REMOVAL OF PHARMACEUTICALS FROM WATER USING NANO - ENGINEERED POROUS CERAMIC MEDIA AND EXPRESSION OF ANTIBIOTIC RESISTANCE OF E.COLI EXPOSED TO TETRACYCLINES By Qi Xu A THESIS Submitted to Michigan State University in partial fulfi llment of the requirements for degree of Crop and Soil Sciences - Master of Science 2015 ABSTRACT REMOVAL OF PHARMACEUTICALS FROM WATER USING NANO - ENGINEERED POROUS CERAMIC MEDIA AND EXPRESSION OF ANTIBIOTIC RESISTANCE OF E.COLI EXPOSED TO TETRAC YCLINES By Qi Xu H ighly porous ceramic media can be effective to remove contaminants from agricultural waters. In this study, these sorbent media were tested for their efficiency to remove three representative pharmaceuticals (i.e., lincomycin, sulfamet hazine and tetracycline) from water. Results reveal that the tested media demonstrated relatively effective sorption for the selected pharmaceuticals from water. Tetracycline manifested the greatest sorption, followed by lincomycin and sulfamethazine. A g r anul ar medium was identified to have great removal efficiency for the selected pharmaceuticals, and could be potentially employed to remov e pharmaceuticals from agricultural drainage water . The widespread tetracyclines in the environment have been consider ed to be responsible for the development of antibiotic resistance in microorganisms. At contaminated sites multiple tetracyclines are commonly found , and could collectively impact microbial populations. T he uptake of different types of tetracyclines by Esc herichia coli exerted varying levels of selective pressure on the bacteria to express antibiotic resistance , following t he order of tetracycline > chlortetracycline > oxytetracycline. Linear relation s between promoter activity and intracellular tetracyclin e concentration were observed except for anhydrotetracycline. The mixture of tetracyclines generally demonstrated additive effects on the E.coli bio reporter for expression of antibiotic resistance. These results suggest that the risk levels of antibiotic r esistance invoked by exposure to tetracycl ine mixtures could be additive. iii ACKNOWLEDGEMENTS First, I would like express my deepest appreciation to my major professor Dr. Hui Li for his guidance, patience and encouragement over these 2 years. Thank you very much for forcing me, to look at my research and work in different ways. Without your help, I could not finish my study. I would also like to thank Dr. Wei Zhang and Dr. Steve Safferman for serving on my committee and providing advice, information, and support on different aspects of my project. I thank Yingjie Zhang, Ya - Hui Chuang, Zeyou Chen, Sangho Jeon, Cheng - H ua Liu and all members in our lab, you guys give me support and generous help during my experiment and my thesis. Of course, I would like to thank our department, Department of Plant, Soil and Microbial Sciences, all the faculty, staff, and st udents. and become a spartan . Finally, I would like to thank my parents Liang Xu and Ying Ji . They were always supporting me and encouraging me with their best wishes. iv TABLE OF CONTENTS LIST OF TABLES ................................ ................................ ................................ ............. v LIST OF FIGURES ................................ ................................ ................................ ......... vi CHAPTER I REMOVAL OF PHARMACEUTICALS FROM WATER USING NANO - ENGINEERED POROUS CERAMIC MEDIA ................................ ................ 1 INTRODUCTION ................................ ................................ ................................ ...... 2 OBJECTIVE ................................ ................................ ................................ ............... 3 MATERIALS AND METHODS ................................ ................................ ................ 3 RESULTS AND DISCUSSION ................................ ................................ .................. 6 CONCLUSIONS ................................ ................................ ................................ ....... 24 REFERENCES ................................ ................................ ................................ ......... 25 CHAPTER II EXPRESSION OF ANTIBIOTIC RESISTANCE OF E.COLI EXPOSED TO TETRACYCLINES ................................ ................................ .............. 28 INTRODUCTION ................................ ................................ ................................ .... 29 MATERIALS AND METHODS ................................ ................................ .............. 31 RESULTS AND DISCUSSION ................................ ................................ ................ 36 CONCLUSION ................................ ................................ ................................ ......... 48 REFERENCES ................................ ................................ ................................ ......... 50 v LIS T OF TABLES Table 1.1 Properties of selected three pharmaceuticals ................................ ...................... 4 Table 1.2 Selected properties of sorbent media ................................ ................................ .. 8 Table 1.3 Pharmaceutical removal percentage by selected sorbent media ......................... 9 Table 1.4 Pharmaceutical removal percentage by sorbent m edia µg/L). ................................ ................................ ................................ ................................ . 11 Table 1.5 Fitting Freundlich isotherm parameters for sorption of lincomycin and sulfamethazine by sorbent media. ................................ ................................ ..................... 12 Table 2.1 Properties of selected four tetracyclines ................................ ........................... 32 Table 2.2 Measured Promoter Activity and Predicted Promoter Activity in the Binary Tetracycline + Chlortetr acycline System ................................ ................................ .......... 45 Table 2.3 Measured Promoter Activity and Predicted Promoter Activity in Binary Tetracycline + Oxytetracycline System ................................ ................................ ............ 46 Table 2.4 Measured Promoter Activity and Predicted Promoter Activity in Trinary Tetracycline + Chlortetracycline + Oxytetracycline System ................................ ............ 47 vi LIST OF FIGURES Figure 1.1 Pharmaceutical removal percentage by selected sorbent media ...................... 10 Figure 1.2 Sorption isotherm of lincomycin by hybrid media #1 ................................ ..... 14 Figure 1.3 Sorption isotherm of lincomycin by hybrid media #2 ................................ ..... 14 Figure 1.4 Sorption isotherm of lincomycin by hybrid media #3 ................................ ..... 15 Figure 1.5 Sorption isotherm of lincomycin by hybrid media #13A ................................ 15 Figure 1.6 Sorption isotherm of lincomycin by hybrid media #14A ................................ 16 Figure 1.7 Sorption isotherm of lincomycin by hybrid media #15A ................................ 16 Figure 1.8 Sorption isotherm of lincomycin by hybrid media #16A ................................ 17 Figure 1.9 Sorption isotherm of lincomycin by iron - modified media (new) .................... 17 Figure 1.10 Sorption isotherm of lincomycin by iron - modified media (old) ................... 18 Figure 1.11 Sorption isotherm of lincomycin by phosphorus - saturated iron - modified media (old) ................................ ................................ ................................ ................................ ... 18 Figure 1.12 Sorption isotherm of sulfamethazine by hybrid media #1 ............................. 19 Figure 1.13 Sorption isotherm of sulfamethazine by hybrid media #2 ............................. 19 vii Figure 1.14 Sorption isotherm of sulfamethazine by hybrid media #3 ............................. 20 Figure 1.15 Sorption isotherm of sulfamethazine by hybrid media #13A ........................ 20 Figure 1.16 Sorption isotherm of sulfamethazine by hybrid media #14A ........................ 21 Figure 1.17 Sorption isotherm of sulfamethazine by hybrid media #15A ........................ 21 Figure 1.18 Sorption isotherm of sulfamethazine by hybrid media #16A ........................ 22 Figure 1.19 Sorption isotherm of sulfamethazine by iron - modified media (new) ........... 22 Figure 1.20 Sorption isotherm of sulfamethazine by iron - modified media (old) ............. 23 Figure 1.21 Sorption isotherm of sulf amethazine by phosphorus - saturated iron - modified media (old) ................................ ................................ ................................ ........................ 23 Figure 2.1 Relationship between bacterial biomass in culture suspension and opt ical density measured at 600 nm ................................ ................................ ................................ .......... 36 Figure 2.2 Relationship between intracellular tetracycline concentration and the expressed promoter activity of the E. coli bioreporter. ................................ ................................ ...... 38 Figure 2.3 Relationship between intracellular chlortetracycline concentration and the expressed promoter activity of the E. coli bioreporter. ................................ ..................... 39 Figure 2.4 Relationship between intracellular oxytetracycline concentration and the expressed promoter activity of the E. coli bioreporter. ................................ ..................... 40 Figure 2.5 Anhydrotetracycline uptake by E.coli bioreporter and calculated promoter activity. ................................ ................................ ................................ .............................. 42 viii Figure 2.6 Comparison between tetracycline and anhydrotetracycline uptake by E.coli bioreporter associated promoter activity. ................................ ................................ .......... 43 Figure 2.7 Measured Promoter Activity vs Predicted Promoter Activity in binary and trinary systems and linear fitting. ................................ ................................ ...................... 48 1 CHAPTER I REMOVAL OF PHARMACEUTICALS FROM WATER USING NANO - ENGINEERED POROUS CERAMIC MEDIA 2 INTRODUCTION Agricultural drainage has been widely applied to crop production systems in the United States (Pavelis, 1987). However, agricultural drainage water discharged to recipient surface waters m ay adversely impact water quality due to contaminants carried in the drainage water. Typical contaminants commonly present in agricultural drainage water are nutrients (e.g., nitrogen and phosphorous), heavy metals (e.g., As, Se, Cd, Pb, Cu, and Zn) and or ganic chemicals (e.g., pharmaceuticals, hormones, and pesticides). Thus, the treatment of agricultural drainage water may be needed to prevent contamination of surface waters. In this study we attempt to test the removal of pharmaceuticals from water using recently developed novel porous ceramic media. Veterinary pharmaceuticals are commonly administered to animals for disease control and improving feeding efficiency, livestock growth, and animal health (Song et al,. 2010). These pharmaceuticals are widely used in concentrated animal feeding operations to annual domestic sales and distribution of antimicrobials approved for use in food - producing animals were approximately 14.6 million kilograms (FDA, 2014). Large fractions of the antimicrobials used in animal feeding operation are excreted to animal manures either as parent compounds or as bioactive metabolites (Jacobsen et al., 2004; Kay et al., 2004; Aga et al., 2005). T hese animal manures contain certain levels of pharmaceuticals; after a short period of storage/treatment (3 - 6 months), they are commonly land - applied as fertilizers for crop production. As a result, pharmaceuticals are introduced to agricultural ecosystems (Kolpin et al., 2002; Hamscher et al., 2005; Snow et al., 2008), which can potentially enter crop or vegetable produce. 3 In order to remove the pharmaceuticals from agricultural drainage water, sorption process can be a viable treatment option. In the past , many filter media have been used to remove phosphorus from water, such as recently developed highly porous ceramic media by MetaMateria. These materials are expected a priori to strongly adsorb pharmaceuticals, and hence reduce the contamination in agric ultural surface waters. MetaMateria media are characterized as unique and highly porous ceramic adsorbents that provide exceptional performance and cost - effective removal approach for contaminants in wastewater. These media have been shown to be able to ef fectively remove phosphorus from drainage water. The large surface areas of the porous media could be modified with either loading reactive nanomaterials or hosting beneficial bacterial colonies in order to achieve effective chemical or biological treatmen ts of contaminated water. The hierarchical pore structure helps maintain high water flow rate into or through the media, and the surfaces can be engineered to provide the desired functionality for adsorbing targeted contaminants from wastewater. For exampl e, the surfaces could be modified via coating polymers, anionic or cationic surfactants iron oxide (e.g. FeOOH), manganese oxide (MnO 2 ), zinc oxide (ZnO) and silver (Ag) nanomaterials on the surfaces ( Boujelben a et al., 2008) . OBJECTIVE The objective of this study was to test if the sorbent media (provided by MetaM ateria) could effectively remove selected pharmaceuticals from water. The goal was to identify the appropriate sorbent media that could achieve the high efficiency of pharmaceuticals removal from agricultural drainage water. MATERIALS AND METHODS 4 Lincomyc - Aldrich Chemical Company. The sorbent media were provided by MetaMateria Technologies. The media included hybrid media (hybrid#1, 2, 3, 13A, 14A, 15A, 16A, 17A, 17B, 17C, 17D and 22), granular media from 1" column (without and with hydrophobic coatings), large - surface - area media (HSA - 0), hydrophilic iron - modified media (new and old), and phosphorus - saturated iron - modified media. In addi tion, granular activated carbon (FILTRASORB 300, coal based, 0.8 - 1.0 mm) was obtained from Calgon Carbon Corporation. The selected properties of the three pharmaceuticals are listed in Table 1.1. Table 1. 1 Properties of selected three pharmaceuticals Phar maceuticals Molecular Weight (g mol - 1 ) Chemical Structure Water Solubility (mg L - 1 ) pK a logK ow Lincomycin 406.54 927 7.6 0.2 Sulfamethazine 278.34 1500 2.6, 7.6 0.l4 Tetracycline 444 .43 231 3.3, 8.3, 10.2 - 1.37 From TOXNET database: http://toxnet.nlm.nih.gov/index.html 5 Pharmaceutical sorption by MetaMaterial media was measured using a single - or multi - point batch sorption experiment method. For the multi - point sorption isotherm experiments , 50 .0 mg of sorbent media was weighed to glass centrifuge tube s containing 5 .0 mL of pharmaceutical solution with the initial concentration s of 50, 100, 200, 400, 600, 800, and 1000 µg/L. The glass tubes were wrapped with Al - foil to prevent the potential of photodegradation. Then the centrifuge tubes were shaken on a platform shaker at 20 rpm for 48 hours for lincomycin and sulfamethazine , and 4 hours for tetracycline. The shorter shaking time (4 hours) selected for tetracycline was tested to be sufficient t o approach sorption equilibration. After approaching sorption equilibration, the tubes were centrifuged at 3500 rpm for 20 min. The supernatant pH value was measured with a pH meter. The pharmaceutical concentration in the supernatant was analyzed with a S himadzu Prominence high - performance liquid chromatograph coupled to an Applied Biosystems Sciex 3200 triple quadrupole mass spectrometer (LC - MS/MS). Control experiments free of MataMaterial sorbents were also conducted, and the sorbate loss during experime nt was negligible. S orbed pharmaceutical concentration by sorbent media was calculated as: (1.1) where S is the sorbed pharmaceutical concentration by media ( µg /kg) , C 0 is the initial pharmaceutical concentration in the aqueous phase as measured in the media - free control treatment ( µg/L ) , C is the pharmaceutical concentration in the aqueous phase after equilibration ( µg/L ) , V is the solution volume (mL) , and M is the mass of sorbent media (kg). 6 The sorption isotherms for lincomy cin and sulfamethazine were fitted with the Freundlich model to estimate sorption parameters. The Freundlich sorption i sotherm is mathematically expressed as: (1.2) In this equation, K f and n are fitting constants fo r a given sorbate and sorbent combination at a given temperature (room temperature in this study) . The sorption efficiencies of tetracycline by all tested media approached to 99% at the majority of the initial concentrations. Thus, it is inappropriate to g enerate sorption isotherms for tetracycline. Instead, the remova was reported. In the single - point sorption experiment, pharmaceutical solution s (5.0 mL) of the initial concentration of 500 µg/L w ere prepared , and mixed with a pproximately 50 .0 mg of sorbent media i n the experiments with lincomycin and sulfamethazine, and 10 .0 mg of sorbent media for tetracycline. Before placing the tubes on the platform shaker, hydrochloric acid (HCl) was added to adjust the final pH range of 6 - 7. The experimental protocol was the s ame to that previously described . The pharmaceutical removal percentage by sorbent media was calculated as : (1.3) RESULTS AND DISCUSSION The selected properties of the sorbent media provided by MetaMateria are shown in Table 1. 2 . The surface area of the sorbent could reach 90 m 2 /g after hydrophobic modification. Thes e materials are shown to have reasonably good retention for water and for hydrophobic organic compounds (e.g. toluene). 7 Table 1. 3 shows the removal percentage of pharmaceuticals by MataMetrial media using the one - point sorption experiment. The correspond ing results are also presented in Figure s 1.1 . The removal percentage ranged from 32.1 % to 97.4 % for lincomycin, 13.2 % to 86.4 % for sulfamethazine , and 48.7 % to 98.0 % for tetracycline. Among these media tested, the granul ar media 1 - inch column consistently demonstrated the highest pharmaceutical removal percentage, i.e. 97. 4 % for lincomycin, 86. 4 % for sulfamethazine, and 97. 7 % for tetracycline. This media performed better than other media including those with surface - hydrophobic - modification media (hybrid media) . For activated carbon, the removal rate was 98.2 % for lincomycin, 99.9 % for sulfamethazine, and 96.9 % for tetracycline. The performance of the granul ar - could achieve a removal percentage close to that of activated carbon (Table 1. 3 ). Among the hybrid media, hybrid #22 o verall demonstrated better performance than other hybrid media, with 92.3 % for lincomycin, 60.0 % for sulfamethazine, and 9 5 . 7 % for tetracycline. The pharmaceutical removal rate was found to depend on solution pH for lincomycin and sulfamethazine . G reater removal percentage was achieved in slightly acidic solution than that in alkaline pH (Table 1. 3 and Table 1 .4 ). For the example of Hybrid#3, at pH 6.3 - 6.5 the removal percentage was measur ed at 83.7 % for lincomycin and 59.6 % for sulfamethazine, but 38.8 % for lincomycin and 17.4 % for sulfamethazine at pH 10.1 - 10.5. However, this pH effect was not apparent for tetracycline since a ll tested sorbent media manifested a very high removal perc entage (> 95.1 % for all tested media expect HAS - 0) . The strong sorption of tetracycline could be due to the fact that Al or Fe hydrous oxides are the major components of these media. Formation of tetracycline - Fe/Al complexes could substantially enhance so rption of tetracycline by the media from water. 8 Table 1. 2 Selected properties of sorbent media Sorbents Surface area (m 2 /g), as prepared Surface area (m 2 /g), after modification Surface area (m 2 /g), after hydrophobic coating Water retention Toluene retentio n Hybrid#1 20.5 77.1 91.2 123% 108% Hybrid#2 19.8 80.4 97.9 86% 98% Hybrid#3 18.1 68.1 66.8 94% 85% Hybrid#13A 23.2 67.5 72.5 115% 97% Hybrid#14A 24.3 57.4 65.3 111% 97% Hybrid#15A 39.4 70.2 89.6 140% 98% Hybrid#16A 26.2 44.8 74.8 108% 105% Hybrid# 17A 37.6 92.6 72.5 Hybrid#17B 36.5 91.2 65.3 Hybrid#17C 18.4 60.1 89.6 Hybrid#17D 21 69.9 74.8 Hybrid#22 150 - - Granules from 1" column - without hydrophobic coating - - - Granules from 1" column - with hydrophobic coating - - - High Surface Area - 0 (HSA - 0) - - - Iron - modified media (new) - - - Iron - modified media (old) - - - P - saturated Iron - modified media - - - 9 Table 1.3 Pharmaceutical removal percentage by selected sorbent media Sample Name Lincomycin Sulfamethazine Tetracycline Removal percentage (%) Final pH Removal percentage (%) Final pH Removal percentage (%) Final pH Hybrid#3 83.7 6.3 59.6 6.4 - 6.5 97.9 5.9 Iron - modified media (new) 79.9 6.0 67.5 6.1 - 6.2 95.6 5.4 - 5.5 P - saturated Iron - modified media (old) 32 .1 6.1 - 6.2 22.0 6.2 - 6.3 97.2 5.7 - 5.8 Hybrid#17A 56.7 6.1 - 6.2 43.2 6.0 - 6.2 95.1 6.1 Hybrid#17B 64.1 6.3 - 6.4 44.8 6.3 - 6.4 96.6 6.0 - 6.1 Hybrid#17C 64.0 6.4 - 6.5 41.4 6.5 - 6.6 98.0 5.9 Hybrid#17D 68.7 6.3 44.3 6.3 - 6.4 97.8 5.8 - 5.9 Hybrid#22 92.3 5.7 - 5.8 60. 0 5.9 - 6.2 95.7 5.5 - 5.6 Granules from 1" column - without hydrophobic coating 97.4 5.9 86.4 5.9 97.7 5.6 - 5.7 Granules from 1" column - with hydrophobic coating 92.3 5.9 - 6.0 53.1 5.9 - 6.0 94.4 5.5 - 5.7 HAS - 0 32.2 9.6 13.2 9.9 - 10.0 48.7 3.0 - 3.2 Activate Car bon 98.2 7.3 - 7.5 99.9 7.0 - 7.1 96.9 6.6 - 6.7 10 Figure 1.1 Pharmaceutical removal percentage by selected sorbent media 11 µg/L). Sorbents Lincomycin Sulfamethazine Tetracycline Final pH Removal percentage (%) Final pH Removal percentage (%) Final pH Removal percentage (%) Hybrid#1 9.7 - 9.9 31.6 9.6 - 10.0 12.8 9.0 - 9.5 99.4 Hybrid#2 9.0 - 9.9 31.3 9.8 - 10.0 9.3 8.6 - 9.1 99.0 Hybrid#3 10.1 - 10.5 38.8 10.2 - 10.5 17.4 9.0 - 9.7 97.7 Hybrid#13 A 10.1 - 10.5 29.0 10.1 - 10.5 12.7 8.8 - 9.8 99.5 Hybrid#14 A 8.7 - 10.0 28.2 9.6 - 10.0 25.9 8.5 - 9.0 99.8 Hybrid#1 5 A 9.2 - 9.7 35.2 9.4 - 10.0 13.9 8.0 - 8.5 99.8 Hybrid#16 A 8.8 - 9.0 35.1 8.8 - 9.4 11.5 7.8 - 8.0 99.9 Iron - modified media (new) 8.1 - 9.4 38.7 8.4 - 8.9 35.7 8.7 - 9.6 99.9 Iron - modified media (old) 7.2 - 8.3 25.5 7.3 - 8.3 28.5 7.2 - 8.2 99.8 P - saturated Iron - modified med ia (old) 5.9 - 6.4 25.8 6.2 - 6.5 18.4 5.8 - 6.4 99.8 12 Table 1.5 Fitting Freundlich isotherm parameters for sorption of lincomycin and sulfamethazine by sorbent media. a Unit of K f is µg/kg (µg/L) . S orption isotherms were measured for lincomycin and sulfamethazine by MetaMaterial media in relatively alkaline solution (Figures 1.2 to 1.21). The Freundlich fitting results are reported in Table 1.5. For lincomycin the sorption isotherms appeared relatively nonlinear and linear, with n values range from 0.66 to 1.16 (Figures 1.2 to 1.11). At the aqueous concentration at 400 g/L, the estimated sorption concentration (using Sorbents Lincomycin Final pH K f a n R 2 Hybrid#1 9.7 - 9.9 214 0.76 0.988 Hybrid#2 9.0 - 9.9 155 0.80 0.995 Hybrid#3 10.1 - 10.5 225 0.80 0.990 Hybrid#13A 10.1 - 10.5 34.8 1.01 0.981 Hybrid#14A 8.7 - 10.0 23.7 1.08 0.982 Hybrid#15A 9.2 - 9.7 34.8 1.08 0.990 Hybrid#16A 8.8 - 9.0 14.5 1.16 0.753 Iron - modified media (new) 8.1 - 9.4 264 0.77 0.985 Iron - mo dified media (old) 7.2 - 8.3 200 0.71 0.984 P - saturated Iron - modified media (old) 5.9 - 6.4 329 0.66 0.951 Sulfamethazine Final pH K f a n R 2 Hybrid#1 9.6 - 10.0 65.4 0.78 0.845 Hybrid#2 9.8 - 10.0 27.9 0.86 0.842 Hybrid#3 10.2 - 10.5 84.1 0.79 0.927 Hybrid# 13A 10.1 - 10.5 254 0.49 0.738 Hybrid#14A 9.6 - 10.0 50.9 0.87 0.885 Hybrid#15A 9.4 - 10.0 143 0.55 0.852 Hybrid#16A 8.8 - 9.4 174 0.53 0.614 Iron - modified media (new) 8.4 - 8.9 103 0.90 0.960 Iron - modified media (old) 7.3 - 8.3 117 0.79 0.871 P - saturated Iron - m odified media (old) 6.2 - 6.5 50.1 0.88 0.936 13 Freun dlich fitting results) was 20322 .6 g/kg for Hybrid#1, 18705.9 g/kg for Hybrid#2, 27153.8 g/kg for Hybrid#3 , 14779.5 g/kg for Hybrid# 1 3 A, 15309.9 g/kg for Hybrid# 14A, 22480.4 g/kg for Hybrid# 15A, 15127.2 g/kg for Hybrid# 16A, 26618.9 g/kg for i ron - mo dified media (new) , 14076.4 g/kg for i ron - modified media (old) and 17161.5 g/kg for P - saturated i ron - modified media (old) . For sulfamethazine, the isotherms demonstrated essentially nonlinear sorption with n values from 0.49 to 0.9 (Figures 1.12 to 1.21) . At 400 g/L , the estimated sorption was 7001.4 g/kg for Hybrid#1, 4823.7 g/kg for Hybrid#2, 9559.2 g/kg for Hybrid#3 , 4784.6 g/kg for Hybrid# 1 3 A, 9343.5 g/kg for Hybrid# 14A, 3858.9 g/kg for Hybrid# 15A, 4165.2 g/kg for Hybrid# 16A, 22630.3 g/kg for i ron - modified media (new) , 13298.8 g/kg for i ron - modified media (old) and 9764.5 g/kg for P - saturated i ron - modified media (old) . Overall the sorption capacity for lincomycin and sulfamethazine was modest , which could be utilized for pharmaceutical remov al in agricultural drainage system, but may not be the best sorbent used for this purpose. Since the MetaMaterial media have been successfully applied for phosphorus removal from agricultural drainage water, we compared sorption of pharmaceuticals by P - sat urated i ron - modified media vs. iron - modified media (Figure 1 .1 0 vs. Figure 1.11 for lincomycin, and Figure 1.20 vs. 1.21 for sulfamethazine). The results reveal that P - saturated i ron - modified media did not substantially reduce sorption by the media for lin comycin and sulfamethazine . These results indicate that P - adsorption on this medium did not affect s orption of the tested pharmaceuticals, which could be due to the fact that phosphorus and lincomycin/sulfamethazine have different sorption sites on the med ium surfaces. 14 Figure 1.2 Sorption isotherm of lincomycin by hybrid media #1 Figure 1.3 Sorption isotherm of lincomycin by hybrid media #2 15 Figure 1.4 Sorption isotherm of linco mycin by hybrid media #3 Figure 1.5 Sorption isotherm of lincomycin by hybrid media #13A 16 Figure 1.6 Sorption isotherm o f lincomycin by hybrid media #14 A Figure 1. 7 Sorption is otherm of lincomycin by hybrid media #15A 17 Figure 1. 8 Sorption isotherm of lincomycin by hybrid media #16A Figure 1. 9 Sorption isotherm of lincomycin by iron - modified media (new) 18 Figure 1.1 0 Sorption isotherm of lincomycin by iron - modified media (old) Figure 1.1 1 Sorption isotherm of lincomycin by phosphorus - saturated iron - modified media (old) 19 Figure 1.1 2 Sorption isotherm of sulfamethazine by hybrid media #1 Figure 1.1 3 Sorption isotherm of sulfamethazine by hybrid media #2 20 Figure 1.1 4 Sorption isotherm of sulfamethazine by hybrid media #3 Figure 1. 15 Sorption isotherm of sulfamethazine by hybrid media #13A 21 Figure 1. 16 Sorption isotherm of sulfamethazine by hybrid media #14A Figure 1. 17 Sorption isotherm of sulfamethazine by hybrid media #15A 22 Figure 1. 18 Sorption isotherm of sulfamethazine by hybrid media #16A Figure 1. 19 Sorption isotherm of sulfamethazine by iron - modified media (new) 23 Figure 1.2 0 Sorption isotherm of sulfamethazine by iron - modified media (old) Figure 1.2 1 Sorption isotherm of sulfamethazine by phosphorus - saturated iron - modified media (old) 24 CONCLUSIONS Overall, the MetaMaterial media demonstrated relatively effective sorpti on for the select ed pharmaceuticals from water. Tetracycline manifested the greatest sorption, followed by lincomycin and then sulfamethazine. G ranul ar medi um from t he 1 - inch column manifested the greatest removal efficiency for the selected pharmaceutical s, and the sorption was comparable to that by activated carbon. This medium could potentially employed to removal pharmaceuticals from agricultural drainage water, along with the major function of phosphorus removal . Among the modified hybrid media, hybrid #22 overall demonstrated a higher sorption for the selected pharmaceuticals . S orption of the selected pharmaceuticals in neutral or slight acid ic solution (pH from 6 to 7) was greater than that in alkaline solution. T he surface area s of the MetaMaterial m edia fell within the same order of magnitude ; modification o n the surface properties could plausibly improve sorption of pharmaceuticals from water. 25 REFERENCES 26 REFERENCES 1. Pavelis G.A. 1987. Economic survey of farm drainage. I n Farm drainage in the United States: History, status and prospects, ed. Pavelis G.A., p 110 - 136. USDA Economic Research Service Miscellaneous Publication 1455. Washington, DC: US Government Printing Office. 2. Frankenberger, J., E. Kladivko, G. Sands, D. Ja ynes, N. Fausey, M. Helmers, R. Cooke, J. Strock, K. Nelson, and L. Brown. 2006. Drainage water management for the Midwest: Questions and answers about drainage water management for the Midwest. Purdue Ext., p. 8. 3. FDA. FDA Annual Summary Report on Antimic robials Sold or Distributed in 2012 for Use in Food - Producing Animals . Rep. FDA, Center for Veterinary Medicine, Oct. 2014. Web. 4. Wenlu Song, Yunjie Ding, Cary T.Chiou, Hui Li. 2010. Selected Veterinary Pharmaceuticals in Agricultural Water and Soil from L and Application of Animal Manure. Environ Qual. 2010 Jul - Aug; 39(4):1211 - 7. 5. Jacobsen, A.M., B. Halling - Sorensen, F. Ingerslev, and S.H. Hansen. 2004. Simult aneous extraction of tetracycline, macrolide and sulfonamide antibiotics from agricultural soils using pressurized liquid extraction, followed by soil - phase extraction and liquid chromatography - tandem mass spectrometry . J. Chromatogr. A 1038:157 170. 6. Kay P, Blackwell PA, Boxall ABA. Fate of veterinary antibiotics in a macroporous tile drained clay soil . Environ. Toxicol. Chem. 2004; 23:1136 1144. 7. 2005. Determination of the persis tence of tetracycline antibiotics and their degradates in manure - amended soil using enzyme - linked immunosorbent assay and liquid chromatography - mass spectrometry . J. Agric. Food Chem. 53:7165 7171. 8. Kolpin, D.W., E.T. Furlong, M.T. Meyer, E.M. Thurman, S.D . Zaugg, L.B. Barber, and H.T. Buxton. 2002. Pharmaceuticals, hormones, and other organic wastewater contaminants in U.S. streams, 1999 2000 : A national reconnaissance. Environ. Sci. Technol. 36:1202 1211. 9. Hamscher, G., H.T. Pawelzick, H. Hoper, and H. Na u. 2005. Different behavior of tetracyclines and sulfonamides in sandy soils after repeated fertilization with liquid manure. Environ. Toxicol. Chem. 24:861 868. 27 10. Snow, D.D., S.L. Bartelt - Hunt, S.E. Saunders, S.L. Devivo, and D.A. Cassada. 2008. Detection, occurrence and fate of emerging contaminants in agricultural environments. Water Environ. Res. 10:868 897. 11. Boujelben a , N., Bouzid a , J., Elouear a , Z., Feki b , M., Jamoussi c , F., Montiel d , A. 2008. Phosphorus removal from aqueous solution using iron coated natural and engineered sorbents . Journal of Hazardous Materials , 03/2008; 151(1):103 - 10. 12. Chuang, YH., Zhang, Y., Zhang W., Boyd, S.A., Li, H. 2015. Comparison of accelerated solvent extraction and quick, easy, cheap, effective, rugged and safe method for extraction and determination of pharmaceuticals in vegetables. Journal of Chromatography A , 1404(2015), p. 1 13. Gu, C., and K.G. Karthikeyan. 2 005. Interaction of tetracycline with aluminum and iron hydrous oxides. Environ. Sci. Technol. 39:2660 2667. 14. Lertpaitoonpan, W, Ong, S K, Moorman, T B. 2009. Effect of organic carbon and pH on soil sorption of sulfamethazine. Chemosphere 76 (2009) 558 564 . 15. Wang, C., B.J. Teppen, S.A. Boyd, C. Song, and H. Li. 2011. Sorption of Lincomycin at Low Concentrations from Water by Soils. Soil Sci. Soc. Am. J. 76:1222 1228 16. Sun, H., Shi, X., Mao, J., Zhu, D. 2010. Tetracycline sorption to coal and soil humic a cids: an examination of humic structural heterogeneity. Environmental Toxicology and Chemistry, Vol. 29, No. 9, pp. 1934 1942, 2010. 28 CHAPTER II EXPRESSION OF ANTIBIOTIC RESISTANCE OF E.COLI EXPOSED TO TETRACYCLINES 29 INTRODUCTION A ntibiotics have been widely used in animal production to treat disease s and to improve feed efficiency (Allen, 2014; Durso , et al., 201 4 ). They have been used extensively in livestock industries since the 1950s (Barton, 2014; Tilman , et al., 2002) . The lo ng - term utilization of antimicrobial drugs in animal production and microbial exposure to low - dosed antibiotic s in the environment contribute to the development of antimicrobial resistance in microorganisms, and recently draw substantial attention to this global healthy issue ( Khachatourians , 1998; Jensen, et al., 2014; Smalla, et al., 2014) . Tetracyclines are a class of a broad - spectrum polyketide antibiotic s, and are among the most commonly used anti biotics in animal feeding operations and disease control. In 201 2, approximately 5.9 million kilograms of tetracyclines were used for both food - producing animals (e.g., cattle and swine) and nonfood - producing animals (e.g., dogs and cats) in United States, which accounted for 40.7% of the total antibiotics use for animals (FDA, 2012) . Tetracycline, chlortetracycline and oxytetracycline are the main active members in the tetracycline family. Since l arge amount o f tetracyclines are used in agricultural livestock production, tetracyclines have been frequently detected in soil s, surface waters, and even in groundwater (Hu et al., 2010; Jacobsen et al., 2004; Batt and Aga, 2005; Christian et al., 2003; Kolpin et al., 2002; Luo et al., 2011; Wei et al., 2011 Chee - Sanford et al., 2001; Gottschall et al. , 2012). The pres ence of ant ibiotics such as tetracylines in the environment has been related to the emergence and ever - increasing abundance of antibiotic resistance genes (ARGs) in natural microbial populations. In several recent studies, tetracycline ARGs were more frequently detec ted in the sites where tetracyclines were present, compared to the sites 30 free of tetracyclines ( Pei , et al., 2006; Storteboom , et al., 2010; Knapp et al., 2010) . Thus, the concurrent presen ce of tetracyclines in the environment and the increasing abundance of tetracycline ARGs suggests that tetracyclines might pose selective pressure on the exposed microbial communities for the development and proliferation of antibiotic resistance . In order for tetracyclines to exert selective pressure and other biologica l effects on bacteria, they should first become available to bacterial for uptake into the cells. Previous studies have demonstrated that zwitterionic tetracycline is the most effective species to enter E. coli from aqueous solution, resulting in the expre ssion of antibiotic resistance genes ( Zhang , et al., 2014). However, in the environment it is more common that multiple types of tetracyclines are present at the contaminated sites. However, little is known about how these tetracyclines collectively contri bute to the uptake and resistance gene expression of tetracyclines by the bacteria. In this study, we aimed to investigate the expression of antibiotic resistance genes of E. coli exposed to different combinations of tetracyclines. The goal was to examine any potential synergistic or antagonistic effects among the combinations of common tetracyclines on the E. coli for expression of antibiotic resistance. An E . coli bioreporter was used for quantifying ARGs responses exposed to tetracycline. The bioreporter was E. coli strain MC4100 containing the plasmid pTGM with a transcriptional fusion between tetracycline inducible promoter and fluorescence - assisted cell sorting optimized gfp gene. Tetracycline uptake by the bioreporter ( determined as intracellular tetr acycline concentration) was quantified by using a high - performance liquid chromatography integrated with a tandem mass spectromet er (LC - MS/MS) . The degree of expression of antibiotic resistance gene s was 31 quantified through promoter activity by integrating bacterial growth rate and fluorescence emitted from the E. coli bioreporter at a steady state . MATERIALS AND METHODS T , c hlortetracycline hydrochloride 97 %) , oxytetracycline hydrochloride , anhydrotetracycline hydrochloride 98 %) , methanol, and 3 - (N - morpholino) propanesulfonic acid (MOPS, buffer re obtained from Sigma - Aldrich Chemical Company. The selected properties of the four tetracycline s are listed in Table 2.1. Sodium chloride, potassium chloride, ethylenediaminetetraacetic acid (EDTA), citric acid, formic acid, sodium phosphate dibasic, and potassium phosphate monobasic were obtained from J.T. Baker . Bacto tryptone and Bacto yeast extracts were obtained fro m Becton, Dickinson and Company. Acetonitrile (HPLC grade) and hydrochloric acid were obtained from EMD Chemicals . The E. coli strain MC 4100/pTGM used as bacterial bioreporter was provided by Dr. S. J. Sørensen at the University of Copenhagen. The E. coli strain was constructed by inserting tet (M) gene (encoding tetracycline resistance by ribosomal protection) into plasmid pTGM, which con tained a transcriptional fusion between a tet R - regulated promoter and flow cytometry - optimized gfp gene ( gfp mut3) encoding green fluorescence protein (GFP) . As tetracycline s enter the E. coli bioreporter cell, they deactivate the tet R repressor protein in the P tet promoter and activate gfp gene transcription. The pTGM construct contains tetracycline resistance gene tet (M) that inhibits tetracyclines from killing the cells, and maintains the intracellular tetracycline concentration. Meanwhile, the GFP trans lated from the expression of gfp gene emits the fluorescence signal with the 32 intensity proportional to the P tet activity that drives antibiotic resistance gene expression in natural settings. Table 2.1 Properties of selected four tetracyclines Pharmaceuti cals Molecular Weight (g mol - 1 ) Chemical Structure Water Solubility (mg L - 1 ) pK a logK ow Tetracycline 444.43 231 3.3, 8.3, 10.2 - 1.37 Chlortetracycline 478.88 630 3.3,7.4,9.3 - 0.62 Oxytetracycline 460.43 313 3.6, 7.5, 9.4 - 0.9 Anhydrotetracycli ne 426.42 2299 a 3.2, 6.0, 8.5 b 0.63 a From TOXNET database: http://toxnet.nlm.nih.gov/index.html a from database: http://www.ch emspider.com/Chemical - Structure.20117965.html b The E. coli bioreporter was cultured in a low - salt lysogeny broth (LB) medium, and pH was adjusted to 7.0 using 50 mM of MOPS buffer. The LB medium was autoclaved at 121 °C for 30 min; E. coli bioreporter was inoculated and cultivated in 25.0 mL of LB medium amended with 100 mg L ampicillin. The culture was incubated on a horizontally moving shaker at 150 rpm and at 30 . When the bacterial culture grew to 33 the mid - log phase as indicated by optical density at 600 nm (OD 60 0 ) approaching 0.7, 0.5 mL of the culture was diluted 100 fold in 50.0 mL of freshly prepared LB media amended with 100 mg L - 1 of ampicillin. The LB media were prepared to contain single tetracycline, chlortetracycline, oxytetracycline and anhydrotetracyc line at the concentration of 0, 50, 100, 200, 300 and 400 nmol L - 1 , binary tetracyclines (tetracycline + chlortetracycline, and tetracycline + oxytetracycline) with the concentration of 0, 50, 100, 150, 200 nmol L - 1 for each tetracycline, and trinary tetra cyclines (tetracycline + chlortetracycline + oxytetracycline) with the concentration at 0, 66.7, 100, 133.3 nmol L - 1 for each. All culture samples were prepared in triplicate. One milliliter of the culture sample was collected every 30 min for each treatme nt, and measured the emitted fluorescence ( gfp mut3 excitation wavelength = 488 nm, emission wavelength = 511 nm) using a SpectraMax M2 spectrofluorometer. The expression of antibiotic resistance is quantified as the promoter activity of tet (M) in the E. coli bioreporter at a steady state according to the model developed by Leveau and Lindow (2001). (2.1) where P is promoter activity (relative unit of immature GFP per OD unit per hour, RU OD h ), f ss represents the fluorescence at the steady state during bacterial growth (relative unit of fluorescent matu re GFP per OD unit, RU OD ). µ (h ) is bacterial growth rate, and m (1.54 h for gfp mut3) is the maturation constant for GFP to develop into fluorescent GFP. The f ss value is obtained from a plot of fluorescence against OD 600 . from the slope of linear plot of natural logarithm of OD 600 values against time (h). 34 (2.2) where t is culture time (h), and OD 600 and OD 600,0 are the measured optical density at 600 nm at time t and t = 0 , respectively. Integration of fluorescence at the steady state and the growth rate to obtain the promoter activity can circumvent the effects of dilution of GFP contents and GFP maturation during the growth of bacteria, which allows comparisons of antibio tic resistance responses among different experimental settings. For the tetracycline - free controls, a small intensity of fluorescence could be measured with the averaged promoter activity of 181 RU OD h . These promoter activity values were relative constant with standard deviation 27.4 RU OD h (n = 10). Therefore, the reported promoter activity values in this study were corrected by subtracting the promoter activity of tetracycline - free control as background. When the OD 600 value of the E. coli bioreporter cultures approached 0.5, the bacteria were separated from the culture media by centrifugation at 15000 g and 4 °C for 15 min. After the centrifugation, the bacterial cell pellets were rinsed twice with 20 mL of 10 - time diluted phosphate - buffered saline solution (PBS solution pH 7.4). Then 10 mL of McIlvain buffer (12.9 g of citric acid monohydrate, 10.9 g of Na 2 HPO 4 and 37.2 g of EDTA dissolved in 1 L of water) was used to suspend the cell pel lets and remove tetracycline from the cells. Then mixture was vortexed for 1 min, sonicated fo r 15 min, (Waters Corporation, Milford, MA) was used in solid phase extraction to extract tetracycline from aqueous phase. The cell extract containing tetracycline (20 mL ) was passed through the preconditioned HLB cartridge. The cartridges were then washed with 1:9 (v/v) methanol/water solution (5 mL). Tetracycline retained on the HLB cartridges 35 was eluted with 1:1 (v/v) methanol/water solution (5.0 mL) containing 150 mg L of EDTA, then with additional 5.0 mL of methanol containing 1% (v/v) formic acid. The tetracycline concentration was analyzed with a Shimadzu Prominence high - performance liquid chromatograph coupled to an Applied Biosystems Sciex 4500 triple quadrupole mass spectrometer (LC - MS/MS). To obtain tetracyclines concentration in bacteria, the measured amount of tetracycline was normalized on the basis of dry bacterial biomass to - 1 ). The dry bacterial biomasses were estima ted using OD 600 values. The relationship between bacterial biomass and optical density was reported by Zhang et al. (2014). The weights of bacterial biomass in culture suspensions obtained by freeze drying were plotted against the corresponding OD 600 value s. The linear relationship (Figure 2.1) was utilized to estimate the bacterial biomass present in culture suspension from the measured OD 600 values. 36 Figure 2.1 Relationship between bacterial biomass in culture suspension and optical density measured at 60 0 nm. (Figure from Zhang et al. 2014, Supporting Information Figure S2) RESULTS AND DISCUSSION Tetracycline can enter the E. coli bioreporter and evoke the expression of antibiotic resistance genes. With increasing initial tetracycline concentrations, th e measured intracellular tetracycline concentrations increased, along with the increasing intensity of fluorescence as indicated by the promoter activity values. For example, when the E. coli bioreporter exposed to aqueous tetracycline concentration at 50, 100, 200, 300 and 400 nmol L - 1 , the intracellular tetracycline concentration was 24.8 ± 1.8, 56.3 ± 2.2, 96.8 37 ±1.2, 151.8 ± 29.1 and 206.2 ± 9.5 nmol g - 1 , respectively. Correspondingly, the estimated promoter activity values increased proportionally yield ing 164.6 ± 5.9, 531.7 ± 23.6, 1683.5 ±54.5, 2047.1 ± 259.8 and 2458.3 ± 110.4 RU OD h , respectively. Similar results were also observed for the bioreporter exposed to oxytetracycline and chlortetracycline, but with varying levels of intracellular uptake and expression of antibiotic resistance genes. For tetracycline, oxytetracycline and chlortetracycline, the promoter activity expressed in the E. coli bioreporter generally manifested a positive linear relation with intracellular tetracycline concentration (Figures 2.2, 2.3 and 2.4). These results suggest that the amount of tetracycli nes entering the E. coli bioreporter is the determinant for the evoked antibiotic resistance. A previous study indicates that tetracycline zwitterion species in solution is responsible for the uptake by the E. coli ; and the same aqueous tetracycline concen tration may not result in the similar selective pressure on the expression of antibiotic resistance depending on the fractional tetracycline zwitterion (Zhang et al., 2014). Therefore, the intracellular concentration is the appropriate parameter to be used for predicting the expression of antibiotic resistance genes. Different types of tetracyclines could vary their uptake by the E. coli bioreporter. In general, under the exposure of the same initial concentration, the uptake by the E. coli bioreporter foll owed the order of oxytetracycline > chlortetracycline > tetracycline. When exposed to 300 nmol L - 1 of tetracyclines, the measured intracellular concentrations were 288.3 ± 36.5 nmol g - 1 for oxytetracycline, 214.4 ± 14.0 nmol g - 1 for chlortetracycline, and 151.8 ± 29.1 nmol g - 1 for tetracycline. 38 Figure 2.2 Relationship between intracellular tetracycline concentration and the expressed promoter activity of the E. coli bioreporter. In Figures 2.2, 2.3 and 2.4, the slope of the linear fitting represents the increase of promoter activity on the increase of per unit of intracellular concentration. The slope value was 12.66 for tetracycline, 8.90 for chlortetracycline, and 6.33 for oxytetracycline, respectively. This result indicates that under per unit of increasing antibiotic intracellular concentration, tetracycline could exert higher selective pressure on the E. coli bioreporter to develop antibiotic resistance, followed by chlortetracycline and oxytetracycline. This 39 result implies tha t at the similar intracellular concentration, bacteria exposed to tetracycline plausibly develop more intensity of antibiotic resistance compared to that exposed to chlortetracycline or oxytetracycline. Figure 2.3 Relationship bet ween intracellular chlortetracycline concentration and the expressed promoter activity of the E. coli bioreporter. 40 Figure 2.4 Relationship between intracellular oxytetracycline concentration and the expressed promoter activity o f the E. coli bioreporter. In contrast to tetracycline, oxytetracycline and chlortetracycline, the increasing uptake of anhydrotetracycline did not demonstrate enhanced promoter activity (Figure 2.5). The evoked promoter activity approached a relative hig h level even at a low intracellular concentration. At the intracellular anhydrotetracycline concentration of 100 nmol g - 1 , the promoter activity was estimated at 2100 RU OD h . At the similar intracellular concentration the promoter activity values were 1200 RU OD h for tetracycline, 800 41 RU OD h for chlortetracycline, and 500 RU OD h for oxytetracycline. In Figure 2.6, the promoter activity of anhydrotetracycl ine reached the highest level even at the lowest initial concentration (50 nmol L - 1 ). Comparing with tetracycline, anhydrotetracycline can more efficiently evoke tetracycline resistance gene. Anhydrotetracycline is a degradation product of tetracycline for med by photolysis under acidic condition (Halling - Sorensen et al., 2002; Hasan et al., 1985; Oka et al., 1989) . T he formed degradation product anhydrotetracycline has been detected in animal m anure composting (Wu et al., 2011) and manure - amended soil (Aga et al., 2005) . Although anhydrotetracycline demonstrates reduced antibacterial activity compared to tetracycline, it can bind to tetracycline repressor protein ( TetR ) in bacteria with 500 - fold higher affinity than tetracycline (Scholz et al., 2000) , which could enhance the activation of resistance genes associated with TetR . In our study, the E. coli bioreporter consists a TetR - regulated tetracycline promoter which is more sensitive to anhydrotetracycline than tetracycline. As a result, the bioreporter manifested very strong promoter activit y when exposed to anhyrotetracycline. 42 Figure 2.5 Anhydro tetracycline uptake by E.coli bioreporter and calculated promoter activity. 43 Figure 2.6 Comparison between tetracycline and anhydrotetracycline uptake by E.coli bioreporter associated promoter activity. To examine the biological effects of tetracycline mixtures, binary tetracycline and trinary tetracycline systems were prepared to measure the invoked promoter activity of the E. coli bioreporter. In the binary experiments, molar equivalent initial concentrations of 0, 50, 100, 200 nmol L - 1 for each tetracycline (tetracycline + chlortetracycline; tetracycline + oxytetracycline) and the total initial tetracyclines concentrations were prepared at 0, 100, 200, 300 and 400 nmol L - 1 . The intracellular concentration for each type of tetra cycline was quantified using LC - MS/MS. The promoter activity was estimated using equation (2.1). 44 The predicted promoter activity for each tetracycline was estimated using the measured intracellular concentration and the linear fitting equations shown in Fi gures 2.2, 2.3 and 2.4. The overall predicted promoter activity in binary and trinary systems was obtained by summing up the individually estimated promoter activity. For example, in the binary tetracycline + chlortetracycline system, at the total initial concentration of 300 nmol L - 1 (150 nmol L - 1 for each), the measured intracellular concentration was 82.4 nmol g - 1 for tetracycline and 73.6 nmol g - 1 for chlortetracycline. The estimated promoter activity was 1043.7 RU OD h from tetracycline (tetracycline: promoter activity = 12.66 × intracellular concentration, Figure 2.2) and 655.2 RU OD h from chlortetracycline (chlortetracycline: promoter activity = 8.90 × intracellular concentration, Figure 2.3). The sum of 16 98.9 RU OD h was the predicted promoter activity assuming that these two tetracyclines function individually and the resultant biological effects are additive in the E.coli cells. Table 2.2 summarizes the measured and predicted promoter activity values in - value which - test method. The results shows statistically significant difference with the significant levels of 0 .05 or 0.01 (Table 2.2). At P < 0.05, the measured and predicted promoter activities demonstrated no statistically significant difference for the higher total initial concentrations (300 and 400 nmol L - 1 ), but manifested significant difference for the syst ems containing lower levels of the total initial concentration (100 and 200 nmol L - 1 ). When selecting P < 0.01, the tetracycline + chlortetracycline system was considered as no significant difference at the total initial concentration of 200, 300 and 400 n mol L - 1 . 45 Table 2.2 Measured Promoter Activity and Predicted Promoter Activity in the Binary Tetracycline + Chlortetracycline System Tetracycline + Chlortetracycline Initial Total Tetracyclines Concentration (nmol L - 1 ) Measured Promoter Activity (RU OD - 1 h - 1 ) Predicted Promoter Activity (RU OD - 1 h - 1 ) P (T t) Statistically Significant Difference (P < 0.05) Statistically Significant Difference (P < 0.01) 100 480.6 379.0 0.002 Yes Yes 487.3 400.7 508.3 357.2 200 1227.2 42 4.0 0.022 Yes No 1140.0 584.1 1199.2 730.5 300 1473.9 1698.9 0.359 No No 1506.3 1396.3 1489.6 2134.0 400 1577.5 1781.0 0.090 No No 1590.8 2395.2 1519.2 2301.7 Table 2.3 summarizes the analytical results of the binary tetracycline + oxytetracycline system. Among the four datasets of the total initial concentration, only the system with 200 nmol L - 1 displayed statistically significant difference. At the other three initial concentration levels, all p - values are > 0.05 indicating that there is no significant difference between the measured promoter activity values and the predicted values. The tetracyclines in the bacterial cells function individually and the antibiotic response effects are additive. 46 Table 2.3 Me asured Promoter Activity and Predicted Promoter Activity in Binary Tetracycline + Oxytetracycline System Tetracycline + Oxy tetracycline Initial Total Tetracyclines Concentration (nmol L - 1 ) Measured Promoter Activity (RU OD - 1 h - 1 ) Predicted Promoter Activ ity (RU OD - 1 h - 1 ) P(T t) Statistically Significant Difference (P<0.05) Statistically Significant Difference (P<0.01) 100 319.9 305.6 0.385 No No 288.7 795.4 305.9 338.2 200 1157.9 576.7 0.001 Yes Yes 1142.8 461.6 10 78.4 410.9 300 1924.2 1537.4 0.574 No No 1929.5 2047.4 1750.3 1716.0 400 2216.0 1925.6 0.803 No No 2141.2 2744.3 2301.6 2197.4 In the trinary system (tetracycline + chlortetracycline + oxytetracycline), the initial concentrat ion was 0, 66.7, 100, 133.3 nmol L - 1 for each type of tetracycline, and the total concentration was 0, 200, 300, 400 nmol L - 1 . The predicted promoter activity was also estimated by the summation of individual promoter activity contributed from tetracycline , chlortetracycline and oxytetracycline (Figures 2.2, 2.3 and 2.4). The statistical analysis results indicate no significant difference between the measured and predicted promoter activity in the trinary systems. The three tetracyclines presented in the sy stem functioned individually to contribute to the evoked antibiotic resistance. 47 Table 2.4 Measured Promoter Activity and Predicted Promoter Activity in Trinary Tetracycline + Chlortetracycline + Oxytetracycline System Tetracycline + Chlortetracycline + Oxy tetracycline Initial Total Tetracyclines Concentration (nmol L - 1 ) Measured Promoter Activity (RU OD - 1 h - 1 ) Predicted Promoter Activity (RU OD - 1 h - 1 ) P(T<=t) Statistically Significant Difference (P<0.05) Statistically Significant Difference (P<0.01) 200 1122.3 950.7 0.068 No No 1231.4 1005.0 1046.9 755.9 300 1604.6 1288.4 0.137 No No 1662.3 1577.3 1597.8 1331.1 400 1738.1 2090.6 0.755 No No 1814.2 1436.2 1781.1 1594.0 Taken together, we plotted all data from binary and trinary systems with the measured promoter activity as x - axis and the predicted values as y - axis. The linear fitting obtain the slope of 0.99 (r 2 = 0.90) indicting the ratio of predicted to measured promo tor activity is nearly 1:1. In the prediction, we assume that when tetracyclines enter the E. coli bioreporter, they function individually with the antibiotic resistance genes, and the resultant effects could be additive. The statistical analysis and compa rison of measured vs. predicted promoter activity suggest that the combined effects of tetracyclines to E. coli could be considered as additive effects; neither major synergistic nor antagonistic effects is applicable to the tetracyclines in evaluating the ir combined impact to the expression of antibiotic resistance genes in the E.coli bioreporter. 48 Figure 2.7 Measured Promoter Activity vs Predicted Promoter Activity in binary and trinary systems and linear fitting. CONCLUSION I n this study , different tetracycline species show different capacity to expression of antibiotic resistant gene for E.coli bioreporter. A linear relationship is showed between promoter activity and intracellular tetracycline concentration for tetracycline, chlortetracycline and oxytetracycline. The antibiotic resistance response is in the order of tetracycline > chlortetracycline > oxytetracycline. Bacterial uptake amount is in the order 49 of oxytetracycline > chlortetracycline > tetracycline. Anhydrotetracyc line has the highest antibiotic resistance response although it did not show a linear relationship between promoter activity and intracellular concentration. In th e environment, multiple tetra c yclines are commonly found at the contaminated sites which cou ld influence the microbial populations collectively. At the same uptake by E. coli different tetracyclines exerted varying levels of selective pressure on the bacteria for expression of antibiotic resistance, with the order of tetracycline > chlortetracycl ine > oxytetracycline. Linear relations between promoter activity and intracellular tetracycline concentration were observed for tetracycline, chlortetracycline and oxytetracycline, but not for anhydrotetracycline, one of metabolites of tetracycline in the environment. Anhydrotetracycline evoked a very high antibiotic resistance response of the E. coli bioreporter due to the fact that it could strongly interact the tetR in the bacteria. The mixture of tetracyclines generally demonstrated additive effects on the E.coli reporter for expression of antibiotic resistance. These results suggest that the risk levels of antibiotic resistance invoked by exposure to tetracycline mixtur es in the environment could be additive . The effects of formed metabolite from tetra cycline such as anhydrotetracycline should be included in the assessment of potential risks of antibiotic resistance to microbial populations. 50 REFERENCES 51 REFERENCES 1. Allen, H. K. 2014. 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