The use of probiotics in aquaculture

This study aims to present comprehensive notes for the use of probiotics in aquaculture. Probiotics have been proven to be positive promoters of aquatic animal growth, survival and health. In aquaculture, intestines, gills, the skin mucus of aquatic animals, and habitats or even culture collections and commercial products, can be sources for acquiring appropriate probiotics, which have been identified as bacteria (Gram‐positive and Gram‐negative) and nonbacteria (bacteriophages, microalgae and yeasts). While a bacterium is a pathogen to one aquatic animal, it can bring benefits to another fish species; a screening process plays a significant role in making a probiotic species specific. The administration of probiotics varies from oral/water routine to feed additives, of which the latter is commonly used in aquaculture. Probiotic applications can be either mono or multiple strains, or even in combination with prebiotic, immunostimulants such as synbiotics and synbiotism, and in live or dead forms. Encapsulating probiotics with live feed is a suitable approach to convey probiotics to aquatic animals. Dosage and duration of time are significant factors in providing desired results. Several modes of actions of probiotics are presented, while some others are not fully understood. Suggestions for further studies on the effects of probiotics in aquaculture are proposed.     

The influence of polyunsaturated fatty acids on probiotic growth and adhesion

The establishment of the intestinal microflora, and probiotic bacteria, may control the inflammatory conditions in the gut. As polyunsaturated fatty acids (PUFA) possess antimicrobial activities, they may deter the action of probiotics. We assessed whether free linoleic, gamma-linolenic, arachidonic, alpha-linolenic and docosahexaenoic acids at physiological concentrations in the growth media would influence the growth and adhesion of Lactobacillus GG (probiotic), Lactobacillus casei Shirota (probiotic) and Lactobacillus bulgaricus (dairy strain). Higher concentrations of PUFA (10-40 microg PUFA ml(-1)) inhibited growth and mucus adhesion of all tested bacterial strains, whilst growth and mucus adhesion of L. casei Shirota was promoted by low concentrations of gamma-linolenic acid and arachidonic acid (at 5 microg ml(-1)), respectively. PUFA also altered bacterial adhesion sites on Caco-2 cells. Caco-2 cells grown in the presence of arachidonic acid were less adhered to by all three bacterial strains. Yet, L. casei Shirota adhered better on Caco-2 cells grown in the presence of alpha-linolenic acid. As the adhesion to mucosal surfaces is pivotal in health promoting effects by probiotics, our results indicate that the action of probiotics in the gut may be modulated by dietary PUFA.     

Evaluation of the cytoprotective effects of bovine lactoferrin against intestinal toxins using cellular model systems

This study investigated the effects of bovine lactoferrin (BLf) on the growth of different groups of bacteria in vitro. BLf showed a significant inhibitory effect on the growth of selected pathogens but not probiotics. BLf, in combination with probiotics, has the potential to influence the composition of the gut microflora via inhibition of intestinal pathogens with no significant effect on probiotic bacteria.     

Probiotic Strains and Their Combination Inhibit <Emphasis Type="Italic">In Vitro</Emphasis> Adhesion of Pathogens to Pig Intestinal Mucosa

The aim of this study was to investigate in vitro the protective effect of commercial probiotic strains (Bifidobacterium lactis Bb12 and Lactobacillus rhamnosus LGG) alone and in combination on the adhesion of pathogenic strains as Salmonella, Clostridium, and Escherichia coli to pig intestinal mucus obtained from different intestinal regions. In combination, probiotic strains enhanced each other’s adhesion, mainly in large intestinal mucus. Treatment of intestinal mucus with Bb12 and LGG, alone or in combination, significantly reduced (P < 0.05) the adhesion of the tested pathogens. The ability to inhibit pathogen adhesion appears to depend on the specific probiotics and pathogens and on the mucosal site. B. lactis Bb12 and L. rhamnosus LGG in combination revealed a better ability to inhibit adhesion of all pathogens tested to pig intestinal mucus than probiotic strains. Probiotic combinations could be useful for counteracting disease-associated aberrations in intestinal microbiota. Specific protective probiotics could be selected for particular pig pathogens. Probiotic strains from human origin and intended for human use also adhere to pig intestinal mucus and are able to displace and inhibit pathogens.     

Characterization of the properties of human- and dairy-derived probiotics for prevention of infectious diseases in fish

The present study aimed to investigate the potential probiotic properties of six lactic acid bacteria (LAB) intended for human use, Lactobacillus rhamnosus ATCC 53103, Lactobacillus casei Shirota, Lactobacillus bulgaricus, L. rhamnosus LC 705, Bifidobacterium lactis Bb12, and Lactobacillus johnsonii La1, and one for animal use, Enterococcus faecium Tehobak, for use as a fish probiotic. The strains for human use were specifically chosen since they are known to be safe for human use, which is of major importance because the fish are meant for human consumption. The selection was carried out by five different methods: mucosal adhesion, mucosal penetration, inhibition of pathogen growth and adhesion, and resistance to fish bile. The adhesion abilities of the seven LAB and three fish pathogens, Vibrio anguillarum, Aeromonas salmonicida, and Flavobacterium psychrophilum, were determined to mucus from five different sites on the surface or in the gut of rainbow trout. Five of the tested LAB strains showed considerable adhesion to different fish mucus types (14 to 26% of the added bacteria). Despite their adhesive character, the LAB strains were not able to inhibit the mucus binding of A. salmonicida. Coculture experiments showed significant inhibition of growth of A. salmonicida, which was mediated by competition for nutrients rather than secretion of inhibitory substances by the probiotic bacteria as measured in spent culture liquid. All LAB except L. casei Shirota showed tolerance against fish bile. L. rhamnosus ATCC 53103 and L. bulgaricus were found to penetrate fish mucus better than other probiotic bacteria. Based on bile resistance, mucus adhesion, mucus penetration, and suppression of fish pathogen growth, L. rhamnosus ATCC 53103 and L. bulgaricus can be considered for future in vivo challenge studies in fish as a novel and safe treatment in aquaculture.     

Detection and Specific Enumeration of Multi-Strain Probiotics in the Lumen Contents and Mucus Layers of the Rat Intestine After Oral Administration

Although the detection of viable probiotic bacteria following their ingestion and passage through the gastrointestinal tract (GIT) has been well documented, their mucosal attachment in vivo is more difficult to assess. In this study, we investigated the survival and mucosal attachment of multi-strain probiotics transiting the rat GIT. Rats were administered a commercial mixture of the intestinal probiotics Lactobacillus acidophilus LA742, Lactobacillus rhamnosus L2H and Bifidobacterium lactis HN019 and the oral probiotic Streptococcus salivarius K12 every 12 h for 3 days. Intestinal contents, mucus and faeces were tested 6 h, 3 days and 7 days after the last dose by strain-specific enumeration on selective media and by denaturing gradient gel electrophoresis. At 6 h, viable cells and DNA corresponding to all four probiotics were detected in the faeces and in both the lumen contents and mucus layers of the ileum and colon. Viable probiotic cells of B. lactis and L. rhamnosus were detected for 7 days and L. acidophilus for 3 days after the last dose. B. lactis and L. rhamnosus persisted in the ileal mucus and colon contents, whereas the retention of L. acidophilus appeared to be relatively higher in colonic mucus. No viable cells of S. salivarius K12 were detected in any of the samples at either day 3 or 7. The study demonstrates that probiotic strains of intestinal origin but not of oral origin exhibit temporary colonisation of the rat GIT and that these strains may have differing relative affinities for colonic and ileal mucosa.     

Role of commercial probiotic strains against human pathogen adhesion to intestinal mucus

AIMS: The aims of this study present were to assess and to evaluate in vitro the abilities of commercial probiotic strains derived from fermented milk products and related sources currently marketed in European countries, to inhibit, compete and displace the adhesion of selected potential pathogens to immobilized human mucus. METHODS AND RESULTS: The adhesion was assessed by measuring the radioactivity of bacteria adhered to the human mucus. We tested 12 probiotic strains against eight selected pathogens. All strains tested were able to adhere to mucus. All probiotic strains tested were able to inhibit and displace (P<0.05) the adhesion of Bacteroides, Clostridium, Staphylococcus and Enterobacter. In addition, the abilities to inhibit and to displace adhered pathogens depended on both the probiotic and the pathogen strains tested suggesting that several complementary mechanisms are implied in the processes. CONCLUSIONS: Our results indicate the need for a case-by-case assessment in order to select strains with the ability to inhibit or displace a specific pathogen. Probiotics could be useful to correct deviations observed in intestinal microbiota associated with specific diseases and also, to prevent pathogen infections. SIGNIFICANCE AND IMPACT OF THE STUDY: The competitive exclusion properties of probiotics as well as their ability to displace and inhibit pathogens are the most importance for therapeutic manipulation of the enteric microbiota. The application of such strategies could contribute to expand the beneficial properties on human health against pathogen infection.     

Specific probiotic strains and their combinations counteract adhesion of Enterobacter sakazakii to intestinal mucus

Enterobacter sakazakii is an opportunistic pathogen and an occasional contaminant in powdered infant formula. Interaction between specific probiotics and E. sakazakii may reduce the risk of infection. The aim of this study was to characterize in vitro the ability of probiotics (alone and in combinations) to inhibit, compete with and displace the adhesion of E. sakazakii to immobilized human mucus and to assess their capacity to aggregate with pathogen. Specific probiotic strains have proved to aggregate E. sakazakii cells and, through competitive exclusion, inhibition and displacement of the adhered pathogen, were able to inhibit E. sakazakii action on intestinal mucus. The ability to inhibit and to displace adhered pathogen depended on both the probiotic and the pathogen, suggesting that several complementary mechanisms are involved in the processes. We suggest that the selection of specific probiotic strains and their combinations may be a useful means of counteracting E. sakazakii contamination in infant formula and thus to reduce the risk of emerging infection. This approach may also allow the development of new probiotic combinations to counteract the risks associated with other pathogens by improving the intestinal barrier against pathogens.     

Correlation between in vitro mucus adhesion and the in vivo colonization ability of lactic acid bacteria: screening of new candidate carp probiotics

We measured the adhesion of candidate probiotic lactic acid bacteria (LAB) to carp intestinal mucus. The percentage of adherent bacteria varied among strains. Four strains, two with high adhesion and two with low adhesion in vitro, were tested for in vivo colonization ability. Carp were fed LAB-containing feed for 12 d, and then unsupplemented feed until day 33, and the numbers and compositions of intestinal LAB were analyzed during the entire period. LAB with lower in vitro adhesion disappeared quickly from the intestine after LAB feeding stopped. LAB with higher in vitro adhesion remained in the intestine 3 weeks after LAB feeding stopped, indicating a strong correlation between mucus adhesion in vitro and colonization ability in vivo. Next we isolated nine candidate probiotic LAB with high in vitro mucus-binding ability. Three of them were fed to carp, and all three were stably maintained in the intestine.     

The effect of probiotic bacteria on the adhesion of pathogens to human intestinal mucus

Human intestinal glycoproteins extracted from faeces were used as a model for intestinal mucus to investigate adhesion of pathogenic Escherichia coli and Salmonella strains, and the effect of probiotics on this adhesion. S-fimbriated E. coli expressed relatively high adhesion in the mucus model, but the other tested pathogens adhered less effectively. Probiotic strains Lactobacillus GG and L. rhamnosus LC-705 as well as a L. rhamnosus isolated from human faeces were able to slightly reduce S-fimbria-mediated adhesion. Adhesion of S. typhimurium was significantly inhibited by probiotic L. johnsonii LJ1 and L. casei Shirota. Lactobacillus GG and L. rhamnosus (human isolate) increased the adhesion of S. typhimurium suggesting that the pathogen interacts with the probiotic.     

Bacterial biota of shrimp intestine is significantly modified by the use of a probiotic mixture: a high throughput sequencing approach

The use of probiotics is a common practice of current shrimp aquaculture. Despite the immunophysiological responses that have been measured in shrimp exposed to probiotics, no information is currently available on the effect of this practice on the intestinal microbiota. The objective of this work was to evaluate the effect of a probiotic mixture on the intestinal microbiota of shrimp cultured under farm conditions. A culture-independent method based on high-throughput-sequencing (16S rRNA) was used to examine intestinal bacterial communities. A traditional system (without probiotics) was used as the reference. Targeted metagenomics analysis revealed that the probiotic mixture was based on bacteria in the phyla Proteobacteria and Firmicutes. A total of 23 species of bacteria were detected in the probiotic mixture; of these, 11 were detected in the intestine of shrimp reared in both systems, and 12 were novel for the system. Eight of the novel species were detected in shrimp cultured with the probiotic mixture; however, none of these novel species were related to marine or inclusively aquacultural environments, and only one (Bacillus subtilis) was recognized as probiotic for shrimp. The use of the probiotic mixture modified the bacterial profile of the shrimp intestine; however, most of the bacteria incorporated into the intestine were nonindigenous to the marine environment with no previous evidence of probiotic effects on any marine organism. The use of this probiotic mixture may represent a risk of causing environmental imbalances, particularly because farms using these types of probiotic mixtures discharge their effluents directly into the ocean without prior treatment.     

Updating the importance of lactic acid bacteria in fish farming: natural occurrence and probiotic treatments

Many recent papers have deepened the state of knowledge about lactic acid bacteria (LAB) in fish gut. In spite of high variability in fish microbiota, LAB are sometimes abundant in the intestine, notably in freshwater fish. Several strains of Streptococcus are pathogenic to fish. Streptococcus iniae and Lactococcus garvieae are major fish pathogens, against which commercial vaccines are available. Fortunately, most LAB are harmless, and some strains have been reported for beneficial effects on fish health. A major step forward in recent years was the converging evidence that LAB can stimulate the immune system in fish. An open question is whether viability can affect immunostimulation. The issue is crucial to commercialize live probiotics rather than inactivated preparations or extracts. There has been a regain of interest in allochthonous strains used as probiotics for terrestrial animals or humans, due to economical and regulatory constraints, but the short survival in sea water may limit application to marine fish. If viability is required, alternative treatments may include the incorporation of prebiotics in feed, and other dietary manipulations that could promote intestinal LAB. Antagonism to pathogens is the other main feature of candidate probiotics, and there are many reports concerning mainly carnobacteria and Enterococcus. Some bacteriocins were characterized which may be of interest not only for aquaculture, but also for food preservation.     

Identification and characterization of starter lactic acid bacteria and probiotics from Columbian dairy products

AIMS: Considering the significant rise in the probiotic market in Columbia, and given the lack of reports concerning the microbial population and strain performance in products from different producers, this study aims at determining the number of viable starter bacteria and probiotics in bio-yoghurts available at the Columbian market, identifying the species and analysing the performance of the isolated strains in bile acid resistance, antagonistic activity against pathogens, and adherence capacity to human intestinal epithelial cells. METHODS AND RESULTS: Seven bio-yoghurts were analysed for the bacterial species present. Species identification was carried out using 16S rRNA gene targeted PCR. The cultured bacteria were tested for bile acid resistance, adherence to a human intestinal epithelial cell line, and antagonism against the pathogen Salmonella enterica serovar Typhimurium. A total of 17 different strains were identified. Based on plate counting, all bio-yoghurts have at least total viable cells of approximately 10(7) CFU ml(-1). Streptococcus thermophilus and Lactobacillus delbrueckii ssp. bulgaricus were the most frequently isolated bacteria. Viable Bifidobacterium was only recovered from one product. However, after PCR analysis, DNA of this genus was confirmed in five out of seven products. Major differences were found for S. typhimurium antagonism. The adherence capacity to Caco-2 cells was observed in 10 of the isolated strains. In general, low survival to simulated gastric juice was observed. CONCLUSIONS: Some of the isolated strains have probiotic potential, although not all of them were present in the advised amount to exert beneficial health effects. However, the full correct scientific name of the isolated bacteria and their viable counts were not included on the product label. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first report describing the identification and functionality of starter bacteria and probiotics present in dairy products on the Columbian market.     

In vitro evaluation on adherence and antimicrobial properties of a candidate probiotic Clostridium butyricum CB2 for farmed fish

Aims: To characterize the antimicrobial and adhesion ability of candidate probiotic Clostridium butyricum CB2 for farmed fish in vitro. Methods and Results: The potential probiotic Cl. butyricum CB2 had been evaluated for its adhesion capacity and antagonistic effect against two fish pathogens Aeromonas hydrophila and Vibrio anguillarum by the intestinal cell model. In addition, the aggregation ability and antimicrobial property on agar plate were assayed. The results indicated that the candidate probiotic Cl. butyricum CB2 have strong adhesion property and a higher antagonistic activity to Aer. hydrophila and V. anguillarum both on agar plate and cell model. Clostridium butyricum showed a higher aggregation which might be the reasons for bacteria adhesion and antimicrobial activity. Conclusions: The strain Cl. butyricum CB2 could be used as potential probiotic to inhibit pathogens growth and prevent their colonization in fish intestinal tract. Significance and Impact of the Study: This study revealed the antimicrobial and adhesion characteristic of Cl. butyricum CB2 which was selected as the potential probiotic to farmed fish.     

Culturable autochthonous gut bacteria in rohu, <Emphasis Type="Italic">Labeo rohita</Emphasis>. In vitro growth inhibition against pathogenic <Emphasis Type="Italic">Aeromonas</Emphasis> spp., stability in gut,bio-safety and identification by 16S rRNA gene sequencing

Autochthonous endosymbiotic gut bacteria antagonistic against pathogenic Aeromonas spp. have been evaluated in rohu, Labeo rohita for characterization of putative probiotics. Four promising pathogen inhibitory bacteria (23 strains out of 225 isolates showed antagonism) were selected by double layer assay, following which inhibition pattern was examined through in vitro growth curve and statistical analyses. Cell free supernatant (CFS) of the selected gut bacteria significantly inhibited the growth of pathogenic aeromonads. While, CFS of strain LR3FG26 was the most efficient among them. Selected strains were γ haemolytic and susceptible to most of the common antibiotics that demonstrated their likely non-pathogenic and eco-friendly nature. Additionally, selected bacteria produced different exo-enzymes (digestive and antinutritional factors degrading), could grow better in skin mucus than intestinal mucus (exceptionally, LR3FG26 grew better in skin mucus) and tolerated diluted bile juice (2–20%). 16S rRNA partial gene sequence analyses and Blast search in NCBI GenBank unveiled that the strains LR1FG1, LR2HG13, LR3FG26 and LR3HG4 were similar with the type strains of Bacillus methylotrophicus (NR116240), Bacillus amyloliquefaciens (NR117946), Pseudomonas fluorescens (NR113647) and Bacillus licheniformis (NR118996), respectively. Application of these symbiotic pathogen-inhibitory bacteria in pathogen challenge studies is required to appraise their probiotic effects in vivo.     

The Yin and Yang of Bacterial Resilience in the Human Gut Microbiota

The human gut is home to trillions of microbes that form a symbiotic relationship with the human host. During health, the intestinal microbiota provides many benefits to the host and is generally resistant to colonization by new species; however, disruption of this complex community can lead to pathogen invasion, inflammation, and disease. Restoration and maintenance of a healthy gut microbiota composition requires effective therapies to reduce and prevent colonization of harmful bacteria (pathogens) while simultaneously promoting growth of beneficial bacteria (probiotics). Here we review the mechanisms by which the host modulates the gut community composition during health and disease, and we discuss prospects for antibiotic and probiotic therapy for restoration of a healthy intestinal community following disruption.     

Probiotics in marine larviculture

Owing to the problem of antibiotic resistance and subsequent reluctance of using antibiotics, the use of probiotics in larviculture is becoming increasingly popular. During the early stages of development, manipulation of the larval digestive system seems possible through the addition of probiotics either through the culture water or via the livefood. Well-studied probiotics used in human medicine and terrestrial agriculture have proved to be successful in aquaculture and therefore reduce the need for extensive biosafety trials. The selection of probiotics requires various in vitro screening experiments, which assay for the production of antagonist compounds, their growth in and attachment to fish intestinal mucus, and the production of other beneficial compounds such as vitamins, fatty acids and digestive enzymes. Further information regarding probiont suitability can be obtained from its identification, interaction with livefood and host pathogenicity. Finally, pilot-scale in vivo tests need be performed, after which a production cost-benefit analysis to determine its economic viability needs to be undertaken.     

Intestinal mucus alters the ability of probiotic bacteria to bind aflatoxin B1 in vitro

Several probiotics are known to bind aflatoxin B(1) (AFB(1)) to their surfaces and to adhere to intestinal mucus. In this study, preincubation of two probiotic preparations with either AFB(1) or mucus reduced the subsequent surface binding of mucus and AFB(1), respectively, in a strain-dependent manner.     

Survival and therapeutic potential of probiotic organisms with reference to Lactobacillus acidophilus and Bifidobacterium spp

The present paper provides an overview on the use of probiotic organisms as live supplements, with particular emphasis on Lactobacillus acidophilus and Bifidobacterium spp. The therapeutic potential of these bacteria in fermented dairy products is dependent on their survival during manufacture and storage. Probiotic bacteria are increasingly used in food and pharmaceutical applications to balance disturbed intestinal microflora and related dysfunction of the human gastrointestinal tract. Lactobacillus acidophilus and Bifidobacterium spp. have been reported to be beneficial probiotic organisms that provide excellent therapeutic benefits. The biological activity of probiotic bacteria is due in part to their ability to attach to enterocytes. This inhibits the binding of enteric pathogens by a process of competitive exclusion. Attachment of probiotic bacteria to cell surface receptors of enterocytes also initiates signalling events that result in the synthesis of cytokines. Probiotic bacteria also exert an influence on commensal micro-organisms by the production of lactic acid and bacteriocins. These substances inhibit growth of pathogens and also alter the ecological balance of enteric commensals. Production of butyric acid by some probiotic bacteria affects the turnover of enterocytes and neutralizes the activity of dietary carcinogens, such as nitrosamines, that are generated by the metabolic activity of commensal bacteria in subjects consuming a high-protein diet. Therefore, inclusion of probiotic bacteria in fermented dairy products enhances their value as better therapeutic functional foods. However, insufficient viability and survival of these bacteria remain a problem in commercial food products. By selecting better functional probiotic strains and adopting improved methods to enhance survival, including the use of appropriate prebiotics and the optimal combination of probiotics and prebiotics (synbiotics), an increased delivery of viable bacteria in fermented products to the consumers can be achieved.