly reduce the number of bacteria on the cow’s body surface and in the farm environment compared with PAA used alone.Conclusion The combined disinfection prepared by phage and PAA can effectively improve the disinfection effect in dairy cows,suggesting that phage cocktail spray disinfections have the potential to prevent and control dairy cow mastitis.