益生蜡样芽孢杆菌可转移四环素耐药基因tet(45)特征分析

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

蜡样芽孢杆菌作为益生菌常用菌种之一被广泛应用于人类医疗保健、畜禽养殖、作物促生长和生物防控、环境水质净化等多个领域.益生菌携带的可转移性耐药基因对公共卫生构成潜在威胁.本团队前期发现1株分离自动物源微生态制剂的蜡样芽孢杆菌(Bacillus cereus 9i,BC 9i)携带可转移的四环素耐药基因.通过生物信息学分析发现,BC 9i携带的四环素耐药基因为tet(45)基因变体,同属于膜转运蛋白超家族基因.BC 9i表达的Tet(45)变体蛋白结构与原Tet(45)蛋白结构类似,均由14个跨膜螺旋核心构成,但在环状区域和α-螺旋中表现出细微差异.通过克隆tet(45)基因变体,研究表明其功能是通过四环素外排泵介导耐药性产生,外排泵抑制剂可以有效抑制该作用.本研究从基因、蛋白和功能等方面揭示可转移tet(45)基因变体的特征,为探究益生菌耐药基因来源以及益生菌候选菌株的筛选和合理使用提供科学依据.

Probiotic microbes conferring health benefits to the hosts have attracted great concerns recently.Particularly,sporeforming 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 9 i(BC 9 i)derived of animal feed additive.The aim of the present work was to systematically characterize this tet(45).The draft assembled genome of BC 9 i 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 3 D protein structures were predicted to identify differences between Tet(45)and the original Tet(45).Lastly,the tet(45)gene in BC 9 i 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 9 i 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 andα-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 9 i 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.