Tag: Pollutants

After a long wait (1), Science of the Total Environment has finally decided to make our paper on selection of antibiotic resistance genes in sewage treatment plants (STPs) available (2). STPs are often suggested to be “hotspots” for emergence and dissemination of antibiotic-resistant bacteria (3-6). However, we actually do not know if the selection pressures within STPs, that can be caused either by residual antibiotics or other co-selective agents, are sufficiently large to specifically promote resistance. To better understand this, we used shotgun metagenomic sequencing of samples from different steps of the treatment process (incoming water, treated water, primary sludge, recirculated sludge and digested sludge) in three Swedish STPs in the Stockholm area to characterize the frequencies of resistance genes to antibiotics, biocides and metal, as well as mobile genetic elements and taxonomic composition. In parallel, we also measured concentrations of antibiotics, biocides and metals.

We found that only the concentrations of tetracycline and ciprofloxacin in the influent water were above those that we predict to cause resistance selection (7). However, there was no consistent enrichment of resistance genes to any particular class of antibiotics in the STPs, neither for biocide and metal resistance genes. Instead, the most substantial change of the bacterial communities compared to human feces (sampled from Swedes in another study of ours (8)) occurred already in the sewage pipes, and was manifested by a strong shift from obligate to facultative anaerobes. Through the treatment process, resistance genes against antibiotics, biocides and metals were not reduced to the same extent as fecal bacteria were.

Worryingly, the OXA-48 beta-lactamase gene was consistently enriched in surplus and digested sludge. OXA-48 is still rare in Swedish clinical isolates (9), but provides resistance to carbapenems, one of our most critically important classes of antibiotics. However, taken together metagenomic sequencing did not provide clear support for any specific selection of antibiotic resistance. Rather, since stronger selective forces affect gross taxonomic composition, and thereby also resistance gene abundances, it is very hard to interpret the metagenomic data from a risk-for-selection perspective. We therefore think that comprehensive analyses of resistant vs. non-resistant strains within relevant species are warranted.

Taken together, the main take-home messages of the paper (2) are:

• There were no apparent evidence for direct selection of resistance genes by antibiotics or co-selection by biocides or metals
• Abiotic factors (mostly oxygen availability) strongly shape taxonomy and seems to be driving changes of resistance genes
• Metagenomic and/or PCR-based community studies may not be sufficiently sensitive to detect selection effects, as important shifts towards resistant may occur within species and not on the community level
• The concentrations of antibiotics, biocides and metals were overall reduced, but not removed in STPs. Incoming concentrations of antibiotics in Swedish STPs are generally low
• Resistance genes are overall reduced through the treatment process, but far from eliminated

References and notes

1. Okay, those who takes notes know that I have already complained once before on Science of the Total Environment’s ridiculously long production handling times. But, seriously, how can a journal’s production team return the proofs for after three days of acceptance, and then wait seven weeks before putting the final proofs online? I still wonder what is going on beyond the scenes, which is totally obscure because the production office also refuses to respond to e-mails. Not a nice publishing experience this time either.
2. Bengtsson-Palme J, Hammarén R, Pal C, Östman M, Björlenius B, Flach C-F, Kristiansson E, Fick J, Tysklind M, Larsson DGJ: Elucidating selection processes for antibiotic resistance in sewage treatment plants using metagenomics. Science of the Total Environment, in press (2016). doi: 10.1016/j.scitotenv.2016.06.228 [Paper link]
3. Rizzo L, Manaia C, Merlin C, Schwartz T, Dagot C, Ploy MC, Michael I, Fatta-Kassinos D: Urban wastewater treatment plants as hotspots for antibiotic resistant bacteria and genes spread into the environment: a review. Science of the Total Environment, 447, 345–360 (2013). doi: 10.1016/j.scitotenv.2013.01.032
4. Laht M, Karkman A, Voolaid V, Ritz C, Tenson T, Virta M, Kisand V: Abundances of Tetracycline, Sulphonamide and Beta-Lactam Antibiotic Resistance Genes in Conventional Wastewater Treatment Plants (WWTPs) with Different Waste Load. PLoS ONE, 9, e103705 (2014). doi: 10.1371/journal.pone.0103705
5. Yang Y, Li B, Zou S, Fang HHP, Zhang T: Fate of antibiotic resistance genes in sewage treatment plant revealed by metagenomic approach. Water Research, 62, 97–106 (2014). doi: 10.1016/j.watres.2014.05.019
6. Berendonk TU, Manaia CM, Merlin C, Fatta-Kassinos D, Cytryn E, Walsh F, et al.: Tackling antibiotic resistance: the environmental framework. Nature Reviews Microbiology, 13, 310–317 (2015). doi: 10.1038/nrmicro3439
7. Bengtsson-Palme J, Larsson DGJ: Concentrations of antibiotics predicted to select for resistant bacteria: Proposed limits for environmental regulation. Environment International, 86, 140–149 (2016). doi: 10.1016/j.envint.2015.10.015
8. Bengtsson-Palme J, Angelin M, Huss M, Kjellqvist S, Kristiansson E, Palmgren H, Larsson DGJ, Johansson A: The human gut microbiome as a transporter of antibiotic resistance genes between continents. Antimicrobial Agents and Chemotherapy, 59, 10, 6551–6560 (2015). doi: 10.1128/AAC.00933-15
9. Hellman J, Aspevall O, Bengtsson B, Pringle M: SWEDRES-SVARM 2014. Consumption of antimicrobials and occurrence of antimicrobial resistance in Sweden. Public Health Agency of Sweden and National Veterinary Institute, Solna/Uppsala, Sweden. Report No.: 14027. Available from: http://www.folkhalsomyndigheten.se/publicerat-material/ (2014)

So, on Thursday (May 26th) I will defend my thesis, titled “Antibiotic resistance in the environment: a contribution from metagenomic studies”. I will not dwell into this by writing a novel text, but will instead shamelessly reproduce the press release, which should give a reasonable overview of what I have been doing:

More and more people are infected with antibiotic resistant bacteria. But how do bacteria become resistant? A doctoral thesis from the Centre for Antibiotic Resistance Research at University of Gothenburg has investigated the role of the environment in the development of antibiotic resistance.

“An important question we asked was how low concentrations of antibiotics that can favour the growth of resistant bacteria in the environment”, says Johan Bengtsson-Palme, author of the thesis.

“Based on our analyses, we propose emission limits for 111 antibiotics that should not be exceeded in order to avoid that environmental bacteria become more resistant.”

A starting point to regulate antibiotic pollution
A recent report, commissioned by the British Prime Minister David Cameron, proposes that the emission limits suggested in Johan’s thesis should be used as a starting point to regulate antibiotic pollution from, for example, pharmaceutical production – globally.

“Many people are surprised that such regulations are not already in place, but today it is actually not a crime to discharge wastewater contaminated with large amounts of antibiotics, not even in Europe”, says Johan Bengtsson-Palme.

Resistance genes
In one of the studies in the thesis, the researchers show that resistance genes against a vast range of antibiotics are enriched in an Indian lake polluted by dumping of wastewater from pharmaceutical production.

“It’s scary. Not only do the bacteria carry a multitude of resistance genes. They are also unusually well adapted to share those genes with other bacteria. If a disease-causing bacterium ends up in the lake, it may quickly pick up the genes it needs to become resistant. Since the lake is located close to residential areas, such spread of resistant bacteria to humans is not hard to imagine”, says Johan Bengtsson-Palme.

Spreading by travelers
The thesis also shows that resistant bacteria spread in the intestines of travelers who have visited India or Central Africa, even if the travelers themselves have not become sick.

“That resistant bacteria spread so quickly across the planet highlights that we must adopt a global perspective on the resistance problem”, says Johan Bengtsson-Palme. “Furthermore, it is not enough to reduce the use of antibiotics in healthcare. We must also reduce the use of antibiotics for animals, and try to limit the releases of antibiotics into the environment to try to get control over the growing antibiotic resistance problem before it is too late”.

The thesis Antibiotic resistance in the environment: a contribution from metagenomic studies will be defended on a dissertation on May 26th.

In the most recent issue of the Medicine Maker (#0416), there is a short opinion piece by me and Joakim Larsson, in which we argue for that pharmaceutical companies should live up to their ethical responsibilities, and may actually benefit from doing so (1). We were invited to write for the Medicine Maker based on our recent papers on proposed limits for antibiotic discharges into the environment (2) and minimal selective concentrations (3).

We argue that now as PNECs for resistance selection are available, they should be applied in regulatory contexts. The recent O’Neill report on antimicrobial resistance (commissioned by the British Government) specifically highlighted the urgent need for enforceable regulations on antibiotic discharges (4). The concentrations we reported in our Environment International paper (2) can be used by local, national and international authorities to define emission limits for antibiotic-producing factories, but also for pharmaceutical companies to assess and manage risks for resistance selection associated with their own discharges.

The Medicine Maker can be read for free, but requires registration to access its full content. The full opinion piece can be found here.

References

1. Bengtsson-Palme J, Larsson DGJ: Time to limit antibiotic pollution. The Medicine Maker, 0416, 302, 17–18 (2016). [Paper link]
2. Bengtsson-Palme J, Larsson DGJ: Concentrations of antibiotics predicted to select for resistant bacteria: Proposed limits for environmental regulation. Environment International, 86, 140–149 (2016). doi: 10.1016/j.envint.2015.10.015 [Paper link]
3. Lundström S, Östman M, Bengtsson-Palme J, Rutgersson C, Thoudal M, Sircar T, Blanck H, Eriksson KM, Tysklind M, Flach C-F, Larsson DGJ: Minimal selective concentrations of tetracycline in complex aquatic bacterial biofilms. Science of the Total Environment, 553, 587–595 (2016). doi: 10.1016/j.scitotenv.2016.02.103 [Paper link]
4. Review on Antimicrobial Resistance: Antimicrobials in agriculture and the environment: Reducing unnecessary use and waste (J O’Neill, Ed,) (2015). [Link].

After a long wait (1) Sara Lundström’s paper establishing minimal selective concentrations (MSCs) for the antibiotic tetracycline in complex microbial communities (2), of which I am a co-author, has gone online. Personally, I think this paper is among the finest work I have been involved in; a lot of good science have gone into this publication. Risk assessment and management of antibiotics pollution is in great need of scientific data to underpin mitigation efforts (3). This paper describes a method to determine the minimal selective concentrations of antibiotics, and investigates different endpoints for measuring those MSCs. The method involves a testing system highly relevant for aquatic communities, in which bacteria are allowed to form biofilms in aquaria under controlled antibiotic exposure. Using the system, we find that 1 μg/L tetracycline selects for the resistance genes tetA and tetG, while 10 μg/L tetracycline is required to detect changes of phenotypic resistance. In short, the different endpoints studied (and their corresponding MSCs) were:

• CFU counts on R2A plates with 20 μg/mL tetracycline – MSC = 10 μg/L
• MIC range – MSC ~ 10-100 μg/L
• PICT, leucine uptake after short-term TC challenge – MSC ~ 100 μg/L
• Increased resistance gene abundances, metagenomics – MSC range: 0.1-10 μg/L
• Increased resistance gene abundances, qPCR (tetA and tetG) – MSC ≤ 1 μg/L
• Changes to taxonomic diversity – no significant changes detected
• Changes to taxonomic community composition – MSC ~ 1-10 μg/L

This study confirms that the estimated PNECs we reported recently (4) correspond well to experimentally determined MSCs, at least for tetracycline. Importantly, the selective concentrations we report for tetracycline overlap with those that have been reported in sewage treatment plants (5). We also see that tetracycline not only selects for tetracycline resistance genes, but also resistance genes against other classes of antibiotics, including sulfonamides, beta-lactams and aminoglycosides. Finally, the approach we describe can be used for improved in risk assessment for (also other) antibiotics, and to refine the emission limits we suggested in a recent paper based on theoretical calculations (4).

References and notes

1. Okay, seriously: how can a journal’s production team return the proofs for a paper within 24 hours of acceptance, and then wait literally five weeks before putting the final proofs online? Nothing against STOTEN, but I honestly wonder what was going on beyond the scenes here.
2. Lundström SV, Östman M, Bengtsson-Palme J, Rutgersson C, Thoudal M, Sircar T, Blanck H, Eriksson KM, Tysklind M, Flach C-F, Larsson DGJ: Minimal selective concentrations of tetracycline in complex aquatic bacterial biofilms. Science of the Total Environment, 553, 587–595 (2016). doi: 10.1016/j.scitotenv.2016.02.103 [Paper link]
3. Ågerstrand M, Berg C, Björlenius B, Breitholtz M, Brunstrom B, Fick J, Gunnarsson L, Larsson DGJ, Sumpter JP, Tysklind M, Rudén C: Improving environmental risk assessment of human pharmaceuticals. Environmental Science and Technology (2015). doi:10.1021/acs.est.5b00302
4. Bengtsson-Palme J, Larsson DGJ: Concentrations of antibiotics predicted to select for resistant bacteria: Proposed limits for environmental regulation. Environment International, 86, 140-149 (2016). doi: 10.1016/j.envint.2015.10.015
5. Michael I, Rizzo L, McArdell CS, Manaia CM, Merlin C, Schwartz T, Dagot C, Fatta-Kassinos D: Urban wastewater treatment plants as hotspots for the release of antibiotics in the environment: a review. Water Research, 47, 957–995 (2013). doi:10.1016/j.watres.2012.11.027

Yesterday was an intensive day for typesetters apparently, since they put two of my papers online on the same day. This second paper was published in Environment International, and focuses on predicting minimal selective concentrations for all antibiotics present in the EUCAST database (1).

Today (well, up until yesterday at least), we have virtually no knowledge of which environmental concentrations that can exert a selection pressure for antibiotic resistant bacteria. However, experimentally determining minimal selective concentrations (MSCs) in complex ecosystems would involve immense efforts if done for all antibiotics. Therefore, efforts to theoretically determine MSCs for different antibiotics have been suggested (2,3). In this paper we therefore estimate upper boundaries for selective concentrations for all antibiotics in the EUCAST database, based on the assumption that selective concentrations a priori must be lower than those completely inhibiting growth. Data on Minimal Inhibitory Concentrations (MICs) were obtained for 122 antibiotics and antibiotics combinations, the lowest observed MICs were identified for each of those across all tested species, and to compensate for limited species coverage, we adjusted the lowest MICs for the number of tested species. We finally assessed Predicted No Effect Concentrations (PNECs) for resistance selection using an assessment factor of 10 to account for the differences between MICs and MSCs. Since we found that the link between taxonomic similarity between species and lowest MIC was weak, we have not compensated for the taxonomic diversity that each antibiotic was tested against – only for limited number of species tested. In most cases, our PNECs for selection of resistance were below available PNECs for ecotoxicological effects retrieved from FASS. Also, concentrations predicted to be selective have, for some antibiotics, been detected in regular sewage treatment plants (4), and are greatly exceeded in environments polluted by pharmaceutical pollution (5-7), often with drastic consequences in terms of resistance gene enrichments (8-10). This is a central issue since in principle a transfer event of a novel resistance determinant from an environmental bacteria to an (opportunistic) human pathogen only need to occur once to become a clinical problem (11). Once established, the gene could then spread through human activities, such as trade and travel (7,13). Importantly, this paper:

• Provides upper boundaries for selective concentrations (MSCs) for 111 antibiotics
• Predicts no effect concentrations (PNECs) for resistance selection
• Can guide implementation of compound-specific emission limits based on the provided concentrations

The paper is available under open access here. We hope, and believe, that the data will be of great use in environmental risk assessments, in efforts by industries, regulatory agencies or purchasers of medicines to define acceptable environmental emissions of antibiotics, in the implementation of environmental monitoring programs, for directing mitigations, and for prioritizing future studies on environmental antibiotic resistance.

References:

1. Bengtsson-Palme J, Larsson DGJ: Concentrations of antibiotics predicted to select for resistant bacteria: Proposed limits for environmental regulation. Environment International, 86, 140-149 (2016). doi: 10.1016/j.envint.2015.10.015 [Paper link]
2. Ågerstrand M, Berg C, Björlenius B, Breitholtz M, Brunstrom B, Fick J, Gunnarsson L, Larsson DGJ, Sumpter JP, Tysklind M, Rudén C: Improving environmental risk assessment of human pharmaceuticals. Environmental Science and Technology (2015). doi:10.1021/acs.est.5b00302
3. Tello A, Austin B, Telfer TC: Selective pressure of antibiotic pollution on bacteria of importance to public health. Environmental Health Perspectives, 120, 1100–1106 (2012). doi:10.1289/ehp.1104650
4. Michael I, Rizzo L, McArdell CS, Manaia CM, Merlin C, Schwartz T, Dagot C, Fatta-Kassinos D: Urban wastewater treatment plants as hotspots for the release of antibiotics in the environment: a review. Water Research, 47, 957–995 (2013). doi:10.1016/j.watres.2012.11.027
5. Larsson DGJ, de Pedro C, Paxeus N: Effluent from drug manufactures contains extremely high levels of pharmaceuticals. Journal of Hazardous Materials, 148, 751–755 (2007). doi:10.1016/j.jhazmat.2007.07.008
6. Fick J, Söderström H, Lindberg RH, Phan C, Tysklind M, Larsson DGJ: Contamination of surface, ground, and drinking water from pharmaceutical production. Environmental Toxicology and Chemistry, 28, 2522–2527 (2009). doi:10.1897/09-073.1
7. Larsson DGJ: Pollution from drug manufacturing: review and perspectives. Philosophical Transactions of the Royal Society London, Series B Biological Sciences, 369 (2014). doi:10.1098/rstb.2013.0571
8. Bengtsson-Palme J, Boulund F, Fick J, Kristiansson E, Larsson DGJ: Shotgun metagenomics reveals a wide array of antibiotic resistance genes and mobile elements in a polluted lake in India. Frontiers in Microbiology, Volume 5, Issue 648 (2014). doi: 10.3389/fmicb.2014.00648 [Paper link]
9. Kristiansson E, Fick J, Janzon A, Grabic R, Rutgersson C, Weijdegård B, Söderström H, Larsson DGJ: Pyrosequencing of antibiotic-contaminated river sediments reveals high levels of resistance and gene transfer elements. PLoS ONE, Volume 6, e17038 (2011). doi:10.1371/journal.pone.0017038.
10. Marathe NP, Regina VR, Walujkar SA, Charan SS, Moore ERB, Larsson DGJ, Shouche YS: A Treatment Plant Receiving Waste Water from Multiple Bulk Drug Manufacturers Is a Reservoir for Highly Multi-Drug Resistant Integron-Bearing Bacteria. PLoS ONE, Volume 8, e77310 (2013). doi:10.1371/journal.pone.0077310
11. Bengtsson-Palme J, Larsson DGJ: Antibiotic resistance genes in the environment: prioritizing risks. Nature Reviews Microbiology, 13, 369 (2015). doi: 10.1038/nrmicro3399-c1 [Paper link]
12. Bengtsson-Palme J, Angelin M, Huss M, Kjellqvist S, Kristiansson E, Palmgren H, Larsson DGJ, Johansson A: The human gut microbiome as a transporter of antibiotic resistance genes between continents. Antimicrobial Agents and Chemotherapy, 59, 10, 6551-6560 (2015). doi: 10.1128/AAC.00933-15 [Paper link]

I am very happy to announce that our paper on the metagenomes of periphyton communities (1) have been accepted in Frontiers in Microbiology (Aquatic Microbiology section). This project has been one of my longest running, as it started as my master thesis in 2010 and has gone through several metamorphoses before hitting its final form.

Briefly, our main findings are that:

1. Periphyton communities harbor an extraordinary diversity of organisms, including viruses, bacteria, algae, fungi, protozoans and metazoans
2. Bacteria are by far the most abundant
3. We find functional indicators of the biofilm form of life in periphyton involve genes coding for enzymes that catalyze the production and degradation of extracellular polymeric substances
4. Genes encoding enzymes that participate in anaerobic pathways are found in the biofilms suggesting that anaerobic or low-oxygen micro-zones within the biofilms exist

Most of this work has been carried out by my colleague Kemal Sanli, who have been doing a wonderful job pulling this together, with the help of Henrik Nilsson and Martin Eriksson. It also deserves to be noted that this work was the starting point for the Metaxa software (2,3), which recently reached version 2.1.1.

References

1. Sanli K, Bengtsson-Palme J, Nilsson RH, Kristiansson E, Alm Rosenblad M, Blanck H, Eriksson KM: Metagenomic sequencing of marine periphyton: Taxonomic and functional insights into biofilm communities. Frontiers in Microbiology, 6, 1192 (2015). doi: 10.3389/fmicb.2015.01192 [Paper link]
2. Bengtsson J, Eriksson KM, Hartmann M, Wang Z, Shenoy BD, Grelet G, Abarenkov K, Petri A, Alm Rosenblad M, Nilsson RH: Metaxa: A software tool for automated detection and discrimination among ribosomal small subunit (12S/16S/18S) sequences of archaea, bacteria, eukaryotes, mitochondria, and chloroplasts in metagenomes and environmental sequencing datasets. Antonie van Leeuwenhoek, 100, 3, 471-475 (2011). doi:10.1007/s10482-011-9598-6. [Paper link]
3. Bengtsson-Palme J, Hartmann M, Eriksson KM, Pal C, Thorell K, Larsson DGJ, Nilsson RH: Metaxa2: Improved identification and taxonomic classification of small and large subunit rRNA in metagenomic data. Molecular Ecology Resources, 15, 6, 1403–1414 (2015). doi: 10.1111/1755-0998.12399 [Paper link]

A couple of days ago a paper was published in Environmental Sciences Europe summarizing the EU report on effect-based tools for use in toxicology in the aquatic environment I have been involved in (1). This report was officially published last spring (2), and can be found here, with the annex available on the European Commission document website. My contribution to the paper was, as with the report, in the genomics and metagenomics section. The paper briefly presents modern bioassays, biomarkers and ecological methods that can be used for aquatic monitoring of the environment.

References:

1. Wernersson A-S, Carere M, Maggi C, Tusil P, Soldan P, James A, Sanchez W, Dulio V, Broeg K, Reifferscheid G, Buchinger S, Maas H, Van Der Grinten E, O’Toole S, Ausili A, Manfra L, Marziali L, Polesello S, Lacchetti I, Mancini L, Lilja K, Linderoth M, Lundeberg T, Fjällborg B, Porsbring T, Larsson DGJ, Bengtsson-Palme J, Förlin L, Kienle C, Kunz P, Vermeirssen E, Werner I, Robinson CD, Lyons B, Katsiadaki I, Whalley C, den Haan K, Messiaen M, Clayton H, Lettieri T, Negrão Carvalho R, Gawlik BM, Hollert H, Di Paolo C, Brack W. Kammann U, Kase R: The European technical report on aquatic effect-based monitoring tools under the water framework directive. Environmental Sciences Europe, 27, 7 (2015). doi: 10.1186/s12302-015-0039-4 [Paper link]
2. Wernersson A-S, Carere M, Maggi C, Tusil P, Soldan P, James A, Sanchez W, Broeg K, Kammann U, Reifferscheid G, Buchinger S, Maas H, Van Der Grinten E, Ausili A, Manfra L, Marziali L, Polesello S, Lacchetti I, Mancini L, Lilja K, Linderoth M, Lundeberg T, Fjällborg B, Porsbring T, Larsson DGJ, Bengtsson-Palme J, Förlin L, Kase R, Kienle C, Kunz P, Vermeirssen E, Werner I, Robinson CD, Lyons B, Katsiadaki I, Whalley C, den Haan K, Messiaen M, Clayton H, Lettieri T, Negrão Carvalho R, Gawlik BM, Dulio V, Hollert H, Di Paolo C, Brack W (2014). Technical Report on Aquatic Effect-Based Monitoring Tools. European Commission. Technical Report 2014-077, Office for Official Publications of European Communities, ISBN: 978-92-79-35787-9. doi:10.2779/7260

Our paper describing the bacterial community of a polluted lake in India has now been typeset and appears in its final form in Frontiers in Microbiology. If I may say so, I think that the paper turned out to be very goodlooking and it is indeed nice to finally see it in print. The paper describes an unprecedented diversity and abundance of antibiotic resistance genes and genes enabling transfer of DNA between bacteria. We also describe a range of potential novel plasmids from the lake. Finally, the paper briefly describes a new approach to targeted assembly of metagenomic data — TriMetAss — which can be downloaded here.

Reference:
Bengtsson-Palme J, Boulund F, Fick J, Kristiansson E, Larsson DGJ: Shotgun metagenomics reveals a wide array of antibiotic resistance genes and mobile elements in a polluted lake in India. Frontiers in Microbiology, 5, 648 (2014). doi: 10.3389/fmicb.2014.00648

The first work in which I have employed metagenomics to investigate antibiotic resistance has been accepted in Frontiers in Microbiology, and is (at the time of writing) available as a provisional PDF. In the paper (1), which is co-authored by Fredrik Boulund, Jerker Fick, Erik Kristiansson and Joakim Larsson, we have used shotgun metagenomic sequencing of an Indian lake polluted by dumping of waste from pharmaceutical production. We used this data to describe the diversity of antibiotic resistance genes and the genetic context of those, to try to predict their genetic transferability. We found resistance genes against essentially every major class of antibiotics, as well as large abundances of genes responsible for mobilization of genetic material. Resistance genes were estimated to be 7000 times more abundant in the polluted lake than in a Swedish lake included for comparison, where only eight resistance genes were found. The abundances of resistance genes have previously only been matched by river sediment subject to pollution from pharmaceutical production (2). In addition, we describe twenty-six known and twenty-one putative novel plasmids from the Indian lake metagenome, indicating that there is a large potential for horizontal gene transfer through conjugation. Based on the wide range and high abundance of known resistance factors detected, we believe that it is plausible that novel resistance genes are also present in the lake. We conclude that environments polluted with waste from antibiotic manufacturing could be important reservoirs for mobile antibiotic resistance genes. This work further highlights previous findings that pharmaceutical production settings could provide sufficient selection pressure from antibiotics (3) to drive the development of multi-resistant bacteria (4,5), resistance which may ultimately end up in pathogenic species (6,7). The paper can be read in its entirety here.

References:

1. Bengtsson-Palme J, Boulund F, Fick J, Kristiansson E, Larsson DGJ: Shotgun metagenomics reveals a wide array of antibiotic resistance genes and mobile elements in a polluted lake in India. Frontiers in Microbiology, Volume 5, Issue 648 (2014). doi: 10.3389/fmicb.2014.00648
2. Kristiansson E, Fick J, Janzon A, Grabic R, Rutgersson C, Weijdegård B, Söderström H, Larsson DGJ: Pyrosequencing of antibiotic-contaminated river sediments reveals high levels of resistance and gene transfer elements. PLoS ONE, Volume 6, e17038 (2011). doi:10.1371/journal.pone.0017038.
3. Larsson DGJ, de Pedro C, Paxeus N: Effluent from drug manufactures contains extremely high levels of pharmaceuticals. J Hazard Mater, Volume 148, 751–755 (2007). doi:10.1016/j.jhazmat.2007.07.008
4. Marathe NP, Regina VR, Walujkar SA, Charan SS, Moore ERB, Larsson DGJ, Shouche YS: A Treatment Plant Receiving Waste Water from Multiple Bulk Drug Manufacturers Is a Reservoir for Highly Multi-Drug Resistant Integron-Bearing Bacteria. PLoS ONE, Volume 8, e77310 (2013). doi:10.1371/journal.pone.0077310
5. Johnning A, Moore ERB, Svensson-Stadler L, Shouche YS, Larsson DGJ, Kristiansson E: Acquired genetic mechanisms of a multiresistant bacterium isolated from a treatment plant receiving wastewater from antibiotic production. Appl Environ Microbiol, Volume 79, 7256–7263 (2013). doi:10.1128/AEM.02141-13
6. Pruden A, Larsson DGJ, Amézquita A, Collignon P, Brandt KK, Graham DW, Lazorchak JM, Suzuki S, Silley P, Snape JR., et al.: Management options for reducing the release of antibiotics and antibiotic resistance genes to the environment. Environ Health Perspect, Volume 121, 878–885 (2013). doi:10.1289/ehp.1206446
7. Finley RL, Collignon P, Larsson DGJ, McEwen SA, Li X-Z, Gaze WH, Reid-Smith R, Timinouni M, Graham DW, Topp E: The scourge of antibiotic resistance: the important role of the environment. Clin Infect Dis, Volume 57, 704–710 (2013). doi:10.1093/cid/cit355

I just got word from BMC Genomics that my most recent paper has just been published (in provisional form; we still have not seen the edited proofs). In this paper (1), which I have co-authored with Anders Blomberg, Magnus Alm Rosenblad and Mikael Molin, we utilize metagenomic data from the GOS-expedition (2) together with fully sequenced bacterial genomes to show that:

1. Detoxification genes in general are underrepresented in marine planktonic bacteria
2. Surprisingly, the detoxification that show a differential distribution are more abundant in open ocean water than closer to the coast
3. Peroxidases and peroxiredoxins seem to be the main line of defense against oxidative stress for bacteria in the marine milieu, rather than e.g. catalases
4. The abundance of detoxification genes does not seem to increase with estimated pollution.

From this we conclude that other selective pressures than pollution likely play the largest role in shaping marine planktonic bacterial communities, such as for example nutrient limitations. This suggests substantial streamlining of gene copy number and genome sizes, in line with observations made in previous studies (3). Along the same lines, our findings indicate that the majority of marine bacteria would have a low capacity to adapt to increased pollution, which is relevant as large amounts of human pollutants and waste end up in the oceans every year. The study exemplifies the use of metagenomics data in ecotoxicology, and how we can examine anthropogenic consequences on life in the sea using approaches derived from genomics. You can read the paper in its entirety here.

References:

1. Bengtsson-Palme J, Alm Rosenblad M, Molin M, Blomberg A: Metagenomics reveals that detoxification systems are underrepresented in marine bacterial communities. BMC Genomics. Volume 15, Issue 749 (2014). doi: 10.1186/1471-2164-15-749 [Paper link]

2. Yooseph S, Sutton G, Rusch DB, Halpern AL, Williamson SJ, Remington K, Eisen JA, Heidelberg KB, Manning G, Li W, Jaroszewski L, Cieplak P, Miller CS, Li H, Mashiyama ST, Joachimiak MP, Van Belle C, Chandonia J-M, Soergel DA, Zhai Y, Natarajan K, Lee S, Raphael BJ, Bafna V, Friedman R, Brenner SE, Godzik A, Eisenberg D, Dixon JE, Taylor SS, et al: The Sorcerer II Global Ocean Sampling expedition: expanding the universe of protein families. PLoS Biology. 5:e16 (2007).
3. Yooseph S, Nealson KH, Rusch DB, McCrow JP, Dupont CL, Kim M, Johnson J, Montgomery R, Ferriera S, Beeson KY, Williamson SJ, Tovchigrechko A, Allen AE, Zeigler LA, Sutton G, Eisenstadt E, Rogers Y-H, Friedman R, Frazier M, Venter JC: Genomic and functional adaptation in surface ocean planktonic prokaryotes. Nature. 468:60–66 (2010).