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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. The bacterial species was established by 16S rRNA sequence analysis of 125 representative bacterial isolates. MIC testing was done for a range of antimicrobials and whole genome sequencing of six multiple antimicrobial resistant Bacillus spp. used to identify resistance genes and genetic elements associated with horizontal gene transfer. Thirteen bacterial species declared on the probiotic products could not be identified and 11 non-declared Bacillus spp. were identified. 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. Only 6/60 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 not known structures associated with horizontal gene transfer. Probiotic bacterial strains used in Vietnamese shrimp culture seem to contribute with very limited types and numbers of resistance genes compared to the naturally occurring bacterial species in aquaculture environments. 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.
Highlights
Aquaculture is the fastest growing animal protein production sector worldwide and Asia contributes annually with about 90% of the global seafood production [1]
Rico et al [6] further reported that Bacillus subtilis, B. licheniformis, B. thuringiensis and Lactobacillus acidophilus were the main bacterial species included in probiotic products used in Vietnamese shrimp culture with bacterial species composition and concentration normally listed on the product labels
All seven probiotic products were approved by the Vietnamese authorities but still contained bacterial strains that were not declared on the product labels, i.e. a total of 11 Bacillus species identified were not declared
Summary
Aquaculture is the fastest growing animal protein production sector worldwide and Asia contributes annually with about 90% of the global seafood production [1]. The intensive culture and rapid expansion of shrimp farming in Vietnam and elsewhere have been negatively affected by various diseases, e.g. White Spot Syndrome Virus (WSSV), Yellow Head Virus (YHV), White Feces Syndrome (WFS) and Early Mortality Syndrome (EMS) [3] and water quality problems [4]. The popularity of probiotic usage in shrimp aquaculture has grown worldwide, mainly because farmers often experience limited effect of antimicrobial treatment, and because of continued problems and reports of antimicrobial residue findings in exported shrimp. Aquaculture farmers are generally uncertain about the effect of the many different types of marketed probiotics, e.g. those used as feed supplement, whereas a change in water colour after application of probiotics is seen as a sign of improved water quality (Tran Minh Phu, unpublished data)
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