Background Over the past few decades,microorganisms have developed a greater ability to withstand the effect of antibiotics due to several factors,including the increasing size of the human population and the overuse of antibiotics.The ineffectiveness of antibiotics has continued to pose serious health problems,typified by the existence of methicillin-resistant Staphylococcus aureus (MRSA).Studies have shown that,once the antibiotic resistance gene is generated,bacteria can then transfer the genetic information in a horizontal fashion,by plasmid exchange within the natural environment.Moreover,studies have indicated that conjugative transfer is an important method of horizontal transfer for multiresistance genes.Nanomaterials,which often possess novel physical,chemical,and biological properties,have been applied to an increasing number of fields.Concern about the potential risks of nanomaterials,and the need to manage those risks at an early stage of development,has captured the interest of the world.Some studies have indicated that nanomaterials can deliver DNA or RNA molecules into animal or plant cells.However,the impacts of nanomaterials in water on aquatic microorganisms are largely an unknown.Based on the results described above,we hypothesized that nanomaterials in the water could promote the horizontal transfer of multidrug-resistant genes between bacteria,by acting on cell membranes and/or DNA.Methods The conjugation test between the donor bacteria E.coli HB101 (contained RP4 plasmids) and the receptor bacteria Salmonella Aberdeen Kauffmann 50312 (acquired high drug-resistant streptomycin (strR) through induction mutation in the laboratory) was conducted with final concentration nano-alumina as50,25,10,5,2.5,1,0.5,0.1,0.05,0.005 and 0mmol/L.Transmission electron microscopy (TEM) and atomic force microscope (AFM) was used to characterize the morphology,including nano-alumina distribution in cell,cell membranes appearance and bacteria conjugation.The anti-oxidative system of bacteria was tested using an assay kit from the Najing Jiancbeng Bioengineering Institute (Nanjiag,China).Promoter fusion technology was used to detect the transcription activity of the RP4 conjugation genes trbBp and trfAp in the mating pair formation (Mpf) system.And Real-time PCR technology was carried out to quantitative the RP4 e over expression of the global regulatory factor genes using SYBR Green Ⅰ (Tiangen Biotechnology Co.,Ltd.,Beijing,China).Results: Our study shows that when the concentration of the parent bacteria was at 106-109 cfu/mL,there was a notable increase in the number of conjugants with increasing nano-alumina concentration.The number of transconjugants in each group increased along with conjugation time,at a concentration of parent bacteria of 106 cfu/mL.Temperature and pH do not have an effect on the conjugative transfer of RP4.Upon observing the bacteria in the 5 mmol/L nano-alumina treatment group,we found that the cytoplasm of the bacteria was agglomerated and that parts of the cell membranes were undefined.When the concentration of nano-alumina was increased to 50 mmol/L,portions of the cell membranes of the parent bacteria were severely damaged,and the borders of the cell membranes were likewise unclear.Furthermore,a large number of round,highly dense particles were observed in the cells,indicating that the nano-alumina not only damaged the cell membrane structure of the bacteria,but also intruded into the bacterial cells.AFM images of the bacteria also showed that the cell membranes in the 5 and 50 mmol/L nano-alumina groups displayed wrinkles and fissures,and membranes in the latter group were severely damaged.It showed that nano-alumina can damage bacterial cell membranes.After treatment with nano-alumina,the OH . yield of the bacteria increased along with the increase in nano-alumina concentration.At the same time,there was a significant increase in the total anti-oxidative capacity (T-AOC),superoxide dismutase (SOD) glutathione reductase (GR) and catalase (CAT) activities of the bacteria.When 5 or 50 mmol/L nano-alumina was used for treatment,we observed by TEM that conjugation took place among numerous bacteria and that a single bacterium may have even conjugated with multiple bacteria.This showed that nano-alumina significantly promotes bacterial conjugation.The promoter fusions results indicated that high concentrations of nano-alumina are able to enhance the transcriptional activity of the trbBp and trfAp RP4 conjugation genes.With higher levels of nano-alumina (5 and 50 mmol/L),the mRNA expression levels of both korB and trbA significantly increased.It concentrated nano-alumina can significantly increase the expression of two of RP4(s) global regulatory genes,korB and trbA.Conclusion: For the first time,the effect of nano-alumina on the horizontal transfer of antibiotic-resistant genes mediated by RP4 conjugation among bacteria has been studied.It was observed that the transfer frequency increased with the concentration of nano-alumina,the initial concentration of parent bacteria,and the conjugation time,but was unaffected by water quality conditions.The mechanisms related to the transfer of drug-resistant genes may be related to the discovery that nano-alumina could damage the bacteria(s) cell membranes,which consequently allowed drug-resistant plasmids to directly enter the receptor bacteria.Nano-alunina could promote the conjugation efficiency between donor and receptor bacteria and enhance the RP4 expression of the global regulatory factor genes.The findings in this study support the notion that nano-alumina in the environment could result in ecological hazards.