Microbiology, Metagenomics and Bioinformatics

Johan Bengtsson-Palme, University of Gothenburg

Browsing Posts tagged Joakim Larsson

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)

Earlier today, my most recent paper (1) became available online, describing resistance gene patterns in the gut microbiota of Swedes before and after travel to the Indian peninsula and central Africa. In this work, we have used metagenomic sequencing of the intestinal microbiome of Swedish students returning from exchange programs to show that the abundance of antibiotic resistance genes in several classes are increased after travel. This work reiterates the findings of several papers describing uptake of resistant bacteria (2-8) or resistance genes (9-11) after travel to destinations with worse resistance situation.

Our paper is important because it:

  1. Addresses the abundance of a vast range of resistance genes (more than 300).
  2. Finds evidence for that the overall relative abundance of antibiotic resistance genes increased after travel, without any intake of antibiotics.
  3. Shows that the sensitivity of metagenomics was, despite very deep sequencing efforts, not sufficient to detect acquisition of the low-abundant (CTX-M) resistance genes responsible for observed ESBL phenotypes.
  4. Reveals a “core resistome” of resistance genes that are more or less omnipresent, and remain relatively stable regardless of travel, while changes seem to occur in the more variable part of the resistome.
  5. Hints at increased abundance of Proteobacteria after travel, although this increase could not specifically be linked to resistance gene increases.
  6. Uses de novo metagenomic assembly to physically link resistance genes in the same sample, giving hints of co-resistance patterns in the gut microbiome.

The paper was a collaboration with Martin Angelin, Helena Palmgren and Anders Johansson at Umeå University, and was made possible by bioinformatics support from SciLifeLab in Stockholm. I highly recommend reading it as a complement to e.g. the Forslund et al. paper (12) describing country-specific antibiotic resistance patterns in the gut microbiota.

Taken together, this study offers a broadened perspective on how the antibiotic resistance potential of the human gut microbiome changes after travel, providing an independent complement to previous studies targeting a limited number of bacterial species or antibiotic resistance genes. Understanding how resistance genes travels the globe is hugely important, since resistance in principle only need to appear in a pathogen once; improper hygiene and travel may then spread novel resistance genes across continents rapidly (13,14).

References

  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. Antimicrob Agents Chemother Accepted manuscript posted online (2015). doi: 10.1128/AAC.00933-15 [Paper link]
  2. Gaarslev K, Stenderup J: Changes during travel in the composition and antibiotic resistance pattern of the intestinal Enterobacteriaceae flora: results from a study of mecillinam prophylaxis against travellers’ diarrhoea. Curr Med Res Opin 9:384–387 (1985).
  3. Paltansing S, Vlot JA, Kraakman MEM, Mesman R, Bruijning ML, Bernards AT, Visser LG, Veldkamp KE: Extended-spectrum β-lactamase-producing enterobacteriaceae among travelers from the Netherlands. Emerging Infect. Dis. 19:1206–1213 (2013).
  4. Ruppé E, Armand-Lefèvre L, Estellat C, El-Mniai A, Boussadia Y, Consigny PH, Girard PM, Vittecoq D, Bouchaud O, Pialoux G, Esposito-Farèse M, Coignard B, Lucet JC, Andremont A, Matheron S: Acquisition of carbapenemase-producing Enterobacteriaceae by healthy travellers to India, France, February 2012 to March 2013. Euro Surveill. 19 (2014).
  5. Kennedy K, Collignon P: Colonisation with Escherichia coli resistant to “critically important” antibiotics: a high risk for international travellers. Eur J Clin Microbiol Infect Dis 29:1501–1506 (2010).
  6. Tham J, Odenholt I, Walder M, Brolund A, Ahl J, Melander E: Extended-spectrum beta-lactamase-producing Escherichia coli in patients with travellers’ diarrhoea. Scand. J. Infect. Dis. 42:275–280 (2010).
  7. Östholm-Balkhed Å, Tärnberg M, Nilsson M, Nilsson LE, Hanberger H, Hällgren A, Travel Study Group of Southeast Sweden: Travel-associated faecal colonization with ESBL-producing Enterobacteriaceae: incidence and risk factors. J Antimicrob Chemother 68:2144–2153 (2013).
  8. Kantele A, Lääveri T, Mero S, Vilkman K, Pakkanen SH, Ollgren J, Antikainen J, Kirveskari J: Antimicrobials increase travelers’ risk of colonization by extended-spectrum betalactamase-producing enterobacteriaceae. Clin Infect Dis 60:837–846 (2015).
  9. von Wintersdorff CJH, Penders J, Stobberingh EE, Oude Lashof AML, Hoebe CJPA, Savelkoul PHM, Wolffs PFG: High rates of antimicrobial drug resistance gene acquisition after international travel, The Netherlands. Emerging Infect. Dis. 20:649–657 (2014).
  10. Tängdén T, Cars O, Melhus A, Löwdin E: Foreign travel is a major risk factor for colonization with Escherichia coli producing CTX-M-type extended-spectrum beta-lactamases: a prospective study with Swedish volunteers. Antimicrob Agents Chemother 54:3564–3568 (2010).
  11. Dhanji H, Patel R, Wall R, Doumith M, Patel B, Hope R, Livermore DM, Woodford N: Variation in the genetic environments of bla(CTX-M-15) in Escherichia coli from the faeces of travellers returning to the United Kingdom. J Antimicrob Chemother 66:1005–1012 (2011).
  12. Forslund K, Sunagawa S, Kultima JR, Mende DR, Arumugam M, Typas A, Bork P: Country-specific antibiotic use practices impact the human gut resistome. Genome Res 23:1163–1169 (2013).
  13. Bengtsson-Palme J, Larsson DGJ: Antibiotic resistance genes in the environment: prioritizing risks. Nat Rev Microbiol 13:396 (2015).
  14. Larsson DGJ: Antibiotics in the environment. Ups J Med Sci 119:108–112 (2014).

Late last year, an opinion paper by José Martínez, Teresa Coque and Fernando Baquero was published in Nature Reviews Microbiology (1). In this paper, the authors present a system – resistance readiness conditions (RESCon) – for ranking the risks associated with the detection of antibiotic resistance genes. They also outline the obstacles associated with determining risks presented by antibiotic resistance genes in environmental microbial communities in terms of their potential to transfer to human pathogens. Generally, I am very positive about this paper, which I think is a must-read for anyone who works with antibiotic resistance genes in metagenomes, regardless of it they stem from the human gut or the external environment.

There is, however, one very important aspect that struck me and many other members of our research group as curious: the proposed system assign antibiotic resistance genes already present on mobile genetic elements in human pathogens to the highest risk category (RESCon 1), while resistance genes encoding novel resistance mechanisms not yet been found on mobile elements in a pathogen are considered to be part of lower risk categories. We believe that this system will overestimate the risks associated with well-known resistance factors that are already circulating among human pathogens and under-appreciate the potentially disastrous consequences that the transfer of previously unknown resistance determinants from the environmental resistome could have (exemplified by the rapid clinical spread of the NDM-1 metallo-beta-lactamase gene (2,3)).

With this in mind me and Joakim Larsson wrote a response letter to Nature Reviews Microbiology that went online last monday (4), together with the authors’ reply to us (5). (I strongly suggest that you read the entire original paper (1) before you read the reply (5) to our response letter (4), since Martinez et al. changes the scope slightly from the original paper in their response letter, and these clarifications may (or may not) have been in response to our arguments.)

In our response, we also stress that the abundances of resistance genes, and not only their presence, should be accounted for when estimating risks (although that last point might have been slightly obscured due to the very low word limit). In other words, we think that identifying environmental hotspots for antibiotic resistance genes, where novel resistance genes could be selected for (6,7,8), is of great importance for mitigating public health risks related to environmental antibiotic resistance. Please read our full thoughts on the matter in Nature Reviews Microbiology.

Similar issues will be touched upon in my talk at the EDAR2015 conference later in May. Hope to see you there!

References

  1. Martinez JL, Coque TM, Baquero F: What is a resistance gene? Ranking risk in resistomes. Nat Rev Microbiol 2015, 13:116–123.
  2. Kumarasamy KK, et al.: Emergence of a new antibiotic resistance mechanism in India, Pakistan, and the UK: a molecular, biological, and epidemiological study. Lancet Infect Dis 2010, 10:597–602.
  3. Walsh TR, Weeks J, Livermore DM, Toleman MA: Dissemination of NDM‐1 positive bacteria in the New Delhi environment and its implications for human health: an environmental point prevalence study. Lancet Infect Dis 2011, 11:355–362.
  4. Bengtsson-Palme J, Larsson DGJ: Antibiotic resistance genes in the environment: prioritizing risks. Nat Rev Microbiol 2015, Advance online publication. doi:10.1038/nrmicro3399‐c1
  5. Martinez JL, Coque TM, Baquero F: Prioritizing risks of antibiotic resistance genes in all metagenomes. Nat Rev Microbiol 2015, Advance online publication. doi:10.1038/nrmicro3399‐c2
  6. Kristiansson E, et al.: Pyrosequencing of antibiotic‐contaminated river sediments reveals high levels of resistance and gene transfer elements. PLoS ONE 2011, 6:e17038.
  7. 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. Front Microbiol 2014, 5:648.
  8. Marathe NP, et al.: A treatment plant receiving waste water from multiple bulk drug manufacturers is a reservoir for highly multi‐drug resistant integron‐bearing bacteria. PLoS ONE 2013, 8:e77310.

I will be giving a talk at the Third International symposium on the environmental dimension of antibiotic resistance (EDAR2015) next month (five weeks from now. The talk is entitled “Turn up the signal – wipe out the noise: Gaining insights into antibiotic resistance of bacterial communities using metagenomic data“, and will deal with handling of metagenomic data in antibiotic resistance gene research. The talk will highlight the some particular pitfalls related to interpretation of data, and exemplify how flawed analysis practices can result in misleading conclusions regarding antibiotic resistance risks. I will particularly address how taxonomic composition influences the frequencies of resistance genes, the importance of knowledge of the functions of the genes in the databases used, and how normalization strategies influence the results. Furthermore, we will show how the context of resistance genes can allow inference of their potential to spread to human pathogens from environmental or commensal bacteria. All these aspects will be exemplified by data from our studies of environments subjected to pharmaceutical pollution in India, the effect of travel on the human resistome, and modern municipal wastewater treatment processes.

The talk will take place on Monday, May 18, 2015 at 13:20. The full scientific program for the conference can be found here. Registration for the conference is still possible, although not for the early-bird price. I look forward to see a lot of the people who will attend the conference, and hopefully also you!

It is nice to see that Indian media has picked up the story about antibiotic resistance genes in the heavily polluted Kazipally lake. In this case, it is the Deccan Chronicle who have been reporting on our findings and briefly interviewed Prof. Joakim Larsson about the study. The issue of pharmaceutical pollution of the environment in drug-producing countries is still rather under-reported and public perception of the problem might be rather low. Therefore, it makes me happy to see an Indian newspaper reporting on the issue. The scientific publication referred to can be found here.

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

Science for Life Laboratories (SciLifeLab) in Stockholm will host a metagenome data analysis workshop on May 21-23, in which I will participate as a tutorial assistant. Additionally, our group leader Joakim Larsson will be giving a lecture about how we use metagenomics to assess the environmental reservoir of antibiotic resistance genes (much of my recent work will likely go into that). I hope to meet you there, so don’t forget to register!

Confirmed speakers:
Lex Nederbragt, Oslo University, Norway
Saskia Smits, Erasmus University Rotterdam, Netherlands
Joakim Larsson, Göteborg University, Sweden
Paul Wilmes, University of Luxembourg, Luxembourg
Anders Andersson, SciLifeLab, Sweden
Noan Le Bescot, UPMC (Tara expedition), France

The workshop is part of the AllBio Bioinformatics initiative.

If you are thinking about doing a PhD and think that bioinformatics and antibiotic resistance is a cool subject, then now is your chance to come and join us for the next four years! There is a PhD position open i Joakim Larsson’s group, which means that if you get the job you will work with me, Joakim Larsson, Erik Kristiansson, Ørjan Samuelsen and Carl-Fredrik Flach on a super-interesting project relating to discovery of novel beta-lactamase genes (NoCURE). The project aims to better understand where, how and under what circumstances these genetic transfer events take place, in order to provide opportunities to limit or delay resistance development and thus increase the functional lifespan of precious antibiotics. The lion’s share of the work will be related to interpreting large-scale sequencing data generated by collaborators within the project; both genome sequencing and metagenomic data.

This is a great opportunity to prove your bioinformatics skills and use them for something urgently important. Full details about the position can be found here.

Our new home

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Last Friday, our research group moved into our new facilities at the Department of Infectious Diseases. I am very happy with my new room and my new view, both depicted below.

Our new affiliation is:

Department of Infectious Diseases, Institute of Biomedicine, Sahlgrenska Academy, University of Gothenburg

Postal address:
Avd. för Klinisk bakteriologi/Virologi
Guldhedsgatan 10
SE-413 46 Göteborg

So, last week I started my Ph.D. in Joakim Larsson’s group at the Sahlgrenska Academy. While I am very happy about how things have evolved, I will also miss the ecotox group and the functional genomics group a lot (though both do their research within 10 minutes walking distance from my new place…) I spent last week getting through the usual administrative hassle; getting keys and cards, signing papers, installing bioinformatics software on my new monster of a computer etc. Slowly, the new room starts to feel like it is mine (after nailing phylogenetic trees, my favorite map of the amino acids, and my remember-why-Cytoscape-visualisation-might-not-be-a-good-idea-for-all-network-like-structures poster to the billboard).

So what will this change of positions mean? Will I quit doing research on microbial communities? Of course not! In my new position, my subject of investigation will be bacterial communities subjected to antibiotics. We will look for resistance genes in such communities, and try to answer questions like: How do a high antibiotic selection pressure affect abundance of resistance genes and mobile elements that could facilitate their transfer between bacteria? Can resistance genes found in environmental bacteria be transferred to the microbes of the human gut? Can the environmental bacteria tell us what resistance genes that will be present in clinical situations in the near future? All these questions could, at least partially, be answered by metagenomic approaches and good bioinformatics tools, and my role will be to come up with the solutions provide answers to them.

I am excited over this new project, which involves my favorite subject – metagenomics and community analysis – as well as important factors, such as the clinical connections, the possibility to add pieces to the antibiotic resistance puzzle, and the role of gene and species transfer in resistance development. I also like the fact that I will need to handle high-throughput  sequence data, meaning that there will be many opportunities to develop tools, a task I highly enjoy. I think the next couple of years will be an exciting time.