Environ. Sci. Technol. 2009, 43, 3455–3460
Tetracycline Resistance Genes and Tetracycline Resistant Lactose-Fermenting Enterobacteriaceae in Activated Sludge of Sewage Treatment Plants TONG ZHANG,* MING ZHANG, XUXIANG ZHANG, AND HERBERT HANPING FANG Environmental Biotechnology Laboratory, Department of Civil Engineering, The University of Hong Kong, Hong Kong SAR, China
Received November 21, 2008. Revised manuscript received March 16, 2009. Accepted March 26, 2009.
Activated sludges were sampled from five sewage treatment plants (STPs) distributed in three geographically isolated areas, i.e., Hong Kong (Shatin, Stanley), Shanghai (Minhang) in China, and the bay area in California (Palo Alto and San Jose) of the United States. Among the tested 14 tetracycline resistance (tet) genes, nine genes encompassing efflux pumps (tetA, tetC, tetE, and tetG), ribosomal protection proteins (tetM, tetO, tetQ, and tetS), and enzymatic modification (tetX) were commonly detected in the STP sludge samples, whereas five genes encompassing efflux pumps [tetB, tetD, tetL, tetK, and tetA(P)] were not detected in any sludge sample. Additionally, 109 lactose-fermenting Enterobacteriaceae (LFE) strains were isolated from the activated sludge of the Shatin STP. Tetracycline-resistant (TR) LFE accounted for 32% of the total 109 LFE strains. The occurrence frequencies of tet genes among all TR-LEF strains varied from 0 to 91%, i.e., tetC (91%), tetA (46%), tetE (9%), tetG (6%), and tetD (6%). Finally, quantitative real-time polymerase chain reaction was used to quantify the change of tetC and tetA genes as the indicator of TR-LEF in the Shatin and Stanley STPs. The results showed that the concentrations of tetC and tetA genes in STP effluent ranged from 104 to 105 copies/mL, significantly lower than those in the influent by 3 orders of magnitude.
Introduction Antibiotic resistance, conferred by a series of antibiotic resistance genes (ARGs), is one of the major global public health issues that need urgent action (1, 2). ARGs have been found in various environments, including sediments (3), river (4), and groundwater (5), plus influent, effluent, and activated sludge of sewage treatment plants (6). ARGs are considered as one group of emerging contaminants, as their widespread dissemination is clearly undesirable (7, 8). In order to protect public health, it is important to address the critical problem of ARGs in the various environments. The tetracycline class of antibiotics is one of the most commonly used therapeutics in human and veterinary medicine (9). Tetracycline-resistant bacteria were found to emerge environments with the introduction of tetracycline * Corresponding author e-mail:
[email protected]. 10.1021/es803309m CCC: $40.75
Published on Web 04/14/2009
2009 American Chemical Society
(10). There have been at least 39 different tetracycline resistance (tet) genes characterized to date (4, 11), including 24 genes encoding energy-dependent efflux proteins (efflux pump mechanism), 11 genes encoding ribosomal protection proteins (RPPs) (target modification mechanism), 3 genes encoding an inactivating enzyme, and 1 gene with an unknown mechanism of resistance (4, 11). Among them, more than 26 tet genes have been found in bacteria isolates from various water and soil environments. Several recent studies have used molecular techniques to study the occurrence of tet genes in waste lagoons (5), groundwater near swine farms (5, 12), river sediment (8), and activated sludge (6, 13). Their results showed the universal occurrence of tet genes in various environments. The efflux genes of tetA, B, C, D, and E frequently appeared in various environmental compartments, including activated sludge of STPs (sewage treatment plants) (13), fish farming ponds (14, 15), surface waters (16), and swine lagoons (17). Recently, the tetracycline resistance genes including tetM, O, S, Q, and W, encoding ribosomal protection proteins, have also been detected in sewage (6) and hospital or animal production wastewater (18, 19) and even in natural water environments (20). A qantitative real-time polymerase chain reaction (qRTPCR) has been used to quantify the tetW and tetO in river sediment (8) and tetO and tetQ in activated sludge (6). However, few studies have investigated the phenotype and genotype of tetracycline resistance in the common human pathogens such as lactose-fermenting Enterobacteriaceae (LFE). In this study, activated sludges were sampled from five STPs distributed in three geographically isolated areas, i.e., Hong Kong (Shatin), Shanghai (Minhang) of China, and bay area in California (Palo Alto and San Jose) of the United States. PCR was used to determine what tet genes are typically present in these samples, with primer sets specific for 14 tet genes encompassing efflux pumps [tetA, tetB, tetC, tetD, tetE, tetG, tetK, tetL, and tetA(P)], RPPs (tetM, tetO, tetQ, and tetS), and enzymatic modification (tetX). These 14 tet genes have been studied and reported to occur in the STP environments (21). Additionally, 109 LFE strains were isolated from the activated sludge of the Shatin STP and checked for their tet gene profiles. Finally, quantitative real-time PCR (qRT-PCR) was used to quantify the change of tetC and tetA genes in the whole process (activated sludge and chlorination disinfection) of the Shatin and Stanley STPs.
Experimental Section Sewage Treatment Plants. Samples were collected from five activated sludge STPs in Hong Kong, Shanghai, and the bay area (Palo Alto and San Jose) in California. The Shatin (define as ST) STP in Hong Kong, with a daily average flow rate of 150000 m3, treats domestic wastewater from a population of about 370000. Stanley (SL), with a daily average flow rate of 8478 m3, treats domestic wastewater from a population about 19000. The Minhang (MH) STP in Shanghai, with a daily average flow rate of 50000 m3, treats mainly domestic wastewater with about 20% industrial input, serving the two nearby towns with a population of 150000. The daily flow rates of the San Jose (SJ) STP and Palo Alto (PA) STP are 450000 m3 and 114000 m3, respectively. More operational data of these two STPs were described by Park et al. (22). Sample Collection and DNA Extraction. At each STP, activated sludge samples were collected from aeration tanks. MH, SJ, and PA sludges were sampled in October of 2007. ST sludges were sampled monthly for a year between March 2007 and March 2008. Samples were kept on ice and transported to the laboratory for immediate processing VOL. 43, NO. 10, 2009 / ENVIRONMENTAL SCIENCE & TECHNOLOGY
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TABLE 1. Occurrence of tet Genes in Activated Sludge from Four STPs STa
STP
SJb
PAc
MHd
2007 2008 2007 sampling time mechanisme Mar May June July August September October November December January February Mar October October October tetA tetC tetE tetG tetM tetO tetS tetQ tetX
E E E E R R R R M
+ + + + + + +
+ + + + + + +
+ + + + + + +
a Shatin b San Jose c Palo Alto enzymatic modification.
+ + + + + + + d
+ + + + + + +
Minhang
+ + + + + + + e
+ + + + + + +
9
+ + + + + + +
+ + + + + + +
+ + + + + + +
+ + + + + + +
+ + + + + + + + +
+ + + + + + + + +
+ + + + + + + + +
E is the efflux pump. R is the ribosomal protection proteins. M is the
(within 2-4 h). Activated sludge samples of 10 mL were centrifuged at 4000 rpm for 10 min at 4 °C. Two-hundred milligrams of the pellet was used for DNA extraction, using the FastDNA soil kit (Q-biogene, CA). PCR. All PCR amplifications were conducted in 30 µL of a pH 8.3 buffer (Pharmacia Biotech Inc., Piscataway, NJ), containing 200 µM each of the four deoxynucleotide triphosphates, 15 mM MgCl2, 0.1 µM of individual primers, and 1 U of Taq polymerase (Pharmacia Biotech, Inc., Piscataway, NJ) using an iCycler thermal cycler (BioRad, Hercules, CA). Primers targeting 14 different tet genes, tetA, tetB, tetC, tetD, tetE, tetG, tetK, tetL, tetM, tetO, tetS, tetA(P), tetQ, and tetX were described previously (22). The thermal cycle was initially denaturated at 94 °C for 5 min, followed by 35 cycles of 94 °C for 1 min, annealing for 1 min at different temperatures (23), then 72 °C for 1.5 min, with a final extension of 72 °C for 7 min, and then stored at 4 °C. PCR products were analyzed by gel electrophoresis using 1% (w/v) agarose in 1 × TAE buffer. To ensure reproducibility, duplicate PCR reactions were performed for each sample. Sterile water was used as the negative control for each assay. Counting and Isolation of LFE. Ten milliliters of a sludge sample from ST STP was mixed with 90 mL of PBS (pH 7.2) and 5 mL of 5 mm glass beads, and the mixture was shaken at 200 rpm for 2 h. Then the suspension was used to count total cultivatable bacteria cells using LB media and lactosefermenting Enterobacteriaceae (LFE), an important group of human pathogens, on MacConkey agar, using the spread plate method after serial dilution (18). Colonies with pink color (defined as LFE) were further purified and screened according to a 16S rDNA RFLP (restriction fragment length polymorphism) profile. In detail, almost the full sequence of 16S rDNA was amplified using primer set EUB8F/Univ1392R and digested using RspI. Strains were type based on a RFLP profile on 1% agarose gel. One to three representative sequences of each RFLP type were sequenced to identify the taxonomy affiliation. All strains were further tested for their resistance to tetracycline. According to the National Antimicrobial Resistance Monitoring System (24), strains were classified as tetracycline “resistant” if they form colonies on agar with 20 mg/L tetracycline. qRT-PCR. Two tet genes (tetC and tetA) were selected for quantitative detection using SYBR Green I real-time PCR (23). In detail, the PCR product of the tetC gene was first cloned using the TA Cloning Kit (Invitrogen Corporation, Carlsbad, CA). Then the plasmid carrying the tetC gene was extracted and purified using PureLink Quick Plasmid Miniprep Kit (Invitrogen Corporation, Carlsbad, CA). Plasmid concentrations were determined through microspectrophotometry (NanoDrop ND-1000, NanDrop Technologies, Willmington, DE). The copy number of tetC gene per µL of plasmid solution was calculated (25) by 3456
+ + + + + + +
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Copy number of tetC gene ⁄ µL ) (L × C) ⁄ (N × M×109) (1) where, M is the molecular weight of an average base pair of DNA (660 g/mol) (26), L is Avogadro’s constant (6.02 × 1023/ mol), N is the length of template containing the tetC gene (418 bps) and pCR 2.1 vector (3929 bps), and C is the mass concentration of plasmid in nanogram per microliter. Reactions were conducted in 96-well plates with a final volume of 20 µL using iQ SYBR Green Supper Mix (BioRad, Hercules, CA), with an annealing temperature of 55 °C. Thermal cycling and fluorescence detection were conducted on a BioRad iCycler with the software iCycler iQ version 3.0 (BioRad, Hercules, CA), using the following protocol: 94 °C for 3 min, followed by 40 cycles of 94 °C for 45 s, 55 °C for 45 s, and 72 °C for 45 s. Each reaction was run in duplicate. Results are reported as the mean of measurements with standard deviations. Five-point calibration curves (Ct value versus log of initial tetC copy number) for qPCR were generated using 10-fold serial dilutions of the plasmid carrying tetC gene (25, 27), from 107 to 103 target copies per reaction. The PCR efficiency for tetC was on average 99% (96-102%). R2 values were 0.993-0.999 for all calibration curves. On the basis of the calibration curves, the Ct value of a test sample was used to calculate the number of tetC gene copies, and then the latter was normalized against mass (ng) of the extracted DNA and the volume (mL) of the original samples. The quantification of tetA was conducted in the same way as tetC, except that the size of the tetA fragment is 219 bps, and average PCR efficiency was 97% (93-102%).
Results and Discussion Occurrence of tet Genes. Among the 14 tet genes tested, five genes encoding efflux pumps, tetB, tetD, tetK, tetL, and tetA(P) were not detected in any of the 15 sludge samples in this study. Table 1 summarizes the occurrence of nine tet genes in the sludge samples from four STPs. All activated sludge samples contained several different tetracycline resistance genes. The results showed that the detection frequency of tet genes had no preference for the resistance mechanism. Three efflux pump genes (tetA, tetC, and tetG), three RPP genes (tetM, tetO, and tetQ) and one enzymatic modification gene (tetX) were the most frequent tet genes. These seem to be the most common tet genes in the activated sludge of STPs. Also there was no significant difference in tet gene patterns in four STPs that are geographically isolated from each other, except for tetB and tetE, which were not found in the STP of Hong Kong, while they were detected in the other three STPs from the United States and China. Among the samples of ST STP taken at different time points, there was no change of tet gene patterns.
Overall, activated sludge in the STP of Hong Kong harbored less tet genes than the sludge from the United States and China (Shanghai). The reason and implication is still not clear at this time. Auerbach et al. (6) reported similar findings in four activated sludge samples of two STPs in the United States. Among the sludges in the United States, the only difference between this study and their study is that tetD was not detected here, whereas its frequency of occurrence was high in their study (6). Genes tetM, tetO, and tetQ were detected in all of the sludge samples in this and other studies (6). In addition to their frequent occurrence in activated sludge, tetM, tetO, and tetQ were also dominant in the microbial communities in the lagoons and groundwater of the state of Illinois (5). These genes may come from the gastrointestinal tracts of pigs and steers (28). Especially among the RPP encoding genes, the tetM gene is the most common and frequently encountered gene in both Gram-positive, e.g, Enterococcus (28), and Gramnegative pathogens in clinical and terrestrial environments (9, 18). So, it is not surprising to detect tetM at high frequencies in activated sludge. Several studies have shown tetE to be frequently present in the aquatic environments (6, 14, 30). As shown in Table 1, it was also widely detected in activated sludges from three STPs, except the one in Hong Kong. The occurrence of three tet genes (tetX, tet34, and tet37) related to the third resistance mechanism, enzymatic modification, have not been studied before in activated sludges. One of the three genes, tetX, was detected universally in all of the four sludges and all ST sludges sampled at different time points. This indicates its importance in the sewage system and warrants further study. Tetracycline-Resistant LFE. The results on tet genes in activated sludges cannot tell whether these genes are carried by pathogens or nonpathogens, although such information may have significant implication for public health as pathogens carrying resistant genes are more dangerous. In order to investigate the tet genes carried by pathogens, strains of LFE, an important group containing many pathogen species, were isolated from activated sludge of Hong Kong and tested for their tet gene profile. On the basis of the results of this study, LFE accounted for 3.1% ( 3.2% of the total cultivable bacteria cells on LB medium. Among a total of 109 LFE strains isolated, 70 (64.2%) were identical to Escherichia coli with a 100% similarity to 16S rDNA. The rest, 39 strains (35.8%), were identical to another common LFE species, Klebsiella pneumonia, with a 100% similarity of 16S rDNA. The strains able to grow on MacConkey agar with 20 mg/L tetracycline were defined as tetracycline resistant (TR) LFE (18), accounting for 32% of the total LFE strains (31 out of 70 E. coli-like strains and 4 out of 39 K. pneumonia-like strains) isolated in this study. The TR-LFE percentage (32%) of total LFE obtained in this study is comparable to a few previous studies showing that up to 57% of E. coli isolates from the wastewater treatment system (31) and up to 54% of E. coli from different animal species (32) were resistant to tetracycline, but this percentage is higher than the tetracycline resistance percentage (25%) of E. coli isolated from dairy cows with mastitis (33). Some TR-LFE isolates in this study were multiple antibiotic-resistant bacteria, with 10 strains showing reduced susceptibility to kanamycin (50 mg/L) and 21 strains showing reduced susceptibility to ampicillin (50 mg/L). On the basis of PCR verification results (Table S1 of the Supporting Information), 34 out of 35 TR-LFE strains contained at least one type of tetracycline resistance gene, while the rest did not contain any tetracycline genes tested in this study. Some of the TR-LFE isolates were found to
FIGURE 1. Relative abundance of tetC and tetA genes (normalized to per nanogram-extracted DNA) in activated sludge samples. carry more than one and often up to four different tet genes. Almost all of these genes (except for tetM of strain MK9) were only related to the efflux enzyme, showing the simplex resistance mechanism. The occurrence frequencies of different tet genes among all TR-LEF strains varied from 0 to 91%. Gene tetC had the highest frequency, 91%, followed by tetA (46%), tetE (9%), tetG (6%), and tetD (6%). This is in agreement with the report in a review paper on the occurrence of tet genes in LFE species (9) and a recent research paper showing that 98% of 32 TR E. coli isolates obtained in the United States carried tetC, followed by tetA (43.8%) (33). But there are also a few controversial reports. Genes tetA and tetB were reported as the most dominant tet genes in LFEs (Klebsiell, Escherichia, and Shigella), and tetC was rarely detected in LFEs isolated in the United States (34). Another report showed that the tetracycline resistance of 90% of E. coli strains isolated from a river basin in China was related to tetA, tetB, tetM, or their combination, while tetC was not detected at high frequency (35). TR Escherichia strains obtained in Japan may also mainly carry tetH (29), which has never been reported to be carried by Escherichia before but may have been acquired through horizontal gene transfer recently. The reasons for these controversial results are complicated, but geographical isolation may be the most important reason. The frequency of tetD was only 6% (2 out of 35) in the LFE strains isolated in this study, which is slightly higher than the frequency (0%) in Pseudoalteromonas (one LFE genus) (15) and much less than that in Vibrio (also a LFE genus) (100%) (15). The frequency of tetE was also low (9%) in LFE from STP, while it is common among Aeromonas (also LFE) strains from Danish fish farms (36). The other eight tet genes, i.e., tetM, tetK, tetL, tetO, tetA(P), tetQ, tetS, and tetX, were not carried by the TR-LFE strains isolated in this study. Among these eight tet genes, tetM, tetO, tetQ, and tetX genes were detected commonly in activated sludge samples in this study. They may be carried by the other species in the sludge, e.g., Campylobacter, Lactobacillus, Streptococcus, Clostridium, and Enterococcus (9). Gene tetM was evident in Gram-negative and Grampositive bacteria from various genera, e.g., Shewanella and Vibrio (19), Alcaligenes, Arthrobacter, and Pseudomonas (37). Gene tetM (50%) was the most common determinant, followed by tetE (45%), tetA (35%), and tetD (15%), in all of the bacteria from aquaculture sources in Australia (38). But tetM was never reported to be carried by LFE, except for in two reports (35, 39). VOL. 43, NO. 10, 2009 / ENVIRONMENTAL SCIENCE & TECHNOLOGY
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TABLE 2. Abundance of tetC and tetA Normalized to Sample Volumea Shatin sample influent × 10 second clarifier effluent × 105 disinfection effluent × 104 activated sludge × 108 biosolid × 108 8
a
tetC 1.35 2.27 NA 0.67 3.06
( 0.20 ( 0.78 ( 0.04 ( 0.05
Units are copy per milliliter of sample.
b
Stanley
tetA
tetC/tetA
tetC
tetA
tetC/tetA
0.60 0.24 ( 0.08 NA 0.26 ( 0.08 1.22 ( 0.03
2.26 9.66 NA 2.58 2.51
1.90 ( 0.37 3.68 ( 0.13 1.33 ( 0.39 8.06 ( 0.60 9.31 ( 0.30b
1.59 ( 0.44 0.65 ( 0.08 2.12 ( 0.39 2.19 ( 0.43 13.0 ( 1.20b
1.19 5.66 0.63 3.68 0.72
Unit is copy per gram of sample.
TABLE 3. Abundance of tetC and tetA Normalized to Mass of DNAa Shatin
Stanley
sample
tetC
tetA
tetC/tetA
tetC
tetA
tetC/tetA
influent second clarifier effluent disinfection effluent activated sludge biosolid
280 ( 42.2 47.1 ( 16.2 NA 12.1 ( 0.69 17.4 ( 0.29
124 4.88 ( 0.17 NA 4.69 ( 0.14 6.96 ( 0.19
2.26 9.66 NA 2.58 2.51
449 ( 8.68 120 ( 4.31 3.75 ( 1.11 68.6 ( 5.13 4.86 ( 0.15
375 ( 10.4 21.3 ( 2.58 6.00 ( 1.09 18.7 ( 0.36 6.80 ( 0.62
1.19 5.66 0.63 3.68 0.72
a
Units are 103 copy per nanogram of DNA.
Gene tetC is only limitedly distributed in a few confined genera, i.e., most of LFE genera, including Klebsiella, Escherichia, Citrobacter, Serratia (18), Shigella, and Enterobacter (9), and a few other Gram-negative genera, including Aeromonas, Vibrio, Proteus, Pseudomonas (9), and Chlamydia (11). It has rarely been found in Gram-positive bacteria, except for Brevibacterium (18). Additionally, tetC always occurs with other tet genes and confers multiple resistance (9). The above two aspects are in favor of using tetC as an indicator or biomarker of the LEF pathogen carrying tetracycline resistance. Therefore, tetC in influent, effluent, activated sludge, and biosolids from the ST STP was quantified using qRTPCR. For comparison purposes, tetA was also measured simultaneously. Quantification of tetC and tetA Genes in Activated Sludge of ST STP. Genes tetC and tetA in activated sludge sampled monthly from the ST STP were initially quantified to investigate the time variation. In order to compare the relative abundance in different samples, the tetC and tetA gene copies detected per qRT-PCR reactions were normalized to the mass of extracted DNA (Figure 1 and Table S2 of the Supporting Information). Gene concentrations of tetC and tetA in ST sludge showed significant variation over sampling time points, from 0.193 × 103 to 14.4 × 103 copies/ng DNA (about 75 times difference) and from 0.279 × 103 to 11.0 × 103 copies/ng DNA (about 39 times difference), respectively. The abundance of tetA was comparable among the sludges from the five STPs. However, tetC abundance in the MH sludge was extremely high compared to that of the sludges from Hong Kong and the United States. This may reflect the application of tetracycline in different areas. Most values of tetC and tetA (except for the tetC of MH) were in the range of 103 to 104 copies/ng, which is lower than that of the tetG gene (104 to 105 copies/ng DNA) (6) and comparable to that of the tetQ gene (103 to 104 copies/ng DNA) detected in activated sludge (6). Fate of tetC and tetA in Wastewater Treatment Processes. To investigate the fate of tet genes in wastewater treatment processes, samples of influent, activated sludge, second clarifier effluent, disinfection effluent, and biosolids from digested sludges from the ST STP and another STP, Stanley (SL) in Hong Kong, were taken for DNA extraction. Then, tetC and tetA were quantified for these samples (Tables 2 and 3). 3458
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As shown in Table 2, effluent samples contained the lowest concentrations of tetC and tetA genes, with typical values in the range of 104-105 copies/mL, which were lower than the levels in the influent sample by 3 orders of magnitude. Biosolid samples contained the highest copy number per volume, presumably because biosolid samples had the most concentrated biomass. On the volumetric basis, tetC and tetA gene levels in the influent samples were always higher than the levels observed in the activated sludge. These results were in agreement with the results of the tetQ gene previously reported (6). In order to conduct a more direct comparison of tetC and tetA gene relative abundance across samples, the detected copy number was normalized per mass of extracted DNA (Table 3). The DNA mass-normalized concentrations of tetC and tetA showed that influent samples contained 1 to 2 orders of magnitude more tetC and tetA copies per mass of DNA than the other samples. This indicated tetC and tetA elimination in the activated sludge wastewater treatment process. Although effluent samples contained the lowest concentrations of tetC and tetA on the volume basis, they had almost the same relative tetC and tetA gene abundance normalized to the extracted DNA mass as those in activated sludge and biosolids (Tables 2 and 3). Auerbach et al. (6) showed similar results for tetQ. This indicated that the low tet resistance gene concentration in treated effluent was primarily attributable to the low biomass in comparison to activated sludge and biosolids. The percentage of tetracycline-resistant bacteria in the effluent is not much lower than those in activated sludge and digested sludge. As shown in Tables 2 and 3, chlorination disinfection processes, using sodium hypochlorite after the second clarifier eliminated tetC and tetA genes significantly, not only on the volume basis (64%), but also on the biomass basis (97%). This result implies that chlorination not only reduced the concentration of the total microbial population, but also selectively destroyed those microbes carrying tetC and tetA genes. In summary, nine tet genes (A, C, E, G, M, O, Q, S, and X) were commonly detected in the STP sludge samples in this study, whereas five other tet genes (B, D, L, K, and A(P)) were not detected in any sludge samples. Tetracyclineresistant (TR) LFE accounted for 32% of the total 109 LFE
strains isolated from the activated sludge of Hong Kong. The occurrence frequencies of tet genes among all TR-LEF strains varied from 0 to 91%, i.e., tetC (91%), tetA (46%), tetE (9%), tetG (6%), and tetD (6%). qRT-PCR results of of tetC and tetA genes, as the indicator of TR-LEF, showed that the concentration of tetC and tetA genes in STP effluent ranged from 104 to 105 copies/mL, significantly lower than those in the influent by 3 orders of magnitude. The results of this study reveal the universal existence of the nine tet genes in STPs and suggest the importance of tetC as an indicator or biomarker of the LEF carrying tetracycline resistance.
Acknowledgments This study was partially supported by the General Research Fund (GRF) of Hong Kong (7195/06E). M. Zhang and X. Zhang thank The University of Hong Kong (HKU) for the fellowship. We all thank Prof. Craig Criddle and Mr. George Wells of Stanford University for the DNA samples from the two STPs in the United States.
Supporting Information Available Multiple tetracycline resistance genes in TR-LFE strains (Table S1) and relative abundance of tetC and tetA genes normalized to extracted DNA (Table S2). This material is available free of charge via the Internet at http://pubs.acs.org.
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