Characteristics of a transferable <italic>tet</italic>(45) gene conferring resistance to tetracyclines in probiotic<italic> Bacillus cereus</italic>

Abstract

Probiotic microbes conferring health benefits to the hosts have attracted great concerns recently. Particularly, spore-forming Bacillus cereus ( B. cereus ), Gram-positive, aerobic or facultative anaerobic bacteria, have been widely used in probiotic preparations for different purposes, due to their high stability to harsh conditions. Probiotics are generally recognized as safe (GRAS) and showing satisfactory overall safety records till now. However, the safety of each probiotic isolate is not guaranteed. The verification of the absence of mobile antimicrobial resistance genes and virulence factors is a prerequisite for the approval of probiotic candidates, according to the guidelines suggested by the Food and Agriculture Organization (FAO) and World Health Organization (WHO). The role of B. cereus and other members of the B. cereus group in gastrointestinal and non-gastrointestinal diseases has not always gained sufficient attention in the past, particularly in view of their use as probiotics. Therefore, the presence of transferrable antimicrobial resistance genes will endow probiotics as a reservoir with increased ability of emergence and dissemination, during their wide and inadvertent use. Our previous study found that the presence of transferable tetracycline resistance gene tet (45) in a probiotic isolate B. cereus 9i (BC 9i) derived of animal feed additive. The aim of the present work was to systematically characterize this tet (45). The draft assembled genome of BC 9i was first used to analyze the genetic environment of tet (45). Meanwhile, multiple alignment and a maximum likelihood tree were built to conduct the phylogenetic relationship between tet (45) and othertetracycline efflux resistance genes. In addition, the 3D protein structures were predicted to identify differences between Tet(45) and the original Tet(45). Lastly, the tet (45) gene in BC 9i was cloned and transferred into Staphylococcus aureus RN4220 to the minimum inhibitory concentrations, in the presence and absence of classic inhibitors of efflux pumps. The phylogenetic analysis showed that the gene tet (45) in BC 9i was classified as a gene variant of tet (45), belonging to the major facilitator superfamily of membrane transporters. Additionally, the predicted structure of Tet(45) variant was similar to the original Tet(45), both sharing a 14-transmembrane helix core, although they exhibited subtle structural differences in the loop regions and α-helixes. Lastly, the resistance to tetracyclines in tet (45) variant was suppressed in the presence of efflux pump inhibitors. These results indicated that the tet (45) variant in BC 9i encoded a efflux pump against tetracyclines. Collectively, our findings suggest that such probiotic isolates harboring transferable antimicrobial resistance genes may consist a potential risk for public health. It sheds light on the subsequent screening of probiotic candidates and the appropriate application of probiotics for various purposes, to promote and improve One Health.