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Antibiotics and antibiotic resistance in water environments

-Luis Mart n1,3 nez2,3 and Rafael Canto Fernando Baquero1,3, Jose
Antibiotic-resistant organisms enter into water environments from human and animal sources. These bacteria are able to spread their genes into water-indigenous microbes, which also contain resistance genes. On the contrary, many antibiotics from industrial origin circulate in water environments, potentially altering microbial ecosystems. Risk assessment protocols for antibiotics and resistant bacteria in water, based on better systems for antibiotics detection and antibioticresistance microbial source tracking, are starting to be discussed. Methods to reduce resistant bacterial load in wastewaters, and the amount of antimicrobial agents, in most cases originated in hospitals and farms, include optimization of disinfection procedures and management of wastewater and manure. A policy for preventing mixing human-originated and animal-originated bacteria with environmental organisms seems advisable.
Addresses 1 n y Cajal University Hospital, Department of Microbiology, Ramo CIBER-ESP, Spain 2 National Center for Biotechnology, CSIC, Spain 3 Joint Unit for Antimicrobial Resistance and Virulence, 28034 Madrid, Spain Corresponding author: Baquero, Fernando (baquero@bitmailer.net)

lation) in the environment of antimicrobial agents, detergents, disinfectants, and residues from industrial pollution, as heavy metals, contributes to the evolution and spread of such resistant organisms in the water environment. The heavy use of prophylactic antibiotics in aquaculture [2] can be particularly relevant. On the contrary, environmental bacteria act as an unlimited source of genes that might act as resistance genes when entering in pathogenic organisms. Note that many of these genes are not primarily resistance genes, but belong to the hidden resistome [3], the set of genes able to be converted in antibiotic-resistance genes. Human health risk assessment protocols for antibiotic and resistant bacteria in water are starting to be discussed [4]. Certainly it is difcult to believe why public health ofcers and ecotoxicologists have failed for more than a century to seriously propose the absolute need of preventing the mix between microorganisms from humananimal and environmental compartments.

The four genetic reactors in antibiotic resistance

Antibiotic resistance evolves in bacteria because of the effect of industrially produced antimicrobial agents on bacterial populations and communities. Genetic reactors are places in which the occasion occurs for genetic evolution, particularly because of high biological connectivity, generation of variation, and presence of specic selection. Beyond mutational events, signicant genetic variation occurs as a consequence of recombinatorial events, frequently resulting from genetic exchanges among organisms inside populations and communities. There are four main genetic reactors in which antibiotic resistance evolves (Figure 1). The primary reactor is constituted by the human and animal microbiota, with more than 500 bacterial species involved, in which therapeutic or preventive antibiotics exert their actions. The secondary reactor involves the hospitals, long-term care facilities, farms, or any other place in which susceptible individuals are crowded and exposed to bacterial exchange. The tertiary reactor corresponds to the wastewater and any type of biological residues originated in the secondary reactor, including for instance lagoons, sewage treatment plants, or compost toilets, in which bacterial organisms from many different individuals have the opportunity to mix and genetically react. The fourth reactor is the soil and the surface or ground water environments, where the bacterial organisms originated in the previous reactors mix and counteract with environmental organisms. Water is involved as a crucial agent in all four genetic reactors, but particularly in the last ones. The possibility of reducing the evolvability of antibiotic resistance depends on
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Current Opinion in Biotechnology 2008, 19:260265 This review comes from a themed issue on Environmental Biotechnology Edited by Carla Pruzzo and Pietro Canepari Available online 4th June 2008 0958-1669/$ see front matter # 2008 Elsevier Ltd. All rights reserved. DOI 10.1016/j.copbio.2008.05.006

Introduction
Human and animal pathogenic and potentially pathogenic bacteria are constantly released with wastewater into the water environment. Many of these organisms harbor antibiotic-resistance genes, eventually inserted into genetic mobile platforms (plasmids, transposons, integrons) able to spread among water and soil bacterial communities [1]. Water constitutes not only a way of dissemination of antibiotic-resistant organisms among human and animal populations, as drinking water is produced from surface water, but also the route by which resistance genes are introduced in natural bacterial ecosystems. In such systems, nonpathogenic bacteria could serve as a reservoir of resistance genes and platforms. Moreover, the introduction (and progressive accumuCurrent Opinion in Biotechnology 2008, 19:260265

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Figure 1

probably changing the reactivity of uoroquinolones in the soilwater interphase. It is to be noticed that general alterations in water or in soil (as pH changes, or ionic strength) might alter these antibioticsoilwater interactions, producing different levels of antibiotic release (dissolution) from soil particles. In a study, half-lives in soil have been estimated in 2030 days for erythromycin or oleandomycin.

Industrial antibiotics in watersludge environments

Antimicrobial agents as sulfonamides, macrolides, trimethoprim, cephalosporins, or uoroquinolones can be found at potentially active concentrations in activated sludge treatment, and the antibiotic load along the year correlates with the variation in annual consumption data, being higher in the winter [6]. The wastewater concentration of antimicrobials depends on the sludgewastewater partition coefcient, but with uoroquinolones eld experiments of sludge application to agricultural land conrmed long persistence of these compounds, but with limited mobility into the subsoil [7]. Very high concentrations of sulfonamides (20 103 ng/ml) have been found in pig farm wastewater, and detection of sulfamethazine has been suggested to serve as a marker for livestock-source contamination in Vietnam [8]. In Japanese urban rivers a high number of antibiotic agents can be detected, including sulfonamides, trimethoprim, and macrolides. In Hong Kong and Shenzhen sewage samples, penicillin levels (as penicillin V) were undetectable, but that was not the case for cephalosporins, as cefalexin or cefotaxime reached concentrations exceeding 1 mg/ml [9], probably sufcient to select organisms producing extended-spectrum beta-lactamases, as CTXM enzymes. If selection of ESBL organism will produce a reduction in the antibiotic concentration is controversial. In compost toilets, amoxicillin decay is negligible, even in the presence of beta-lactamase producing bacteria. Hydrophobic antibiotics, as tetracycline or ciprooxacin were detected in all sludge samples from two Oslo city hospitals, but not in the collected inuent samples, suggesting binding to efuent particles [10]. Similarly, uoroquinolones were consistently found in hospital efuents [11]. The extensive use of antibiotics in human medicine, animal farming, and agriculture leads to antibiotic contamination of manure, which can be used as fertilizer. Leaching tests indicate that in general less than 1% of uoroquinolones in the sludge reached the aqueous phase, which might indicate a relatively reduced mobility when sludge is used to fertilize soil [7]. Nevertheless, that does not exclude localized biological effects on particulated material. Indeed high concentrations of uoroquinolones were found in secondary sludge (sorption). Macrolides were frequently resistant to the processes carried out in sewage treatment plants in South China, and even higher concentrations were found in the nal efuents than in the raw sewages [12].
Current Opinion in Biotechnology 2008, 19:260265

The four genetic reactors in antibiotic resistance, where genetic exchange and recombination shapes the future evolution of resistance determinants. Particularly in the lowest reactors, bacteria from humanassociated or animal-associated microbiota (in black) mix with environmental bacteria (in white), increasing the power of genetic variation and possible emergence of novel mechanisms of resistance that are re-introduced in human or animal environments (back arrows).

the ability of humans to control the ow of active antimicrobial agents, bacterial clones, and genetically based biological information along these genetic reactors.

Industrial antibiotics in soilwater environments

Water dissolves industrial antibiotics that are bound to environmental matrices. Binding to soil particles (and sediments) delays its biodegradation and explains longterm permanence of the drugs in the environment. Of course, soil particles also remove antibiotics from water, so that a kind of watersoil pharmacokinetics might be considered. Antimicrobial agents are retained in soil by its association with soil chemicals. For instance, Elliot soil humic acids produce complexation of antibiotics [5]. Interestingly, heavy metals (as methyl-mercury) also associate with humic acids, so that in the water lm associated with soil organic particles several antimicrobial effects might be simultaneously present. Indeed it appears that in the presence of humic substances, in both dissolved and mineral-bound forms, environmental mobility of antibiotics [5] might increase. Aluminum and iron oxides might alter these interactions by changing surface charge. For instance, sorption to such oxides results in different types of ciprooxacin-surface complexes [5]
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262 Environmental Biotechnology

Water disinfection and wastewater treatment inuence antibiotic concentrations

Water disinfection by ClO(2) might contribute to the removal to beta-lactam agents. The water-degradation of beta-lactams (penicillin G) has been recently explored, being penicilloic acid the main degradation product [13]. Aqueous chlorination of drinking water and wastewater removes trimethoprim activity [14]. Wastewater treatment might eliminate nearly 80% of uoroquinolones or tetracyclines before they enter rivers, and are susceptible to photodegradation [7,9]. Antibiotic removal efciencies by wastewater treatment are less effective for macrolides that are relatively persistent in the environment [9]. The application of techniques for antibiotic removal by coagulation and granular activated carbon ltration, ionic treatment or micelleclay systems are promising for the removal of tetracycline and sulfonamides [15].

In these cases effective chlorine doses might require concentrations of 100 mg/L. Filtering technologies including surface-modied ones activated carbon lter media are promising (more than 6 logs reduction). The results of some studies [20] indicate the possibility that chlorination might result in the alteration of wastewater populations, with the selection of chlorine-resistant bacteria (related to Bacillus subtilis and Bacillus licheniformis), which might contribute to the selection of particular resistance genes and genetic platforms. A number of studies have addressed the inuence of different types of manure management into the environmental fate of resistance genes. High-intensity manure management (with amending, watering, and turning) was more effective in reducing permanence of resistance genes than lowintensity management. Different genes had diverse kinetics of maintenance [21,22,23], probably in relation to the organisms harboring them.

Measuring water-antibiotic concentrations

The progress in instrumental analytical chemistry, using electrophoretic and chromatographic techniques, as liquid chromatographytandem mass spectrometry enables to detect many different types of antibiotics at concentrations of nanograms/liter, after solid-phase extraction [16,17]. Alternatively, immunochemical approaches are also useful for inexpensive quick screening [17]. Voltammetry and amperometric detection of tetracyclines using multiwall carbon nanotube modied electrodes have been recently proposed for monitoring water samples [18]. Even commercially available test kits have been successfully used for rapid screening of antibiotic activity in efuents and surface water samples [19]. It is to be noticed that natural organic matter might signicantly impact the results of the analysis of some antibiotics.

Inuence of water-antibiotics on antibiotic resistance

Land application of manure can result in the dispersion of resistant bacteria to water sources. The effect of a concentrated swine feeding operation on surface water and groundwater situated up and down gradient of the swine facility was studied [24]. The proportion of macrolide and tetracycline-resistant enterococci was signicantly increased in down-gradient surface waters. Similarly, samples of surface water sites near wastewater treatment plants in Australia had a signicant increase of antibioticresistant Escherichia coli [25]. Nevertheless, the quantitation of the effects of antibiotics on water bacteria remains a difcult question, which has been addressed by using model systems as mesocosms, soil-lled lysimeters representing the leacheld of a septic system. Under these conditions, the local release of tetracycline (5 mg/ml) had a very small effect in this model on the development of antibiotic resistance [26]. In another model, using river water in continuous ow chemostat system, similar results were found, but with high tetracycline concentrations a greater diversity of tetracyclineresistance genes was detected [27]. In a third model, using a soil microcosm supplemented with pig manure slurry and an Enterococcus faecalis strain harboring a tet(M) resistance gene, stable tetracycline concentrations were unable to inuence the local prevalence of antibiotic-resistant bacteria, but tetracycline-resistance genes persisted for a long time, probably because of horizontal gene transfer to other organisms. Obviously these models do not take into account the possible concentration of drugs and bacteria in particulated surfaces or sediments. A nal concern regards the utilization of prophylactic antibiotics in aquaculture. The heavy use of these compounds, several of which are nonbiodegradable increases antibiotic selective pressure in water, facilitates the transfer of antibioticresistance determinants between aquatic bacteria, including sh and human pathogens, and allows the
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Reducing antibiotic-resistant bacteria in wastewater

Antibiotic-resistant organisms from humans and animals are released into the sewage by contaminated sites (including urine), feces, eventually corpses and manure. In particular, wastewater from hospitals and intensive farming facilities (under concentrated animal feeding operations) is probably a major source of pathogenic and antibiotic-resistant organisms and antibiotic-resistance genes that are released into the environment. It is essential to increase our knowledge on effective barrier measures preventing the incorporation of resistant and pathogenic bacteria into the environment (see Introduction). Wastewater could be disinfected in many ways, including chlorine (23 logs bacterial reduction with chlorine dose of 30 mg/L), ozone (34 logs reduction at 100 mg/L), or ultraviolet light (effective but expensive) [20]. These treatments might differ in different circumstances, as for instance ammonia present in wastewaters might compete for free chlorine to form monochloramine.
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presence of residual antibiotics in commercialized sh and shellsh products [2].

Antibiotic-resistant bacteria in water

Water bacteria might be indigenous to aquatic environments, or exogenous, transiently and occasionally present in the water as a result of shedding from animal, vegetal, or soil surfaces. More than 90% of bacterial strains originated in seawater are resistant to more than one antibiotic, and 20% are resistant at least to ve [28]. The study of antibiotic resistance in indigenous water organisms is important, as it might indicate the extent of alteration of water ecosystems by human action. Aeromonas strains from Portuguese estuarine water carry less frequently beta-lactamase genes than Enterobacteriaceae (10% versus 78%) [29]. In water reservoirs a-half of Aeromonas strains might present multiple antibiotic resistance [30]. Resistance proles of aquatic pseudomonads depend on the species composition, but also from the site in which they were isolated, being more antibiotic-resistant along shorelines and in sheltered bays than in the open water, indicating the inuence of nonaquatic organisms or pollutants. Nevertheless, such inuences can be found in the more remote water environments; psychrotrophic bacteria from Antarctic show various degrees of resistance to industrial antibiotics and metals [31]. The association of antibiotic-resistance and resistance to heavy metals is very frequent in the same organism (also in the same plasmid, transposon, or integron) so that industrial pollution probably selects for antibiotic-resistance and vice versa [32]. Indeed metal contamination represents a long-standing, widespread, and recalcitrant selection pressure for multiresistant organisms. For the nonaquatic organisms, obviously the density of antibiotic-resistance organisms and antibiotic-resistance genes in fresh water varies with the proximity to areas with increased antibiotic consumption, metal pollution, and between seasons, being more frequently found in rainy seasons [23]. Very little work has been done to elucidate the role of bacterial biolms in water environments and its role in antibiotic resistance. Phenotypic antibiotic resistance in bacterial biolms might indeed protect the water environment from selective events caused by the antibiotic release, which probably are acting more effectively on planktonic bacteria.

platforms involved in antibiotic resistance, but it is clear that genetic techniques are providing a much more accurate image of the real diversity and complexity of antibiotic resistance in water-borne bacteria, if compared with cultivation-depending approaches [29]. Nevertheless, for local purposes, phenotypic techniques, as those based on carbon-utilization and antibiotic-resistance patterns might still be useful for bacterial source tracking [34].

Horizontal gene transfer of antibiotic resistance in water environments

Estuarine water-borne Aeromonas strains carry almost as frequently as Enterobacteriaceae class 1 integron platforms carrying antibiotic-resistance genes [29]. Exclusively environmentally based organisms, as Delftia, also harbor class 3 integrons [35]. The persistence of such genetic structures cannot probably be explained solely by antibiotic selection, suggesting that activities resulting in antibiotic resistance might have other physiological roles, or that they are placed in multifunctional plasmids. The most frequent gene cassette found involves aminoglycoside-resistance genes, rarely under positive selection in our days, and there is a suspicion that some other resistance genes, as integron sul genes, might provide benets for the bacteria, unrelated with resistance. However, some of these mobile gene cassettes in Aeromonas might involve important mechanisms of resistance, as Qnr, involved in uoroquinolone resistance, which might be horizontally propagated by IncU-type plasmids [36]. Certainly the dense bacterial populations in sewage treatment plants favor genetic exchange among bacterial populations and communities, integrons predating transposons and plasmid dissemination. Multiresistance plasmids of broad host-range are consistently recovered in sewage [37]. Interestingly, antibiotic-resistance genes from manure inuence the lagoons and groundwater gene pool, but this pool also contains antibiotic-resistance genes from indigenous bacteria [38]. Aeromonas from aquaculture water systems (sh, eel farming) are particularly resistant to antibiotics [39], and frequently contain plasmids and integrons with multiple genes for antibiotic resistance [40], and the association with heavy-metal resistance is not uncommon [32]. Water originated in transgenic plant elds may constitute a matter of concern, but no signicant differences have been found in bacterial antibiotic-resistance levels between transgenic and nontransgenic corn elds [41].

Tracking the sources for antibiotic-resistant organisms in water

The accessibility of modern molecular techniques for subspecic characterization of bacterial organisms (clonal detection) should readily increase our possibilities for antibiotic-resistance microbial source tracking. Such approach will provide only useful results after a much more comprehensive knowledge of population biology of bacterial organisms, as the genetic diversity of potential organisms entering in water is very high [33]. The same is true for tracking the plasmids and other genetic mobile
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Environmental damage mediated by antibiotics in water environments

Pharmaceuticals are introduced in the environment from human and veterinary applications at volumes comparable with total pesticide loadings [42]. Antibiotic resistance is not the only possible adverse effect of antibiotic release in water environments, and ecotoxicity tests are starting to be introduced to document these effects [43]. Antibiotics might act, at very low concentrations, as signaling agents
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(a kind of hormones) in microbial environments [4446]. Common receptors have been identied in plants for a number of antibiotics and disinfectants affecting chloroplast replication (uoroquinolones), transcriptiontranslation (tetracyclines, macrolides, lincosamides, aminoglycosides, pleuromutilins), folate biosynthesis (sulfonamides, and probably trimetoprim), fatty acid synthesis (triclosan), and sterol biosynthesis (azoles, statins) [42]. During the past years the environmental consequences of the release of triclosan in freshwater environment has been considered [47]. Ciprooxacin affects stream microbial communities, including those colonizing senesced leaf materials [48]. A matter of major future concern is the effect of antibiotics and disinfectants released into the environment on Cyanobacteria, largely susceptible to antimicrobial agents, as such type of organisms accounts for more than 70% of the total phytoplankton mass, and are responsible for more than a third of the total free O2 production, or CO2 xation. Amazonia is green, visible, and attractive, but there are much bigger microscopic Amazonias! What seems certain is that such alterations in microbial ecosystems, either produced by antimicrobial release or by the unexpected effective dispersal in water environments of resistant pathogenic organisms [49] might be relevant for public health. Future prediction and prevention of antibiotic resistance [50] depends on the research investments in the ecology, including water ecology, of antibiotic-resistant microorganisms.

3. 4.

DAcosta VM, McGrann KM, Hughes DW, Wright GD: Sampling the antibiotic resistome. Science 2006, 311:374-377. Kim S, Aga DS: Potential ecological and human health impacts of antibiotics and antibiotic-resistant bacteria from wastewater treatment plants. J Toxicol Environ Health B: Crit Rev 2007, 10:559-573. Gu C, Karthikeyan KG: Sorption of the antibiotic tetracycline to humicmineral complexes. J Environ Qual 2008, 37:704-711. bel A, Thomsen A, McArdell CS, Joss A, Giger W: Occurrence Go and sorption behavior of sulfonamides, macrolides, and trimethoprim in activated sludge treatment. Environ Sci Technol 2005, 39:3981-3989. Sukul P, Spiteller M: Fluoroquinolone antibiotics in the environment. Rev Environ Contam Toxicol 2007, 191:131-162. Managaki S, Murata A, Takada H, Tuyen BC, Chiem NH: Distribution of macrolides, sulfonamides and trimethoprim in tropical waters: ubiquitous occurrence of veterinary antibiotics in the Mekong delta. Environ Sci Technol 2007, 41:8004-8010. Gulkowska A, Leung HW, So MK, Taniyasu S, Yamashita N, Yeung LW, Richardson BJ, Lei AP, Giesy JP, Lam PK: Removal of antibiotics from wastewater by sewage treatment facilities in Hong Kong and Shenzhen, China. Water Res 2008, 42:395-403.

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10. Thomas KV, Dye C, Schlabach M, Langford KH: Source to sink tracking of selected human pharmaceuticals from two Oslo city hospitals and a wastewater treatment works. J Environ Monit 2007, 12:1410-1418. rklund K, Rendahl P, Johansson MI, Tysklind M, 11. Lindberg RH, Bjo  Andersson BA: Environmental risk assessment with emphasis on sewage treatment plants. Water Res 2007, 41:613-619. A synthetic view of the environmental effects of antibiotics and its potential risks. 12. Xu W, Zhang G, Li X, Zou S, Li P, Hu Z, Li J: Occurrence and elimination of antibiotics at four sewage treatment plants in the Pearl River Delta, South China. Water Res 2007, 19:45264534. 13. Li D, Yang M, Hu J, Zhang Y, Chang H, Jin F: Determination of penicillin G and its degradation products in a penicillin production wastewater treatment plant and the receiving river. Water Res 2008, 42:307-317. 14. Dodd MC, Huang CH: Aqueous chlorination of the antibiotic agent trimethoprim: reaction kinetics and pathways. Water Res 2007, 41:647-655. 15. Choi KJ, Kim SG, Kim SH: Removal of antibiotics by coagulation and granular activated carbon ltration. J Hazard Mater 2008, 151:38-43. D: Determination of antimicrobial az-Cruz MS, Barcelo 16. D residues and metabolites in the aquatic environment by liquid ndem mass spectrometry. Anal Bioanal chromatography ta Chem 2006, 386:973-985. 17. Buchberger WW: Novel analytical procedures for screening of drug residues in water, wastewater, sediment, and sludge. Anal Chim Acta 2007, 19:129-139. s A, Ya n i L, Gonzalez-Corte ez-Seden o P, 18. Vega D, Agu Pingarron JM: Voltammetry and amperometric detection of tetracyclines at multi-wall carbon nanotube modied electrodes. Anal Bioanal Chem 2007, 389:951-958. 19. Smith AJ, Balaam JL, Ward A: The development of a rapid screening technique to measure antibiotic activity in efuents and surface water samples. Mar Pollut Bull 2007, 54:1940-1946. 20. Macauley JJ, Quiang Z, Adams CD, Surampalli R, Mormile MR: Disinfection of swine wastewater using chlorine, ultraviolet light and ozone. Water Res 2006, 10:2017-2026. 21. Pei R, Cha J, Carlson KH, Pruden A: Response of antibiotic resistance genes to biological treatment in dairy lagoon water. Environ Sci Technol 2007, 41:5108-5113. www.sciencedirect.com

Conclusions
An important part of the dispersal and evolution of antibiotic-resistant bacterial organisms depends on water environments. In water, bacteria from different origins (human, animal, environmental) are able to mix, and resistance evolves as a consequence of promiscuous exchange and shufing of genes, genetic platforms, and genetic vectors. At the same time, antibiotics, disinfectants, and heavy metals are released in water, and might exert selective activities, as well as ecological damage in water communities, resulting in antibiotic resistance. Methods should be developed for cheap and reliable: rst, bacterial clones and resistance genes source tracking; second, detection of antibiotics in water environments; third, disinfection of water from antibiotic-resistant populations and the resistance gene pool, and removal of antibiotics from wastewater; and fourth, prevention policies for mixing humananimal-originated and soilwater bacteria.

References and recommended reading

Papers of particular interest, published within the period of review, have been highlighted as:  of special interest 1. 2. Alonso A, Sanchez P, Martinez JL: Environmental selection of antibiotic resistance genes. Environ Microbiol 2001, 3:1-9. Cabello FC: Heavy use of prophylactic antibiotics in aquaculture: a growing problem for human and animal health and for the environment. Environ Microbiol 2006, 8:1137-1144.

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22. Auerbach EA, Seyfried EE, McMahon KD: Tetracycline resistance genes in activated sludge wastewater treatment plants. Water Res 2007, 41:1143-1151. 23. Peak N, Knapp CW, Yang RK, Hanfelt MM, Smith MS, Aga DS,  Graham DW: Abundance of six tetracycline resistance genes in wastewater lagoons at cattle feedlots with different antibiotic use strategies. Environ Microbiol 2007, 9:143-151. The approach of this paper is to explore gene diversity instead of organism diversity in contaminated water, and how this gene population biology is altered by the use of antibiotics. 24. Sapkota AR, Curriero FC, Gibson KE, Schwab KJ: Antibioticresistant enterococci and fecal indicators in surface water and groundwater impacted by a concentrated Swine feeding operation. Environ Health Perspect 2007, 115:1040-1045. 25. Watkinson AJ, Micalizzi GR, Bates JR, Costanzo SD: Novel method for rapid assessment of antibiotic resistance in Escherichia coli isolates from environmental waters by use of a modied chromogenic agar. Appl Environ Microbiol 2007, 73:2224-2229. 26. Atoyan JA, Patenaude EL, Potts DA, Amador JA: Effects of  tetracycline on antibiotic resistance and removal of fecal indicator bacteria in aerated and unaerated leacheld mesocosms. J Environ Sci Health A: Tox Hazard Subst Environ Eng 2007, 42:1571-1578. This paper proposes the use of model systems to predict the environmental effect of antibiotic release in wastewater treatment facilities. oz-Aguayo J, Lang KS, LaPara TM, Gonzalez G, Singer RS: 27. Mun Evaluating the effects of chlortetracycline on the proliferation of antibiotic-resistant bacteria in a simulated river water ecosystem. Appl Environ Microbiol 2007, 17:5421-5425. 28. Martinez JL: Recent advances on antibiotic resistance genes. In Recent Advances in Marine Biotechnology. Molecular Genetics of Marine Organisms, vol 10. Edited by Fingerman, Nagabhushanam. 2003:13-32. 29. Henriques IS, Fonseca F, Alves A, Saavedra MJ, Correia A: Occurrence and diversity of integrons and beta-lactamase genes among ampicillin-resistant isolates from estuarine waters. Res Microbiol 2006, 157:938-947. 30. Blasco MD, Esteve C, Alcaide E: Multiresistant waterborne pathogens isolated from water reservoirs and cooling systems. J Appl Microbiol 2008. 31. De Souza MJ, Nair S, Loka Bharati PA, Chandramohan D: Metal and antibiotic resistance in psychotrophic bacteria from Antarctic marine waters. Ecotoxicology 2006, 15:379-384. 32. Baker-Austin C, Wright MS, Stepanauskas R, McArthur JV: Co selection of antibiotic and metal resistance. Trends Microbiol 2006, 14:176-182. This is an excellent review of the role of metal pollution in selecting antibiotic resistance and vice versa. 33. Olivas Y, Faulkner BR: Fecal source tracking by antibiotic resistance analysis on a watershed exhibiting low resistance. Environ Monit Assess 2008, 139:15-25. 34. Moussa SH, Massengale RD: Identication of the sources of Escherichia coli in a watershed using carbon-utilization patterns and composite data sets. J Water Health 2008, 6:197-207. 35. Xu H, Davies J, Miao V: Molecular characterization of class 3  integrons from Delftia spp. J Bacteriol 2007, 189:6276-6283. The investigation of the integron dispersal in environmental bacterial communities is crucial to understand the antibiotic-resistance gene ow among free-leaving and human-associated or animal-associated bacteria. 36. Cattoir V, Poirel L, Aubert C, Soussy CJ, Nordmann P:  Unexpected occurrence of plasmid-mediated quinolone

resistance determinants in environmental Aeromonas spp. Emerg Infect Dis 2008, 14:231-237. This group of authors explore in this and other papers the origin of clinically relevant antibiotic resistances in environmental bacteria. ter A, Szczepanowski R, Kurz N, Schneiker S, Krahn I, 37. Schlu hler A: Erythromycin resistance-conferring plasmid Pu pRSB105, isolated from a sewage treatment plant, harbors a new macrolide resistance determinant, an integroncontaining Tn402-like element, and a large region of unknown function. Appl Environ Microbiol 2007, 73:1952-1960. 38. Koike S, Krapac IG, Oliver HD, Yannarell AC, Chee-Sanford JC, Aminov RI, Mackie RI: Monitoring and source tracking of tetracycline resistance genes in lagoons and groundwater adjacent to swine production facilities over a 3-year period. Appl Environ Microbiol 2007, 73:4813-4823. 39. Penders J, Stobbering EE: Antibiotic resistance of motile aeromonads in indoor catsh and eel farms in the southern part of The Netherlands. Int J Antimicrob Agents 2007, 31:261-265. 40. Jacobs L, Chenia HY: Characterization of integrons and tetracycline resistance determinants in Aeromonas spp. isolated from South African aquaculture systems. Int J Food Microbiol 2007, 114:295-306. che S, Sanguin H, Pote J, Navarro E, Bernillon D, 41. Demane Mavingui P, Wildi W, Vogel TM, Simonet P: Antibiotic-resistant soil bacteria in transgenic plant elds. Proc Natl Acad Sci U S A 2008, 105:3957-3962. 42. Brain RA, Hanson ML, Solomon KR, Brooks BW: Aquatic plants  exposed to pharmaceuticals: effects and risks. Rev Environ Contam Toxicol 2008, 192:67-115. This paper deals with larger ecological and environmental effects of pharmaceuticals, and particularly antibiotics. 43. Yamashita N, Yasojima M, Nakada N, Miyajima K, Komori K, Suzuki Y, Tanaka H: Effects of antibacterial agents, levooxacin and clarithromycin, on aquatic organisms. Water Sci Technol 2006, 53:65-72. nez JL: Antibiotics as signals that trigger 44. Fajardo A, Mart specic bacterial responses. Curr Opin Microbiol 2008, 11:161-167. 45. Linares JF, Gustafsson I, Baquero F, Martinez JL: Antibiotics as intermicrobial signaling agents instead of weapons. Proc Natl Acad Sci U S A 2006, 103:19484-19489. n JC, Ghysels B, nez-Mart n N, Mercadillo M, Gala 46. Fajardo A, Mart mmler, Baquero F, Matthijs S, Cornelis P, Wiehlmann L, Tu nez JL: The neglected intrinsic resistome of bacterial Mart pathogens. PLoS ONE 2008, 3:e1619. This paper reveals the wealth of genes potentially involved not only in antibiotic resistance in bacterial pathogens but also in environmental organisms, genes able to contribute to the evolution of antibiotic resistance. 47. Capdevielle M, van Egmond R, Whelan M, Versteeg D, HofmannKamensky M, Inauen J, Cunningham V, Woltering D: Consideration of exposure and species sensitivity of triclosan in the freshwater environment. Integr Environ Assess 2008, 4:15-23. 48. Maul JD, Schuler LJ, Beiden JB, Whiles MR, Lydy MJ: Effects of the antibiotic ciprooxacin on stream microbial communities and detritivorous macroinvertebrates. Environ Toxicol Chem 2006, 25:1598-1606. 49. Quinteira S, Peixe L: Multiniche screening reveals the clinically relevant metallo-beta-lactamase VIM-2 in Pseudomonas aeruginosa far from the hospital setting: an ongoing dispersion process? Appl Environ Microbiol 2006, 72:3743-3745. nez JL, Baquero F, Andersson DI: Predicting antibiotic 50. Mart resistance. Nat Rev Microbiol 2007, 5:958-965.

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