Use of 16S rRNA gene sequencing analysis to characterize culturable intestinal bacteria in Atlantic salmon (Salmo salar) fed diets with cellulose or non-starch polysaccharides from soy

Duplicate groups of Atlantic salmon (Salmo salar L.), kept in seawater, were fed fish meal‐based cold‐pelleted diets. Diets with non‐starch polysaccharides (NSP), either cellulose, purified soybean NSP or extruded purified soybean NSP at a dietary level of 100 g kg^?1, were compared with a diet without supplemental NSP and a diet with soybean meal in a 28‐day feeding trial. Isolation and characterizations were limited to culturable bacteria and population levels of aerobic and facultative aerobic heterotrophic autochthonous (adherent) and allochthonous (transient) bacteria present in the mid and distal intestines of Atlantic salmon fed the five different diets estimated using traditional bacteriological techniques. The presence of an autochthonous microbiota was demonstrated using transmission electron microscopy. No significant effects of diet composition were observed on total population levels of culturable bacteria present in the digestive tract, but the study showed that the composition of the gut microbiota (autochthonous or allochthonous) was sensitive to dietary changes. A total of 752 culturable isolates from the intestines were characterized by biochemical and physiological properties. Of these, 188 isolates were further characterized by partial sequencing the 16S rRNA genes. Among these, 146 isolates belonged to 31 phylotypes that were >94% identical to previously described species. However, 42 isolates showed similarity <94% to species available at the National Center of Biotechnology Information. Several of the phylotypes identified in the present study have not been reported previously in the gastrointestinal (GI) tract of fish, including the Gram‐negative bacteria Gelidibacter salicanalis, Pseudoalteromonas elyakovii, Psychrobacter aquimaris, Psychrobacter cibarius, Psychrobacter fozii, Psychrobacter maritimus, Psychrobacter okhotskensis and Psychrobacter psychrophilus. Among the Gram‐positive bacteria identified were Arthrobacter bergeri, Arthrobacter psychrolactophilus, Arthrobacter rhombi, Bacillus pumilus, Bacillus subtilis, Exiguobacterium spp., Microbacterium oxydans, Planococcus maritimus, Sporosarcina ginsengisoli and several bacteria that have been described as unculturable previously. In addition, we identified Carnobacterium inhibens, a lactic acid bacterium that is not frequently isolated from the GI tract of fish. Psychrobacter cibarius was the dominant bacterial species and was isolated from the digestive tract of all fish investigated.     

Characterisation of the microbiota associated with intestine of Atlantic cod (Gadus morhua L.): The effect of fish meal, standard soybean meal and a bioprocessed soybean meal

Populations of aerobic heterotrophic bacteria present in the gastrointestinal (GI) tract of healthy Atlantic cod (Gadus morhua L.) fed three different diets, fish meal, standard or a bioprocessed soybean meal (BPSBM), were estimated using the dilution plate technique. A total of 944 isolates were characterised by biochemical and physiological properties and 425 isolates were identified further by sequencing the 16S rRNA genes. Our results showed that gut microbiota were affected by dietary manipulation. The GI tract of fish fed fish meal was dominated by Gram-positive bacteria of the genera Brochothrix and Carnobacterium. The Gram-negative bacteria Chryseobacterium spp. and Psychrobacter glacincola, and Gram-positive bacteria belonging to Carnobacterium, dominated in the digestive tract of fish fed soybean meal. In contrast to these results, genus Psychrobacter dominated in the GI tract when fish were fed BPSBM.Until recently, it was generally suggested that the gut microbiota of fish were less diverse than in homoeothermic animals. However, the present study identified several “new” bacterial species isolated from the alimentary tract of Atlantic cod. These “new” bacterial species are not normally isolated from the GI tract of fish. Based on our finding we suggest that the GI tract microbiota of fish might not be as simple as believed.Antagonistic activity of carnobacteria regarding inhibition of growth of two fish pathogens (Aeromonas salmonicida ssp. salmonicida and Vibrio anguillarum) was observed. However, some difference in the antibacterial activity of Carnobacterium spp. was observed. Whether this antagonistic activity has any effect in challenge studies will be discussed, especially in relation to the finding that the digestive tract is one of the major infection routes in fish.     

Role of gastrointestinal microbiota in fish

The gastrointestinal (GI) tract of an animal consists of a very complex and dynamic microbial ecosystem that is very important from a nutritional, physiological and pathological point of view. A wide range of microbes derived from the surrounding aquatic environment, soil/sediment and feed are found to colonize in the GI tract of fish. Among the microbial groups, bacteria (aerobic, facultative anaerobic and obligate aneraobic forms) are the principal colonizers in the GI tract of fish, and in some fish, yeasts are also reported. The common bacterial colonizers in the GI tract of freshwater and marine fish include Vibrio, Aeromonas, Flavobacterium, Plesiomonas, Pseudomonas, Enterobacteriaceae, Micrococcus, Acinetobacter, Clostridium, Fusarium and Bacteroides, which may vary from species to species as well as environmental conditions. Besides, several unknown bacteria belonging to Mycoplasma, Arthrobacter, Brochothrix, Jeotgailbacillus, Ochrobactrum, Psychrobacter and Sejongia species in the GI tract of different fish species have now been identified successfully using culture‐independent techniques. Gnotobiotic and conventional studies indicate the involvement of GI microbiota in fish nutrition, epithelial development, immunity as well as disease outbreak. This review also highlights the need for manipulating the gut microbiota with useful beneficial microbes through probiotic, prebiotic and synbiotic concepts for better fish health management.     

营养代谢过程中黄条鰤幼鱼消化道菌群的演变趋势

为认识消化道菌群结构在宿主营养代谢过程中的变化规律,并探讨饵料菌群对其的影响,采用16SrDNA高通量测序技术,对黄条鰤(Seriola lalandi)幼鱼一个完整摄食周期内的消化道菌群结构特征进行研究,并解析其与饵料菌群的相关性.结果显示,乳杆菌属(Lactobacillus)、假单胞菌属(Pseudomonas)...     

Lactic acid bacteria vs. pathogens in the gastrointestinal tract of fish: a review

Intensive fish production worldwide has increased the risk of infectious diseases. However, before any infection can be established, pathogens must penetrate the primary barrier. In fish, the three major routes of infection are the skin, gills and gastrointestinal (GI) tract. The GI tract is essentially a muscular tube lined by a mucous membrane of columnar epithelial cells that exhibit a regional variation in structure and function. In the last two decades, our understanding of the endocytosis and translocation of bacteria across this mucosa, and the sorts of cell damage caused by pathogenic bacteria, has increased. Electron microscopy has made a valuable contribution to this knowledge. In the fish‐farming industry, severe economic losses are caused by furunculosis (agent, Aeromonas salmonicida spp. salmonicida) and vibriosis [agent, Vibrio (Listonella) anguillarum]. This article provides an overview of the GI tract of fish from an electron microscopical perspective focusing on cellular damage (specific attack on tight junctions and desmosomes) caused by pathogenic bacteria, and interactions between the ‘good’ intestinal bacteria [e.g. lactic acid bacteria (LAB)] and pathogens. Using different in vitro methods, several studies have demonstrated that co‐incubation of Atlantic salmon (Salmo salar L.) foregut (proximal intestine) with LAB and pathogens can have beneficial effects, the cell damage caused by the pathogens being prevented, to some extent, by the LAB. However, there is uncertainty over whether or not similar effects are observed in other species such as Atlantic cod (Gadus morhua L.). When discussing cellular damage in the GI tract of fish caused by pathogenic bacteria, several important questions arise including: (1) Do different pathogenic bacteria use different mechanisms to infect the gut? (2) Does the gradual development of the GI tract from larva to adult affect infection? (3) Are there different infection patterns between different fish species? The present article addresses these and other questions.     

Identification and partial characterization of potential probiotic lactic acid bacteria in freshwater Labeo rohita and Cirrhinus mrigala

Carps are the most diversified freshwater fish belonging to family Cyprinidae. Numerous probiotic and pathogenic lactic acid bacteria (LAB) have been characterized from carps. However, the diversity of these ecologically important bacteria is entirely unknown in freshwater fish of Pakistan. The present study aimed to characterize and identify the lactic acid bacteria from two carps viz. Laboe rohita and Cirrhinus mrigala and determine their antagonistic activity. Seventeen bacterial isolates were purified from the gastrointestinal tract and gills of these fish and characterized morphologically. Initially, seven isolates were screened as LAB using agar supplemented with CaCO₃. Subsequently, only two isolates CILB2 and RIL10 were selected as LAB after high‐performance liquid chromatography analysis for lactic acid production. Isolates CILB2 and RIL10 were genetically identified as Enterococcus faecalis and Weissella sp., respectively after 16S rRNA gene sequence analysis. Both strains exhibited significant antagonistic activity against common fish pathogens Streptococcus agalactiae, Staphylococcus aureus and Escherichia coli. Enterococcus faecalis CILB2 and Weissella sp. RIL10 were also found negative for haemolysis and gelatinase activities and were sensitive to ampicillin, amoxicillin, doxycycline, erythromycin, chloramphenicol and co‐trimoxazole antibiotics. The identified LAB strains may further be investigated for their potential probiotic application in fish feed and food preservation techniques.     

Dietary effects of soybean products on gut microbiota and immunity of aquatic animals: A review

Soybean meal (SBM) is one of the most commonly used vegetable ingredient to replace fish meal in fish diets. However, SBM is limiting in some essential amino acids and contains numerous antinutritional factors and antigens that can affect intestinal microbiota and innate immune system in several finfish species and crustaceans and compromise health. The impact of SBM on health and gut microbiota of aquatic animals is not only affected by SBM in general, but also on the degree of treatment of the meal and exposure. Recently, many studies are actively seeking ways to complement or balance those adverse responses induced by high inclusion of SBM in aquaculture diets. These include advanced processing and mixture of feed with other feed components to balance antinutritional factors. The impact of dietary soybean oil on gut microbiota has also been investigated but to a lesser extent than SBM. As the gastrointestinal tract has been suggested as one of the major routes of infection in finfish species and crustaceans, the effect of soybean products on the gut microbiota is important to investigate. Several studies have focus on supplementation of SBM on the adverse responses of the innate immune system as immunological mechanisms are likely involved in the underlying pathology. However, the precise cause of the inflammatory process has not yet been clarified, even though some investigations have suggested that alcohol‐soluble antinutritional factors, especially soy saponins, are potential causative factors. Possible interactions between soybean products and innate immune system in several finfish species and crustaceans are discussed.     

Composition of the microbial communities in the gastrointestinal tract of perch (Perca fluviatilis L. 1758) and cestodes parasitizing the perch digestive tract

Using the approach of sequencing the V3-V4 region of the 16S rRNA gene, we have analysed the bacterial diversity associated with the distinct compartments of the gastrointestinal tract of perch (Perca fluviatilis) and cestodes (Proteocephalus sp.) parasitizing their digestive tract. The dominant microbiota associated with cestodes (Proteocephalus sp.) was represented by bacteria from the genera Serratia, Pseudomonas and Mycoplasma. By comparing the associated microbiota of perch and cestodes, a clear difference in bacterial composition and diversity was revealed between the community from the stomach content and other parts of the gastrointestinal tract of fish. Microbiota associated with cestodes was not significantly different in comparison with microbiota of different subcompartments of perch (mucosa and content of intestine and pyloric caeca) (ADONIS, p > .05) excluding microbiota of stomach content (ADONIS, p ≤ .05). PICRUSt-based functional assessments of the microbial communities of perch and cestodes indicated that they mainly linked in terms of metabolism and environmental information processing and could play an important role in the nutrition and health of host.     

Enhancement of rotifer (Brachionus plicatilis) growth by using terrestrial lactic acid bacteria

One of the pathways for the entry of bacteria, both pathogenic and probiotic, into the larvae of fish hatcheries is via live prey. As a preventive measure against infections, live prey may be cultured, supplementing the food with probiotics. Some lactic acid bacteria (LAB) have been successfully used in the larviculture. In this study, the nutritional effect of seven terrestrial LAB has been studied regarding the growth of the rotifer Brachionus plicatilis. The cultures were carried out without partial renewal of the culture medium, feeding the rotifers on baker's yeast and adding some of the species of bacteria. In all cases, the addition of the bacteria increased both the specific maximum growth rate and the maximum density obtainable in the cultures. However, the best results were obtained with the addition of Lactococcus casei ssp. casei, Pediococcus acidilactici, or Lactobacillus lactis spp lactis. The rates of growth obtained with the individual or joint addition of these three bacteria were 8–13 times greater than those obtained with the control cultures after 4–5 days' culture. In this study, a series of kinetic models has been applied (logistic modified—Gompertz, logistic–logistic, and generalised logistic) which describes the experimental data, obtaining a set of parameters of biological significance which facilitate the optimisation of the use of these bacterial strains in the mass production of rotifers.     

两种养殖模式下黄条鰤幼鱼消化道菌群对生长的微生态调控作用

探究了工厂化、网箱养殖模式对黄条鰤(Seriola lalandi)幼鱼生长性能及消化道菌群的影响, 通过 16S rRNA 高通量测序和生物信息学方法, 分析了两种养殖模式下黄条鰤幼鱼消化道(胃、幽门盲囊、肠道)、饵料及养殖水中的菌群结构特征及其互作关系。结果显示, 本实验条件下, 网箱养殖黄条鰤幼鱼较工厂化养殖鱼生长性能显著提升; 在黄条鰤幼鱼消化道菌群方面, 门水平上的拟杆菌门(Bacteroidetes)、厚壁菌门(Firmicutes)和属水平上的拟普雷沃氏菌属(Alloprevotella)、拟杆菌属(Bacteroides)在网箱养殖模式中其丰度值高于工厂化养殖模式, 其中拟杆菌属仅出现于网箱养殖模式且呈现显著性差异; Beta 多样性分析显示消化道菌群更多受到饵料菌群的影响, 受水环境影响相对较小。KEGG 注释分析表明两种养殖模式的差异菌群中, 网箱养殖幼鱼消化道菌群主要参与磷酸转移酶系统 (PTS)和 NOD 样受体信号通路, 而工厂化养殖幼鱼为碳水化合物代谢和类胡萝卜素合成通路。本研究表明, 环境菌群中, 饵料菌群对消化道菌群的影响大于养殖水体菌群; 消化道微生物群落通过调整其组成结构从而改变菌群功能通路的方式, 积极参与两种养殖模式下黄条鰤幼鱼生长机能差异的调控。因此, 网箱养殖鱼表现出更快速的生长性能可能是由于机体内的菌群产生了更多的短链脂肪酸并诱导 IGF-1 等生长相关功能基因的表达, 从而促进机体的营养吸收和生长。本研究预期结果将为黄条鰤专用配合饲料的研制和健康养殖技术开发提供微生态理论支撑。