Bioavailibility Of Tetracycline In Water And Soil To Escherichia Coli Bioreporter For Expression Of Antibiotic Resistance

The proliferation of tetracycline-resistant bacteria has become an ever-increasing global threat to human health.This has been attributed to the aggressive use of antibiotics to humans and animals,which results in the widespread distributions of antibiotics in the environment.Tetracyclines are the class of antibiotics most widely utilized for livestock production and for infection therapy.Tetracyclines in water and soil could exert selective pressure on indigenous microbial communities and increase the prevalence of antibiotic-resistant bacteria in the environment.A prerequisite for selective pressure is that bacteria have to access the tetracycline present in varying environmental matrices;but little is known about the degree to which tetracyclines bound to soil and/or dissolved organic matter(DOM)versus those dissolved in water are bioavailable for uptake by bacteria.In addition,certain groups of naturally-occurring chemicals may exert co-selective pressure on bacteria for development and enrichment of antibiotic resistance.Consequently,a fundamental understanding of the bioavailability of tetracycline in soil and water to bacteria is crucial for protecting human health and developing sustainable waste management practices.In this study,we use a tetracycline-responsive whole-cell bioreporter,Escherichia coli MC4100/pTGM constructed by inserting plasmid pTGM containing tetracycline resistant gene tetM and green florescence protein gene,to both detect and quantify the bioavailable fraction of tetracycline.The primary results are listed below:1.Influence of dissolved organic matter on tetracycline bioavailability to Escherichia coli bioreporter.Complexation of tetracycline with dissolved organic matter(DOM)in aqueous solution could alter the bioavailability of tetracycline to bacteria,thereby alleviating selective pressure for development of antibiotic resistance.In this study,an E.coli whole-cell bioreporter was exposed to tetracycline in the presence of DOM derived from humic acids.Complexation between tetracycline and DOM diminished tetracycline bioavailability to E.coli,as indicated by reduced expression of antibiotic resistance genes.Increasing DOM concentration resulted in decreasing bioavailability of tetracycline to the bioreporter.Freely dissolved tetracycline(not complexed with DOM)was identified as the major fraction responsible for the rate and magnitude of antibiotic resistance genes expressed.Furthermore,adsorption of DOM on bacterial cell surfaces inhibited tetracycline diffusion into the bioreporter cells.The magnitude of the inhibition was related to the amount of DOM adsorbed and tetracycline affinity for the DOM.These findings provide novel insights into the mechanisms by which the bioavailability of tetracycline antibiotics to bacteria is reduced by DOM present in water.Agricultural lands receiving livestock manures commonly have elevated levels of both DOM and antibiotics;the DOM could suppress the bioavailability of antibiotics,hence reducing selective pressure on bacteria for development of antibiotic resistance.2.Effect of naturally-occurring compounds on tetracycline activity to Escherichia coli bioreporter.The occurrence and proliferation of antibiotic resistance in microbial communities has become a global issue of public health.The presence of trace levels of antibiotics in the environment exerts selective pressure on susceptible microbes for development and enrichment of antibiotic resistance.Environmental conditions may alter the selective pressure of antibiotics on bacteria due to the changes of antibiotic chemistry or bacterial physiology.In this study,we found that certain groups of phenolic compounds commonly found in the environment could alter the bioavailability of tetracycline in water hence selective pressure on an E.coli bioreporter construct carrying tetracycline resistance genes.In the solution,the presence of phenolic compounds was observed to enhance the response of antibiotic resistance genes in an E.coli bioreporter,and the magnitude of the enhancement increased with the amount of phenolic compounds present in water.The results of increased intercellular concentration of tetracycline in the bacterium suggest that phenolic compounds could facilitate bacterial uptake and accumulation of tetracycline.The experimental results of bacterial stain with propidium iodide indicated that phenolic compounds did not damage the integrity of bacterial membrane.The increase of bacterial uptake of tetracycline could be attributed to the impairment of bacterial efflux pump activity by phenolic acids.3.Bioavailability of soil-sorbed tetracycline to Escherichia coli bioreporter:agar diffusion assay and direct microscopic observation.Environmental residues of antibiotics could be responsible for the ever-increasing antibiotic resistance in microbial communities.A large proportions of antibiotics administered to animals are discharged to manure;land application of animal manure as auxiliary plant fertilizers renders a predominant portion of antibiotics bound to soils/sediments.The bioavailability of soil-sorbed antibiotics to bacteria for development of antibiotic resistance in soil bacterial communities is largely unknown.In this study,a whole-cell E.coli bioreporter was employed to detect and quantify the bioavailable fraction of tetracycline sorbed by three surface soils.The results of agar diffusion assay indicate that soil-sorbed tetracycline was desorbed and still bioavailable to activate the antibiotic resistance genes in the E.coli bioreporter.Desorption of soil-sorbed tetracycline to agar medium manifested a concentration gradient for tetracycline.The combination effects of uptake of tetracycline by E.coli bioreporter and the growth of bacterial numbers along the concentration gradient resulted in the formation of a bright fluorescent ring with the center of the tetracycline-sorbed soils.As for the bacteria residing at the soil surfaces,they could still access the soil-sorbed tetracycline,and the amount of tetracycline associated with the soil determined the extent of the antibiotic response of the bacteria.Overall,soil types,incubation time and water contents could put combined effects on the bioavailability of soil-sorbed tetracycline to E.coli bioreporter.