Influences Of Extracellular Polymeric Substances And Several Environmental Pollutants On The Lateral Transfer Of Antibiotic Resistance Genes

The environmental abundance of antibiotic resistance genes(ARGs)has increased significantly since the 1940s,which coincided with the increased production and use of antibiotics in medicine and agriculture,and presents severe threats to human health.Compared to clonal expansion that passes genes from parent cells to offspring cells,lateral gene transfer(LGT)can spread ARGs across organisms of similar or different species,which may substantially increase the risk of ARGs.Extracellular polymeric substances(EPS),as the first interface between microorganisms and the external environment,can protect microorganisms against external stress or damage.Moreover,antibiotics could exert a stimulating effect on both ARG abundance and EPS amount in the same microenvironment.However,it is unclear whether EPS could affect the LGT of ARGs into bacteria,which awaits further exploration.Additionally,there are many other types of pollution co-existing in the same environment,e.g.,polycyclic aromatic hydrocarbons(PAHs),metal oxide nanoparticles(MONPs)and microplastics.Previous studies have mainly focused on the adsorption or degradation of vector DNA carrying ARGs by these environmental pollutants.However,the lateral transfer of ARGs has been rarely studied.Antibiotic resistance plasmids(e.g.,pUC19,pHSG298,and pHSG396)and bacterium Escherichia coli as the receipt cells were applied to investigate the effect of EPS,PAHs,MONPs and microplastics on the lateral ARG transfer(i.e.,transformation),and the mechanisms were also explored.1.Extracellular polymeric substances hinder the lateral transfer of antibiotic resistance genes into Escherichia coli.This study investigated the lateral transfer of ARGs carried by plasmids(pUC19,pHSG298,and pHSG396)into competent Escherichia coli cells with and without EPS.Transformant numbers and transformation efficiency for E.coli without EPS were upto 29 folds higher than those with EPS.The EPS removal further increased cell permeability in addition to the enhanced cell permeability by Ca2+,which could be responsible for the enhanced lateral transfer of ARGs.The fluorescence quenching experiments showed that EPS could bind strongly to plasmid DNA,in the presence of Ca2+.Also,the binding strength(LogKA=10.60-15.80 L mol-1)between EPS and plasmids was positively correlated with the enhancement percentage of transformation efficiency due to the EPS removal.X-ray photoelectron spectroscopy(XPS)analyses and model computation further showed that Ca2+could bind with EPS mainly through the carboxyl group,the hydroxyl group and the RC-O-CR in glucoside,thus bridging the plasmid and EPS.These results indicated that EPS could trap plasmids and make them difficult to enter E.coli cells,which showed an inhibitation on the LGT of ARGs.2.Noncovalent binding of PAHs with genetic bases reducing the in vitro lateral transfer of antibiotic resistant genes.In this investigation,we examined the association of some widely occurring PAHs in different sizes(phenanthrene,pyrene,benzo[g,h,i]perylene,and other congeners)with antibiotic resistance plasmid(pUC19).PAHs in different sizes had different binding capacities with the pUC19 plasmid.Smaller PAHs(e.g.,phenanthrene and pyrene)bind effectively with plasmids to form a loosely clew-like plasmid-PAH complex(16.5-49.5 nm),their corresponding binding strength were 14.4 and 12.8 L mol-1,respectively.Larger PAHs(e.g.,benzo[g,h,i]perylene)bind with plasmid weakly and their binding strength was merely 2.9 L mol-1.Fourier transform infrared spectroscopy(FTIR),and theoretical interaction models showed that adenine in plasmid has a stronger capacity to bind with phenanthrene and pyrene molecules via a ?-? attraction.Changes in Gibbs free energy(?G)suggest that the CT-PAH model reliably depicted the plasmid-PAH interaction through a noncovalently physical sorption mechanism.Binding with smaller PAHs inhibited the LGT of ARGs significantly.The inhibiting rate of phenanthrene and pyrene on transformation were 17.4%and 11.2%,respectively.The effect of larger PAHs was insignificant,and the inhibiting rate was merely 0.5%.The in vitro transcription analysis demonstrated that the reduced transformation of ARGs in plasmids stemmed from the PAH-inhibited ARGs transcription to RNA.3.Plasmid binding to metal oxide nanoparticles inhibited lateral transfer of antibiotic resistance genes.This study explored the lateral transfer of ARG-carrying plasmids to Escherichia coli(i.e.,transformation)in the presence of metal oxide nanoparticles(MONPs).The presence of ZnO,Al2O3 and TiO2 nanoparticles(ZnONP,Al2O3NP,and TiO2NP,respectively)at concentrations less than 50 mg L-1 inhibited the lateral transfer of pUC19 plasmid carrying ampicillin resistance gene into E.coli DH5? in a decreasing degree of Al2O3NP>ZnONP>TiO2NP.Compared to the control group,inhibiting rates of Al2O3NP,ZnONP and TiO2NP(2.5-50 mg L-1)on the transformation were 25.5%-79%,13.2%-72%and 10.1%-64%,respectively.Metal ions released from MONPs had no significant effect on the LGT of ARGs.Results of the plasmid adsorption experiment on MONPs suggested that MONPs could bind with pUC19,and the binding capacity decreased in the order Al2O3NP>ZnONP>TiO2NP.Additionally,the atomic force microscope(AFM)images illuminated the aggregation of MONPs and plasmids.FTIR analyses and computation modeling indicated that MONPs bound to phosphate groups in plasmids via a chemical bond interaction or strong noncovalent interaction and bound to bases in plasmids via strong noncovalent interaction or van der Waals interaction.The amount of plasmids bound to MONPs showed a significantly negative correlation with the efficiency of ARG transfer,demonstrating that the binding to MONPs hindered the LGT of ARGs.Finally,the scanning electron microscopy(SEM)and transmission electron microscopy(TEM)revealed that aggregates of plasmids and MONPs were blocked out of the cell surface,thus preventing their lateral transfer into E.coli.4.Nano-sized microplastics enhanced lateral transfer of antibiotic resistance genes.In order to explore the effect of microplastics on the lateral transfer of antibiotic resistance genes,polystyrene microspheres with sizes of 0.1,1 and 1 ?m,pUC19 plasmid and E.coli DH5? competence cells were used.It was detected that microplastics with sizes of 1 and 10?m showed no apparent influence on the LGT of ARGs.Microplastics sized 0.1 ?m at concentrations ranging from 10-500 mg L-1 exerted a significant enhancing effect on the ARG transfer.The transformation efficiency and frequency were even increased to 1.6 and 1.5 times of those in the control group,respectively.The results in the adsorption experiments and the agarose gel electrophoresis research suggested that the microplastics barely adsorbed pUC19 plasmids and could not cause any degradation or breakage of the plasmids.In the SEM images,the intact cell membrane could be seen in the treatment using 1 and 10 ?m sized microplastics.However,for 0.1 ?m,some nanopores were shown on the cell membrane of E.coli,and plasmids could enter the E.coli cells more easily through these nanopores,thus increasing the transformation efficiencies or frequencies.Results of the intracellular reactive oxygen species(ROS)quantification showed a significant increase of ROS concentrations induced by 0.1 ?msized microplastics,which might modify the structure of cell membranes.The antioxidant was applied in the transformational experiment system.In this situation,no nanopores on the membrane were shown,and the transformation enhancement disappeared in the treatment of 0.1 ?m-sized microplastics.The findings revealed that microplastics in the size of 0.1 ?m could increase the intracellular ROS,which caused nanopores formation on the cell membranes,thus,hindering the lateral ARG transfer into E.coli cells.