| The development of antibiotic resistance in bacteria is one of the most serious threats to global public health, as exemplified by the appearance of a new ’super bug’ in2010in South of Asia and our country. The spread of antibiotic resistance genes is due to the selective pressures caused by increases in the use and misuse of antibiotics in medicine and animal feedstuffs. Also important is the presence of increasing amounts of these substances in the environment by horizontal transfer between bacteria, rather than by the sequential modification of gene function by the accumulation of point mutations. Of all the mechanisms and mobile elements that mediate horizontal gene transfer between bacteria, conjugation by self-transferable plasmids may be the most important. The critical role that plasmids play in the rapid spread of antibiotic resistance genes is particularly salient. Aquatic environments and water treatment processes are able to affect the efficiency of antibiotic resistance gene transfer.Nanomaterials, which often possess novel physical, chemical, and biological properties, are being utilised in an increasing number of fields, especially in water treatment as efficient adsorbents and oxidants. Their use in water treatment may result in a considerable amount of nanomaterial residue in the water, and cause negative impact on the environment, biological and human. Otherwise, nanosized materials at the same time they may, in fact, become new environmental hazards themselves. Some studies have indicated that nano-materials can cause disruption to bacterial membranes by the production of reactive oxygen species (ROS), can affect protein and gene, and can deliver DNA or RNA molecules into animal or plant cells. Recently, there are a large number of drug-resistant bacteria and nano materials in water environment. The extent to which nano-materials are able to cause an increase in antibiotic resistance by the regulation of the conjugative transfer of antibiotic resistance genes in bacteria is still unknown.Based on the above data, we hypothesised that nano-materials that are present in water may promote the horizontal transfer of multidrug-resistance genes by acting on cell membranes and/or regulating genes involved in plasmid transfer. And on basis of the construction of the conjugative transfer models mediated by plasmids, we studied the effect of nano-materials in water environment on the conjugative transfer, and analyzed the effect of various factors on conjugative transfer under nano-materials induced, found out the main factors with Orthogonal experiment, and explored the mechanisms by which nano-alumina promote the horizontal transfer of antibiotic multiresistance features by morphological, biochemical, and molecular biological methods.The main findings are as follows:We established four kinds conjugative transfer models with RP4plasmid, including the resistance gene conjugative transfer in E. coli species, from E. coli to Salmonella, from E. coli to Enterococcus faecalis, and from Enterococcus faecalis to Enterococcus faecalis. Another two conjugative transfer models, including transfer in E. coli species with RK2plasmid and transfer in to Enterococcus faecalis with PCF10plasmid were also be established. These six models include the resistance genes transferring within species and across specie. The results showed that the six conjugative transfer models were relatively stable and could be used to evaluate the effect of nano-materials on the conjugative transfer of resistance genes.Nano-TiO2, nano-SiO2, nano-Fe2O3and nano-Al2O3could promote the transfer of the RP4plasmid from E. coli to Salmonella. At5mmol/L, these four anomaterials could promote the conjugative transfer of RP4by up to89times,10times,20times and142times respectively compared with blank control and bulk materials. Nano-alumina gave the most significant effect. And in other conjugative transfer systems, nano-materials also could promote the resistance genes transfer.5mmol/L nano-alumina can promote the conjugative transfer of RP4between bacteria of the same genus, more specifically from E. coli to E. coli, and increase the transfer by200-fold. Nano-alumina also can significantly promote the conjugative transfer of RP4from Gram-negative bacteria to Gram-positive bacteria by more than50-fold. In other mating systems, from Enterococci to Enterococci, the conjugative transfer of RP4could also be promoted by5mmol/L nano-alumina which increased the transfer by100-fold. Nano-alumina also could promote the horizontal transfer of other conjugative plasmids, such as an unrelated conjugation system in the Gram-negative bacteria, RK2and the enterococcal pheromone-mediated conjugation system involving an endogenous enterococcal plasmid, pCF10. The results showed that nano-alumina could significantly promote the RK2and pCF10conjugational transfer when the concentration of nano-alumina ranged from0.05to50mmol/L. The conjugative transfer frequencies of RK2and pCF10were also the highest in the5mmol/L nano-alumina group, which induced an increase in the transfer of more than170-fold and120-fold compared to the control group respectively. These data showed that it might be a common phenomenon that nano-materials could promote the conjugative transfer of resistance genes. And the results that bulk nano-materials did not affect the conjugative transfer of resistance genes suggested that nanostructures rather than the chemical composition of these nano-materials played a major role in the promoting transfer.Nano Al2O3and RP4plasmid were chosen as the indicators to evaluate the effect of factors on the transfer of resistance genes. Bulk alumina at any concentration had no significant effect on conjugative transfer of RP4, but nano-alumina could significantly promote conjugative transfer, and this transfer increased with increasing of nano-alumina concentrations the conjugative transfer was the highest in the5mmol/L nano-alumina group, which was more than100-fold higher than that of control group. When the nano-alumina concentration was higher than5mmol/L, the transfer rate decreased, as higher nano-alumina concentrations were found to damage the bacterial membrane severely and kill most bacteria. Furthermore, the nanoparticles may have aggregated, and thus become relatively less bioavailable at these concentrations compared with lower concentrations.The conjugative transfer of RP4increased with bacterium concentration. The conjugative transfer in each group increased with the prolongation of mating time, and the conjugative transfer in5mmol/L nano-alumina group was markedly higher than that of the control group at each time point. In order to determine the impact of nano-alumina on RP4transfer accurately, a simple mass action model was used to explain the kinetics of RP4conjugative transfer. The results showed that the conjugational transfer rate constant of5mmol/L nano-alumina group was approximately5orders magnitude higher than that of the control group. The conjugation was lower at4℃and15℃than that at20℃or higher, but showed no further increase above20℃. The pH had no significant effect on plasmid transfer. We used orthogonal design (L64(421)) with the four variables to observe the effect on the conjugative transfer of RP4. The statistical results showed that every one of these four variables had a significant effect on the conjugation, while there was an interaction between the bacterial concentration and nano-alumina concentration. The ranking of these factors in the order of importance of affecting the conjugative transfer was as follows:bacteria concentration> nano-alumina concentration> mating temperature> mating time> the interaction between bacteria concentration and nano-alumina concentration. The effect on promotion of conjugative transfer of nano-alumina was greater than the effects of mating temperature and mating time. Under the optimum conditions, the conjugative transfers of RP4across genera in PBS and within species in LB were more than200-fold and250fold higher than that when no nano-alumina was added.We also explored the potential mechanisms of nano-alumina in promoting conjugative transfer of RP4. Our study showed that nano-alumina caused the SOS response of parent bacteria and also damage the integrity of cell membranes, as was seen from the results of TEM and AFM. The productions of hydroxyl free radicals (OH·) in bacteria were increased with the increasing of nano-alumina concentration. And other inductors of bacterial oxidative stress-response systems increased with increase in nano-alumina levels and significantly higher than those in the control group and bulk alumina groups. The cytoplasm of the bacteria agglomerated, and the parts of the cell membranes were undefined, and the cell membranes displayed wrinkles and fissures when nano-alumina induced. Nano-alumina can enhance the conjugation efficiency through the regulation of conjugative gene expression. We observed that nano-alumina significantly promoted bacteria conjugation. This phenomenon results from the promotion of RP4conjugation gene expression by nano-alumina. The trbBp expression was enhanced after treatment with nano-alumina, which led to the formation of more conjugants. In addition, nano-alumina could promote the expression of trfAp, which is a gene that plays an important role in the transfer and replication of RP4. Nano-alumina may promote the horizontal transfer by repressing the expression of global regulatory genes that are involved in RP4conjugation. We found in this study that korA, korB and trbA mRNA expression were repressed significantly by nano-alumina. These results mean that trfA and trbB are activated and increase this expression, which provides the sequential steps that enhance the efficiency of transfer.In conclusion, this study reported that nano-materials in water increased the conjugative transfer of resistance genes involve the damage of bacterial membranes by oxidative stresses, an enhancement of the expression of conjugative genes, and the repression of the global regulatory factor genes for RP4plasmid conjugation. This study will not only open up a new field of study on the environmental safety of nanomaterials, but also further enrich our knowledge about the the transfer of resistance genes. The results will also help us to control the generation of drug resistance bacteria, to lay theoretical and technical foundation for us to in-depth study the potential risks of nanomaterials and provide us an guidline of nano-materials production and security applications. |
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