Phylogenetic analysis and in situ identification of the intestinal microbial community of rainbow trout (Oncorhynchus mykiss, Walbaum)
Phylogenetic analysis and in situ identification of the intestinal microbial community of rainbow trout (Oncorhynchus mykiss, Walbaum)
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DOI：
10.1046/j.1365-2672.2003.02109.x
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发表时间：
2004-01-01
影响因子：
4
通讯作者：
Gram, L
中科院分区：
文献类型：
--
作者：
Huber, I;Spanggaard, B;Gram, L
目的：鉴定虹鳟鱼肠道的优势可培养菌群和不可培养菌群。方法与结果：采用直接显微镜计数法（4(')，6-二氨基-2-苯基吲哚，DAPI）和色氨酸大豆琼脂（TSA）培养法测定虹鳟鱼肠道微生物密度。对肠道样本的细菌DNA进行差异梯度凝胶电泳分析、条带再扩增和序列分析，以确定好氧计数小于或等于DAPI计数2%的样品中占主导地位的细菌。鉴定了146株分离菌的16S rDNA基因序列和3个非培养菌的16S rDNA基因序列。构建了一套寡核苷酸探针，应用荧光原位杂交（FISH）技术对虹鳟鱼胃肠道细菌群落结构进行检测和枚举。变形菌科的γ亚类成员（主要是气单胞菌和肠杆菌科）在细菌群体结构中占主导地位。不动杆菌、假单胞菌、希瓦氏菌、Plesiomonas和变形杆菌、变形杆菌属β亚纲和革兰氏阳性菌的DNA G + C含量有高有低。在大多数样品中，有氧计数（在TSA上）是直接（DAPI）计数的50-90%。在肠道样本中检测到一种细菌，该细菌代表了一种以前未知的系统发育谱系，其16S rRNA基因序列与戊氧厌氧菌只有89%的相似性，其中需氧计数小于或等于直接（DAPI）计数的2%。在低需氧计数的样品中，有10 - 75%的微生物种群与针对这种尚未培养的细菌构建的探针杂交（FISH）。结论：虹鳟鱼肠道微生物群以γ亚纲的变形菌群为主。然而，在一些样品中，微生物群主要是未经培养的，假定的厌氧微生物。虹鳟鱼肠道的细菌种群结构以及细菌总数因鱼而异。本研究的意义和影响：培养结果与原位分析结果具有良好的相关性，但对于TSA上未培养生物的鉴定，分子方法至关重要。
Aims: To identify the dominant culturable and nonculturable microbiota of rainbow trout intestine.Methods and Results: Microbial density of rainbow trout intestine was estimated by direct microscopic counts (4('),6-diamidino-2-phenylindole, DAPI) and by culturing on tryptone soya agar (TSA). Differential gradient gel electrophoresis analysis of bacterial DNA from intestinal samples, re-amplification of bands and sequence analysis was used to identify the bacteria that dominated samples where aerobic counts were less than or equal to2% of the DAPI counts. 16S rDNA gene sequences of 146 bacterial isolates and three sequences of uncultured bacteria were identified. A set of oligonucleotide probes was constructed and used to detect and enumerate the bacterial community structure of the gastrointestinal tract of rainbow trout by fluorescence in situ hybridization (FISH). Members of the gamma subclass of Proteobacteria (mainly Aeromonas and Enterobacteriaceae) dominated the bacterial population structure. Acinetobacter, Pseudomonas, Shewanella, Plesiomonas and Proteus were also identified together with isolates belonging to the beta subclass of Proteobacteria and Gram-positive bacteria with high and low DNA G + C content. In most samples, the aerobic count (on TSA) was 50-90% of the direct (DAPI) count. A bacterium representing a previously unknown phylogenetic lineage with only 89% 16S rRNA gene sequence similarity to Anaerofilum pentosovorans was detected in intestinal samples where aerobic counts were less than or equal to2% of direct (DAPI) counts. Ten to 75% of the microbial population in samples with low aerobic counts hybridized (FISH) with a probe constructed against this not-yet cultured bacterium.Conclusions: Proteobacteria belonging to the gamma subclass dominated the intestinal microbiota of rainbow trout. However, in some samples the microflora was dominated by uncultivated, presumed anaerobic, micro-organisms. The bacterial population structure of rainbow trout intestine, as well as total bacterial counts, varied from fish to fish.Significance and Impact of the Study: Good correlation was seen between cultivation results and in situ analysis, however, a molecular approach was crucial for the identification of organisms uncultivated on TSA.