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July 12, 2015

Identification and Antimicrobial Resistance of Bacteria Isolated from Probiotic Products Used in Shrimp Farming


This study was published in its entirety on-line by PLOS ONE, a peer-reviewed, open access (free) scientific journal published by the Public Library of Science (PLOS) under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution and reproduction in any medium, provided the original author and source are credited.  Operating under a pay-to-publish model, all submissions go through a pre-publication review by a member of the board of academic editors, who can elect to seek an opinion from an external reviewer.  As of 2014, PLOS ONE was the world’s largest journal by number of papers published.



Study’s Summary


With reference to the increasing use of probiotics in shrimp and other types of aquaculture, the objective of this study was to determine the bacterial species composition in probiotic products commonly used in Vietnamese shrimp culture.  Furthermore, the phenotypic antimicrobial susceptibility and genetic basis for antimicrobial resistance in the isolated probiotic strains was determined to assess likelihood of possible transfer of antimicrobial resistance.


What follows are some excerpts from a study that contains a wealth of information on the probiotics used in shrimp farming.  You can view and download the entire study, including its lengthy tabular data, by clicking here.


Abstract: Probiotics are increasingly used in aquaculture to control diseases and improve feed digestion and pond water quality; however, little is known about the antimicrobial resistance properties of such probiotic bacteria and to what extent they may contribute to the development of bacterial resistance in aquaculture ponds.  Concerns have been raised that the declared information on probiotic product labels are incorrect and information on bacterial composition are often missing.  We therefore evaluated seven probiotics commonly used in Vietnamese shrimp culture for their bacterial species content, phenotypic antimicrobial resistance and associated transferable resistance genes.  ...Thirteen bacterial species declared on the probiotic products could not be identified and 11 non-declared Bacillus species were identified.  Only 6 of 60 isolates were resistant to more than four antimicrobials and whole genome sequencing showed that they contained macrolide (ermD), tetracycline (tetL), phenicol (fexA) and trimethoprim (dfrD, dfrG and dfrK) resistance genes, but no known structures associated with horizontal gene transfer.  ...Approval procedures of probiotic products must be strengthened through scientific-based efficacy trials and product labels should allow identification of individual bacterial strains and inform the farmer on specific purpose, dosage and correct application measures.


From the Body of the Study: One researcher reported that 19% of the intensive shrimp farms in Vietnam used oxytetracycline, ciprofloxacin and/or enrofloxacin to treat mainly early mortality syndrome (EMS), also called acute hepatopancreatic necrosis syndrome (AHPNS).  The popularity of probiotic usage in shrimp aquaculture has grown worldwide, mainly because farmers often experience limited effect of antimicrobial treatment, but also because of continued problems and reports of antimicrobial residue findings in exported shrimp.  Probiotic bacterial strains are thought to inhibit pathogens by colonization of the gut-intestinal environment and to the production of compounds that have a biocidal effect on shrimp pathogens.  Furthermore, probiotic bacteria incorporated into shrimp feed may enhance uptake of various nutrients and increase yield.  The popularity of probiotic use is highlighted by a recent study showing that 91% of surveyed shrimp farmers used probiotics.  Among them, 84% applied probiotic products directly into the pond water to improve water quality and to reduce environmental stress and 16% of them mixed probiotics with pelleted feed.  One researcher reported that Bacillus subtilis, B. licheniformis, B. thuringiensis and Lactobacillus acidophilus were the main bacterial species included in the probiotic products used by Vietnamese shrimp farmers.  Bacterial species composition and concentration were normally listed on the product labels.  Shrimp farmers, however, are generally uncertain about the effect of the many different types of probiotics.


In contrast to antimicrobials, it is generally believed that probiotics do not play a role in the development of resistance among aquatic animal bacterial pathogens or the general aquatic microflora.  Resistance developed through antimicrobial selection pressure may spread by different mechanisms, for example, horizontal gene transfer via plasmids and other genetic elements.  Probiotic bacterial strains used in livestock production have actually been found to contain tetracycline resistance genes.  Also, the presence and expression of resistance genes located on plasmids and transposons in Lactobacillus species and Bacillus species used as probiotics in foods have been reported.  In recent guidelines provided by authorities approving the use of antimicrobials, it is clearly stated that the presence of antimicrobial resistance in probiotic bacterial strains are not allowed.  Furthermore, bacterial species composition and a measurable beneficial effect and the associated mechanism(s) accounting for such effects seem often not documented for many marketed probiotic products.


Seven probiotic products commonly used in Vietnamese shrimp culture were purchased from aquaculture chemical shops in Soc Trang and Bac Lieu provinces in the Mekong Delta.  Three of the probiotics were marketed as feed supplements and four for water treatment.  Four probiotics were in powder form and three were pellets.  Two probiotic products were imported and distributed by Vietnamese companies while the remaining products were produced and distributed by local companies.  The information on product labels was read for a description of contents and formula, the bacterial genus and species and their concentrations, expiry date and expected outcomes.  After purchase, the probiotics were stored at room temperature and transported to the University of Copenhagen, Denmark, for bacteriological analysis.  The products were analyzed at least one year before the declared date of expiration.


Based on the product labels of the seven products, the following bacterial species were found: Bacillus licheniformis, B. subtilis, B. megaterium, B. laterrosporus, B. amyloliquefaciens, B. thuringiensis, B. mesentericus, B. circulans, B. azotoformans, B. pantothenticus, B. coagulans, Lactobacillus acidophilus, Pediococcus acidilactici, L. helveticus, L. lactic, L. sporogenes, Nitrobacter species, Nitrosomonas species, Alcaligenes denitrificans and Pseudomonas denitrificans.  Microbiological analysis was done to identify the bacterial species, not to determine the concentration of the individual probiotic strains. 


As Bacillus species were by far the most commonly declared and isolated bacterial species, six representative isolates including B. licheniformis, B. amyloliquefaciens, B. tequilensis, B. cereus, B. aerius and B. nealsonii were selected for whole genome sequencing to determine antimicrobial resistance genes.  The isolates were selected based on their multiple antimicrobial resistance patterns mainly to antimicrobials used therapeutically in humans, for example, ciprofloxacin, tetracycline, sulfamethoxazole/trimethoprim, erythromycin and clindamycin.


Overall, we were unable to isolate all bacterial species declared in the probiotic products.  Bacterial species identified, but not declared included several Bacillus species, Aerococcus urinaeequi and Klebsiella species.  A total of 15 Bacillus species were identified compared to the 11 species declared.  However, only four of the 11 declared species were isolated.


A total of 65 isolates were selected for antimicrobial susceptibility testing including Bacillus species (60), Klebsiella species (4) and Aerococcus urinaeequi (1).  In general, the Bacillus species strains were susceptible to a wide range of the antimicrobials tested, for example, 9 of 60 (15%) were fully susceptible to all antimicrobials tested.  Twelve (20%) isolates of Bacillus species were resistant to more than three antimicrobials.


The three Klebsiella species strains were all resistant to ampicillin only, whereas the Aerococcus urinaeequi strain showed resistance to ampicillin, chloramphenicol, clindamycin, erythromycin, oxacillin, penicillin and sulfamethoxazole/trimethoprim.


In the present study, 125 bacterial strains were isolated and identified from the selected seven commercial probiotic products commonly used in Vietnamese shrimp farming.  They included 118 strains of Bacillus, six strains of Klebsiella and one strain of Aerococcus urinaeequi.  All seven probiotic products contained bacterial strains that were not declared on the product labels; a total of 11 Bacillus species identified were not declared.  Further, seven Bacillus species declared could not be isolated.  In addition, Lactobacillus species were declared for some products and Pseudomonas denitrificans was listed in another product; however, none of these bacterial species were identified.


We were occasionally able to isolate only less than five colonies of each type seen on the individual culture media despite doing subculture from the lowest dilution of the probiotic product, which indicates a lower concentrations of probiotic strains than declared.  Although we found that this does not represent a major bias on the diversity of species identified, it may be that some declared species were actually present in the product, but that they were not identified, due to different species showing identical colony morphology.


It should be noted that we did not identify any Vibrio species, for example, V. parahaemolyticus, which are associated with early mortality syndrome (EMS).  Because the production of various beneficial compounds and other positive properties of probiotic bacteria are quite strain specific and such properties can vary significantly among strains of the same bacterial species, the product labels should correctly state the bacterial species included.  However, further details on the actual strains, for example, a unique identification number, should also be provided allowing users and others to obtain specific information about the specific strains used.  None of the products analyzed provided information allowing the identification of the specific probiotic strains used.


According to the Vietnamese Ministry of Agriculture and Rural Development (MARD), probiotic products used in aquaculture must be registered before being sold to shrimp farmers.  The Directorate of Fisheries under MARD is responsible for the formal approval of probiotic products for use in aquaculture.  Such approval is based on performance as documented through on-farm trials, bacterial species composition and concentration analyses performed by laboratories approved by MARD, evaluation of information provided on product labels and on-site inspection of production and storage facilities at the manufacturer.  All seven probiotic products analyzed in this study were on the list of approved products.  It should be noted that the total number of registered products for use in Vietnamese aquaculture in 2012 was 2,913, including 813 so-called veterinary drugs and 2,100 chemicals, including probiotic products.  Clearly, the approval of such a high number of registered products would demand vast amount of resources and be highly costly.  We do not know if the inadequate information provided about the bacterial species included and declared on the products tested in our study may be due to inadequate testing before the products were approved or if bacterial species composition may have been changed after product approval.  Also, it seems that a company that wants to market a new probiotic product with an identical bacterial species composition and concentration to an already approved product does not need to document a positive effect in on-farm trials before being put on the market.  Our observations of product labels revealed that only two products provided information about how the product should be handled when mixing it with the feed.  Information provided on some labels were unclear and seemed to exaggerate the effectiveness of the product, for example, one product declared that it could increase oxygen level.


Most of the microorganisms in probiotics used in aquaculture are Bacillus species, Lactobacillus species and yeast, although other bacterial species like Nitrosomonas species may also be included.  We identified a total of 15 Bacillus species in the seven products tested and Bacillus species, including B. clausii, B. licheniformis, B. cereus, B. pumilus and B. thuringiensis, are often included in probiotic products because they are reported to produce antimicrobial compounds inhibitory to pathogens and stimulate the immune system.  Equally important is that Bacillus species can be kept in the spore form and therefore stored at ambient temperatures for long periods.  The problems with misidentification and labeling of Bacillus species described in the current study are supported by other researchers who found that Bacillus species used for oral bacteriotherapy and prophylaxis of gastroenteritis were mislabeled as B. subtilis.  Researchers have reported that more than 28% of the commercial cultures intended for human and/or animal probiotic use were misidentified at the genus or species level.


Several commonly used Bacillus species have been shown resistant to antimicrobials like chloramphenicol, tetracycline, erythromycin, lincomycin, penicillin and streptomycin.


Conclusion: All seven probiotic products were approved by the Vietnamese authorities, but still contained bacterial strains that were not declared on the product labels, and a total of 11 Bacillus species identified were not declared.  Further, Bacillus species and other bacterial species declared could not be isolated.  Although our culture-based isolation and identification may have missed a few bacterial species present in the tested products this would represent minor bias, but future studies may apply culture independent identification methods like pyro sequencing.  Probiotic product label information should instruct on correct use and dosage as well as allowing identification on the individual bacterial strains included rather than just informing about the bacterial species.  The approval of probiotic products needs to be strengthened and should include documentation from scientific-based efficacy trials and that antibiotic resistance is not present in probiotic strains.  Overall, the Bacillus species showed limited phenotypic antimicrobial resistance.  Whole genome sequencing of selected multiple antimicrobial resistant Bacillus species showed that they contained a low number of resistance genes to macrolides, tetracycline, phenicol and trimethoprim, but not any genetic structures associated with horizontal gene transfer.  With reference to the recently proposed ranking of public health risks associated with antimicrobial resistance genes found in metagenomic studies, the genes found in our study would be ranked as the lowest risks.  In comparison with natural occurring bacterial species in aquaculture environments, our study documents that the probiotic bacterial strains used in Vietnamese shrimp culture contain very limited types and numbers of resistance genes.


Sources: 1. PLOS ONE.  Identification and Antimicrobial Resistance of Bacteria Isolated From Probiotic Products Used in Shrimp Culture.  Gazi Md. Noor Uddin, Marianne Halberg Larsen, Henrik Christensen, Frank M. Aarestrup, Tran Minh Phu, Anders Dalsgaard (email, Department of Veterinary Disease Biology, Faculty of Health and Medical Sciences, University of Copenhagen, Copenhagen, Denmark).  July 6, 2015. 2. Bob Rosenberry, Shrimp News International, July 12, 2015.

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