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Quantification of genes encoding resistance to aminoglycosides, β-lactams and tetracyclines in wastewater environments by real-time PCR
Linköping University, Department of Clinical and Experimental Medicine, Medical Microbiology . Linköping University, Faculty of Health Sciences.
Department of Clinical Microbiology, County Hospital Ryhov, Jönköping, Sweden.
Department of Infectious Diseases, Kalmar County Hospital, Kalmar, Sweden.
Department of Natural Science, University of Kalmar, Kalmar, Sweden/Department of Clinical Sciences, Uppsala University Hospital, Uppsala, Sweden.
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2009 (English)In: International Journal of Environmental Health Research, ISSN 0960-3123, 1-12 p.Article in journal (Refereed) Published
Abstract [en]

In this study real-time PCR assays, based on the LUX-technique, were developed for quantification of genes mediating resistance to aminoglycosides [aac(6 ')-Ie + aph(2 ' ')], beta-lactams (mecA), and tetracyclines (tetA and tetB), for use in wastewater environments. The developed assays were applied on DNA extracted from three wastewater-associated environments: soil from an overland flow area treating landfill leachates, biofilm from a municipal wastewater treatment plant, and sludge from a hospital wastewater pipeline. The highest concentration of all genes was observed in the hospital pipeline and the lowest in the overland flow system. TetA and aac(6 ')-Ie + aph(2 ' ') could be detected in all environments. The tetB gene was detected in the overland flow area and the hospital wastewater pipeline and mecA was detected in the wastewater treatment plant and the hospital pipeline. The developed LUX real-time PCR assays were shown to be fast and reproducible tools for detection and quantification of the four genes encoding antibiotic resistance in wastewater.

Place, publisher, year, edition, pages
Taylor & Francis , 2009. 1-12 p.
Keyword [en]
Water pollutants; sewage pollution; water quality; aac(6')-Ie + aph(2''); mecA; tetA; tetB; LUX™ real-time PCR
National Category
Medical and Health Sciences
URN: urn:nbn:se:liu:diva-18293DOI: 10.1080/09603120802449593PubMedID: 19370439OAI: diva2:217643
Available from: 2009-05-15 Created: 2009-05-15 Last updated: 2009-05-15Bibliographically approved
In thesis
1. Antibiotic Resistance in Wastewater: Methicillin-resistant Staphylococcus aureus (MRSA)and antibiotic resistance genes
Open this publication in new window or tab >>Antibiotic Resistance in Wastewater: Methicillin-resistant Staphylococcus aureus (MRSA)and antibiotic resistance genes
2009 (English)Doctoral thesis, comprehensive summary (Other academic)
Alternative title[sv]
Resistenta gula stafylokocker (MRSA) och antibiotikaresistensgener förekommer i svenskt kommunalt avloppsvatten
Abstract [en]

A large part of the antibiotics consumed ends up in wastewater, and in the wastewater the antibiotics may exert selective pressure for or maintain resistance among microorganisms. Antibiotic resistant bacteria and genes encoding antibiotic resistance are commonly detected in wastewater, often at higher rates and concentrations compared to surface water. Wastewater can also provide favourable conditions for the growth of a diverse bacterial community, which constitutes a basis for the selection and spread of antibiotic resistance. Therefore, wastewater treatment plants have been suggested to play a role in the dissemination and development of antibiotic resistant bacteria. Methicillin-resistant Staphylococcus aureus (MRSA) is a large problem worldwide as a nosocomial pathogen, but knowledge is limited about occurrence in non-clinical environments, such as wastewater, and what role wastewater plays in dissemination and development of MRSA.


In this thesis we investigated the occurrence of MRSA in a full-scale wastewater treatment plant (WWTP). We also investigated the concentration of genes encoding resistance to aminoglycosides (aac(6’)-Ie+aph(2’’)), β-lactam antibiotics (mecA) and tetracyclines (tetA and tetB) in three wastewater-associated environments: (1) soil from an overland flow area treating landfill leachates, (2) biofilm from a municipal wastewater treatment plant, and (3) sludge from a hospital wastewater pipeline. In addition, concentrations of mecA, tetA and tetB were investigated over the treatment process in the WWTP. These investigations were performed to determine how the prevalence and concentration of MRSA and the antibiotic resistence genes are affected in wastewater and wastewater treatment processes over time. The occurrence of MRSA was investigated by cultivation and a commercially available real-time PCR assay. In order to determine concentrations of the genes aac(6’)-Ie+aph(2’’), mecA, tetA and tetB in wastewater we developed a LUXTM real-time PCR assay for each gene.


Using cultivation and real-time PCR we could for the first time describe the occurrence of MRSA in wastewater and show that it had a stable occurrence over time in a WWTP. MRSA could mainly be detected in the early treatment steps in the WWTP, and the wastewater treatment process reduced the number and diversity of cultivated MRSA. However, our results also indicate that the treatment process selects for strains with more extensive resistance and possibly higher virulence. The isolated wastewater MRSA strains were shown to have a close genetic relationship to clinical isolates, and no specific wastewater lineages could be detected, indicating that they are a reflection of carriage in the community. Taken together, these data indicate that wastewater may be a potential reservoir for MRSA and that MRSA are more prevalent in wastewater than was previously thought.


The real-time PCR assays, for aac(6’)-Ie+aph(2’’), mecA, tetA, and tetB that we developed, were shown to be sensitive, fast, and reproducible methods for detection and quantification of these genes in wastewater environments. The highest concentrations of all genes were observed in the hospital pipeline, and the lowest in the overland flow system, with tetA and aac(6´)-Ie+aph(2´´) detected in all three environments. In the full-scale WWTP, we continuously detected mecA, tetA and tetB over the treatment process and over time. In addition, it was shown that the treatment process reduces concentrations of all three genes. The data presented in this thesis also indicate that the reduction for all three genes may be connected to the removal of biomass, and in the reduction of tetA and tetB, sedimentation and precipitation appear to play an important role.

Place, publisher, year, edition, pages
Linköping: Linköping University Electronic Press, 2009. 61 p.
Linköping University Medical Dissertations, ISSN 0345-0082 ; 1128
MRSA, SCCmec, spa typing, Staphylococcus aureus, methicillin, B-lactam, aminoglycoside, tetracycline, antibiotic, wastewater, wastewater treatment plant
National Category
urn:nbn:se:liu:diva-17709 (URN)978-91-7393-629-3 (ISBN)
Public defence
2009-05-14, Berzeliussalen, Hälsouniversitetet, Campus US, Linköpings universitet, Linköping, 13:00 (English)
Available from: 2009-05-15 Created: 2009-04-15 Last updated: 2009-05-20Bibliographically approved

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Börjesson, StefanLindgren, Per-Eric
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