Tag: Joakim Larsson

After the usual (1,2) long wait between acceptance and publication, Science of the Total Environment today put a paper online in which I have played a role in the bioinformatic analysis. In the paper, we investigate whether antifouling paint containing copper and zinc could co-select for antibiotic resistance, using microbiological methods and metagenomic sequencing (3).

In this work, we have studied marine microbial biofilms allowed to grow on surfaces painted with antifouling paint submerged in sea water. Such antifouling paints often contain metals that potentially could co-select for antibiotic resistance (4). Using microbiological culturing, we found that the heavy-metal based paint co-selected for bacteria resistant to tetracycline. However, the paint did not enrich neither the total abundance of known mobile antibiotic resistance genes nor the abundance of tetracycline resistance genes in the biofilm communities. Rather, the communities from the painted surfaces were enriched for bacteria with genetic profiles suggesting increased capacity for extrusion of antibiotics via RND efflux systems. In addition, these communities were also enriched for genes involved in mobilization of DNA, such as ISCR transposases and integrases. Finally, the biofilm communities from painted surfaces displayed lower taxonomic diversity and were at the same time enriched for Gammaproteobacteria. The paper builds on our previous work in which we identify certain co-occurences between genes conferring metal and antibiotic resistance (4). However, the findings of this paper do not lend support for that mobile resistance genes are co-selected for by copper and zinc in the marine environment – rather the increase in antibiotic resistance seem to be due to taxonomic changes and cross-resistance mechanisms. The entire paper can be read here.

References

1. Bengtsson-Palme J: Published paper: Community MSCs for tetracycline. https://microbiology.se/2016/03/22/published-paper-community-mscs-for-tetracycline/
2. Bengtsson-Palme J: Published paper: Antibiotic resistance in sewage treatment plants . https://microbiology.se/2016/08/17/published-paper-antibiotic-resistance-in-sewage-treatment-plants/
3. Flach C-F, Pal C, Svensson CJ, Kristiansson E, Östman M, Bengtsson-Palme J, Tysklind M, Larsson DGJ: Does antifouling paint select for antibiotic resistance? Science of the Total Environment, in press (2017). doi: 10.1016/j.scitotenv.2017.01.213 [Paper link]
4. Pal C, Bengtsson-Palme J, Kristiansson E, Larsson DGJ: Co-occurrence of resistance genes to antibiotics, biocides and metals reveals novel insights into their co-selection potential. BMC Genomics, 16, 964 (2015). doi: 10.1186/s12864-015-2153-5 [Paper link]

So 2017 has begun, and this year will bring new challenges and exciting opportunities. First of all, my application for a 3.5 year grant from the Swedish Research Council for Environment, Agricultural Sciences and Spatial Planning (FORMAS) to go to Prof. Jo Handelsman‘s lab in the US was granted. Since Prof. Handelsman in is moving her lab to University of Wisconsin in Madison, where she will be heading the Wisconsin Institute of Discovery (after returning from the White House), it means that this summer I will be moving to Wisconsin. I will retain a link to the University of Gothenburg and the Joakim Larsson lab though, and part of the grant is actually for covering my salary after returning from the US, so Gothenburg won’t get rid of me so easily.

The granted project will use high-throughput sequencing techniques to identify genes improving colonization and invasion ability or resistance to invasion in microbial communities, using a model system developed by the Handelsman lab. The project will focus on genes important for colonization, invasion and resistance to invasion under exposure to sub-lethal antibiotics concentrations. The project will contribute important knowledge towards the understanding of microbial colonization and invasion and highlight disturbances to the interactions in microbial communities caused by anthropogenic activities. In addition, the results of the project will hopefully allow for prediction of secondary effects of antibiotic exposure in the environment, and the preparation for future challenges related to infections with pathogenic bacteria. The project has already been highlighted by CARe (although this was before Jo announced her move from Yale) and a FORMAS press release (in Swedish).

The project will go under the acronym InSiDER, and I intend to write about it in a special section of the website, called the Wisconsin Blog. My intention is to include personal reflections on life in Wisconsin and work in the Handelsman lab there, but we’ll see how those plans turn out. Anyway, I am very thankful for FORMAS funding this project and giving me the opportunity to work with one of the leading scientists within microbial ecology in the world!

Late yesterday, Microbiome put online our most recent work, covering the resistomes to antibiotics, biocides and metals across a vast range of environments. In the paper (1), we perform the largest characterization of resistance genes, mobile genetic elements (MGEs) and bacterial taxonomic compositions to date, covering 864 different metagenomes from humans (350), animals (145) and external environments such as soil, water, sewage, and air (369 in total). All the investigated metagenomes were sequenced to at least 10 million reads each, using Illumina technology, making the results more comparable across environments than in previous studies (2-4).

We found that the environment types had clear differences both in terms of resistance profiles and bacterial community composition. Humans and animals hosted microbial communities with limited taxonomic diversity as well as low abundance and diversity of biocide/metal resistance genes and MGEs. On the contrary, the abundance of ARGs was relatively high in humans and animals. External environments, on the other hand, showed high taxonomic diversity and high diversity of biocide/metal resistance genes and MGEs. Water, sediment and soil generally carried low relative abundance and few varieties of known ARGs, whereas wastewater and sludge were on par with the human gut. The environments with the largest relative abundance and diversity of ARGs, including genes encoding resistance to last resort antibiotics, were those subjected to industrial antibiotic pollution and air samples from a Beijing smog event.

A paper investigating this vast amount of data is of course hard to describe in a blog post, so I strongly suggest the interested reader to head over to Microbiome’s page and read the full paper (1). However, here’s a ver short summary of the findings:

• The median relative abundance of ARGs across all environments was 0.035 copies per bacterial 16S rRNA
• Antibiotic-polluted environments have (by far) the highest abundances of ARGs
• Urban air samples carried high abundance and diversity of ARGs
• Human microbiota has high abundance and diversity of known ARGs, but low taxonomic diversity compared to the external environment
• The human and animal resistomes are dominated by tetracycline resistance genes
• Over half of the ARGs were only detected in external environments, while 20.5 % were found in human, animal and at least one of the external environments
• Biocide and metal resistance genes are more common in external environments than in the human microbiota
• Human microbiota carries low abundance and richness of MGEs compared to most external environments

Importantly, less than 1.5 % of all detected ARGs were found exclusively in the human microbiome. At the same time, 57.5 % of the known ARGs were only detected in metagenomes from environmental samples, despite that the majority of the investigated ARGs were initially encountered in pathogens. Still, our analysis suggests that most of these genes are relatively rare in the human microbiota. Environmental samples generally contained a wider distribution of resistance genes to a more diverse set of antibiotics classes. For example, the relative abundance of beta-lactam resistance genes was much larger in external environments than in human and animal microbiomes. This suggests that the external environment harbours many more varieties of resistance genes than the ones currently known from the clinic. Indeed, functional metagenomics has resulted in the discovery of many novel ARGs in external environments (e.g. 5). This all fits well with an overall much higher taxonomic diversity of environmental microbial communities. In terms of consequences associated with the potential transfer of ARGs to human pathogens, we argue that unknown resistance genes are of greater concern than those already known to circulate among human-associated bacteria (6).

This study describes the potential for many external environments, including those subjected to pharmaceutical pollution, air and wastewater/sludge, to serve as hotspots for resistance development and/or transmission of ARGs. In addition, our results indicate that these environments may play important roles in the mobilization of yet unknown ARGs and their further transmission to human pathogens. To provide guidance for risk-reducing actions we – based on this study – suggest strict regulatory measures of waste discharges from pharmaceutical industries and encourage more attention to air in the transmission of antibiotic resistance (1).

References

1. Pal C, Bengtsson-Palme J, Kristiansson E, Larsson DGJ: The structure and diversity of human, animal and environmental resistomes. Microbiome, 4, 54 (2016). doi: 10.1186/s40168-016-0199-5
2. Durso LM, Miller DN, Wienhold BJ. Distribution and quantification of antibiotic resistant genes and bacteria across agricultural and non-agricultural metagenomes. PLoS One. 2012;7:e48325.
3. Nesme J, Delmont TO, Monier J, Vogel TM. Large-scale metagenomic-based study of antibiotic resistance in the environment. Curr Biol. 2014;24:1096–100.
4. Fitzpatrick D, Walsh F. Antibiotic resistance genes across a wide variety of metagenomes. FEMS Microbiol Ecol. 2016. doi:10.1093/femsec/fiv168.
5. Allen HK, Moe LA, Rodbumrer J, Gaarder A, Handelsman J. Functional metagenomics reveals diverse β-lactamases in a remote Alaskan soil. ISME J. 2009;3:243–51.
6. Bengtsson-Palme J, Larsson DGJ: Antibiotic resistance genes in the environment: prioritizing risks. Nature Reviews Microbiology, 13, 369 (2015). doi: 10.1038/nrmicro3399-c1

Me and Joakim Larsson wrote an opinion/summary piece for the APUA Newsletter, issued by the Alliance for Prudent Use of Antibiotics, that was published yesterday (1). The paper is essentially a summary of work included in my PhD thesis, and discusses how to establish minimal selective concentrations of antibiotics for microbial communities (2-4), how to identify risk environments for resistance selection (5-9), and which mitigation strategies that can be implemented (10-12). Partially, we also discussed these issues earlier in our paper in the Medicine Maker (10), but this paper goes deeper into why limiting antibiotic pollution is important to mitigate the accelerating antibiotic resistance problem. I recommend this short summary piece to anyone who would like a brief overview of our research on antibiotic resistance, and think that it can serve as a great starting point for further reading! In addition, this issue of the newsletter features very interesting pieces on reducing antibiotics use (and disposal) outside of the clinics (13) and revival of old antibiotics (14). Please go ahead to the APUA web site and read the entire newsletter!

References

1. Bengtsson-Palme J, Larsson DGJ: Why limit antibiotic pollution? The role of environmental selection in antibiotic resistance development. APUA Newsletter, 34, 2, 6-9 (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. Gullberg E, Cao S, Berg OG, Ilbäck C, Sandegren L, Hughes D, et al.: Selection of resistant bacteria at very low antibiotic concentrations. PLoS Pathogens 7, e1002158 (2011).
4. 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
5. 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
6. 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
7. 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
8. Martinez JL, Coque TM, Baquero F: What is a resistance gene? Ranking risk in resistomes. Nature Reviews Microbiology 2015, 13:116–123. doi:10.1038/nrmicro3399
9. Bengtsson-Palme J, Larsson DGJ: Antibiotic resistance genes in the environment: prioritizing risks. Nature Reviews Microbiology, 13, 369 (2015) doi:10.1038/nrmicro3399‐c1
10. Bengtsson-Palme J, Larsson DGJ: Time to limit antibiotic pollution. The Medicine Maker, 0416, 302, 17–18 (2016). [Paper link]
11. Ashbolt NJ, Amézquita A, Backhaus T, Borriello P, Brandt KK, Collignon P, et al.: Human Health Risk Assessment (HHRA) for Environmental Development and Transfer of Antibiotic Resistance. Environmental Health Perspectives, 121, 993–1001 (2013)
12. Pruden A, Larsson DGJ, Amézquita A, Collignon P, Brandt KK, Graham DW, et al.: Management options for reducing the release of antibiotics and antibiotic resistance genes to the environment. Environmental Health Perspectives, 121, 878–85 (2013).
13. Theuretzbacher U: Optimizing the Use of Old Antibiotics — A Global Health Agenda. APUA Newsletter, 34, 2, 10-13 (2016). [Paper link].
14. Amábile-Cuevas CF: Antibiotics and Antibiotic Resistance All Around Us. APUA Newsletter, 34, 2, 3-5 (2016). [Paper link].

I just want to highlight that the paper on strategies to improve database accuracy and usability we recently published in Proteomics (1) has been included in their most recent issue, which is a special issue focusing on Data Quality Issues in Proteomics. I highly recommend reading our paper (of course) and many of the other in the special issue. Happy reading!

On another note, I will be giving a talk next Wednesday (October 5th) on a seminar day on next generation sequencing in clinical microbiology, titled “Antibiotic resistance in the clinic and the environment – There and back again“. You are very welcome to the lecture hall at floor 3 in our building at Guldhedsgatan 10A here in Gothenburg if you are interested! (Bear in mind though that it all starts at 8.15 in the morning.)

Finally, it seems that I am going to the Next Generation Sequencing Congress in London this year, which will be very fun! Hope to see some of you dealing with sequencing there!

References

1. Bengtsson-Palme J, Boulund F, Edström R, Feizi A, Johnning A, Jonsson VA, Karlsson FH, Pal C, Pereira MB, Rehammar A, Sánchez J, Sanli K, Thorell K: Strategies to improve usability and preserve accuracy in biological sequence databases. Proteomics, 16, 18, 2454–2460 (2016). doi: 10.1002/pmic.201600034 [Paper link]

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

The Royal Swedish Academy of Sciences (KVA) is, together with Joakim Larsson, arranging a conference on the mechanisms and evidence for the involvement of metals and biocides in selection of antibiotic resistant bacteria. Several experts from around Europe will attend and give talks, including for example Dan Andersson, Kristian Brandt, Teresa Coque, Will Gaze, Åsa Melhus and Chris Rensing. The symposium is open to everyone and is free of charge (although registration is binding).

The conference take place between 15th and 16th of March, at The Royal Swedish Academy of Sciences’ facilities at Lilla Frescativägen in Stockholm. And you should join us; register here!