烧伤患者泛耐药肺炎克雷伯菌噬菌体的生物学特性与基因组信息和对细菌生物膜的作用

Biological characteristics and genomic information of a bacteriophage against pan-drug resistant Klebsiella pneumoniae in a burn patient and its effects on bacterial biofilm

目的分离可裂解烧伤患者泛耐药肺炎克雷伯菌的噬菌体,研究其生物学特性、基因组信息及对细菌生物膜的作用。方法(1)2018年,取分离自上海交通大学医学院附属瑞金医院(下称瑞金医院)烧伤患者血液的泛耐药肺炎克雷伯菌UA168(下称宿主菌)液,采用污水共培养法及滴板法和双层琼脂平板法,从瑞金医院污水中分离纯化得到烧伤患者泛耐药肺炎克雷伯菌噬菌体,将其命名为噬菌体KP168,观察其噬菌斑形态。(2)取噬菌体KP168液行氯化铯密度梯度离心和透析后,采用磷钨酸负染法通过透射电子显微镜观察噬菌体KP168形态。(3)取噬菌体KP168液,采用滴板法检测其对从瑞金医院烧伤患者血液中分离得到的20株泛耐药肺炎克雷伯菌的裂解能力,计算裂解率。(4)分别以感染复数为10.000、1.000、0.100、0.010和0.001共同常规振荡培养初始效价为9.3×10^11噬菌斑形成单位(PFU)/mL噬菌体KP168液(每管400μL)和浓度为1×10^9集落形成单位(CFU)/mL宿主菌液(每管4 mL)4 h(每种感染复数1管),采用双层琼脂平板法测算噬菌体KP168效价(测算方法下同)以筛选最佳感染复数,实验重复3次。(5)以感染复数为0.005混合浓度为1×10^9 CFU/mL宿主菌液(每管4 mL)和效价调整为5×10^7 PFU/mL噬菌体KP168液(每管400μL),分别于混合后0(即刻)、1、2、3、4、5、15、30 min(每个时间点1管)采用双层琼脂平板法计数噬菌斑(计数方法下同),计算已吸附噬菌体百分比以筛选最佳吸附时间,实验重复3次。(6)以感染复数为0.005混合浓度为1×10^9 CFU/mL宿主菌液(每管300μL)和效价调整为5×10^8 PFU/mL噬菌体KP168液(每管60μL),静置最佳吸附时间后常规振荡培养,分别于培养0(即刻)、10、20、30、40、50、60、70、80、90、100 min测算噬菌体KP168效价,绘制一步生长曲线,实验重复3次。(7)取效价为2.5×10^10 PFU/mL噬菌体KP168液,分别于37、40、50、60、70℃静置培养1 h(每个时间点3管,每管1 mL),计数噬菌斑并绘制热稳定曲线。取效价为3.0×10^10 PFU/mL噬菌体KP168液,分别加入pH值为5.0、6.0、7.0、7.4、8.0、9.0、10.0的SM缓冲液于37℃静置培养1 h(每种pH值3管,每管含100μL噬菌体KP168液、900μL SM缓冲液),计数噬菌斑并绘制酸碱稳定曲线。(8)取噬菌体KP168液同实验(2)透析后行DNA抽提与测序,用Prokka对全基因组进行注释以获得噬菌体KP168编码序列,使用Nucleotide BLAST功能进行核酸序列比对以寻找与噬菌体KP168核酸序列相似度最高的已知噬菌体,使用Blastx功能将编码序列翻译成蛋白质后进行功能预测,比对抗性基因数据库和毒力因子数据库。(9)于96孔板中,分别以感染复数为1.000、0.100、0.010和0.001,在浓度为1.5×10^8 CFU/mL(浓度下同)的200μL宿主菌液中加入初始效价为5.8×10^10 PFU/mL噬菌体KP168液20μL(每种感染复数3孔)共同培养48 h;将200μL宿主菌液静置培养48 h后,分别加入效价为1×106、1×10^7、1×10^8、1×10^9、1×10^10 PFU/mL的噬菌体KP168液20μL(每种效价3孔)静置作用4 h。2种实验均以200μL宿主菌液中加入SM缓冲液20μL(3孔)为阴性对照,以220μL LB培养液(3孔)为空白对照,均采用酶标仪测定吸光度值并分别计算生物膜抑制率和生物膜破坏率,实验均重复3次。结果(1)成功分离纯化噬菌体KP168,其噬菌斑透亮,呈圆形,直径约1.5 mm。(2)噬菌体KP168呈正多面体结构,直径约为50 nm,无尾部结构。(3)噬菌体KP168可裂解20株烧伤患者泛耐药肺炎克雷伯菌中的13株,裂解率为65.0%。(4)感染复数为1.000时,噬菌体KP168与宿主菌共同培养4 h后,效价最高,以此为最佳感染复数。(5)噬菌体KP168与宿主菌混合后4 min,已吸附噬菌体百分比最高,以此为最佳吸附时间。(6)一步生长曲线表明噬菌体KP168裂解宿主菌时,潜伏期约10 min,裂解期约40 min。(7)噬菌体KP168在40℃、pH值为7.4时,噬菌斑数最多,活性最大。(8)噬菌体KP168基因组是线状双链DNA,长度为40114 bp;有48个可能编码序列;与Klebsiella phage_vB_Kp1相似度最高;编码序列翻译蛋白对应的已知最相似蛋白中包含23个假设蛋白和25个已知功能蛋白;未见抗性基因和毒力因子基因;获得GeneBank登录号MN585130。(9)以各感染复数共同培养噬菌体KP168与宿主菌48 h后,生物膜抑制率相近,平均约为45%;效价为1×10^9 PFU/mL噬菌体KP168作用于宿主菌已形成的生物膜4 h后,生物膜破坏率最高,达平均42%。结论本研究自污水中分离得到1株烧伤患者泛耐药肺炎克雷伯菌噬菌体——噬菌体KP168,该株噬菌体属于无尾科噬菌体,宿主谱宽、吸附时间短、潜伏期短,具有一定的热和酸碱稳定性;其基因组信息明确,不含抗性基因及毒力因子基因;对细菌生物膜的形成有抑制作用,对已形成的细菌生物膜有破坏作用。

Objective To isolate a bacteriophage against pan-drug resistant Klebsiella pneumoniae in a burn patient,and to study its biological characteristics,genomic information,and effects on bacterial biofilm.Methods(1)In 2018,pan-drug resistant Klebsiella pneumoniae UA168(hereinafter referred to as the host bacteria)solution isolated from the blood of a burn patient in Ruijin Hospital Affiliated to Shanghai Jiao Tong University School of Medicine(hereinafter referred to as Ruijin Hospital)was used to isolate and purify the bacteriophage against pan-drug resistant Klebsiella pneumoniae from the sewage of Ruijin Hospital with sewage co-culture method,drip plate method,and double-agar plate method.The bacteriophage was named as phage KP168 and the plaque morphology was observed.(2)The phage KP168 solution was taken for cesium chloride density gradient centrifugation and dialysis,and then the morphology of phage KP168 was observed through transmission electron microscope after phosphotungstic acid negative staining.(3)The phage KP168 solution was taken to determine the lytic ability of the phage KP168 against 20 strains of pan-drug resistant Klebsiella pneumoniae isolated from the burned patients′blood in Ruijin Hospital by the drip plate method,and then the lysis rate was calculated.(4)The phage KP168 solution at a initial titer of 9.3×10^11 plaque-forming unit(PFU)/mL(400μL per tube)and the host bacteria solution at a concentration of 1×10^9 colony-forming unit(CFU)/mL(4 mL per tube)were conventionally shaking cultured together for 4 hours at multiplicity of infection(MOI)of 10.000,1.000,0.100,0.010,or 0.001,respectively(1 tube per MOI).The titer of phage KP168 was measured by the double-agar plate method(the measurement method was the same below)to select the optimal MOI.The experiment was repeated three times.(5)The host bacteria solution at a concentration of 1×10^9 CFU/mL(4 mL per tube)and the phage KP168 solution at an adjusted titer of 5×10^7 PFU/mL(400μL per tube)were mixed at the MOI of 0.005.The plaques were counted 0(immediately),1,2,3,4,5,15,and 30 minutes(1 tube at each time point)after mixing by the double-agar plate method(the counting method was the same below),and the percentage of adsorbed phages was calculated to screen for the optimal adsorption time.The experiment was repeated three times.(6)The host bacteria solution at a concentration of 1×10^9 CFU/mL(300μL per tube)and the phage KP168 solution at a titer of 5×10^8 PFU/mL(60μL per tube)were mixed at MOI of 0.005 and conventionally shaking cultured after standing for the optimal adsorption time.The phage KP168 titer was measured 0(immediately),10,20,30,40,50,60,70,80,90,and 100 minutes after culture,and a one-step growth curve was drawn.The experiment was repeated three times.(7)The phage KP168 solution at a titer of 2.5×10^10 PFU/mL was left to stand for 1 hour at 37,40,50,60,or 70℃(3 tubes at each time point,1 mL per tube)for counting the plaques,and then the thermal stability curve was drawn.SM buffer at a pH values of 5.0,6.0,7.0,7.4,8.0,9.0,or 10.0 were added to the phage KP168 solution at a titer of 3.0×10^10 PFU/mL,respectively.The mixed solution was left to stand for 1 hour at 37℃(3 tubes of each pH,each tube containing 100μL phage KP168 solution and 900μL SM buffer),and then the plaques were counted,and an acid-base stability curve was drawn.(8)The phage KP168 solution was taken for DNA extraction and sequencing after dialysis as in experiment(2).The whole genome was annotated with Prokka to obtain the coding sequence of phage KP168.Nucleotide′s BLAST function was used to proceed nucleic acid sequence alignment for finding a known phage with the highest similarity to the phage KP168 nucleic acid sequence,and Blastx function was used to translate the coding sequence into protein for its function prediction.The comparison with Antibiotic Resistance Genes Database and Virulence Factors Database was proceeded.(9)In a 96-well plate,at a MOI of 1.000,0.100,0.010 or 0.001(3 wells per MOI),20μL phage KP168 solution at a initial titer of 5.8×10^10 PFU/mL was added to 200μL host bacteria solution at a concentration of 1.5×10^8 CFU/mL(the same concentration below)for co-cultivation for 48 hours.After 200μL host bacteria solution was left to stand for 48 hours,20μL phage KP168 solution at a titer of 1×10^6,1×10^7,1×10^8,1×10^9,or 1×10^10 PFU/mL(3 wells per titer)was added respectively for action for 4 hours.In both experiments,200μL host bacteria solution added with 20μL SM buffer(3 wells)acted as a negative control,and 220μL LB culture medium(3 wells)acted as a blank control.Absorbance values were measured by a microplate reader,and inhibition/destruction rates of biofilm were calculated.The experiments were both repeated three times.Results(1)The plaques of phage KP168 successfully isolated and purified were transparent and round,and its diameter was approximately 1.5 mm.(2)The phage KP168 has a regular polyhedron structure with a diameter of about 50 nm and without a tail.(3)The phage KP168 could lyse 13 of 20 strains of Klebsiella pneumoniae from burned patients,with a lysis rate of 65.0%.(4)When MOI was 1.000,the titer was the highest after co-culturing the phage KP168 with the host bacteria for 4 hours,which was the optimal MOI.(5)After the mixing of the phage KP168 with the host bacteria for 4 minutes,the percentage of the adsorbed phage reached the highest,which was the optimal adsorption time.(6)The one-step growth curve showed that during the lysis of the host bacteria by phage KP168,the incubation period was about 10 minutes,and the lysis period was about 40 minutes.(7)With the condition of 40℃ or pH 7.4,the number of plaques and the activity of phage KP168 reached the highest.(8)The genome of phage KP168 was a linear double-stranded DNA with a length of 40114 bp.There were 48 possible coding sequences.It had the highest similarity to Klebsiella phage_vB_Kp1.The most similar known proteins corresponding to the translated proteins of coding sequences contained 23 hypothetical proteins and 25 proteins with known functions.No resistance genes or virulence factor genes were found.The GeneBank accession number was KT367885.(9)After 48 hours of co-cultivation of the phage KP168 and the host bacteria at each MOI,the inhibition rates of biofilm were similar,with an average of about 45%.After the phage KP168 with a titer of 1×10^9 PFU/mL acted on the biofilm formed by the host bacteria for 4 h,the destruction rate of biofilm was the highest,reaching an average of 42%.Conclusions In this study,a bacteriophage against pan-drug resistant Klebsiella pneumoniae from a burn patient,phage KP168,is isolated from sewage,which belongs to the tailless phage.It has a wide host spectrum,short adsorption time,and short incubation period,with certain thermal and acid-base stability.Its genomic information is clear,and it does not contain resistance genes or virulence factor genes.It also has an inhibitory effect on the formation of bacterial biofilm and a destructive effect on the formed bacterial biofilm.