Microbiology, Metagenomics and Bioinformatics

Johan Bengtsson-Palme, University of Gothenburg | Wisconsin Institute for Discovery

Browsing Posts tagged Antibiotics

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

I have been asked to give a short talk on the metals and biocides and antibiotic resistance co-selection conference I mentioned in February. My presentation will take place late Tuesday afternoon, and is entitled “Elucidating biocide and metal co-selection for antibiotic resistance in sewage treatment plants using metagenomics“. I hope to see you there!

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!

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:

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 DGJAntibiotic 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]

Yesterday, a paper I co-authored with my colleagues Chandan Pal, Erik Kristiansson and Joakim Larsson on the co-occurences of resistance genes against antibiotics, biocides and metals in bacterial genomes and plasmids became published in BMC Genomics. In this paper (1) we utilize the publicly available, fully sequenced, genomes and plasmids in GenBank to investigate the co-occurence network of resistance genes, to better understand risks for co-selection for resistance against different types of compounds. In short, the findings of the paper are that:

  • ARGs are associated with BMRG-carrying bacteria and the co-selection potential of biocides and metals is specific towards certain antibiotics
  • Clinically important genera host the largest numbers of ARGs and BMRGs and those also have the highest co-selection potential
  • Bacteria isolated from human and domestic animal origins have the highest co-selection potential
  • Plasmids with co-selection potential tend to be conjugative and carry toxin-antitoxin systems
  • Mercury and QACs are potential co-selectors of ARGs on plasmids, however BMRGs are common on chromosomes and could still have indirect co-selection potential
  • 14 percent of bacteria and more than 70% of the plasmids completely lacked resistance genes

This analysis was possible thanks to the BacMet database of antibacterial biocide and metal resistance genes, published about two years ago (2). The visualization of the plasmid co-occurence network we ended up with can be seen below. Note the strong connection between the mercury resistance mer operon and the antibiotic resistance genes to the right.

On a side note, it is interesting to note that the underrepresentation of detoxification systems in marine environments we noted last year (3) still seems to hold for genomes (and particularly plasmids), supporting the genome streamlining hypothesis (4).

References:

  1. 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]
  2. Pal C, Bengtsson-Palme J, Rensing C, Kristiansson E, Larsson DGJ: BacMet: Antibacterial Biocide and Metal Resistance Genes Database. Nucleic Acids Research, 42, D1, D737-D743 (2014). doi: 10.1093/nar/gkt1252 [Paper link]
  3. Bengtsson-Palme J, Alm Rosenblad M, Molin M, Blomberg A: Metagenomics reveals that detoxification systems are underrepresented in marine bacterial communities. BMC Genomics, 15, 749 (2014). doi: 10.1186/1471-2164-15-749 [Paper link]
  4. Giovannoni SJ, Cameron TJ, Temperton B: Implications of streamlining theory for microbial ecology. ISME Journal, 8, 1553-1565 (2014).

UPDATE: This post has been updated to reflect a valid comment that I messed up in the original post. Specific use of antibiotics as feed additives has been forbidden for years in the EU. The petition was about cutting the prophylactic use of antibiotics in animals, but that was very unclear from the original post. I thank my readers for pointing this unclarity out.

I’m going to do something unusual and ask you to sign a petition targeted at European Union ministers to support new EU laws to drastically cut the prophylactic use of antibiotics in agriculture as growth promoters. The problem is that if ministers don’t feel the public pressure to act, the laws may be delayed or not be implemented. Examples from Denmark, Sweden, Norway and the Netherlands show that it is possible to produce meat with little or no antibiotics, but since bacteria can travel across borders (1), we need to bring the rest of the world onboard, and the EU is good first step. Therefore I ask you to sign the Avaaz petition here.

  1. 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]

The paper we published in August on travelers carrying resistance genes with them in their gut microbiota has now been typeset and got proper volume and issue numbers assigned to it in Antimicrobial Agents and Chemotherapy. Take a look at it, I personally think it’s quite good-looking.

Also, if you understand Swedish, here is an interview with me broadcasted on Swedish Radio last month about this study and the consequences of it.

The new citation for the paper is:

  • 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]

There have been quite a lot of buzz this week around the travel paper we published earlier this month. Twitter aside, the findings of the paper has also been covered by a range of news outlets, both in Sweden and internationally. Today, I was on Swedish radio talking resistance problems for about ten minutes (listen here; in Swedish). Here’s a few takes on the story I gathered around the web:

Science Daily
Business Standard
Z News
Englemed Health News
Läkemedelsvärlden (in Swedish)
Sveriges Radio (in Swedish)
Göteborgs-Posten (in Swedish)