The Effect Of DHA-modified Molybdenum Disulfide Nanosheets On Conjugative Transfer Of Antibiotic-resistant Genes In Enterococcus Faecalis

Antibiotics resistance has become a major threat to human health.Antibiotics resistance bacteria and antibiotics resistance genes（ARGs）can be detected in various environments such as hospitals,waste water,and foods.Antibiotics resistance has become a global problem and is expected to overtake cancer as the leading cause of death by 2050 without intervention.The misuse of antibiotics is the main cause of antibiotics resistance.A large number of antibiotics are applied in medicine,agriculture and animal husbandry,and a large proportion of these antibiotics enter the environment directly or as metabolites,which leads to increased levels of antibiotics in the environment,and bacteria evolve resistance under the selective pressure of antibiotics.The increasing antibiotics resistance of Enterococcus faecalis（E.faecalis）has become one of the major bacterial strains causing nosocomial infections.E.faecalis is mainly distributed in the gastrointestinal tract of humans and animals and in various environments,it is prone to induce resistance and ARGs can be transmitted at the interspecies level by conjugative transfer.Various nanomaterials have been shown to affect the plasmid-mediated conjugative transfer process,such as nano-Al₂O₃,nano-Ti O₂,nano-Fe₂O₃,etc.In recent years,the emerging two-dimensional nanomaterials,represented by graphene,are hot research topics and have been widely used in various industries.In this study,molybdenum disulfide nanosheets（MoS₂）and E.faecalis were selected to investigate the effects of MoS₂ nanosheets,docosahexaenoic acid（DHA）,and functionally modified MoS₂-DHA on the plasmid-mediated conjugative transfer of ARGs in E.faecalis,and to elucidate the mechanism.Firstly,nano-MoS₂ was added into the conjugative system of E.faecalis,and it was found that 25 mg/L of nano-MoS₂ could increase the conjugative transfer frequency5～8 times after 2 h,and it did not affect the growth of donor and recipient bacteria.The mechanism behind the phenomenon was elucidated by measuring the level of reactive oxygen species,cell membrane permeability,expression of conjugative transfer regulatory genes and observing bacterial morphology after nano-MoS₂ treatment.Nano-MoS₂ had no significant effects on the reactive oxygen levels,cell membrane permeability,and genes expression of bacteria.Scanning electron microscopy observation of bacterial morphology revealed that nano-MoS₂ would adhere to the surface of bacteria and increase bacterial aggregation thereby promoting the conjugative transfer.Next,the inhibitory effect of DHA,an essential unsaturated fatty acid to human body,on conjugative transfer in E.faecalis was investigated.After adding DHA to the conjugative system for 2 h,it was found that the inhibition rate of conjugative transfer could reach more than 95%when the DHA concentration was greater than 5 mg/L.At the same time,the growth of the recipient bacteria in the conjugative system was inhibited by the bacteriostatic effect of DHA,which was one of the reasons for the decrease of conjugative transfer frequency,but the main reason was the inhibition of conjugative transfer process.By measuring the biofilm production,it was found that DHA significantly inhibited the formation of biofilm in the conjugative system and reduced the adhesion of bacteria,which was also confirmed by the results observed by scanning electron microscopy.Quantification of the expression of conjugative transfer regulatory genes revealed that the expression of prg Z gene,which is responsible for pheromone transport,was significantly decreased,this would affect the transport of pheromones between bacteria.The result of intracellular and extracellular pheromone content detected by liquid chromatography mass spectrometry revealed that the extracellular i CF10 pheromone content decreased and the intracellular i CF10pheromone content increased after DHA treatment.A significant decrease in the expression of the prg Y gene,which is responsible for regulating donor self-induction,also indicated changes in the intracellular pheromone content of the donor,and changes in the intracellular and extracellular pheromone content demonstrated the effect of DHA on the pheromone transport process.The intracellular accumulation of the i CF10pheromone resulted in a significant decrease in the expression of the downstream prg A gene encoding a surface rejection protein,and the prg B gene encoding an adhesion protein,these were consistent with the results of reduced biofilm formation and reduced bacterial adhesion.Finally,nano-MoS₂ was functionally modified with DHA to obtain MoS₂-DHA composites.Then MoS₂-DHA was added into the conjugative system and found to inhibit the conjugative transfer by up to 60%.By measuring the biofilm production,it was found that MoS₂-DHA had no significant effect on the biofilm formation;Scanning electron microscopy observed that MoS₂-DHA adhered to the bacterial surface like MoS₂ nanosheets,but MoS₂-DHA did not promote bacterial aggregation,and made the bacteria less adherent;the results of the expression of conjugative transfer regulatory genes revealed that MoS₂-DHA decreased the expression of prg Z gene,and affected pheromone transport between bacteria,leading to intracellular accumulation of i CF10pheromone;decreased expression of the prg Y gene,also indicated a change in the intracellular pheromone content of the donor;reduced bacterial adhesion also resulted in a significant decrease in the expression of the downstream prg A gene;the expression of the prg B gene,which encodes an adhesion protein and is closely related to biofilm formation,did not change significantly because biofilm formation did not change significantly.In marked contrast to DHA,MoS₂-DHA down-regulated the expression of genes encoding pheromones and inhibited the synthesis of pheromones.The above results indicate that MoS₂-DHA inhibits conjugative transfer by a similar mechanism as DHA,and also exhibits novel properties.This is of great significance for controlling the propagation of ARGs and the modification of MoS₂ nanosheets.Most of the current studies on conjugative transfer have focused on gram-negative bacteria,and there are few studies on gram-positive bacteria.In this study,gram-positive bacteria were selected to investigate the effect of emerging two-dimensional nanomaterials on the conjugative transfer process,the role of unsaturated fatty acids as conjugative inhibitors in gram-positive bacteria,and the effect of composites on conjugative transfer after functionalized modification of two-dimensional nanomaterials using conjugative inhibitors.The potential hazards of emerging two-dimensional nanomaterials were elucidated,and technical means were provided to control the horizontal transfer of ARGs and the modification of new materials.