摘要
为探究针对多重耐药铜绿假单胞菌(P.aeruginosa,PA)裂解性噬菌体生物学特性及其基因组结构特征,本研究以一株人源多重耐药PA 3099-2aT株为宿主菌,从养殖场污水中分离纯化到一株噬菌体,命名为p PA-3099-2aT.2。噬菌体经磷钨酸染色后采用透射电镜观察形态,经双层平板法测定其对温度和酸碱的稳定性、绘制一步生长曲线,通过双层平板法测定该噬菌体对各试验菌株的裂解情况确定其宿主谱,通过测定不同感染复数(MOI)细菌和噬菌体混合液的OD_(595nm)分析该噬菌体体外抑菌效果,通过不同浓度的PA 3099-2a T对大蜡螟幼虫致病性及攻毒保护率试验检测噬菌体体内抑菌效果。结果显示,噬菌体p PA-3099-2aT.2头部直径约为76.69 nm,有尾鞘,尾长123.89 nm,形态学分类为肌尾噬菌体;在-20℃~60℃和p H值为4~10的环境中稳定性良好,最适p H值为6,最适温度为37℃,潜伏期小于10 min,裂解期持续至100 min,裂解量约为178 pfu/cell;能裂解ST198型、ST274型、ST3683型、ST3875型、ST3216型等8株不同来源的PA;体外抑菌效果良好,其中MOI 0.01抑菌效果最好,能在0~15 h内有效抑制细菌生长;在体内也具有良好的抑菌效果,该噬菌体对大蜡螟幼虫的保护率与其MOI呈正相关,24 h内该噬菌体的MOI为100时对大蜡螟幼虫的保护率为100%,MOI 1时对大蜡螟幼虫的保护率为60%,MOI 0.01时对其的保护率为40%。采用高通量测序技术对p PA-3099-2aT.2进行全基因组测序,并于GenBank数据库下载32条噬菌体N-乙酰胞壁酰胺酶氨基酸序列,分析其遗传演化规律,采用DNAMAN v6对p PA3099-2aT.2、噬菌体JG004 lysozyme(Pae87)和v B_PaeM_C2-10_Ab1的N-乙酰胞壁酰胺酶的氨基酸序列进行比对分析。全基因组测序结果显示,p PA-3099-2a T.2基因组为ds DNA,长度为93031 bp,GC含量49.4%,含有穿孔素、N-乙酰胞壁酰胺酶、细胞壁水解酶等裂解相关蛋白;N-乙酰胞壁酰胺酶进化分析结果显示,该蛋白�
In order to explore the biological characteristics and genome structure of multidrug-resistant Pseudomonas aeruginosa lytic phage,a multidrug-resistant Pseudomonas aeruginosa from human was used as host bacteria and a bacteriophage named pPA-3099-2aT.2 was isolated and purified from aquaculture wastewater samples.The morphology of pPA-3099-2aT.2 was observed by transmission electron microscopy after phosphotungstic acid staining;the stability of temperature and acid base was measured by double-layer plate method;the one-step growth curve was drawn,and the host spectrum was determined by whether the phage can lyse the test strains.The antibacterial effect of bacteria and bacteriophage mixture at different MOI in vitro was measured.The results showed that the head diameter of pPA-3099-2aT.2 was about 76.69nm,with a tail sheath and a tail length of 123.89nm,and pPA-3099-2aT.2 was morphologically classified as Myococcygeal phage.It had good stability in the environment of-20℃-60℃and pH value of 4.0-10.0.The optimum pH value was 6.0,and the optimum temperature was 37℃.The incubation period was less than 10min,and the lytic period could continue to 100min.It could lyse 8 strains of Pseudomonas aeruginosa from different sources,including ST198,ST274,ST3683,ST3875 and ST3216.The lytic capacity was about 178pfu/cell.It had a good antibacterial effect within 0-15 hours in vitro,of which MOI 0.01 had the best antibacterial effect.There was a positive correlation between the protection rate of MOI and MOI larvae.The protection rates of MOI 100,MOI 1 and MOI 0.01 within 24 hours were 100%,60%and 40%,respectively.The whole genome sequencing of pPA-3099-2aT.2 was performed by high-throughput sequencing technology,and 32 amino acid sequences of N-acetylmuraminase were downloaded from GenBank database to analyze its genetic evolution,the amino acid sequences of N-acetylmuramidase from pPA3099-2aT.2,phage JG004 lysozyme(Pae87)and vB_PaeM_C2-10_Ab1 were analyzed by DNAMAN v6.The whole genome sequencing results showed that the
作者
曾君
王雪
王猛
左君豪
郭志良
赵佳男
季芳
邵建立
张立敏
徐莉莉
赵瑞利
王承民
ZENG Jun;WANG Xue;WANG Meng;ZUO Jun-hao;GUO Zhi-liang;ZHAO Jia-nan;JI Fang;SHAO Jian-li;ZHANG Li-min;XU Li-li;ZHAO Rui-li;WANG Cheng-min(College of Animal Science and Veterinary Medicine,Tianjin Agricultural University,Tianjin 300384,China;Guangdong Provincial Key Laboratory of Animal Protection and Resource Utilization,Guangdong Provincial Public Laboratory of Wildlife Protection and Utilization,Institute of Zoology,Guangdong Academy of Sciences,Guangzhou 510260,China;College of Veterinary Medicine,Agricultural University of Hebei,Baoding 071000,China;The First Affiliated Hospital of Jinan University,Guangzhou 510260,China;Beidaihe Rehabilitation Center of Joint Logistic Support Force,Qinhuangdao 066100,China;Union Biotechnology(Shanghai)Co.,LTD.,Shanghai 201103,China)
出处
《中国预防兽医学报》
CAS
CSCD
北大核心
2023年第10期1008-1018,共11页
Chinese Journal of Preventive Veterinary Medicine
基金
优宜邦生物科技(上海)有限公司资助产业化项目:微生物资源与产业化利用(UYB20220001)。
