冷处理对单增李斯特菌生物膜形成能力的影响

胡安妥 王娉 张彩霞 胡秋辉 陈颖

胡安妥, 王娉, 张彩霞, 胡秋辉, 陈颖. 冷处理对单增李斯特菌生物膜形成能力的影响[J]. 疾病监测, 2019, 34(8): 760-766. doi: 10.3784/j.issn.1003-9961.2019.08.018
引用本文: 胡安妥, 王娉, 张彩霞, 胡秋辉, 陈颖. 冷处理对单增李斯特菌生物膜形成能力的影响[J]. 疾病监测, 2019, 34(8): 760-766. doi: 10.3784/j.issn.1003-9961.2019.08.018
Antuo Hu, Ping Wang, Caixia Zhang, Qiuhui Hu, Ying Chen. Effect of cold treatment on biofilm formation ability of Listeria monocytogenes[J]. Disease Surveillance, 2019, 34(8): 760-766. doi: 10.3784/j.issn.1003-9961.2019.08.018
Citation: Antuo Hu, Ping Wang, Caixia Zhang, Qiuhui Hu, Ying Chen. Effect of cold treatment on biofilm formation ability of Listeria monocytogenes[J]. Disease Surveillance, 2019, 34(8): 760-766. doi: 10.3784/j.issn.1003-9961.2019.08.018

冷处理对单增李斯特菌生物膜形成能力的影响

doi: 10.3784/j.issn.1003-9961.2019.08.018
基金项目: “十三五”国家重点研发计划重点专项(2017YFC1601202)
详细信息
    作者简介:

    胡安妥,男,河南省信阳市人,硕士研究生,主要从事食源性致病微生物研究,Email:18305187793@163.com

    通讯作者:

    陈颖,Tel:010-53897910,Email:chenyingcaiq@163.com

  • 中图分类号: R517.7

Effect of cold treatment on biofilm formation ability of Listeria monocytogenes

Funds: This study was supported by the fund for Key National Research and Development Program of China (No. 2017YFC1601202) during the 13th Five-Year Plan Period
More Information
  • 摘要: 目的研究冷处理对不同食品来源及不同血清型的单增李斯特菌生物膜形成能力的影响,为食品中单增李斯特菌的防治提供基础信息。方法使用结晶紫染色法对单增李斯特菌食品分离株在37 ℃、4 ℃冷胁迫及4 ℃冷适应条件下生物膜形成能力进行测定,并对代表菌株的生物膜相关基因hptluxSsigB进行基因序列分析及表达量差异分析。结果37 ℃条件下,1/2c血清型单增李斯特菌生物膜形成能力较强,不存在生物膜形成能力弱株。 37 ℃条件下的生物膜形成能力强组(3株)以及中组(10株)菌株中,9株菌的hpt基因发生非同义突变。 冷处理条件下4株中组菌株中,1株菌的sigB基因表达量增多,3株菌的hpt基因表达量增多。结论单增李斯特菌生物膜形成能力与血清型相关,1/2c血清型菌株在37 ℃条件下具有较强生物膜形成能力。 hpt基因突变株在37 ℃下具有较强生物膜形成能力,冷处理使得病原菌生物膜形成能力降低,但hptsigB基因的过量表达会增强4 ℃条件下单增李斯特菌生物膜形成能力。
  • 图  1  冷适应和阳性对照单增李斯特菌菌株的菌落计数结果

    Figure  1.  Colony count of cold-adapted strains and positive control strains of Listeria monocytogenes

    图  2  冷胁迫和冷适应单增李斯特菌菌株生物膜生成情况

    Figure  2.  Biofilm formation by cold-stressed strains and cold-adapted strains of L. monocytogenes

    表  1  本研究中食源性单增李斯特菌菌株信息

    Table  1.   Information about foodborne Listeria monocytogenes isolates in study

     菌株编号 血清型 分离基质
    LM16081407 1/2c 生肉  
    LM16081408 1/2c 生肉  
    LM16081409 1/2a 生肉  
    LM16081412 1/2c 生肉  
    LM16081413 1/2a 生肉  
    LM16081414 1/2b 即食食品
    LM16081419 1/2b 即食食品
    LM16081422 1/2b 即食食品
    LM16081423 1/2b 即食食品
    LM16081426 1/2b 即食食品
    LM16081428 1/2b 即食食品
    LM16081432 1/2b 即食食品
    LM16081433 1/2a 生肉  
    LM16101202 1/2a 即食食品
    LM16101204 1/2a 即食食品
    LM16111706 1/2c 生肉  
    LM17041901 1/2a 即食食品
    LM17041903 1/2a 即食食品
    LM17061701 1/2a 即食食品
    LM17061706 1/2c 生肉  
    LM17061711 1/2c 即食食品
    LM17081501 1/2c 粮谷  
    LM17081502 1/2c 粮谷  
    LM17081503 1/2c 粮谷  
    LM17081504 1/2b 生肉  
    LM17081505 1/2b 生肉  
    LM17081506 1/2b 生肉  
    LM17081507 1/2c 生肉  
    LM17081510 1/2a 生肉  
    LM17081512 1/2a 生肉  
    ATCC 19111 1/2a 标准菌株
    ATCC 19112 1/2c 标准菌株
    下载: 导出CSV

    表  2  用于PCR扩增的生物膜相关基因及16S rRNA基因的引物

    Table  2.   Primers for PCR amplification of biofilm-related genes and 16S rRNA gene

    基因 引物序列(5′ ~ 3′) 扩增片
    段长度
    (bp)
    参考
    文献
    hpt F:ATGTCATTATTCAGTTTAAAAAG 1 386 21
    R:TTATAGATGTAAAACTTTTGC
    luxS F:ATGGCAGAAAAAATGAATGTAG 468 21
    R:TTATTCACCAAACACATTTTTCC
    sigB F:ATGCCAAAAGTATCTCAACCTG 780 21
    R:TTACTCCACTTCCTCATTCTG
    16S rRNA F:AGAGTTTGATCCTGGCTCAG 1 465 26
    R:GGTTACCTTGTTACGACTT
      注:F. 正向引物;R. 反向引物
    下载: 导出CSV

    表  3  用于qPCR扩增的3个生物膜相关基因及16S rRNA基因的引物

    Table  3.   Primers for qPCR amplifications of 3 biofilm-related genes and 16S rRNA gene

    基因
    名称
    序列(5′~3′) 扩增片段长度(bp)
    hpt F:ATTGCTAGGTTCATTATCGTGG 144
    R:ACCGTATACATGCTATAAACAC
    P:FAM-CAGATATTATGAAAGGTCGCC
    GTA-TAMRA
    luxS F:CCTTCTAAACTATGGCTTGCT 101
    R:GAAATCATCGCCACAACACT
    P:FAM-CTGTCGCAGCTAAAACATCCG-TAMRA
    sigB F:ATCAAAGAGTTAGGTCCGAA 97
    R:CAATAAAATCAGCAATGTCGCTA
    P:FAM-AAATGCCGTAGAAGAGCTAAC
    GA-TAMRA
    16S rRNA F:TCGCTAGTAATCGTGGATCAGCA 118
    R:CTCCATAAAGGTTACCCTACCGAC
    P:FAM-CCGCCCGTCACACCACGAG-TAMRA
      注:F. 正向引物;R. 反向引物;P. 探针
    下载: 导出CSV

    表  4  单增李斯特菌阳性对照菌株生物膜生成情况

    Table  4.   Biofilm formation of positive control strain of L.monocytogenes

     菌株编号 A 生物膜形成能力分组 血清型 分离基质
    LM17081501 0.9607 1/2c 粮谷  
    LM17081502 1.0777 1/2c 粮谷  
    LM17081503 1.0857 1/2c 粮谷  
    LM16081407 0.7313 1/2c 生肉  
    LM16081408 0.7775 1/2c 生肉  
    LM16081409 0.7584 1/2a 生肉  
    LM16081412 0.7509 1/2c 生肉  
    LM16111706 0.7793 1/2c 生肉  
    LM17061706 0.6266 1/2c 生肉  
    LM17061711 0.7908 1/2c 即食食品
    LM17081507 0.6061 1/2c 生肉  
    LM17081510 0.6554 1/2a 生肉  
    LM17081512 0.8509 1/2a 生肉  
    ATCC 19111 0.8197 1/2a 标准菌株
    ATCC 19112 0.7809 1/2c 标准菌株
    LM16081413 0.2608 1/2a 生肉  
    LM16081414 0.5252 1/2b 即食食品
    LM16081419 0.4402 1/2b 即食食品
    LM16081422 0.5055 1/2b 即食食品
    LM16081423 0.4906 1/2b 即食食品
    LM16081426 0.3665 1/2b 即食食品
    LM16081428 0.3545 1/2b 即食食品
    LM16081432 0.4721 1/2b 即食食品
    LM16081433 0.3729 1/2a 生肉  
    LM16101202 0.4614 1/2a 即食食品
    LM16101204 0.4899 1/2a 即食食品
    LM17041901 0.4995 1/2a 即食食品
    LM17041903 0.2805 1/2a 即食食品
    LM17061701 0.4948 1/2a 即食食品
    LM17081504 0.1909 1/2b 生肉  
    LM17081505 0.1552 1/2b 生肉  
    LM17081506 0.1475 1/2b 生肉  
      注:A. 600 nm波长下的吸光度
    下载: 导出CSV

    表  5  部分单增李斯特菌的生物膜形成相关基因在冷处理条件下的表达量

    Table  5.   Expression levels of biofilm-forming genes of some L.monocytogenes strains under cold treatment condition

     菌株编号 基因 CT(平均值±标准差) 表达比率(2−ΔΔCT
    阳性对照 冷胁迫 冷适应 冷胁迫 冷适应
    LM16111706 16SrRNA 19.54±0.36 24.14±1.57 20.57±1.69 1.00 1.00
    sigB 31.69±0.56 32.76±0.83 30.59±0.28 11.50 4.36
    hpt 24.98±0.80 35.45±0.76 33.13±1.28 0.02 0.01
    LM17081506 16SrRNA 22.64±1.01 26.38±1.19 20.57±2.64 1.00 1.00
    sigB 34.98±0.48 38.75±0.24 34.67±1.65 0.98 0.30
    hpt 37.82±1.34 40.06±0.88 29.82±0.58 2.83 60.86
    LM17081510 16SrRNA 22.32±0.67 24.80±0.22 20.60±1.11 1.00 1.00
    sigB 30.40±0.79 34.32±0.79 31.24±1.28 0.37 0.17
    hpt 34.93±1.19 36.33±0.33 31.62±0.15 2.11 3.00
    LM17081512 16SrRNA 22.05±1.63 24.28±0.21 20.52±0.39 1.00 1.00
    sigB 27.74±1.34 32.31±1.25 30.82±0.31 0.20 0.04
    hpt 32.49±0.66 34.61±0.84 29.34±0.24 1.07 3.05
    ATCC19111 16SrRNA 20.52±0.65 21.44±0.74 24.16±0.45 1.00 1.00
    sigB 30.00±0.34 34.34±0.58 30.51±0.61 0.09 8.75
    hpt 31.46±0.98 41.34±1.90 39.57±0.39 0.00 0.05
    ATCC19112 16SrRNA 25.00±0.45 27.08±1.58 18.65±1.55 1.00 1.00
    sigB 35.14±0.71 40.77±0.96 26.43±1.93 0.09 5.13
    hpt 37.76±0.33 40.65±0.18 36.99±1.88 0.57 0.02
      注:表达比率大于(小于)1表示基因表达量高于(低于)阳性对照菌株
    下载: 导出CSV
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    [33] Sofos JN, Geornaras I. Overview of current meat hygiene and safety risks and summary of recent studies on biofilms, and control of Escherichia coli O157∶H7 in nonintact, and Listeria monocytogenes in ready-to-eat, meat products[J]. Meat Sci, 2010,86(1):2–14. DOI: 10.1016/j.meatsci.2010.04.015.
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  • 收稿日期:  2019-03-13
  • 网络出版日期:  2019-07-03
  • 刊出日期:  2019-08-01

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