紫花苜蓿种带阪崎克罗诺杆菌的分离鉴定及其致病性初步研究

Preliminary study on isolation and identification of seed-borne Cronobacter sakazakii from lucerne(Medicago sativa)and determination of its pathogenicity

采用稀释平板法从6种表面消毒的紫花苜蓿种子中分离和纯化出3株阪崎克罗诺杆菌疑似分离物。选取代表分离物1gF3室内条件下通过菌悬液(≈109 cfu·mL-1)浸种紫花苜蓿种子,皿内发芽;腹腔注射洁净级昆明小白鼠试验;测定阪崎克罗诺杆菌疑似分离物对紫花苜蓿和小鼠的致病性。结果表明,阪崎克罗诺杆菌疑似分离物对紫花苜蓿不致病;小鼠在接种分离物1gF3 12h后病理特征显示化脓性脑炎、心肌水肿扩张,局部心肌纤维有断裂、肝脏局部坏死、急性脾炎、肺部急性弥散性出血以及肾脏弥散性血管内凝血等症状,1gF3对小鼠具有致病性。发病小鼠血液涂板分离到阪崎克罗诺杆菌疑似分离物TM2。结合表型特征和16SrDNA鉴定,确定分离物1gF3和TM2为阪崎克罗诺杆菌。在此基础上,测定了菌株1gF3的生物学特性,该菌对数生长期为24~36h,之后衰退;干旱胁迫加重其生长量线性下降;菌株1gF3能在较宽温度(10~41℃)和pH(3~11)范围内生长;该菌不需盐生长,NaCl浓度上升该菌的生长量受到抑制;黑暗交替的光照有利其生长。研究材料为具有动物和植物跨域寄主特点的一类病原菌资源,研究结果对隐藏于植物或动物材料中跨域病原菌侵染传播特征及相关检疫防控提供了理论基础。

Cronobacter sakazakii is an emerging opportunistic Gram-negative pathogen,and it is associated with various neonatal infections such as meningitis,septicemia,bacteremia,and necrotizing enterocolitis.The aims of this work were to explore the contents of seed-borne C.sakazakii in lucerne,to determine its pathogenicity against animals and plants,and to understand its bacterial characteristics.Using the dilution plate method,three suspected C.sakazakii bacterial isolates were isolated from surface-sterilized seeds of six lucerne varieties.The representative bacterial isolate 1 gF3 was used to prepare a bacterial suspension（about 109 cfu·mL^-1）,which was used in a germination test of alfalfa seeds in Petri dishes under controlled laboratory conditions.In addition,intraperitoneal injection（I/P）into pathogen-free Kunming mice was conducted to understand the animal pathogenicity of 1 gF3.The results showed that 1 gF3 was not pathogenic to lucerne under laboratory conditions.At 12 hafter I/P injection,mice showed suppurative encephalitis,myocardial edema dilatation and local myocardial fiber rupture,partial liver necrosis,acute splenitis,and acute diffuse hemorrhage of the lungs and kidneys.These results indicated that 1 gF3 showed significant pathogenicity to mice after I/P injection under laboratory conditions.The suspected C.sakazakii bacterial isolate TM2 was isolated from the blood of injected diseased mice.Using phenotypic and 16 SrDNA identification methods,1 gF3 and TM2 were identified as C.sakazakii.The biological characteristics of 1 gF3 were verified.The bacterium was in the logarithmic growth phase from 24 to 36 h,followed by a recession.Drought stress aggravated the decline of bacterial linear growth.Strain 1 gF3 could grow at a wide range of temperatures（10-41 ℃）and pH（3-11）.It grew well without salt,but its growth was inhibited by increased NaCl concentrations.A 12-h light∶12-h dark photoperiod favored bacterial growth.This research has provided a theoretical basis and new ideas about bacteria that could be pathogenic to animals and plants and could be hidden in plant or animal materials,thus avoiding animal and plant quarantine measures.It also provides a theoretical basis for the prevention and control of the transport and spread of C.sakazakii.