曼氏杆菌(Mannheimia pernigra)的分离鉴定及基因组序列分析

Isolation,identification and genome sequence analysis of Mannheimia pernigra

为了对云南3起奶牛和山羊呼吸道疾病进行病原检测,本研究在5%CO_(2)的培养条件下从呼吸道疾病奶牛和山羊的肺脏中分离到3株革兰阴性嗜二氧化碳小杆菌YN21118、YN22011和ZY171111,对3株分离株进行16S r DNA基因的PCR扩增及同源性和遗传进化分析,结果显示3株分离株与曼氏杆菌(Mannheimia pernigra)模式菌株CCUG 74657T及其它M.pernigra分离株的16S r DNA基因序列的同源性达99.6%以上,且3株分离株与M.pernigra参考株形成同一个进化分支,表明3株分离株均为M.pernigra。采用琼脂扩散法检测3株分离株对10类32种抗菌药物的敏感性,结果显示3株分离株对青霉素类的阿莫西林克拉维酸、头孢菌素类的头孢噻吩、喹诺酮类的氧氟沙星等29种抗菌药物敏感。将3株分离株感染小鼠进行致病性试验,结果显示3株分离株均能引起小鼠呼吸道疾病。采用Illumina Miseq和PacBio高通量测序平台对分离株ZY171111进行全基因组高通量测序并预测其毒力基因和耐药基因,基因组序列分析结果显示,分离株ZY171111基因组全长2 197 384 bp,G+C摩尔含量为39.1%,基因组携带1 978个蛋白编码基因,其中包括溶血素基因hlyB、黏附相关基因htpB、生存胁迫相关基因clpP和katA等32个毒力基因和磺胺类耐药基因folP、氨基糖苷类耐药基因StrA、喹诺酮类耐药基因gyr A和parC、甲氧苄啶耐药基因dfr A3等39个耐药相关基因。本研究在我国首次分离到M.pernigra,证实我国存在奶牛和山羊M.pernigra感染,并于国内外首次报道M.pernigra的耐药性、耐药基因及其对小鼠的致病性,上述结果为深入研究M.pernigra的致病性和耐药性提供菌种资源和基因组数据。

To diagnose the pathogens in three cases of respiratory diseases in dairy cows and goats in Yunnan Province,three strains of Gram-negative,capnophilic,rod-shaped bacteria,designated as YN21118,YN22011 and ZY171111,respectively,were isolated from lungs of dairy cows and goats with respiratory disease under 5% CO2.The 16S r DNA gene homology and genetic evolution of the three isolates were analyzed,and the results showed that the three strains shared the highest 16S r DNA sequence similarities of ≥99.6% with Mannheimia pernigra type strain CCUG 74657Tand other M.pernigra strains,and fell into the cluster with M.pernigra strains in the 16S r DNA phylogenetic tree,indicating that the three isolates were all M.pernigra.The antimicrobial resistance to 32 antibiotics in 10 categories was detected using the Kirby-Bauer method,and the results showed that the three isolates were sensitive to 29 kinds of antibiotics,such as amoxicillin-clavulanic acid of β-lactams,cephalothin of cephalosporins and ofloxacin of quinolones.The pathogenicity test of the three isolates infected mice showed that the three isolates could induce respiratory diseases in mice.The whole genome sequencing of strain ZY171111 was performed on the Illumina Miseq and Pac Bio high-throughput sequencing platforms to identify the virulence genes and antibiotic resistance genes,and the genome sequence analyses showed that the complete genome size of strain ZY201224Twas 2197384 bp with G+C molar content of39.1%,which contained 1978 protein coding genes,including 32 virulence genes,such as hemolysin gene hly B,adhesion-related gene htp B,survival stress-related genes clp P and kat A,and 39 drug-resistant genes,such as sulfonamides resistance gene fol P,aminoglycoside resistance gene Str A,quinolone resistance genes gyr A and par C,trimethoprim resistance gene dfr A3.To our knowledge,this was the first isolation of M.pernigra in China,which confirms the presence of M.pernigra infections in dairy cows and goats in China,and the antibiotic resistance,antibiotic resistance genes and pathogenicity to mice of M.pernigra were reported for the first time in China and abroad.The above results provide strain resources and genomic data for further study of its pathogenicity and antibiotic resistance.