In the present study, metagenomic sequencing was employed to evaluate claims regarding bacterial composition on the labels of five commercial aquaculture probiotics sold in India. A probiotic product is said to be reliable if bacterial strains are present in right composition and concentration as claimed on the label. Culture-based isolation and identification of probiotic bacterial strains may result in missing out of few bacterial species26. The traditional method of culture-based enumeration of specific organisms will only identify bacteria that are able to grow and reproduce on synthetic media under definite conditions27. High Throughput DNA sequencing can offer resolution required for examining all microbial communities including the contaminant and unculturable organisms in mixed samples to the preferred taxonomy up to strain level28. In this study, the microbial quality of probiotic products were evaluated through NGS technique by means of the discriminative power of V3 region sequencing of 16S rDNA. Identification of bacteria were done only up-to species level as the disclosure on labels were up to that level. NGS sequencing of the V2 and V3 regions of 16SrRNA were found to be most suitable for distinguishing most bacterial species up to the genus level29. NGS has the potential to detect unexpected microbes30 with the advantage of being high-throughput having ability to generate large sequence data.
It was evident that all the probiotic products tested contained several contaminant bacteria and the microbial composition of the product were not in agreement with the label information of the product. Aquaculture probiotic products approved in Vietnam were found to contain bacterial strains not declared on the product labels. Also, many bacterial species including Bacillus spp. declared on the label were not isolated26. In our study, only 2 products (1 and 4) contained all bacterial species claimed by the manufacturer. The relative abundance of the sole declared organism, B. subtilis was just 8.9 % in Product 4. Bacillus is one of the most widely used bacteria incorporated in majority of the aquaculture probiotics. When Bacillus spp., is used in aquaculture pond, it can easily become part of the culture environment by competing with pathogenic or non-pathogenic bacteria31. Bacillus spp. was reported to act as a growth promoter32, inhibit pathogen33, improve nutrient digestibility34, enhance water quality35 and ameliorate reproduction36. Bacillus spores are able to survive under various conditions including desiccation, freezing, elevated temperatures, chemicals, increased pressure and even radiation37. The ability of Bacillus spp. to form spores will help in the long-term storage of probiotic products.
In the present study, absence of B. licheniformis and B. coagulans in product 2, B. subtilis and Lactobacillus acidophilus in product 3, Alkaligenes faecalis, B. amyloliquefaciens, B. megaterium and B. polymyxa in product 5 and Nitrobacter sp., and Nitrosomonas sp. in products 2 and 5 highlighted the poor correlation between the label claims and actual content of the products. The nitrifying bacteria are beneficial as they increase the number of microbial species in water and improve the water quality by eliminating ammonia and nitrate toxicity38,39. The accumulation of ammonia nitrogen and nitrite are two major toxic factors that directly affect the economic benefits of aquaculture40,41,42.
One of the foremost concerns to be considered while evaluating the quality of commercial probiotics is the existence of unspecified organisms within the product. P. acne, an anaerobic bacterium causing acne and one of the most abundant organisms found on human skin43, is a common taxonomic group found in three out of the five probiotic products analyzed. This organism was totally unexpected in the products and might have got incorporated into the product due to lack of good manufacturing practices and proper quality control during production process. Based on sequence analysis it was found that there were many microorganisms that were not declared in the labels but were found in the product. E. faecium was present in product 5 as a contaminant and this bacteria was found to decrease the mortality of European eel due to edwardsiellosis44. Even though Enterococci are Lactic Acid Bacteria, their application in animal and human health are hindered by occurrence of virulence and antibiotic resistant genes and pathogenicity45,46. Enterococcus spp. is neither recommended for the QPS status by the European Food Safety Authority (EFSA)47 nor have GRAS status48, in spite of recent scientific knowledge allowing differentiation of commensal from pathogenic strains49,50. Due to limited therapeutic options, vancomycin-resistant Enterococcus faecium (VREF) is of great clinical significance. The clinical E. faecium isolates in Germany exhibiting resistance to vancomycin was 26.1% in 201751. Arthospira platensis was present in product 3, a filamentous cyanobacterium widely used as a prebiotic in aquaculture because of its best-known nutritional qualities52. Interestingly in Product 4, an organism that was not claimed on the label, Streptococcus thermophiles, was found to be present. S. thermophiles is a moderate thermophile and a widely used human probiotic53. The presence of E. faecium, A. platensis and S. thermophilus in the products cannot be considered as unintentional. It is suggested that the product label should specify all the organisms that are incorporated in a product and their intended use. Several strains of S. thermophilus have also been shown to have several in vitro and in vivo probiotic properties ranging from the production of a range of short-chain fatty acids to moderately high survivability levels under simulated gastrointestinal tract conditions54,55,56.
The existence of human infectious bacteria; A. baumannii, A. junii and P. acne, human and fish pathogen; Pseudomonas aeruginosa and faecal contaminants; E. coli, E. cloacae and E. faecium in probiotic was unanticipated. The use of probiotics with contaminant bacteria carrying potential risks and of unknown provenance in aquaculture farms could cause a major shift in natural microbial community57. There is a high risk due to human exposure to the pathogenic contaminant bacteria present in aquaculture probiotics, during handling of such probiotics or probiotic fed aquatic animals and by dissemination to surrounding environment and terrestrial animals.
To our knowledge, this is the first study that has employed NGS for quality analysis of commercial aquaculture probiotics. Inconsistencies in the bacterial composition against the label declarations and occurrence of Bacillus as the integral constituent of products observed in the current study were also observed in similar reports on the culture dependent evaluation of aquaculture probiotics5,26. The microbiological analysis of seven probiotics commonly used in Vietnamese shrimp culture, revealed that all the bacteria as stated on the label could not be detected while few bacteria that were not declared; Aerococcus sp., Klebsiella spp., and Bacillus spp. were present in the products26.
Since knowledge about the heterogeneity and growth requirements of contaminant bacteria in a probiotic are limited, isolation and identification of these bacteria will be difficult with traditional microbiological techniques or even with molecular methods. High-throughput based metagenomics studies can identify entire range of organisms that were not declared on the label. However, some closely related species would have little divergence among the variable regions of 16S rDNA. Therefore, discrimination of such species that belongs to the same group as Bacillus subtilis group will be difficult and the categorization will be terminated at the genus level25. Since 16S database is incomplete, bacterial species present in the tested products which were unclassified and recorded as unknown could not be identified. However, this weakness of the technique has not affected the results since the major focus of our work was to isolate contaminant microorganisms.
It is mandatory that all aquaculture probiotics sold in the Indian market have to be registered with Coastal Aquaculture Authority (CAA). The approval is solely based on the absence of antibiotics in the product. Many probiotics sold in the Indian market donot carry information on the bacterial composition and concentration of each species, the method or the frequency of application. The claims on the effectiveness of the products is often highly exaggerated. Aquaculture farmers purchase the probiotic products based on the claims of the merchandise, and hence misleading, incorrect, or deceptive labeling on products need to be avoided. The findings from the current study raises concerns regarding the quality of aquaculture probiotics in the interest of the stakeholders including the farmers and the final consumer. It is evident that all the probiotics screened in this study do not fulfill the basic requirement of labeling the exact content of the probiotic product. Besides, the occurrence of potential pathogenic organisms in the product is an added public health concern. This underlines the need for stringent quality control and regular screening by competent authorities to monitor the manufacture of aquaculture probiotics and implementing a legislation system for such an emerging and growing industry. The effect due to application of probiotic product shall clearly be mentioned on the label and the information provided on the label shall be evaluated based on on-farm trials before the product is approved for use.