金黄色葡萄球菌噬菌体的生物学特性分析及其对小鼠乳腺炎的治疗效果评估

Biological Characterization and Therapeutic Evaluation of Staphylococcus aureus Bacteriophages in the Treatment of Mastitis in Mice

噬菌体作为一种天然裂解细菌的病毒,可用于细菌疾病治疗。旨在筛选出有效治疗小鼠乳腺炎的噬菌体,为奶牛乳腺炎防治提供参考。以实验室保存的金黄色葡萄球菌AA292菌株为宿主菌,分别从污水样、粪样中分离纯化出2株噬菌体,命名为φFJ04、φAH12。对2株噬菌体的生物学特性进行了分析,结果表明,φFJ04最佳感染复数为1,裂解量为149,温度耐受范围在40~50℃,pH耐受范围在3~10,宿主谱为63/90株;φAH12最佳感染复数为1,裂解量为448,耐受温度在40℃,pH耐受范围在4~12,宿主谱为28/90株。通过全基因组测序分析,发现φFJ04全长38 540 bp,(G+C)%=33.79%,含有48个ORF;φAH12全长264 664 bp,(G+C)%=24.7%,含有282个ORF。φFJ04、φAH12基因组内均不含整合元件、毒力基因和耐药基因,可用于体内治疗。对噬菌体杀菌效果进行评估,发现由φFJ04、φAH12组成的噬菌体鸡尾酒在6 h可以将10^(4) CFU/mL的金黄色葡萄球菌全部杀灭。施用10^(8) PFU/mL噬菌体鸡尾酒对10^(8) CFU/mLAA292所致的小鼠乳腺炎有明显改善作用,能使治疗组乳腺组织细菌载量下降2.8 log10CFU/g,杀菌效果约为99.84%。通过观察HE染色切片发现,治疗组乳腺腺泡结构较为完整,乳腺上皮细胞脱落显著减少,为奶牛乳腺炎防治提供了参考。

Bacteriophages(phages),natural viruses that lyse bacteria,are utilized to treat bacterial diseases.This work aims to identify bacteriophages effective in the treatment of mouse mastitis,providing a reference for the prevention and control of bovine mastitis.Two bacteriophages,φFJ04 andφAH12,were isolated and purified from sewage and fecal samples,using laboratory-preserved Staphylococcus aureus AA292 as the host bacterium.The biological characteristics of the phages were analyzed:φFJ04 exhibited an optimal multiplicity of infection(MOI)of 1,a burst size of 149,temperature tolerance between 40℃and 50℃,pH tolerance from 3 to 10,and lysed 63 out of 90 strains.φAH12 showed an optimal MOI of 1,a burst size of 448,temperature tolerance at 40℃,pH tolerance from 4 to 12,and lysed 28 out of 90 strains.Whole genome sequencing revealed thatφFJ04 genomic DNA has a total length of 38540 bp with a G+C content of 33.79%,containing 48 ORFs,whileφAH12 genomic DNA has a total length of 264664 bp with a G+C content of 24.7%,containing 282 ORFs.The genomes ofφFJ04 andφAH12 lack integrative elements,virulence genes,and antibiotic resistance genes,making them suitable for in vivo therapy.The bactericidal efficacy of the phages was evaluated and showed that a cocktail composed ofφFJ04 andφAH12 completely eradicated 10^(4) CFU/mL of S.aureus within 6 h.Administration of a cocktail containing 10^(8) PFU/mL of phages significantly improved mouse mastitis induced by 10^(8) CFU/mL of strain AA292,resulting in a reduction of the bacterial load in the mammary tissue of the treatment group by 2.8 log10 CFU/g,achieving a bactericidal effect of approximately 99.84%.Histologic examination(HE staining)revealed that the treatment group had more intact mammary alveolar structures and significantly reduced mammary epithelial cell desquamation,providing insight into the prevention and treatment of bovine mastitis.