Article pubs.acs.org/est
Bioavailability of Soil-Sorbed Tetracycline to Escherichia coli under Unsaturated Conditions Zeyou Chen,†,‡ Wei Zhang,‡ Gang Wang,§ Yingjie Zhang,‡ Yanzheng Gao,*,† Stephen A. Boyd,‡ Brian J. Teppen,‡ James M. Tiedje,‡ Dongqiang Zhu,∥ and Hui Li*,‡ †
Institute of Organic Contaminant Control and Soil Remediation, College of Resource and Environmental Sciences, Nanjing Agricultural University, Nanjing 210095, China ‡ Department of Plant, Soil and Microbial Sciences, Michigan State University, East Lansing, Michigan 48824, United States § Department of Water and Soil Sciences, China Agricultural University, Beijing 100193, China ∥ School of Urban and Environmental Sciences, Peking University, Beijing 100871, China S Supporting Information *
ABSTRACT: Increasing concentrations of anthropogenic antibiotics in soils are partly responsible for the proliferation of bacterial antibiotic resistance. However, little is known about how soil-sorbed antibiotics exert selective pressure on bacteria in unsaturated soils. This study investigated the bioavailability of tetracycline sorbed on three soils (Webster clay loam, Capac sandy clay loam, and Oshtemo loamy sand) to a fluorescent Escherichia coli bioreporter under unsaturated conditions using agar diffusion assay, microscopic visualization, and model simulation. Tetracycline sorbed on the soils could be desorbed and become bioavailable to the E. coli cells at matric water potentials of −2.95 to −13.75 kPa. Bright fluorescent rings were formed around the tetracycline-loaded soils on the unsaturated agar surfaces, likely due to radial diffusion of tetracycline desorbed from the soils, tetracycline uptake by the E. coli cells, and its inhibition on E. coli growth, which was supported by the model simulation. The bioavailability of soil-sorbed tetracycline was much higher for the Oshtemo soil, probably due to faster diffusion of tetracycline in coarse-textured soils. Decreased bioavailability of soil-sorbed tetracycline at lower soil water potential likely resulted from reduced tetracycline diffusion in soil pore water at smaller matric potential and/or suppressed tetracycline uptake by E. coli at lower osmotic potential. Therefore, soil-sorbed tetracycline could still exert selective pressure on the exposed bacteria, which was influenced by soil physical processes controlled by soil texture and soil water potential.
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INTRODUCTION Discovery and application of antibiotics in healthcare have saved millions of lives.1,2 Unfortunately, imprudent use of antibiotics in human medicine and animal agriculture has led to the proliferation of antibiotics, antibiotic resistant bacteria (ARB), and antibiotic resistance genes (ARGs) in the environment.3,4 More alarmingly, because ARGs can be horizontally transferred among bacterial populations, acquisition of functional ARGs in bacterial pathogens is diminishing the effectiveness of antibiotics for treating bacterial infections. Currently, antibiotic resistance in bacteria has become a global health threat.5,6 The Centers for Disease Control and Prevention estimated that infections by ARB cause over two million illnesses and 23,000 deaths annually in the US alone.7 This situation could be even worse in developing countries because of more aggressive use of antibiotics and insufficient healthcare systems.8−10 Among the plethora of antibiotics administered to humans and animals, tetracyclines are the most used antibiotics in animal production.11,12 Large fractions of these tetracyclines are © XXXX American Chemical Society
excreted in animal urine and feces, and land application of animal manure and agricultural lagoon effluents results in considerable concentrations of tetracyclines in soils.13−17 Tetracyclines are often strongly bound to soils,18,19 where soil bacteria may access either the soil-sorbed fraction or the dissolved fraction in soil pore water. Understanding bacterial access and uptake of tetracyclines in soils (i.e., bioavailability) is critical to linking their environmental concentrations to the selective pressure on soil microbial communities. Previous studies have investigated the bioavailability of organic contaminants to soil microorganisms, primarily focusing on hydrophobic organic contaminants such as polyaromatic hydrocarbons (PAHs) and polychlorinated biphenyls (PCBs). It is widely accepted that hydrophobic organic contaminants are not bioavailable to microorganisms Received: January 31, 2017 Revised: May 2, 2017 Accepted: May 5, 2017
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DOI: 10.1021/acs.est.7b00590 Environ. Sci. Technol. XXXX, XXX, XXX−XXX
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Environmental Science & Technology unless desorbed into aqueous solution.20−24 However, several studies have documented that certain bacteria can access pools of soil-sorbed PAHs and PCBs,25,26 likely because biosurfactants or extracellular polymeric substances produced by soil microorganisms facilitate the desorption of soil-sorbed hydrophobic organic contaminants into bulk solution.27−30 For antibiotics sorbed by soils, Chander et al. found that tetracycline and tylosin spiked into Webster clay loam and Hubbard loamy sandy soils at 185−2209 mg kg−1 could inhibit the growth of Salmonella sp. and E. coli.31 However, Subbiah et al. reported that ciprofloxacin, neomycin, and tetracycline sorbed on silt loam, sandy loam, and sandy soils did not apparently inhibit E. coli growth in saturated soil slurries, in contrast to β-lactams and florfenicol, which was attributed to their different affinities for these soils.32 Soil bacteria tend to either be trapped in soil pores or attach to soil surfaces and form biofilms.33,34 Thus, it is hypothesized that soil-sorbed antibiotics may be bioavailable to soil bacteria via desorption to soil water and/or direct diffusion through the cell membranes of the bacteria attached on the same soil surfaces. However, studies on the bioavailability of soil-sorbed antibiotics to bacteria have been sparse. Natural soils are often under unsaturated conditions with soil pores filled with both water and air. Most bacteria live in fragmented aqueous microhabitats with negative water potentials,35,36 composed of a matric potential due to capillarity, and an osmotic potential due to aqueous solute concentration. The majority of previous studies focused on the bioavailability of soil-borne contaminants to microorganisms in water-saturated systems.22,23,32,37−40 Under saturated conditions, soil pores are filled with water, and chemical mass transfer via diffusion or convection is much faster than that under unsaturated conditions.41,42 Bacterial movements, whether passive or active, are also faster at more positive water potentials (e.g., greater soil water content).43,44 Under unsaturated conditions, the mass transfer of contaminants in soil pores is limited, and bacterial motility and dispersal are physically constrained within small water-filled pores or water films on soil particles.44,45 Therefore, the bioavailability of soilsorbed antibiotics to bacteria in unsaturated soils is expected to be drastically reduced compared to that in saturated systems. This study aimed at investigating the bioavailability of soilsorbed tetracycline to a tetracycline-responsive cell bioreporter (E. coli MC4100/pTGM) under unsaturated conditions using a combination of agar diffusion assays, microscopic visualizations, and model simulations. The E. coli bioreporter was constructed by inserting the plasmid pTGM, containing both the tetracycline resistance gene tet(M) and a green florescence protein (gf p) gene, into E. coli cells.46 This bioreporter has been successfully used to measure the bioavailable fraction of tetracycline in aqueous solution.47−49 Unsaturated conditions were achieved by depositing soils on unsaturated agar surfaces, where E. coli growth and fluorescence patterns were experimentally determined and also simulated by an agentbased model. To the best of our knowledge, this was the first study to systematically investigate the bioavailability of soilsorbed tetracycline to bacteria under unsaturated conditions.
physicochemical properties are summarized in Table S1. These soils were autoclaved for 20 min prior to use in order to eliminate the interference of native soil-borne bacteria. Sorption of Tetracycline by Soils. Batch sorption and desorption experiments were carried out to obtain soils loaded with a known range of tetracycline concentrations and to characterize tetracycline interactions with the soils. Detailed experimental procedures are provided in the SI. The tetracycline-loaded soils were freeze-dried before use. Agar Diffusion Assay. The bioavailability of soil-sorbed tetracycline to the E. coli bioreporter was examined using an agar diffusion assay. To do so, lysogeny broth (LB) medium containing 2% of agar (w/v) was autoclaved and cooled to approximately 45 °C. Seven milliliters of the agar solution containing 100 mg L−1 of ampicillin was aseptically poured into sterilized Petri dishes (100 × 15 mm) and solidified. The E. coli bioreporter was incubated in the LB medium at pH 7.0 and 30 ± 0.2 °C for 5 h to reach the mid log growth phase (OD600 ≈ 0.5). Then 100 μL of E. coli suspension was spread evenly over each agar surface using a sterilized bent glass rod. All agar plates were dried in a laminar flow cabinet for 10 min, and then 10 mg of tetracycline-loaded dry soil was placed on each agar surface. The plates were incubated at 30 °C for 12 h, and examined for fluorescence emission from the bioreporters using an UltraBright LED Transilluminator. The captured images were analyzed using the ImageJ software. The E. coli colonies that grew on the soils or the agar media were collected for measuring their green fluorescence intensities. To collect the E. coli attached to soils, the soils were suspended in 0.5 mL of a detachment solution containing sodium pyrophosphate (0.1%) and Tween 20 (0.5%) in a phosphate buffer solution (PBS, pH 7.4). The suspension was shaken at 150 rpm for 30 min at 30 °C. The nonionic densitygradient medium Histodenz (0.5 mL) was carefully added at the bottom of the suspension, followed by centrifugation at 14000g for 30 min at 25 °C using an Eppendorf 5415D centrifuge. The entire supernatant above the Histodenz medium was collected and vortexed for 1 min, then a supernatant sample of 50 μL was diluted with 3.0 mL of PBS, and analyzed for mean fluorescence intensity per cell using a BD LSR II flow cytometer. A preliminary experiment demonstrated that this bacterial detachment procedure did not affect the fluorescence emission from the E. coli. The E. coli colonies on the agar media were directly scraped off from the locations at 0.5, 0.75, 1.0, and 1.5 cm from the center of deposited soils using an aseptic toothpick, and then dispersed in 3 mL PBS for the flow cytometry analyses. The percentage of fluorescent bacteria among the total 10,000 bacteria counts was calculated after each flow cytometry analysis. To closely examine the densities and morphologies of fluorescent bacteria, the images of bacteria on the soils and agar media were recorded using a Zeiss LSM 5 Pascal laser scanning confocal microscope. Bacterial green fluorescence (488 nm excitation and 511 nm emission) was detected using a bandpass 505−530 emission filter set under excitation by a 488 nm argon-ion laser line. The images were captured using a 20 × plan-Neofluar objective (NA 0.5) and analyzed with the LSM 5 Pascal software. Influence of Soil Water Potential on Bioavailability of Soil-Sorbed Tetracycline. To obtain varying degrees of water potential, polyethylene glycol (PEG) solutions of 250−700 g L−1 were poured on the solidified agar surface to reduce water potential in the agar media.50,51 After 24 h, excess PEG solution
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MATERIALS AND METHODS Chemicals and Soils. Chemicals used in this study are described in the Supporting Information (SI). Three soils of contrasting textures (Webster clay loam, Capac sandy clay loam, and Oshtemo loamy sand) were used, and selected B
DOI: 10.1021/acs.est.7b00590 Environ. Sci. Technol. XXXX, XXX, XXX−XXX
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with the parameters for bacterial growth and metabolism in Table S3. The simulation was performed on the surface-roughness network at a matric potential of −3.0 kPa. The initial inoculations of 1000 E. coli cells were randomly distributed on the entire simulation domain. We assumed that the amount of tetracycline desorbed from soils to the solution phase decreased over time, following a first-order decay. This simulation attempted to capture the salient processes in the agar diffusion assay, including tetracycline diffusion in the agar media, cell reproduction, uptake of tetracycline (i.e., fluorescence emission), and inhibition of bacterial growth by tetracycline. The simulation results were not expected to quantitatively match the experimental results but to qualitatively estimate bacterial response patterns to tetracycline stress in the agar media.
remaining on each agar surface was decanted. PEG (molecular weight of 8000 Da) was selected because it does not pass through bacterial membrane or inhibit the growth of E. coli.52,53 Solidified agars are porous materials with circular pores ranging from a few tens of nanometers to a few micrometers, depending on the agar concentration and composition.54,55 Thus, the added PEG penetrates the agar media, reducing water activity and hence osmotic potential. The matric potential of the agar media could also be adjusted, as shown in previous studies.56,57 The osmotic potential of the agar media was measured by a vapor pressure osmometer (Wescor Vapro Model 5600, Logan, UT) and the matric potential by a filter-paper technique,56,58 as described in SI. The prepared PEG-infused agar plates were then used in the agar diffusion assays as described above. Since soil water potential was in equilibrium with the water potential of the agar media, soil water conditions were thus manipulated by PEG infusion in the agar. The matric potential was measured at −2.95 kPa in the PEG-free agar and decreased to −13.75 kPa at the lowest (Table S2). According to the van Genuchten equation relating volumetric soil water content with soil matric potential, the volumetric soil water content (i.e., the level of water saturation) was estimated for the clay loam, sandy clay loam, and loamy sand soils from the measured matric potentials.59,60 The Webster, Capac, and Oshtemo soils on the PEG-free agar were under unsaturated conditions with 89%, 84%, and 71% of water-filled soil pores, respectively (Table S2). The soils deposited on the PEG-infused agars had substantially lower volumetric soil water contents. At the matric potential of −13.75 kPa, the volumetric soil water content was 0.32 for the Webster soil, 0.23 for the Capac soil, and 0.11 for the Oshtemo soil, equivalent to soil water saturation of 69%, 57%, and 28%, respectively. Clearly, these experiments represented a range of unsaturated soil water conditions. Model Simulation. To elucidate the mechanism controlling the formation of fluorescent rings as discussed later, tetracycline diffusion and bacterial growth at a given soil matric potential were simulated using a previously developed agentbased model.44,61 Since the agar surface is microscopically rough and contains nano- and micrometer-sized pores,54 the agar surface can be represented as a network of identical surface-roughness elements partially filled with water, and water film thickness is controlled by the matric potential.44,62 The model simulates tetracycline diffusion patterns, bacterial uptake of tetracycline, and the spatial distribution of fluorescent E. coli cells within the surface-roughness network, similar to the previous studies.44,62 In the model, cell motion was selfpropelled in the water film on the agar media, and the maximum cell velocity was 0.05 μm s−1 in bulk water. The diffusivity of nutrients and tetracycline was controlled by the hydraulic connectivity in the network.61 By considering bacterial chemotactic behavior, an actual cell displacement was calculated as a function of the local water film thickness and chemical concentration gradient. Tetracycline diffusion and its uptake by E. coli were solved for each single roughness element according to the well-established reaction-diffusion method.63 The permeability coefficient of tetracycline through cell membrane was 8.0 × 10−6 mm s−1,64 and the fluorescence intensity of an E. coli cell was proportional to its intracellular tetracycline concentration.47 It was assumed that the E. coli would be deactivated when intracellular tetracycline concentration reached 5% of dry cellular biomass.61A detailed description of the simulation model is provided in SI along
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RESULTS AND DISCUSSION Sorption and Desorption of Tetracycline in Soils. Figure 1 shows that sorption isotherms of tetracycline were
Figure 1. Sorption and desorption isotherms of tetracycline in soils.
linear with distribution coefficients of 1869, 154, and 8653 L kg−1 for the Webster, Capac, and Oshtemo soils, respectively. Tetracycline can be strongly sorbed to soils via cation exchange, cation-bridging interaction, and hydrogen bonding.18,19,65,66 The greatest sorption was found for the Oshtemo soil, probably due to its lower pH of 5.3 (Table S1), which could increase tetracycline sorption via enhanced cation exchange.18 High clay contents could be responsible for the relatively higher sorption by the Webster soil, which is dominated by smectite clays.19 The sorption and desorption isotherms were nearly overlapped for the Oshtemo and Webster soils (Figure 1), suggesting relatively homogeneous sorption sites for tetracycline in these soils. The Capac soil had a weaker affinity for tetracycline, compared to the other two soils. There was an apparent desorption hysteresis (Figure 1), where the distribution coefficient (154 L kg−1) of the sorption isotherm was significantly less than that of the desorption isotherm (586 L kg−1). This hysteresis may indicate that tetracycline sorption sites in the Capac soil were heterogeneous, and tetracycline was first desorbed from the weaker sorption sites. Overall, tetracycline demonstrated a strong affinity for these three soils. Only the Webster soil > the Oshtemo soil. Bioavailability of Soil-Sorbed Tetracycline to E. coli Bioreporter. The images of the agar plates after the incubation are shown in Figure S1. The fluorescent rings formed around the soils with relatively higher soil-sorbed tetracycline concentrations, i.e., 104 and 148 mg kg−1 for the Webster soil, 96 and 136 mg kg−1 for the Capac soil, and 45, 105, and 150 mg kg−1 for the Oshtemo soil. No visible fluorescent rings were observed for the tetracycline-free soil controls or the soils with lower soil-sorbed tetracycline concentrations (≤44 mg kg−1 for the Webster soil, ≤ 41 mg kg−1 for the Capac Soil, and ≤15 mg kg−1 for the Oshtemo soil). Exploring the mechanisms of fluorescent ring formation would shed light on the behaviors of bacteria upon exposed to tetracycline and will be further discussed below. To examine the influence of soil-sorbed tetracycline concentration on the size of the fluorescent ring, the soils with a range of tetracycline concentration (79−757 mg kg−1) were used in the agar diffusion assays. With increasing soilsorbed tetracycline concentrations, the fluorescent rings were enlarged (Figure S2). The diameters of fluorescent rings around Oshtemo soils were approximately 0.98, 1.36, 1.66, and 2.01 cm for the soil-sorbed tetracycline concentration of 86, 160, 460, and 757 mg kg−1, respectively. Similar patterns were also observed for the Webster and Capac soils. The agar diffusion assay is commonly used to determine the bacterial susceptibility to antibiotics, and the larger inhibition zone usually corresponds to the stronger antibiotics or higher antibiotic concentration.67 Thus, larger sizes of florescent rings (i.e., larger inhibition zones) were expected at greater soilsorbed tetracycline concentrations, likely due to greater concentrations of desorbed tetracycline. Mean fluorescence intensity of the E. coli at 0.75 cm from the soil center was minimal in the tetracycline-free soil controls, and increased with increasing soil-sorbed tetracycline concentration (Figure 2A). The E. coli isolated from the soils also had increased fluorescence intensity at greater soil-sorbed tetracycline concentrations (Figure 2B), demonstrating that the E. coli on the soils could access the soil-sorbed tetracycline. In fact, the E. coli in the soils still emitted a measurable fluorescence (>750 counts) at the lowest tetracycline concentration of 6.9 mg kg−1 in the Webster soil, 6.4 mg kg−1 in the Capac soil, and 1.0 mg kg−1 in the Oshtemo soil. Clearly, tetracycline could be desorbed from each soil and become bioavailable to the E. coli. At the similar tetracycline loadings in the soils, the mean fluorescence intensity from the E. coli in the soils decreased in the order of the Oshtemo soil > the Webster soil > the Capac soil (Figure 2B), whereas it was the greatest for the Oshtemo soil, but indistinguishable for the Webster and Capac soils for the E. coli on the agar (Figure 2A). These trends seem contradictory to the decreasing tetracycline desorption in the order of the Capac soil > the Webster soil > the Oshtemo soil (in water, Figure 1), because the mean fluorescence intensity should have been the highest for the Capac soil due to the greatest amount of desorbed tetracycline. This discrepancy suggests that the bioavailability of tetracycline in the soils could not be simply explained by chemical interactions of tetracycline with the soils, such as simple desorption into the water phase. It was also likely influenced by physical processes of soil pore structure and solute diffusion (or mass transfer) that are largely controlled by soil texture and structure. Under the slightly unsaturated conditions (Table S2), water-filled pores would be
Figure 2. Relationship between soil-sorbed tetracycline concentration and mean fluorescence intensity of E. coli sampled at (A) 0.75 cm from soil center and (B) on the soil center.
larger in the Oshtemo loamy sand soil, comparing to those of the Webster clay loam and the Capac sandy clay loam soils. Therefore, the desorbed tetracycline could presumably diffuse more rapidly through pore water and subsequently into the agar media, thus contributing to the greater mean fluorescence intensity for the Oshtemo soil, despite its lowest tetracycline desorption in water. The lower mean fluorescence intensity of the E. coli in the Capac soil seemed inconsistent with its coarser texture and greater tetracycline desorption compared with the Webster soil; this effect plausibly resulted from the greater organic matter content of the Capac soil, since greater tetracycline complexation with dissolved organic matter (DOM) could diminish the bioavailability of tetracycline to the E. coli.49 There was no distinguishable difference in the mean fluorescence intensity of bacteria on the agar between the Webster and Capac soils (Figure 2A), likely due to a decrease in this DOM effect further out in the agar media. Fluorescent Ring. The mean fluorescence intensity and the percentage of fluorescent bacteria were measured for the E. coli located at varying distances from the soil center (Figures 3). For the soils with the higher tetracycline loadings (136−150 mg kg−1), the fluorescent rings were formed with a diameter of 1.13 cm for the Capac soil, 1.26 cm for the Webster soil, and 1.56 cm for the Oshtemo soil (Figure S3). The mean fluorescence intensity remained essentially unchanged up to a threshold distance from the soil center beyond which it decreased substantially (Figure 3A). This plateau of fluorescence intensity might be because the free tetracycline concentration in the agar was out of the linear response range of the bioreporter (i.e., generally 10−500 μg L−1).46,47 The D
DOI: 10.1021/acs.est.7b00590 Environ. Sci. Technol. XXXX, XXX, XXX−XXX
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Figure 3. Mean fluorescence intensity of E. coli collected from the soils and agar media at varying distances from the soil center and the percentage of fluorescent bacteria. Visible fluorescent rings (A and C) and no visible fluorescent rings (B and D) were noted in the agar plates in Figure S3.
Figure 4. Confocal scanning laser microscope images of fluorescent E. coli on the soils and agar media. Scale bar in each image is 20 μm. Sample 0 refers to tetracycline-free controls (soil + E. coli); sample 1 refers to the soils with the lower tetracycline loading + E. coli, and the images were positioned at the soil center; sample 2 refers to the soils with the higher tetracycline loading+ E. coli, and the images were positioned at the soil center; sample 3 refers to the soils loaded with more tetracycline + E. coli, and the images were positioned between the soil and the formed fluorescent ring; sample 4 refers to the soils with the higher tetracycline loading + E. coli, and the images were positioned on the fluorescent rings; sample 5 refers to E. coli free controls of the soils with the higher tetracycline loadings. The soils with the higher tetracycline loading were 148 mg kg−1 of tetracycline for the Webster soil, 136 mg kg−1 for the Capac soil, and 150 mg kg−1 for the Oshtemo soil. The soils with the lower tetracycline loadings were 6.9 mg kg−1 of tetracycline for the Webster soil, 6.4 mg kg−1 for the Capac soil, and 7.0 mg kg−1 for the Oshtemo soil.
percentage of fluorescent bacteria increased monotonically with distance from the soil center. Within distances of 1.0 cm from the soil center, the fluorescent bacteria numbered less than 10% of the total bacterial count (Figure 3C) but emitted the strongest fluorescence (Figure 3A). In the vicinity of the fluorescent rings (about 1.1−1.6 cm from the soil center), a larger percentage of the E. coli cells were fluorescent but manifested relatively weak intensities (Figure 3A,C). This could possibly be explained as a result of tetracycline radial diffusion, for which we would expect decreasing tetracycline concentrations with increasing distance from the soil center. Near the soils, the desorbed tetracycline concentration might be greater than minimal inhibitory concentration (MIC) of the E. coli,
resulting in effective inhibition of bacterial growth. Perhaps only a small number of E. coli cells could access the desorbed tetracycline near the soils, but showed a strong mean fluorescence intensity. The E. coli cells further away from the soils could grow into a dense population, due to lower tetracycline concentrations. In turn, the dense E. coli population could actually form the bright fluorescent rings, despite their low average fluorescence emission per cell. For the soils with the lower tetracycline loading (13−15 mg kg−1), no apparent fluorescent ring was observed (Figure S3). The mean fluorescence intensity decreased with distance from the soil (Figure 3B), suggesting that the tetracycline concentration was within the linear response range of the E
DOI: 10.1021/acs.est.7b00590 Environ. Sci. Technol. XXXX, XXX, XXX−XXX
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Environmental Science & Technology bioreporter. The desorbed tetracycline concentration was expected to be low in the agar media, which might be unable to effectively inhibit the bacterial growth. For the Webster soil, the percentage of fluorescent bacteria decreased with the distance. For the Oshtemo and Capac soils, the percentage of fluorescent bacteria increased to 25% at 0.75 cm away from the soil center and then decreased with the distance (Figure 3D). The reasons for this discrepancy could be that at 0.75 cm from the two soils there may be a relatively dense population of cells due to low tetracycline concentrations that allow E. coli growth but are apparently high enough to induce 25% of cells to turn on their gf p (Figure 3D). Despite the high percentage of fluorescent E. coli, no visible fluorescent ring was formed due to their very low fluorescence intensity (645−2427 counts) per cell (Figure 3B). The above-mentioned phenomena were further corroborated by the microscope images of fluorescent bacteria on the agar plates (Figure 4). No green fluorescence was observed in the tetracycline-free samples (sample 0) or the E. coli-free controls (sample 5), indicating negligible autofluorescence of the soils or tetracycline. The green fluorescence emitted from the bioreporters was clearly visible in the samples with soil-sorbed tetracycline and E. coli (samples 1−4). The bacterial fluorescence emission from the soils with the higher tetracycline loading (sample 2) demonstrated a greater intensity than that from the soils with the lower tetracycline loading (sample 1). Clearly, the soil-sorbed tetracycline was somehow bioavailable to the E. coli, which in turn triggered the tetracycline resistance genes. Samples 2−4 showed that the fluorescent bacterial populations became denser with increasing distances away from the soil, and the maximal total fluorescence intensity was reached at the areas of the fluorescent rings (sample 4). The formation of fluorescent rings might be due to the convergence of tetracycline desorption from the soils, its radial diffusion into the agar media, its inhibition on E. coli growth, and bacterial uptake of tetracycline and gfp activation. Model Simulation. The mechanism underlying the fluorescent ring formation was further revealed by the model simulation. The simulated E. coli growth dynamics under the influence of tetracycline point sources is shown in SI video 1 and in a snapshot of the simulation (Figure 5). In the simulation, fluorescent E. coli cells were represented in yellow and tetracycline concentrations in blue. The video clearly showed the changing tetracycline concentration zones, the multiplication of E. coli cells, and the emergence of fluorescent rings. As shown in Figure 5, the E. coli cells had a color of light or bright yellow (i.e., equivalent to lower or higher fluorescence intensity), in agreement with the tetracycline diffusion concentration from the source. The bacteria population density close to the tetracycline source was generally less than that at the further distance. The model described tetracycline diffusion, inhibition of bacterial growth, and bacterial fluorescence emission (proportional to intracellular tetracycline concentration) in the absence of soil. Thus, the similar patterns between simulated and observed results suggest that the formation of fluorescent ring resulted primarily from the diffusion of tetracycline in the agar media and tetracyclinemediated bacteria growth with induction of bacterial fluorescence emission. Influence of Water Potential on Bioavailability of SoilSorbed Tetracycline. Water is the major carrier responsible for tetracycline desorption from soil particles and diffusion in soils. The water potential was controlled primarily by the
Figure 5. Snapshot of simulated tetracycline diffusion, bacteria growth, and fluorescent bacteria populations at 10 h incubation. The yellow dots represent individual fluorescent cells with the brighter yellow indicating a stronger fluorescence intensity. The black circles represent the point source of tetracycline.
osmotic potential and to a less extent by the matric potential (Figure 6). However, decrease of soil matric potential tends to
Figure 6. Effects of soil water potential on fluorescence emission of the bacteria attached to soil surfaces.
reduce the diffusion of solutes due to decreased water-filled pore space and increased tortuosity of the pore network.67,68 Mean fluorescence intensities from the E. coli growing on the soil surfaces decreased with decreasing water potential in all three soils (Figure 6). As the matric potential decreased from −2.95 to −13.75 kPa, and the osmotic potential decreased from −0.83 to −2.46 MPa, the mean fluorescence intensity decreased by approximately 97.4%, 98.8%, and 76.0% for the Webster, Capac, and Oshtemo soils, respectively. The decrease in the osmotic potential presumably reduced the water flux across cell membranes and thus the uptake of tetracycline by E. coli. The decrease in the matric water potential could diminish the diffusion of desorbed tetracycline in soil pore water (i.e., lower mass transfer of tetracycline), resulting in an overall lower exposure of E. coli to dissolved tetracycline.13,69 In F
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Environmental Science & Technology addition, as soil matric potential decreased, the water films in the soils became thinner, which could reduce the contact area between water and bacteria, and subsequently lessen the bacterial uptake of tetracycline.44,70 These factors collectively contributed to the decreasing bioavailability of soil-sorbed tetracycline to the E. coli at lower soil water potential in the unsaturated soils. Environmental Implications. In the environment, the majority of tetracycline is held by soils due to its strong affinity for soil sorbents, and soil bacteria live in aqueous microhabitats of unsaturated soils primarily in the form of biofilms.33,34 Our results indicate that tetracycline sorbed by soils is still bioavailable to E. coli to invoke antibiotic resistance genes. The mass transfer of desorbed tetracycline in soils and the close contact between E. coli and soils could be key factors governing the bacterial uptake of soil-sorbed tetracycline and the induction of bacterial antibiotic resistance. For soils with relatively high tetracycline concentrations (e.g., hundreds of mg kg−1) resulting from continuous land applications of animal manure and sewer sludge,71 the desorbed tetracycline near the contaminant source could inhibit the bacteria growth, leading to lower bacterial populations in that immediate vicinity. But at several centimeters away from the point source, our study implies that tetracycline concentrations could decrease to levels that allow lush bacteria growth, but still exert selective pressure on the bacteria to maintain and develop antibiotic resistance. For soils with lower tetracycline concentrations (e.g., several mg kg−1) found in manured agricultural fields,72 the tetracycline concentrations are also too low to effectively inhibit bacteria growth, but our study still shows widespread induction of bacterial antibiotic resistance. Therefore, lower levels of tetracycline in soils plausibly pose a greater risk via exerting stress on larger bacteria populations, compared to higher concentrations (e.g., near MIC) at which bacterial growth is more effectively inhibited. The bioavailability of tetracycline in unsaturated soils is apparently controlled by complex physicochemical processes including sorption/desorption, along with tetracycline diffusion modulated by soil texture/ structure and soil matric potential. In moist sandy soils, the soilsorbed tetracycline may exert stronger selective pressure on bacteria because of faster mass transfer in soil pore water, compared with finer-textured soils (e.g., loam or clay loam). Decreased water potential in soils reduced the bioavailability of soil-sorbed tetracycline to bacteria, implying that lowering water content in soils may mitigate the impact of soil-sorbed tetracycline to bacteria. Conversely, rainfall or irrigation events will increase soil water potential and hence the bioavailability of tetracycline to soil bacteria. Finally, this study was limited in a narrow range of soil matric potentials (−2.95 to −13.75 kPa), so future studies should explore the bioavailability of soilsorbed antibiotics under a wider range of soil matric potential such as dryer soils.
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Additional experimental details, modeling description, and supplementary figures and tables (PDF) Video of simulated E. coli growth dynamics under the influence of tetracycline point sources (AVI)
AUTHOR INFORMATION
Corresponding Authors
*Phone: +86-25-84395019; fax: +86-25-84395019; e-mail:
[email protected]. *Phone: +1-517-353-0151; fax: +1-517-355-0270; e-mail:
[email protected]. ORCID
Wei Zhang: 0000-0002-2937-1732 Dongqiang Zhu: 0000-0001-6190-5522 Hui Li: 0000-0003-3298-5265 Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS This work was partly funded by National Science Foundation (CBET-1438105), National Natural Science Foundation of China (21428701, 21225729, 21024093), and National Key R&D Program of China (2016YFD0200306). We also thank Yingmei Ma, Sanalkumar Krishnan, and Sangho Jeon for their assistance in water potential measurement. Z.C. acknowledges the Chinese Scholarship Council (No. 201306850037) for financial support.
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ASSOCIATED CONTENT
S Supporting Information *
Additional experimental details, modeling description, supplementary figures, tables and video are provided in the Supporting Information. These materials are available free of charge via the Internet at The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.est.7b00590. G
DOI: 10.1021/acs.est.7b00590 Environ. Sci. Technol. XXXX, XXX, XXX−XXX
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