一株牛源致病性卡氏变形杆菌的分离鉴定与敏感药物筛选

doi:10.3969/j.issn.1004-1524.2021.10.06

浙江农业学报 ›› 2021, Vol. 33 ›› Issue (10): 1836-1843.DOI: 10.3969/j.issn.1004-1524.2021.10.06

一株牛源致病性卡氏变形杆菌的分离鉴定与敏感药物筛选

马志宇¹(), 黄方园¹, 王巍², 易军², 左之才¹^,^*()

1.四川农业大学动物医学院/动物疫病与人类健康四川省重点实验室,四川雅安 625014
2.四川省畜牧科学研究院,四川成都 610066

收稿日期:2020-12-07 出版日期:2021-10-25 发布日期:2021-11-02
通讯作者: 左之才
作者简介:*左之才,E-mail: zzcjl@126.com
马志宇(1999—),男,四川宜宾人,本科生,主要从事临床兽医学研究。E-mail: 1042767565@qq.com
基金资助:
国家重点研发计划(2018YFD0501800);四川省科技计划(2018NZ0002);四川省科技计划(2019YFQ0012);国家现代农业产业技术体系四川肉牛创新团队项目(SCCXTD-2020-13)

Isolation, identification and sensitive drug screening of a pathogenic Proteus cibarius strain from beef cattle

MA Zhiyu¹(), HUANG Fangyuan¹, WANG Wei², YI Jun², ZUO Zhicai¹^,^*()

1. College of Veterinary Medicine/Key Laboratory of Animal Disease and Human Health of Sichuan Province, Sichuan Agricultural University, Ya’an 625014, China
2. Sichuan Academy of Animal Husbandry, Chengdu 610066, China

Received:2020-12-07 Online:2021-10-25 Published:2021-11-02
Contact: ZUO Zhicai

摘要/Abstract

摘要：

为确定引起某养殖场肉牛体温升高、流涎、流鼻液、呼吸困难等症状疾病的病原菌,采集病牛鼻腔拭子进行细菌分离鉴定和敏感药物筛选。对分离的优势菌株ZZC-JM-1进行群集运动特性、生化特性、16S rRNA测序等试验。结果表明,该优势菌为卡氏变形杆菌,该菌的生长在含1%和2%琼脂的培养基上呈现群集运动,而琼脂含量达到3%时生长被抑制;除麦芽糖和蔗糖试验为阳性外,其余生化特性均与卡氏变形杆菌一致;ZZC-JM-1的16S rRNA基因与卡氏变形杆菌的同源性达99.7%,并与卡氏变形杆菌聚为一支。小鼠腹腔注射7.35×10⁷ CFU·mL^-1 ZZC-JM-1菌液(每只小鼠注射0.2 mL),7~14 h全部死亡;病理剖检可见肺、肝、脾等器官充血、肿大。ZZC-JM-1对常见抗菌药物耐药,对庆大霉素和环丙沙星、左氧氟沙星高度敏感。综上,该研究从体温升高、呼吸道症状为主症的病牛分离到一株对小鼠具有强致死性的卡氏变形杆菌,该卡氏变形杆菌可能为肉牛呼吸道感染的主要致病菌或继发感染病原菌,应引起高度重视。

关键词: 卡氏变形杆菌, 肉牛, 呼吸道感染, 耐药性

Abstract:

To determine the pathogen causing symptoms such as increased body temperature, salivation, runny nose and dyspnea of beef cattle in a cattle farm, nasal swabs of sick cattle were collected for bacterial isolation and identification and sensitive drug screening. The isolated strain ZZC-JM-1 was identified as Proteus cibarius by determination of swarming motility, biochemical identification and 16S rRNA sequencing. The strain showed swarming motility on the ordinary plate containing 1% and 2% agar, and when the agar concentration reached 3%, the growth was inhibited. Biochemical characteristics were consistent with Proteus cibarius except that maltose and sucrose test showed positive. Similarity between 16S rRNA gene of ZZC-JM-1 and Proteus cibarius reported on Genbank was 99.7%, and ZZC-JM-1 and Proteus cibarius were clustered together by constructing phylogenetic tree.When mice were intraperitoneally injected with 7.35×10 ⁷ CFU·mL^-1 bacterial solution (0.2 mL per mouse), they all died within 7-14 h. Anatomical examination showed hemorrhage and swelling of lung, liver and spleen. Drug sensitivity test showed that ZZC-JM-1 was resistant to common antibiotics, and highly sensitive to ciprofloxacin, levofloxacin and gentamicin. In conclusion, a strain of Proteus cibarius was isolated from diseased cattle with high body temperature and respiratory symptoms, and Proteus cibarius might be the main pathogen or the secondary pathogen of respiratory tract infection in beef cattle, which should be paid highly attention.

Key words: Proteus cibarius, beef cattle, respiratory tract infection, tolerance

中图分类号:

S852.61

马志宇, 黄方园, 王巍, 易军, 左之才. 一株牛源致病性卡氏变形杆菌的分离鉴定与敏感药物筛选[J]. 浙江农业学报, 2021, 33(10): 1836-1843.

MA Zhiyu, HUANG Fangyuan, WANG Wei, YI Jun, ZUO Zhicai. Isolation, identification and sensitive drug screening of a pathogenic Proteus cibarius strain from beef cattle[J]. Acta Agriculturae Zhejiangensis, 2021, 33(10): 1836-1843.

图/表 9

表1 GenBank公布的参考菌株信息

Table 1 Information of the reference strains published in GenBank

菌株 Strain	来源 Source	登录号 Accession No.	分离地 Separated area	分离时间 Separated time
N5/687T	硝酸氨化的土壤Nitrate-ammonifying soil	LN680103.1	德国Germany	2015 year
DSM 14437	未知Unknown	NR_104767.1	德国Germany	2013 year
ZF2	猪粪便Swine faeces	CP047349.1	中国江苏Jiangsu, China	2018 year
JS9	发酵海鲜Fermented seafood	FJ796245.2	韩国South Korea	2016 year
ATCC 29906	未知Unknown	NR_114419.1	意大利Italy	1999 year
JCM 1669	未知Unknown	NR_113344.1	日本Japan	2011 year
08MAS2615	鸽子肉Pigeon flesh	MF143629.1	中国安徽Anhui, China	2008 year
ATCC 29905	未知Unknown	DQ885257.1	意大利Italy	1999 year
NCTC 12737	未知Unknown	NR_043998.1	意大利Italy	1999 year
08MAS0041	猪肉Pork	NR_163665.1	中国安徽Anhui, China	2008 year

图1 分离菌株ZZC-JM-1在TSA培养基上的生长情况和革兰氏染色镜检形态(1 000×)

Fig.1 Growth of isolated strain ZZC-JM-1 on tryptic soy agar medium and its Gram staining morphology (1 000×)

图2 不同琼脂浓度下菌株的群集运动 a、b和c分别为1%、2%和3%琼脂培养基。

Fig.2 Swarming motility of strains under different agar concentrations a, b and c were ordinary plate containing 1%, 2% and 3% agar, respectively.

表2 分离菌株ZZC-JM-1生化试验结果

Table 2 Biochemical results of isolated strain ZZC-JM-1

底物 Substrate	实验结果 Result	底物 Substrate	实验结果 Result
葡萄糖Glucose	+	枸橼酸盐Citrate	+
木糖Xylose	+	丙二酸盐Malonate	+
蔗糖Sucrose	+	苯丙氨酸Phenylalanine	+
乳糖 Lactose	-	赖氨酸脱羧酶 Lysine decarboxylase	-
棉子糖 Raffinose	-	硫化氢 H₂S	+
甘露糖 Mannose	-	吲哚 Indole	-
血清菊糖 Seruminulin	-	精氨酸脱羧酶 Arginine decarboxylase	-
阿拉伯糖 Arabinose	-	鸟氨酸脱羧酶 Ornithine decarboxylase	-
甘露醇 Mannitol	-	麦芽糖 Maltose	+
水杨素Salicin	-	明胶Gelatin	+

图3 分离菌株ZZC-JM-1的16S rRNA序列PCR扩增结果 M,DNA分子量标准;1,阴性对照;2,细菌16S rRNA扩增产物。

Fig.3 Amplification result of 16S rRNA from isolated strain ZZC-JM-1 by PCR M, DNA marker; 1, Negative control; 2, 16 S rRNA amplification products of strain.

图4 基于分离菌株ZZC-JM-1与变形杆菌属各菌种的16S rRNA序列构建的系统进化树

Fig.4 Phylogenetic tree of the 16S rRNA sequences of isolated strain ZZC-JM-1 and the other proteus species

图5 试验小鼠的剖检病变 a, 左侧为试验组小鼠肺,表现为充血、鲜红色;右侧为对照组小鼠肺,无明显病变。b,左侧为试验组小鼠肝,表现为肿大、土黄色,表面有出血点;右侧为对照组小鼠肝,无明显病变。c,左侧为试验组小鼠脾,表现为淤血肿大;右侧为对照组小鼠脾,无明显病变。

Fig.5 Anatomical pathological changes of the test mice a, The left lung from the test group of mice showed congestion and bright red color, the right lung from the control mice showed no obvious lesions. b, The left liver from the test group mice showed enlargement, earthy yellow color and with hemorrhagic spots on the surface, the right liver from the control mice showed no obvious lesions. c, The left spleen from the test group mice showed bruising and enlargement, the right spleen from the control mice showed no obvious lesions.

图6 小鼠器官病理组织学观察 a,肺充血(→),肺泡壁增厚、淋巴细胞等炎症细胞浸润(←);b,肝中央静脉淤血(↑),肝索紊乱与增宽、肝窦变窄(→),肝细胞变性、发生坏死(←);c,脾淤血,有大量红细胞(→),细胞空泡变性(←)。

Fig.6 Histopathological observation of mice organs a, Pulmonary tissue hyperemia(→), alveolar wall thickening, lymphocytes and other inflammatory cells infiltrate(←); b, Congestion in the central vein of the liver(↑), liver cord disorder and broadening and liver antrum narrows(→), liver cell degeneration and necrosis(←);c, Spleen was congested, with numerous red blood cells(→) and vacuolization(←).

表3 分离菌株ZZC-JM-1药敏试验结果

Table 3 Drug susceptibility test results of the isolated strain ZZC-JM-1

药物 Drug		抑菌圈直径 Inhibition zone diameter	敏感性 Susceptibility
头孢类Cephalosporins	头孢曲松Ceftriaxone	13	R
	头孢噻肟Cefotaxime	11	R
	头孢吡肟Cefepime	14	R
	头孢氨苄Cephalexin	0	R
	头孢呋辛Cefuroxime	0	R
青霉素类Penicillins	苯唑西林Oxacillin	0	R
	阿莫西林Amoxicillin	0	R
碳青霉烯类Carbapenems	亚胺培南Imipenem	7	R
	美罗培南Meropenem	10	R
喹诺酮类Quinolones	环丙沙星Ciprofloxacin	23	S
	恩诺沙星Enrofloxacin	8	R
	左氧氟沙星Levofloxacin	21	S
氨基糖苷类Aminoglycosids	庆大霉素Gentamicin	15	S
	卡那霉素Kanamycin	0	R
林克酰胺类Lincolnamids	克林霉素Clindamycin	0	R
酰胺醇类 Amphenicols	氯霉素Chloramphenicol	0	R
	氟苯尼考Florfenicol	0	R
四环素类Tetracyclines	多西环素Doxycycline	7	R
大环内酯类Macrolactons	红霉素Erythromycin	0	R
硝基呋喃类Nitrofurans	痢特灵 Furazolidin	11	R

参考文献 23

[1]	LEE C C, WANG J L, LEE C H, et al. Clinical benefits of antimicrobial de-escalation in adults with community-onset monomicrobial Escherichia coli, Klebsiella species and Proteus mirabilis bacteremia[J]. International Journal of Antimicrobial Agents, 2017, 50(3):371-376. DOI URL
[2]	ZHANG W W, NIU Z L, YIN K, et al. Quick identification and quantification of Proteus mirabilis by polymerase chain reaction (PCR) assays[J]. Annals of Microbiology, 2013, 63(2):683-689. DOI URL
[3]	CHRISTOPOULOS G, CHRISTOPOULOU V, ROUTSIAS J G, et al. Greek rheumatoid arthritis patients have elevated levels of antibodies against antigens from Proteus mirabilis[J]. Clinical Rheumatology, 2017, 36(3):527-535. DOI URL
[4]	周湘华, 陆国忠. 临床药师参与1例重症社区获得性奇异变形杆菌肺炎治疗[J]. 北方药学, 2019, 16(7):191-192.
	ZHOU X H, LU G Z. The experience of clinical pharmacists participating in the treatment of a case of serious community acquired pneumonia caused by Proteus mirabills[J]. Journal of North Pharmacy, 2019, 16(7):191-192.(in Chinese with English abstract)
[5]	WANG S, ZHANG Y, ZHANG X, et al. An evaluation of multidrug-resistant (MDR) bacteria in patients with urinary stone disease: data from a high-volume stone management center[J]. World Journal of Urology, 2020, 38(2):425-432. DOI URL
[6]	TAVICHAKORNTRAKOOL R, PRASONGWATTANA V, SUNGKEEREE S, et al. Extensive characterizations of bacteria isolated from catheterized urine and stone matrices in patients with nephrolithiasis[J]. Nephrology Dialysis Transplantation, 2012, 27(11):4125-4130. DOI URL
[7]	王丹阳, 王旭荣, 张康, 等. 牛病毒性腹泻病毒、大肠杆菌和奇异变形杆菌混合感染致犊牛腹泻的研究[J]. 中国畜牧兽医, 2018, 45(1):189-195.
	WANG D Y, WANG X R, ZHANG K, et al. Study on calves diarrhea caused by mixed infection of bovine viral diarrhea virus, Escherichia coli and Proteus mirabilis[J]. China Animal Husbandry & Veterinary Medicine, 2018, 45(1):189-195.(in Chinese with English abstract)
[8]	路振香, 孙坤, 郭伟娜, 等. 变形杆菌的分离鉴定及其毒力与耐药相关基因检测[J]. 畜牧与兽医, 2017, 49(12):97-101.
	LU Z X, SUN K, GUO W N, et al. Isolation and identification of Proteus and detection of virulence and resistant-associated genes[J]. Animal Husbandry & Veterinary Medicine, 2017, 49(12):97-101.(in Chinese with English abstract)
[9]	曾邦权, 段博芳, 郑欢莉, 等. 牛奇异变形杆菌的分离鉴定及16S rRNA基因系统发育分析[J]. 中国畜牧兽医文摘, 2018, 34(4):71-73.
	ZHENG B Q, DUAN B F, ZHENG H L, et al. Isolation and identification of Proteus mirabilis and phylogenetic analysis of 16S rRNA gene[J]. Zhongguo Xumu Shouyi Wenzhai, 2018, 34(4):71-73. (in Chinese)
[10]	陈星, 胡松华, 曹立亭. 奶牛子宫内膜炎病原菌的分离鉴定及ε-多聚赖氨酸的抑菌作用[J]. 中国兽药杂志, 2008, 42(10):1-5.
	CHEN X, HU S H, CAO L T. Isolation and identification of pathogenic bacteria from endometritis in dairy cows and bacteriostasis of ε-polylysine[J]. Chinese Journal of Veterinary Drug, 2008, 42(10):1-5. (in Chinese with English abstract)
[11]	HYUN D W, JUNG M J, KIM M S, et al. Proteus cibarius sp. nov., a swarming bacterium from Jeotgal, a traditional Korean fermented seafood, and emended description of the genus Proteus[J]. International Journal of Systematic and Evolutionary Microbiology, 2016, 66(6):2158-2164. DOI URL
[12]	PENG K, LI R C, HE T, et al. Characterization of a porcine Proteus cibarius strain co-harbouring tet(X6) and cfr[J]. Journal of Antimicrobial Chemotherapy, 2020, 75(6):1652-1654. DOI URL
[13]	邵让. 耐锑卡氏变形杆菌筛选及其改性除锑研究[D]. 湘潭: 湖南科技大学, 2018.
	SHAO R. The selection of Proteus cibarius and its modifition for treating Sb(V)[D]. Xiangtan: Hunan University of Science and Technology, 2018. (in Chinese with English abstract)
[14]	ADESINA T D, NWINYI O C, DE N, et al. First detection of Proteus cibarius sp. of clinical significance[J]. IOP Conference Series: Earth and Environmental Science, 2018, 210:012001. DOI URL
[15]	东秀珠, 蔡妙英. 常见细菌系统鉴定手册[M]. 北京: 科学出版社, 2001.
[16]	谢秀兰, 黎帅, 马小明, 等. 羊奇异变形杆菌的分离鉴定及药敏试验[J]. 动物医学进展, 2017, 38(7):115-119.
	XIE X L, LI S, MA X M, et al. Isolation, identification and drug sensitivity test of Proteus mirabilis from lambs[J]. Progress in Veterinary Medicine, 2017, 38(7):115-119.(in Chinese with English abstract)
[17]	尹有勤, 马德慧, 李岩松, 等. 鹅源奇异变形杆菌的分离鉴定及毒力基因检测[J]. 微生物学通报, 2018, 45(10):2152-2163.
	YIN Y Q, MA D H, LI Y S, et al. Isolation, identification of goose Proteus mirabilis and it’s virulence genes detection[J]. Microbiology China, 2018, 45(10):2152-2163.(in Chinese with English abstract)
[18]	董萌萌, 徐彦召, 杭柏林, 等. 猪源奇异变形杆菌的分离鉴定与耐药性分析[J]. 中国兽医杂志, 2018, 54(5):82-85.
	DONG M M, XU Y Z, HANG B L, et al. Isolation, identification and drug-resistance analysis of Proteus mirabilis from swine[J]. Chinese Journal of Veterinary Medicine, 2018, 54(5):82-85.(in Chinese with English abstract)
[19]	杨移斌, 余琳雪, 杨秋红, 等. 斑点叉尾鮰源普通变形杆菌的分离、鉴定及药敏特性[J]. 微生物学通报, 2017, 44(10):2391-2397.
	YANG Y B, YU L X, YANG Q H, et al. Isolation, identification and antimicrobial susceptibility of Proteus vulgaris isolated from Ictalurus punctatus[J]. Microbiology China, 2017, 44(10):2391-2397.(in Chinese with English abstract)
[20]	李槿年, 余为一, 祖国掌, 等. 中华鳖普通变形杆菌的分离鉴定及其致病性研究[J]. 中国兽医科技, 1999, 29(3):28-30.
	LI J N, YU W Y, ZU G Z, et al. Isolation, identification and pathogenicity of Proteus vulgaris from Trionyx sinensis[J]. Chinese Journal of Veterinary Science and Technology, 1999, 29(3):28-30.(in Chinese)
[21]	任怡飞, 颜家坤, 操江民, 等. 扬子鳄源变形杆菌生物学特性研究[J]. 畜牧与兽医, 2016, 48(5):82-85.
	REN Y F, YAN J K, CAO J M, et al. Analysis of biological characteristics of Proteus isolated from Alliator sinensis[J]. Animal Husbandry & Veterinary Medicine, 2016, 48(5):82-85.(in Chinese)
[22]	范克伟, 余佳, 杨守深, 等. 华南虎源奇异变形杆菌的分离鉴定及其系统发育分析[J]. 中国兽医科学, 2018, 48(1):69-75.
	FAN K W, YU J, YANG S S, et al. Isolation, identification and phylogenetic analysis of Proteus mirabilis from South China tiger[J]. Chinese Veterinary Science, 2018, 48(1):69-75.(in Chinese with English abstract)
[23]	任梅渗, 王印, 杨泽晓, 等. 猪源奇异变形杆菌的分离鉴定与生物学分析[J]. 中国人兽共患病学报, 2015, 31(12):1093-1097.
	REN M S, WANG Y, YANG Z X, et al. Isolation, identification and biological characteristics analysis of Proteus mirabilis from swine[J]. Chinese Journal of Zoonoses, 2015, 31(12):1093-1097.(in Chinese with English abstract)

一株牛源致病性卡氏变形杆菌的分离鉴定与敏感药物筛选

Isolation, identification and sensitive drug screening of a pathogenic Proteus cibarius strain from beef cattle

RichHTML

PDF (PC)

可视化

摘要/Abstract

引用本文

使用本文

图/表 9

参考文献 23

相关文章 8

编辑推荐

Metrics

本文评价

[1]	张玉龙, 马志宇, 崔耀成, 谭天宇, 姚彩霞, 樊利虹, 左之才, 才冬杰. 肉牛呼吸道感染主要细菌性病原多重PCR检测方法的建立[J]. 浙江农业学报, 2020, 32(2): 210-217.
[2]	唐标, 陈凌云, 罗怡, 施杏芬, 胡骥, 陈怡飞, 钱鸣蓉, 杨华. 2019年浙江和福建沙门氏菌和弯曲杆菌的耐药性分析[J]. 浙江农业学报, 2020, 32(12): 2218-2225.
[3]	戴宝玲, 杨华, 戴贤君, 杨桂玲, 汪雯, 肖英平. 杨梅污染肺炎克雷伯氏菌的分离及其耐药特征和毒力基因[J]. 浙江农业学报, 2018, 30(9): 1513-1518.
[4]	王婧, 刁小龙, 杨茂义, 王全方, 陈晓兰, 张龙. 致仔猪腹泻大肠埃希菌的分离及耐药性分析[J]. 浙江农业学报, 2018, 30(4): 568-575.
[5]	廖倡宇, 张鹏飞, 王印, 杨泽晓, 姚学萍, 姜睿姣, 邬旭龙, 张博, 周丽军, 宋勇. 猪源莫拉菌的分离鉴定与部分生物学特性分析[J]. 浙江农业学报, 2018, 30(1): 36-42.
[6]	杨华, 何祥祥, 肖英平, 钱鸣蓉, 张巧艳, 唐标. 浙江省三地区冷鲜鸡中大肠埃希菌耐药谱测定及MLST分子分型分析[J]. 浙江农业学报, 2017, 29(6): 888-893.
[7]	邢朝斌;孟春燕;修乐山;劳凤云;庄鹏宇;柴丽花. 刺五加多向耐药性转运蛋白基因的克隆及其表达对皂苷含量的影响[J]. , 2014, 26(2): 0-351355.
[8]	章明奎;普锦成;. 土霉素诱导土壤可培养细菌产生耐药性的研究[J]. , 2010, 22(1): 0-72.