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Efficient removal of antibiotics in surface-flow constructed wetlands, with no observed impact on antibiotic resistance genes.
Linköping University, Department of Clinical and Experimental Medicine, Division of Microbiology and Molecular Medicine. Linköping University, Faculty of Health Sciences.
Department of Chemistry, Umeå University, Umeå, Sweden.
Wetland Research Centre, Halmstad University, Halmstad, Sweden.
Wetland Research Centre, Halmstad University, Halmstad, Sweden.
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2014 (English)In: Science of the Total Environment, ISSN 0048-9697, E-ISSN 1879-1026, Vol. 476-477, 29-37 p.Article in journal (Refereed) Published
Abstract [en]

Recently, there have been growing concerns about pharmaceuticals including antibiotics as environmental contaminants. Antibiotics of concentrations commonly encountered in wastewater have been suggested to affect bacterial population dynamics and to promote dissemination of antibiotic resistance. Conventional wastewater treatment processes do not always adequately remove pharmaceuticals causing environmental dissemination of low levels of these compounds. Using constructed wetlands as an additional treatment step after sewage treatment plants have been proposed as a cheap alternative to increase reduction of wastewater contaminants, however this means that the natural microbial community of the wetlands becomes exposed to elevated levels of antibiotics. In this study, experimental surface-flow wetlands in Sweden were continuously exposed to antibiotics of concentrations commonly encountered in wastewater. The aim was to assess the antibiotic removal efficiency of constructed wetlands and to evaluate the impact of low levels of antibiotics on bacterial diversity, resistance development and expression in the wetland bacterial community. Antibiotic concentrations were measured using liquid chromatography-mass spectrometry and the effect on the bacterial diversity was assessed with 16S rRNA-based denaturing gradient gel electrophoresis. Real-time PCR was used to detect and quantify antibiotic resistance genes and integrons in the wetlands, during and after the exposure period. The results indicated that the antibiotic removal efficiency of constructed wetlands was comparable to conventional wastewater treatment schemes. Furthermore, short-term treatment of the constructed wetlands with environmentally relevant concentrations (i.e. 100-2000 ng×l(-1)) of antibiotics did not significantly affect resistance gene concentrations, suggesting that surface-flow constructed wetlands are well-suited for wastewater treatment purposes.

Place, publisher, year, edition, pages
Elsevier, 2014. Vol. 476-477, 29-37 p.
Keyword [en]
Antibiotic resistance genes; Antibiotics; Quantitative real-time PCR; Constructed wetlands
National Category
Medical and Health Sciences
Identifiers
URN: urn:nbn:se:liu:diva-105870DOI: 10.1016/j.scitotenv.2013.12.128ISI: 000333772500004PubMedID: 24448029OAI: oai:DiVA.org:liu-105870DiVA: diva2:711730
Available from: 2014-04-11 Created: 2014-04-11 Last updated: 2017-12-05Bibliographically approved
In thesis
1. Deliberations on the impact of antibiotic contamination on dissemination of antibiotic resistance genes in aquatic environments
Open this publication in new window or tab >>Deliberations on the impact of antibiotic contamination on dissemination of antibiotic resistance genes in aquatic environments
2014 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

The great success of antibiotics in treating bacterial infectious diseases has been hampered by the increasing prevalence of antibiotic resistant bacteria. Not only does antibiotic resistance threaten to increase the difficulty in treating bacterial infectious diseases, but it could also make medical procedures such as routine surgery and organ transplantations very dangerous to perform. Traditionally, antibiotic resistance has been regarded as a strictly clinical problem and studies of the problem have mostly been restricted to a clinical milieu. Recently, non-clinical environments, and in particular aquatic environments, have been recognised as important factors in development and dissemination of antibiotic resistance. Elevated concentrations of antibiotics in an environment are likely to drive a selection pressure which favours resistant bacteria, and are also believed to promote horizontal gene transfer among the indigenous bacteria. Antibiotic resistance genes are often located on mobile genetic elements such as plasmids and integrons, which have the ability to disseminate among taxonomically unrelated species. The environmental bacteria can thus serve as both reservoirs for resistance and hot spots for the development of new antibiotic resistance determinants.

There is still a lack of data pertaining to how high antibiotic concentrations are necessary to drive a selection pressure in aquatic environments. The aim of this thesis is to determine the effect of high and low concentrations of antibiotics on environmental bacterial  communities from different aquatic environments. In the studies performed, antibiotics were measured using liquid chromatography-mass spectrometry. Bacterial diversity and evenness were assessed using molecular fingerprints obtained with 16S rRNA gene-based denaturing gradient gel electrophoresis, and antibiotic resistance genes and class 1 integrons were quantified using real-time PCR.

Water and sediment samples were collected from different rivers and canals in Pakistan. The environments differed in anthropogenic exposure from undisturbed to heavily contaminated. A general trend could be observed of high concentrations of antibiotics correlating to elevated concentrations of antibiotic resistance genes and integrons. Extremely high concentrations of antibiotic resistance genes and integrons were found in the sediments downstream of an industrial drug formulation site, which likely correlated to the high load of antibiotics found in the water. Antibiotic and antibiotic resistance gene concentrations were also shown to increase downstream of Ravi river, which flows through Lahore, a city of more than 10 million inhabitants. Rivers not impacted by anthropogenic contamination were found to contain antibiotics and resistance gene concentrations of similar levels as in Europe and the U.S. Similar measurements were performed in the Swedish river Stångån. The concentrations of antibiotic resistance genes and class 1 integrons were shown to increase in the river after it had passed, and received urban wastewater effluent from the city of Linköping.

A series of constructed wetlands were exposed to a mixture of different antibiotics at environmentally relevant concentrations over a few weeks. The antibiotic exposure did not observably affect the bacterial diversity or integron concentrations. Antibiotic resistance genes were found at low background concentrations, but the antibiotic exposure did not observably affect the concentrations. The constructed wetlands were also found to reduce most antibiotics at levels comparable to conventional wastewater treatment schemes, suggesting that constructed wetlands may be useful supplementary alternatives to conventional wastewater treatment.

To investigate the effect of antibiotics on an uncontaminated aquatic environment in a more controlled setting, microcosms were constructed from lake water and sediments and subsequently exposed to varying concentrations of antibiotics (ranging from wastewater-like concentrations to 1,000 times higher). The water and sediments were gathered from the lake Nydalasjön, near Umeå, which is not exposed to urban waste. While antibiotic resistance genes and class 1 integrons were found in the lake sediments, no increase in the concentrations of these genes could be observed due to the antibiotic additions.

In conclusion, although antibiotic resistance genes and integrons are part of the environmental gene pool, low concentrations of antibiotics do not seem to immediately impact their prevalence. However, aquatic environments exposed to anthropogenic waste do exhibit elevated levels of antibiotic resistance genes and integrons. Aquatic environments heavily polluted with antibiotics also clearly display correspondingly high concentrations of antibiotic resistance genes and integrons. These results clearly indicate the necessity to keep down pollution levels as well as the need to establish the range of antibiotic concentrations which do promote resistance. This must be done in order to enable risk assessments and to establish acceptable levels of antibiotic pollution. It should also be stressed that more research is required to elucidate what effect low levels of antibiotic exposure has on environmental bacterial communities.

Place, publisher, year, edition, pages
Linköping: Linköping University Electronic Press, 2014. 63 p.
Series
Linköping University Medical Dissertations, ISSN 0345-0082 ; 1402
National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:liu:diva-105872 (URN)10.3384/diss.diva-105872 (DOI)978-91-7519-361-8 (ISBN)
Public defence
2014-05-12, Berzeliussalen, Hälsouniversitet, Campus Valla, Linköpings universitet, Linköping, 13:00 (Swedish)
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Available from: 2014-04-11 Created: 2014-04-11 Last updated: 2014-04-14Bibliographically approved

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