Isolation of Bifidobacteria from Breast Milk and Assessment of the Bifidobacterial Population by PCR-Denaturing Gradient Gel Electrophoresis and Quantitative Real-Time PCR
Isolation of Bifidobacteria from Breast Milk and Assessment of the Bifidobacterial Population by PCR-Denaturing Gradient Gel Electrophoresis and Quantitative Real-Time PCR
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DOI：
10.1128/aem.02063-08
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发表时间：
2009-02-15
影响因子：
4.4
通讯作者：
Rodriguez, Juan M.
中科院分区：
文献类型：
--
作者：
Martin, Rocio;Jimenez, Esther;Rodriguez, Juan M.
这项工作的目的是阐明母乳中是否含有双歧杆菌以及它们是否可以通过母乳喂养传播到婴儿肠道。 23 名妇女及其各自的婴儿分别在出生后第 4 至 7 天提供了母乳和粪便样本。通过 F6PPK（6-磷酸果糖磷酸酮醇酶）测定，从特定培养基中分离出具有典型双歧杆菌形状的革兰氏阳性和过氧化氢酶阴性菌株，并通过 16S rRNA 基因测序鉴定到种属水平。通过 PCR 变性梯度凝胶电泳 (PCR-DGGE) 评估母乳中的双歧杆菌群落，并通过定量实时 PCR (qRTi-PCR) 估计其水平。 8 个牛奶样本和 21 个粪便样本中存在双歧杆菌。从牛奶样品中分离出短双歧杆菌、青春双歧杆菌和双歧双歧杆菌，而婴儿粪便中还含有长双歧杆菌和假链状双歧杆菌。 PCR-DGGE 显示 22 个牛奶样品中存在一到四个显性双歧杆菌条带。相似度超过98%的序列被鉴定为短双歧杆菌、青春双歧杆菌、长双歧杆菌、双歧双歧杆菌和齿双歧杆菌。通过 qRTi-PCR 在相同的 22 个牛奶样品中检测到双歧杆菌 DNA，每毫升 16S rRNA 基因拷贝数在 40 到 10,000 个之间。总之，母乳似乎是婴儿肠道活双歧杆菌的来源。
The objective of this work was to elucidate if breast milk contains bifidobacteria and whether they can be transmitted to the infant gut through breastfeeding. Twenty-three women and their respective infants provided samples of breast milk and feces, respectively, at days 4 to 7 after birth. Gram-positive and catalase-negative isolates from specific media with typical bifidobacterial shapes were identified to the genus level by F6PPK (fructose-6-phosphate phosphoketolase) assays and to the species level by 16S rRNA gene sequencing. Bifidobacterial communities in breast milk were assessed by PCR-denaturing gradient gel electrophoresis (PCR-DGGE), and their levels were estimated by quantitative real-time PCR (qRTi-PCR). Bifidobacteria were present in 8 milk samples and 21 fecal samples. Bifidobacterium breve, B. adolescentis, and B. bifidum were isolated from milk samples, while infant feces also contained B. longum and B. pseudocatenulatum. PCR-DGGE revealed the presence of one to four dominant bifidobacterial bands in 22 milk samples. Sequences with similarities above 98% were identified as Bifidobacterium breve, B. adolescentis, B. longum, B. bifidum, and B. dentium. Bifidobacterial DNA was detected by qRTi-PCR in the same 22 milk samples at a range between 40 and 10,000 16S rRNA gene copies per ml. In conclusion, human milk seems to be a source of living bifidobacteria for the infant gut.