鸽源肺炎克雷伯菌的分离鉴定及其毒力和耐药性分析  

Isolation and identification of Klebsiella pneumoniae from pigeons and analysis of virulence and drug resistance

作　　者：兰雪琴 黄颖 白怡馨 靳婉婧 王金泉[1] 苏战强[1] 谢金鑫 佟盼盼 LAN Xue-qin;HUANG Ying;BAI Yi-xin;JIN Wan-jing;WANG Jin-quan;SU Zhan-qiang;XIE Jin-xin;TONG Pan-pan(College of Veterinary Medicine,XinjiangAgricultural University,Urumqi 830052,China)

机构地区：[1]新疆农业大学动物医学学院,新疆乌鲁木齐830052

出　　处：《中国预防兽医学报》2024年第8期812-818,共7页Chinese Journal of Preventive Veterinary Medicine

基　　金：中央引导地方科技发展专项资金项目“鸽病防控和高质量肉品保障技术集成与示范”(ZYYD2022C13)。

摘　　要：为探究喀什某鸽场致种鸽腹泻和雏鸽死亡的病原,本研究于2023年2~3月采集71份腹泻种鸽粪便和43份死亡雏鸽肝、肾、脾和肠样品,进行细菌的分离培养,采用16S rRNA和ITS(16S rDNA-23S rDNA内部转录间隔序列)对分离菌进行PCR扩增及测序,采用PCR鉴定分离菌的毒力基因;经K-B纸片扩散法检测分离菌的药物敏感性,并采用PCR检测其耐药基因;通过肠杆菌科基因间重复序列-聚合酶链式反应(ERIC-PCR)分析菌株间的亲缘关系及多位点序列分型(MLST)分析分离菌的ST型。利用小鼠致病性试验检测分离菌的致病性。结果显示:经分离培养和PCR鉴定114份样品中共分离到83株肺炎克雷伯菌(Kp)(72.8%,83/114);毒力基因uge、mrkD、rmpA、wabG、ureA和magA的检出率在85.5%~98.8%,fimH的检出率为1.2%;83株Kp对阿莫西林、氨苄西林、头孢噻肟和头孢他啶耐药的菌株占73.0%~100%,对头孢曲松、阿米卡星、左氧氟沙星、四环素、链霉素、阿奇霉素、氟苯尼考、多黏菌素B和庆大霉素耐药的菌株占1.2%~25.0%,其中4株为多重耐药Kp;耐药基因blaSHV-11(72.3%,60/83)和bla_CTX-M-1G)43.4%,36/83)的携带率较高,而bla_(CTX-M-9G)14.5%,12/83)和tet(A)(6.0%,5/83)的携带率较低,但未检出耐药基因bla_(TEM)、bla_(CTX-M-2G)、aac、aadA1、strA、strB、tet(E)、tet(G)和mph(A),耐药基因和耐药表型间基本呈正相关。ERIC-PCR和MLST分析显示,种鸽和雏鸽Kp分离株亲缘关系较近,且以ST23型为主。致病性试验结果显示,鸽源Kp对小鼠的LD_(50)为3.6×10^(3)cfu/mL。本研究首次在腹泻种鸽和死亡雏鸽体内分离到Kp,其致病性较强,携带丰富的毒力基因,且对β-内酰胺类药物耐药严重,该结果为肉鸽养殖合理用药及Kp感染的有效控制提供了参考依据。To investigate the pathogens of diarrhea and death of youn pigeons in a pigeon farm in Kashgar.In this study,71 stool samples from diarrhea breeding pigeons and 43 liver,kidney,spleen and intestine samples from dead young pigeons were collected from February to March 2023,and the bacteria were isolated and cultured,and the Klebsiella pneumoniae(Kp)was amplified and*Corresponding author;†Equal contributors sequenced by PCR using 16S rRNA and ITS(16S rDNA-23S rDNA internal transcription interval sequence).The virulent gene of Kp was detected by PCR.The drug sensitivity of Kp was determined by using a Kirby-Bauer disk diffusion method and the drug resistance genee were detected by PCR.Epidemiological typing was performed by enterobacterial repetitive intergenic consensus-polymerase chain reaction(ERIC-PCR),and Multi-Locus Sequence Typing(MLST)was implemented to detect the sequence type(ST).The pathogenicity of Kp was detected by mouse pathogenicity test.The results showed that a total of 83 Kp isolates were isolated from 114 samples.The detection rates of virulence gene uge,mrkD,rmpA,wabG,ureA and magA were 85.5%-98.7%,and the detection rates of fimH were 1.2%.The drug resistance rates of 83 Kp isolates to amoxicillin,ampicillin,cefotaxime and ceftazidime ranged from 73.0%to 100%,that to ceftriaxone,amikacin,levofloxacin,tetracycline,streptomycin,azithromycin,florfenicol,polymyxin B and gentamicin ranged from 1.2%to 25.0%,among of which 4 isolates were multiple drug resistance.The prevalence of drug resistance genes blaSHV-11(72.2%),blaCTX-M-1G(43.3%)were high,while the carrying rates of blaCTX-M-9G(14.5%,12/83)and tet(A)(6.0%,5/83)were low.blaTEM,blaCTX-M-2G,aac,aadA1,strA,strB,tet(E),tet(G)and mph(A)were not detected.ERIC-PCR and MLST typing showed that the Kp genetic relationship between breeding pigeons and young pigeons were closed relatively,and ST23 was the main type.The median lethal dose(LD_(50))for mice was 3.6×10^(3)cfu/mL.In this study,Kp was isolated from diarrhea breeding pigeons and dead young pige

关 键 词：鸽 肺炎克雷伯菌 致病性 耐药性 分子分型 

分 类 号：S852.61[农业科学—基础兽医学]