Transfer Of The Chlortetracycline Resistant Gene From Bacillus Subtilis

Antibiotics which are used as growth promoters have brought great economic benefits.But at the same time they have resulted in huge known or potential risk for human health,especially drug residue and bacterial resistance.Probiotics have been widely used as growth promoters in recent years to substitute the antibiotics for its low toxicity and no drug residue.But the problems of bacterial resistance which result from the use of probiotics have not been dealt with seriously.The Bacillus subtilis and chlortetracycline which are widely used in china were selected as the material in this study.The selection of resistant strains at the presence of chlortetracycline was studied, and then the transfer of chlortetracycline resistant gene from Bacillus subtilis to other bacteria was also studied.The data of this study provide evidences for the government to take scientific measures.The Bacillus subtilis ATCC6633 and the chlortetracycline were selected as research materials.The in vitro model which consisted of nutrient broth within chlortetracycline the concentration of which was gradually increased was used to study the selection of resistant Bacillus subtilis,which began at a concentration of 1/2 MIC values of Bacillus subtilis to chlortetracycline.The Antibiotic Susceptibility Testing of Bacillus subtilis to 10 antibiotics were performed before and after the selection according to the methods recommended by CLSI.Three strains of chlortetracycline resistant Bacillus subtilis were obtained after 41 generations.And they were named BS1,BS2 and BS3 whose MIC values to chlortetracycline were 128μg/mL,32μg/mL and 32μg/mL respectively.The MIC values of the three strains to tetracyclines were much higher compared with that of Bacillus subtilis before selected.And all of the three strains are resistant to four tetracyclines(Chlortetracycline,Doxycycline,Oxytetracycline,Tetracyclin).But their sensitivities to other antibiotics didn't change.Four primers were designed according to the gene sequences in Genebank which were relative to the tetracycline resistance of Bacillus subtilis.The plasmid and genome DNA of the three resistant strains were extracted respectively,and PCR amplifications were performed.After that,the products were detected by agarose gel electrophoresis.The tet(K) gene was found on the plasmids of BS1 and BS2,but no target genes on other plasmids or genomes.This shows that the tet(K) could be the major gene which is responsible for the resistance of Bacillus subtilis to chlortetracycline and the other tetracyclines.The competent cells of E.coli DH5αand BL21 were prepared by calcium chloride method.The plasmids of each resistant strain were extracted,and then the transformation was performed respectively.But no transformants were got.This shows that resistant Bacillus subtilis perhaps can not transfer its resistance to E.coli by transformation.Conjugation transfer experiments were carried out using filter conjugation method. There resistant strains were used as donors,E.coli NK5449 and E.faecalis JH2-2 as recipients.Three Conjugatants which were named BS1-N,BS2-N,BS3-N were obtained when the resistant Bacillus subtilis conjugated with NK5449.BS1,BS2,BS3 conjugated with NK5449 at the frequency of 4.57×10^-7,1.4×10^-7,1.3×10^-8respectively.The results of Antibiotic Sensitivity Testing showed that all the conjugants were resistant to tetracyclines,but not to the other kinds of antibiotics.The tet(K) was found on the plasmids of BS1-N after PCR amplification and the detection of agarose gel electrophoresis.None targets genes were found on the genomes or the plasmids of BS2-N and BS3-N.It could be concluded from the result above that tet(K) gene can be transferred to E.coli by conjugation.And besides this,there may be unknown genes which code chlortetracycline resistance or other transfer mechanisms by which chlortetracycline resistance of Bacillus subtilis can transfered to E.coli.All the three strains of resistant Bacillus subtilis couldn't conjugate with E.faecalis JH2-2.This suggests that resistant Bacillus subtilis perhaps couldn't transfer its resistance to E. faecalis by conjugation.According to the experiment data,some conclusions could be made that the chlortetracycline resistance Bacillus subtilis can be selected in the long term of contact with chlortetracycline,and it has cross resistance to other kinds of tetracyclines.The chlortetracycline resistantance of Bacillus subtilis can be transfered to E.coli by conjugation.The use of tetracyclines may be confined in the treatment of relative diseases, and the risk of therapeutic failure may increase.This shows that the use of Bacillus subtilis as growth promoters has potential risk to animal health.People should pay more attention to the risks which result from the use of probiotics and some measures should be made to guarantee the public health.