1株朱鹮源红斑丹毒丝菌的分离鉴定及生物学特征分析

solation,identification and biological characteristics of Erysipelothrix rhusiopathiae from crested ibis

为确定浙江省德清县朱鹮抢救保护基地一只死亡朱鹮的发病原因及病原特征,本研究采集其肝脏、心脏等组织样品,通过TSA平板划线培养,观察菌落、革兰氏染色、生化鉴定和16S rRNA基因的PCR扩增与序列分析。结果显示,从朱鹮体内分离的细菌为红斑丹毒丝菌(E.rhusiopathiae),并命名为E.rhusiopathiae-716株。采用纸片扩散(K-B)法检测该菌对8类10种药物的敏感性;采用微量结晶紫染色法鉴定分离菌的生物被膜(BF)形成能力,结果显示该菌对青霉素类的氨苄西林和青霉素、喹诺酮类的诺氟沙星等5类6种抗生素敏感,对四环素类的四环素、大环内酯类的红霉素、氨基糖苷类的庆大霉素和阿米卡星等3类4种抗生素耐药,且其BF形成能力适中。采用Illumina二代测序平台对分离菌的全基因组测序,并采用SPAdes拼接序列;利用Prokka和PATRIC 3.6.9分析该菌基因组的分子特征。利用CARD数据库预测分离菌的耐药基因;采用VFDB数据库预测分离菌的毒力基因。采用ParSNP软件绘制该分离菌与GenBank中不同国家不同宿主来源的红斑丹毒丝菌全基因组的系统发育树。全基因组的分子特征分析结果显示,该分离菌基因组全长1.78×10^(3)kb,GC含量36.5%,含1694个蛋白编码基因、55个tRNA、6个rRNA、1个tmRNA,无质粒、无前噬菌体。经软件预测结果显示,该分离菌携带3个耐药基因,分别是耐四环素类药物的tetM、耐大环内酯类药物的mefA及兼具大环内酯类、林可酰胺类以及原霉素B类的msrD基因,这与其对四环素、红霉素的耐药表型相符,但未携带庆大霉素和阿米卡星耐药相关基因;其携带76个与菌毛、荚膜、铁摄取、溶胞素、分泌、转运等毒力相关基因;系统发育树结果显示分离株E.rhusiopathiae-716与韩国海豚源红斑丹毒丝菌分离株KC-Sb-R1处于同一分支,进一步表明分离菌为红斑丹毒丝菌。将不同剂量的E.rhusiopathiae-716株分别经腹腔感染雏鸭及小鼠后在7 d观察期内评估其致病性。结果显示,中高剂量组(1×10^(5)cfu/mL~1×10^(8)cfu/mL)雏鸭和小鼠均出现厌食,被毛杂乱现象,部分雏鸭久卧不起,低剂量(1×10^(4)cfu/mL)组及阴性对照组雏鸭和小鼠均无明显异常。经计算,该分离菌对雏鸭和小鼠的LD50分别为1×10^(4.94)cfu/mL和1×10^(5.86)cfu/mL;病死鸭及小鼠剖检后观察到二者消化道有出血点,雏鸭气囊浑浊等剖检病变,并再次从这两种动物的肝脏中分离到该菌。上述结果表明该菌株对禽类和哺乳动物均具有潜在致病性。本研究首次分离鉴定到一株朱鹮源致病性红斑丹毒丝菌,并证实该菌为朱鹮的致病菌,该结果为进一步研究红斑丹毒丝菌的致病机制及朱鹮相关疾病的防治提供了用药参考。

To determine the etiology and pathogenic characteristics of a dead crested ibis(nipponia nippon)in the rescue and protection base of crested ibis in Deqing County,Zhejiang Province,the liver,heart and other tissue were sampled aseptically.Bacterial colonies were observed through streak culture on TSA plates,followed by Gram staining,biochemical identification,and PCR amplification sequencing and analysis of the 16S rRNA gene.The results showed that one Erysipelothrix rhusiopathiae(E.rhusiopathiae)isolate were obtained from the crested ibis,which were named as E.rhusiopathiae-716.The sensitivity of the isolate to 10 antibiotics in 8 categories was detected by the universal Kirby Bauer(K-B)test,and its biofilm formation(BF)ability was detected by the microplate crystal violet staining method.The results showed that the strain was sensitive to 5 categories and 6 antibiotics such as ampicillin and penicillin of penicillins and norfloxacin of quinolones,and resistant to 3 categories and 4 antibiotics such as tetracycline of tetracyclines,erythromycin of macrolides,gentamicin of aminoglycosides and amikacin,and it had moderate BF ability.The whole genome of the isolate was sequenced using Illumina second-generation sequencing platform,and the sequence was assembled using SPAdes.Prokka and PATRIC 3.6.9 were used to analyze the functional characteristics of the genome,and the CARD database and VFDB database were used to predict the antibiotics resistance genes and the virulence genes of the isolate,respectively.A phylogenetic tree of the whole genome of the isolate and E.rhusiopathiae from different countries and host sources in GenBank was constructed using ParSNP.The functional annotation results of the whole genome showed that the genome of the isolate was 1.78×10^(3)kb in length,with a GC content of 36.5%,containing 1694 protein-coding gene sequences,55 tRNAs,6 rRNAs,1 tmRNA,and no plasmid,no prophagosome.The results of software prediction showed that the isolate carried three antibiotics resistance genes,namely tetM resistant to tetracycline,mefA resistant to macrolide,and msrD gene resistant to macrolide,lincomonamide,and protomycin B.This was consistent with its resistance phenotypes to tetracycline and erythromycin,but did not carry gentamicin and amikacin resistance-related genes;It carried 76 virulence-related genes such as pili,capsule,iron uptake,cytolysin,secretion,and transport.The phylogenetic tree showed that the isolate E.rhusiopathiae-716 was in the same branch as the strain KC-Sb-R1 from dolphin in South Korea,which further indicated that the isolate was E.rhusiopathiae.Ducklings and mice were infected intraperitoneally with different doses of E.rhusiopathiae-716,and the pathogenicity of the strain was evaluated during a 7-day observation period.The results showed that the ducklings and mice in the medium and high dose groups(1×10^(5)cfu/mL-1×10^(8)cfu/mL)exhibited anorexia,messy fur,and some ducklings were unable to lie up for a long time.No significant abnormalities were observed in ducklings and mice of the low-dose group(1×10^(4)cfu/mL)and negative control group.After calculation,The LD50 of the isolate to ducklings and mice were 1×10^(4.94)cfu/mL and 1×10^(5.86)cfu/mL,respectively.After autopsy,bleeding spots were observed in the digestive tract of dead ducklings and mice,as well as turbidity was observed in the air sacs of the ducklings.The bacteria were recovered from the livers of these two animals.This indicated that the strain had potential pathogenicity to both poultry and mammals.In this study,a strain of pathogenic E.rhusiopathiae from crested ibis was isolated and identified for the first time,and it was confirmed that this strain was the pathogen to crested ibis,providing a basis for further research on the pathogenesis of E.rhusiopathiae and the prevention and treatment of crested ibis related diseases.