Occurrence, Abundance, and Diversity of Tetracycline Resistance

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Occurrence, Abundance, and Diversity of Tetracycline Resistance Genes in 15 Sewage Treatment Plants across China and Other Global Locations Xu-Xiang Zhang and Tong Zhang* Environmental Biotechnology Lab, Department of Civil Engineering, The University of Hong Kong, Hong Kong SAR, China

bS Supporting Information ABSTRACT: Activated sludge was sampled from 15 sewage treatment plants (STPs) across China and other global locations to investigate the occurrence, abundance and diversity of tetracycline resistance genes (tet) in the STPs. Occurrence and abundance of 14 tet genes were determined using polymerase chain reaction (PCR) and quantitative real time PCR. Six genes (tet(A), tet(C), tet(G), tet(M), tet(S), and tet(X)) were detected in all the STPs, while no sludge sample contained tet(Q). Total concentration of the 14 genes was significantly different among the STPs and average tet abundance of the STPs varied greatly among the tet types (p < 0.05). Tet(G) had the highest concentration in the STPs, followed by tet(C), tet(A) and tet(S). Phylogenetic diversity of the genes was investigated using DNA cloning. BLAST analysis showed that all of the 450 cloned sequences matched known tet genes, except for tet(G). The 56 tet(G) clones were grouped into 14 genotypes, among which type G24 had an identical sequence to tet(G) carried by Salmonella enterica or Acinetobacter baumannii, while the other sequences had low similarity to the known genes in GenBank. The results of this study might be useful to understand the diversity of these resistance genes in STPs.

’ INTRODUCTION Various antibiotics have been widely used for human and animals, and misuse of antibiotics in many countries potentially contributes to the emerging and spread of antibiotic resistant bacteria (ARB) and antibiotics resistance genes (ARGs) in the environments.1 Tetracycline is one of the most commonly used therapeutics in human and veterinary medicine,2 and at least 40 different tetracycline resistance genes (tet) have been characterized to date.3 Many mechanisms are involved in tetracycline resistance and three different specific mechanisms have been identified so far: antibiotic efflux pumps, target modification with ribosomal protection protein (RPP) and antibiotic inactivation.4,5 Tetracycline resistance is not conferred by exposure to tetracycline alone but also other antibiotics as well as chemical stressors.3 So far, more than 22 tet genes have been detected in environmental samples.6 The efflux genes (tet(A), tet(B), tet(C), tet(D), and tet(E)) occur in various environmental compartments including sewage treatment plants (STPs),7-10 lagoon,11 fish farming ponds,12 river or lake water,9,13 seawater,14 and soil.15 Recently, RPP genes (tet(M), tet(O), tet(S), tet(Q), and tet(W)) have also been detected in STPs,8,16 animal lagoons,17-20 river water and sediments,13,21,22 marine sediments,23,24 soil,15,25 groundwater,26 and even treated drinking water.22 r 2011 American Chemical Society

STPs receive antibiotics and ARGs with the inflow sewage water originating from hospitals, private households, industry and animal husbandry, and play important roles in recombination, exchange, and spread of environmental ARGs.27 STPs are well-known to serve as important reservoirs for various ARGs encoding resistances to aminoglycoside,28,29 tetracycline,8,30 quinolone,31 and β-lactam antibiotics.27,32 Recently, quantitative real time polymerase chain reaction (qPCR) has also been used to quantify the tet genes, which are more readily transmit through STPs.8-10 However, few studies were conducted to investigate diversity of the environmental ARGs, including tet, although some types have been detected in STPs with high abundance.8-10 In this study, activated sludge samples were collected from the 15 STPs across China and other global locations. PCR and qPCR were used to determine the occurrence and abundance of 14 tet genes in the sludge samples. Furthermore, DNA cloning, sequencing and phylogenetic analyses were conducted to reveal diversity of the tet genes. A total of 14 genes (tet(A), tet(B), tet(C), tet(D), tet(E), Received: October 31, 2010 Accepted: February 23, 2011 Revised: February 11, 2011 Published: March 09, 2011 2598

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Table 1. PCR Detection Results of Tetracycline Resistance Genes in Activated Sludge from 15 Sewage Treatment Plantsa AS sampled from STPs sample name

A

C

D

E

G

K

L

M

O

Harbin, China

HEB

þ

Beijing, China

JXQ

þ

þ

þ

-

þ

þ

-

þ

-

þ

þ

þ

þ

þ

-

-

þ

Beijing, China

XBH

þ

-

þ

-

þ

þ

-

-

Qingdao, China

QD

þ

þ

þ

-

-

þ

-

Wuhan, China

WH

þ

-

þ

-

-

þ

Nanjing, China

NJ

þ

þ

þ

-

þ

Shanghai, China

MH

þ

þ

þ

-

Shanghai, China Shanghai, China

TS DQ

þ þ

þ þ

þ þ

þ þ

Guangzhou, China Hong Kong, China

GZ

þ

-

þ

-

ST

þ

þ

þ

-

Columbia, USA

COL

þ

-

þ

-

Griffin, USA

PC

þ

þ

þ

Guelph, Canada

CAN

þ

þ

Singapore

SIN

þ

þ

15/15

10/15

geographical location

occurrence a

tetracycline resistance gene (tet) B

A(P)

Q

S

X

þ

þ

-

þ

þ

þ

-

-

þ

þ

þ

þ

þ

-

þ

þ

-

þ

-

-

-

þ

þ

-

-

þ

þ

-

-

þ

þ

þ

-

-

þ

þ

-

-

þ

þ

þ

þ

þ

þ

þ

þ

-

-

þ

þ

þ þ

þ þ

þ þ

þ þ

þ þ

þ þ

þ þ

-

þ þ

þ þ

þ

þ

-

-

þ

þ

þ

-

þ

þ

-

þ

-

þ

þ

þ

þ

-

þ

þ

þ

þ

þ

þ

þ

þ

þ

-

þ

þ

-

-

þ

-

þ

þ

þ

þ

-

þ

þ

þ

-

þ

þ

þ

þ

þ

þ

þ

-

þ

þ

þ

-

þ

þ

þ

þ

þ

þ

þ

-

þ

þ

15/15

2/15

11/15

15/15

7/15

9/15

15/15

14/15

10/15

0/15

15/15

15/15

þ: positive result; -: negative result.

tet(G), tet(K), tet(L), tetA(P), tet(M), tet(O), tet(Q), tet(S), and tet(X)) were selected in this survey, since these genes have been reported to frequently occur in STPs and are closely related to tetracycline resistance although presence of tet genes does not mean at all that they are being expressed.

’ EXPERIMENTAL SECTION Sewage Treatment Plants and Sample Collection. Activated sludge samples were collected from 15 sewage treatment plants (STPs) geographically located in China (11 STPs), Singapore (one STP defined as SIN), USA (two STPs defined as COL and PC), and Canada (one STP defined as CAN) (Supporting Information (SI) Figure S1). The 11 STPs of China were distributed at different cities including Harbin (HEB STP), Beijing (JXQ and XBH STPs), Qingdao (QD STP), Wuhan (WH STP), Nanjing (NJ STP), Shanghai (MH, TS and DQ STPs), Guangzhou (GZ STP) and Hong Kong (ST STP). SI Table S1 shows the detailed information of the STPs. DNA Extraction and PCR. Activated sludge was sampled from aeration tank of the STPs and mixed with 100% ethanol immediately at a ratio of 1:1 for DNA protection. The mixture was centrifuged to collect approximately 200 mg of the pellet for DNA extraction. For each sludge sample, the extraction was conducted in duplicate using the FastDNA Soil kit (Q-biogene, CA) and DNA concentration was determined by microspectrophotometry (NanoDrop ND-1000, NanDrop Technologies, Willmington, DE). Using the primers listed in SI Table S2,33 PCRs of the 14 tet genes were conducted in a 30-μL volume containing 1  PCR buffer, 100 μM dNTP, 2 pmol of each primer, 150 ng of template DNA and 0.8 U of EXTaq polymerase (TaKaRa, Japan). Tet(O) and tet(S) were amplified using the following conditions: initial denaturation at 95 °C for 7 min, followed by 40 cycles of 94 °C for 15 s, 50.3 °C (tet(O)) or 56 °C (tet(S)) for 30 s and 72 °C for 30 s, with a final extension of 72 °C for 7 min. For the other 12 tet genes, the thermal cycle was: initial denaturation at 94 °C for 5 min, followed by 35 cycles of 94 °C for 1 min, annealing for 1 min at different temperatures (SI Table S2) and 72 °C for 1.5 min, and a final extension of 72 °C for 10 min. PCR products were

analyzed by gel electrophoresis using 1% (w/v) agarose in 1  TAE buffer. To check reproducibility, duplicate PCR reactions were performed for each sample. Sterile water was used as the negative control for each assay. qPCR. Thirteen tet genes (except tet(Q)) were selected for quantitative detection using SYBR Green I qPCR. Tet genes were cloned to plasmids to generate qPCR standard curves to determine the tet abundance in per ng of extracted DNA. In detail, the PCR product of each tet gene was purified using PCRquick-spin PCR Product Pufication Kit (iNtRON, China) and cloned using pMD18-T Vector (TaKaR, Japan). Plasmids carrying each tet gene were extracted and purified using MiniBest Plasmid Purification Kit (TaKaRa, Japan). Plasmid concentrations were determined by NanoDrop and the abundance of tet gene per μL plasmid solution was calculated according to Zhang et al.10 Five- or six-point calibration curve (Ct value versus log of initial tet gene copy) were generated for qPCR using 10-fold serial dilution of the plasmid carrying tet gene.34,35 Based on the calibration curves, Ct value of a test sample was used to calculate the abundance of tet gene, and then the latter was normalized against mass (ng) of the extracted DNA. In order to correct for potential variations in extraction efficiencies, eubacterial 16S rRNA genes were quantified using the method recommended by Lopez-Gutierrez et al.,36 so that tet abundance could be normalized to the total bacterial community. qPCRs were conducted in 96-well plates with a final volume of 20 μL, containing 10 μL iQ SYBR Green SuperMix (BioRad, Hercules, CA), plus 1 μL each primer (10 μM) and 1 μL template DNA. 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 30 s, annealing at different temperatures for 45 s (SI Table S2) and 72 °C for 45 s. Each reaction was run in duplicate for each of the two DNA samples from one STP. PCR efficiency of each gene ranged from 85.9% to 105.5% with R2 values more than 0.991 for all calibration curves. In addition, 5  105 copies of the tet-carrying plasmid were added in serial dilutions of environmental DNA to check for real time PCR inhibition according 2599

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Figure 1. Abundance of tetracycline resistance genes in activated sludge samples collected from 15 sewage treatment plants across China and other three countries (U.S., Canada, and Singapore). The abundance was normalized to the total 16S rRNA genes.

to Pei et al.21 Final concentration of template DNA in reaction volumes was controlled at 98%) to the known tetA(P) genes carried by Clostridium sp. (SI Figure S13), although 15 base alteration sites and 4 genotypes were observed. Compared to other 12 genes, tet(G) showed an extremely higher diversity in STPs. The 56 tet(G) clones were grouped into 14 types (Table 2 and Figure 2), among which type G24 had a 100% identity to the corresponding sequence of Salmonella enterica (AY963803.5) or Acinetobacter baumannii (CU459141.1), while most of the other sequences had low similarity to the known genes. Each of the types G7, G11 and G36 had a similarity of only 93% to the most closely related known gene: Ochrobactrum sp. LM2-P1-53 tet(G)

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Figure 2. Neighbor-joining phylogenetic analysis of tet(G) diversity in activated sludge sampled from 15 sewage treatment plants. The tree was constructed using MEGA version 4.1 and bootstrap analysis with 1000 replicates was used to evaluate the significance of the nodes.

(EF055280.1). Types G9, G15 and G33 were most similar to tet(G) of Pseudomonas aeruginosa (GQ388247.1) or Mannheimia hemolytica (AJ276217.1), but each had a sequence identity of only 96%. The 468-bp tet(G) fragment contained a total of 97 nucleotide alteration locations including 59 multialteration ones, meaning that these changes are pretty common among different bacterial species (SI Figure S10). The changes of genetic codes resulted in 32 potential AA alteration locations on the corresponding polypeptide chains and 14 different types of AA sequences produced in the 56 tet(G) clones (Table 2). Our study indicated that tet(G) had a high genetic diversity in STPs, which can be confirmed by the comparisons of the known sequences deposited in GenBank. For example, Stenotrophomonas sp. tet(G) (EF055281.1) had a very low similarity (92%) to the corresponding gene of both Pasteurella sp. (AY232670.1) and Ochrobactrum sp. (EF055280.1) (SI Figure S10). Currently, limited information is available about the molecular diversity of ARGs in the environment, especially in STPs. Wu et al.15 carried out a phylogenetic analysis of tet(M) in soil and found that all the five tet(M) types obtained matched known genes in GenBank, with sequence identities ranging from 98% to 100%. Storteboom et al.13 found Poudre River water contained several types of tet(W) significantly different from lagoons of upstream animal feeding operations. The diversity of tet genes in STPs probably results from a broad host range of the genes carried by various environmental genera since the ARGs are usually located on large horizontally transferable plasmids and are capable of interspecies transfer.6,19,40 First, the activated sludge environment of high microbial density and diversity may facilitate genetic exchange.44 Additionally, presence of various mobile elements at high densities in STPs may accelerate gene recombination and transfer.44 Finally, tetracycline is preferentially absorbed by activated sludge to produce a microenvironment containing high-level tetracycline,45 which subsequently contributes to nucleotide alterations on tet fragments. In conclusion, this study revealed that tet(A), tet(C), tet(G), tet(M), tet(S), and tet(X) were universal in the STPs with high abundance, indicating that these genes deserve more attention in future work. Tet(G) showed extremely high diversity in the 2602

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Environmental Science & Technology STPs, while the other tet genes were relatively more conservative. The results of this study may be of use and improtance to provide baseline information to understand diversity of ARGs in STPs.

’ ASSOCIATED CONTENT

bS

Supporting Information. Information of the 15 STPs (Table S1), PCR primers used in this study (Table S2), average abundance of tet genes in the STPs of China and other three countries (Table S3), geographical locations of the STPs (Figure S1), electrophoresis image of PCR products (Figure S2) and selected qPCR products (Figure S3) of tet genes, abundance of tet genes normalized to extracted DNA mass (Figure S4), polygenetic analysis of tet fragments and base alterations on DNA sequences (Figures S5S15). This material is available free of charge via the Internet at http://pubs.acs.org.

’ AUTHOR INFORMATION Corresponding Author

*Phone: þ852-2857 8551; fax: þ852-2859 8987; e-mail: [email protected].

’ ACKNOWLEDGMENT This study was financially supported by the Research Grants Council of Hong Kong (HKU7202/09E), Small Project Fund (201007176069), and SPACE Research Fund of the University of Hong Kong. We would like to thank Prof. Gao DW, Prof. Deng BL, Dr. Huang QG, Dr. Zhu HG, Dr. Liang DW, Dr. Duan JZ, Dr. Zhang M, Mr. Yu K, Miss Yang Y, and Mr. Ye L for the sludge sampling. ’ REFERENCES (1) Davies, J.; Davies, D. Origins and evolution of antibiotic resistance. Microbiol. Mol. Biol. Rev. 2010, 74, 417–433. (2) Chopra, I.; Roberts, M. Tetracycline antibiotics: Mode of action, applications, molecular biology, and epidemiology of bacterial resistance. Microbiol. Mol. Biol. Rev. 2001, 65, 232–260. (3) Roberts, M. C. Update on acquired tetracycline resistance genes. FEMS Microbiol. Lett. 2005, 245, 195–203. (4) Kumar, A.; Schweizer, H. P. Bacterial resistance to antibiotics: Active efflux and reduced uptake. Adv. Drug Delivery Rev. 2005, 57, 1486–1513. (5) Lambert, P. A. Bacterial resistance to antibiotics: Modified target sites. Adv. Drug Delivery Rev. 2005, 57, 1471–1485. (6) Zhang, X. X.; Zhang, T.; Fang, H. H. P. Antibiotic resistance genes in water environment. Appl. Microbiol. Biotechnol. 2009, 82, 397–414. (7) Guillaume, G.; Verbrugge, D.; Chasseur-Libotte, M. L.; Moens, W.; Collard, J. M. PCR typing of tetracycline resistance determinants (TetA-E) in Salmonella enterica serotype Hadar and in the microbial community of activated sludges from hospital and urban wastewater treatment facilities in Belgium. FEMS Microbiol. Ecol. 2000, 32, 77–85. (8) Auerbach, E. A.; Seyfried, E. E.; McMahon, K. D. Tetracycline resistance genes in activated sludge wastewater treatment plants. Water Res. 2007, 41, 1143–1151. (9) Zhang, X. X.; Wu, B.; Zhang, Y.; Zhang, T.; Yang, L. Y.; Fang, H. H. P.; Cheng, S. P. Class 1 integronase gene and tetracycline resistance genes tetA and tetC in different water environments of Jiangsu Province, China. Ecotoxicology 2009, 18, 652–660. (10) Zhang, T.; Zhang, M.; Zhang, X. X.; Fang, H. H. P. Tetracycline resistance genes and tetracycline resistant lactose-fermenting

ARTICLE

Enterobacteriaceae in activated sludge of sewage treatment plants. Environ. Sci. Technol. 2009, 43, 3455–3460. (11) Macauley, J. J.; Adams, C. D.; Mormile, M. R. Diversity of tet resistance genes in tetracycline resistant bacteria isolated from a swine lagoon with low antibiotic impact. Can. J. Microbiol. 2007, 53, 1307–1315. (12) Dang, H. Y.; Zhang, X. X.; Song, L.; Chang, Y. Q.; Yang, G. P. Molecular determination of oxytetracycline-resistant bacteria and their resistance genes from mariculture environments of China. J. Appl. Microbiol. 2007, 103, 2580–2592. (13) Storteboom, H.; Arabi, M.; Davis, J.; Crimi, B.; Pruden, A. Tracking antibiotic resistance genes in the South Platte River Basin using molecular signatures of urban, agricultural, and pristine sources. Environ. Sci. Technol. 2010, 44, 7397–7404. (14) Kim, Y. H.; Jun, L. J.; Park, S. H.; Yoon, S. H.; Chuang, J. K.; Kim, J. C.; Jeong, H. D. Prevalence of tet(B) and tet(M) genes among tetracycline-resistant Vibrio spp. in the aquatic environments of Korea. Dis. Aquat. Org. 2007, 75, 209–216. (15) Wu, N.; Qiao, M.; Zhang, B.; Cheng, W. D.; Zhu, Y. G. Abundance and diversity of tetracycline resistance genes in soils adjacent to representative swine feedlots in China. Environ. Sci. Technol. 2010, 44, 6933–6939. (16) Kim, S.; Park, H.; Chandran, K. Propensity of activated sludge to amplify or attenuate tetracycline resistance genes and tetracycline resistant bacteria: A mathematical modeling approach. Chemosphere 2010, 78, 1071–1077. (17) McKinney, C.; Loftin, K.; Meyer, M.; Davis, J.; Pruden, A. Tet and sul antibiotic resistance genes in livestock lagoons of various operation type, configuration, and antibiotic occurrence. Environ. Sci. Technol. 2010, 44, 6102–6109. (18) Peak, N.; Knapp, C. W.; Yang, R. K.; Hanfelt, M. M.; Smith, M. S.; Aga, D. S.; Graham, D. W. Abundance of six tetracycline resistance genes in wastewater lagoons at cattle feedlots with different antibiotic use strategies. Environ. Microbiol. 2007, 9, 143–151. (19) Engemann, C. A.; Keen, P.; Knapp, C. W.; Hall, K. J.; Graham, D. W. Fate of tetracycline resistance genes in aquatic systems: Migration from the water column to peripheral biofilms. Environ. Sci. Technol. 2008, 42, 5131–5136. (20) Smith, M. S.; Yang, R. K.; Knapp, C. W.; Niu, Y. F.; Peak, N.; Hanfelt, M. M.; Galland, J. C.; Graham, D. W. Quantification of tetracycline resistance genes in feedlot lagoons by real-time PCR. Appl. Environ. Microbiol. 2004, 12, 7372–7377. (21) Pei, R. T.; Kim, S. C.; Carlson, K. H.; Pruden, A. Effect of river landscape on the sediment concentrations of antibiotics and corresponding antibiotic resistance genes (ARG). Water Res. 2006, 40, 2427–2435. (22) Pruden, A.; Pei, R. T.; Storteboom, H.; Carlson, K. H. Antibiotic resistance genes as emerging contaminants: Studies in northern Colorado. Environ. Sci. Technol. 2006, 40, 7445–7450. (23) Nonaka, L.; Ikeno, K.; Suzuki, S. Distribution of tetracycline resistance gene, tet(M), in Gram-positive and Gram-negative bacteria isolated from sediment and seawater at a coastal aquaculture site in Japan. Microb. Environ. 2007, 22, 355–364. (24) Rahman, M. H.; Nonaka, L.; Tago, R.; Suzuki, S. Occurrence of two genotypes of tetracycline (TC) resistance gene tet(M) in the TCresistant bacteria in marine sediments of Japan. Environ. Sci. Technol. 2008, 42, 5055–5061. (25) Knapp, C. W.; Dolfing, J.; Ehlert, P. A. I.; Graham, D. W. Evidence of increasing antibiotic resistance gene abundances in archived soils since 1940. Environ. Sci. Technol. 2010, 44, 580–587. (26) Mackie, R. I.; Koike, S.; Krapac, I; Chee-Sanford, J; Maxwell, S.; Aminov, R. I. Tetracycline residues and tetracycline resistance genes in groundwater impacted by swine production facilities. Anim. Biotechnol. 2006, 17, 157–176. (27) Szczepanowski, R.; Krahn, I.; Linke, B.; Goesmann, A.; P€uhler, A.; Schl€uter, A. Antibiotic multiresistance plasmid pRSB101 isolated from a wastewater treatment plant is related to plasmids residing in phytopathogenic bacteria and carries eight different 2603

dx.doi.org/10.1021/es103672x |Environ. Sci. Technol. 2011, 45, 2598–2604

Environmental Science & Technology

ARTICLE

resistance determinants including a multidrug transport system. Microbiology 2004, 150, 3613–3630. (28) Tennstedt, T.; Szczepanowski, R.; Krahn, I.; P€uhler, A.; Schl€uter, A. Sequence of the 68,869 bp IncP-1a plasmid pTB11 from a waste-water treatment plant reveals a highly conserved backbone, a Tn402-like integron and other transposable elements. Plasmid 2005, 53, 218–238. (29) Moura, A.; Henriques, I.; Ribeiro, R.; Correia, A. Prevalence and characterization of integrons from bacteria isolated from a slaughterhouse wastewater treatment plant. J. Antimicrob. Chemother. 2007, 60, 1243–1250. (30) Mispagel, H.; Gray, J. T. Antibiotic resistance from wastewater oxidation ponds. Water Environ. Res. 2005, 77, 2996–3002. (31) B€onemann, G.; Stiens, M.; P€uhler, A.; Schl€uter, A. Mobilizable IncQ-related plasmid carrying a new quinolone resistance gene, qnrS2, isolated from the bacterial community of a wastewater treatment plant. Antimicrob. Agents Chemother. 2006, 50, 3075–3080. (32) Taviani, E.; Ceccarelli, D.; Lazaro, N.; Bani, S.; Cappuccinelli, P.; Colwell, R. R.; Colombo, M. M. Environmental Vibrio spp., isolated in Mozambique, contain a polymorphic group of integrative conjugative elements and class 1 integrons. FEMS Microbiol. Ecol. 2008, 64, 45–54. (33) Ng, L.-K.; Martin, I.; Alfa, M.; Mulvey, M. Multiplex PCR for the detection of tetracycline resistant genes. Mol. Cell. Probe. 2001, 15, 209–215. (34) Zhang, T.; Fang, H. H. P. Applications of real-time polymerase chain reaction for quantification of microorganisms in environmental samples. Appl. Microbiol. Biotechnol. 2006, 70, 281–289. (35) Liang, D. W.; Zhang, T.; Fang, H. H. P. Real-time quantifications of dominant anaerobes in an upflow reactor by polymerase chain reaction using a TaqMan probe. Water Sci. Technol. 2008, 57, 1851–1855. (36) Lopez-Gutierrez, J.; Henry, S.; Hallet, S.; Martin-Laurent, F.; Catroux, G.; Philippot, L. Quantification of a novel group of nitratereducing bacteria in the environment by real-time PCR. J. Microbiol. Meth. 2004, 57, 399–407. (37) Thompson, J. D.; Higgins, D. G.; Gibson, T. J. Clustal-W improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice. Nucleic Acids Res. 1994, 22, 4673–4680. (38) Patterson, A. J.; Colangeli, R.; Spigaglia, P.; Scott, K. P. Distribution of specific tetracycline and erythromycin resistance genes in environmental samples assessed by microarray detection. Environ. Microbiol. 2007, 9, 703–715. (39) Akinbowale, O. L.; Peng, H.; Barton, M. D. Diversity of tetracycline resistance genes in bacteria from aquaculture sources in Australia. J. Appl. Microbiol. 2007, 103, 2016–2025. (40) Agersø, Y.; Bruun, M. S.; Dalsgaard, I.; Larsen, J. L. The tetracycline resistance gene tet(E) is frequently occurring and present on large horizontally transferable plasmids in Aeromonas spp. from fish farms. Aquaculture 2007, 266, 47–52. (41) Shen, X. X.; Lu, Q.; Deng, L.; Yu, S. J.; Zhang, H.; Deng, Q. L.; Jiang, M.; Hu, Y. Y.; Yang, Y. H. Surveillance of antimicrobial susceptibility of Haemophilus influenzae isolates from children in Beijing, Shanghai, and Guangzhou 2000-2002. Natl. Med. J. China 2004, 84, 1335–1339. (42) Ding, H.; Yang, Y. H.; Chen, Y.; Wang, Y.; Fan, S. Z.; She, S. Z. Antimicrobial usage in paediatric intensive care units in China. Acta Pediatr. 2008, 97, 100–104. (43) Grohskopf, L. A.; Huskins, W. C.; Sinkowitz-Cochran, R. L.; Levine, G. L.; Goldmann, D. A.; Jarvis, W. R. Use of antimicrobial agents in United States neonatal and pediatric intensive care patients. Pediatr. Infect. Dis. J. 2005, 24, 766–773. (44) Schl€uter, A.; Szczepanowski, R.; P€uhler, A.; Top, E. M. Genomics of IncP-1 antibiotic resistance plasmids isolated from wastewater treatment plants provides evidence for a widely accessible drug resistance gene pool. FEMS Microbiol. Rev. 2007, 31, 449–477. (45) Li, B.; Zhang, T. Biodegradation and adsorption of antibiotics in the activated sludge process. Environ. Sci. Technol. 2010, 44, 3468–3473. 2604

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