Horizontal Transfer Of Multiresistance Genes Mediated By Plasmid PYN1 Across Species And Genera

The wide use of antibiotics,especially unreasonable abuse,has accelerated the development of resistant bacteria,which is becoming amajor threat to global public health.Due to some hazardous characteristics of resistant genes,（such as,difficult to degreadation,high-frequency horizontal transfer,etc.）,resistant genes have been considered to be a contaminant.The horizontal transfer of resistant genes in the environment mainly mediated by plasmids,transposons and phage.Among them,gene transfer mediated by plasmid is the most important transmission route.Based on the previous study in our labrotory,the transformed ability of multi-drug plasmid pYN1 in different bacterial species and genera were studied.Moreover,the effect of antibiotic and heavy metal on dissemination were also studied.The main results are as follows:（1）The plasmid used in this study comes from E.coli Ls1 which preserved in our laboratory.The total plasmid isolated from E.coli Ls1 with amount of 1.5 ug were co-cultured with the foure recipient strain（Bacillus subtilis,Bacillus cereus,Bacillus thuringiensis and Proteus vulgaris）for different treating time.As the results,the transformants were detected on selected medium supplied with antibolics;the bands which sizes were same to multi-resistant plasmid（about 15 Kb）were appeared in all recipient strain.However,the transfer capability of plasmid pYN1 in the four recipient strains were different in order to sort as follows: Bacillus cereus(8.5 x 10^-4)> Proteus vulgaris(4.7 x 10^-6)> Bacillus thuringiensis(4.5 x 10^-6)> Bacillus subtilis（2 x 10-7）.（2）Then,the effect of the kinds and concentrations of antibiotics on transformation frequence were studied.And the results showed that both kinds and concentrations of antibiotics affected the plasmid transformation in recepient bacteria.In the range of 0-0.15 ug/ml cefalexin,0-0.3 ug/ml tetracycline,or 0-0.1.5 ug/ml kanamycin,the transformation efficiency increased of 10²-10³ fold than control.in Bacillus subtilis and Proteus vulgaris,and 10-10² fold in.Bacillus cereus and Bacillus thuringiensis.The growth curves of recepient strain under different processing conditionswere drawed The trend is was decrease;The results of SDS-page showed that a protein bands more than 245 kD appeared in Bacillus subtilis under the teatment of 0.5 ug/ml kanamycin and 0.2 ug/ml of tetracycline In addition,the HPLC results showed that two new peak were found in 15.057 and 15.057 min retention time,respectively Presumably,these proteins may be associated with stress resistant.（3）At last,the effect of the kinds and concentrations of heavy metal ion on transformation frequence were studiedAnd the results showed that both the kinds and concentrations of heavy metal ion affected the plasmid transformation in receptor recepient bacteria.Among them,in the range of 0-4 mmol/L copper ion,the transfromation efficiency of Bacillus subtilis,Bacillus cereus and Bacillus Proteus increased 10²-10³ times,but Bacillus thuringiensis decreased 10³;in the range of 0-9 mmol/L manganese ion,the transfromation efficiency of Bacillus subtilis and Proteus vulgaris decreased,but Bacillus thuringiensis and Bacillus cereus increased;in the range of 0-4 mmol/l zinc ion,the transfromation efficiency of Bacillus subtilis and Bacillus cereus increased 10-10² times;in the range of 0-4 mmol/l iron ion,the transfromation efficiency of Bacillus thuringiensis,Bacillus cereus and Bacillus Proteus increased,but Bacillus subtilis decreased.The growth curve of receptor strain,showed the heavy metal were beneficial to the growth of recepint strains.The results of SDS-page found showed that a protein bands about 180 kDa appeared in Bacillus subtilis under the teatment of copper,manganese,or zinc ion.In addition,the HPLC experiments results were found that a new peak was found in the retention time of 15.072 min compared with control.Presumably,this protein may be associated with stress resistant.