Article pubs.acs.org/est
Multiple Discharges of Treated Municipal Wastewater Have a Small Effect on the Quantities of Numerous Antibiotic Resistance Determinants in the Upper Mississippi River Timothy M. LaPara,*,†,‡ Matthew Madson,† Spencer Borchardt,† Kevin S. Lang,§ and Timothy J. Johnson§ †
Department of Civil, Environmental, and Geo- Engineering, University of Minnesota , Minneapolis, Minnesota 55455, United States Biotechnology Institute and §Department of Veterinary and Biomedical Sciences, University of Minnesota St. Paul, Minnesota 55108, United States
‡
S Supporting Information *
ABSTRACT: This study evaluated multiple discharges of treated wastewater on the quantities of antibiotic resistance genes (ARGs) in the Upper Mississippi River. Surface water and treated wastewater samples were collected along the Mississippi River during three different periods of 4 days during the summer of 2012, and quantitative real-time PCR (qPCR) was used to enumerate several ARGs and related targets. Even though the wastewater effluents contained 75- to 831-fold higher levels of ARGs than the river water, the quantities of ARGs in the Mississippi River did not increase with downstream distance. Plasmids from the incompatibility group A/C were detected at low levels in the wastewater effluents but not in the river water; synthetic DNA containing an ampicillin resistance gene (bla) from cloning vectors was not detected in either the wastewater effluent or river samples. A simple 1D model suggested that the primary reason for the small impact of the wastewater discharges on ARG levels was the large flow rate of the Mississippi River compared to that of the wastewater discharges. Furthermore, this model generally overpredicted the ARG levels in the Mississippi River, suggesting that substantial loss mechanisms (e.g., decay or deposition) were occurring in the river.
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INTRODUCTION
these studies is to identify the key reservoirs of ARGs and then to suggest strategies to ameliorate these reservoirs. There is a growing consensus that untreated municipal wastewater, treated municipal wastewater, and residual wastewater biosolids (also known as sludge) are pertinent environmental reservoirs of ARGs.13−19 In addition, although treated municipal wastewater contains substantially fewer ARGs than untreated wastewater, numerous studies have reported that the quantities of ARGs in treated wastewater effluents are significantly higher than in unperturbed surface waters.20−26 Previous research has demonstrated, for example, that the release of tertiary-treated wastewater resulted in elevated concentrations of ARGs in the receiving body of water at relatively short distances (600 miles) of the Upper Mississippi River. These wastewater treatment facilities varied substantially with respect to design, flow rate (from 0.1 MGD
Antibiotic-resistant bacterial infections are common, expensive, and deadly.1,2 For example, one specific type of antibacterialresistant infection, methicillin-resistant Staphylococcus aureus (MRSA), is responsible for more deaths in the United States than emphysema, HIV/AIDS, Parkinson’s disease, and homicide combined.2,3 The overall cost of additional medical treatments necessitated by antibiotic resistance is estimated to be $20 to $40 billion each year in the United States.2,3 To help solve this problem, there are ongoing investigations to discover new antibiotics4,5 as well as various initiatives to reduce inappropriate antibiotic use.6 Historically, antibiotic resistance has been strictly viewed as a medical problem in which antibiotic use was typically identified as the primary cause of the development and evolution of antibiotic resistance.6 Although antibiotic use is certainly a pertinent factor that helps select for antibiotic resistance, there is now considerable interest in gaining a broader understanding of the factors controlling the proliferation of antibiotic resistance. Many researchers, therefore, have begun investigating environmental factors in the spread of antibiotic resistance, with particular interest in tracking and understanding the fate of antibiotic resistance genes (ARGs).7−12 The explicit goal of © XXXX American Chemical Society
Received: June 8, 2015 Revised: August 21, 2015 Accepted: September 1, 2015
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then extracted and purified from these samples using the FastDNA Spin Kit (MP Biomedicals; Solon, OH) according to the manufacturer’s instructions. All genomic DNA extractions were performed in triplicate and stored at −20 °C until needed. Quantitative PCR. qPCR was used to quantify several ARGs, including three different tetracycline resistance genes (tet(A), tet(W), and tet(X)), an erythromycin resistance gene (erm(B)), a sulfonamide resistance gene (sul1), and a fluoroquinolone resistance gene (qnrA). Prior work has demonstrated that all six of these ARGs are present at substantial concentrations in wastewater or wastewater solids.18,21,23,28−30 In addition, qPCR was used to quantify the integrase gene of class 1 integrons (intI1), plasmids from incompatibility group A/C (Inc A/C plasmids), and synthetic DNA containing an ampicillin resistance gene (bla) from cloning vectors. Prior work has detected intI1 genes in wastewater and surface water samples,23,31 and the synthetic bla genes were detected in six Chinese rivers.32 In contrast, Inc A/C plasmids have been primarily detected only in enteric bacteria associated with humans and animals;33 this study represents an early attempt to quantify its presence in surface water and in treated wastewater samples. The sequence of an Inc A/C plasmid can be found at GenBank under accession number NC_012692.1. Additional information regarding the use of qPCR to quantify these genes can be found in Table S3. The qPCR analysis was conducted using an Eppendorf Mastercycler ep realplex thermal cycler (Eppendorf; Westbury, NY). Each qPCR run consisted of 10 min of denaturation at 95 °C, followed by 40 cycles of denaturation at 95 °C for 15 s and a combined annealing and extension at a specified temperature for 1 min. A 25 μL sample of reaction mixture contained 12.5 μL of BioRad iTaq SYBR Green Supermix with ROX (Life Science Research, Hercules, CA), 25 μg of bovine serum albumin, optimized quantities of forward and reverse primers, and approximately 1 ng of template genomic DNA. Each qPCR result reported herein consists of single qPCR assays performed on triplicate genomic DNA extractions. The quantity of target DNA in unknown samples was calculated based on a standard curve generated using known quantities of template DNA. Standards for qPCR were prepared by PCR amplification of genes from positive controls, followed by ligation into pGEM-T Easy vectors following the manufacturer’s instructions (Promega; Madison, WI), and transformation into E. coli JM109. Plasmids were purified using the alkaline lysis procedure.34 Plasmid DNA was quantified by staining with Hoechst 33258 dye and measured on a TD-700 fluorometer (Turner Designs; Sunnyvale, CA) using calf thymus DNA as a standard. Then, 10-fold serial dilutions of plasmid DNA were prepared and run on the thermal cycler to generate standard curves (r2 > 0.99). Amplification efficiencies were calculated based on the slope of the standard curve; amplification efficiencies were typically 100% ± 15%. The inhibition of amplification for individual samples was monitored by visually comparing the amplification curves of the unknown samples versus the standards. Following qPCR, the melting curves were generated and analyzed to verify that nonspecific amplification did not occur. Data Analysis. All statistical analysis was performed using JMP 11.2.0 (SAS; Cary, North Carolina). Preliminary analysis of the qPCR data suggested that it was described by a lognormal distribution. Statistical comparisons between pairs of data were made via the Student’s t-test of log-transformed data;
to 180 MGD), treatment facility design, and (presumably) treatment quality. Similarly, the terrain and land use through which the Mississippi River meanders ranges from undeveloped northern forest (starting at its headwaters) to a highly urbanized area (the Twin Cities metropolitan region) to the Driftless Area of southeast Minnesota, which is less urbanized but also includes the Minnesota River basin, which is heavily impacted by agricultural activity.
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MATERIAL AND METHODS Sample Collection. Surface water and treated wastewater samples were collected from different locations along the Mississippi River (Figure 1) during three separate periods of 4
Figure 1. Map of the State of Minnesota showing the locations where wastewater effluents are discharged to the Mississippi River, as well as locations along the Mississippi River where samples were collected for this study. Letters identify the locations of the river water samples; numbers identify the locations where the wastewater effluents enter the Mississippi River. Additional information on these sites can be found in Tables S1 and S2.
days in May, July, and August of 2012 using sterile polystyrene bottles. Surface water samples were collected manually at a distance of 0.5 m below the water surface; treated wastewater samples were collected from within the treatment facilities after the disinfection process but prior to discharge. Samples were stored on ice until they could be processed. Brief descriptions of each wastewater treatment facility and of each sample location on the Mississippi River are provided in Tables S1 and S2. Ancillary water quality data from the Mississippi River can be found elsewhere in the literature.27 As soon as possible after collection (2−6 h), samples were passed through a 47 mm diameter membrane filter (pore size of 0.22 μm) to concentrate the microbial biomass. Filters were then immersed in 0.5 mL of lysis buffer (120 mM phosphate buffer, pH of 8.0, 5% sodium dodecyl sulfate) and stored at −20 °C to preserve the sample until the genomic DNA could be extracted and purified. Genomic DNA Extraction. Samples (preserved in lysis buffer) underwent three consecutive freeze−thaw cycles and an incubation of 90 min at 70 °C to lyse cells. Genomic DNA was B
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as well as the integrase gene of Class 1 integrons (intI1) were also measured in the wastewater effluent and river samples (Figure 3; Tables S7−S34). In each, the levels of ARGs and
analogous comparisons of pairs within a matrix of different pairs were made using the Tukey HSD test. Modeling. The Mississippi River was mathematically simulated using a 1D plug-flow model. Flow rates in the Mississippi River were obtained for 11 (July and August of 2012) or 13 (May of 2012) stations maintained by the United States Geological Survey (waterdata.usgs.gov) or the United States Army Corps of Engineers (www.mvp-wc.usace.army.mil) for the dates during which samples were collected; this information is included in Table S4. A linear increase in flow rates was assumed between stations. All increases in flow to the Mississippi River, except for the wastewater treatment inputs, were assumed to have a uniform background concentration of ARGs equal to the geometric mean of the values measured at the three northernmost sample locations. ARGs released with treated wastewater were assumed to be conserved (i.e., no decay, deposition into the river sediment, or other loss mechanisms were assumed) because there is no information in the technical literature, to our knowledge, to suggest the rates at which these loss mechanisms occur.
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Figure 3. Comparison of the quantities of numerous ARGs and the integrase gene of Class 1 integrons (intI1) in wastewater effluent samples (n = 42) and in Mississippi River samples (n = 35). Numbers show the ratio of each target gene in the wastewater effluent samples compared to that in the Mississippi River samples.
RESULTS Bacterial Biomass. The quantity of bacterial biomass (measured as 16S rRNA genes) was measured at 12 different locations along the Upper Mississippi River and in the treated wastewater effluents from 14 different wastewater treatment facilities that discharge to the Upper Mississippi River in May, July, and August of 2012 (Figure 2 and Tables S5−S6).
intI1 were significantly higher in the wastewater effluents compared to the river samples (P < 0.0001). In contrast to the bacterial biomass levels, in which only a 4-fold difference was observed between wastewater effluents and river samples, there were much greater differences in the quantities of ARGs in the wastewater effluents compared to those in the river samples. These differences ranged from as low as 75-fold higher levels of qnrA in wastewater effluents compared to those in the river samples to as high as 831-fold levels of tet(X) in the wastewater effluents compared to those in the river samples. In pairwise comparisons of all ARGs and intI1 quantities in all of the wastewater effluent samples (n = 3), a handful of wastewater effluents were significantly higher or lower in quantity than other treatments for specific gene targets, but no wastewater treatment facility was consistently higher or lower than all of the other facilities. In addition to the aforementioned gene targets, qPCR was also used to quantify the levels of Inc A/C plasmids and a synthetic plasmid−ampicillin resistance gene (bla) that had been previously detected in six Chinese rivers.32 Inc A/C plasmids were detected in 28 of the 35 treated wastewater samples at generally low concentrations (arithmetic mean ± standard deviation of log10-transformed values =2.5 ± 0.7), but no Inc A/C plasmids were detected in any of the river samples (Table S35). Similarly, no bla targets were detected in any of the wastewater effluent or river samples. Effect of Wastewater Effluents on ARG Levels in the Mississippi River. A simple 1D model was developed and applied on each of the sample collection dates to better understand the relative impact of the discharge of ARGs in the treated wastewater effluents into the Mississippi River for each of the three sample collection periods (Figure 4). For the May of 2012 sample collection period, the model described the ARG levels in the Mississippi River downstream of the Twin Cities metropolitan region (>500 miles) reasonably well. The model, however, typically overestimated the quantities of ARGs just upstream of the Twin Cities, where the measured levels of ARGs were typically lowest for this sample collection period
Figure 2. Quantities of 16S rRNA genes in wastewater effluent and Mississippi River samples collected in May (red), July (green), and August (blue) of 2012 . Open symbols identify Mississippi River samples; closed symbols identify wastewater effluent samples.
Applying ANOVA to each of the sample locations (i.e., the wastewater effluents and the Mississippi River) suggested that no sample type (i.e., wastewater effluent versus river), location, or time was significantly higher or lower in quantity than any other sample location or time, probably caused by the limited number of samples (n = 3) collected at each location or time. In contrast, when grouping samples together, the treated wastewater effluent samples (n = 42; arithmetic mean ± standard deviation of log10-transformed values =7.9 ± 1.0) had subtle yet significantly higher quantities of bacterial biomass than the Mississippi River samples (n = 35; arithmetic mean ± standard deviation of log10-transformed values = 7.3 ± 1.0) (P = 0.004). There was also a strong seasonal effect, as the quantities of biomass in both the wastewater effluents and in the river samples were significantly higher in July and August of 2012 than in May of 2012 (P < 0.0001). Antibiotic Resistance Genes. The concentrations of 6 different ARGs (tet(A), tet(W), tet(X), sul1, erm(B), and qnrA) C
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Figure 4. Model predictions (solid lines) and measured quantities of numerous ARGs and the integrase gene of Class 1 integrons (intI1) in Mississippi River samples on three different dates. A, May of 2012; B, July of 2012; C, August of 2012.
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(Figure 4A). In contrast, the model generally predicted much higher levels of ARGs in the Mississippi River than were measured in July of 2012 (Figure 4B), when river flow rates were about half of their May of 2012 levels. The model predictions for this period were heavily influenced by very high levels of ARGs released from the Brainerd wastewater treatment facility, which was experiencing a catastrophic process upset at the time. The model predictions for the August of 2012 sample period were also generally higher than the measured values (Figure 4C). For this sample period, the river flow rates were approximately half of that reported in July of 2012, such that the predicted impact of each wastewater effluent was much more pronounced than what was empirically measured. In conclusion, in spite of the multiple inputs of wastewater effluents, ARGs levels in the Mississippi River were relatively static as a function of downstream distance, such that model predictions of ARG levels in the Mississippi River again exceeded measured values.
DISCUSSION
There is growing acceptance that wastewater treatment effluents are significant point sources of ARGs to surface waters.20−26 Most of these studies to date, however, have examined only a small number of wastewater treatment outfalls over relatively short distances. In contrast, the goal of the present study was to measure the impact of multiple wastewater treatment discharges over more than 600 miles of the Mississippi River. Although wastewater effluents contained very high levels of ARGs, empirical evidence showed that these inputs of ARGs had little or no effect on the ARG quantities in the river. We speculate that there are two primary reasons for this surprising result. First, the flow rate of the Mississippi River was very large compared to the wastewater discharge rates (the largest wastewater effluent flow rate was 30) had been collected. Additional research, therefore, is needed to identify the best operational strategies and treatment technologies for limiting the release of ARGs in municipal wastewater treatment effluents. In this vein, additional research is particularly needed to limit the discharge of ARGs (via disinfection, filtration, etc.) from municipal wastewater treatment facilities.10 This study is one of the first to quantify the presence of plasmids belonging the A/C incompatibility group (Inc A/C) in surface waters and in treated municipal wastewater. These plasmids are large, broad-host-range plasmids, which contain genes that confer resistance to many different classes of antibiotics, including phenicols, tetracyclines, and extendedspectrum β-lactam antibiotics, including third-generation cephalosporins and carbapenems.33,36 To our knowledge, only one previous study has detected Inc A/C plasmids in wastewater and surface waters.37 Our research confirms that treated municipal wastewater is a potential source of these plasmids, but it is unclear whether these plasmids remain viable or propagate in the environment because none of the Mississippi River water samples contained detectable levels of Inc A/C plasmids. Similarly, we decided to investigate the quantities of synthetic bla genes that had been previously detected at relatively high levels (103 copies per mL) in six Chinese rivers.32 The authors of that study suggested that laboratory waste that had been improperly disposed of was the source of this material. Our failure to detect any of these genes in any of the wastewater effluent or river water samples is consistent with this interpretation, assuming that stringent regulations on destroying laboratory waste containing recombinant DNA are followed throughout the State of Minnesota. A pertinent limitation of this research is that the identity and physiological state of the organisms harboring ARGs remains unknown. Although there is increasing evidence that nonpathogenic microorganisms are a pertinent reservoir of ARGs,38,39 the presence of ARGs in pathogens is far more worrisome. Similarly, the ARGs detected in this study could have been in intact but nonviable cells. The concern of ARGs in nonviable cells is particularly likely for disinfected wastewater effluents, in which bacteria are exposed to lethal levels of chlorine or ultraviolet (UV) radiation, although a recent study suggested that UV disinfection directly damaged cellular DNA such that it was not available for detection by qPCR.40
drought periods.35 Second, ARGs discharged from wastewater treatment facilities undoubtedly have significant loss mechanisms, such as natural decay and deposition. The rates and mechanisms by which ARGs would decline in a riverine system, such as that studied herein, are poorly understood at the present time and need further investigation; it is likely that a better understanding of these loss mechanisms would allow our simple model to accurately predict ARG levels in the Mississippi River and other water bodies affected by multiple wastewater effluents. Our research demonstrates that municipal wastewater treatment facilities discharge water of similar quality to the receiving stream with respect to the quantity of bacterial biomass. This is particularly pertinent because the wastewater treatment facilities investigated herein are not currently regulated with respect to the discharge of ARGs, but they are regulated with respect to the levels of suspended solids and of pathogens in their effluents, which presumably correlate to the quantities of total bacteria. These regulations on suspended solids and pathogenic microorganisms have been established under the general paradigm of using wastewater treatment to produce effluent water that does not adversely affect the quality of the surface water to which it is discharged; our data supports the conclusion that the wastewater treatment facilities in this study satisfy this general paradigm for wastewater treatment with respect to quantities of bacterial biomass (except for the catastrophic process upset that occurred in Brainerd in July 2012). In spite of having relatively similar levels of 16S rRNA genes, our research unequivocally demonstrates that municipal wastewater treatment discharges are extraordinarily rich in ARGs compared to Mississippi River water. This study is relatively unique in that it queried a relatively large number of wastewater treatment effluents (n = 42) and surface water samples (n = 35), generating a relatively large data set that was particularly powerful for statistical analysis. Because it is unlikely that treatment technologies will be developed that specifically target ARGs, our research suggests that the total numbers of bacteria in wastewater effluents would need to be substantially lower than those in the receiving water body to eliminate wastewater treatment facilities as a pertinent point source of ARGs. An implicit assumption of the research performed herein is that the Mississippi River flowed from a relatively pristine area, beginning from its headwaters, into an urban area and then into an area heavily impacted by agricultural activity. In contrast, the quantities of ARGs were relatively static as a function of location along the Mississippi River. As such, it seems that neither wastewater effluents nor agricultural activity have a significant effect on the levels of ARGs in the Mississippi River. The input of ARGs to rivers from agriculture, however, could occur in short-term, hot moments following the application of manure, unlike municipal wastewater effluents that are discharged continuously; as such, the three relatively short sample periods used in the present study could have missed the effects of ARG inputs from agriculture. Alternatively, our assumption that the northernmost area of the Mississippi River was relatively pristine could be erroneous. Graham et al.22 suggested that pristine areas exhibit a ratio of tetracycline resistance determinants to 16S rRNA genes of 10−6 to 10−8, whereas for highly contaminated sites, this ratio is >10−4. In our study, these ratios for tet(A), tet(X), and tet(W) were 10−5.1,
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ASSOCIATED CONTENT
S Supporting Information *
The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.est.5b02803. E
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Additional information on the wastewater treatment facilities (Table S1), locations along the Mississippi River from which samples were collected (Table S2), methods for qPCR (Table S3), flow rates in the Mississippi River on the dates of sample collection (Table S4), qPCR data for 16S rRNA genes in the wastewater effluents and Mississippi River samples (Tables S5−S6), and qPCR data for all other ARGs and related gene targets (Tables S7−S35). (PDF)
AUTHOR INFORMATION
Corresponding Author
*Phone: (612) 624-6028; fax: (612) 626-7750; e-mail:
[email protected]. Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS This work was financially supported by the National Science Foundation (CBET-0967176). REFERENCES
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