中华流行病学杂志  2018, Vol. 39 Issue (5): 669-672   PDF    
http://dx.doi.org/10.3760/cma.j.issn.0254-6450.2018.05.025
中华医学会主办。
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文章信息

李文彬, 纪凌云, 徐冬蕾, 刘海灿, 赵秀芹, 吴移谋, 万康林.
Li Wenbin, Ji Lingyun, Xu Donglei, Liu Haican, Zhao Xiuqin, Wu Yimou, Wan Kanglin.
乳腺炎奶牛双重感染抗热分枝杆菌和象分枝杆菌的鉴定及药敏试验研究
Identification and drug susceptibility testing of Mycobacterium thermoresistibile and Mycobacterium elephantis isolated from a cow with mastitis
中华流行病学杂志, 2018, 39(5): 669-672
Chinese Journal of Epidemiology, 2018, 39(5): 669-672
http://dx.doi.org/10.3760/cma.j.issn.0254-6450.2018.05.025

文章历史

收稿日期: 2017-12-30
乳腺炎奶牛双重感染抗热分枝杆菌和象分枝杆菌的鉴定及药敏试验研究
李文彬1, 纪凌云2,3, 徐冬蕾2, 刘海灿2, 赵秀芹2, 吴移谋1, 万康林2     
1. 421001 衡阳, 南华大学医学院病原生物研究所;
2. 102206 北京, 中国疾病预防控制中心传染病预防控制所;
3. 200123 上海, 同济大学附属东方医院检验科
摘要: 目的 对疑似分枝杆菌感染乳腺炎奶牛进行病原及药敏谱调查。方法 采集1头患乳腺炎奶牛的牛奶,采用4% NaOH预处理,接种于L-J培养基分离培养。阳性培养物利用抗酸染色和多位点PCR方法进行初步鉴定,采用16S rRNAhsp65ITSSodA基因的多位点序列分析进行种的鉴定,利用Alamar blue显色法对分离菌株进行27种药物的药敏试验。结果 从1头患乳腺炎奶牛的牛奶中同时分离获得2株抗酸染色阳性培养物,经PCR鉴定为非结核分枝杆菌,多位点序列分析鉴定为抗热分枝杆菌和象分枝杆菌。药敏试验表明这2株菌对利福平和异烟肼等大多数抗结核药物耐药,但对阿米卡星、莫西沙星、左氧氟沙星、乙胺丁醇、链霉素、妥布霉素、环丙沙星和利奈唑胺等敏感。结论 乳腺炎奶牛中分离了抗热分枝杆菌和象分枝杆菌,有其独特的药敏谱特征,为其感染防治提供了科学依据。
关键词: 非结核分枝杆菌     抗热分枝杆菌     象分枝杆菌     奶牛乳腺炎    
Identification and drug susceptibility testing of Mycobacterium thermoresistibile and Mycobacterium elephantis isolated from a cow with mastitis
Li Wenbin1, Ji Lingyun2,3, Xu Donglei2, Liu Haican2, Zhao Xiuqin2, Wu Yimou1, Wan Kanglin2     
1. Institution of Pathogenic Biology, Medical College, University of South China, Hengyang 421001, China;
2. National Institute for Communicable Disease Control and Prevention, Chinese Center for Disease Control and Prevention, Beijing 102206, China;
3. Department of Laboratory Medicine, Shanghai East Hospital, Tongji University, Shanghai 200123, China
Corresponding author: Wu Yimou, Email: yimouwu@sina.com; Wan Kanglin, Email: wankanglin@icdc.cn
Fund program: Project of the State Key Laboratory for Infectious Disease Control and Prevention (2014SKLID104); National Basic Research Program of China (973 Program)(2015CB554202); National Science and Technology Major Project of China (2013ZX10003002-001)
Abstract: Objective To understand the etiological characteristics and drug susceptibility of Mycobacterium thermoresistibile and Mycobacterium elephantis isolated from a cow with mastitis and provide evidence for the prevention and control of infectious mastitis in cows. Methods The milk sample was collected from a cow with mastitis, which was pretreated with 4% NaOH and inoculated with L-J medium for Mycobacterium isolation. The positive cultures were initially identified by acid-fast staining and multi-loci PCR, then Mycobacterium species was identified by the multiple loci sequence analysis (MLSA) with 16S rRNA, hsp65, ITS and SodA genes. The drug sensitivity of the isolates to 27 antibiotics was tested by alamar blue assay. Results Two anti-acid stain positive strains were isolated from the milk of a cow with mastitis, which were identified as non-tuberculosis mycobacterium by multi-loci PCR, and multi-loci nucleic acid sequence analysis indicated that one strain was Mycobacterium thermoresistibile and another one was Mycobacterium elephantis. The results of the drug susceptibility test showed that the two strains were resistant to most antibiotics, including rifampicin and isoniazid, but they were sensitive to amikacin, moxifloxacin, levofloxacin, ethambutol, streptomycin, tobramycin, ciprofloxacin and linezolid. Conclusions Mycobacterium thermoresistibile and Mycobacterium elephantis were isolated in a cow with mastitis and the drug susceptibility spectrum of the pathogens were unique. The results of the study can be used as reference for the prevention and control the infection in cows.
Key words: Non-tuberculosis mycobacterium     Mycobacterium thermoresistibile     Mycobacterium elephantis     Cow with mastitis    

非结核分枝杆菌(non-tuberculosis mycobacterium,NTM)由结核分枝杆菌复合群和麻风分枝杆菌以外的一大群分枝杆菌组成[1]。据不完全统计NTM共有160多种,其中致病或条件致病的至少有30种。NTM对多种抗结核药物有耐药现象,常规药物治疗效果欠佳。本研究从中国乳腺炎奶牛的牛奶中分离抗热分枝杆菌和象分枝杆菌并对其药敏性进行分析。

材料与方法

1.牛奶来源:牛奶标本由陕西省一家牛奶公司患乳腺炎的奶牛中收集,该牛奶标本新鲜,未经任何消毒灭菌处理。

2.可疑感染菌鉴定:参考菌株包括结核分枝杆菌标准株(H37Rv),象分枝杆菌参考菌株(95080),抗热分枝杆菌参考菌株(95124),由中国CDC传染病预防控制所结核病研究室传代培养、保藏。

(1)细菌培养:牛奶分离重悬后接种于罗氏培养基和960液体培养基。水煮法提取DNA。

(2)PCR鉴定:利用16S rRNARv0577IS1561Rv1510Rv1970Rv3877Rv3120 7个位点进行PCR扩增,扩增产物用1.5%琼脂糖凝胶电泳25 min,EB染色20 min后,紫外灯下观察结果。初步鉴定分离株为结核分枝杆菌复合群或者NTM,所用到的引物见参考文献[2]。

(3)多位点序列分析(MLSA):hsp65扩增、测序分析引物为hsp65Fhsp65R[2]ITS扩增、测序分析引物为16sF23sR[3]SodA扩增、测序分析引物为SodlgFSodlgF[4],PCR反应体系:引物各1 μl,PCR-mix 12.5 μl,DNA模板2 μl,纯水8.5 μl。引物由北京擎科新业生物技术有限公司合成,PCR-mix购自北京擎科新业生物技术有限公司,PCR扩增产物也由该公司测序。16S rRNASodAhsp65ITS测序结果提交到美国国立生物技术信息中心的GenBank数据库进行同源性比对,同源性达到98%以上可鉴定为相应的分枝杆菌种[5]。使用MEGA 6.01软件进行聚类分析。

(4)药敏试验:Alamar blue显色试验。对参考菌株95080、95124、牛奶标本分离株ShannX15001和ShannX15002使用包括异烟肼、利福平、氧氟沙星等27种药物进行药敏试验。结合现有药物的界值判断菌株对药物的敏感性。从L-J培养基上刮取单克隆化的菌落至提前加入两滴高压过的5%吐温80的磨菌瓶中,充分振荡使菌分散开来,使用生理盐水将磨好的菌比浊到1个麦氏浓度,然后用7H9培养基将其稀释20倍,使得菌悬液终浓度接近107 CFU/ml。在除了边缘孔的96孔板中提前加入100 μl的7H9培养基,对抗菌药物进行倍比稀释。最终,在每个孔加入100 μl菌悬液。设置3个阴性对照:①7H9培养基+菌悬液,用以确定显色剂加入时间;②单纯7H9培养基,用以观察培养基对显色剂是否有干扰;③7H9培养基+抗菌药物,用以观察培养基和抗菌药物对显色剂的影响。最后将96孔板装入密封袋中,置于37 ℃培养箱中孵育。24 h后,先给第一个没有药物的生长对照孔加入显色剂(20 μl Alamar blue和50 μl高压过的5%吐温80混合物),将96孔板继续孵育24 h,若生长对照孔液体由蓝色变成粉色,则在试验孔中加入显色剂,若第一个生长对照孔没有变粉色,继续在第二个生长对照孔加显色剂,重复上述步骤。没有变色的孔所对应的药物浓度最小值和变色孔所对应的药物浓度最大值之间的范围为最小抑菌浓度范围,数值越小,表明抑菌活性越大。最小抑菌浓度临界值判断参考文献[6-9]。

结果

1.培养结果:培养1周后,L-J培养基上出现两种可疑菌落,一种为光滑湿润的黄色菌落,一种为相对干燥粗糙的淡黄色菌落,960液体培养基培养4日显示阳性。

2. PCR鉴定:仅16S rRNA所对应的位点出现了相应条带,初步鉴定2株分离株均为NTM。见图 1

注:M:DNA Marker;1~8代表16S rRNARv0577IS1561Rv1510Rv1970Rv3877Rv3120和阴性对照;H37Rv为结核分枝杆菌标准株;ShannX15001、ShannX15002为奶牛分离株 图 1 乳腺炎奶牛分离的分枝杆菌多位点PCR结果

3. MLSA:将ShannX15001和ShannX15002的16S rRNASodAhsp65ITS基因序列与美国国立生物技术信息中心的GenBank数据库进行比对,ShannX15001与象分枝杆菌在4个位点的同源性分别为99%、99%、99%和98%,ShannX15002与抗热分枝杆菌在SodAhsp65基因位点的序列同源性分别为99%、100%。

4.聚类分析:利用hsp65基因进行聚类分析,结果显示,分离株ShannX15001与象分枝杆菌在同一个分支,ShannX15002与抗热分枝杆菌在一个分支。见图 2

图 2 部分非结核分枝杆菌hsp65基因序列聚类分析

5.药敏试验:ShannX15001与象分枝杆菌参考菌株95080药敏情况相似,分离株ShannX15002与抗热分枝杆菌参考菌株95124药敏情况相似,进一步验证分离株ShannX15001与ShannX15002分别为象分枝杆菌和抗热分枝杆菌。药敏试验显示,这2株分离株对大多数一线抗结核药如利福平、异烟肼耐药,对阿米卡星、莫西沙星、左氧氟沙星、乙胺丁醇、链霉素、妥布霉素、环丙沙星、利奈唑胺等敏感。见表 1

表 1 新分离的象分枝杆菌和抗热分枝杆菌药敏试验
讨论

长期以来,分枝杆菌菌种鉴定一直通过表型特征及生化试验等多项指标进行综合分析,其操作过程繁琐且费时(≥4周),重复性差,且对新出现的菌种用传统方法已不能得到准确、高效的鉴定[10]。如对硝基苯甲酸、噻吩-2-羧酸肼鉴别培养基进行菌种鉴定,虽然已被常规应用,但其为临床提供信息效率低,且只能区分结核分枝杆菌、NTM和牛分枝杆菌,不能进行进一步详细鉴定[11]

随着分子生物学理论和技术的发展,以PCR为基础的各种分子鉴定技术已经为分枝杆菌菌种鉴定开辟了新的途径。这些技术在临床实验室的应用,为结核病提供快速、准确的实验室诊断[10]。本研究中选用的hsp65基因广泛地存在于分枝杆菌中,该基因核苷酸序列的多态性总体上大于16S rRNA基因的多态性,其核苷酸位点的变化具有菌种特异性,适用于对分枝杆菌菌种类型进行区分。自1993年开始,hsp65基因就被报道用来鉴定NTM[7],所有的分枝杆菌均含hsp65基因,因其高度保守性和种间差异性被广泛用于分枝杆菌的鉴定。

NTM广泛分布于自然界,在水、土壤、尘埃、人及动物体内普遍存在[12]。象分枝杆菌是一种快速生长分枝杆菌,最初是在2000年从一头患肺脓肿的大象中分离出来,该菌常从临床痰标本中分离获得[13-14]。抗热分枝杆菌是在1966年从土壤中分离得到,属于快速生长型分枝杆菌,该菌在52 ℃仍可生长,对热具有很好的抵抗性[15]。1981年,抗热分枝杆菌首次从肺炎患者中分离出来,被认为是人类致病菌[16]。随后又有抗热分枝杆菌引起肺肉芽肿以及引起心脏移植后的皮肤感染等的报道[17]

奶牛乳腺炎是奶牛养殖中的常见病,不但使奶牛产奶量下降,牛奶品质下降,而且牛奶中的细菌毒素还会影响人体健康。众所周知,牛奶公司主要采取巴氏消毒的方法对牛奶来进行消毒,为了保留牛奶中的营养成分和活性物质,可能会采取低温(低于60 ℃)长时间的消毒方法。据文献报道,象分枝杆菌的最适生长温度是42 ℃,在52 ℃不生长[15]。抗热分枝杆菌在52 ℃仍可生长,对热具有很好的抵抗性[13]。因此推测抗热分枝杆菌甚至是象分枝杆菌可能因不彻底的巴氏消毒法而残留在牛奶中。


利益冲突:
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