ISOLATION AND IDENTIFICATION OF ANAEROBES IN THE INTESTINAL MUCOSA OF GRASS CARP CTENOPHARYNGODON IDELLUS
-
摘要: 研究以草鱼(Ctenopharyngodon idellus)为实验对象, 运用厌氧培养的方法, 研究了饥饿状态下草鱼肠道黏膜固有微生物的类群及其在不同肠段的分布。实验结果显示草鱼前肠、中肠与后肠中细菌的数量分别是3.17×103、1.63×104和1.79×107 cfu/g。研究共分离到274株单菌落, 经16S rRNA鉴定, 分别属于拟杆菌属(Bacteroides spp.)、鲸杆菌属(Cetobacterium spp.)、梭形杆菌属(Fusobacterium spp.)、气单胞菌属(Aeromonas spp.)、希瓦氏菌属(Shewanella spp.)、芽孢杆菌属(Bacillus spp.)、泛菌属(Pantoea spp.)和柠檬酸杆菌属(Citrobacter spp.)8个种类, 其中专性厌氧细菌的数量占9.1%, 兼性厌氧细菌的数量占90.9%。进一步分析发现, 前肠中只分离到兼性厌氧细菌, 中肠与后肠专性厌氧细菌和兼性厌氧细菌都有分布。专性厌氧细菌Bacteroides paurosaccharolyticus和Bacteroides luti在中肠与后肠都有分布, 而Cetobacterium somerae和Fusobacterium ulcerans只在后肠有发现。兼性厌氧细菌是草鱼肠道黏膜的优势菌群, 其中嗜水气单胞菌Aeromonas hydrophila占73.7%。草鱼肠道不同部位固有厌氧微生物组成存在差异, 细菌数量也明显不同, 后肠中具有更高的细菌丰度和多样性。Abstract: The bacteria community in the gastrointestinal tract of fish has been the subject of numerous studies. However, most of those researches have focused on the molecular ecology of intestinal microbiota. Few studies pay attention to the bacterial culture, especially to the culture of the anaerobic intestinal bacteria, which leads to poor description in anaerobic autochthonous bacteria of gut. In this study, we investigated the autochthonous microflora of intestinal mucosa in grass carp (Ctenopharyngodon idellus) with the culture dependent method. The bacterial numbers in foregut, midgut and hindgut of the fish were 3.17×104, 1.63×105 and 1.79×108 cfu/g, respectively. A total of 274 bacterial isolations were sequenced and identified as genus Bacteroides, Cetobacterium, Fusobacterium, Aeromonas, Shewanella, Bacilluss, Pantoea and Citrobacter. 9.1% of which were obligate anaerobic bacteria and the others were facultative bacteria. The anaerobic bacteria, Bacteroides paurosaccharolyticus and Bacteroides. Eggerthii, were only found in midgut and hindgut, while Cetobacterium somerae and Fusobacterium varium were only found in hindgut. The facultative bacteria existed in the whole gut, among which Aeromonas hydrophila was dominant. Our results suggest that species and quantities of intestinal bacterial vary with the segments of C. idellus, and hindgut presents higher bacterial abundance and diversity.
-
Key words:
- Ctenopharyngodon idellus /
- Intestinal mucosa /
- Anaerobic bacteria /
- Bacteria abundance
-
-
图 1 基于草鱼肠道黏膜厌氧细菌16S rRNA基因序列构建的系统发育树
Figure 1. Neighbor-joining phylogenetic tree of 16S rRNA gene sequences of intestinal mucosa anaerobes of grass carp
表 1 不同肠段厌氧细菌数量
Table 1. Numbers of anaerobic bacteria in different intestinal segment (cfu/g)
不同重复
Different repetition前肠
Foregut中肠
Midgut后肠
Hindgut1 2.40×103 2.35×104 1.60×107 2 4.00×103 1.08×104 2.03×107 3 3.10×103 1.46×104 1.73×107 平均值Mean 3.17×103 1.63×104 1.79×107 
下载: 导出CSV
表 2 草鱼肠道可培养专性厌氧细菌数目、种类及分布
Table 2. Numbers, species and distribution of cultivable obligate anaerobes in intestine of grass carp
菌株编号
Strain No.专性厌氧细菌
Obligate anaerobe前肠
Foregut中肠
Midgut后肠
Hindgut总和
TotalOA 1 Bacteroides luti 0 8 7 15 OA 2 Bacteroides paurosaccharolyticus 0 4 2 6 OA 3 Cetobacterium somerae 0 0 3 3 OA 4 Fusobacterium ulcerans 0 0 1 1 总和Total 0 12 13 25
下载: 导出CSV
表 3 草鱼肠道可培养兼性厌氧细菌数目、种类及分布
Table 3. Numbers, species and distribution of cultivable facultative anaerobes in intestine of grass carp
菌株编号Strain No. 兼性厌氧细菌Facultative anaerobe 前肠Foregut 中肠Midgut 后肠Hindgut 总和Total FA 1 Aeromonas hydrophila 78 69 55 202 FA 2 Aeromonas allosaccharophila 0 0 1 1 FA 3 Aeromonas aquatica 6 1 0 7 FA 4 Aeromonas encheleia 4 3 1 8 FA 5 Aeromonas piscicola 1 0 0 1 FA 6 Bacillus licheniformis 5 2 1 8 FA 7 Citrobacter youngae 2 0 0 2 FA 8 Pantoea ananatis 0 2 1 3 FA 9 Shewanella oneidensis 0 0 1 1 FA 10 Shewanella xiamenensis 1 3 10 14 FA 11 Flavobacterium acidificum 0 0 2 2 总和Total 97 80 72 249
下载: 导出CSV
-
Ringø E, Strøm E, Tabachek J A. Intestinal microflora of salmonids: a review [J]. Aquaculture Research, 1995, 26(10): 773—789
Trust T, Sparrow R. The bacterial flora in the alimentary tract of freshwater salmonid fishes [J]. Canadian Journal of Microbiology, 1974, 20(9): 1219—1228
Ramirez R F, Dixon B A. Enzyme production by obligate intestinal anaerobic bacteria isolated from oscars (Astronotus ocellatus), angelfish (Pterophyllum scalare) and southern flounder (Paralichthys lethostigma) [J]. Aquaculture, 2003, 227(1): 417—426
Rajilić-Stojanović M, Heilig H G, Molenaar D, et al. Development and application of the human intestinal tract chip, a phylogenetic microarray: analysis of universally conserved phylotypes in the abundant microbiota of young and elderly adults [J]. Environmental Microbiology, 2009, 11(7): 1736—1751
Huber I, Spanggaard B, Appel K, et al. Phylogenetic analysis and in situ identification of the intestinal microbial community of rainbow trout (Oncorhynchus mykiss, Walbaum) [J]. Journal of Applied Microbiology, 2004, 96(1): 117—132
Xia J, Lin G, Fu G, et al. The intestinal microbiome of fish under starvation [J]. BMC Genomics, 2014, 15(1): 266
Chinese Fishery Statistical Yearbook [M]. Beijing: Chinese Agricultural Press. 2016, 31
Wu S, Wang G, Angert E R, et al. Composition, diversity, and origin of the bacterial community in grass carp intestine [J]. PloS One, 2012, 7(2): e30440
Tran N T, Xiong F, Hao Y T, et al. Two biomass preparation methods provide insights into studying microbial communities of intestinal mucosa in grass carp (Ctenopharyngodon idellus) [J]. Aquaculture Research, 2017, DOI: 10.1111/are.13248
Han S, Liu Y, Zhou Z, et al. Analysis of bacterial diversity in the intestine of grass carp (Ctenopharyngodon idellus) based on 16S rDNA gene sequences [J]. Aquaculture Research, 2010, 42(1): 47—56
Wang W W, Wu S G, Zou H, et al. Characterization of cellulose-decomposing bacteria in the intestine of grass carp, Ctenopharyngodon idellus [J] Acta Hydrobiologica Sinica, 2014, 38(2): 291—297
Sugita H, Tokuyama K, Deguchi Y. The intestinal microflora of carp Cyprinus carpio, grass carp Ctenopharyngodon idella and tilapia Sarotherodon niloticus [J]. Bulletin of the Japanese Society for the Science of Fish, 1985, 51(8): 1325—1329
Trust T, Bull L, Currie B, et al. Obligate anaerobic bacteria in the gastrointestinal microflora of the grass carp (Ctenopharyngodon idella), goldfish (Carassius auratus), and rainbow trout (Salmo gairdneri) [J]. Journal of the Fisheries Board of Canada, 1979, 36(10): 1174—1179
Ringø E, Olsen R E, Mayhew T M, et al. Electron microscopy of the intestinal microflora of fish [J]. Aquaculture, 2003, 227(1-4): 395—415
Ni D S, Wang J G. Biology and Disease of Grass Carp [M]. Beijing: Science Press. 1999, 29—33
Wu S, Gao T, Zheng Y, et al. Microbial diversity of intestinal contents and mucus in yellow catfish (Pelteobagrus fulvidraco) [J]. Aquaculture, 2010, 303(1): 1—7
Yu Z, Morrison M. Improved extraction of PCR-quality community DNA from digesta and fecal samples [J]. Biotechniques, 2004, 36(5): 808—813
Cheng Y Y, Wu S G, Zheng Y Z, et al. Microbial diversity in the sediment of a pond mainly stocked with Ctenopharyngodon idellus [J]. Freshwater Fisheries, 2011, 41(6): 43—49
Cole J R, Wang Q, Fish J A, et al. Ribosomal Database Project: data and tools for high throughput rRNA analysis [J]. Nucleic Acids Research, 2013, 42: D633—42
Kumar S, Stecher G, Tamura K. MEGA7: Molecular Evolutionary Genetics Analysis version 7.0 for bigger datasets [J]. Molecular Biology and Evolution, 2016, msw054
Kimura M. A simple method for estimating evolutionary rates of base substitutions through comparative studies of nucleotide sequences [J]. Journal of Molecular Evolution, 1980, 16(2): 111—120
Saitou N, Nei M. The neighbor-joining method: a new method for reconstructing phylogenetic trees [J]. Molecular Biology and Evolution, 1987, 4(4): 406—425
Xu Y F, Song D J. The structure and function of fish intestinal tissue [J]. Jiangxi Feed, 2004, 4: 16—19
Ren B Z, Tang Y, Li H Z. Progress of studies on the mechanism and control of activated sludge bulking [J] Journal of University of South China (Science and Technology), 2010, 2: 25
Cruden D, Markovetz A. Microbial ecology of the cockroach gut [J]. Annual Reviews in Microbiology, 1987, 41(1): 617—643
Clements K D. Fermentation and Gastrointestinal Microorganisms in Fishes [M]. Springer: Gastrointestinal Microbiology. 1997, 156—198
Finegold S M, Shepherd W E, Spaulding E H. Practical Anaerobic Bacteriology. Cumitech 5 [M]. American Society for Microbiology, 1977
Sugita H, Miyajima C, Deguchi Y. The vitamin B12-producing ability of the intestinal microflora of freshwater fish [J]. Aquaculture, 1991, 92: 267—276
Ray A, Ghosh K, Ringø E. Enzyme-producing bacteria isolated from fish gut: a review [J]. Aquaculture Nutrition, 2012, 18(5): 465—492
Jiang Y, Xie C, Yang G, et al. Cellulase-producing bacteria of Aeromonas are dominant and indigenous in the gut of Ctenopharyngodon idellus (Valenciennes) [J]. Aquaculture Research, 2011, 42(4): 499—505
Sugita H, Tanaka K, Yoshinami M, et al. Distribution of Aeromonas species in the intestinal tracts of river fish [J]. Applied and Environmental Microbiology, 1995, 61(11): 4128—4130
Lee C, Kim J, Hwang K, et al. Fermentation and growth kinetic study of Aeromonas caviae under anaerobic conditions [J]. Applied Microbiology and Biotechnology, 2009, 83(4): 767—773
Sugita H, Shibuya K, Shimooka H, et al. Antibacterial abilities of intestinal bacteria in freshwater cultured fish [J]. Aquaculture, 1996, 145(1): 195—203
Tindall B J, Rosselló-Móra R, Busse H-J, et al. Notes on the characterization of prokaryote strains for taxonomic purposes [J]. International Journal of Systematic and Evolutionary Microbiology, 2010, 60(1): 249—266
Olmos J, Ochoa L, Paniagua-Michel J, et al. Functional feed assessment on Litopenaeus vannamei using 100% fish meal replacement by soybean meal, high levels of complex carbohydrates and Bacillus probiotic strains [J]. Marine Drugs, 2011, 9(6): 1119—1132
Cutting S M. Bacillus probiotics [J]. Food Microbiology, 2011, 28(2): 214—220
Zhang C N, Li X F, Xu W N, et al. Combined effects of dietary fructooligosaccharide and Bacillus licheniformis on innate immunity, antioxidant capability and disease resistance of triangular bream (Megalobrama terminalis) [J]. Fish & Shellfish Immunology, 2013, 35(5): 1380—1386
Wu Y C, Gong Q. Effect of feeding microorganisms on growth performance and the activitiesdigestive enzymes of Oreochromis niloticus×O. aureus [J] Acta Hydrobiologica Sinica, 2011, 35(6): 998—1004
图(1)
表(3)
计量
- 文章访问数: 1188
- PDF下载数: 1
- 施引文献: 0
