胸骨正中切口感染患者耐甲氧西林金黄色葡萄球菌噬菌体的基因组学信息及生物学特性分析

Analysis of genomic information and biological characteristics of a bacteriophage against methicillin-resistant Staphylococcus aureus in patients with median sternal incision infection

目的分离提纯1株新型耐甲氧西林金黄色葡萄球菌(MRSA)的噬菌体,并对其基因组学信息和生物学特性进行分析。方法采用实验研究方法。取分离自陆军军医大学(第三军医大学)第二附属医院收治的1例胸骨正中切口感染的63岁女性患者创面的MRSA(下称宿主菌)液,采用污水共培养法和双层琼脂平板法从该院污水中分离提纯得到噬菌体,并命名为噬菌体SAP23,观察噬菌斑形态。采用磷钨酸负染法将噬菌体SAP23染色,采用透射电子显微镜观察其形态。采用十二烷基磺酸钠/蛋白酶裂解方案制备噬菌体SAP23 DNA,在Illumina NovaSeq PE150平台下进行全基因组测序,并完成序列组装、注释、系统发生树等基因组学分析。将噬菌体SAP23液分别按10.0000、1.0000、0.1000、0.0100、0.0010、0.0001感染复数与宿主菌液共培养4 h后,采用点滴法测定噬菌体效价,筛选最佳感染复数,此处及以下样本数均为3。按测得的最佳感染复数取噬菌体SAP23液与宿主菌液分别共同孵育5、10、15 min后,同前测定噬菌体效价,筛选最佳吸附时间。按测得的最佳感染复数取噬菌体SAP23液与宿主菌液按最佳吸附时间孵育后,分别于培养0(即刻)、5、10、15、20、30、40、50、60、80、100、120 min,同前测定噬菌体效价,绘制一步生长曲线。取噬菌体SAP23液分别在温度为4、37、50、60、70、80℃下,在pH值为2、3、4、5、6、7、8、9、10、11、12下孵育1 h,测定稳定性。取陆军军医大学(第三军医大学)微生物教研室储存的41株MRSA,完成噬菌体SAP23的宿主谱范围检测。结果噬菌体SAP23能在宿主菌双层琼脂板上形成透明噬菌斑。噬菌体SAP23头部是直径为(88±4)nm的多面体,其尾部长度为(279±21)nm、宽度为(22.6±2.6)nm。噬菌体SAP23基因组为全长151618 bp的线状双链DNA,序列两端有11681 bp的长末端重复序列,预测出220个开放阅读框,噬菌体可编码4个转运RNA,未预测出毒力因子或抗性基因,注释功能的噬菌体SAP23基因可分为5个组,GenBank登录号为MZ427930,噬菌体SAP23全基因组序列与共线性分析中的6个葡萄球菌噬菌体全基因组序列有5个局部共线区域,但在局部共线区域内部或外部存在差异。噬菌体SAP23属于Herelleviridae科Twortvirinae亚科Kayvirus病毒属。噬菌体SAP23的最佳感染复数为0.0100,最佳吸附时间为10 min,潜伏期约为20 min,裂解期约为80 min;在4~37℃温度条件及pH值为4~9的条件中,稳定性较好。噬菌体SAP23可裂解41株MRSA中的3株。结论噬菌体SAP23为Herelleviridae科Twortvirinae亚科Kayvirus病毒属成员,潜伏期短,其对温度和酸碱耐受性好,可有效裂解MRSA,为不含毒力因子和抗性基因的新型烈性窄谱噬菌体。

Objective To isolate and purify a bacteriophage against methicillin-resistant Staphylococcus aureus(MRSA),and to analyze its genomic information and biological characteristics.Methods The experimental research methods were adopted.MRSA(hereinafter referred to as host bacteria)solution was collected from the wound of a 63-year-old female patient with the median sternum incision infection admitted to the Second Affiliated Hospital of Army Medical University(the Third Military Medical University).The bacteriophage,named bacteriophage SAP23 was isolated and purified from the sewage of the Hospital by sewage co-culture method and double-layer agar plate method,and the plaque morphology was observed.The morphology of bacteriophage SAP23 was observed by transmission electron microscope after phosphotungstic acid negative staining.The whole genome of bacteriophage SAP23 was sequenced with NovaSeq PE15 platform after its DNA was prepared by sodium dodecyl sulfonate/protease cleavage scheme,and genomic analysis including sequence assembly,annotation,and phylogenetic tree were completed.The bacteriophage SAP23 solution was co-incubated with the host bacterial solution for 4 h at the multiplicity of infection(MOI)of 10.0000,1.0000,0.1000,0.0100,0.0010,and 0.0001,respectively,and then the bacteriophage titer was measured by the drip plate method to select the optimal MOI,with here and the following sample numbers of 3.The bacteriophage SAP23 solution was co-incubated with the host bacterial solution at the optimal MOI for 5,10,and 15 min,respectively,and the bacteriophage titer was measured by the same method as mentioned above to select the optimal adsorption time.After the bacteriophage SAP23 solution was co-incubated with the host bacterial solution at the optimal MOI for the optimal adsorption time,the bacteriophage titers were measured by the same method as mentioned above at 0(immediately),5,10,15,20,30,40,50,60,80,100,and 120 min after culture,respectively,and a one-step growth curve was drawn.The bacteriophage SAP23 solution was incubated at 4,37,50,60,70,and 80℃and pH 2,3,4,5,6,7,8,9,10,11,and 12 for 1 h,respectively,to determine its stability.A total of 41 MRSA strains stored in the Department of Microbiology of Army Medical University(the Third Military Medical University)were used to determine the host spectrum of bacteriophage SAP23.Results The bacteriophage SAP23 could form a transparent plaque on the host bacteria double-layer agar plate.The bacteriophage SAP23 has a polyhedral head with(88±4)nm in diameter and a tail with(279±21)nm in length and(22.6±2.6)nm in width.The bacteriophage SAP23 has a linear,double-stranded DNA with a full length of 151618 bp and 11681 bp long terminal repeats sequence in the sequence ends.There were 220 open reading frames predicted and the bacteriophage could encode 4 transfer RNAs,while no resistance genes or virulence factors were found.The annotation function of bacteriophage SAP23 genes could be divided into 5 groups.The GenBank accession number was MZ427930.According to the genomic collinearity analysis,there were 5 local collinear blocks in the whole genome between the bacteriophage SAP23 and the chosen 6 Staphylococcus bacteriophages,while within or outside the local collinear region,there were still some differences.The bacteriophage SAP23 belonged to the Herelleviridae family,Twortvirinae subfamily,and Kayvirus genus.The optimal MOI of bacteriophage SAP23 was 0.0100,and the optimal adsorption time was 10 min.The bacteriophage SAP23 had a latent period of 20 min,and a growth phase of 80 min.The bacteriophage SAP23 was able to remain stable at the temperature between 4 and 37℃and at the pH values between 4 and 9.The bacteriophage SAP23 could lyse 3 of the 41 tested MRSA strains.Conclusions The bacteriophage SAP23 is a member of the Herelleviridae family,Twortvirinae subfamily,and Kayvirus genus.The bacteriophage SAP23 has a good tolerance for temperature and acid-base and a short latent period,and can lyse MRSA effectively.The bacteriophage SAP23 is a new type of potent narrow-spectrum bacteriophage without virulence factors and resistance genes.