Increased Pollution-Induced Bacterial Community Tolerance to

Mar 18, 2009 - of pollution-induced community tolerance (PICT) to SDZ concentration levels in bulk soil and nutrient amended soil hotspots. Agricultur...
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Environ. Sci. Technol. 2009, 43, 2963–2968

Increased Pollution-Induced Bacterial Community Tolerance to Sulfadiazine in Soil Hotspots Amended with Artificial Root Exudates K R I S T I A N K . B R A N D T , * ,† OLE R. SJØHOLM,† KRISTINE A. KROGH,‡ BENT HALLING-SØRENSEN,‡ AND OLE NYBROE† Department of Agriculture and Ecology, Faculty of Life Sciences, University of Copenhagen, Thorvaldsensvej 40, DK-1871 Frederiksberg C, Denmark, and Department of Pharmaceuticals and Analytical Chemistry, Faculty of Pharmaceutical Sciences, University of Copenhagen, Universitetsparken 2, DK-2100 Copenhagen, Denmark

Received December 15, 2008. Revised manuscript received February 19, 2009. Accepted February 24, 2009.

Sulfadiazine (SDZ) residues constitute an important pollutant in soils that may increase environmental reservoirs of antibiotic resistance. Our primary aim was to compare the development of pollution-induced community tolerance (PICT) to SDZ concentration levels in bulk soil and nutrient amended soil hotspots. Agricultural soil microcosms were amended with different concentrations of SDZ with or without weekly additions of artificial root exudates corresponding to realistic rhizodeposition rates. Bacterial community tolerance to SDZ residues, as determined by the [3H]leucine incorporation technique, increased progressively with elevated SDZ exposure, and was significantly increased in soil hotspots (LOEC of 1 µg kg-1). An alternative PICT approach based on single-cell esterase probing by flow cytometry failed to demonstrate SDZ impacts. Bacterial growth rates ([3H]leucine incorporation) were significantly reduced in both bulk soil and hotspots 24 h after amendment with environmentally relevant concentrations of SDZ, while soil respiration remained unaffected even at 100 µg SDZ g-1. Our study for the first time demonstrates a drastically increased PICT response of a soil bacterial community due to increased carbon substrate amendment per se. Hence, hotspot soil environments such as rhizosphere and manure-soil interfaces may comprise key sites for proliferation of bacteria that are resistant or tolerant to antibiotics.

Experimental section

Introduction The extensive use of antibiotics in modern agriculture and livestock industries (1-3) may constitute a key driver for the expansion of environmental reservoirs of antibiotic resistance. A substantial fraction of the administered antibiotic dosage reaches the environment in active or partly active forms (1, 3) that may select for antibiotic resistant microbial communities (1). * Corresponding author e-mail: [email protected]. † Department of Agriculture and Ecology. ‡ Department of Pharmaceuticals and Analytical Chemistry. 10.1021/es803546y CCC: $40.75

Published on Web 03/18/2009

Among the different classes of veterinary antibiotics, sulfonamides constitute a particularly important group of pollutants due to their frequent use, high excretion rates (4), and their persistence in soil (5). Furthermore, they serve as potent broad spectrum bacteriostatic agents by virtue of their ability to bind to dihydropteroate synthase thereby inhibiting the folic acid biosynthesis pathway in both gram-negative and gram-positive bacteria (6). The expansion of environmental reservoirs of antibiotic resistance as caused by anthropogenic activities may be studied using the pollution-induced community tolerance (PICT) approach (7, 8). PICT experiments consist of two phases: a selection phase, which may include long-term field exposure or controlled laboratory exposure to the studied toxicant, and a subsequent detection phase for evaluation of tolerance/resistance levels in the studied biotic communities. Studies of antimicrobial resistance levels in soil microbial communities typically rely on isolation of soil microorganisms by cultivation-dependent techniques, but these techniques typically only retrieve a tiny proportion of environmental bacteria belonging to relatively few wellknown taxonomic groups (9). Hence, we know very little about the uncultured microbial majority that is likely to be important for the spread of antibiotic resistance. Another problem with cultivation-dependent PICT determination including Biolog-PICT approaches (8, 10) is the typical requirement for a long detection phase of several days before growth can be measured. A long detection phase is unwanted for PICT experiments as it may provide ample time for community adaptation (i.e., selective growth of tolerant bacteria) to the toxicant under study, thereby obscuring treatment impacts during the PICT selection phase (7, 10, 11). The primary aim of the current study was to compare the impact of the frequently used sulfonamide antibiotic sulfadiazine (SDZ) on the bacterial PICT response in soil and compare observations in bulk soil to nutrient amended soil hotspots with elevated microbial activity. Due to its specific mode of action, we hypothesized that the observed PICT response following SDZ exposure would be stimulated by the increased growth activity of bacteria present in soil amended with pulses of artificial root exudates (ARE). A secondary aim was to provide the first evaluation of a recently proposed fluorescence viability staining-flow cytometry (FCM) PICT protocol (12) against a more well-established protocol (Leu-PICT) based on short-term [3H]leucine incorporation into growing cells (13). Both methods allow for cultivation-independent PICT detection and were investigated for their ability to provide a comprehensive evaluation of the bacterial community response to antibiotic exposure using short-term PICT detection phases.

 2009 American Chemical Society

Soil Sampling and Characteristics. An agricultural soil used for cultivation of cereals was sampled during late winter (February 2007) from the CRUCIAL long-term fertilization experiment located at the University of Copenhagen Experimental Farm in Taastrup, Denmark (14). The soil pH(KCl) was 6.6, and the soil texture was classified (U.S. Department of Agriculture classification) as a sandy loam. Prior to use the soil was air-dried, sieved (2 mm mesh size) and homogenized by manual mixing. Determination of Heterotrophic Microbial Activity in Soil. Replicated (n ) 3) microcosms (5 g dry wt soil) for nondestructive measurement of microbial activity (i.e., VOL. 43, NO. 8, 2009 / ENVIRONMENTAL SCIENCE & TECHNOLOGY

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mineralization of organic C) were set up in sealed 324 mL infusion bottles outgassed with CO2-free air using the same experimental soil treatments as for the PICT selection phase experiments (see text below). Soil respiration was determined during 81 days as accumulated CO2 by gas chromatography (GC) using the same equipment and procedure as described previously (15). When required, the headspace was outgassed with CO2-free air to ensure that headspace CO2 never exceeded concentrations of 2% vol/vol. Determination of Bacterial Growth Rates Using [3H]leucine Incorporation Assay. The [3H]leucine incorporation method was also used to estimate impacts of SDZ (CAS 54732-0, Fluka, Switzerland) on bacterial growth activity shortly after SDZ amendment to soil. For these experiments, airdried soil (10 g dry wt) was amended with 0.1, 1, 10, or 100 µg SDZ per g of soil in the absence (bulk soil) or presence (hotspot soil) of ARE (937 µg organic C g-1 soil; see Supporting Information). All SDZ treatments were compared to a control treatment receiving water only (∼55% of water-holding capacity). The soils were incubated in the dark for 24 h at 22 °C. Following incubation, soil bacteria were extracted from soil, and their relative [3H]leucine incorporation rates were measured using a microcentrifugation method for shortterm evaluation of soil bacterial growth activity (13). In brief, soil samples (10 g dry wt) were mixed with 100 mL MOPSbuffer (3-N-morpholinopropanesulfonic acid, 5 mM in demineralized water, pH 6.8) and incubated on a horizontal shaker (250 rpm, 15 min, 22 °C). Following centrifugation (1000g, 10 min, 22 °C) soil bacterial suspensions were collected as the resulting supernatants. The suspensions (1.5 mL) were transferred to 2 mL microtubes and preincubated for 30 min at 22 °C. Incubations were subsequently initiated at 22 °C by adding radiolabeled [3H]leucine (2.59 TBq mmol-1, 35 MBq mL-1, Amersham, Hillerød, Denmark) and nonlabeled L-leucine to give a final radioactivity of 6 kBq per microtube and a leucine concentration of 200 nM. Incubations were stopped after 2.5 h by addition of ice-cold 50% trichloroacetic acid. Finally, [3H]leucine incorporated into precipitated proteins was physically separated from nonincorporated [3H]leucine through a series of washing and centrifugation steps (13) and quantified by scintillation counting as described previously (10). Microcosm Setup for Determination of Bacterial Community Tolerance to SDZ (PICT Selection Phase). Triplicate microcosms were set up with 500 g dry wt soil. Two series of microcosms were established using air-dried bulk soil (22 °C; 24 h) as the starting material. One series of microcosms (hotspot soil) was amended with ARE (16) at weekly intervals at an amendment rate of 937 µg organic C per g of dry wt soil, whereas another series (bulk soil) received Milli Q water (Milli Q water purification system, Millipore, Bedford, MA). Both series of microcosms were established with the following SDZ amendments (µg SDZ g-1 dry wt soil): 0, 1, 10, and 100. The appropriate sterile-filtered (0.22 µm pore size) solutions were sprayed into thin layers of soil resulting in soil moisture of ∼55% of the water-holding capacity. The soils were repeatedly mixed during the spiking process to ensure homogeneous distribution of SDZ and ARE. All soil microcosms were incubated in the dark at 15 °C. Soil was air-dried for ∼24 h at 22 °C before each weekly amendment with ARE to prevent build-up of excessive moisture and to further activate the living microbial biomass. After 5 and again after 10 weeks, all soils from both series of microcosms (with or without ARE) were air-dried (∼24 h; 22 °C) and reamended with the same concentration of SDZ and ARE as described above. In parallel, new microcosms were set up with various concentrations of SDZ with or without ARE after five and ten weeks, respectively. After 10 and 15 weeks, bacterial tolerance to SDZ was determined for 2964

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all soil microcosms using cultivation-independent protocols (see below). A graphical presentation of the experimental design is available in Supporting Information Figure S1. Detection of Bacterial Community Tolerance Using [3H]leucine Incorporation Method (Leu-PICT Detection Phase). Bacterial community tolerance to SDZ was determined using the same microcentrifugation protocol as described above with the following modification. Bacterial community tolerance to SDZ was determined by preincubating extracted bacterial communities (1.5 mL supernatant suspensions) for 0.5 or 24 h (see results) at 22 °C with different concentrations of SDZ-Na salt before the incubation (2.5 h) with radiolabeled leucine. Bacterial community tolerance to SDZ was determined using tolerance (T) indices (17). A T-index (Tleu) for each triplicate bacterial soil suspension was calculated as follows: Tleu ) Linc_SDZ/Linc_control, Linc_SDZ equals the amount of incorporated [3H]leucine (dpm mL-1) in the sample amended with SDZ and Linc_control equals the corresponding amount of incorporated [3H]leucine (dpm mL-1) in the sample not amended with SDZ. Detection of Bacterial Community Tolerance Using Single-Cell Esterase Activity Probing and Flow Cytometry (FCM-PICT Assay). A flow cytometry protocol developed for detection of metal and antibiotic tolerance in aquatic bacterial communities (12) was adapted for soil by inclusion of an optimized Nycodenz density gradient centrifugation-based cell extraction step (18) prior to flow cytometric analysis. The FCM protocol is based on the use of the fluorogenic substrate carboxyfluorescein diacetate (CFDA), which is converted into the fluorochrome carboxyfluorescein inside bacterial cells possessing active esterases. A complete flowchart and a detailed description of the procedure for the optimized Nycodenz extraction protocol are available as Supporting Information. Bacterial community tolerance to SDZ was determined using tolerance (T) indices as described for the Leu-PICT assay. A T index (TFCMabs) for each triplicate bacterial soil suspension was calculated as described previously (12): TFCMabs ) CSDZ/Ccontrol, where CSDZ equals the absolute number of esterase positive cells (cells mL-1) in the sample amended with SDZ and Ccontrol equals the number of esterase positive cells (cells mL-1) in the corresponding sample not amended with SDZ. In parallel, another T index (TFCMrel) was also calculated as follows: TFCMrel ) FCSDZ/FCcontrol, where FCSDZ equals the fraction of esterase positive cells (i.e., the number of CFDA stained cells divided by the number of SyBR green I stained cells) in the sample amended with SDZ and FCcontrol equals the corresponding fraction of esterase positive cells in the sample not amended with SDZ. Extraction and Chemical Analysis of SDZ. SDZ was extracted from soil using pressurized liquid extraction (PLE) followed by solid-phase extraction (SPE) procedure and final chemical analyzes by liquid chromatography tandem mass spectrometry (LC-MS/MS) as described previously (see ref (19) and the Supporting Information). Statistics. SigmaStat Version 3.5 (Systat Software, Point Richmond, CA) was used for statistical significance testing. If not stated otherwise, significance testing was carried out by one-way or two-way ANOVA. The Holm-Sidak method was used for all pairwise multiple comparisons and multiple comparisons versus the control group (i.e., treatments without SDZ).

Results Impacts of SDZ and ARE on Heterotrophic Microbial Activity and Bacterial Growth Rates. To determine the influence of different SDZ concentration levels on heterotrophic microbial activity and to validate the hotspot nature of soil amended with ARE, accumulating CO2 was

FIGURE 1. Impacts of sulfadiazine (SDZ) on cumulated soil respiration (CO2 accumulation) in soil microcosms during the first 81 days of the experimental period. Hotspot soil (open symbols), soil receiving weekly pulses of artificial root exudates (ARE). Bulk soil (closed symbols), corresponding soil without ARE. Arrows indicate times of repeated SDZ amendments (µg g-1 dry wt soil): circles (0), triangles (1), squares (10), and diamonds (100). Means ( one standard deviation (n ) 3) are shown. Soil respiration during 24 h periods of soil drying prior to amendments with SDZ and ARE (see text) was neglected.

FIGURE 2. Relative bacterial growth rates (i.e., [3H]leucine incorporation rates) 24 h after amendment with various concentrations of sulfadiazine (SDZ) and artificial root exudates (ARE) to soil. Closed symbols, bulk soil. Open symbols, hotspot soil amended with ARE. Mean ( standard deviation (n ) 3). measured in soil microcosms. The microbial activity was approximately 20-fold higher for soil hotspots as compared to corresponding bulk soil conditions whereas SDZ had no impact on the accumulated microbial activity (Figure 1). The lacking SDZ impact on microbial CO2 production was verified by detailed studies of the CO2 accumulation kinetics during the first 48 h for all experimental treatments (Supporting Information Figure S3). The influence of SDZ and ARE on bacterial growth measured as [3H]leucine incorporation was determined in microcosms exposed to the compounds for 24 h (Figure 2). Interestingly, significant growth inhibition (P < 0.05) was evident for both ARE-amended and unamended soil even at the lowest tested concentration level of 0.1 µg SDZ g-1. However, growth was more strongly inhibited by SDZ (P < 0.001; log-transformed data) in the presence of ARE than in bulk soil indicating that SDZ exerted a stronger selection pressure in soil hotspots. Bacterial growth rates were 9.9 times higher in hotspot soil than in bulk soil (control treatment; data not shown). Optimization of the Leu-PICT Assay. A 24-h preincubation period with SDZ before addition of [3H]leucine was necessary and used for all subsequent PICT detection experiments to make soil bacteria sensitive to SDZ exposure (Supporting Information Figure S4). Under these assay conditions, SDZ concentration levels between 10 and 250 mg L-1 led to a ∼70-90% inhibition of [3H]leucine incorporation depending on the soil used. The dose-response pattern for a nonadapted bacterial community extracted

FIGURE 3. Sulfadiazine (SDZ) dose-response curve for nonadapted bacterial community extracted from bulk soil as measured by the [3H]leucine incorporation method. Mean ( standard deviation (n ) 3); values for the control treatment were 100 ( 8.9. from unamended field soil is depicted in Figure 3. The doseresponse curve showed a biphasic pattern suggesting the presence of three main groups of bacteria with regard to their sensitivity to SDZ: extremely sensitive bacteria exhibiting inhibition at the lowest tested concentration (0.01 mg L-1), moderately sensitive bacteria inhibited between 0.1 and 10 mg L-1 and resistant bacteria. Based on our optimization experiments, we chose to estimate bacterial community tolerance in soil extracts with or without a high concentration of SDZ (250 mg L-1) to ensure that the concentration of SDZ remained within the 10-250 mg L-1 range causing growth inhibition of sensitive bacteria (Supporting Information Figure S4). Development of Bacterial Community Tolerance. Bacterial community tolerance to SDZ, as measured by the LeuPICT assay, in the different soil microcosms harvested after 15 weeks is depicted in Figure 4A-C. Corresponding data obtained after 10 weeks were in excellent agreement with the findings presented below (data not shown). Bacterial community tolerance increased progressively with elevated SDZ exposure level (1, 10, or 100 µg SDZ g-1; P < 0.001). Interestingly, the PICT response was also significantly affected by the ARE amendment (P < 0.001). Hence, community tolerance developed already at the lowest SDZ exposure level (1 µg SDZ g-1) in soil hotspots, whereas a significant PICT response in bulk soil was only observed at 10 and 100 µg SDZ g-1 (P < 0.05). Moreover, Tleu values were significantly higher for soil amended with ARE than for bulk soil at the two highest SDZ loadings (10 and 100 µg g-1) in all cases (P < 0.05). Tleu increased to ∼100% in hotspot soil amended with 100 µg SDZ g-1. In most cases, bacterial community tolerance did not differ significantly depending on the number of SDZ additions to the soil (Figure 4A-C). In conclusion, our data show that bacterial community tolerance was significantly increased by ARE amendment to soil. Furthermore tolerance increased progressively with elevated SDZ exposure, and developed rapidly following just one single amendment with SDZ. The impact of SDZ amendment on the absolute rates of [3H]leucine incorporation measured during the PICT detection phase (PICT detection phase incubations without added SDZ) is depicted in Figure 4D-F. [3H]leucine incorporation rates remained unaffected by SDZ amendment rate in all hotspot soil microcosms, whereas it was progressively reduced with increasing SDZ amendment rate in bulk soil microcosms. These findings are thus consistent with the increased degree of bacterial community adaptation to SDZ observed for the ARE-amended hotspots. Bacterial Community Tolerance As Determined by Flow Cytometry (FCM-PICT). The FCM assay scored a relatively high percentage of the extracted bacterial cells as being active. Hence the frequency of CFDA esterase positive VOL. 43, NO. 8, 2009 / ENVIRONMENTAL SCIENCE & TECHNOLOGY

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FIGURE 4. Pollution-induced community tolerance (PICT) and relative bacterial growth rates (i.e., [3H]leucine incorporation rates) five weeks after preceding amendment with sulfadiazine (SDZ) in soil microcosms subjected to one (A, D), two (B, E), or three (C, F) repeated 5-week SDZ exposure periods. A-C, [3H]leucine incorporation PICT assay; the dotted line represents mean background community tolerance in control soil treatments without SDZ during the PICT selection phase. Background Tleu values for control soil did not differ significantly between hotspot and bulk soil and averaged 21.6 ( 0.8%. D-F, [3H]leucine incorporation rates in control treatments of the PICT detection phase incubations. Tleu, bacterial community tolerance index (see text for details). Closed symbols, bulk soil. Open symbols, hotspot soil amended with artificial root exudates (ARE). Mean ( standard deviation (n ) 3). cells in all control treatments without SDZ averaged 12.1 ( 3.3% (mean ( standard deviation) of the total SyBR green bacterial count. Initial experiments designed to verify the CFDA esterase activity - FCM method confirmed that this type of viability assessment was indeed sensitive to toxicants. Hence, high doses of ethanol led to almost 100% loss of CFDA esterasepositive cells within a few minutes, whereas only 34% of cells extracted from soil remained CFDA esterase-positive following incubation with 600 µM CuSO4 for 3 h (data not shown). However, even the very high SDZ concentration used for community tolerance detection (400 mg l-1) did not decrease the number of esterase positive cells substantially (see Supporting Information Figure S5). TFCMrel values averaged 92% with no apparent trend as related to SDZ levels during the selection phase in soil microcosms. Similar results were obtained when reporting bacterial community tolerance based on counts of CFDA esterase positive cells only as TFCMabs averaged 88% again with no apparent trend as related to SDZ levels (data not shown). Attempts to increase the sensitivity of the extracted soil bacteria to SDZ by adding 0.22 mM glucose were also unsuccessful (data not shown). Bacterial cell numbers as determined by automated flow cytometry cell counting of SyBR green-stained soil bacteria increased by a factor of 3.1 ( 0.9 ((one standard deviation) during the 24 h preincubations without SDZ, whereas the numbers were 3.0 ( 0.6 for the corresponding preincubations with 400 mg SDZ L-1. Hence, SDZ did not significantly inhibit the number of cell divisions taking place during the 24 h period. Fate of SDZ during Soil Microcosm Incubations. Most of the added SDZ (>74%) dissipated during the five weeks following each SDZ amendment. However, the extractable 2966

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pool of SDZ remaining five weeks after the first SDZ amendment was significantly lower in soil receiving ARE than in bulk soil (P < 0.05; log-transformed data; Supporting Information Table S1). The fraction of amended SDZ that subsequently could be extracted from soil five weeks after the amendment was also significantly affected by initial SDZ loading (P < 0.001; log-transformed data). Hence a significantly higher (P < 0.05; log-transformed data) percentage of added SDZ remained at the highest initial SDZ loading.

Discussion Stimulated PICT Response in Soil Hotspots. We here provide first experimental evidence for a significantly increased bacterial community tolerance to an antibiotic following pulse addition of ARE, simulating the scenario of bulk soil getting into contact with exudates from growing roots. The weekly addition of ARE corresponded to an organic carbon amendment rate of 134 µg C g-1 d-1, which constitutes a realistic rhizodeposition rate (16, 20). Further, pulse amendments with ARE may provide for a more realistic model system than continuous ARE loading when simulating soil conditions near the surface of a growing root (20). Our study is also the first to explicitly demonstrate accelerated development of PICT to antibiotics in hotspot soil environments due to increased loading with bacterial growth substrates per se. A few previous studies have reported stimulation of PICT responses following exposure to antibiotics in soils amended with manure (11, 21) and to a lesser degree alfalfa (11). However, apart from supplying increased nutrient input to soil, manure may also provide a source of resistant microorganisms and resistance genes that may spread by horizontal gene transfer (21). The manure impact on bioavailability and thus on the selective force imposed by

toxic antibiotics in soil constitutes another confounding factor. Hence, manure will affect both chemical speciation and the complex sorption behavior of sulfonamide antibiotics in soil (22, 23) with subsequent derived effects on toxicity (24). Finally, it must be mentioned that the previous reports on stimulation of PICT responses in hotspot soil environments were based on cultivation techniques (11, 21) relying on long PICT detection phases and a low, and most likely highly variable, coverage of the soil bacterial community. It is thus interesting to note that more recent studies based on the Leu-PICT approach have failed to show significant stimulation of the PICT response by manure and alfalfa amendment to soil (25, 26). The observed increased PICT response in soil hotspots generated by ARE was not due to increased SDZ exposure. By contrast, SDZ appeared to dissipate faster under hotspot than under bulk soil conditions (Supporting Information Table S1). Considering that abiotic SDZ removal processes (27, 28) most likely were similar for all soil treatments under the prevailing experimental conditions, we assume that the observed differences between soil treatments could be explained mainly by differences in microbial activity. Hence, our data suggest that SDZ-transforming microorganisms were most active under soil hotspot conditions leading to increased microbial degradation or microbial formation of nonextractable SDZ residues (5). Comparison of PICT Methodologies. The Leu-PICT assay proved to be well-suited for sensitive detection of the PICT response following SDZ exposure, whereas the FCM-PICT assay proved insufficient of scoring SDZ sensitive cells. The used leucine incorporation protocol provides a measure of bacterial growth (protein synthesis) and has previously been successfully used for sensitive and reliable PICT determination of antibiotics and other toxicants (17, 25, 26). Growth constitutes an integrating-effect parameter that is relatively independent of the toxicant’s mode of action and may be affected at much lower toxicant concentrations than a specific metabolic activity such as an enzyme activity (29). By contrast, the FCM-PICT assay measures intracellular esterase activity, which may remain unaffected even under growth limiting conditions. The FCM-PICT protocol used in our study has previously been published as a high-throughput and sensitive approach for antibiotic tolerance testing in aquatic bacterial communities using a 24 h preincubation step that closely matched the PICT detection conditions used in our study (12). However, our data clearly show that the FCM-PICT assay is not suited for SDZ tolerance testing. In the original FCMPICT study no sulfonamide antibiotics were used and it is thus likely that a delayed growth inhibition caused by sulfonamides as compared to other antibiotics explains this difference. Most heterotrophic bacteria are capable of taking up leucine during growth (30) and the Leu-PICT method thus targets most heterotrophic soil bacteria provided that they can be extracted from soil. However, only ∼12% of Nycodenz extracted soil bacteria were esterase positive as evaluated by the FCM-PICT approach. Considering that some of the Nycodenz-extracted bacteria present in soil extracts were likely to represent dead or inactive cell stages, this nevertheless implies that the FCM-based CFDA-staining approach targeted a fairly large proportion of the extractable soil bacterial community as compared to the cultivation-dependent PICT detection methods (e.g., Biolog-PICT), which are known to be highly biased toward fast-growing, copiotrophic, bacteria (31). Bioavailability of SDZ in Soil. Tleu values up to 100% was obtained for high SDZ treatments amended with ARE (Figure 4) indicating the exclusive presence of SDZ tolerant bacteria. This strongly suggests that all extractable soil bacteria were exposed to sufficient bioavailable SDZ to selectively inhibit

the growth of sensitive soil bacteria during the PICT selection phase. However, it should be mentioned that we only studied the easily extractable fraction of the soil bacterial community and that this fraction contains more growth-active bacteria (18) and may be exposed to higher levels of bioavailable toxicants than the strongly attached fraction of the soil bacterial community (32). Comparison of PICT with Other Ecotoxicological End Points. Our results indicate the presence of extremely sensitive bacterial populations in nonadapted soil that show growth inhibition below predicted environmental SDZ concentrations for soils receiving different types of manures (33). Hence, we observed a lowest observed effect concentration (LOEC) of 0.1 µg SDZ g-1 of dry wt soil for bacterial growth 24 h after SDZ amendment to soil (Figure 2). However, the initial bacterial growth inhibition caused by SDZ did not necessarily lead to a significant PICT response as shown for the bulk soil microcosms receiving 1 µg SDZ g-1. Still, the Leu-PICT method was >100-fold more sensitive than soil respiration which was unaffected by even extremely high SDZ exposure levels (Figure 1). Apart from the differential impact of bacteriostatic agents on growth and overall energy metabolism (i.e., CO2 production) in susceptible bacteria, the lacking impact of SDZ on soil respiration may also be explained by stimulation of fungal respiration due to fungalbacterial niche overlap (34). Perspectives and Outlook. Collectively, our results suggest a marked potential for SDZ impacts on soil bacterial communities in soil hotspots with increased loading of carbon substrates. These soil hotspots may therefore also be hotspots for expansion of environmental reservoirs of bacterial resistance from where antibiotic resistance may ultimately spread to animals and humans (35). As bacteria in bulk soil are often carbon limited (36) we thus suggest that future studies of the soil bacterial resistome (37) should be directed to soil microenvironments with increased carbon substrate loading such as rhizosphere and manure-soil interfaces. Further, our study highlights the need for new standard protocols incorporating treatments with increased substrate loadings for testing impacts of bacteriostatic toxicants in soil. Specifically, we suggest the use of ARE for microbial ecotoxicity testing because of their ecological relevance and less complex side effects on soil chemistry and microbiology as compared to manure or other complex nutrient sources that have been suggested previously (11). We further suggest that the Leu-PICT assay holds potential as a future standard method for PICT detection in soil as recently suggested also by other authors (25). Finally, the [3H]leucine incorporation technique comprises a highly sensitive, relevant and economical method that may replace or at least complement the highly insensitive soil respiration tests extensively used for laboratory testing of chemicals in soil.

Acknowledgments This work was supported by Center for Environmental and Agricultural Microbiology (CREAM), funded by the Villum Kann Rasmussen Foundation. Jakob Magid is acknowledged for providing access to the CRUCIAL field site and for providing soil characterization data. Susanne Iversen and Dorthe Thybo Ganzhorn are acknowledged for excellent technical assistance.

Supporting Information Available Soil sampling and characteristics (Appendix 1). Composition of artificial root exudate solution (Appendix 2). Graphical presentation of the experimental design for the PICT selection phase (Appendix 3; Figure S1). Detailed description of FCMPICT protocol including experimental flowchart (Appendix 4; Figure S2). Detailed description of protocol for solid phase VOL. 43, NO. 8, 2009 / ENVIRONMENTAL SCIENCE & TECHNOLOGY

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extraction and subsequent chemical analysis of extracted SDZ (Appendix 5). Dynamics of heterotrophic microbial activity following addition of SDZ and ARE (Figure S3). Optimization of Leu-PICT detection assay (Figure S4). FCMPICT response of Nycodenz extracted soil bacteria (Figure S5). Representative FCM dot plot for CFDA-stained soil bacteria (Figure S6). Solid phase extractable SDZ in soil microcosms five weeks after preceding SDZ amendment (Table S1). This material is available free of charge via the Internet at http://pubs.acs.org.

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