Author: Johan

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

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!

From today, I will shut down my activities on the website and over mail to spend the Christmas holidays with my family. I will likely not read e-mails until the first week of January, and as I might then have a large pile of mail to go through, please re-send any messages to me after January 3.

I apologise to everyone who might have outstanding support issues for Metaxa2 or ITSx. If you feel that I have neglected your e-mail, please re-send it after January 3 as well, to make sure that I have not missed it.

I wish you all a very Merry Christmas and a Happy new year!

As the 8th Next Generation Sequencing Congress in London is drawing to a close as I write this, I have a few reflections that might warrant sharing. The first thing that has been apparent this year compared to the two previous times I have visited the event (in 2012 and 2013) is that there was very little talk about where Illumina sequencing is heading next. Instead the discussion was about the applications of Illumina sequencing in the clinical setting; so apparently this is now so mainstream that we only expect slow progress towards longer reads. Apart from that, Illumina is a completed, mature technology. Instead, the flashlight is now pointing entirely towards long-read sequencing (PacBio, NanoPore) as the next big thing. However, the excitement around these technologies has also sort of faded compared to in 2013 when they were soon-to-arrive. Indeed, it seems like there’s not much to be excited about in the sequencing field at the moment, or at least Oxford Global (who are hosting the conference) has failed to get these technologies here.

What also strikes me is the vast amounts of talk about RNAseq of cancer cells. The scope of this event has narrowed dramatically in the past three years. Which makes me substantially less interested in returning next year. If there is not much to be excited about, and the focus is only on cancer sequencing – despite the human microbiota being a very hot topic at the moment – what is the reason for non-cancer researchers to come to the event? There will need to be a stark shift towards another direction of this event if the arrangers want it to remain a broad NGS event. Otherwise, they may just as well go all in and rename the event the Next Generation Sequencing of Cancer Congress. But I hope they choose to widen the scope again; conferences discussing technology as a foundation for a variety of applications are important meeting points and spawning grounds for novel ideas.

Yesterday, Molecular Ecology Resources put online an unedited version of a recent paper which I co-authored. This time, Rodney Richardson at Ohio State University has made a tremendous work of evaluating three taxonomic classification software – the RDP Naïve Bayesian Classifier, RTAX and UTAX – on a set of DNA barcoding regions commonly used for plants, namely the ITS2, and the matK, rbcL, trnL and trnH genes.

In the paper (1), we discuss the results, merits and limitations of the classifiers. In brief, we found that:

• There is a considerable trade-off between accuracy and sensitivity for the classifiers tested, which indicates a need for improved sequence classification tools (2)
• UTAX was superior with respect to error rate, but was exceedingly stringent and thus suffered from a low assignment rate
• The RDP Naïve Bayesian Classifier displayed high sensitivity and low error at the family and order levels, but had a genus-level error rate of 9.6 percent
• RTAX showed high sensitivity at all taxonomic ranks, but at the same time consistently produced the high error rates
• The choice of locus has significant effects on the classification sensitivity of all tested tools
• All classifiers showed strong relationships between database completeness, classification sensitivity and classification accuracy

We believe that the methods of comparison we have used are simple and robust, and thereby provides a methodological and conceptual foundation for future software evaluations. On a personal note, I will thoroughly enjoy working with Rodney and Reed again; I had a great time discussing the ins and outs of taxonomic classification with them! The paper can be found here.

References and notes

1. Richardson RT, Bengtsson-Palme J, Johnson RM: Evaluating and Optimizing the Performance of Software Commonly Used for the Taxonomic Classification of DNA Sequence Data. Molecular Ecology Resources, Early view (2016). doi: 10.1111/1755-0998.12628 [Paper link]
2. This is something that several classifiers also showed in the evaluation we did for the Metaxa2 paper (3). Interestingly enough, Metaxa2 is better at maintaining high accuracy also as sensitivity is increased.
3. Bengtsson-Palme J, Hartmann M, Eriksson KM, Pal C, Thorell K, Larsson DGJ, Nilsson RH: Metaxa2: Improved identification and taxonomic classification of small and large subunit rRNA in metagenomic data. Molecular Ecology Resources, 15, 6, 1403–1414 (2015). doi: 10.1111/1755-0998.12399 [Paper link]

I will give a short talk on our findings related to antibiotic resistance associated with pharmaceutical production facilities in India at a one-hour webinar arranged by Healthcare Without Harm, taking place on Thursday, November 3rd, 10.00 CET. The webinar will discuss “hot-spot” environments in which antimicrobial resistance can emerge, such as areas in which there are poor pharmaceutical manufacturing practices, where expired or unused drugs are disposed of in an inappropriate way (i.e. by flushing them down the toilet or sink, or disposing them in household rubbish), and areas in which pharmaceuticals are used for aquaculture or agriculture. This is an important aspect of the resistance problem, but to date most of the actions taken to tackle the spread of AMR don’t take into account this aspect of antimicrobials released into the environment. The webinar is co-organised by HCWH Europe and HCWH Asia, and aims to raise awareness about the issue of AMR and its environmental impact. It features, apart from myself, Lucas Wiarda (Global Marketing Director & Head of Sustainable Antibiotics Program at DSM Sinochem Pharmaceuticals) and Sister Mercilyn Jabel (Pharmacist at Saint Paul Hospital Cavite, Philippines).

Sign up here to learn about:

• Antibiotic pollution and waste
• Recent findings from India regarding antibiotic discharges in rivers from manufacturers and new mechanisms by which resistance spreads in the environment
• Sustainable antibiotics – how to support the proper and effective use of antibiotics and their responsible production
• How the pharmaceutical industry is addressing the environmental pollution that leads to AMR
• The best practices in managing infectious waste at hospital level

Today the 15th Swedish Bioinformatics Workshop starts in Linköping. For the first time in many many years, I will not be there, which feels super-strange. (I even took part in organizing the workshop two years ago, in Gothenburg.) I would have thoroughly enjoyed the schedule (the workshops look amazing), and hope that everyone will have a very good time there without me. I do miss you! Please make sure I get another opportunity next year!

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

I am part of the organizing committee for the newly invented annual meeting for GOTBIN – the Gothenburg Bioinformatics Network. We will arrange a meeting on December 6 to get the networking activities for 2017 kickstarted, and every bioinformatician in Gothenburg is invited!

GOTBIN was launched to bridge and bring together all researchers in Gothenburg who fully or partially dealt with bioinformatics in their research. Through the network it should be possible to quickly find other local researchers tackling the same research problems as you are; to find appropriate resources to run your analyses; and to discuss research or infrastructure problems as they arise. To keep the network alive and kicking, it is crucial to keep relations active. Furthermore, it is also crucial to interact with key persons in the GOTBIN network to keep the lists of active researchers, resources and discussion forums up to date.

To facilitate future communication, we invite everyone who works with bioinformatics in Gothenburg to participate in a get-together workshop. The purpose of this workshop is to find out who is working with what and where, and to better get to know each other. This is a great opportunity to meet your next collaboration partner, post-doc or supervisor! The event will take place in Birgit Thilander at Medicinareberget (just next to the large lecture hall Arvid Carlsson) on December 6th, from 9.00 to 12.00. Fika will be provided and we will arrange ice-breaker activities suitable for the number of participants, so please register at: https://goo.gl/forms/KYdiiZMBDf0F9hvp2 by the 16th of November.

We hope to find everyone with a research interest in bioinformatics there and that this will be the launch of the next era of GOTBIN in 2017! See you there!

In the area of random notes, I turned 1 gigasecond today. That is, this morning it was 1 billion seconds since I was born. As good reasons as any to celebrate with cake at work! You’re all welcome to the 2 gigasecond celebration in ~31 years. Cheers!