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Effect of wastewater colloids on membrane removal of antibiotic resistance genes
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Available online at www.sciencedirect.com
journal homepage: www.elsevier.com/locate/watres
Effect of wastewater colloids on membrane removal of antibiotic resistance genes Maria V. Riquelme Breazeal, John T. Novak, Peter J. Vikesland, Amy Pruden* Via Department of Civil and Environmental Engineering, Virginia Tech, Blacksburg, VA 24061, USA
article info
abstract
Article history:
Recent studies have demonstrated that wastewater treatment plants (WWTPs) signifi-
Received 21 June 2012
cantly alter the magnitude and distribution of antibiotic resistance genes (ARGs) in
Received in revised form
receiving environments, indicating that wastewater treatment represents an important
21 September 2012
node for limiting ARG dissemination. This study examined the potential for membrane
Accepted 22 September 2012
treatment of microconstituent ARGs and the effect of native wastewater colloids on the
Available online 4 October 2012
extent of their removal. Plasmids containing vanA (vancomycin) and blaTEM (b-lactam) ARGs were spiked into three representative WWTP effluents versus a control buffer and
Keywords:
tracked by quantitative polymerase chain reaction through a cascade of microfiltration and
Antibiotic resistance genes
ultrafiltration steps ranging from 0.45 mm to 1 kDa. Significant removal of ARGs was ach-
Wastewater colloids
ieved by membranes of 100 kDa and smaller, and presence of wastewater colloids resulted
Microfiltration
in enhanced removal by 10 kDa and 1 kDa membranes. ARG removal was observed to
Ultrafiltration
correlate significantly with the corresponding protein, polysaccharide, and total organic carbon colloidal fractions. Alumina membranes removed ARGs to a greater extent than polyvinylidene fluoride membranes of the same pore size (0.1 mm), but only in the presence of wastewater material. Control studies confirmed that membrane treatment was the primary mechanism of ARG removal, versus other potential sources of loss. This study suggests that advanced membrane treatment technology is promising for managing public health risks of ARGs in wastewater effluents and that removal may even be enhanced by colloids in real-world wastewaters. ª 2012 Elsevier Ltd. All rights reserved.
1.
Introduction
Antibiotic resistance and, particularly, multidrug resistance, is an increasingly critical problem affecting human health (Davies and Davies, 2010). Globally, antibiotic resistance has been reported to be widespread (Allen et al., 2010) and on the rise (Zhang et al., 2006). Although most attention has been focused on nosocomial (i.e., hospital acquired) infections, environmental factors are also important contributors to the transport and spread of antibiotic resistance in the community (Larson, 2007). Recent studies have demonstrated that wastewater treatment
plants can significantly alter the magnitude and distribution of antibiotic resistance genes (ARGs) in receiving environments (Storteboom et al., 2010). Given that antibiotic resistant bacteria (ARB) and ARGs of clinical concern have been documented to arise from environmental sources (Martinez, 2008), there is growing interest in limiting dissemination pathways between humans and the environment (Baquero et al., 2008). Thus, wastewater treatment plants may represent an important node for limiting the spread of antibiotic resistance. The presence of low levels of ARB and ARGs in wastewater is inevitable and several studies have documented their
* Corresponding author. Tel.: þ1 540 231 3980. E-mail addresses: [email protected] (M.V. Riquelme Breazeal), [email protected] (J.T. Novak), [email protected] (P.J. Vikesland), apruden@engr. colostate.edu, [email protected] (A. Pruden). 0043-1354/$ e see front matter ª 2012 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.watres.2012.09.044
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proliferation and persistence in wastewater treatment plants (Auerbach et al., 2007; Borjesson et al., 2010; Kim et al., 2010; Szczepanowski et al., 2009; Zhang et al., 2009). Traditionally the objective of disinfection following water or wastewater treatment is to kill or inactivate bacterial cells. However, to address the challenge of antibiotic resistance, DNA removal or destruction is ideal prior to discharge, reuse, or other applications. This is because ARGs are typically carried on highly transmissible DNA elements, such as plasmids and integrons, which can remain functional and be assimilated by downstream bacteria. Because of this ability of ARGs to transcend their bacterial host, ARGs themselves are considered to be the primary “contaminant” of concern (Pruden et al., 2006). Once released, ARGs can persist in the environment (Biyela et al., 2004; Storteboom et al., 2010), and have even been observed to establish and proliferate in drinking water biofilms (Schwartz et al., 2003; Xi et al., 2009). Although the effect of some disinfection techniques on the fate of ARGs has been investigated in a few studies (Munir et al., 2011; Oncu et al., 2011), the fate of these microconstituents after wastewater disinfection and release is still largely unknown. The fate of ARGs through membrane removal processes is of particular interest as membranes are growing in popularity for enhancing sustainability of water resources and addressing concerns for other emerging contaminants (Dolar et al., 2009, 2012; Munir et al., 2011; Wintgens et al., 2004). The present study is the first to our knowledge to examine the potential for membrane removal of ARGs. Membrane filtration processes are commonly used for the removal of a wide variety of contaminants, but most would not be expected to impart significant removal of DNA based on its molecular properties. However, wastewater effluents are complex in their chemistries and could significantly affect membrane removal of DNA. Thus, the focus of this study was on the potential for the advanced membrane treatment of ARGs within real-world wastewater effluent matrices. Of specific interest to this study was the effect of DNA interactions with wastewater effluent colloidal particles. DNA is well known to interact with clay minerals and various soil colloidal particles (Cai et al., 2007, 2006a, 2006b; Crecchio and Stotzky, 1998; Nguyen et al., 2010; Nguyen and Elimelech, 2007a,b; Romanowski et al., 1991; Saeki et al., 2010). Considering that significant amounts of suspended colloidal particles are present in wastewaters, these interactions represent a promising and unexploited approach for enhanced removal of ARGs from wastewater. However, most of the available studies concerning DNAecolloid interactions have been carried out in simplified or artificial systems (Mitra et al., 2001; Nguyen and Elimelech, 2007a,b; Romanowski et al., 1991); thus, there is a need for studies that emphasize the behavior and fate of DNA in situ. In addition, the relationship between the removal of ARGecolloid complexes and membrane pore size has not been investigated in natural systems. The overall objective of this project was to investigate the effect of wastewater colloids on the removal of ARGs by membrane filtration. It was hypothesized that colloidal interactions with DNA would enhance membrane removal of ARGs. To test this hypothesis, plasmids containing vanA and blaTEM ARGs were spiked into three representative WWTP effluents and a control buffer, and subjected to a cascade of
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microfiltration (MF) and ultrafiltration (UF) steps ranging from 0.45 mm to 1 kDa. The vanA and blaTEM ARGs encode resistance to vancomycin and b-lactam antibiotics (e.g., ampicillin), respectively, with the former used typically as a last-resort antibiotic when other treatments fail. Other potential sources of DNA loss, such as degradation, were also monitored to verify that observed ARG removal was due to the membrane treatment and also to examine the potential for wastewater colloids to protect ARGs from deoxyribonuclease (DNase), a naturally-occurring DNA-degrading enzyme. The effect of membrane composition (alumina versus polyvinylidene fluoride) was compared for the 0.1 mm pore size. Building a knowledge base of the effect of membrane treatment on ARGs is critical for enabling comprehensive management strategies for emerging contaminants in wastewater effluents.
2.
Materials and methods
A cascade of MF and UF steps was used to compare the removal of ARGs from colloid-containing wastewater effluent versus colloid-free aqueous buffer samples. Effluents from three representative WWTPs were employed in order to test the occurrence of DNAecolloid interactions in a complex environmental matrix, as well as the reproducibility of the results. Tris buffer (pH 8) was used as colloid-free material in order to maintain DNA stability in the controls throughout the experiment, as low salt concentrations could cause DNA instability, while high salt concentrations can promote the sorption of the genetic material to the glass containers. The key characteristics of the WWTPs selected for this study and effluent quality are summarized in Table 1. An additional experiment was carried out to further explore the potential for DNAecolloid interactions to protect ARGs from degradation by DNase.
2.1.
Plasmid solution preparation
The pCR 4-TOPO plasmids (Invitrogen, Carlsbad, CA) containing the ARGs of interest were used to spike the appropriate test solutions. This plasmid contains blaTEM and was modified to contain vanA. A 732 bp portion of the vanA gene was PCRamplified from vancomycin-resistant Enterococcus genomic DNA using previously described primers (Dutka-Malen et al., 1995). The 25 mL PCR amplification reaction contained 1 Master Taq reaction buffer (5 Prime, Gaithersburg, MD), 1 Taq Master PCR enhancer (5 Prime), 1.5 mM Mg2þ solution (5 Prime), 0.05 mM of each deoxynucleoside triphosphate (Promega, Madison, WI), 0.2 mM of each primer, 1.75 U Taq DNA polymerase (5 Prime), 1 mL template DNA, and autoclaved nanopure water to a final volume of 25 mL. The thermal cycle consisted of an initial 3 min denaturing step at 95 C; followed by 50 cycles of a 30 s denaturing step at 95 C, a 30 s annealing step at 54 C, and a 30 s extension step at 72 C; and a 7 min final extension step at 72 C. The vanA PCR product was ligated to the pCR 4-TOPO plasmid and cloned using the TOPO TA cloning kit (Invitrogen), following the manufacturer’s instructions. Plasmids were extracted using the Qiagen Plasmid Mega kit (Qiagen, Valencia, CA) and quantified by qPCR targeting blaTEM and vanA ARGs.
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Table 1 e General WWTP and effluent characteristics and effluent sampling dates. WWTP
Capacity (MGD)
Key processesb
Influent composition
TSSb (mg/L)
pHb
Otherb (mg/L)
Collection date 4/21/11
4/7/11
A
6
Residential, industrial
Conventional,a UV disinfection
7.7
7.1
B
9
Conventional,a chlorine disinfection
2
7
C
6
Residential, hospital, industrial Residential, hospital, high Na2SO4 industrial
DO (8.1) BOD (19.6) DO (9.5)
Conventional,a nitrification, chlorine disinfection
7
6.7
BOD (<5)
3/1/11
An additional sampling event occurred on 1/30/12 from all WWTPs, for DNase-degradation test. a Conventional treatment: primary clarification, aeration, secondary clarification, and disinfection. b WWTP and effluent characteristics obtained by phone from WWTP operators.
2.2.
WWTP effluent collection and initial filtration
Grab samples of final effluent were collected from each of the test wastewater treatment facilities (WWTPs A, B, or C) at most 6 h before the experiment was carried out. Effluent collection occurred during cool (2e15 C) working week mornings, avoiding rain or snow events taking place during the previous 24 h. The effluent was immediately transported to the lab where it was kept at 4 C prior to use. As a pretreatment to eliminate particles above the colloid size range, the effluent was vacuum-filtered through a 1.2 mm pore-size mixed cellulose ester membrane (Millipore, Billerica, MA) using a sterilized Nalgene polysulfone filter holder (Thermo Fisher Scientific, Rochester, NY).
2.3.
Cascading filtration experiments
Each of the WWTP effluents was subjected to cascading filtration experiments and compared with appropriate controls: (1) Autoclaved 10 mM Tris buffer pH 8 (blank); (2) Autoclaved 10 mM Tris pH 8 spiked with vanA gene-containing plasmids to a final concentration of 1 107 to 1 108 plasmid copies/mL based on blaTEM gene concentrations (no-colloid control); (3) filtered WWTP effluent (background); and (4) filtered WWTP effluent spiked with vanA gene-containing plasmids to a final concentration of 1 107 to 1 108 plasmid copies/mL based on blaTEM gene concentrations (wastewater colloid treatment). Four 1000-mL flasks were filled with 600 mL of corresponding solution and lightly agitated for 4e5 h using a Burrell wrist action shaker (Burrell Scientific, Pittsburg, PA) to enable DNA interactions with wastewater colloids prior to filtration. 10-mL subsamples were collected from each of the four flasks during this stage to test for alternative sources of DNA loss. Sequential cascading filtration steps were carried out for each of the four flask contents. First, 500 mL were filtered through the membrane with the largest pore size (0.45 mmpore size membrane). Next, 10-mL sub-samples of the filtrate were pipetted into previously baked 30-mL Qorpak (Bridgeville, PA) clear graduated glass bottles and immediately frozen at 80 C for subsequent lyophilization and DNA extraction. Filtrate sub-samples originating from the unspiked WWTP effluent were also collected for colloid characterization and stored at 4 C. The remaining water/buffer filtrate was then
filtered through the subsequent membrane (0.1 mm-pore size). The process was repeated for each of the filtrates, applying the membranes in order of decreasing pore size: 0.45 mm / 0.1 mm / 100 kDa / 10 kDa / 1 kDa. To minimize carryover, the flask contents were processed in the following order: (1) buffer blank, (2) unspiked background wastewater, (3) spiked no-colloid control buffer, (4) spiked wastewater containing colloids. The latter was processed after the 4-h period allotted for DNAecolloid interactions to occur. MF was carried out using 0.45 mm and 0.1 mm pore size Durapore hydrophilic polyvinylidene fluoride (PVDF) membranes (Millipore, Billerica, MA), and a Nalgene polysulfone filter holder (Thermo Fisher Scientific, Rochester, NY). To compare the effect of membrane type, Anodisc 47 alumina 0.1 mm pore size membranes (Whatman GmbH, Germany) were compared to the Durapore membrane of the same pore size. UF was carried out using 100 kDa, 10 kDa, and 1 kDa MWCO Ultracel regenerated cellulose membranes (Millipore, Billerica, MA) and an 8200 Ultrafiltration Stirred Cell (Millipore). UF filtrates flowed into baked glass flasks shielded by baked aluminum foil. Because the UF membranes were reusable, they were cleansed between usages using DNA Away (Molecular Bio Products, San Diego, CA) followed by a thorough rinsing step that included filtering water through the membrane for at least 10 min. A list of the membrane types and pore sizes is provided in Table S1.
2.4.
DNA stability in pretreated wastewater and buffer
To determine the extent to which membrane filtration accounted for removal of ARGs in the above experiments, changes in gene concentrations in pretreated wastewater and buffer were monitored over the time allotted for DNAecolloid interactions prior to filtration. A 10-mL sample was collected immediately after plasmid spiking, and 2e3 additional samples were collected during the following 4e5 h. Gene fractions at times t > 0 were calculated with respect to the initial concentration measured at t ¼ 0 h.
2.5.
DNase-degradation experiment
The potential for colloideDNA interactions to protect ARGs from degradation was investigated using DNase-degradation experiments. All three WWTP effluents were sampled on the
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same day and compared in parallel to the colloid-free Tris buffer control. 500 mL of either Tris buffer (pH 8) or the filtered effluents were collected in fluorinated HDPE Nalgene bottles (Thermo Fisher Scientific, Rochester, NY) and spiked to a concentration of 1 107 plasmids/mL, and placed in a shaking incubator at 125 rpm and room temperature for 4 h to allow DNAecolloid interactions. Sub-samples were collected at 0, 2, and 4 h to test for background DNA losses. After 4 h, 200 mL of the contents of each bottle (i.e., buffer or WWTP effluent) were split into two new bottles (100 mL each), one of which was spiked with 10.2 U of RQ1 RNase-free DNase (Promega) and subject to 2.5 mM MgCl2 and 0.5 mM CaCl2 at 37 C, and the other only subject to the same salt and temperature conditions used to activate the DNase. The bottles were incubated at 37 C and shaken at 125 rpm. This approach allowed direct comparison of the effect of the DNase versus the background salt and temperature conditions required to activate the DNase. 10 mL sub-samples were collected after light manual vortexing, immediately after salt and/or DNase addition (i.e., shortly after hour 4), and at 5 and 7 h from the beginning of the experiment. All collected samples were immediately stored at 80 C for further analysis.
2.6.
DNA concentration and extraction
All samples were concentrated by lyophilization using a FreeZone Plus 2.5 L Benchtop Freeze Dry System (Labconco, Kansas City, Missouri). Isolation of DNA originating from WWTP effluent samples was carried out using the FastDNA Spin Kit for Soil (MP Biomedicals, Solon, OH) as recommended in the kit manual. Isolation of DNA originating from buffer samples was carried out using the PowerClean DNA Cleanup Kit (MoBio Laboratories, Carlsbad, CA).
concentrations in 10 reaction blanks and multiplying that value by 3. The background gene concentrations detected in each analytical blank was subtracted from the measured sample concentration. Each individual measurement greater than the detection limit was then multiplied by the dilution factor (10) and the extraction volume, and divided by the volume of each sample in microliters (1 104 mL) to obtain the final concentration in gene copies/mL of each sample. The three analytical measurements per sample were then averaged to obtain the estimated concentration for that sample.
2.8.
Colloid characterization
Non-purgeable organic carbon concentrations were measured using the Shimadzu TOC-VCSN analyzer (Shimadzu Scientific Instruments, Columbia, MD) and potassium hydrogen phthalate (KHP) as the standard. Polysaccharide concentrations were measured by the phenolesulfuric acid method (Dubois et al., 1956) using dextrose as standard. Protein concentrations were measured by the Lowry method (Lowry et al., 1977) using bovine serum albumin (BSA) as the standard. Briefly, five reagents were prepared as follows e Reagent 1: 143 mM NaOH and 270 mM Na2CO3; Reagent 2: 57 mM CuSO4; Reagent 3: 124 mM Na-tartrate; Reagent 4: mix of reagents 1, 2, and 3 at a ratio of 100:1:1 (made daily); and Reagent 5 contained folin ciocalteus reagent diluted 5:6 with distilled water. 1-mL samples were added by sequential addition and mixing of Reagents 4 and 5, respectively. The samples containing the reagents were incubated for 45 min at room temperature before measuring the absorbance at 750 nm.
2.9. 2.7.
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Statistical analyses
DNA quantification by qPCR
Quantification of vanA and blaTEM was carried out using realtime quantitative polymerase chain reaction (qPCR) on a CFX96 Touch Real-Time PCR Detection System (Bio-Rad, Hercules, CA). 1:10 DNA extract dilutions were analyzed in triplicate reactions containing 40 nM of each primer, 1 SsoFast EvaGreen Supermix (Bio-Rad), 1 mL template DNA, and sterile nanopure to a final volume of 10 mL. The primers used for amplification of the vanA gene were vanAstF (Bo¨ckelmann et al., 2009) and vana3RP (Volkmann et al., 2004). The blaTEM gene was amplified using previously described primers (Bibbal et al., 2007). The temperature programs consisted of a 95 C initial denaturing step for 30 s; 40 cycles of denaturing at 95 C for 5 s, annealing and extension at 60 C for 5 s, and an additional extension step at 72 C for 5 s for vanA amplifications only. A melt curve was incorporated in every reaction to verify specificity by ramping the temperature from 65 C to 95 C by 0.5 C every 5 s. A calibration curve was constructed for each set of reactions using at least five standards. The qPCR standards were prepared by serially diluting M13 PCR products obtained from cloned positive controls (vanA or blaTEM). Concentrations were determined by gel densitometry and converted to gene copies using the relationship described by Pei et al. (2006). A limit of detection (LOD) was calculated for each gene by averaging gene
Statistical analyses were performed using R statistics software (www.r-project.org). Statistical differences between the membrane-removal of ARGs in the presence versus the absence of colloids were calculated using a two-way analysis of variance (ANOVA). The two-way ANOVA was applied to the log of the filtrate gene fraction from spiked WWTP effluent or spiked buffer as the dependent variable. The filtrate gene fraction was calculated as C/C0, where C was the gene concentration in the filtrate and C0 was the average gene concentration in the unfiltered samples used to test for degradation. The presence/absence of colloids and the membrane pore size or type were independent variables. The analyses were repeated individually for each of the genes monitored. To test the significance of the DNA degradation in the water or buffer samples prior to treatment (i.e., prior to filtration or application of DNase-degrading conditions), sampling times after the addition of the plasmid spike were binned as A (1e2 h), B (2.5e3 h), and C (4e5 h). A one-way ANOVA test was applied to the log of the gene fractions present in the water or buffer samples for each bin. Statistical significance was set at a ¼ 0.05 ( p < 0.05). Correlation analysis between the filtrate fraction of each of the colloidal components and the logarithm of the filtrate gene fraction was conducted using R, applying a one-sided Pearson correlation test.
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3.
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Results
3.1. Membrane removal of ARGs and effect of wastewater colloids Sequential filtration across decreasing membrane pore sizes resulted in increasing removal of plasmid-associated ARGs (Fig. 1). Specifically, filtration through the 0.45 and 0.1 mm pore-size microfiltration membranes resulted in less than 1-log removal of ARGs, either in the presence or absence of colloidal material. On the other hand, filtration through the 100, 10, and 1 kDa membranes resulted in an average of 0.9, 3.6, and 4.2 e log reductions across all no-colloid controls and ARGs, and 1.7, 4.9, and >5.9 (most concentrations were below the LOD) e log reductions across all WWTP effluents and ARGs. A summary of the log reductions achieved across each membrane and for each WWTP effluent is shown in Table 2. As expected, membrane pore size had a significant effect on the removal of ARGs ( p < 0.001). Further, the presence of colloidal material in the aqueous matrix was found to have a significant effect on the removal of blaTEM ( p ¼ 0.004) and
vanA ARGs ( p ¼ 0.002). Additional analysis revealed that as membrane pore size decreased, the influence of colloids on the removal of ARGs became more apparent (Table 3). Specifically, the effect of the presence of colloidal material in the removal of blaTEM and vanA ARGs became significant at 10 kDa ( p < 0.05 and <0.01, respectively), and reached a maximum at 1 kDa membrane pore size ( p < 0.001 and <0.01, respectively).
3.2.
Accounting for other sources of DNA loss
blaTEM and vanA ARGs were monitored over 4e5 h to account for alternative sources of DNA loss, such as degradation (Fig. 2). ARG losses were found not to be significant ( p > 0.05) in either wastewater or buffer samples, suggesting that any observed DNA removal during filtration experiments was attributable to retention by the membranes either by size exclusion of the DNA itself, exclusion of DNAecolloid complexes, or interactions with the membrane material. The gene concentrations measured in the unspiked buffer blank confirmed absence of contamination during the experiments.
Fig. 1 e Effect of membrane pore size on ARG removal from spiked WWTP effluents and no-colloid buffer controls. Error bars represent standard deviation of analytical triplicates. 1 kDa data not shown was below LOD. Letters on the left indicate WWTP effluent source.
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Table 2 e Log reduction of plasmid-associated ARGs across all WWTPs and ultrafiltration membranes. WWTP
Log reduction of blaTEM gene 100 kDa
A B C No-colloid control (average)
1.1 2.4 1.6 1
10 kDa
Log reduction of vanA gene 1 kDa a
4.2 4.9 5.5 3.7
a a
4.2
100 kDa
10 kDa
1.2 2.3 1.7 0.9
4.3 4.7 5.8 3.5
1 kDa a a
5.9 4.2
a Gene concentrations were below the LOD.
ARG concentrations in unspiked WWTP effluent samples were tracked to monitor existing gene concentrations and, if possible, to characterize the behavior of native sorbed DNA. Background concentrations of blaTEM and vanA were generally very low and near the LOD. Specifically, average blaTEM and vanA concentrations in unspiked 1.2 mm-filtered effluents were
3.3.
Table 3 e Relative influence of colloids and membrane pore size on the removal of ARGs. Membrane pore size
Gene
Colloids
Membrane
0.1 mm
blaTEM vanA blaTEM vanA blaTEM vanA blaTEM vanA
* e e e * ** *** **
e e *** *** *** *** * *
100 kDa
1 kDa
Colloid characterization
TOC, protein, and polysaccharide measurements are provided in Fig. S2. Results of one-sided Pearson correlation analysis of the filtrate fraction of each of the colloidal components and the logarithm of the filtrate gene fraction are shown in Fig. S3. ARG fractions in the filtrates were found to significantly correlate with all three colloidal components. The highest correlation was observed between ARGs and proteins [r ¼ 0.78 and 0.81 for blaTEM and vanA ( p < 0.001)]. Pearson correlation coefficients were slightly strengthened when considering the sum of the protein and polysaccharide components [r ¼ 0.79 and 0.82 for blaTEM and vanA, respectively ( p < 0.001)].
Effect of colloids on degradation by DNase
The blaTEM and vanA gene fractions measured in the aqueous samples before and after the application of DNase and DNaseactivating conditions (T and salt) are shown in Fig. S1. Under the conditions of these experiments, DNA interactions with wastewater colloids were not found to protect ARGs from DNase degradation. Conditions were confirmed to be amenable to DNase activity, as apparent by the significant difference in ARG concentrations when DNase was present or absent. However, no significant difference was observed between the end point concentrations in the presence/ absence of colloidal material.
10 kDa
3.4.
Symbols represent significance of p-values obtained from two-way ANOVA test: p > 0.05 (e), p < 0.05 (*), p < 0.01 (**), p < 0.001 (***). The two-way ANOVA was applied to the log of the filtrate gene fraction of adjacent membranes. The filtrate gene fraction was calculated as C/C0, where C was the gene concentration in the filtrate and C0 was the average gene concentration in the unfiltered samples used to test for degradation.
3.5. Comparison between PVDF and alumina membranes A comparison of ARG removal achieved by MF (0.1 mm-pore size) hydrophilic PVDF Durapore (Millipore, Billerica, MA) versus alumina Anodisc 47 (Whatman GmbH, Germany) filtration of the WWTP effluents is presented in Fig. 3. There was less than 1-log removal of plasmid-associated ARGs from the no-colloid control by either membrane. While less than 1log ARG removal was also observed across all wastewater samples and ARGs when the Durapore PVDF membrane was applied, significant removal (3.2 log; p < 0.05) was observed when the alumina Anodisc 47 membrane was applied. The blaTEM and vanA log removals achieved using the alumina membrane were 3.2 and 3.1 for WWTP A, 2.2 and 2.1 for WWTP B, and 4.5 and 4.2 for WWTP C, respectively. The combined effect of colloids and the alumina membrane was also found to be significant ( p < 0.05). There was no significant difference in the removal of TOC, protein, or polysaccharides by the two membrane types ( p > 0.05).
4.
Discussion
The results of this study suggest that membrane treatment is a promising technology for the removal of ARGs from treated wastewater effluents. An encouraging finding was that greater removal was achieved than expected. While the molecular weight of the spiked plasmid was w2600 kDa, in reality, the practical effective size of DNA for membrane retention is smaller than predicted by molecular weight. This is because DNA is a long, thin, and flexible molecule that could hypothetically squeeze length-wise through a membrane pore
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Fig. 2 e Stability of vanA and blaTEM ARGs in spiked WWTP effluents and no-colloid buffer controls. Error bars represent standard deviation of analytical triplicates, but are smaller than the symbols in most cases. Values in parentheses indicate minimum (in gene copies/mL), maximum (in gene copies/mL), and coefficient of variation. Letters on the left indicate WWTP effluent source.
(Marko et al., 2011; Travers, 2004). Correspondingly, the 1 kDa membranes did not completely exclude passage of the plasmid in the spiked buffer control (Fig. 1). The overall findings highlight the importance of DNA conformation and behavior, which can vary widely depending on water chemistry and composition. To confirm that DNA removal was actually a result of membrane filtration, ARG concentrations were monitored to account for alternative sources of DNA loss, such as biodegradation or sorption to the glass container. Interestingly, the ARG concentrations did not change significantly, which supports the conclusion that removal was driven by the membranes. These control experiments also bring to light the overall persistence of ARGs in treated wastewater effluent. This result was somewhat unforeseen as the extracellular DNA was expected to be more susceptible to biodegradation given the numerous enzymes and other cellular components present within a wastewater effluent matrix. This observation further emphasizes the potential importance of advanced treatment of wastewater effluents to
remove ARGs that can be transported and protected by larger molecules. Previous studies have noted the persistence of ARGs under environmentally-relevant conditions, highlighting the potential for DNA protection through interactions with environmental substrates. Specifically, the presence of ARB and ARGs, including vanA, has been reported in drinking water sources, treated drinking water, and treated wastewater (Auerbach et al., 2007; Pruden et al., 2006; Schwartz et al., 2003; Xi et al., 2009; Zhang et al., 2009; LaPara et al., 2011). The present study found trace levels of vanA in all three WWTP effluents. In addition, several studies have previously shown that water disinfection processes such as chlorination can select for ARB (Armstrong et al., 1982; Murray et al., 1984; Shrivastava et al., 2004). Previous studies have shown that DNA can be protected against DNase degradation when it is bound to other materials (Table 4). Contrary to these studies, the wastewater colloidassociated ARGs remained susceptible to DNase degradation. It is worth noting that the DNase degradation experiments in
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Fig. 3 e Comparison of DNA removal between 0.1 mm PVDF and alumina membranes. Error bars associated with WWTPs represent standard deviations of analytical measurements. Error bars associated with the control represent standard deviations of experimental measurements done independently in parallel with the experiments involving each of the WWTPs.
this study were performed under conditions of excess enzyme, and it is not certain whether the DNA was completely sorbed to suspended material in the wastewater. Further investigations of varying concentrations of DNA and DNase, including environmentally-relevant concentrations, within the wastewater matrix would be of interest. Importantly, this study demonstrated that membrane removal of ARGs is actually enhanced in the presence of
wastewater. Filtration experiment results revealed a significant effect of 1.2 mm-filtered wastewater effluent matrix on the removal of blaTEM and vanA genes present on the spiked plasmids. This effect is presumably a result of interactions with colloidal material present in the effluent, and is supported by the correlations observed with the TOC, protein, and polysaccharide fractions present in the filtrates. Previous reports provide evidence of interactions between DNA and organic and inorganic materials that are likely present in wastewaters and also in natural waters (Table 4). However, these studies were carried out only in simplified artificial systems. Thus, the results of the present study provide insight into the potential for membranes to remove ARGs from actual wastewater effluents. Correlations with TOC, proteins, and polysaccharides highlight the possibility that the interactions taking place in the wastewater are complex and involve agglomeration with various materials rather than the simple interaction between DNA and discrete particles. The overall results indicate that wastewater colloideARG interactions could facilitate ARG removal by larger pore size membranes, thus reducing the energy requirement for membrane filtration. The findings are congruent with those of a recent study that demonstrated significantly higher ARB and ARG removals from wastewater treated with a membrane bioreactor compared to conventional treatment (Munir et al., 2011). This finding further substantiates the hypothesis of DNAecolloid interactions because, based on the molecular properties of DNA, a conventional MBR system would not be expected to significantly remove extracellular ARGs. Effluents from three WWTPs were collected and used as natural colloid-containing media in this study. According to the literature, DNA has been found to bind to a variety of organic and inorganic materials including different soil colloidal particles, different types of clay, and NOM (Cai et al., 2007, 2006a, 2006b; Crecchio and Stotzky, 1998; Nguyen et al., 2010; Nguyen and Elimelech, 2007a,b). However, DNA sorption behavior not only varies with type of sorbent, but also with pH, ionic strength, type of cations present and DNA conformation (Cai et al., 2006a; Mitra et al., 2001; Nguyen et al., 2010; Nguyen and Elimelech, 2007b; Saeki et al., 2010). For this reason, it is not possible to generate adsorption isotherms that would be consistent across effluents. Even within one WWTP effluent,
Table 4 e Summary of materials observed to interact with and/or protect DNA against enzymatic degradation, and transformation ability of sorbed DNA. Material
Sorption
Protection against DNase degradation
Transformation ability
References
þ
þ
þ
Cai et al. (2007), Cai et al. (2006a), Cai et al. (2006b), Khanna and Stotzky (1992) Cai et al. (2007), Cai et al. (2006a), Cai et al. (2006b), Saeki et al. (2010) Lorenz and Wackernagel (1987), Mitra et al. (2001), Nguyen et al. (2010), Nguyen and Elimelech (2007b), Romanowski et al. (1991) Mitra et al. (2001) Crecchio and Stotzky (1998) Nguyen et al. (2010)
Organic and inorganic clays Soil colloids and particles Silica/sand
þ
þ
þ
þ
þ
a
Charcoal Humic acids NOM
þ þ þ
a
a
þ
þ
a
a
a Data not available.
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the composition would likely change seasonally or even daily. Based on adsorption times reported in the literature (Mitra et al., 2001) and on typical WWTP retention times, a 4-h sorption time was chosen to allow the DNA to interact with colloidal material in the wastewater effluent. Recent studies have indicated trends of DNA transmission through UF membranes at different ionic strengths, pH, and pressure (Arkhangelsky et al., 2008; Latulippe and Zydney, 2008). Thus, the ionic strength of the no-colloid control and the working pressure for all UF membranes were kept constant throughout this study. Comparison of the membrane composition provided further insight into mechanisms of ARG removal. The removal of ARGs was most significantly enhanced by the combination of the alumina membrane and the presence of wastewater material. Interestingly, reductions observed in TOC, protein, and polysaccharide colloidal components following alumina membrane filtration (Fig. S2) were not significant ( p > 0.05). While DNA has been previously reported to interact with alumina and aluminum species at intermediate pH (Karlik et al., 1980; Ma and Yeung, 2010), the removal of DNA in the buffer control was negligible (Fig. 3). Although this finding suggests that DNAealumina interactions may not be a significant factor affecting the enhanced DNA removal by alumina membranes, it is also possible that DNA retention was enhanced by DNAealumina interactions within the wastewater matrix, but perhaps not necessarily by the presence of colloidal material. One of the reported attributes of the Anodisc alumina membrane is that it is made of a non-deformable material with no crossover between individual pores. It is possible that although the average reported pore size of the PVDF membrane was equivalent (0.1 mm), either the actual range of pore sizes was higher and reached pore sizes above 0.1 mm, or there were crossovers among the pores. It is also possible that the PVDF membrane was slightly deformable, thus allowing the transmission of larger particles. These observations highlight the importance of investigating the differences among potential membranes prior to wider-scale use, as certain membranes could have the potential to remove a wider range of contaminants under similar or even lower energy demands. Fouling is an important factor to consider for any membrane treatment technology. Membranes subjected to colloids and wastewater effluents are expected to foul as these materials build up at the membrane surface (Ang et al., 2011). The present study was purposefully not designed to examine the implications of membrane fouling on ARG removal; however, based upon the observed enhancement in ARG removal in the presence of wastewater colloids it can be hypothesized that ARGs will be more efficiently removed by membranes that are partially fouled with organic material than pristine membranes alone. This latter hypothesis, however, requires additional experimental testing under a variety of operating conditions.
5.
Conclusions
Overall, this study demonstrates that membranes are a promising treatment technology for the removal of ARGs from wastewater effluents. Four main findings are highlighted from this study:
ARGs could be effectively removed by membrane filtration, with best performance observed for 10 kDa membranes (UF) and smaller. Membrane removal of ARGs was better than expected based on the molecular properties of DNA. ARGs spiked into the pre-filtered wastewater effluent matrix remained stable during the 4-h monitoring period, indicating that ARGs can persist in wastewater effluents and supporting the need to investigate treatment technologies. Associations of ARGs with natural colloidal material present in wastewater effluent actually resulted in enhanced removal of ARGs. These results are encouraging, and suggest that cost and energy requirements for ARG treatment may be less than would be predicted by molecular properties alone. Membrane material and other characteristics also have an impact on ARG removal and should be optimized in future studies. Membrane treatments are likely to grow in their implementation for advancing water reuse and the results provide important baseline information for the development of a comprehensive water sustainability strategy. In particular, membranes have been demonstrated to be effective for the removal of other emerging contaminants, such as pharmaceuticals and personal care products. Benefits of membrane treatment could be extended to ARGs as WWTPs consider upgrades. Considering the growing problem of antibiotic resistance and the demonstrated links between WWTPs and ARGs in receiving environments, incorporating membrane treatment of WWTP effluents may be advantageous for minimizing potential risk to public health.
Acknowledgments Financial support for this project was provided by the Water Environment Research Foundation Project INFR8SG09, by the NSF CBET CAREER award #0547342, and by the Institute for Critical Technology and Applied Science award TSTS-11-26. The findings do not reflect the opinions of the sponsors. Special thanks to Dr. Dan Gallagher at Virginia Tech for his insights regarding statistical analyses and Dr. Tony Fane of the Singapore Membrane Technology Centre for consultation in experimental design.
Appendix A. Supplementary data Supplementary data related to this article can be found at http://dx.doi.org/10.1016/j.watres.2012.09.044.
references
Allen, H.K., Donato, J., Wang, H.H., Cloud-Hansen, K.A., Davies, J., Handelsman, J., 2010. Call of the wild: antibiotic resistance genes in natural environments. Nature Reviews Microbiology 8 (4), 251e259. Ang, W.S., Tiraferri, A., Chen, K.L., Elimelech, M., 2011. Fouling and cleaning of RO membranes fouled by mixtures of organic
w a t e r r e s e a r c h 4 7 ( 2 0 1 3 ) 1 3 0 e1 4 0
foulants simulating wastewater effluent. Journal of Membrane Science 376 (1e2), 196e206. Arkhangelsky, E., Steubing, B., Ben-Dov, E., Kushmaro, A., Gitis, V., 2008. Influence of pH and ionic strength on transmission of plasmid DNA through ultrafiltration membranes. Desalination 227 (1e3), 111e119. Armstrong, J.L., Calomiris, J.J., Seidler, R.J., 1982. Selection of antibiotic-resistant standard plate-count bacteria during water treatment. Applied and Environmental Microbiology 44 (2), 308e316. Auerbach, E.A., Seyfried, E.E., McMahon, K.D., 2007. Tetracycline resistance genes in activated sludge wastewater treatment plants. Water Research 41 (5), 1143e1151. Baquero, F., Martinez, J.L., Canton, R., 2008. Antibiotics and antibiotic resistance in water environments. Current Opinion in Biotechnology 19 (3), 260e265. Bibbal, D., Dupouy, V., Ferre, J.P., Toutain, P.L., Fayet, O., Prere, M.F., Bousquet-Melou, A., 2007. Impact of three ampicillin dosage regimens on selection of ampicillin resistance in Enterobacteriaceae and excretion of blaTEM genes in swine feces. Applied and Environmental Microbiology 73 (15), 4785e4790. Biyela, P.T., Lin, J., Bezuidenhout, C.C., 2004. The role of aquatic ecosystems as reservoirs of antibiotic resistant bacteria and antibiotic resistance genes. Water Science and Technology 50 (1), 45e50. Bo¨ckelmann, U., Dorries, H.H., Ayuso-Gabella, M.N., de Marcay, M.S., Tandoi, V., Levantesi, C., Masciopinto, C., Van Houtte, E., Szewzyk, U., Wintgens, T., Grohmann, E., 2009. Quantitative PCR monitoring of antibiotic resistance genes and bacterial pathogens in three European artificial groundwater recharge systems. Applied and Environmental Microbiology 75 (1), 154e163. Borjesson, S., Matussek, A., Melin, S., Lofgren, S., Lindgren, P.E., 2010. Methicillin-resistant Staphylococcus aureus (MRSA) in municipal wastewater: an uncharted threat? Journal of Applied Microbiology 108 (4), 1244e1251. Cai, P., Huang, Q., Chen, W., Zhang, D., Wang, K., Jiang, D., Liang, W., 2007. Soil colloids-bound plasmid DNA: effect on transformation of E. coli and resistance to DNase I degradation. Soil Biology & Biochemistry 39 (5), 1007e1013. Cai, P., Huang, Q., Zhang, X., Chen, H., 2006a. Adsorption of DNA on clay minerals and various colloidal particles from an Alfisol. Soil Biology & Biochemistry 38 (3), 471e476. Cai, P., Huang, Q.Y., Zhang, X.W., 2006b. Interactions of DNA with clay minerals and soil colloidal particles and protection against degradation by DNase. Environmental Science & Technology 40 (9), 2971e2976. Crecchio, C., Stotzky, G., 1998. Binding of DNA on humic acids: effect on transformation of Bacillus subtilis and resistance to DNase. Soil Biology & Biochemistry 30 (8e9), 1061e1067. Davies, J., Davies, D., 2010. Origins and evolution of antibiotic resistance. Microbiology and Molecular Biology Reviews 74 (3), 417e433. Dolar, D., Kosutic, K., Pavlovic, D.M., Kunst, B., 2009. Removal of emerging contaminants of industrial origin by NF/RO e a pilot scale study. Desalination and Water Treatment 6 (1e3), 197e203. Dolar, D., Zokic, T.I., Kosutic, K., Asperger, D., Pavlovic, D.M., 2012. RO/NF membrane treatment of veterinary pharmaceutical wastewater: comparison of results obtained on a laboratory and a pilot scale. Environmental Science and Pollution Research 19 (4), 1033e1042. Dubois, M., Gilles, K.A., Hamilton, J.K., Rebers, P.A., Smith, F., 1956. Colorimetric method for determination of sugars and related substances. Analytical Chemistry 28 (3), 350e356. Dutka-Malen, S., Evers, S., Courvalin, P., 1995. Detection of glycopeptide resistance genotypes and identification to the
139
species level of clinically relevant Enterococci by PCR. Journal of Clinical Microbiology 33 (1), 24e27. Karlik, S.J., Eichhorn, G.L., Lewis, P.N., Crapper, D.R., 1980. Interaction of aluminum species with deoxyribonucleic-acid. Biochemistry 19 (26), 5991e5998. Khanna, M., Stotzky, G., 1992. Transformation of Bacillus subtilis by DNA bound on montmorillonite and effect of DNase on the transforming ability of bound DNA. Applied and Environmental Microbiology 58 (6), 1930e1939. Kim, S., Park, H., Chandran, K., 2010. Propensity of activated sludge to amplify or attenuate tetracycline resistance genes and tetracycline resistant bacteria: a mathematical modeling approach. Chemosphere 78 (9), 1071e1077. LaPara, T.M., Burch, T.R., McNamara, P.J., Tan, D.T., Yan, M., Eichmiller, J.J., 2011. Tertiary-treated municipal wastewater is a significant point source of antibiotic resistance genes into Duluth-Superior harbor. Environmental Science & Technology 45 (22), 9543e9549. Larson, E., 2007. Community factors in the development of antibiotic resistance. Annual Review of Public Health 28, 435e447. Latulippe, D.R., Zydney, A.L., 2008. Salt-induced changes in plasmid DNA transmission through ultrafiltration membranes. Biotechnology and Bioengineering 99 (2), 390e398. Lorenz, M.G., Wackernagel, W., 1987. Adsorption of DNA to sand and variable degradation rates of adsorbed DNA. Applied and Environmental Microbiology 53 (12), 2948e2952. Lowry, O.H., Rosebrough, N.J., Farr, A.L., Randall, R.J., 1977. Protein measurement with Folin phenol reagent. Current Contents (1), 7. Ma, C.B., Yeung, E.S., 2010. Entrapment of individual DNA molecules and nanoparticles in porous alumina membranes. Analytical Chemistry 82 (2), 654e657. Marko, A., Denysenkov, V., Margraft, D., Cekan, P., Schiemann, O., Sigurdsson, S.T., Prisneet, T.F., 2011. Conformational flexibility of DNA. Journal of the American Chemical Society 133 (34), 13375e13379. Martinez, J.L., 2008. Antibiotics and antibiotic resistance genes in natural environments. Science 321 (5887), 365e367. Mitra, A., Chakraborty, P., Chattoraj, D.K., 2001. Kinetics of adsorption of DNA at solideliquid interfaces. Journal of the Indian Chemical Society 78 (10e12), 689e696. Munir, M., Wong, K., Xagoraraki, I., 2011. Release of antibiotic resistant bacteria and genes in the effluent and biosolids of five wastewater utilities in Michigan. Water Research 45 (2), 681e693. Murray, G.E., Tobin, R.S., Junkins, B., Kushner, D.J., 1984. Effect of chlorination on antibiotic-resistance profiles of sewagerelated bacteria. Applied and Environmental Microbiology 48 (1), 73e77. Nguyen, T.H., Chen, K.L., Elimelech, M., 2010. Adsorption kinetics and reversibility of linear plasmid DNA on silica surfaces: influence of alkaline earth and transition metal ions. Biomacromolecules 11 (5), 1225e1230. Nguyen, T.H., Elimelech, M., 2007a. Adsorption of plasmid DNA to a natural organic matter-coated silica surface: kinetics, conformation, and reversibility. Langmuir 23 (6), 3273e3279. Nguyen, T.H., Elimelech, M., 2007b. Plasmid DNA adsorption on silica: kinetics and conformational changes in monovalent and divalent salts. Biomacromolecules 8 (1), 24e32. Oncu, N.B., Menceloglu, Y.Z., Balcioglu, I.A., 2011. Comparison of the effectiveness of chlorine, ozone, and photocatalytic disinfection in reducing the risk of antibiotic resistance pollution. Journal of Advanced Oxidation Technologies 14 (2), 196e203. Pei, R.T., Kim, S.C., Carlson, K.H., Pruden, A., 2006. Effect of river landscape on the sediment concentrations of antibiotics and
140
w a t e r r e s e a r c h 4 7 ( 2 0 1 3 ) 1 3 0 e1 4 0
corresponding antibiotic resistance genes (ARG). Water Research 40 (12), 2427e2435. Pruden, A., Pei, R.T., Storteboom, H., Carlson, K.H., 2006. Antibiotic resistance genes as emerging contaminants: studies in northern Colorado. Environmental Science & Technology 40 (23), 7445e7450. Romanowski, G., Lorenz, M.G., Wackernagel, W., 1991. Adsorption of plasmid DNA to mineral surfaces and protection against DNaseI. Applied and Environmental Microbiology 57 (4), 1057e1061. Saeki, K., Kunito, T., Sakai, M., 2010. Effects of pH, ionic strength, and solutes on DNA adsorption by andosols. Biology and Fertility of Soils 46 (5), 531e535. Schwartz, T., Kohnen, W., Jansen, B., Obst, U., 2003. Detection of antibiotic-resistant bacteria and their resistance genes in wastewater, surface water, and drinking water biofilms. FEMS Microbiology Ecology 43 (3), 325e335. Shrivastava, R., Upreti, R.K., Jain, S.R., Prasad, K.N., Seth, P.K., Chaturvedi, U.C., 2004. Suboptimal chlorine treatment of drinking water leads to selection of multidrug-resistant Pseudomonas aeruginosa. Ecotoxicology and Environmental Safety 58 (2), 277e283. Storteboom, H., Arabi, M., Davis, J.G., Crimi, B., Pruden, A., 2010. Tracking antibiotic resistance genes in the South Platte river basin using molecular signatures of urban, agricultural, and pristine sources. Environmental Science & Technology 44 (19), 7397e7404. Szczepanowski, R., Linke, B., Krahn, I., Gartemann, K.H., Gutzkow, T., Eichler, W., Puhler, A., Schluter, A., 2009.
Detection of 140 clinically relevant antibiotic-resistance genes in the plasmid metagenome of wastewater treatment plant bacteria showing reduced susceptibility to selected antibiotics. Microbiology-Sgm 155, 2306e2319. Travers, A.A., 2004. The structural basis of DNA flexibility. Philosophical Transactions of the Royal Society of London Series A-Mathematical Physical and Engineering Sciences 362 (1820), 1423e1438. Volkmann, H., Schwartz, T., Bischoff, P., Kirchen, S., Obst, U., 2004. Detection of clinically relevant antibiotic-resistance genes in municipal wastewater using real-time PCR (TaqMan). Journal of Microbiological Methods 56 (2), 277e286. Wintgens, T., Gallenkernper, M., Melin, T., 2004. Removal of endocrine disrupting compounds with membrane processes in wastewater treatment and reuse. Water Science and Technology 50 (5), 1e8. Xi, C.W., Zhang, Y.L., Marrs, C.F., Ye, W., Simon, C., Foxman, B., Nriagu, J., 2009. Prevalence of antibiotic resistance in drinking water treatment and distribution systems. Applied and Environmental Microbiology 75 (17), 5714e5718. Zhang, R.F., Eggleston, K., Rotimi, V., Zeckhauser, R.J., 2006. Antibiotic resistance as a global threat: evidence from China, Kuwait and the United States. Globalization and Health 2. Zhang, Y.L., Marrs, C.F., Simon, C., Xi, C.W., 2009. Wastewater treatment contributes to selective increase of antibiotic resistance among Acinetobacter spp. Science of the Total Environment 407 (12), 3702e3706.
Available online at www.sciencedirect.com
journal homepage: www.elsevier.com/locate/watres
Effect of wastewater colloids on membrane removal of antibiotic resistance genes Maria V. Riquelme Breazeal, John T. Novak, Peter J. Vikesland, Amy Pruden* Via Department of Civil and Environmental Engineering, Virginia Tech, Blacksburg, VA 24061, USA
article info
abstract
Article history:
Recent studies have demonstrated that wastewater treatment plants (WWTPs) signifi-
Received 21 June 2012
cantly alter the magnitude and distribution of antibiotic resistance genes (ARGs) in
Received in revised form
receiving environments, indicating that wastewater treatment represents an important
21 September 2012
node for limiting ARG dissemination. This study examined the potential for membrane
Accepted 22 September 2012
treatment of microconstituent ARGs and the effect of native wastewater colloids on the
Available online 4 October 2012
extent of their removal. Plasmids containing vanA (vancomycin) and blaTEM (b-lactam) ARGs were spiked into three representative WWTP effluents versus a control buffer and
Keywords:
tracked by quantitative polymerase chain reaction through a cascade of microfiltration and
Antibiotic resistance genes
ultrafiltration steps ranging from 0.45 mm to 1 kDa. Significant removal of ARGs was ach-
Wastewater colloids
ieved by membranes of 100 kDa and smaller, and presence of wastewater colloids resulted
Microfiltration
in enhanced removal by 10 kDa and 1 kDa membranes. ARG removal was observed to
Ultrafiltration
correlate significantly with the corresponding protein, polysaccharide, and total organic carbon colloidal fractions. Alumina membranes removed ARGs to a greater extent than polyvinylidene fluoride membranes of the same pore size (0.1 mm), but only in the presence of wastewater material. Control studies confirmed that membrane treatment was the primary mechanism of ARG removal, versus other potential sources of loss. This study suggests that advanced membrane treatment technology is promising for managing public health risks of ARGs in wastewater effluents and that removal may even be enhanced by colloids in real-world wastewaters. ª 2012 Elsevier Ltd. All rights reserved.
1.
Introduction
Antibiotic resistance and, particularly, multidrug resistance, is an increasingly critical problem affecting human health (Davies and Davies, 2010). Globally, antibiotic resistance has been reported to be widespread (Allen et al., 2010) and on the rise (Zhang et al., 2006). Although most attention has been focused on nosocomial (i.e., hospital acquired) infections, environmental factors are also important contributors to the transport and spread of antibiotic resistance in the community (Larson, 2007). Recent studies have demonstrated that wastewater treatment
plants can significantly alter the magnitude and distribution of antibiotic resistance genes (ARGs) in receiving environments (Storteboom et al., 2010). Given that antibiotic resistant bacteria (ARB) and ARGs of clinical concern have been documented to arise from environmental sources (Martinez, 2008), there is growing interest in limiting dissemination pathways between humans and the environment (Baquero et al., 2008). Thus, wastewater treatment plants may represent an important node for limiting the spread of antibiotic resistance. The presence of low levels of ARB and ARGs in wastewater is inevitable and several studies have documented their
* Corresponding author. Tel.: þ1 540 231 3980. E-mail addresses: [email protected] (M.V. Riquelme Breazeal), [email protected] (J.T. Novak), [email protected] (P.J. Vikesland), apruden@engr. colostate.edu, [email protected] (A. Pruden). 0043-1354/$ e see front matter ª 2012 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.watres.2012.09.044
w a t e r r e s e a r c h 4 7 ( 2 0 1 3 ) 1 3 0 e1 4 0
proliferation and persistence in wastewater treatment plants (Auerbach et al., 2007; Borjesson et al., 2010; Kim et al., 2010; Szczepanowski et al., 2009; Zhang et al., 2009). Traditionally the objective of disinfection following water or wastewater treatment is to kill or inactivate bacterial cells. However, to address the challenge of antibiotic resistance, DNA removal or destruction is ideal prior to discharge, reuse, or other applications. This is because ARGs are typically carried on highly transmissible DNA elements, such as plasmids and integrons, which can remain functional and be assimilated by downstream bacteria. Because of this ability of ARGs to transcend their bacterial host, ARGs themselves are considered to be the primary “contaminant” of concern (Pruden et al., 2006). Once released, ARGs can persist in the environment (Biyela et al., 2004; Storteboom et al., 2010), and have even been observed to establish and proliferate in drinking water biofilms (Schwartz et al., 2003; Xi et al., 2009). Although the effect of some disinfection techniques on the fate of ARGs has been investigated in a few studies (Munir et al., 2011; Oncu et al., 2011), the fate of these microconstituents after wastewater disinfection and release is still largely unknown. The fate of ARGs through membrane removal processes is of particular interest as membranes are growing in popularity for enhancing sustainability of water resources and addressing concerns for other emerging contaminants (Dolar et al., 2009, 2012; Munir et al., 2011; Wintgens et al., 2004). The present study is the first to our knowledge to examine the potential for membrane removal of ARGs. Membrane filtration processes are commonly used for the removal of a wide variety of contaminants, but most would not be expected to impart significant removal of DNA based on its molecular properties. However, wastewater effluents are complex in their chemistries and could significantly affect membrane removal of DNA. Thus, the focus of this study was on the potential for the advanced membrane treatment of ARGs within real-world wastewater effluent matrices. Of specific interest to this study was the effect of DNA interactions with wastewater effluent colloidal particles. DNA is well known to interact with clay minerals and various soil colloidal particles (Cai et al., 2007, 2006a, 2006b; Crecchio and Stotzky, 1998; Nguyen et al., 2010; Nguyen and Elimelech, 2007a,b; Romanowski et al., 1991; Saeki et al., 2010). Considering that significant amounts of suspended colloidal particles are present in wastewaters, these interactions represent a promising and unexploited approach for enhanced removal of ARGs from wastewater. However, most of the available studies concerning DNAecolloid interactions have been carried out in simplified or artificial systems (Mitra et al., 2001; Nguyen and Elimelech, 2007a,b; Romanowski et al., 1991); thus, there is a need for studies that emphasize the behavior and fate of DNA in situ. In addition, the relationship between the removal of ARGecolloid complexes and membrane pore size has not been investigated in natural systems. The overall objective of this project was to investigate the effect of wastewater colloids on the removal of ARGs by membrane filtration. It was hypothesized that colloidal interactions with DNA would enhance membrane removal of ARGs. To test this hypothesis, plasmids containing vanA and blaTEM ARGs were spiked into three representative WWTP effluents and a control buffer, and subjected to a cascade of
131
microfiltration (MF) and ultrafiltration (UF) steps ranging from 0.45 mm to 1 kDa. The vanA and blaTEM ARGs encode resistance to vancomycin and b-lactam antibiotics (e.g., ampicillin), respectively, with the former used typically as a last-resort antibiotic when other treatments fail. Other potential sources of DNA loss, such as degradation, were also monitored to verify that observed ARG removal was due to the membrane treatment and also to examine the potential for wastewater colloids to protect ARGs from deoxyribonuclease (DNase), a naturally-occurring DNA-degrading enzyme. The effect of membrane composition (alumina versus polyvinylidene fluoride) was compared for the 0.1 mm pore size. Building a knowledge base of the effect of membrane treatment on ARGs is critical for enabling comprehensive management strategies for emerging contaminants in wastewater effluents.
2.
Materials and methods
A cascade of MF and UF steps was used to compare the removal of ARGs from colloid-containing wastewater effluent versus colloid-free aqueous buffer samples. Effluents from three representative WWTPs were employed in order to test the occurrence of DNAecolloid interactions in a complex environmental matrix, as well as the reproducibility of the results. Tris buffer (pH 8) was used as colloid-free material in order to maintain DNA stability in the controls throughout the experiment, as low salt concentrations could cause DNA instability, while high salt concentrations can promote the sorption of the genetic material to the glass containers. The key characteristics of the WWTPs selected for this study and effluent quality are summarized in Table 1. An additional experiment was carried out to further explore the potential for DNAecolloid interactions to protect ARGs from degradation by DNase.
2.1.
Plasmid solution preparation
The pCR 4-TOPO plasmids (Invitrogen, Carlsbad, CA) containing the ARGs of interest were used to spike the appropriate test solutions. This plasmid contains blaTEM and was modified to contain vanA. A 732 bp portion of the vanA gene was PCRamplified from vancomycin-resistant Enterococcus genomic DNA using previously described primers (Dutka-Malen et al., 1995). The 25 mL PCR amplification reaction contained 1 Master Taq reaction buffer (5 Prime, Gaithersburg, MD), 1 Taq Master PCR enhancer (5 Prime), 1.5 mM Mg2þ solution (5 Prime), 0.05 mM of each deoxynucleoside triphosphate (Promega, Madison, WI), 0.2 mM of each primer, 1.75 U Taq DNA polymerase (5 Prime), 1 mL template DNA, and autoclaved nanopure water to a final volume of 25 mL. The thermal cycle consisted of an initial 3 min denaturing step at 95 C; followed by 50 cycles of a 30 s denaturing step at 95 C, a 30 s annealing step at 54 C, and a 30 s extension step at 72 C; and a 7 min final extension step at 72 C. The vanA PCR product was ligated to the pCR 4-TOPO plasmid and cloned using the TOPO TA cloning kit (Invitrogen), following the manufacturer’s instructions. Plasmids were extracted using the Qiagen Plasmid Mega kit (Qiagen, Valencia, CA) and quantified by qPCR targeting blaTEM and vanA ARGs.
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w a t e r r e s e a r c h 4 7 ( 2 0 1 3 ) 1 3 0 e1 4 0
Table 1 e General WWTP and effluent characteristics and effluent sampling dates. WWTP
Capacity (MGD)
Key processesb
Influent composition
TSSb (mg/L)
pHb
Otherb (mg/L)
Collection date 4/21/11
4/7/11
A
6
Residential, industrial
Conventional,a UV disinfection
7.7
7.1
B
9
Conventional,a chlorine disinfection
2
7
C
6
Residential, hospital, industrial Residential, hospital, high Na2SO4 industrial
DO (8.1) BOD (19.6) DO (9.5)
Conventional,a nitrification, chlorine disinfection
7
6.7
BOD (<5)
3/1/11
An additional sampling event occurred on 1/30/12 from all WWTPs, for DNase-degradation test. a Conventional treatment: primary clarification, aeration, secondary clarification, and disinfection. b WWTP and effluent characteristics obtained by phone from WWTP operators.
2.2.
WWTP effluent collection and initial filtration
Grab samples of final effluent were collected from each of the test wastewater treatment facilities (WWTPs A, B, or C) at most 6 h before the experiment was carried out. Effluent collection occurred during cool (2e15 C) working week mornings, avoiding rain or snow events taking place during the previous 24 h. The effluent was immediately transported to the lab where it was kept at 4 C prior to use. As a pretreatment to eliminate particles above the colloid size range, the effluent was vacuum-filtered through a 1.2 mm pore-size mixed cellulose ester membrane (Millipore, Billerica, MA) using a sterilized Nalgene polysulfone filter holder (Thermo Fisher Scientific, Rochester, NY).
2.3.
Cascading filtration experiments
Each of the WWTP effluents was subjected to cascading filtration experiments and compared with appropriate controls: (1) Autoclaved 10 mM Tris buffer pH 8 (blank); (2) Autoclaved 10 mM Tris pH 8 spiked with vanA gene-containing plasmids to a final concentration of 1 107 to 1 108 plasmid copies/mL based on blaTEM gene concentrations (no-colloid control); (3) filtered WWTP effluent (background); and (4) filtered WWTP effluent spiked with vanA gene-containing plasmids to a final concentration of 1 107 to 1 108 plasmid copies/mL based on blaTEM gene concentrations (wastewater colloid treatment). Four 1000-mL flasks were filled with 600 mL of corresponding solution and lightly agitated for 4e5 h using a Burrell wrist action shaker (Burrell Scientific, Pittsburg, PA) to enable DNA interactions with wastewater colloids prior to filtration. 10-mL subsamples were collected from each of the four flasks during this stage to test for alternative sources of DNA loss. Sequential cascading filtration steps were carried out for each of the four flask contents. First, 500 mL were filtered through the membrane with the largest pore size (0.45 mmpore size membrane). Next, 10-mL sub-samples of the filtrate were pipetted into previously baked 30-mL Qorpak (Bridgeville, PA) clear graduated glass bottles and immediately frozen at 80 C for subsequent lyophilization and DNA extraction. Filtrate sub-samples originating from the unspiked WWTP effluent were also collected for colloid characterization and stored at 4 C. The remaining water/buffer filtrate was then
filtered through the subsequent membrane (0.1 mm-pore size). The process was repeated for each of the filtrates, applying the membranes in order of decreasing pore size: 0.45 mm / 0.1 mm / 100 kDa / 10 kDa / 1 kDa. To minimize carryover, the flask contents were processed in the following order: (1) buffer blank, (2) unspiked background wastewater, (3) spiked no-colloid control buffer, (4) spiked wastewater containing colloids. The latter was processed after the 4-h period allotted for DNAecolloid interactions to occur. MF was carried out using 0.45 mm and 0.1 mm pore size Durapore hydrophilic polyvinylidene fluoride (PVDF) membranes (Millipore, Billerica, MA), and a Nalgene polysulfone filter holder (Thermo Fisher Scientific, Rochester, NY). To compare the effect of membrane type, Anodisc 47 alumina 0.1 mm pore size membranes (Whatman GmbH, Germany) were compared to the Durapore membrane of the same pore size. UF was carried out using 100 kDa, 10 kDa, and 1 kDa MWCO Ultracel regenerated cellulose membranes (Millipore, Billerica, MA) and an 8200 Ultrafiltration Stirred Cell (Millipore). UF filtrates flowed into baked glass flasks shielded by baked aluminum foil. Because the UF membranes were reusable, they were cleansed between usages using DNA Away (Molecular Bio Products, San Diego, CA) followed by a thorough rinsing step that included filtering water through the membrane for at least 10 min. A list of the membrane types and pore sizes is provided in Table S1.
2.4.
DNA stability in pretreated wastewater and buffer
To determine the extent to which membrane filtration accounted for removal of ARGs in the above experiments, changes in gene concentrations in pretreated wastewater and buffer were monitored over the time allotted for DNAecolloid interactions prior to filtration. A 10-mL sample was collected immediately after plasmid spiking, and 2e3 additional samples were collected during the following 4e5 h. Gene fractions at times t > 0 were calculated with respect to the initial concentration measured at t ¼ 0 h.
2.5.
DNase-degradation experiment
The potential for colloideDNA interactions to protect ARGs from degradation was investigated using DNase-degradation experiments. All three WWTP effluents were sampled on the
w a t e r r e s e a r c h 4 7 ( 2 0 1 3 ) 1 3 0 e1 4 0
same day and compared in parallel to the colloid-free Tris buffer control. 500 mL of either Tris buffer (pH 8) or the filtered effluents were collected in fluorinated HDPE Nalgene bottles (Thermo Fisher Scientific, Rochester, NY) and spiked to a concentration of 1 107 plasmids/mL, and placed in a shaking incubator at 125 rpm and room temperature for 4 h to allow DNAecolloid interactions. Sub-samples were collected at 0, 2, and 4 h to test for background DNA losses. After 4 h, 200 mL of the contents of each bottle (i.e., buffer or WWTP effluent) were split into two new bottles (100 mL each), one of which was spiked with 10.2 U of RQ1 RNase-free DNase (Promega) and subject to 2.5 mM MgCl2 and 0.5 mM CaCl2 at 37 C, and the other only subject to the same salt and temperature conditions used to activate the DNase. The bottles were incubated at 37 C and shaken at 125 rpm. This approach allowed direct comparison of the effect of the DNase versus the background salt and temperature conditions required to activate the DNase. 10 mL sub-samples were collected after light manual vortexing, immediately after salt and/or DNase addition (i.e., shortly after hour 4), and at 5 and 7 h from the beginning of the experiment. All collected samples were immediately stored at 80 C for further analysis.
2.6.
DNA concentration and extraction
All samples were concentrated by lyophilization using a FreeZone Plus 2.5 L Benchtop Freeze Dry System (Labconco, Kansas City, Missouri). Isolation of DNA originating from WWTP effluent samples was carried out using the FastDNA Spin Kit for Soil (MP Biomedicals, Solon, OH) as recommended in the kit manual. Isolation of DNA originating from buffer samples was carried out using the PowerClean DNA Cleanup Kit (MoBio Laboratories, Carlsbad, CA).
concentrations in 10 reaction blanks and multiplying that value by 3. The background gene concentrations detected in each analytical blank was subtracted from the measured sample concentration. Each individual measurement greater than the detection limit was then multiplied by the dilution factor (10) and the extraction volume, and divided by the volume of each sample in microliters (1 104 mL) to obtain the final concentration in gene copies/mL of each sample. The three analytical measurements per sample were then averaged to obtain the estimated concentration for that sample.
2.8.
Colloid characterization
Non-purgeable organic carbon concentrations were measured using the Shimadzu TOC-VCSN analyzer (Shimadzu Scientific Instruments, Columbia, MD) and potassium hydrogen phthalate (KHP) as the standard. Polysaccharide concentrations were measured by the phenolesulfuric acid method (Dubois et al., 1956) using dextrose as standard. Protein concentrations were measured by the Lowry method (Lowry et al., 1977) using bovine serum albumin (BSA) as the standard. Briefly, five reagents were prepared as follows e Reagent 1: 143 mM NaOH and 270 mM Na2CO3; Reagent 2: 57 mM CuSO4; Reagent 3: 124 mM Na-tartrate; Reagent 4: mix of reagents 1, 2, and 3 at a ratio of 100:1:1 (made daily); and Reagent 5 contained folin ciocalteus reagent diluted 5:6 with distilled water. 1-mL samples were added by sequential addition and mixing of Reagents 4 and 5, respectively. The samples containing the reagents were incubated for 45 min at room temperature before measuring the absorbance at 750 nm.
2.9. 2.7.
133
Statistical analyses
DNA quantification by qPCR
Quantification of vanA and blaTEM was carried out using realtime quantitative polymerase chain reaction (qPCR) on a CFX96 Touch Real-Time PCR Detection System (Bio-Rad, Hercules, CA). 1:10 DNA extract dilutions were analyzed in triplicate reactions containing 40 nM of each primer, 1 SsoFast EvaGreen Supermix (Bio-Rad), 1 mL template DNA, and sterile nanopure to a final volume of 10 mL. The primers used for amplification of the vanA gene were vanAstF (Bo¨ckelmann et al., 2009) and vana3RP (Volkmann et al., 2004). The blaTEM gene was amplified using previously described primers (Bibbal et al., 2007). The temperature programs consisted of a 95 C initial denaturing step for 30 s; 40 cycles of denaturing at 95 C for 5 s, annealing and extension at 60 C for 5 s, and an additional extension step at 72 C for 5 s for vanA amplifications only. A melt curve was incorporated in every reaction to verify specificity by ramping the temperature from 65 C to 95 C by 0.5 C every 5 s. A calibration curve was constructed for each set of reactions using at least five standards. The qPCR standards were prepared by serially diluting M13 PCR products obtained from cloned positive controls (vanA or blaTEM). Concentrations were determined by gel densitometry and converted to gene copies using the relationship described by Pei et al. (2006). A limit of detection (LOD) was calculated for each gene by averaging gene
Statistical analyses were performed using R statistics software (www.r-project.org). Statistical differences between the membrane-removal of ARGs in the presence versus the absence of colloids were calculated using a two-way analysis of variance (ANOVA). The two-way ANOVA was applied to the log of the filtrate gene fraction from spiked WWTP effluent or spiked buffer as the dependent variable. The filtrate gene fraction was calculated as C/C0, where C was the gene concentration in the filtrate and C0 was the average gene concentration in the unfiltered samples used to test for degradation. The presence/absence of colloids and the membrane pore size or type were independent variables. The analyses were repeated individually for each of the genes monitored. To test the significance of the DNA degradation in the water or buffer samples prior to treatment (i.e., prior to filtration or application of DNase-degrading conditions), sampling times after the addition of the plasmid spike were binned as A (1e2 h), B (2.5e3 h), and C (4e5 h). A one-way ANOVA test was applied to the log of the gene fractions present in the water or buffer samples for each bin. Statistical significance was set at a ¼ 0.05 ( p < 0.05). Correlation analysis between the filtrate fraction of each of the colloidal components and the logarithm of the filtrate gene fraction was conducted using R, applying a one-sided Pearson correlation test.
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3.
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Results
3.1. Membrane removal of ARGs and effect of wastewater colloids Sequential filtration across decreasing membrane pore sizes resulted in increasing removal of plasmid-associated ARGs (Fig. 1). Specifically, filtration through the 0.45 and 0.1 mm pore-size microfiltration membranes resulted in less than 1-log removal of ARGs, either in the presence or absence of colloidal material. On the other hand, filtration through the 100, 10, and 1 kDa membranes resulted in an average of 0.9, 3.6, and 4.2 e log reductions across all no-colloid controls and ARGs, and 1.7, 4.9, and >5.9 (most concentrations were below the LOD) e log reductions across all WWTP effluents and ARGs. A summary of the log reductions achieved across each membrane and for each WWTP effluent is shown in Table 2. As expected, membrane pore size had a significant effect on the removal of ARGs ( p < 0.001). Further, the presence of colloidal material in the aqueous matrix was found to have a significant effect on the removal of blaTEM ( p ¼ 0.004) and
vanA ARGs ( p ¼ 0.002). Additional analysis revealed that as membrane pore size decreased, the influence of colloids on the removal of ARGs became more apparent (Table 3). Specifically, the effect of the presence of colloidal material in the removal of blaTEM and vanA ARGs became significant at 10 kDa ( p < 0.05 and <0.01, respectively), and reached a maximum at 1 kDa membrane pore size ( p < 0.001 and <0.01, respectively).
3.2.
Accounting for other sources of DNA loss
blaTEM and vanA ARGs were monitored over 4e5 h to account for alternative sources of DNA loss, such as degradation (Fig. 2). ARG losses were found not to be significant ( p > 0.05) in either wastewater or buffer samples, suggesting that any observed DNA removal during filtration experiments was attributable to retention by the membranes either by size exclusion of the DNA itself, exclusion of DNAecolloid complexes, or interactions with the membrane material. The gene concentrations measured in the unspiked buffer blank confirmed absence of contamination during the experiments.
Fig. 1 e Effect of membrane pore size on ARG removal from spiked WWTP effluents and no-colloid buffer controls. Error bars represent standard deviation of analytical triplicates. 1 kDa data not shown was below LOD. Letters on the left indicate WWTP effluent source.
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Table 2 e Log reduction of plasmid-associated ARGs across all WWTPs and ultrafiltration membranes. WWTP
Log reduction of blaTEM gene 100 kDa
A B C No-colloid control (average)
1.1 2.4 1.6 1
10 kDa
Log reduction of vanA gene 1 kDa a
4.2 4.9 5.5 3.7
a a
4.2
100 kDa
10 kDa
1.2 2.3 1.7 0.9
4.3 4.7 5.8 3.5
1 kDa a a
5.9 4.2
a Gene concentrations were below the LOD.
ARG concentrations in unspiked WWTP effluent samples were tracked to monitor existing gene concentrations and, if possible, to characterize the behavior of native sorbed DNA. Background concentrations of blaTEM and vanA were generally very low and near the LOD. Specifically, average blaTEM and vanA concentrations in unspiked 1.2 mm-filtered effluents were
3.3.
Table 3 e Relative influence of colloids and membrane pore size on the removal of ARGs. Membrane pore size
Gene
Colloids
Membrane
0.1 mm
blaTEM vanA blaTEM vanA blaTEM vanA blaTEM vanA
* e e e * ** *** **
e e *** *** *** *** * *
100 kDa
1 kDa
Colloid characterization
TOC, protein, and polysaccharide measurements are provided in Fig. S2. Results of one-sided Pearson correlation analysis of the filtrate fraction of each of the colloidal components and the logarithm of the filtrate gene fraction are shown in Fig. S3. ARG fractions in the filtrates were found to significantly correlate with all three colloidal components. The highest correlation was observed between ARGs and proteins [r ¼ 0.78 and 0.81 for blaTEM and vanA ( p < 0.001)]. Pearson correlation coefficients were slightly strengthened when considering the sum of the protein and polysaccharide components [r ¼ 0.79 and 0.82 for blaTEM and vanA, respectively ( p < 0.001)].
Effect of colloids on degradation by DNase
The blaTEM and vanA gene fractions measured in the aqueous samples before and after the application of DNase and DNaseactivating conditions (T and salt) are shown in Fig. S1. Under the conditions of these experiments, DNA interactions with wastewater colloids were not found to protect ARGs from DNase degradation. Conditions were confirmed to be amenable to DNase activity, as apparent by the significant difference in ARG concentrations when DNase was present or absent. However, no significant difference was observed between the end point concentrations in the presence/ absence of colloidal material.
10 kDa
3.4.
Symbols represent significance of p-values obtained from two-way ANOVA test: p > 0.05 (e), p < 0.05 (*), p < 0.01 (**), p < 0.001 (***). The two-way ANOVA was applied to the log of the filtrate gene fraction of adjacent membranes. The filtrate gene fraction was calculated as C/C0, where C was the gene concentration in the filtrate and C0 was the average gene concentration in the unfiltered samples used to test for degradation.
3.5. Comparison between PVDF and alumina membranes A comparison of ARG removal achieved by MF (0.1 mm-pore size) hydrophilic PVDF Durapore (Millipore, Billerica, MA) versus alumina Anodisc 47 (Whatman GmbH, Germany) filtration of the WWTP effluents is presented in Fig. 3. There was less than 1-log removal of plasmid-associated ARGs from the no-colloid control by either membrane. While less than 1log ARG removal was also observed across all wastewater samples and ARGs when the Durapore PVDF membrane was applied, significant removal (3.2 log; p < 0.05) was observed when the alumina Anodisc 47 membrane was applied. The blaTEM and vanA log removals achieved using the alumina membrane were 3.2 and 3.1 for WWTP A, 2.2 and 2.1 for WWTP B, and 4.5 and 4.2 for WWTP C, respectively. The combined effect of colloids and the alumina membrane was also found to be significant ( p < 0.05). There was no significant difference in the removal of TOC, protein, or polysaccharides by the two membrane types ( p > 0.05).
4.
Discussion
The results of this study suggest that membrane treatment is a promising technology for the removal of ARGs from treated wastewater effluents. An encouraging finding was that greater removal was achieved than expected. While the molecular weight of the spiked plasmid was w2600 kDa, in reality, the practical effective size of DNA for membrane retention is smaller than predicted by molecular weight. This is because DNA is a long, thin, and flexible molecule that could hypothetically squeeze length-wise through a membrane pore
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Fig. 2 e Stability of vanA and blaTEM ARGs in spiked WWTP effluents and no-colloid buffer controls. Error bars represent standard deviation of analytical triplicates, but are smaller than the symbols in most cases. Values in parentheses indicate minimum (in gene copies/mL), maximum (in gene copies/mL), and coefficient of variation. Letters on the left indicate WWTP effluent source.
(Marko et al., 2011; Travers, 2004). Correspondingly, the 1 kDa membranes did not completely exclude passage of the plasmid in the spiked buffer control (Fig. 1). The overall findings highlight the importance of DNA conformation and behavior, which can vary widely depending on water chemistry and composition. To confirm that DNA removal was actually a result of membrane filtration, ARG concentrations were monitored to account for alternative sources of DNA loss, such as biodegradation or sorption to the glass container. Interestingly, the ARG concentrations did not change significantly, which supports the conclusion that removal was driven by the membranes. These control experiments also bring to light the overall persistence of ARGs in treated wastewater effluent. This result was somewhat unforeseen as the extracellular DNA was expected to be more susceptible to biodegradation given the numerous enzymes and other cellular components present within a wastewater effluent matrix. This observation further emphasizes the potential importance of advanced treatment of wastewater effluents to
remove ARGs that can be transported and protected by larger molecules. Previous studies have noted the persistence of ARGs under environmentally-relevant conditions, highlighting the potential for DNA protection through interactions with environmental substrates. Specifically, the presence of ARB and ARGs, including vanA, has been reported in drinking water sources, treated drinking water, and treated wastewater (Auerbach et al., 2007; Pruden et al., 2006; Schwartz et al., 2003; Xi et al., 2009; Zhang et al., 2009; LaPara et al., 2011). The present study found trace levels of vanA in all three WWTP effluents. In addition, several studies have previously shown that water disinfection processes such as chlorination can select for ARB (Armstrong et al., 1982; Murray et al., 1984; Shrivastava et al., 2004). Previous studies have shown that DNA can be protected against DNase degradation when it is bound to other materials (Table 4). Contrary to these studies, the wastewater colloidassociated ARGs remained susceptible to DNase degradation. It is worth noting that the DNase degradation experiments in
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Fig. 3 e Comparison of DNA removal between 0.1 mm PVDF and alumina membranes. Error bars associated with WWTPs represent standard deviations of analytical measurements. Error bars associated with the control represent standard deviations of experimental measurements done independently in parallel with the experiments involving each of the WWTPs.
this study were performed under conditions of excess enzyme, and it is not certain whether the DNA was completely sorbed to suspended material in the wastewater. Further investigations of varying concentrations of DNA and DNase, including environmentally-relevant concentrations, within the wastewater matrix would be of interest. Importantly, this study demonstrated that membrane removal of ARGs is actually enhanced in the presence of
wastewater. Filtration experiment results revealed a significant effect of 1.2 mm-filtered wastewater effluent matrix on the removal of blaTEM and vanA genes present on the spiked plasmids. This effect is presumably a result of interactions with colloidal material present in the effluent, and is supported by the correlations observed with the TOC, protein, and polysaccharide fractions present in the filtrates. Previous reports provide evidence of interactions between DNA and organic and inorganic materials that are likely present in wastewaters and also in natural waters (Table 4). However, these studies were carried out only in simplified artificial systems. Thus, the results of the present study provide insight into the potential for membranes to remove ARGs from actual wastewater effluents. Correlations with TOC, proteins, and polysaccharides highlight the possibility that the interactions taking place in the wastewater are complex and involve agglomeration with various materials rather than the simple interaction between DNA and discrete particles. The overall results indicate that wastewater colloideARG interactions could facilitate ARG removal by larger pore size membranes, thus reducing the energy requirement for membrane filtration. The findings are congruent with those of a recent study that demonstrated significantly higher ARB and ARG removals from wastewater treated with a membrane bioreactor compared to conventional treatment (Munir et al., 2011). This finding further substantiates the hypothesis of DNAecolloid interactions because, based on the molecular properties of DNA, a conventional MBR system would not be expected to significantly remove extracellular ARGs. Effluents from three WWTPs were collected and used as natural colloid-containing media in this study. According to the literature, DNA has been found to bind to a variety of organic and inorganic materials including different soil colloidal particles, different types of clay, and NOM (Cai et al., 2007, 2006a, 2006b; Crecchio and Stotzky, 1998; Nguyen et al., 2010; Nguyen and Elimelech, 2007a,b). However, DNA sorption behavior not only varies with type of sorbent, but also with pH, ionic strength, type of cations present and DNA conformation (Cai et al., 2006a; Mitra et al., 2001; Nguyen et al., 2010; Nguyen and Elimelech, 2007b; Saeki et al., 2010). For this reason, it is not possible to generate adsorption isotherms that would be consistent across effluents. Even within one WWTP effluent,
Table 4 e Summary of materials observed to interact with and/or protect DNA against enzymatic degradation, and transformation ability of sorbed DNA. Material
Sorption
Protection against DNase degradation
Transformation ability
References
þ
þ
þ
Cai et al. (2007), Cai et al. (2006a), Cai et al. (2006b), Khanna and Stotzky (1992) Cai et al. (2007), Cai et al. (2006a), Cai et al. (2006b), Saeki et al. (2010) Lorenz and Wackernagel (1987), Mitra et al. (2001), Nguyen et al. (2010), Nguyen and Elimelech (2007b), Romanowski et al. (1991) Mitra et al. (2001) Crecchio and Stotzky (1998) Nguyen et al. (2010)
Organic and inorganic clays Soil colloids and particles Silica/sand
þ
þ
þ
þ
þ
a
Charcoal Humic acids NOM
þ þ þ
a
a
þ
þ
a
a
a Data not available.
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the composition would likely change seasonally or even daily. Based on adsorption times reported in the literature (Mitra et al., 2001) and on typical WWTP retention times, a 4-h sorption time was chosen to allow the DNA to interact with colloidal material in the wastewater effluent. Recent studies have indicated trends of DNA transmission through UF membranes at different ionic strengths, pH, and pressure (Arkhangelsky et al., 2008; Latulippe and Zydney, 2008). Thus, the ionic strength of the no-colloid control and the working pressure for all UF membranes were kept constant throughout this study. Comparison of the membrane composition provided further insight into mechanisms of ARG removal. The removal of ARGs was most significantly enhanced by the combination of the alumina membrane and the presence of wastewater material. Interestingly, reductions observed in TOC, protein, and polysaccharide colloidal components following alumina membrane filtration (Fig. S2) were not significant ( p > 0.05). While DNA has been previously reported to interact with alumina and aluminum species at intermediate pH (Karlik et al., 1980; Ma and Yeung, 2010), the removal of DNA in the buffer control was negligible (Fig. 3). Although this finding suggests that DNAealumina interactions may not be a significant factor affecting the enhanced DNA removal by alumina membranes, it is also possible that DNA retention was enhanced by DNAealumina interactions within the wastewater matrix, but perhaps not necessarily by the presence of colloidal material. One of the reported attributes of the Anodisc alumina membrane is that it is made of a non-deformable material with no crossover between individual pores. It is possible that although the average reported pore size of the PVDF membrane was equivalent (0.1 mm), either the actual range of pore sizes was higher and reached pore sizes above 0.1 mm, or there were crossovers among the pores. It is also possible that the PVDF membrane was slightly deformable, thus allowing the transmission of larger particles. These observations highlight the importance of investigating the differences among potential membranes prior to wider-scale use, as certain membranes could have the potential to remove a wider range of contaminants under similar or even lower energy demands. Fouling is an important factor to consider for any membrane treatment technology. Membranes subjected to colloids and wastewater effluents are expected to foul as these materials build up at the membrane surface (Ang et al., 2011). The present study was purposefully not designed to examine the implications of membrane fouling on ARG removal; however, based upon the observed enhancement in ARG removal in the presence of wastewater colloids it can be hypothesized that ARGs will be more efficiently removed by membranes that are partially fouled with organic material than pristine membranes alone. This latter hypothesis, however, requires additional experimental testing under a variety of operating conditions.
5.
Conclusions
Overall, this study demonstrates that membranes are a promising treatment technology for the removal of ARGs from wastewater effluents. Four main findings are highlighted from this study:
ARGs could be effectively removed by membrane filtration, with best performance observed for 10 kDa membranes (UF) and smaller. Membrane removal of ARGs was better than expected based on the molecular properties of DNA. ARGs spiked into the pre-filtered wastewater effluent matrix remained stable during the 4-h monitoring period, indicating that ARGs can persist in wastewater effluents and supporting the need to investigate treatment technologies. Associations of ARGs with natural colloidal material present in wastewater effluent actually resulted in enhanced removal of ARGs. These results are encouraging, and suggest that cost and energy requirements for ARG treatment may be less than would be predicted by molecular properties alone. Membrane material and other characteristics also have an impact on ARG removal and should be optimized in future studies. Membrane treatments are likely to grow in their implementation for advancing water reuse and the results provide important baseline information for the development of a comprehensive water sustainability strategy. In particular, membranes have been demonstrated to be effective for the removal of other emerging contaminants, such as pharmaceuticals and personal care products. Benefits of membrane treatment could be extended to ARGs as WWTPs consider upgrades. Considering the growing problem of antibiotic resistance and the demonstrated links between WWTPs and ARGs in receiving environments, incorporating membrane treatment of WWTP effluents may be advantageous for minimizing potential risk to public health.
Acknowledgments Financial support for this project was provided by the Water Environment Research Foundation Project INFR8SG09, by the NSF CBET CAREER award #0547342, and by the Institute for Critical Technology and Applied Science award TSTS-11-26. The findings do not reflect the opinions of the sponsors. Special thanks to Dr. Dan Gallagher at Virginia Tech for his insights regarding statistical analyses and Dr. Tony Fane of the Singapore Membrane Technology Centre for consultation in experimental design.
Appendix A. Supplementary data Supplementary data related to this article can be found at http://dx.doi.org/10.1016/j.watres.2012.09.044.
references
Allen, H.K., Donato, J., Wang, H.H., Cloud-Hansen, K.A., Davies, J., Handelsman, J., 2010. Call of the wild: antibiotic resistance genes in natural environments. Nature Reviews Microbiology 8 (4), 251e259. Ang, W.S., Tiraferri, A., Chen, K.L., Elimelech, M., 2011. Fouling and cleaning of RO membranes fouled by mixtures of organic
w a t e r r e s e a r c h 4 7 ( 2 0 1 3 ) 1 3 0 e1 4 0
foulants simulating wastewater effluent. Journal of Membrane Science 376 (1e2), 196e206. Arkhangelsky, E., Steubing, B., Ben-Dov, E., Kushmaro, A., Gitis, V., 2008. Influence of pH and ionic strength on transmission of plasmid DNA through ultrafiltration membranes. Desalination 227 (1e3), 111e119. Armstrong, J.L., Calomiris, J.J., Seidler, R.J., 1982. Selection of antibiotic-resistant standard plate-count bacteria during water treatment. Applied and Environmental Microbiology 44 (2), 308e316. Auerbach, E.A., Seyfried, E.E., McMahon, K.D., 2007. Tetracycline resistance genes in activated sludge wastewater treatment plants. Water Research 41 (5), 1143e1151. Baquero, F., Martinez, J.L., Canton, R., 2008. Antibiotics and antibiotic resistance in water environments. Current Opinion in Biotechnology 19 (3), 260e265. Bibbal, D., Dupouy, V., Ferre, J.P., Toutain, P.L., Fayet, O., Prere, M.F., Bousquet-Melou, A., 2007. Impact of three ampicillin dosage regimens on selection of ampicillin resistance in Enterobacteriaceae and excretion of blaTEM genes in swine feces. Applied and Environmental Microbiology 73 (15), 4785e4790. Biyela, P.T., Lin, J., Bezuidenhout, C.C., 2004. The role of aquatic ecosystems as reservoirs of antibiotic resistant bacteria and antibiotic resistance genes. Water Science and Technology 50 (1), 45e50. Bo¨ckelmann, U., Dorries, H.H., Ayuso-Gabella, M.N., de Marcay, M.S., Tandoi, V., Levantesi, C., Masciopinto, C., Van Houtte, E., Szewzyk, U., Wintgens, T., Grohmann, E., 2009. Quantitative PCR monitoring of antibiotic resistance genes and bacterial pathogens in three European artificial groundwater recharge systems. Applied and Environmental Microbiology 75 (1), 154e163. Borjesson, S., Matussek, A., Melin, S., Lofgren, S., Lindgren, P.E., 2010. Methicillin-resistant Staphylococcus aureus (MRSA) in municipal wastewater: an uncharted threat? Journal of Applied Microbiology 108 (4), 1244e1251. Cai, P., Huang, Q., Chen, W., Zhang, D., Wang, K., Jiang, D., Liang, W., 2007. Soil colloids-bound plasmid DNA: effect on transformation of E. coli and resistance to DNase I degradation. Soil Biology & Biochemistry 39 (5), 1007e1013. Cai, P., Huang, Q., Zhang, X., Chen, H., 2006a. Adsorption of DNA on clay minerals and various colloidal particles from an Alfisol. Soil Biology & Biochemistry 38 (3), 471e476. Cai, P., Huang, Q.Y., Zhang, X.W., 2006b. Interactions of DNA with clay minerals and soil colloidal particles and protection against degradation by DNase. Environmental Science & Technology 40 (9), 2971e2976. Crecchio, C., Stotzky, G., 1998. Binding of DNA on humic acids: effect on transformation of Bacillus subtilis and resistance to DNase. Soil Biology & Biochemistry 30 (8e9), 1061e1067. Davies, J., Davies, D., 2010. Origins and evolution of antibiotic resistance. Microbiology and Molecular Biology Reviews 74 (3), 417e433. Dolar, D., Kosutic, K., Pavlovic, D.M., Kunst, B., 2009. Removal of emerging contaminants of industrial origin by NF/RO e a pilot scale study. Desalination and Water Treatment 6 (1e3), 197e203. Dolar, D., Zokic, T.I., Kosutic, K., Asperger, D., Pavlovic, D.M., 2012. RO/NF membrane treatment of veterinary pharmaceutical wastewater: comparison of results obtained on a laboratory and a pilot scale. Environmental Science and Pollution Research 19 (4), 1033e1042. Dubois, M., Gilles, K.A., Hamilton, J.K., Rebers, P.A., Smith, F., 1956. Colorimetric method for determination of sugars and related substances. Analytical Chemistry 28 (3), 350e356. Dutka-Malen, S., Evers, S., Courvalin, P., 1995. Detection of glycopeptide resistance genotypes and identification to the
139
species level of clinically relevant Enterococci by PCR. Journal of Clinical Microbiology 33 (1), 24e27. Karlik, S.J., Eichhorn, G.L., Lewis, P.N., Crapper, D.R., 1980. Interaction of aluminum species with deoxyribonucleic-acid. Biochemistry 19 (26), 5991e5998. Khanna, M., Stotzky, G., 1992. Transformation of Bacillus subtilis by DNA bound on montmorillonite and effect of DNase on the transforming ability of bound DNA. Applied and Environmental Microbiology 58 (6), 1930e1939. Kim, S., Park, H., Chandran, K., 2010. Propensity of activated sludge to amplify or attenuate tetracycline resistance genes and tetracycline resistant bacteria: a mathematical modeling approach. Chemosphere 78 (9), 1071e1077. LaPara, T.M., Burch, T.R., McNamara, P.J., Tan, D.T., Yan, M., Eichmiller, J.J., 2011. Tertiary-treated municipal wastewater is a significant point source of antibiotic resistance genes into Duluth-Superior harbor. Environmental Science & Technology 45 (22), 9543e9549. Larson, E., 2007. Community factors in the development of antibiotic resistance. Annual Review of Public Health 28, 435e447. Latulippe, D.R., Zydney, A.L., 2008. Salt-induced changes in plasmid DNA transmission through ultrafiltration membranes. Biotechnology and Bioengineering 99 (2), 390e398. Lorenz, M.G., Wackernagel, W., 1987. Adsorption of DNA to sand and variable degradation rates of adsorbed DNA. Applied and Environmental Microbiology 53 (12), 2948e2952. Lowry, O.H., Rosebrough, N.J., Farr, A.L., Randall, R.J., 1977. Protein measurement with Folin phenol reagent. Current Contents (1), 7. Ma, C.B., Yeung, E.S., 2010. Entrapment of individual DNA molecules and nanoparticles in porous alumina membranes. Analytical Chemistry 82 (2), 654e657. Marko, A., Denysenkov, V., Margraft, D., Cekan, P., Schiemann, O., Sigurdsson, S.T., Prisneet, T.F., 2011. Conformational flexibility of DNA. Journal of the American Chemical Society 133 (34), 13375e13379. Martinez, J.L., 2008. Antibiotics and antibiotic resistance genes in natural environments. Science 321 (5887), 365e367. Mitra, A., Chakraborty, P., Chattoraj, D.K., 2001. Kinetics of adsorption of DNA at solideliquid interfaces. Journal of the Indian Chemical Society 78 (10e12), 689e696. Munir, M., Wong, K., Xagoraraki, I., 2011. Release of antibiotic resistant bacteria and genes in the effluent and biosolids of five wastewater utilities in Michigan. Water Research 45 (2), 681e693. Murray, G.E., Tobin, R.S., Junkins, B., Kushner, D.J., 1984. Effect of chlorination on antibiotic-resistance profiles of sewagerelated bacteria. Applied and Environmental Microbiology 48 (1), 73e77. Nguyen, T.H., Chen, K.L., Elimelech, M., 2010. Adsorption kinetics and reversibility of linear plasmid DNA on silica surfaces: influence of alkaline earth and transition metal ions. Biomacromolecules 11 (5), 1225e1230. Nguyen, T.H., Elimelech, M., 2007a. Adsorption of plasmid DNA to a natural organic matter-coated silica surface: kinetics, conformation, and reversibility. Langmuir 23 (6), 3273e3279. Nguyen, T.H., Elimelech, M., 2007b. Plasmid DNA adsorption on silica: kinetics and conformational changes in monovalent and divalent salts. Biomacromolecules 8 (1), 24e32. Oncu, N.B., Menceloglu, Y.Z., Balcioglu, I.A., 2011. Comparison of the effectiveness of chlorine, ozone, and photocatalytic disinfection in reducing the risk of antibiotic resistance pollution. Journal of Advanced Oxidation Technologies 14 (2), 196e203. Pei, R.T., Kim, S.C., Carlson, K.H., Pruden, A., 2006. Effect of river landscape on the sediment concentrations of antibiotics and
140
w a t e r r e s e a r c h 4 7 ( 2 0 1 3 ) 1 3 0 e1 4 0
corresponding antibiotic resistance genes (ARG). Water Research 40 (12), 2427e2435. Pruden, A., Pei, R.T., Storteboom, H., Carlson, K.H., 2006. Antibiotic resistance genes as emerging contaminants: studies in northern Colorado. Environmental Science & Technology 40 (23), 7445e7450. Romanowski, G., Lorenz, M.G., Wackernagel, W., 1991. Adsorption of plasmid DNA to mineral surfaces and protection against DNaseI. Applied and Environmental Microbiology 57 (4), 1057e1061. Saeki, K., Kunito, T., Sakai, M., 2010. Effects of pH, ionic strength, and solutes on DNA adsorption by andosols. Biology and Fertility of Soils 46 (5), 531e535. Schwartz, T., Kohnen, W., Jansen, B., Obst, U., 2003. Detection of antibiotic-resistant bacteria and their resistance genes in wastewater, surface water, and drinking water biofilms. FEMS Microbiology Ecology 43 (3), 325e335. Shrivastava, R., Upreti, R.K., Jain, S.R., Prasad, K.N., Seth, P.K., Chaturvedi, U.C., 2004. Suboptimal chlorine treatment of drinking water leads to selection of multidrug-resistant Pseudomonas aeruginosa. Ecotoxicology and Environmental Safety 58 (2), 277e283. Storteboom, H., Arabi, M., Davis, J.G., Crimi, B., Pruden, A., 2010. Tracking antibiotic resistance genes in the South Platte river basin using molecular signatures of urban, agricultural, and pristine sources. Environmental Science & Technology 44 (19), 7397e7404. Szczepanowski, R., Linke, B., Krahn, I., Gartemann, K.H., Gutzkow, T., Eichler, W., Puhler, A., Schluter, A., 2009.
Detection of 140 clinically relevant antibiotic-resistance genes in the plasmid metagenome of wastewater treatment plant bacteria showing reduced susceptibility to selected antibiotics. Microbiology-Sgm 155, 2306e2319. Travers, A.A., 2004. The structural basis of DNA flexibility. Philosophical Transactions of the Royal Society of London Series A-Mathematical Physical and Engineering Sciences 362 (1820), 1423e1438. Volkmann, H., Schwartz, T., Bischoff, P., Kirchen, S., Obst, U., 2004. Detection of clinically relevant antibiotic-resistance genes in municipal wastewater using real-time PCR (TaqMan). Journal of Microbiological Methods 56 (2), 277e286. Wintgens, T., Gallenkernper, M., Melin, T., 2004. Removal of endocrine disrupting compounds with membrane processes in wastewater treatment and reuse. Water Science and Technology 50 (5), 1e8. Xi, C.W., Zhang, Y.L., Marrs, C.F., Ye, W., Simon, C., Foxman, B., Nriagu, J., 2009. Prevalence of antibiotic resistance in drinking water treatment and distribution systems. Applied and Environmental Microbiology 75 (17), 5714e5718. Zhang, R.F., Eggleston, K., Rotimi, V., Zeckhauser, R.J., 2006. Antibiotic resistance as a global threat: evidence from China, Kuwait and the United States. Globalization and Health 2. Zhang, Y.L., Marrs, C.F., Simon, C., Xi, C.W., 2009. Wastewater treatment contributes to selective increase of antibiotic resistance among Acinetobacter spp. Science of the Total Environment 407 (12), 3702e3706.