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Deliberations on the impact of antibiotic contamination on dissemination of antibiotic resistance genes in aquatic environments
Linköping University, Department of Clinical and Experimental Medicine, Division of Microbiology and Molecular Medicine. Linköping University, Faculty of Health Sciences.
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: urn:nbn:se:liu:diva-105872DOI: 10.3384/diss.diva-105872ISBN: 978-91-7519-361-8 (print)OAI: oai:DiVA.org:liu-105872DiVA: diva2:711749
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
List of papers
1. Occurrence and Abundance of Antibiotics and Resistance Genes in Rivers, Canal and near Drug Formulation Facilities – A Study in Pakistan
Open this publication in new window or tab >>Occurrence and Abundance of Antibiotics and Resistance Genes in Rivers, Canal and near Drug Formulation Facilities – A Study in Pakistan
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2013 (English)In: PLoS ONE, ISSN 1932-6203, E-ISSN 1932-6203, Vol. 8, no 6Article in journal (Refereed) Published
Abstract [en]

Antibiotic resistance (AR) is a global phenomenon that has severe epidemiological ramifications world-wide. It has been suggested that antibiotics that have been discharged into the natural aquatic environments after usage or manufacture can promote the occurrence of antibiotic resistance genes (ARG). These environmental ARGs could serve as a reservoir and be horizontally transferred to human-associated bacteria and thus contribute to AR proliferation. The aim of this study was to investigate the anthropogenic load of antibiotics in Northern Pakistan and study the occurrence of ARGs in selected samples from this region. 19 sampling sites were selected; including six rivers, one dam, one canal, one sewage drain and four drug formulation facilities. Our results show that five of the rivers have antibiotic levels comparable to surface water measurements in unpolluted sites in Europe and the US. However, high levels of antibiotics could be detected in the downstream river in close vicinity of the 10 million city Lahore, 1100, 1700 and 2700 ng L−1 for oxytetracycline, trimethoprim, and sulfamethoxazole respectively. Highest detected levels were at one of the drug formulation facilities, with the measured levels of 1100, 4100, 6200, 7300, 8000, 27000, 28000 and 49000 ng L−1 of erythromycin, lincomycin, ciprofloxacin, ofloxacin, levofloxacin, oxytetracycline, trimethoprim and sulfamethoxazole respectively. ARGs were also detected at the sites and the highest levels of ARGs detected, sulI and dfrA1, were directly associated with the antibiotics detected at the highest concentrations, sulfamethoxazole and trimethoprim. Highest levels of both antibiotics and ARGs were seen at a drug formulation facility, within an industrial estate with a low number of local residents and no hospitals in the vicinity, which indicates that the levels of ARGs at this site were associated with the environmental levels of antibiotics.

Place, publisher, year, edition, pages
Public Library of Science, 2013
National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:liu:diva-96426 (URN)10.1371/journal.pone.0062712 (DOI)000321148400001 ()
Note

Funding Agencies|Swedish Research Council for Environment, Agricultural Sciences and Spatial Planning FORMAS|210-2006-2132|Foundation for Strategic Environmental Research (MISTRA)||

Available from: 2013-08-20 Created: 2013-08-19 Last updated: 2017-12-06
2. Detection and Quantification of Antibiotic Resistance Genes in Stångån River, Sweden
Open this publication in new window or tab >>Detection and Quantification of Antibiotic Resistance Genes in Stångån River, 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.

National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:liu:diva-105869 (URN)
Available from: 2014-04-11 Created: 2014-04-11 Last updated: 2014-04-11
3. Efficient removal of antibiotics in surface-flow constructed wetlands, with no observed impact on antibiotic resistance genes.
Open this publication in new window or tab >>Efficient removal of antibiotics in surface-flow constructed wetlands, with no observed impact on antibiotic resistance genes.
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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
Keyword
Antibiotic resistance genes; Antibiotics; Quantitative real-time PCR; Constructed wetlands
National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:liu:diva-105870 (URN)10.1016/j.scitotenv.2013.12.128 (DOI)000333772500004 ()24448029 (PubMedID)
Available from: 2014-04-11 Created: 2014-04-11 Last updated: 2017-12-05Bibliographically approved
4. Abundance and dynamics of antibiotic resistance genes and integrons in lake sediment microcosms
Open this publication in new window or tab >>Abundance and dynamics of antibiotic resistance genes and integrons in lake sediment microcosms
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2014 (English)In: PLoS ONE, ISSN 1932-6203, E-ISSN 1932-6203, Vol. 9, no 9, e108151- p.Article in journal (Refereed) Published
Abstract [en]

Antibiotic resistance in bacteria causing disease is an ever growing threat to the world. Recently, environmental bacteria have become established as important both as sources of antibiotic resistance genes and in disseminating resistance genes. Low levels of antibiotics and other pharmaceuticals are regularly released into water environments via wastewater, and the concern is that such environmental contamination may serve to create hotspots for antibiotic resistance gene selection and dissemination. In this study, microcosms were created from water and sediments gathered from a lake in Sweden only lightly affected by human activities. The microcosms were exposed to a mixture of antibiotics of varying environmentally relevant concentrations (i.e., concentrations commonly encountered in wastewaters) in order to investigate the effect of low levels of antibiotics on antibiotic resistance gene abundances and dynamics in a previously uncontaminated environment. Antibiotic concentrations were measured using liquid chromatography-tandem mass spectrometry. Abundances of seven antibiotic resistance genes and the class 1 integron integrase gene, intL1, were quantified using real-time PCR. Resistance genes sulI and ermB were quantified in the microcosm sediments with mean abundances 5 and 15 gene copies/10(6) 16S rRNA gene copies, respectively. Class 1 integrons were determined in the sediments with a mean concentration of 3.86x10(4) copies/10(6) 16S rRNA gene copies. The antibiotic treatment had no observable effect on antibiotic resistance gene or integron abundances.

National Category
Basic Medicine
Identifiers
urn:nbn:se:liu:diva-105871 (URN)10.1371/journal.pone.0108151 (DOI)000342351800068 ()25247418 (PubMedID)
Available from: 2014-04-11 Created: 2014-04-11 Last updated: 2017-12-05Bibliographically approved

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