Tag: Environment

Myself, Joakim Larsson and Erik Kristiansson have written a review on the environmental factors that influence development and spread of antibiotic resistance, which was published today in FEMS Microbiology Reviews. The review (1) builds on thoughts developed in the latter parts of my PhD thesis (2), and seeks to provide a synthesis knowledge gained from different subfields towards the current understanding of evolutionary and ecological processes leading to clinical appearance of resistance genes, as well as the important environmental dispersal barriers preventing spread of resistant pathogens.

We postulate that emergence of novel resistance factors and mobilization of resistance genes are likely to occur continuously in the environment. However, the great majority of such genetic events are unlikely to lead to establishment of novel resistance factors in bacterial populations, unless there is a selection pressure for maintaining them or their fitness costs are negligible. To enable measures to prevent resistance development in the environment, it is therefore critical to investigate under what conditions and to what extent environmental selection for resistance takes place. Selection for resistance is likely less important for the dissemination of resistant bacteria, but will ultimately depend on how well the species or strain in question thrives in the external environment. Metacommunity theory (3,4) suggests that dispersal ability is central to this process, and therefore opportunistic pathogens with their main habitat in the environment may play an important role in the exchange of resistance factors between humans and the environment. Understanding the dispersal barriers hindering this exchange is not only key to evaluate risks, but also to prevent resistant pathogens, as well as novel resistance genes, from reaching humans.

Towards the end of the paper, we suggest certain environments that seem to be more important from a risk management perspective. We also discuss additional problems linked to the development of antibiotic resistance, such as increased evolvability of bacterial genomes (5) and which other types of genes that may be mobilized in the future, should the development continue (1,6). In this review, we also further develop thoughts on the relative risks of re-recruiting and spreading well-known resistance factors already circulating in pathogens, versus recruitment of completely novel resistance genes from environmental bacteria (7). While the latter case is likely to be very rare, and thus almost impossible to quantify the risks for, the consequences of such (potentially one-time) events can be dire.

I personally think that this is one of the best though-through pieces I have ever written, and since it is open access and (in my biased opinion) written in a fairly accessible way, I recommend everyone to read it. It builds on the ecological theories for resistance ecology developed by, among others, Fernando Baquero and José Martinez (8-13). Over the last year, it has been stressed several times at meetings (e.g. at the EDAR conferences in August) that there is a need to develop an ecological framework for antibiotic resistance genes. I think this paper could be one of the foundational pillars on such an endeavor and look forward to see how it will fit into the growing literature on the subject!

References

1. Bengtsson-Palme J, Kristiansson E, Larsson DGJ: Environmental factors influencing the development and spread of antibiotic resistance. FEMS Microbiology Reviews, accepted manuscript (2017). doi: 10.1093/femsre/fux053
2. Bengtsson-Palme J: Antibiotic resistance in the environment: a contribution from metagenomic studies. Doctoral thesis (medicine), Department of Infectious Diseases, Institute of Biomedicine, Sahlgrenska Academy, University of Gothenburg, 2016. [Link]
3. Bengtsson J: Applied (meta)community ecology: diversity and ecosystem services at the intersection of local and regional processes. In: Verhoef HA, Morin PJ (eds.). Community Ecology: Processes, Models, and Applications. Oxford: Oxford University Press, 115–130 (2009).
4. Leibold M, Norberg J: Biodiversity in metacommunities: Plankton as complex adaptive systems? Limnology and Oceanography, 1278–1289 (2004).
5. Gillings MR, Stokes HW: Are humans increasing bacterial evolvability? Trends in Ecology and Evolution, 27, 346–352 (2012).
6. Gillings MR: Evolutionary consequences of antibiotic use for the resistome, mobilome and microbial pangenome. Frontiers in Microbiology, 4, 4 (2013).
7. Bengtsson-Palme J, Larsson DGJ: Antibiotic resistance genes in the environment: prioritizing risks. Nature Reviews Microbiology, 13, 369 (2015). doi: 10.1038/nrmicro3399-c1
8. Baquero F, Alvarez-Ortega C, Martinez JL: Ecology and evolution of antibiotic resistance. Environmental Microbiology Reports, 1, 469–476 (2009).
9. Baquero F, Tedim AP, Coque TM: Antibiotic resistance shaping multi-level population biology of bacteria. Frontiers in Microbiology, 4, 15 (2013).
10. Berendonk TU, Manaia CM, Merlin C et al.: Tackling antibiotic resistance: the environmental framework. Nature Reviews Microbiology, 13, 310–317 (2015).
11. Hiltunen T, Virta M, Laine A-L: Antibiotic resistance in the wild: an eco-evolutionary perspective. Philosophical Transactions of the Royal Society B: Biological Sciences, 372 (2017) doi: 10.1098/rstb.2016.0039.
12. Martinez JL: Bottlenecks in the transferability of antibiotic resistance from natural ecosystems to human bacterial pathogens. Frontiers in Microbiology, 2, 265 (2011).
13. Salyers AA, Amábile-Cuevas CF: Why are antibiotic resistance genes so resistant to elimination? Antimicrobial Agents and Chemotherapy, 41, 2321–2325 (1997).

Today, Microbiome put online a paper lead-authored by my colleague Fanny Berglund – one of Erik Kristiansson‘s brilliant PhD students – in which we identify 76 novel metallo-ß-lactamases (1). This feat was made possible because of a new computational method designed by Fanny, which uses a hidden Markov model based on known B1 metallo-ß-lactamases. We analyzed over 10,000 bacterial genomes and plasmids and over 5 terabases of metagenomic data and could thereby predict 76 novel genes. These genes clustered into 59 new families of metallo-β-lactamases (given a 70% identity threshold). We also verified the functionality of 21 of these genes experimentally, and found that 18 were able to hydrolyze imipenem when inserted into Escherichia coli. Two of the novel genes contained atypical zinc-binding motifs in their active sites. Finally, we show that the B1 metallo-β-lactamases can be divided into five major groups based on their phylogenetic origin. It seems that nearly all of the previously characterized mobile B1 β-lactamases we identify in this study were likely to have originated from chromosomal genes present in species within the Proteobacteria, particularly Shewanella spp.

This study more than doubles the number of known B1 metallo-β-lactamases. As with the study by Boulund et al. (2) which we published last month on computational discovery of novel fluoroquinolone resistance genes (which used a very similar approach but on a completely different type of genes), this study also supports the hypothesis that environmental bacterial communities act as sources of uncharacterized antibiotic resistance genes (3-7). Fanny have done a fantastic job on this paper, and I highly recommend reading it in its entirety (it’s open access so you have virtually no excuse not to). It can be found here.

References

1. Berglund F, Marathe NP, Österlund T, Bengtsson-Palme J, Kotsakis S, Flach C-F, Larsson DGJ, Kristiansson E: Identification of 76 novel B1 metallo-β-lactamases through large-scale screening of genomic and metagenomic data. Microbiome, 5, 134 (2017). doi: 10.1186/s40168-017-0353-8
2. Boulund F, Berglund F, Flach C-F, Bengtsson-Palme J, Marathe NP, Larsson DGJ, Kristiansson E: Computational discovery and functional validation of novel fluoroquinolone resistance genes in public metagenomic data sets. BMC Genomics, 18, 682 (2017). doi: 10.1186/s12864-017-4064-0
3. Bengtsson-Palme J, Larsson DGJ: Antibiotic resistance genes in the environment: prioritizing risks. Nature Reviews Microbiology, 13, 369 (2015). doi: 10.1038/nrmicro3399-c1
4. Allen HK, Donato J, Wang HH et al.: Call of the wild: antibiotic resistance genes in natural environments. Nature Reviews Microbiology, 8, 251–259 (2010).
5. Berendonk TU, Manaia CM, Merlin C et al.: Tackling antibiotic resistance: the environmental framework. Nature Reviews Microbiology, 13, 310–317 (2015).
6. Martinez JL: Bottlenecks in the transferability of antibiotic resistance from natural ecosystems to human bacterial pathogens. Frontiers in Microbiology, 2, 265 (2011).
7. Finley RL, Collignon P, Larsson DGJ et al.: The scourge of antibiotic resistance: the important role of the environment. Clinical Infectious Diseases, 57, 704–710 (2013).

BMC Genomics today published a paper first-authored by my long-time colleague Fredrik Boulund, which describes a computational screen of genomes and metagenomes for novel qnr fluoroquinolone resistance genes (1). The study makes use of Fredrik’s well-designed and updated qnr-prediction pipeline, but in contrast to his previous publication based on the pipeline from 2012 (2), we here study a 20-fold larger dataset of almost 13 terabases of sequence data. Based on this data, the pipeline predicted 611 putative qnr genes, including all previously described plasmid-mediated qnr gene families. 20 of the predicted genes were previously undescribed, and of these nine were selected for experimental validation. Six of those tested genes improved the survivability under ciprofloxacin exposure when expressed in Escherichia coli. The study shows that qnr genes are almost ubiquitous in environmental microbial communities. This study also lends further credibility to the hypothesis that environmental bacterial communities can act as sources of previously uncharacterized antibiotic resistance genes (3-7). The study can be read in its entirety here.

References

1. Boulund F, Berglund F, Flach C-F, Bengtsson-Palme J, Marathe NP, Larsson DGJ, Kristiansson E: Computational discovery and functional validation of novel fluoroquinolone resistance genes in public metagenomic data sets. BMC Genomics, 18, 682 (2017). doi: 10.1186/s12864-017-4064-0
2. Boulund F, Johnning A, Pereira MB, Larsson DGJ, Kristiansson E: A novel method to discover fluoroquinolone antibiotic resistance (qnr) genes in fragmented nucleotide sequences. BMC Genomics, 13, 695 (2012). doi: 10.1186/1471-2164-13-695
3. Bengtsson-Palme J, Larsson DGJ: Antibiotic resistance genes in the environment: prioritizing risks. Nature Reviews Microbiology, 13, 369 (2015). doi: 10.1038/nrmicro3399-c1
4. Allen HK, Donato J, Wang HH et al.: Call of the wild: antibiotic resistance genes in natural environments. Nature Reviews Microbiology, 8, 251–259 (2010).
5. Berendonk TU, Manaia CM, Merlin C et al.: Tackling antibiotic resistance: the environmental framework. Nature Reviews Microbiology, 13, 310–317 (2015).
6. Martinez JL: Bottlenecks in the transferability of antibiotic resistance from natural ecosystems to human bacterial pathogens. Frontiers in Microbiology, 2, 265 (2011).
7. Finley RL, Collignon P, Larsson DGJ et al.: The scourge of antibiotic resistance: the important role of the environment. Clinical Infectious Diseases, 57, 704–710 (2013).

Mitochondrial DNA Part B today published a mitochondrial genome announcement paper (1) in which I was involved in doing the assemblies and annotating them. The paper describes the mitogenome of Calanus glacialis, a marine planktonic copepod, which is a keystone species in the Arctic Ocean. The mitogenome is 20,674 bp long, and includes 13 protein-coding genes, 2 rRNA genes and 22 tRNA genes. While this is of course note a huge paper, we believe that this new resource will be of interest in understanding the structure and dynamics of C. glacialis populations. The main work in this paper has been carried out by Marvin Choquet at Nord University in Bodø, Norway. So hats off to him for great work, thanks Marvin! The paper can be read here.

Reference

1. Choquet M, Alves Monteiro HJ, Bengtsson-Palme J, Hoarau G: The complete mitochondrial genome of the copepod Calanus glacialis. Mitochondrial DNA Part B, 2, 2, 506–507 (2017). doi: 10.1080/23802359.2017.1361357 [Paper link]

Today, a review paper which I wrote together with Joakim Larsson and Erik Kristiansson was published in Journal of Antimicrobial Chemotherapy (1). We have for a long time used metagenomic DNA sequencing to study antibiotic resistance in different environments (2-6), including in the human microbiota (7). Generally, our ultimate purpose has been to assess the risks to human health associated with resistance genes in the environment. However, a multitude of methods exist for metagenomic data analysis, and over the years we have learned that not all methods are suitable for the investigation of resistance genes for this purpose. In our review paper, we describe and discuss current methods for sequence handling, mapping to databases of resistance genes, statistical analysis and metagenomic assembly. We also provide an overview of important considerations related to the analysis of resistance genes, and end by recommending some of the currently used tools, databases and methods that are best equipped to inform research and clinical practice related to antibiotic resistance (see the figure from the paper below). We hope that the paper will be useful to researchers and clinicians interested in using metagenomic sequencing to better understand the resistance genes present in environmental and human-associated microbial communities.

References

1. Bengtsson-Palme J, Larsson DGJ, Kristiansson E: Using metagenomics to investigate human and environmental resistomes. Journal of Antimicrobial Chemotherapy, advance access (2017). doi: 10.1093/jac/dkx199 [Paper link]
2. 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 [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. 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, 572, 697–712 (2016). doi: 10.1016/j.scitotenv.2016.06.228 [Paper link]
5. 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 [Paper link]
6. 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, 590–591, 461–468 (2017). doi: 10.1016/j.scitotenv.2017.01.213 [Paper link]
7. 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]

In two weeks time, on the 15th of June, I will participate in a seminar organised by Landstingens nätverk för läkemedel och miljö (the Swedish county council network for pharmaceuticals and environment; the seminar will be held in Swedish) in Stockholm. I will give a talk on our proposed emission limits for antibiotics published last year (the paper is available here), but there will also be talks on wastewater treatment, sustainable pharmaceutical usage and environmental standards for pharmaceuticals. The full program can be found here, and you may register here until June 9. The seminar is free of charge.

And if you are interested in this, I can also recommend the webinar given by Healthcare Without Harm next week (on June 8), which will deal with sustainable procurement as a means to deal with pharmaceutical pollution in the environment. I will at least tune in to hear how the discussion goes here.

In March, I attended a workshop on the role of NGS technologies in the coordinated action plan against antimicrobial resistance, organised by JRC in Italy. I was, together with 14 other experts, invited to discuss where and how sequencing can be used to investigate and manage antibiotic resistance. The report from the workshop has just recently been published, and is available here. There will be follow-up activities on this workshop, which I also hope that I will be able to participate in, since this is an important and very interesting pet topic of mine.

Reference

Sorry for the late notice, but if you have half an hour to spare later today I will discuss our findings on resistance genes in Beijing air on a webinar organised by Healthcare Without Harm on “The (un)recognised pathways of AMR: Air pollution and food“. Tune in a few minutes before 16.00 CEST!

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]

I am happy to announce that the opinion/review piece I wrote for Current Opinion in Food Science has been published. The paper (1) extends on some of my thoughts on how high-throughput sequencing and metagenomics can aid in risk assessment of antibiotic resistant bacteria that I outlined in my PhD thesis (2), but specifically focuses on the food supply chain and its role in resistance dissemination and selection.

In the paper, I argue for that the food supply chain is a special type of setting in the resistance puzzle, as it not only serves as a connection between environmental habitats for bacteria and humans, but also sometimes presents a substantial selection for resistance, due to use of antibiotics in agri- and aquaculture. International food standards are clear that both selection and dissemination of foodborne resistance should be considered in the risk analysis of food production (3). However, the current main use of DNA sequencing in food safety is whole genome sequencing to delineate which specific strains that are involved in foodborne disease outbreaks, including the resistance factors they may carry (4,5). Further, I argue that while shotgun metagenomics could be used to screen samples for a large number of genes involved in resistance and virulence in the food supply chain, it would at present be very costly and therefore of doubtful benefit to employ in routine screening programs. Still, metagenomics can contribute knowledge that can be used in quantitative risk assessment of antibiotic resistance in the food supply chain.

The entire paper can be read here.

References

1. Bengtsson-Palme J: Antibiotic resistance in the food supply chain: Where can sequencing and metagenomics aid risk assessment? Current Opinion in Food Science, in press (2017). doi: 10.1016/j.cofs.2017.01.010 [Paper link]
2. Bengtsson-Palme J: Antibiotic resistance in the environment: a contribution from metagenomic studies. Doctoral thesis (medicine), Department of Infectious Diseases, Institute of Biomedicine, Sahlgrenska Academy, University of Gothenburg, 2016. [Link]
3. Codex Alimentarius Commission: Guidelines for risk analysis of foodborne antimicrobial resistance. Food and Agriculture Organization of the United Nations & World Health Organization2011. [Link]
4. Franz E, Gras LM, Dallman T: Significance of whole genome sequencing for surveillance, source attribution and microbial risk assessment of foodborne pathogens. Current Opinion in Food Science, 8, 74-79 (2016). doi: 10.1016/j.cofs.2016.04.004
5. Stasiewicz MJ, Bakker den HC, Wiedmann M: Genomics tools in microbial food safety. Current Opinion in Food Science, 4, 105-110 (2015). doi: 10.1016/j.cofs.2015.06.002