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Antibiotic Resistance in Wastewater: Methicillin-resistant Staphylococcus aureus (MRSA)and antibiotic resistance genes
Linköping University, Department of Clinical and Experimental Medicine, Medical Microbiology . Linköping University, Faculty of Health Sciences. (Per-Eric Lindgren)
2009 (English)Doctoral thesis, comprehensive summary (Other academic)Alternative title
Resistenta gula stafylokocker (MRSA) och antibiotikaresistensgener förekommer i svenskt kommunalt avloppsvatten (Swedish)
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.
Series
Linköping University Medical Dissertations, ISSN 0345-0082 ; 1128
Keyword [en]
MRSA, SCCmec, spa typing, Staphylococcus aureus, methicillin, B-lactam, aminoglycoside, tetracycline, antibiotic, wastewater, wastewater treatment plant
National Category
Microbiology
Identifiers
URN: urn:nbn:se:liu:diva-17709ISBN: 978-91-7393-629-3 (print)OAI: oai:DiVA.org:liu-17709DiVA: diva2:211479
Public defence
2009-05-14, Berzeliussalen, Hälsouniversitetet, Campus US, Linköpings universitet, Linköping, 13:00 (English)
Opponent
Supervisors
Available from: 2009-05-15 Created: 2009-04-15 Last updated: 2009-05-20Bibliographically approved
List of papers
1. Quantification of genes encoding resistance to aminoglycosides, β-lactams and tetracyclines in wastewater environments by real-time PCR
Open this publication in new window or tab >>Quantification of genes encoding resistance to aminoglycosides, β-lactams and tetracyclines in wastewater environments by real-time PCR
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2009 (English)In: International Journal of Environmental Health Research, ISSN 0960-3123, E-ISSN 1369-1619, 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
Keyword
Water pollutants; sewage pollution; water quality; aac(6')-Ie + aph(2''); mecA; tetA; tetB; LUX™ real-time PCR
National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:liu:diva-18293 (URN)10.1080/09603120802449593 (DOI)19370439 (PubMedID)
Available from: 2009-05-15 Created: 2009-05-15 Last updated: 2017-12-13Bibliographically approved
2. A seasonal study of the mecA gene and Staphylococcus aureus including methicillin-resistant S. aureus in a municipal wastewater treatment plant
Open this publication in new window or tab >>A seasonal study of the mecA gene and Staphylococcus aureus including methicillin-resistant S. aureus in a municipal wastewater treatment plant
2009 (English)In: Water Research, ISSN 0043-1354, E-ISSN 1879-2448, Vol. 43, no 4, 925-932 p.Article in journal (Refereed) Published
Abstract [en]

The spread of methicillin-resistant Staphylococcus aureus (MRSA), in which the mecA gene mediates resistance, threatens the treatment of staphylococcal diseases. The aims were to determine the effect of wastewater treatment processes on mecA gene concentrations, and the prevalence of S. aureus and MRSA over time. To achieve this a municipal wastewater treatment plant was investigated for the mecA gene, S. aureus and MRSA, using real-time PCR assays. Water samples were collected monthly for one year, at eight sites in the plant, reflecting different aspects of the treatment process. The mecA gene and S. aureus could be detected throughout the year at all sampling sites. MRSA could also be detected, but mainly in the early treatment steps. The presence of MRSA was verified through cultivation from inlet water. The concentration of the mecA gene varied between months and sampling sites, but no obvious seasonal variation could be determined. The wastewater treatment process reduced the mecA gene concentration in most months. Taken together our results show that the mecA gene, S. aureus and MRSA occur over the year at all sites investigated.

Keyword
Methicillin-resistant, Staphylococcus aureus, mecA, LUX (TM) real-time PCR, spa Typing, Wastewater treatment plant, Seasonal study
National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:liu:diva-17599 (URN)10.1016/j.watres.2008.11.036 (DOI)19084256 (PubMedID)
Note
Original Publication: Stefan Börjesson, Sara Melin, Andreas Matussek and Per-Eric Lindgren, A seasonal study of the mecA gene and Staphylococcus aureus including methicillin-resistant S. aureus in a municipal wastewater treatment plant, 2009, Water Research, (43), 4, 925-932. http://dx.doi.org/10.1016/j.watres.2008.11.036 Copyright: Elsevier Science B.V., Amsterdam. http://www.elsevier.com/ Available from: 2009-07-09 Created: 2009-04-06 Last updated: 2017-12-13Bibliographically approved
3. Methicillin-resistant Staphylococcus aureus (MRSA) in municipal wastewater: An uncharted threat?
Open this publication in new window or tab >>Methicillin-resistant Staphylococcus aureus (MRSA) in municipal wastewater: An uncharted threat?
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(English)Manuscript (Other academic)
Abstract [en]

Methicillin-resistant S. aureus (MRSA) was recently detected in municipal wastewater, why there is a need for further studies to elucidate if MRSA in wastewater constitutes a health risk, and to determine how wastewater treatment processes affects MRSA. We cultivated MRSA from a full-scale wastewater treatment plant to characterise the indigenous MRSA-flora and to investigate how the wastewater treatment process affects the clonal distribution. MRSA isolates were characterised using spa typing, antibiograms, SSCmec typing and detection of Panton Valentine leukocidin (PVL) genes. We found that the wastewater MRSA-flora has a close genetic relationship to clinical isolates, but we also isolated novel spa types, primarily from the activated sludge treatment step. The number of isolates and the diversity of MRSA are reduced by the treatment process, but the process also selects for more extensive antibiotic resistant strains as well as for PVL positive strains.

Keyword
Staphylococcus aureus, MRSA, methicillin, β-lactam, SCCmec, spa typing, Panton Valentine leukocidin, PVL, antibiotic resistance, antibiogram
National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:liu:diva-18295 (URN)
Available from: 2009-05-15 Created: 2009-05-15 Last updated: 2010-01-14Bibliographically approved
4. Genes encoding tetracycline resistance in a full-scale municipal wastewater treatment plant investigated during one year
Open this publication in new window or tab >>Genes encoding tetracycline resistance in a full-scale municipal wastewater treatment plant investigated during one year
(English)Manuscript (Other academic)
Abstract [en]

Tetracycline-resistant bacteria and genes encoding tetracycline resistance are common in anthropogenic environments. We studied how wastewater treatment affects the prevalence and concentration of two genes that encode resistance to tetracycline: tetA and tetB. Using real-time PCR we analysed wastewater samples collected monthly for one year at eight key-sites in a full-scale municipal wastewater treatment plant (WWTP). We detected tetA and tetB at each sampling site and the concentration of both genes, expressed per wastewater volume or per total-DNA, decreased over the treatment process. The reduction of tetA and tetB was partly the result of the sedimentation process. The ratio of tetA and tetB, respectively, to total DNA was lower in or after the biological processes. Taken together our data show that tetracycline resistance genes occur throughout the WWTP and that the concentrations are reduced under conventional operational strategies. However, it is not possible to conclude the eventual risk for humans with respect to resistance spreading.

Keyword
tetA, tetB, tetracycline, LUXTM real-time PCR, wastewater treatment plant
National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:liu:diva-18296 (URN)
Available from: 2009-05-15 Created: 2009-05-15 Last updated: 2010-01-14Bibliographically approved

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