The cross talk occurring between bifidobacteria in the mammalian’s gut microbiota [PRL]. The cross talk occurring between bifidobacteria in the mammalian’s gut microbiota [PRL]

We describe the molecular cross talk established under in vivo conditions between a set of human gut bifidobacterial commensals. Eleven groups of five conventional female 8-wk-old BALB/c mice taking a standard polysaccharide-rich Chow diet were administered a single daily dose of 109 CFU of either B. bifidum PRL2010, B. breve 12L , B. adolescentis 22L , B. longum subsp. infantis ATCC15697, or bifidobacterial couples, i.e., PRL2010-12L, PRL2010-22L, PRL2010-ATCC15696, 12L-22L, 12L-ATCC15697, 22L-ATCC15697, or a combination of all bifidobacterial strains. The transcriptome of bifidobacterial strains under in vivo conditions was analyzed. The transcripts expressed in B. bifidum PRL2010, B. breve 12L, B. adolescentis 22L and B. longum subsp. infantis ATCC15697 were profiled using a custom-made PRL2010-12L-22L-ATCC15697 (multibifido)-array representing 100%, 99%, 96%, 99% of the identified genes of these organisms, respectively.The observed functional changes in the trascriptomes of bifidobacteria might be caused by the possible shifts of the mice cecum microbiota upon colonization with bifidobacteria. Thus, we assessed if the presence of B. bifidum PRL2010, B. breve 12L, B. adolescentis 22L and B. longum subsp. infantis ATCC15697 on mono-, bi- or multi-association in the cecum of mice affects the overall composition of the microbiota of this environment. Overall design: Microarray analysis was performed with an oligonucleotide array based on the B. adolescentis 22L, B. bifidum PRL2010, B. breve 12L and B. longum subsp. infantis ATCC15697 genomes for a total of 45220 oligonucleotide probes of 60 bp in length were designed on 7679 ORFs using eArray5.0 (Agilent Technologies). 5 Oligos were designed for each gene on a 4x44k Agilent Microarrays (Agilent Technologies, Santa Clara, CA, USA). Replicates were distributed on the chip at random, non-adjacent positions. A set of 152 negative control probes designed on phage and plant sequences were also included on the chip. Reverse transcription and amplification of 500 ng of total RNA was performed with ImProm-IITM Reverse Transcriptase (Promega, Madison, USA) according to the manufacturer’s instructions. Five µg of cDNA was then labeled with ULS Labeling kit with Cy5 or Cy3 (Kreatech, The Netherlands). Labeled cDNA was hybridized using the Agilent Gene Transcription hybridization kit (part number 5188-5242) as described in the Agilent Two-Color Microarray-Based Gene Transcription Analysis v4.0 manual (G4140-90050). Following hybridization, microarrays were washed in accordance with Agilent's standard procedures.

本研究阐述了多株人体肠道共生双歧杆菌在体内环境下建立的分子交叉对话。将11组、每组5只饲喂标准富含多糖基础饲料的普通级雌性8周龄BALB/c小鼠，每日单次给予10^9菌落形成单位（Colony-Forming Units, CFU）的下述菌株：两歧双歧杆菌（Bifidobacterium bifidum）PRL2010、短双歧杆菌（Bifidobacterium breve）12L、青春双歧杆菌（Bifidobacterium adolescentis）22L、长双歧杆菌婴儿亚种（Bifidobacterium longum subsp. infantis）ATCC15697，或双歧杆菌双菌株组合（即PRL2010-12L、PRL2010-22L、PRL2010-ATCC15696、12L-22L、12L-ATCC15697、22L-ATCC15697），以及全部双歧杆菌菌株的混合组合。本研究对体内环境下双歧杆菌菌株的转录组进行了分析。针对两歧双歧杆菌PRL2010、短双歧杆菌12L、青春双歧杆菌22L以及长双歧杆菌婴儿亚种ATCC15697的表达转录本，本研究采用定制化的PRL2010-12L-22L-ATCC15697（multibifido）基因芯片进行转录组分析，该芯片分别覆盖上述菌株已注释基因的100%、99%、96%与99%。本研究观测到的双歧杆菌转录组功能变化，可能源于双歧杆菌定植后小鼠盲肠微生物群的组成改变。因此，本研究探究了小鼠盲肠内单菌株、双菌株或多菌株定植的两歧双歧杆菌PRL2010、短双歧杆菌12L、青春双歧杆菌22L以及长双歧杆菌婴儿亚种ATCC15697，是否会改变该环境下微生物群的整体组成。实验整体设计：本研究基于青春双歧杆菌22L、两歧双歧杆菌PRL2010、短双歧杆菌12L以及长双歧杆菌婴儿亚种ATCC15697的基因组设计寡核苷酸芯片，用于微阵列分析：利用Agilent Technologies的eArray5.0软件，针对7679个开放阅读框（Open Reading Frames, ORFs）设计了总计45220条长度为60 bp的寡核苷酸探针。在4×44K Agilent Microarrays（Agilent Technologies, Santa Clara, CA, USA）上，每个基因对应设计5条寡核苷酸探针。重复探针在芯片上随机分布于非相邻位置。芯片中同时包含152条基于噬菌体与植物序列设计的阴性对照探针。按照制造商说明书，使用ImProm-IITM Reverse Transcriptase（Promega, Madison, USA）对500 ng总RNA进行反转录与扩增。随后取5 μg cDNA，采用Cy5或Cy3标记的ULS Labeling kit（Kreatech, The Netherlands）进行荧光标记。按照Agilent Two-Color Microarray-Based Gene Transcription Analysis v4.0手册（货号G4140-90050）中的操作步骤，使用Agilent Gene Transcription hybridization kit（货号5188-5242）对标记后的cDNA进行杂交。杂交完成后，按照安捷伦标准流程对微阵列芯片进行洗涤。