CN117051074B - 一种基于代谢通路分析的四联活菌制品活菌计数用培养基、计数方法及培养基的筛选方法 - Google Patents
一种基于代谢通路分析的四联活菌制品活菌计数用培养基、计数方法及培养基的筛选方法 Download PDFInfo
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Abstract
本发明属于微生物检测技术领域，具体涉及一种基于代谢通路分析的四联活菌制品活菌计数用培养基、计数方法及培养基的筛选方法。嗜酸乳杆菌计数用培养基中含有0.1μg/mL~0.125μg/mL的四环素，婴儿双歧杆菌计数用培养基中含有3μg/mL~5μg/mL的四环素，四联活菌制品由蜡样芽胞杆菌、粪肠球菌、嗜酸乳杆菌和婴儿双歧杆菌制成。本发明为基于代谢通路分析的四联活菌制品活菌计数用培养基的筛选方法，确定具有代谢差异的抗生素种类，并进一步进行浓度筛选，有效消除粪肠球菌对嗜酸乳杆菌活菌计数的干扰，有效消除嗜酸乳杆菌和粪肠球菌对婴儿双歧杆菌活菌计数的干扰，保证计数结果准确有效。
Description
技术领域
本发明属于微生物检测技术领域，具体涉及一种基于代谢通路分析的四联活菌制品活菌计数用培养基、计数方法及培养基的筛选方法。
背景技术
双歧杆菌四联活菌片的主要成分是蜡样芽胞杆菌、粪肠球菌、嗜酸乳杆菌和婴儿双歧杆菌，用于治疗与肠道菌群失调相关的腹泻、便秘、功能性消化不良。本品每片中婴儿双歧杆菌和嗜酸乳杆菌分别应不低于0.5×108CFU；粪肠球菌应不低于0.5×107CFU；蜡样芽孢杆菌应不低于0.5×106CFU。准确计数产品中的活菌数，是重要的产品质量控制内容。现行的活菌计数方法是根据活菌的种属不同，营养需求有差异，建立选择性的培养条件，针对性的培养某一种活菌并进行计数。但是，本品为四联活菌制品，很难针对每一种活菌，建立有效的选择性培养条件，同时能排除其他活菌的干扰。现行标准中收载的检测方法，其主要问题在于：在进行嗜酸乳杆菌计数时，粪肠球菌在该培养条件下也会迅速生长，与嗜酸乳杆菌菌落混合，严重干扰计数；在进行婴儿双歧杆菌计数时，嗜酸乳杆菌和粪肠球菌均为兼性厌氧菌，也可在该培养条件下生长，三种菌落混合，无法计数婴儿双歧杆菌菌落数。目前尚无有效的培养基，用于嗜酸乳杆菌和婴儿双歧杆菌的计数培养。筛选出可以有效抑制干扰微生物的培养基或培养基添加剂，是解决问题的关键。
现有基于药敏试验的筛查方法，多是在标准或试剂给定的有限的抗生素抗性谱上进行分析，其中可用于分析的抗生素数量少，且工作量巨大，步骤繁杂，效果差，这种基于有限研究抗生素的盲筛药敏试验结果不具有前瞻性和预测性。
基因组规模代谢网络模型（Genome-scale Metabolic Network Model，GEM）是基于基因-蛋白-反应（Gene-Protein-Reaction，GPR）三者关联，模拟细胞内复杂代谢通路的一种数学模型，可以预测细胞内的代谢通路和通量分布。目前，基于基因组测序注释和生化数据库已建立了许多微生物及动植物细胞的GEM，其已经成为系统研究生物体内代谢的主要建模分析方法之一。根据GPR关联数据，利用数学模型，GEM系统描述了生物体内一整套基于化学计量学的质量平衡代谢反应，可以通过通量平衡分析（Flux Balance Analysis，FBA）、通量可变性分析（Flux Variability Analysis，FVA）及代谢调节最小化分析（Minimization of Metabolic Adjustment，MOMA）等方法预测代谢反应的通量值。
发明内容
针对现有技术中没有有效的培养基用于嗜酸乳杆菌和婴儿双歧杆菌的计数培养的问题，本发明提供了一种基于代谢通路分析的四联活菌制品活菌计数用培养基、计数方法及培养基的筛选方法，筛选适宜种类和浓度的抗生素加入培养基中，可有效抑制干扰活菌的生长，保证计数结果准确有效。
本发明通过以下技术方案实现：
一种基于代谢通路分析的四联活菌制品活菌计数用培养基，嗜酸乳杆菌计数用培养基中含有0.1μg/mL~0.125μg/mL的四环素，婴儿双歧杆菌计数用培养基中含有3μg/mL~5μg/ml的四环素；
所述四联活菌制品由蜡样芽胞杆菌、粪肠球菌、嗜酸乳杆菌和婴儿双歧杆菌制成。
进一步地，所述的嗜酸乳杆菌计数用培养基中含有0.125μg/mL的四环素，婴儿双歧杆菌计数用培养基中含有3μg/mL的四环素,
进一步地，分别用嗜酸乳杆菌计数用培养基和婴儿双歧杆菌计数用培养基对四联活菌制品进行培养，对嗜酸乳杆菌和婴儿双歧杆菌进行计数。
本发明中，公开了基于代谢通路分析的四联活菌制品活菌计数用培养基的筛选方法，采用代谢通路分析的方法进行计数用培养基的筛选，具体包括以下步骤：
（1）基因组提取：分别培养、纯化蜡样芽胞杆菌、粪肠球菌、嗜酸乳杆菌和婴儿双歧杆菌，提取完整基因组；
（2）基因组序列拼接及序列校正：将基因组测序的得到的碱基序列通过无参序列拼接，组成完整的基因组，然后采用序列矫正对测序产生的低质量环化共有序列进行纠错，使所得序列能够完整正确反映目标菌株的实际遗传序列信息，拼接结果为环状基因组，无碎片；
（3）基因注释及酶功能注释；
（4）抗生素抗性基因筛选及分析：经过注释的基因组与CARD数据库进行比对，采用DIAMOND检索策略，检索收集蜡样芽胞杆菌、粪肠球菌、嗜酸乳杆菌和婴儿双歧杆菌中含有的抗生素抗性基因；
（5）关键代谢通路筛选及分析：检索与抗生素抗性基因相关的代谢通路，并对抗生素抗性基因进行代谢通路归类；
（6）目标抗生素确认：根据筛选获得的抗生素抗性基因和代谢通路分析，选择四联活菌制品中四种菌株共有的抗性基因及代谢通路，根据代谢通路进行培养基成分的优化，筛选出对四种菌存在不同耐药性的抗生素作为培养基添加剂；
（7）抗生素浓度分析：步骤（6）筛选出的抗生素添加剂分别加入至嗜酸乳杆菌培养基和婴儿双歧杆菌培养基中，筛选出计数用培养基中抗生素的浓度。
进一步地，步骤（1）采用磁珠法提取完整基因组。
进一步地，步骤（2）中以二代及三代测序结果互为参考，采用从头测序方法，原始数据通过FastQC进行质量评估，通过Trimmomatic对测序数据进行质量剪切，使用SPAdes拼接二代测序数据，采用GapFiller对拼接得到的contig补GAP，利用PrInSeS-G修正拼接过程中的剪辑错误及小片段的插入缺失。
进一步地，步骤（3）中采用Uniref作为基因注释及酶功能注释的数据库。
进一步地，步骤（4）中蜡样芽胞杆菌、粪肠球菌、嗜酸乳杆菌和婴儿双歧杆菌中含有的抗生素抗性基因为13类，17个基因，分别为rpoB、tetW、poxtA、dfrE、lsaA、emeA、efrA、efrB、vanRA、MCR-4、msrA、FosB、mphB、BcI、vmlR、mphB和arnA。
进一步地，步骤（5）从京都基因和基因组百科全书中检索与抗生素抗性基因相关的通路，并对抗性基因进行代谢通路归类，与抗生素抗性基因相关的通路涉及的代谢类型共15种，分别为K03043、K18220、K03110、K00287、K19350、K01358、K18887、K18888、K18349、K18231、K11210、K06979、K17836、K18231和K10011。
进一步地，步骤（6）筛选出来自蜡样芽胞杆菌的MCR-4、来自粪肠球菌的emeA、来自嗜酸乳杆菌的poxtA和来自婴儿双歧杆菌的tetW产生四环素抗性，在四环素耐药性上，四种菌株存在显著差异。
本发明取得的有益效果为：
本发明公开了一种基于代谢通路分析的四联活菌制品活菌计数用培养基的筛选方法，基于生物信息学的基因功能预测及代谢通路分析是基于微生物细胞内的遗传物质基础上的理论预测，具有极强的前瞻性和专属性，通过基因组分析及代谢通路分析可有效预测微生物的抗生素敏感性及抗性，根据预测结果精准的确定几种微生物具有代谢差异的抗生素种类，并进一步进行浓度筛选。该方法将大大加快实验效率，结果更具有预测性和准确性。本发明基于代谢通路分析的四联活菌制品计数用培养基有效消除粪肠球菌对嗜酸乳杆菌活菌计数的干扰，有效消除嗜酸乳杆菌和粪肠球菌对婴儿双歧杆菌活菌计数的干扰，保证计数结果准确有效。
附图说明
图1 四环素浓度为1/16μg/mL、1/8μg/mL、1/4μg/mL时琼脂培养基上粪肠球菌的生长情况图，上下两个培养基的四环素浓度相同；
图2为四环素浓度为1μg/mL、2μg/mL、4μg/mL时琼脂培养基上嗜酸乳杆菌的生长情况图，上下两个培养基的四环素浓度相同；
图3四环素浓度为0μg/mL、0.1μg/mL、0.125μg/mL时琼脂培养基上嗜酸乳杆菌和粪肠球菌的生长情况图；
图4为四环素浓度为1μg/mL、2μg/mL、4μg/mL时琼脂培养基上婴儿双歧杆菌的生长情况图，上下两个培养基的四环素浓度相同；
图5为四环素浓度为0、3μg/mL、4μg/mL、5μg/mL时琼脂培养基上婴儿双歧杆菌、嗜酸乳杆菌和粪肠球菌的生长情况图。
具体实施方式
为了对本发明进行更进一步的详细描述，给出以下具体实施范例，但仅用于阐明本发明，使步骤更加清晰，而不是为了限制本发明的应用范围。
实施例1 四联活菌制品（双歧杆菌四联活菌片（思连康））活菌计数用培养基的筛选方法，所述的双歧杆菌四联活菌片由蜡样芽胞杆菌、粪肠球菌、嗜酸乳杆菌和婴儿双歧杆菌制成，具体的筛选方法为：
（1）基因组提取：分别培养、纯化蜡样芽胞杆菌、粪肠球菌、嗜酸乳杆菌和婴儿双歧杆菌，采用磁珠法提取完整基因组；
（2）基因组序列拼接及序列校正：将基因组测序的得到的碱基序列通过无参序列拼接，即从头测序（Denovo Genome Sequencing，Denovo），组成完整的基因组，原始数据通过FastQC进行质量评估，通过Trimmomatic对测序数据进行质量剪切，使用SPAdes拼接二代测序数据，采用GapFiller对拼接得到的contig补GAP，利用PrInSeS-G修正拼接过程中的剪辑错误及小片段的插入缺失。三代及二代测序结果互为参考序列，后采用序列矫正（Polish）对测序产生的低质量环化共有序列（Circular Consensus Sequencing，CCS）等进行纠错，所得序列能够完整正确反映目标菌株的实际遗传序列信息，拼接结果为环状基因组，无碎片；
（3）基因注释及酶功能注释：基因注释及酶功能注释采用Uniref（https://ftp.uniprot.org/pub/databases/uniprot/uniref/，2023-05-03）作为数据库（与GO（GeneOntology）、NR（NCBI non-redundant protein sequences）以及COG（Clusters ofOrthologous Groups of proteins）等数据库相比，具有更高的覆盖率及更低的冗余度）；
（4）抗生素抗性基因筛选及分析：经过注释的基因组与CARD数据库（TheComprehensive Antibiotic Resistance Database，更新至2023-05-25）进行比对，根据抗生素分子类型可分为59类，用DIAMOND检索策略（该检索策略的检索速率较BLAST提高20000倍），检索收集蜡样芽胞杆菌、粪肠球菌、嗜酸乳杆菌和婴儿双歧杆菌中含有的抗生素抗性基因涉及13类，共计17个抗生素抗性基因；
17个抗生素抗性基因分别为：婴儿双歧杆菌（Bifidobacterium animalis）基因组中有2种抗生素抗性基因：rpoB（ARO:3004480）、tetW（ARO:3000194）；嗜酸乳杆菌（Lactobacillus paracasei）基因组中有1种抗生素抗性基因：poxtA（ARO:3004470）；粪肠球菌（Enterococcus faecalis）基因组中有5种抗生素抗性基因：dfrE（ARO:3002875）、lsaA（ARO:3000300）、emeA（ARO:3003551）、efrA（ARO:3003948）、efrB（ARO:3003949）；蜡样芽孢杆菌（Bacillus cereus）基因组中有9种抗生素抗性基因：vanRA（ARO:3002919）、MCR-4（ARO:3004325）、msrA（ARO:3000251）、FosB（ARO:3000172）、mphB（ARO:3000318）、BcI（ARO:3002877）、vmlR（ARO:3004476）、mphB（ARO:3000318）以及arnA（ARO:3002985）；
13类抗生素抗性基因分别为：青霉烷酸头孢菌素类抗生素、大环内酯类抗生素、恶唑烷酮抗生素、二氨基嘧啶类抗生素、氟喹诺酮类抗生素、利福霉素抗生素、链菌素抗生素、林可酰胺类抗生素、磷霉素抗生素、氯霉素抗生素、四环素类抗生素、肽类抗生素、胸膜菌素抗生素；
（5）关键代谢通路筛选及分析：从京都基因和基因组百科全书（KyotoEncyclopedia of Genes and Genomes，KEGG）中检索与收集到的抗生素抗性基因相关的通路，并进行代谢通路归类；筛选到的代谢类型共15种，分别为K03043、K18220、K03110、K00287、K19350、K01358、K18887、K18888、K18349、K18231、K11210、K06979、K17836、K18231和K10011；
（6）目标抗生素确认：根据筛选获得的抗生素抗性基因和代谢通路分析，选择四联活菌制品中四种菌株共有的抗性基因及代谢通路，确认来自蜡样芽胞杆菌的MCR-4（ARO:3004325）、来自粪肠球菌的emeA（ARO:3003551）、来自嗜酸乳杆菌的poxtA（ARO:3004470）和来自婴儿双歧杆菌的tetW（ARO:3000194）等抗生素抗性基因产生四环素抗性，涉及包括map02024、map03070、map03060、map04112、map00520、map01503、map01250和map01100在内的7个代谢通路，在四环素耐药性上，四种菌存在显著差异，因此，选择四环素作为计数用培养基添加剂，进一步对其浓度进行筛选。
四联活菌制品中四种菌株的抗生素抗性基因及其代谢途径汇总表1~4所示：
表1
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表2
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表3
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表4
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（7）培养基中抗生素浓度筛选：
a)嗜酸乳杆菌计数用培养基四环素浓度筛选：
分别取粪肠球菌菌粉、嗜酸乳杆菌菌粉、双歧杆菌四联活菌片3g，加入0.9%无菌氯化钠溶液27mL，振荡使其充分混匀，作为10-1稀释液，取10-1稀释液1mL，加入到9mL 0.9%无菌氯化钠溶液中，制成10-2稀释液，用相同方法稀释到10-5，取10-5稀释液100μL滴加到制备好的含有不同浓度四环素的琼脂平皿上，以无菌涂布棒涂布均匀，置37℃下培养；
抗生素敏感性试验表明，四环素对粪肠球菌的MIC在1/16μg/mL~1/8μg/mL之间，首先采用粪肠球菌菌粉考察四环素浓度为1/16μg/mL、1/8μg/mL、1/4μg/mL的培养基上粪肠球菌的生长情况，如图1所示；由图1可知，四环素浓度为1/16μg/mL时，粪肠球菌的生长不会受到抑制，四环素浓度为1/8μg/mL及以上时，粪肠球菌被完全抑制，不生长。四环素对嗜酸乳杆菌的MIC在2~4μg/mL之间，采用嗜酸乳杆菌菌粉考察四环素浓度为1μg/mL、2μg/mL、4μg/mL的培养基上嗜酸乳杆菌的生长情况，如图2所示；由图2可知，四环素浓度达到2μg/mL时，嗜酸乳杆菌的生长受到明显抑制。采用双歧杆菌四联活菌片进行进一步的浓度筛选试验，如图3所示；由图3可知0.1μg/mL时，粪肠球菌虽然有少量生长，但菌落形态较小，不会对嗜酸乳杆菌的计数产生干扰；当四环素浓度在0.125μg/mL时，粪肠球菌被完全抑制，嗜酸乳杆菌可正常生长，可以进行嗜酸乳杆菌的菌落计数。因此，嗜酸乳杆菌计数用培养基中四环素浓度优选为0.1～0.125μg/mL。
进而确定嗜酸乳杆菌计数用培养基成分为：酪蛋白胨：10.0μg/mL，牛肉膏：10.0μg/mL，葡萄糖：20.0μg/mL，酵母浸膏：5.0μg/mL，吐温80：1.0μg/mL，磷酸氢二钾：5.0μg/mL，柠檬酸三胺：2.0μg/mL，乙酸钠：10.0μg/mL，硫酸镁：0.5μg/mL，硫酸锰：0.2μg/mL，琼脂：15.0μg/mL，四环素：0.1～0.125μg/mL。25℃调节pH值至5.2±0.2。
b)婴儿双歧杆菌计数用培养基四环素浓度筛选：
分别称取婴儿双歧杆菌菌粉、双歧杆菌四联活菌片3g，加入0.9%无菌氯化钠溶液27mL，振荡使其充分混匀，作为10-1稀释液，取10-1稀释液1mL，加入到9ML 0.9%无菌氯化钠溶液中，制成10-2稀释液，用相同方法稀释到10-5，取10-5稀释液100μL滴加到制备好的含有不同浓度四环素的琼脂培养基平皿上，以无菌涂布棒涂布均匀，置37℃厌氧培养72h。
抗生素敏感性试验表明，四环素对嗜酸乳杆菌的MIC在2～4μg/mL之间，首先采用婴儿双歧杆菌菌粉考察四环素浓度为1μg/mL、2μg/mL、4μg/mL的琼脂培养基上婴儿双歧杆菌的生长情况，如图4所示；由图4可知，四环素浓度为1μg/mL、2μg/mL时，婴儿双歧杆菌菌落形态生长良好，当四环素浓度为4μg/mL时，婴儿双歧杆菌菌落稍微小于1μg/mL和2μg/mL组，但菌落数不受影响，不会对计数产生干扰。采用双歧杆菌四联活菌片进行进一步的浓度筛选试验，如图5所示，由图5可知，四环素浓度为3μg/mL时，婴儿双歧杆菌可正常生长，嗜酸乳杆菌、粪肠球菌均不生长；当四环素浓度增加至5μg/mL时，婴儿双歧杆菌仍可正常生长，菌落形态、菌落数与4μg/mL组基本一致，嗜酸乳杆菌、粪肠球菌均不生长。因此，婴儿双歧杆菌计数用培养基中四环素浓度为3~5μg/mL。
进而确定婴儿双歧杆菌计数用培养基成分如下为：酪蛋白胨：10.0μg/mL，牛肉膏：2.0μg/mL，大豆蛋白胨：5.0μg/mL，葡萄糖：5.0μg/mL，酵母浸膏：3.0μg/mL，吐温80：1.0μg/mL，L-半胱氨酸：0.3μg/mL，磷酸氢二钾：2.0μg/mL，氯化镁：0.5μg/mL，硫酸锌：0.25μg/mL，氯化钙：0.15μg/mL，三氯化铁：0.15μg/mL，三氯化铁：0.05μg/mL，维生素B1：0.015μg/mL，维生素B6：0.015μg/mL，维生素B2：0.015μg/mL，泛酸钙：0.0015μg/mL，琼脂：15.0μg/mL，四环素：3～5μg/mL，25℃调节pH值至7.2±0.2。
Claims (3)
1.一种基于代谢通路分析的四联活菌制品活菌计数用培养基，其特征在于，嗜酸乳杆菌计数用培养基成分为：酪蛋白胨：10.0 μg/mL，牛肉膏：10.0 μg/mL，葡萄糖：20.0 μg/mL，酵母浸膏：5.0 μg/mL，吐温80：1.0 μg/mL，磷酸氢二钾：5.0 μg/mL，柠檬酸三胺：2.0 μg/mL，乙酸钠：10.0 μg/mL，硫酸镁：0.5 μg/mL，硫酸锰：0.2 μg/mL，琼脂：15.0 μg/mL，四环素：0.1～0.125 μg/mL，25℃调节pH值至5.2±0.2；
婴儿双歧杆菌计数用培养基成分为：酪蛋白胨：10.0 μg/mL，牛肉膏：2.0 μg/mL，大豆蛋白胨：5.0 μg/mL，葡萄糖：5.0 μg/mL，酵母浸膏：3.0 μg/mL，吐温80：1.0 μg/mL，L-半胱氨酸：0.3 μg/mL，磷酸氢二钾：2.0 μg/mL，氯化镁：0.5 μg/mL，硫酸锌：0.25 μg/mL，氯化钙：0.15 μg/mL，三氯化铁：0.05 μg/mL，维生素B1：0.015 μg/mL，维生素B6：0.015 μg/mL，维生素B2：0.015 μg/mL，泛酸钙：0.0015 μg/mL，琼脂：15.0 μg/mL，四环素：3～5 μg/mL，25℃调节pH值至7.2±0.2；
所述四联活菌制品由蜡样芽胞杆菌、粪肠球菌、嗜酸乳杆菌和婴儿双歧杆菌制成。
2.根据权利要求1所述的基于代谢通路分析的四联活菌制品活菌计数用培养基，其特征在于，所述的嗜酸乳杆菌计数用培养基中含有0.125 μg/mL的四环素，婴儿双歧杆菌计数用培养基中含有3 μg/mL的四环素。
3.一种利用权利要求1或2所述的基于代谢通路分析的四联活菌制品活菌计数用培养基进行活菌计数的方法，其特征在于，分别用嗜酸乳杆菌计数用培养基和婴儿双歧杆菌计数用培养基对四联活菌制品进行培养，对嗜酸乳杆菌和婴儿双歧杆菌进行计数。
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