Environ. Sci. Technol. 2011, 45, 386–391
Tetracycline Resistance Genes Persist at Aquaculture Farms in the Absence of Selection Pressure MANU TAMMINEN,† ANTTI KARKMAN,† ˜ HMUS,† ANDRES LO WINDI INDRA MUZIASARI,† HIROYUKI TAKASU,‡ SHIGEKI WADA,‡ S A T O R U S U Z U K I , ‡ A N D M A R K O V I R T A * ,† Department of Food and Environmental Sciences, University of Helsinki, P.O. Box 56, 00014 Helsinki, Finland, Center for Marine Environmental Studies, Ehime University, Matsuyama, Ehime 7908577, Japan
Received September 16, 2009. Revised manuscript received November 15, 2010. Accepted November 29, 2010.
The prophylactic and therapeutic use of tetracyclines in aquaculture has been shown to contribute to the spread of tetracycline resistance in the environment. In this work, the prevalence of four different tetracycline-resistance genes, tetA, tetC, tetH, and tetM, in sediments from four aquaculture farms and their surroundings in the Baltic Sea was monitored by quantitative polymerase chain reaction (qPCR). The presence of three additional tetracycline-resistance genes (tetE, tetG, and tetW) was studied qualitatively by standard PCR, and the amount of bioavailable tetracyclines and total amounts of tetracycline and oxytetracycline in samples were also measured. None of the farms were using tetracycline at the time of the sampling and one of the farms had stopped all antibiotic use six years prior to the first sampling. Two of the farms were sampled over four successive summers and two were sampled once. Our results showed greater copy numbers of tetA, tetC, tetH, and tetM at the farms compared to pristine sites and demonstrated the presence of tetE, tetG, and tetW genes in the sediments under aquaculture farms at most sampling times. However, no resistance genes were found in samples collected 200 m from any of the farms. None of the samples contained therapeutically active concentrations of tetracyclines at any of the sampling times, suggesting that the increase in the prevalence of tetracycline resistance genes is caused by the persistence of these genes in the absence of selection pressure.
Introduction Tetracyclines have been used as a feed additive at Baltic fish farms for four decades, mainly to treat fish illnesses. Tetracyclines end up in the sediment below the farms due to excretion and the accumulation of uneaten feed (1). The emergence of tetracycline-resistant bacterial strains has led many farms to abandon tetracyclines in favor of other antibiotics, such as florfenicol and sulphonamide derivatives, or fish vaccinations. * Corresponding author phone: +358 9 191 57586; fax: +358 9 191 59322; e-mail:
[email protected]. † Department of Applied Chemistry and Microbiology, University of Helsinki. ‡ Center for Marine Environmental Studies, Ehime University. 386
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Among the common practices used to reduce the prevalence of resistance genes at aquaculture farms are switching to other antibiotics or curtailing antibiotic use for an indefinite time. The efficacy of these practices has not been systematically evaluated by studies designed to determine their impact on the stability of resistance genes over a period of several years. Previous studies have primarily concentrated on characterizing antibiotic-resistant bacterial isolates from fish farms (2-4). However, because up to 99% of environmental microbes are uncultivable (5), this approach is unable to account for the prevalence of resistance genes across the entire spectrum of the microbial ecosystem. A full appreciation of the scale of the drug-resistance situation requires a specific quantitative analysis of resistance genes using a culture-free method. In addition, to our knowledge only one study has been published that investigated sediments collected from nearby regions beyond the confines of the aquaculture facilities (6). Because resistant bacteria may be transferred to humans (7), and many resistant bacteria have been reported capable of transferring their resistance elements to opportunistic human pathogens (3, 8), implementation of optimized, efficient strategies to contain and manage resistance-gene emergence and spread is essential. In addition to potential human health impacts, inefficiencies in antibiotic treatment of fish illnesses lead to significant economic losses. In this work, we assessed the presence and persistence of tetracycline-resistance genes at four different fish farms using PCR-based, cultivation-independent methods. We designed PCR primer pairs to detect tetracycline resistance genes tetE, tetG, and tetW, and quantitative PCR primers to detect tetC and tetM genes. Previously designed primers were used for quantification of tetA and tetH (9, 10). These genes include both membrane-bound efflux-pump proteins (tetA, tetC, tetE, tetG, and tetH) and soluble ribosomal protection proteins (tetM and tetW). Sediments were collected from two Finnish aquaculture farms over four successive summers and once from two Swedish aquaculture farms. None of the farms were using any tetracyclines during the sampling period and one of the farms had stopped any antibiotic use six years prior to the first sampling. The sediment DNA was tested by PCR for the presence and copy number of the resistance genes.
Materials and Methods Sampling. Sediment samples were collected during the summers of 2006-2009 from FIN1 and FIN2 farm sites, two medium-scale fish farms in the northern Baltic Sea in the Turku Archipelago. In addition, SWE1 and SWE2 farm sites corresponding to two small-to-medium scale farms in the Stockholm Archipelago were sampled in August 2007. The farms are geographically separated by several kilometers. Pristine reference samples outside the FIN1 farm site were collected at distances of 200, 400, 600, 800, and 1000 m from the farm. Pristine samples from the FIN2, SWE1, and SWE2 farm sites were collected at a distance of 200 m from each farm. Sampling sites are characterized in Table 1. During the first summer, samples were collected using an Ekman Grab probe (ENVCO, Romania). The following summer, a sediment Limnos probe was used (Limnos Ltd., Turku, Finland). In each case, 2-3 cm of surface sediment was collected. Sediment samples were immediately frozen and stored on dry ice until extraction of DNA at the laboratory. Water temperature and depth were measured at the sample site, and water samples were collected (Limnos Ltd., Turku, Finland) along with sediment samples for measurement of 10.1021/es102725n
2011 American Chemical Society
Published on Web 12/15/2010
TABLE 1. Sampling Locations and Their Characteristicsa location FIN1 farm site
production volume (tons year-1)
farmed fish
50
FIN1 200-1000 m
Rainbow Trout, Common Whitefish none
FIN2 farm site
Rainbow Trout
50
FIN2 pristine site SWE1 farm site
none Rainbow Trout
none 5
SWE1 pristine site SWE2 farm site
none Rainbow Trout
none 50
SWE2 pristine site
none
none
none
description of the location A cage farm situated in a long narrow strait between two islands. Use of antibiotics stopped ca. 2000. Sampled 200-1000 m away from FIN1 farm site along the strait at 200-m intervals. A cage farm situated in a wide strait with a strong current. Tetracycline use stopped ca. 2000; florfenicol used occasionally. Sampled behind a small peninsula 200 m from FIN2 farm site. A cage farm situated in a shallow bay. Florfenicol used occasionally. Sampled 200 m toward open water from SWE1 farm site. A cage farm situated in a shallow bay. Florfenicol used occasionally. Sampled 200 m toward open water from SWE2 farm site.
a
All farms are situated in brackish water in the archipelago area in the northern Baltic Sea. FIN1 200-1000m denotes sediments collected from FIN1 farm site at 200-m intervals (200, 400, 600, 800, and 1000 m). FIN2 pristine site, SWE1 pristine site and SWE2 pristine site denote sediments collected 200 m from the respective farms.
TABLE 2. Designed Primer Pairs, Their Melting Temperatures and Lengths of the Resulting Amplicons gene tetE tetG tetW tetA qPCR tetC qPCR tetH qPCR tetM qPCR
sequence 5′-3′
Tm (°C)
length (base pairs)
reference
TTG TTG GAA AGG CTA ATG TTG C GGG TTG CAC TAT ACA AAA ATG C GAT TAC ACG ATT ATG GCA TCA G CAG CAA CAG AAT CGG GAA C GGG AGC GTC GAA AAA GG ACA GCA AAG CGG AAA CAA CC GCT ACA TCC TGC TTG CCT TC CAT AGA TCG CCG TGA AGA GG TGC GTT GAT GCA ATT TCT ATG C GGA ATG GTG CAT GCA AGG AG CAA CCC ATT ACG GTG TGC TA AAG TGT GGT TGA GAA TGC CA GCA ATT CTA CTG ATT TCT GC CTG TTT GAT TAC AAT TTC CGC
60
986
this study
60
671
this study
60
1113
this study
64
210
9
64
335
this study
60
164
10
60
186
this study
pH and oxygen content (Eutech Instruments, Thermo Scientific, U.S.A.). DNA Extraction. DNA was extracted using a commercial FastDNA spin kit for soil (Qbiogene, Morgan Irvine, CA, U.S.A.) according to the instructions provided by the manufacturer. Extractions of two sediment samples (500 mg wet weight) were done in parallel, followed by washing with 5.5 M guanidine thiocyanate to remove any humic acids. DNA concentrations were determined with the Quant-iT PicoGreen reagent (Molecular Probes, Eugene, OR, USA) by measuring fluorescence using a Victor3 fluoroluminometer (Perkin-Elmer, Waltham, MA, USA) with excitation and emission wavelengths of 485 and 535 nm, respectively. Primer Design. Primers were designed to amplify five commonly occurring tetracycline resistance genes, tetC, tetE, tetG, tetM, and tetW. All known sequences of the genes were retrieved from GenBank database and aligned using MUSCLE (11) on Sepeli cluster (Center for Scientific Computing, Espoo, Finland). The sequences were manually processed using the BioEdit program (Tom Hall, Ibis Biosciences, Carlsbad, CA, U.S.A.). The primers were designed to target conserved regions of the genes, have a melting temperature around 60 °C and amplify fragments of the resistance genes ∼1000 base pairs in length. After primer design, a BLAST analysis was performed on the primer sequences to confirm their specificity for the target genes. Quantitative PCR primers for tetC and tetM were similarly designed. The forward primer was the same as that used to amplify the longer product. A reverse primer was designed
based on the sequence alignment to have a melting temperature of 60 °C and amplify a 300 base-pair fragment of the tetC and tetM genes. PCR of the Resistance Genes. PCR reactions contained 1× HF buffer (Finnzymes, Espoo, Finland), 200 µM of each nucleotide (Finnzymes, Espoo, Finland), 10 pmol of each primer (Oligomer, Helsinki, Finland), 0.4 U of Phusion polymerase (Finnzymes, Espoo, Finland) and 1 µL of 1:10 dilutions of environmental DNA in a reaction volume of 20 µL. The PCR program was run on a PTC-200 Thermal Cycler (MJ Research, Waretown, MA, U.S.A.) using the following cycling conditions: 30 s at 98 °C (initial denaturation), followed by 40 cycles of 10 s at 98 °C (denaturation), 10 s at the temperatures indicated in Table 2 (annealing), and 40 s at 72 °C (extension). A final 10-min extension was carried out at 72 °C. Two identical replicates of the reaction were run in parallel. The products from June 2006 (FIN1 and FIN2 farm sites) and August 2007 (SWE1 and SWE2 farm sites) were sequenced by the DNA Sequencing Laboratory, Institute of Biotechnology (University of Helsinki, Finland) to confirm that amplifications were correct. Quantitative PCR of tetA, tetC, tetH, and tetM. Quantitative PCR was performed using a DyNAmo Flash SYBR Green kit (Finnzymes, Espoo, Finland), which responds to increases in double-stranded DNA with an increase in fluorescence. The reaction mixture contained 1× DyNAmo Flash SYBR Green master mix solution, 6 pmol of each primer, 1X ROX reference dye (Finnzymes, Espoo, Finland) and 5 µL of sample DNA. Constructs of pUC57 vector and the fragments of tetA VOL. 45, NO. 2, 2011 / ENVIRONMENTAL SCIENCE & TECHNOLOGY
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and tetH genes were ordered presynthesized (GenScript, Piscataway, NJ, USA). Constructs of pDrive vector and the fragments of tetC and tetM genes were constructed using Qiagen PCR Cloning plus Kit and the protocol provided by the manufacturer (Qiagen, Hilden, Germany). Serial 1:10 dilutions of the plasmids were used as standards for quantification. The sediment DNA extracts were diluted 100fold (tetA and tetH) or 200-fold (tetC and tetM) to contain any inhibition caused by the environmental DNA preparations. Three identical reactions were performed in parallel on each plate, and duplicate plates were assayed. Standard deviation values of the measurements were determined from these parallel runs. The qPCR program was run on a 7300 Real Time PCR system (Applied Biosystems, Foster City, CA, U.S.A.). The program consisted of an initial denaturation of 7 min at 95 °C and 40 cycles of 10 s at 95 °C (denaturation) and 30 s at 60 °C for tetH and tetM and 64 °C (extension) for tetA and tetC, respectively. Melting curves for the amplicons were measured by raising the temperature slowly while monitoring fluorescence. The results were analyzed using Sequence Detection Software v.1.2.3. (Applied Biosystems, Foster City, CA, USA). The copy numbers of tetA, tetC, tetH, and tetM were normalized by dividing by the 16S rRNA gene copy number at the respective time points (Pitka¨nen et al., unpublished results) to take into account any temporal variation in bacterial cell numbers. Bioavailability Measurements. Bioavailable tetracyclines were measured using a genetically modified Escherichia coli strain that responds to bioavailable tetracycline by emitting light in proportion to the tetracycline concentration (12). Constitutively luminescent E. coli cells were used to measure the inhibition of luminescence by the samples (13). Lyophilized cells were rehydrated in Luria-Bertani broth at room temperature for 2 h. Distilled water and dilutions of tetracycline stock solutions were used as blanks and standards, respectively. The samples were diluted 10-fold by adding 9 mL of water to 1 g of wet sediment. Duplicates of sample and standard were measured in parallel. Prior to measurement, 50 µL of each (sample, standard or blank) solution was mixed with 50 µL of lyophilized bacteria and incubated for 2 h at 37 °C. Fluorescence was measured using a Victor3 fluoroluminometer (Perkin-Elmer, Waltham, MA, USA). The limit of detection (LOD) for the samples was calculated according to (14). HPLC Measurements. Tetracycline residues were extracted twice from 2.5 g of wet sediment by centrifugation after shaking with 15 mL of EDTA-containing citric acid buffer (0.07 M citric acid, 0.2 M Na2HPO4, 0.01 M EDTA) as described in ref 15. The cleanup and HPLC analysis were adapted from ref 16. The aqueous phase was extracted twice with 5 mL of hexane to remove hydrophobic impurities. The extract was loaded onto solid-phase extraction columns (Sep-Pak Plus-2 C18, Waters) after conditioning with 10 mL of methanol and 10 mL of EDTA. After loading the samples, the column was washed with 10 mL of Milli-Q water, and the absorbed antibiotics were eluted with methanol. The extract was dried under an N2 stream and dissolved in 500 µL of 1.36% KH2PO4. The sample solution was injected into an HPLC instrument with a fluorescence detector (Hitachi Elite LaChrom; excitation and emission wavelengths: 380 and 520 nm, respectively). The mobile phase solution consisted of 15% methanol in imidazol buffer (1.0 M imidazole, 1.0 mM EDTA, 0.08 M Mgacetate pH 7.2), and the flow rate was 1.0 mL/min. The extracted antibiotics were separated on a C18 column (Bridge C18, Waters; 4.6 mm diameter, 150 mm length, 5 µm pore size) at 40 °C.
Results Primer Design. New primers were designed based on the most comprehensive and most up-to-date sequence data 388
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available to account for the sequence diversity of the resistance genes. All known sequences of the resistance genes tetC, tetE, tetG, tetM, and tetW were retrieved and aligned for manual primer design. The primer sequences are listed in Table 2, together with the amplicon lengths and melting temperatures. All of the designed primers amplified the desired products without side products or primer dimers. The specificity of the primers in qPCR experiments was tested using melting curve analyses. Because the primers were designed based on sequence alignments of all available resistance-gene sequence variants, they can be expected to amplify the largest number of sequence variants of the resistance genes from any environmental setting. Tetracycline Resistance Genes in the Sediments. Between 2006 and 2009, the presence of tetC, tetE, tetG, tetM, and tetW genes in sediments directly under the two Finnish fish farms (FIN1 farm site and FIN2 farm site) was monitored by standard PCR. The presence of these genes was also measured by standard PCR in the single sampling of the two Swedish fish farms (SWE1 farm site and SWE2 farm site) performed in 2007. The prevalence of tetA, tetC, tetH, and tetM was also measured by quantitative PCR in the samples from both Finnish and Swedish sites. Pristine sites outside the farms were subjected to a similar analysis; in FIN1 farm site, a series of samples collected out to 1000 m from the farm boundary at 200 m intervals was analyzed. Sediments under FIN1 farm site contained most of the tested tetracycline-resistance genes at all sampling times (Figure 1A). None of the genes were detected outside the farm. Most of the genes tested were also found at the FIN2 farm site and SWE2 farm site, whereas sediment under SWE1 farm site contained only tetC and tetM (Figure 1B). The samples collected outside of these farms contained no tet genes. The numbers of tetC and tetM genes were clearly elevated under all fish farms and were at a similar level at sites FIN1 farm site, SWE1 farm site, and SWE2 farm site (107 copies g-1 sediment, corresponding to 10-3-10-2 copies per copy of 16S rRNA), and varied slightly at FIN2 farm site (106-107 copies g-1 sediment, corresponding to 10-4-10-2 copies per copy of 16S rRNA). The numbers of tetA and tetH were also elevated under the FIN1 and FIN2 farm sites (the copy numbers ranging from 104-105 copies g-1 sediment, corresponding to 10-5-10-4 copies per copy of 16S rRNA) but were not found at the farm sites SWE1 and SWE2. The limit of detection was 2 × 104 copies of tetC and tetM g-1 sediment and 1 × 104 copies of tetA and tetH g-1 sediment. There was no evidence for a decrease in copy numbers during the 4-year observation period. Bioavailable and Total Amount of Tetracyclines in Sediments. The bioavailable and total amounts of tetracyclines in sediments were analyzed by bacterial bioreporter and HPLC. The bioreporter used responds to bioavailable concentration of several tetracyclines (including tetracycline and oxytetracycline), whereas the HPLC assay was configured to specifically detect tetracycline and oxytetracycline. The concentrations of tetracyclines were below the detection limits of both bioreporter assay (300 ng g-1 sediment wet weight for tetracycline) and HPLC (66 and 25 ng g-1 sediment wet weight for oxytetracycline and tetracycline, respectively) in all sediments.
Discussion Aquaculture is thought to stimulate the spread and stability of antibiotic resistance in the environment (as reviewed by Sapkota et al. (17)). It has been shown that antibioticresistant bacteria are more likely to occur in water and sediment associated with aquaculture (3, 4, 6, 18). However, the long-term effects of antibiotic use on aquaculture facilities and their surroundings have received very little
FIGURE 1. Presence of different tetracycline resistance genes and copy numbers of tetA, tetC, tetH, and tetM normalized to the copy number of 16S rRNA at FIN1 farm site (A) and FIN2, SWE1 and SWE2 farm sites (B). FIN1 and FIN2 farm sites were sampled eight times during four consecutive summers; SWE1 and SWE2 farm sites were sampled once. Sediments collected at FIN1 farm site at 200-m intervals extending from the farm boundary correspond to the observations, FIN1 200-1000 m. FIN2, SWE1, and SWE2 pristine sites correspond to the sediments collected 200 m from the respective farms. “+” denotes the presence of a gene; “-” indicates that levels were below the limit of detection in PCR; and “*” indicates that a sample was not measured. Lack of a symbol in the quantitative PCR plot indicates that the respective gene was below the limit of detection of the quantitative PCR. The error bars represent standard deviation from parallel qPCR measurements. The error bars are not indicated if they are smaller than the symbols used in the plot. attention. Most studies on antibiotic resistance and aquaculture have been based on only one sampling per farm (4, 19), and to our knowledge only one study has been published that investigated sediments from collected
from nearby regions beyond the confines of the aquaculture facilities (6). To address these understudied aspects, we qualitatively monitored for the presence of three tetracycline-resistance genes and quantified the prevalence of VOL. 45, NO. 2, 2011 / ENVIRONMENTAL SCIENCE & TECHNOLOGY
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four tetracycline-resistance genes over four successive summers in sediments of two aquaculture facilities situated in the Baltic Sea. We also collected sediments from outside the aquaculture facilities, including a series of five sediments at 200-m intervals from farm FIN1 farm site. Sediments from two additional farms and their surroundings were sampled at a single time point. Resistance genes were monitored by direct PCR-based methods in order to avoid the problems associated with culturing bacteria from environmental samples. A number of tetracycline determinants were clearly elevated in the sediments under FIN1 and FIN2 farm sites during the study period (Figure 1A,B), despite the fact that the FIN1 farm site had discontinued the use of any antibiotics more than 6 years prior to the first sampling and the FIN2 farm site had not used any tetracyclines during the same period. No tetracycline or oxytetracycline residues could be detected in the sediments by the methods employed. Our results thus suggest that tetracycline resistance genes are highly persistent and do not disappear from aquaculture sites, even after several years without antibiotic use. Studies from nonaquatic environments have yielded inconclusive results with respect to the persistence of tetracyclineresistance genes. A simulated wastewater treatment study showed that the number of tet genes in wastewater from a cattle feedlot decreases rapidly if light is provided (20). In addition, treatment in wastewater treatment plants has been shown to reduce the number of tet genes by several orders of magnitude (21, 22). However, tet genes in groundwater underlying pig farms remained high during a 3-year sampling period; however, in that study, the pigs were frequently given tetracyclines (23). A slow rate of antibiotic resistance gene loss has recently been observed for bacteria living in anoxic biofilms, a habitat that is likely common in the sediments under aquaculture farms (24). Although the number of tetA, tetC, tetH, and tetM genes remained elevated in the sediments under fish farms, the prevalence of these genes decreased rapidly outside farms; even samples collected 200 m from farms contained no detectable levels of any of the tet genes tested (Figure 1A,B). The lack of diffusion of tet genes is somewhat surprising, considering the continuous presence of tet genes at the farms and the free flow of seawater. Our results in the Baltic Sea suggest that, although the tetracycline resistance genes are a serious and persistent problem at the farms, they do not spread to the surrounding environment and therefore are likely not a serious environmental problem. In previous studies, tetracycline-resistant bacteria have been cultivated from Mekong river sediments in Vietnam (25, 26) and marine sediments in Japan, including sediments from pristine sites (27). This result can be due to methodological differences because unlike molecular methods, cultivation experiments have the potential to detect even single culturable microbes from environmental samples (28) while excluding the unculturable majority (5). Nevertheless, spreading of tetracycline resistance genes from swine feedlots to the surrounding environment has recently been observed using only molecular methods (29). The presence of resistance determinants without obvious selection pressure has been attributed to constant influx of resistant bacteria from external sources, such as farmlands and wastewater treatment plants (30), fish hatchlings (7), or fish feed (31, 32). Input of resistance genes from agriculture or communal waste is rather implausible in this work because the archipelago surrounding the farms is sparsely inhabited and largely unused for agriculture. Furthermore, the background level of tetracycline resistance genes in Baltic sediments is very low, as indicated by results from outside sites (Figure 1A,B). Conceivably, the fish feed and fish 390
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hatchlings could serve as a constant source of antibiotic resistance genes to the farm sediments. Because the expression of resistance genes in mobile genetic elements is carefully regulated, these genes are not a metabolic burden to the host organisms (33). As a result, the resistance genes that have been enriched as a result of past selection pressure might not disappear from the sediment microbial ecosystem, even after the selection pressure ceases. Furthermore, subtherapeutic antibiotic levels have been found to select for antibiotic-resistant phenotypes (34). Therefore, synthetic antibiotics at negligible concentrations, or even after their complete disappearance from the environment, may have a long-lasting or irreversible influence on the sediment resistome. Such changes in the sediment resistome could explain the presence of tetE and tetW at the FIN1 farm site and their absence from the FIN2 farm site, despite the two farms receiving their feed and hatchlings from the same source. It may be that the establishment of different tet genes is a process that requires a relatively long period of tetracycline exposure, but once established, the genes persist. An increase in antibiotic resistance genes without the actual presence of the antibiotic has also been attributed to coselection with other antibiotics (35). This could be a plausible explanation at the FIN2 farm site, where florfenicol is occasionally used to treat the fish. However, this explanation does not hold for the FIN1, SWE1, or SWE2 farm sites, where little or no or antibiotics are used. A coselection of antibiotic resistance genes with heavy metal resistance genes has also been observed (36). This explanation does not hold for any of our farm sites as biological and chemical analyses of the samples revealed only very low background concentrations of any heavy metals (Pitka¨nen et al., submitted for publication; unpublished results). In summary, tetracycline-resistance genes are found even at small farms that rarely use antibiotics. The copy numbers of tetA, tetC, tetH, and tetM genes remain elevated at farms over the surveillance period of four years in the absence of any selection pressure from tetracycline or even other antibiotics. The resistance genes therefore appear to be a serious and persistent problem to the fish farming industry in the Baltic Sea. Because the resistance genes appear to be locally restricted, the effects to the surrounding aquatic environment are not likely to be serious. These results call for the development of better management strategies for fish farming to prevent the emergence of resistant gene pools in the sediments of aquaculture facilities and promote the disappearance of established resistant gene pools.
Acknowledgments This study was financially supported by the Maj and Tor Nessling Foundation, Academy of Finland, and the GlobalCOE Program, MEXT, Japan. We would like to thank L. Pitka¨nen for her suggestions and remarks, which considerably improved the quality of the manuscript.
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