引用本文
豆朋朋, 魏勇, 王利. 一株羊源尸毒梭菌的鉴定及生物学特性研究[J]. 畜牧兽医学报, 2020, 51(2): 399-403.
DOU Pengpeng, WEI Yong, WANG Li. Identification and Biological Characteristics of Clostridium cadaveris Isolated from Goat[J]. Acta Veterinaria et Zootechnica Sinica, 2020, 51(2): 399-403.
一株羊源尸毒梭菌的鉴定及生物学特性研究
1. 西南民族大学生命科学与技术学院, 成都 610041;
2. 四川省畜牧科学研究院, 成都 610041;
3. 西南民族大学青藏高原动物遗传资源保护与利用教育部重点实验室, 成都 610041;
4. 西南民族大学青藏高原动物遗传资源保护与利用四川省重点实验室, 成都 610041
2. 四川省畜牧科学研究院, 成都 610041;
3. 西南民族大学青藏高原动物遗传资源保护与利用教育部重点实验室, 成都 610041;
4. 西南民族大学青藏高原动物遗传资源保护与利用四川省重点实验室, 成都 610041
摘要：为探究羊源尸毒梭菌的生物学基本特性，进行生理生化分析、16S rDNA基因扩增、动物回感试验、耐药表型和耐药基因检测等试验。结果显示，菌株LJH-1为革兰阳性菌。PCR扩增获得长度为1 023 bp的片段。LJH-1与尸毒梭菌C4菌株聚类，从而判定LJH-1为尸毒梭菌。试验组小鼠全部死亡。小鼠肝中央静脉充盈大量红细胞，炎性细胞浸润，肾间质血管充血，肠黏膜上皮细胞脱落。LJH-1对头孢噻肟、氨苄西林等敏感，对头孢拉定、阿莫西林等耐药，并检出ant（3″）-Ia、aac（3）-IIa、aph（3'）-IIa、Sul2、Sul3和TEM耐药基因。这为该细菌性疾病的检测和防治提供了科学资料。
关键词：尸毒梭菌 鉴定 致病性 耐药性
Identification and Biological Characteristics of Clostridium cadaveris Isolated from Goat
1. College of Life Sciences and Technology, Southwest Minzu University, Chengdu 610041, China;
2. Sichuan Institute of Animal Husbandry Science, Chengdu 610041, China;
3. Key Laboratory of Qinghai-Tibetan Plateau Animal Genetic Resource Reservation and Utilization of Ministry of Education, Southwest Minzu University, Chengdu 610041, China;
4. Qinghai-Tibetan Plateau Animal Genetic Reservation and Utilization Key Laboratory of Sichuan Province, Southwest Minzu University, Chengdu 610041, China
2. Sichuan Institute of Animal Husbandry Science, Chengdu 610041, China;
3. Key Laboratory of Qinghai-Tibetan Plateau Animal Genetic Resource Reservation and Utilization of Ministry of Education, Southwest Minzu University, Chengdu 610041, China;
4. Qinghai-Tibetan Plateau Animal Genetic Reservation and Utilization Key Laboratory of Sichuan Province, Southwest Minzu University, Chengdu 610041, China
Abstract: To explore the biological characteristics of Clostridium cadaveris isolated from goat, physiological and biochemical analysis, 16S rDNA gene amplification, animal infection test, drug-resistant phenotype and genetic testing for drug resistance of LJH-1 strain were carried out. The results showed that strain LJH-1 was Gram-positive bacteria. A fragment of 1 023 bp was obtained by PCR amplification. LJH-1 and Clostridium cadaveris were clustered, so it was identified as Clostridium cadaveris. All mice in the experimental group died. There were many red blood cells and inflammatory cells in the central hepatic lobular vein of the mice. Blood vessels in the renal stroma were congested. Intestinal mucosa epithelial cells shed. LJH-1 was sensitive to cefotaxime and amoxicillin, and resistant to cefradine and amoxicillin. Ant(3″)-Ia, aac(3)-IIa, aph(3')-IIa, Sul2, Sul3 and TEM were detected. This provided scientific data for the detection, prevention and treatment of the bacterial diseases.
Key words:
Clostridium cadaveris identification pathogenicity drug resistance
尸毒梭菌(Clostridium cadaveris)属于芽胞杆菌科，梭菌属。该菌为厌氧的革兰阳性杆菌，周生鞭毛，孢子卵圆、端生[1]。该菌广泛分布于土壤、海底沉积物和人的粪便中，在腐烂的尸体中较为常见，但在活人身上罕见[2]。至今为止，关于尸毒梭菌的报道尚少。1991年，田军川等[3]从变质兔肉罐头中检出尸毒梭菌产毒株。尸毒梭菌和败毒梭菌等也可以从创伤感染、脓肿及血液样品等临床样品中检出[3]。这表明尸毒梭菌可以危害人和动物的健康。目前尚未见羊源尸毒梭菌的报道，对该菌进行鉴定和其生物学特性研究，对防治该菌导致的疾病有重要意义。
1 材料与方法 1.1 样品来源样品来源于四川省成都市某羊场内发生腹泻的瘫痪羔羊的肛门拭子。
1.2 主要试剂和仪器LB肉汤购自青岛高科技工业园海博生物技术有限公司；药敏纸片购自杭州微生物试剂有限公司；PCR仪购自Eppendorf Germany公司；电泳仪、凝胶成像系统均购自Bio-RAD USA公司。
1.3 细菌分离与鉴定样品于37 ℃培养箱中培养20 h，划线接种于LB固体培养基培养，挑取单菌落进行纯化、革兰染色和镜检。参照李绍戊等[4]和《伯杰氏系统细菌学手册》[1]进行生理生化鉴定。提取DNA进行16S rDNA基因扩增并测序分析。
1.4 动物回感试验及病理观察将20只SPF小鼠(购自四川省成都市中医药研究所)随机分组，试验组、对照组分别注射0.2 mL 9.744×108 CFU·mL-1的菌悬液和生理盐水，连续观察7 d。剖检死亡小鼠，取肝、肾等组织于4%多聚甲醛固定，制作病理切片。
1.5 耐药表型及耐药基因检测参照周海波等[5]的报道进行耐药表型试验，根据CLSI标准判断其敏感程度[6]。参照文献[7-9]设计氨基糖苷类耐药基因4种[aph(3′)-IIa、ant(3″)-Ia、aac(6′)-Ib、aac(3)-IIa]、磺胺类耐药基因3种(Sul1、Sul2、Sul3)和β-内酰胺类耐药基因1种(TEM)引物。
2 结果 2.1 细菌鉴定菌株LJH-1形成圆形、半透明的菌落。革兰染色菌体呈紫色的短杆状，表明该菌为革兰阳性菌。木糖、果糖、葡萄糖、麦芽糖、阿拉伯糖、β-半乳糖、鸟氨酸脱羧酶、赖氨酸脱羧酶、硫化氢、甘露醇、山梨醇、吲哚试验、明胶液化阳性；柠檬酸盐、枸橼酸盐和丙二酸盐阴性，符合梭菌属成员的特征。综合判定LJH-1与《常见细菌系统鉴定手册》[10]中尸毒梭菌描述基本一致，初步鉴定为尸毒梭菌。经过16S rDNA基因序列测定，获得长度为1 023 bp的片段(GenBank：MK968718)，LJH-1与尸毒梭菌C4菌株聚类(图 1)。综合判定LJH-1为尸毒梭菌。
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M.DNA相对分子质量标准；1. 16S rDNA基因PCR产物 M. DL2000 DNA marker; 1. 16S rDNA gene PCR product 图 1 16S rDNA基因扩增(A)及系统发育树(B)结果 Fig. 1 Result of 16S rDNA gene amplification (A) and phylogenetic tree (B) |
动物回感8 h后，试验组均出现精神沉郁、少动等症状，14 h后全部死亡。对照组未出现发病或死亡。试验组小鼠的肝、肾和小肠均出现充血、肿胀等病变，对照组无异常。根据《兽医病理学》[11]观察病理切片，发现：肝小叶中央静脉充血，炎性细胞浸润；肾间质血管充血，可见少量巨噬细胞；肠黏膜上皮细胞坏死、脱落(图 2)。
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A.肝小叶中央静脉有大量红细胞、炎性细胞；B.肾间质血管充血；C.肠黏膜上皮细胞坏死、脱落 A. Many red blood cells and inflammatory cells in the central hepatic lobular vein; B. Renal interstitial vascular congestion; C. Intestinal mucosa epithelial cells necrosis, shedding 图 2 小鼠组织病理学变化(400×) Fig. 2 The histopathological changes of mice (400×) |
结果显示，LJH-1对头孢噻肟、拉氧头孢、头孢唑啉、氨苄西林、氨曲南、阿米卡星和磷霉素等7种抗生素敏感；对头孢哌酮、妥布霉素、米诺环素、阿奇霉素和硫酸黏菌素等5种抗生素中度敏感；对头孢拉定、头孢噻吩、头孢克肟、青霉素、阿莫西林、邻氯西林、氧氟沙星、奥复星、环丙沙星、土霉素、多西环素、克林霉素、林可霉素、氟苯尼考、杆菌肽、复方新诺明、磺胺甲基异唑和氟苯尼考等18种抗生素耐药。耐药基因检测结果与其PCR产物测序结果一致(图 3)。β-内酰胺类和磺胺类耐药基因检测结果与耐药表型结果基本一致，而氨基糖苷类不一致。
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A.氨基糖苷类耐药基因检测结果(M.DNA相对分子质量标准；1. ant(3″)-Ia；2. aac(3)-IIa；3.aph(3′)-IIa)；B.磺胺类耐药基因检测结果(M.DNA相对分子质量标准；1.Sul2；2. Sul3)；C.β-内酰胺类耐药基因检测结果(M.DNA相对分子质量标准；1.TEM) A. Detective results of aminoglycosides resistant genes (M. DL2000 DNA marker; 1. ant(3″)-Ia; 2. aac(3)-IIa; 3. aph (3′)-IIa); B. Detective results of sulfonamide resistant genes (M. DL2000 DNA marker; 1. Sul2; 2. Sul3); C. Detective results of β-lactams resistant genes (M. DL2000 DNA marker; 1. TEM) 图 3 耐药基因检测结果 Fig. 3 Detective results of resistant genes |
至今为止关于尸毒梭菌致病性的报道较少，一般认为在活着的个体中不具有致病性[12]。Morshed等[13-15]发现在医学文献中尸毒梭菌作为人类病原体的报道仅有5次。然而，最近该菌与人类全身疾病密切相关，如腹腔内感染和菌血症等[16]。Poduval等[12]发现尸毒梭菌感染主要发生在免疫功能低下的宿主中。本试验回感试验中，试验组小鼠全部死亡，肝、肾等发生充血、出血等病变，这表明该尸毒梭菌具有极强的致病性。菌株的致病性强弱可能与分离宿主、分离地点等的不同相关。
目前，尚未见关于尸毒梭菌耐药表型和基因型的报道。细菌的耐药性可能因菌株来源的宿主、地区、用药情况等不同而异, 关于耐药机制的研究已进入基因水平[17-18]。本研究中该菌株对18种抗生素耐药，根据耐药表型试验结果选择常见的8种耐药基因进行检测。该菌株的β-内酰胺类和磺胺类耐药表型与耐药基因检测结果大体一致，而氨基糖苷类不一致。这可能是检测到的耐药基因未能表达转化为具有生物活性的蛋白质分子，或者该菌对氨基糖苷类抗生素存在其他尚未发现的耐药机制[19]。
4 结论分离到一株致病性和耐药性较强的尸毒梭菌，可以交叉、轮换使用头孢噻肟、拉氧头孢等敏感抗生素进行防控。
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