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Detection and Quantification of Antibiotic Resistance Genes in Stångån River, Sweden
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.
Linköping University, Department of Clinical and Experimental Medicine, Division of Microbiology and Molecular Medicine. Linköping University, Faculty of Health Sciences. Department of Microbiology, Medical Services, County Hospital Ryhov, Jönköping, Sweden.
2014 (English)Manuscript (preprint) (Other academic)
Abstract [en]

Antibiotic resistant bacteria are an emerging global problem which threatens to undermine important advances in modern medicine. It is becoming increasingly clear that the dynamics of antibiotic resistance are not confined to clinical settings. The environment is likely to play an important role in dissemination of antibiotic resistance genes from and to both environmental and pathogenic bacteria. Wastewater treatment plants accumulate both chemical and biological waste from the surrounding urban milieu and have therefore been viewed as potential hotspots for dissemination and development of antibiotic resistance. To assess the effect of wastewater effluent on a river which flows through a Swedish city, sediment and water samples were collected from Stångån River, both upstream and downstream of an adjacent wastewater treatment plant over three months. Seven antibiotic resistance genes and the integrase gene on class 1 integrons were quantified in the collected sediment using realtime PCR. Furthermore, liquid chromatography-mass spectrometry was used to assess the abundance of ten different antibiotics in the water phase of the samples. The results showed an increase in ARGs and integrons downstream of the wastewater treatment plant as compared to upstream. The measured concentrations of antibiotics were low in the water samples from Stångån River, suggesting that selection for antibiotic resistance genes did not occur in the surface water. Instead, the downstream increase in antibiotic resistance genes is likely to be due to accumulation of genes present in the treated effluent discharged from the wastewater treatment plant.

Place, publisher, year, edition, pages
2014.
National Category
Medical and Health Sciences
Identifiers
URN: urn:nbn:se:liu:diva-105869OAI: oai:DiVA.org:liu-105869DiVA: diva2:711725
Available from: 2014-04-11 Created: 2014-04-11 Last updated: 2014-04-11
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)
Opponent
Supervisors
Available from: 2014-04-11 Created: 2014-04-11 Last updated: 2014-04-14Bibliographically approved

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Berglund, BjörnLindgren, Per-Eric

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