豪猪源福氏志贺菌的分离鉴定及喹诺酮类耐药基因检测
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摘要
为调查四川省某规模化豪猪养殖场中豪猪大面积腹泻死亡的原因,本研究采集病死豪猪的肠道内容物和肺脏等病料样品进行细菌的分离纯化,通过培养特性观察、革兰染色镜检、生化鉴定、16S rRNA基因的克隆测序进行鉴定,并对分离菌株进行致病性、耐药性分析,对其喹诺酮类耐药基因进行PCR检测并测序分析。结果显示,分离得到1株革兰阴性菌,该分离株符合福氏志贺菌的培养特性和生化特性,并且其16S rRNA基因序列与福氏志贺菌该基因序列的同源性为99%;对小鼠具有较强致病性;药敏试验结果显示分离株具有多重耐药性,仅对丁胺卡那敏感,对喹诺酮类、四环素类、头孢类、氨基糖苷类、青霉素类和氯霉素类药物均表现为耐药;分离株的DNA促旋酶Ⅱ和拓扑异构酶Ⅳ的喹诺酮耐药决定区均有突变,且携带有质粒介导的喹诺酮耐药基因qnr A和qnr S。本研究是国内首次关于豪猪源福氏志贺菌病的报道,为豪猪养殖中该细菌性疾病的诊断防治等相关研究提供参考。
The aim of this study was to explore the cause of the death of Hystrix hodgsoni in a large-scale farm in Sichuan province. Intestine contents and lungs of dead H.hodgsoni were collected for bacterial isolation. The staining and culture characteristics of the isolate were observed and identified by biochemical tests. The 16S rRNA gene of isolate was amplified and cloned for sequencing. And mice infection test and drug sensitive test was conducted to analyze pathogenicity and drug resistance of isolate. Moreover, mutations within quinolone resistance gene of the isolate were identified by PCR. The results showed, the culture and biochemical characteristics of the isolate was Shigella flexneri. And the results of the 16S rRNA sequencing of the isolate showed 99% homology with S.flexneri. In addition, the isolate had pathogenicity to mice. The results of antibiotic sensitivity test showed that it was sensitive to amikacin but resistance to quinolone, tetracycline, cephalosporin, aminoglycoside,benzylpenicillin and chloramphenicol. Furthermore, the isolate had mutations in the quinolone resistance determining regions(QRDR) of gyr A and par C and carry plasmid-mediated quinolone resistance genes of qnr A and qnr S. This study is the first report of S.flexneri infestation on the H.hodgsoni breeding industry in China and provides references for the disease prevention and drug treatment of associated Shigella disease.
The aim of this study was to explore the cause of the death of Hystrix hodgsoni in a large-scale farm in Sichuan province. Intestine contents and lungs of dead H.hodgsoni were collected for bacterial isolation. The staining and culture characteristics of the isolate were observed and identified by biochemical tests. The 16S rRNA gene of isolate was amplified and cloned for sequencing. And mice infection test and drug sensitive test was conducted to analyze pathogenicity and drug resistance of isolate. Moreover, mutations within quinolone resistance gene of the isolate were identified by PCR. The results showed, the culture and biochemical characteristics of the isolate was Shigella flexneri. And the results of the 16S rRNA sequencing of the isolate showed 99% homology with S.flexneri. In addition, the isolate had pathogenicity to mice. The results of antibiotic sensitivity test showed that it was sensitive to amikacin but resistance to quinolone, tetracycline, cephalosporin, aminoglycoside,benzylpenicillin and chloramphenicol. Furthermore, the isolate had mutations in the quinolone resistance determining regions(QRDR) of gyr A and par C and carry plasmid-mediated quinolone resistance genes of qnr A and qnr S. This study is the first report of S.flexneri infestation on the H.hodgsoni breeding industry in China and provides references for the disease prevention and drug treatment of associated Shigella disease.
引文
[1]Mathan M M,Mathan V I.Morphology of rectal mucosa of patients with shigellosis[J].Rev Infect Dis,1991,13(4):S314.
[2]邹瑶,张露,陈正礼,等.猴源宋内氏志贺菌的分离鉴定及生物学特性分析[J].中国兽医科学,2017,(1):60-66.
[3]冉丹丹,于学辉,汤承,等.牦牛源鲍氏志贺菌的鉴定及对BALB/c小鼠的致病性[J].畜牧兽医学报,2013,44(1):145-151.
[4]王彦红,苗晓青,周琼,等.奶牛大肠杆菌与鲍氏志贺氏菌混合感染的诊疗[J].中国兽医杂志,2007,43(9):70-71.
[5]许兰菊,王川庆,胡功政,等.鸡志贺氏菌病在我国的发现及其病原特性研究[J].中国预防兽医学报,2004,26(4):281-286.
[6]张兴民,张焕容,黄忍,等.山羊源志贺菌的分离鉴定及其部分生物学特性的研究[J].中国兽医科学,2018,(07):1-9.
[7]张鞠玲,曲芬.福氏志贺菌及其对氟喹诺酮类药物的耐药机制[J].国外医药(抗生素分册),2011,32(1):16-20.
[8]Greisen K,Loeffelholz M,Purohit A,et al.PCR primers and probes for the 16S rR NA gene of most species of pathogenic bacteria,including bacteria found in cerebrospinal fluid[J].JClin Microbiol,1994,32(2):335-351.
[9]朱东安,孙景勇,范惠清.一株福氏志贺菌中同时检出CTX-M-3型超广谱β-内酰胺酶和质粒介导的喹诺酮耐药基因qnrS[J].检验医学,2009,24(7):489-492.
[10]张雯霞,张珏,陈洪友,等.喹诺酮类耐药福氏志贺菌的相关耐药基因研究[J].中国感染与化疗杂志,2017,17(1):46-51.
[2]邹瑶,张露,陈正礼,等.猴源宋内氏志贺菌的分离鉴定及生物学特性分析[J].中国兽医科学,2017,(1):60-66.
[3]冉丹丹,于学辉,汤承,等.牦牛源鲍氏志贺菌的鉴定及对BALB/c小鼠的致病性[J].畜牧兽医学报,2013,44(1):145-151.
[4]王彦红,苗晓青,周琼,等.奶牛大肠杆菌与鲍氏志贺氏菌混合感染的诊疗[J].中国兽医杂志,2007,43(9):70-71.
[5]许兰菊,王川庆,胡功政,等.鸡志贺氏菌病在我国的发现及其病原特性研究[J].中国预防兽医学报,2004,26(4):281-286.
[6]张兴民,张焕容,黄忍,等.山羊源志贺菌的分离鉴定及其部分生物学特性的研究[J].中国兽医科学,2018,(07):1-9.
[7]张鞠玲,曲芬.福氏志贺菌及其对氟喹诺酮类药物的耐药机制[J].国外医药(抗生素分册),2011,32(1):16-20.
[8]Greisen K,Loeffelholz M,Purohit A,et al.PCR primers and probes for the 16S rR NA gene of most species of pathogenic bacteria,including bacteria found in cerebrospinal fluid[J].JClin Microbiol,1994,32(2):335-351.
[9]朱东安,孙景勇,范惠清.一株福氏志贺菌中同时检出CTX-M-3型超广谱β-内酰胺酶和质粒介导的喹诺酮耐药基因qnrS[J].检验医学,2009,24(7):489-492.
[10]张雯霞,张珏,陈洪友,等.喹诺酮类耐药福氏志贺菌的相关耐药基因研究[J].中国感染与化疗杂志,2017,17(1):46-51.