Microbiology, Metagenomics and Bioinformatics

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

Browsing Posts tagged Public information

I have recently started to receive requests for full-text versions of my publications on ResearchGate. That’s great, but I have yet to figure out how to send them over, without breaking any agreements. As I am in a somewhat intensive work-period at the moment, please forgive me for not spending time on ResearchGate right now. And if you would like full-text versions of my publications, please send me an e-mail! I’ll be glad to help!

I was recently involved as an adviser in a report by the County Administrative Board in Västra Götaland (Länsstyrelsen) which has now been published [1]. [UPDATE: The PDF link at Länsstyrelsen's page does not seem to work, but leads to another report in Swedish. I have reported this error to the web admin, we'll see what happens. Once again, the PDF seems to work.] The report aims to identify gaps in the current monitoring system of hazardous substances in the Swedish environment. The report deals with effect based monitoring tools and their usefulness for predicting and/or observing effects of hazardous substances in the environment. The overall conclusion of the report is that there are several gaps in both knowledge and techniques, and a need for developing new resources. However, Sweden still has a good potential to adapt the monitoring system to fill the needs. I have been involved in one of the last chapters, describing the use of metagenomics if study ecosystem function (chapter 30.3). For people with an interest in environmental monitoring, the report is an interesting read in its entirety. For those more interested in applications for metagenomics I recommend turning to page 285 and continue to the end of the report (it’s only five pages on metagenomics, so you’ll manage).

  1. Länsstyrelsen i Västra Götalands län. (2012). Swedish monitoring of hazardous substances in the aquatic environment (No. 2012:23). (A.-S. Wernersson, Ed.) Current vs required monitoring and potential developments (pp. 1–291). Länsstyrelsen i Västra Götalands län, vattenvårdsenheten.

Finally I have gotten around to finish my reply to Amy Pruden, who gave me some highly relevant and well-balanced critique of my previous post on antibiotic resistance genes as pollutants, back in early March. Too much came in between, but now I am more or less content with my answer.

First of all I would like to thank Amy for her response to my post on antibiotic resistance genes as pollutants. Her reply is very well thought-through, and her criticism of some of my claims is highly appropriate. For example, I have to agree on that the extracellular DNA pool is vastly uncharacterized, and that my statement on this likely not being a source of resistance transmission is a bit of a stretch. The role of “free-floating” DNA in gene transfer must be further elucidated, and currently we do not really know whether it is important or not; and if so, to what extent it contributes.

However, I still maintain my view that there are problems with considering resistance genes pollutants, mainly because the blurs the line between cause and effect. If we for example consider photosynthetic microbial communities exposed to the photosynthesis inhibitor Irgarol, the communities develop (or acquires) tolerance towards the compound over time (Blanck et al 2009). The tolerance mechanism has been attributed to changes in the psbA gene sequence (Eriksson et al. 2009). If we address this issue from a “resistance-genes-as-pollutants” perspective, would these tolerance-conveying psbA genes be considered pollutants? It would make sense to do so as they are unwanted in weed control circumstances; much like antibiotic resistance genes are unwanted in clinical contexts. It could be argued here that in these microbes such tolerance-associated psbA genes do not cause any harm. But consider for a moment that they did not occur microbes, but in weeds, would they then be considered pollutants? In weeds they would certainly cause (at least economic) harm. Furthermore, say that the tolerance-conveying psbA genes have the ability to spread (which is possible at least in marine settings assisted by phages (Lindell et al 2005)), would that make these tolerance genes pollutants? It is quite of a stretch but as plants can take up genetic material from bacteria (c.f. Clough & Bent 1998, although this is not my area of expertise), there could be a spreading potential to weeds of these tolerance-conveying psbA genes.

What I am trying to say is that if we start viewing antibiotic resistance genes as pollutants per se, instead of looking at the chemicals (likely) causing resistance development, we start blurring the line between cause and effect. Resistance genes in the environment provide resilience to communities (at least to some species – the issue of ecosystem function responses to toxicants is a highly interesting area one as well). However, in this case the resilience itself is the problem, because we think it can spread into human and animal pathogens. But from my point of view, the causes are still use, overuse, misuse and inappropriate release of antibiotics. Therefore, I maintain that we should be careful with pointing out resistance genes by themselves as pollutants – if we do not have very good reasons to do so.

Nevertheless, that does not mean that I think Pruden, and many other prominent authors, are wrong when they refer to resistance genes as pollutants. All I want to point out is that the statement in itself is a bit dangerous, as it might draw attention towards mitigating the effect of pollution, instead of mitigating the source of pollution itself. The persistence of resistance genes in bacterial genomes is alarming (Andersson & Hughes 2011), as it means that removal of selection pressures may have less effect on resistance gene abundance than anticipated. However, the only way I see out of this darkening scenario is to:

  1. Minimize the selection pressure for resistance genes in the clinical setting
  2. Immediately reduce environmental release of antibiotics, both from manufacturing and use. This primarily has to be done using better treatment technologies
  3. Find the routes that enable environmental bacteria to disseminate resistance genes to clinically relevant species and strains – and close them
  4. Develop antibiotics exploiting new mechanisms to eliminate bacteria

Lastly, I would like to thank Amy for taking my critique seriously – I think we agree on a lot more than we differ on, and I look forward to have this discussion in person at some point. I think we both agree that regardless of our standpoint, the terminology used in this context deserves to be discussed. Nevertheless, the terminology is quite unimportant compared to the values that are at stake – our fundamental ability to treat diseases and perform modern health care.

References

  1. Andersson, D.I. & Hughes, D., 2011. Persistence of antibiotic resistance in bacterial populations. FEMS Microbiology Reviews, 35(5), pp.901–911.
  2. Blanck, H., Eriksson, K. M., Grönvall, F., Dahl, B., Guijarro, K. M., Birgersson, G., & Kylin, H. (2009). A retrospective analysis of contamination and periphyton PICT patterns for the antifoulant irgarol 1051, around a small marina on the Swedish west coast. Marine pollution bulletin, 58(2), 230–237. doi:10.1016/j.marpolbul.2008.09.021
  3. Clough, S. J., & Bent, A. F. (1998). Floral dip: a simplified method for Agrobacterium-mediated transformation of Arabidopsis thaliana. The Plant journal : for cell and molecular biology, 16(6), 735–743.
  4. Eriksson, K. M., Clarke, A. K., Franzen, L.-G., Kuylenstierna, M., Martinez, K., & Blanck, H. (2009). Community-level analysis of psbA gene sequences and irgarol tolerance in marine periphyton. Applied and Environmental Microbiology, 75(4), 897–906. doi:10.1128/AEM.01830-08
  5. Lindell, D., Jaffe, J. D., Johnson, Z. I., Church, G. M., & Chisholm, S. W. (2005). Photosynthesis genes in marine viruses yield proteins during host infection. Nature, 438(7064), 86–89. doi:10.1038/nature04111

I received some well-formulated and very much relevant critique on my post Why viewing antibiotic resistance genes as a pollutant is a problem, which I wrote in January. To encourage the debate on this issue, I have asked the author – Amy Pruden – for her permission to republish it here, to give it the visibility it deserves. I intend to follow up on her comments in a forthcoming post, but I have not had time to formulate my answer yet. Until then, please read and contemplate both the original post by me, and Amy’s highly relevant answer below. I hope that we can continue this discussion in the same fruitful manner!

First of all I thank Johan Bengtsson for initiating a lively and much needed discussion on which pollutant we should precisely be targeting, antibiotics or antibiotic resistance genes (ARGs), in our important war against the spread of antibiotic resistance. As Bengtsson correctly alludes, my perspective comes from that of environmental science and engineering. At the core of these disciplines is defining and predicting the fate of pollutants in the environment, as well as designing appropriate means for their control. For these purposes, the definition of the pollutant of interest is of central importance. In general they may be defined as “undesired or harmful constituents within an environmental matrix, usually of human origin.” Pollutants may be classified in all shapes and sizes, including conservative (i.e., not subject to degradation or growth), non-conservative, biotic, abiotic, dissolved, and suspended (i.e., not dissolved). Thus, the first point, regarding the nature by which ARGs are spread disqualifying them from being considered as pollutants, is inaccurate.

At the same time, I recognize and agree that ARGs are indeed a natural and important aspect of the natural ecosystem. I commend recent work revealing the vast “antibiotic-resistome” in ancient environments (D’Costa et al. 2011; Allen et al. 2009), as it provides an essential understanding of the baseline antibiotic resistance in the pre-antibiotic era, which may serve as contrast for observations in the current antibiotic era. Thus, I agree that not all ARGs are pollutants, rather, anthropogenic sources of ARGs are the agents of interest. Perhaps I and others are guilty of not making this distinction more clear. It should also be pointed out that likewise, the vast majority of antibiotics in use today are derived from natural compounds, yet I agree that they can also serve as important environmental pollutants of concern. Thus, it is not necessarily whether the constituent is naturally occurring that defines the pollutant, rather its magnitude and distribution, as influenced by human activities.

It is agreed that viewing ARGs as contaminants does pose technical challenges. They may amplify within a host, or attenuate due to degradation or diminished selection pressure. However, with appropriate understanding of the mechanisms of transport and persistence, accurate models may be developed. I do contend that the jury is still out regarding the relative importance of extracellular and intracellular ARGs. The pool of extracellular DNA remains vastly uncharacterized, and some studies suggest that it is more extensive than previously thought (Wu et al. 2009; Corinaldesi et al. 2005). Other studies have specifically demonstrated the capability of extracellular ARGs to persist under certain environmental conditions and maintain its integrity for host uptake (Cai et al. 2007). While focusing attention on individual resistant strains of bacteria has merit in some instances, this approach is also greatly limited by the unculturability of the vast majority of environmental microbes. As we have now entered the metagenomic era, we now have the tools to tackle the complexity of resistance elements in the environment and precisely define the human influence. Distribution of ARGs may also be considered in parallel with key genetic elements driving their horizontal gene transfer, such as plasmids, transposons, and integrons.

Regarding the antibiotics themselves, clearly they are important. The direct relationship between clinical use and increasing rates of antibiotic resistance is well-documented and certainly continued vigilance in promoting their appropriate use and disposal is called for. What remains much foggier is the exact role of environmental antibiotics in enabling selection once released into the environment. There is good evidence that even sub-inhibitory levels of antibiotics can stimulate various functions in the cell, especially horizontal gene transfer, as reviewed recently by Aminov (2011). However, environmentally-relevant concentrations driving selection of resistant strains are largely unknown. Further, at what point along a discharge pathway from wastewater treatment plant or livestock lagoon do ARGs persist independently of ambient antibiotic conditions? Indeed, some studies have noted correlations between antibiotics and ARGs in environmental matrices while others have noted an absence of such a correlation. In either case, it appears that ARGs persist and are transported further along pathways than antibiotics, suggesting distinct factors governing transport (McKinney et al. 2010; Peak et al. 2007). Research is needed to better understand the mechanisms at play, such as antibiotics other selectors (e.g. metals and other toxins), in leaving a human foot-print on environmental reservoirs of resistance. Nonetheless, a reasonable approach for mitigating risk seems to be focusing attention on developing appropriate technologies for eliminating both antibiotics and genetic material from wastestreams.

Thanks again for opening this discussion- I hope to meet you at a conference sometime in the future!

References
1. Allen, H.K., Moe, L.A., Rodbumrer, J., Gaarder, A., & Handelsman, J., 2009. Functional metagenomics reveals diverse b-lactamases in a remote Alaskan soil. ISME 3, pp. 243-251.
2. Aminov, R.I., 2011. Horizontal gene exchange in environmental microbiota. Front. Microbiol. 2,158 doi:10.3389/fmicb.2011.00158.
3. Corinaldesi, C., Danovaro, R. & Dell‘Anno, A., 2005. Simultaneous recovery of intracellular and extracellular DNA suitable for molecular studies from marine sediments. Appl. Environ. Microbiol. 71, pp. 46-50.
4. D’Costa, V.M., McGrann, K.M., Hughes, D.W., & Wright, G.D., 2006. Sampling the antibiotic resistome. Science 311, pp. 374-377.
5. McKinney, C.W., Loftin, K.A., Meyer, M.T., Davis, J.G., & Pruden, A., 2010. tet and sul antibiotic resistance genes in livestock lagoons of various operation type, configuration, and antibiotic occurrence. Environ. Sci. Technol. 44 (16), pp. 6102-6109.
6. Peak, N., C.W. Knapp; R.K. Yang; M.M. Hanfelt; M.S. Smith, D.S. Aga, & Graham, D. W., 2007. Abundance of six tetracycline resistance genes in wastewater lagoons at cattle feedlots with different antibiotic use strategies. Environ. Microbiol. 9 (1), pp. 143–151.
7. Wu, J. F. & Xi, C. W., 2009. Evaluation of different methods for extracting extracellular DNA from the biofilm matrix. Appl. Environ. Microbiol. 75, pp. 5390-5395.

It is not uncommon that scientists, especially researchers active within the environmental field, view antibiotic resistance genes (ARGs) as pollutants (e.g. Pruden et al. 2006). While there are practical benefits of doing so, especially when explaining the threat of antibiotic resistance to politicians and the public, this generalization is a little bit problematic from a scientific view. There are several reasons why this view is not as straightforward as one might think.

The first is that ARGs does not spread the same way as pollutants do. ARGs are carried in bacteria. This means that ARGs cannot readily be transferred into, e.g. the human body by themselves. They need to be carried by a bacterial host (ARGs present on free DNA floating around is of course possible, but likely not a major source of ARG transmission into new systems). Therefore, when we find resistance genes in an environment, that is an extremely strong indication of that we also have resistant bacteria. Also, finding ARGs is not necessarily an indication of high levels of antibiotics, as the resistance genes can remain present in the bacterial genome for extended periods of time after exposure (Andersson & Hughes 2011).

The second reason why ARGs should not be viewed as pollutants is that they are not. If anything, the ARGs contribute to the resilience of the ecosystem towards the actual toxicants, which are the antibiotics themselves. Having a resistance gene is an insurance that you will survive antibiotic perturbations. Calling ARGs pollutants just deflects attention from the real problem to nature’s response to our contaminant.

What we have to do is not to try to defeat the resistance itself, but to try to minimize the spread of it. This means that we need to constantly monitor our usage and possible emissions of antibiotics and try to reduce risk environments as much as possible. Emissions from sewage treatment plants (Karthikeyan & Meyer 2006; Lindberg et al. 2007), hospitals (Lindberg et al. 2004), production facilities (Larsson et al. 2007; Fick et al. 2009) and food production (Davis et al. 2011) are obvious starting points, but we need to continuously monitor sources of antibiotic pollutions. Of course, this is only my view of the problem, but I believe that while the problem for our society lies within the resistance genes, the cause lies within the actual pollutants – the antibiotics we use and abuse.

References

  1. Andersson, D.I. & Hughes, D., 2011. Persistence of antibiotic resistance in bacterial populations. FEMS Microbiology Reviews, 35(5), pp.901–911.
  2. Davis, M.F. et al., 2011. An ecological perspective on U.S. industrial poultry production: the role of anthropogenic ecosystems on the emergence of drug-resistant bacteria from agricultural environments. Current Opinion in Microbiology, 14(3), pp.244–250.
  3. Fick, J. et al., 2009. Contamination of surface, ground, and drinking water from pharmaceutical production. Environmental toxicology and chemistry / SETAC, 28(12), pp.2522–2527.
  4. Karthikeyan, K.G. & Meyer, M.T., 2006. Occurrence of antibiotics in wastewater treatment facilities in Wisconsin, USA. The Science of the total environment, 361(1-3), pp.196–207.
  5. Larsson, D.G.J., de Pedro, C. & Paxeus, N., 2007. Effluent from drug manufactures contains extremely high levels of pharmaceuticals. Journal of hazardous materials, 148(3), pp.751–755.
  6. Lindberg, R. et al., 2004. Determination of antibiotic substances in hospital sewage water using solid phase extraction and liquid chromatography/mass spectrometry and group analogue internal standards. Chemosphere, 57(10), pp.1479–1488.
  7. Lindberg, R.H. et al., 2007. Environmental risk assessment of antibiotics in the Swedish environment with emphasis on sewage treatment plants. Water research, 41(3), pp.613–619.
  8. Pruden, A. et al., 2006. Antibiotic resistance genes as emerging contaminants: studies in northern Colorado. Environmental Science & Technology, 40(23), pp.7445–7450.

In December, Alex Bateman, whose opinions on open science I support and have touched upon earlier, wrote a short correspondence letter to Nature [1] in which he again repeated the points of his talk at FEBS last summer. He concludes by the paragraph:

Many in the scientific community will admit to using Wikipedia occasionally, yet few have contributed content. For society’s sake, scientists must overcome their reluctance to embrace this resource.

I agree with this statement. However, as I also touched upon earlier, but like to repeat again – bold statements doesn’t make dreams come true – action does. Rfam, and the collaboration with RNA Biology and Wikipedia is a great example of such actions. So what other actions may be necessary to get researchers to contribute to the Wikipedian wisdom?

First of all, I do not think that the main obstacle to get researchers to edit Wikipedia articles is reluctance to doing so because Wikipedia is “inconsistent with traditional academic scholarship”, though that might be a partial explanation. What I think is the major problem is the time-reward tradeoff. Given the focus on publishing peer-reviewed articles, the race for higher impact factor, and the general tendency of measuring science by statistical measures, it should be no surprise that Wikipedia editing is far down on most scientists to-do lists, so also on mine. The reward of editing a Wikipedia article is a good feeling in your stomach that you have benefitted society. Good stomach feelings will, however, feed my children just as little as freedom of speech. Still, both Wikipedia editing and freedom of speech are extremely important, especially as a scientist.

Thus, there is a great need of a system that:

  • Provides a reward or acknowledgement for Wikipedia editing.
  • Makes Wikipedia editing economically sustainable.
  • Encourages publishing of Wikipedia articles, or contributions to existing ones as part of the scientific publishing process.

Such a system could include a “contribution factor” similar to the impact factor, in which contribution of Wikipedia and other open access forums was weighted, with or without a usefulness measure. Such a usefulness measure could easily be determined by links from other Wikipedia articles, or similar. I realise that there would be severe drawbacks of such a system, similar to those of the impact factor system. I am not a huge fan of impact factors (read e.g. Per Seglen’s 1997 BMJ article [2] for  some reasons why), but I do not see that system changing any time soon, and thus some kind of contribution factor could provide an additional statistical measure for evaluators to consider when examining scientists’ work.

While a contribution factor would be an incitement for  researchers to contribute to the common knowledge, it will still not provide an economic value to do so. This could easily be changed by allowing, and maybe even requiring, scientists to contribute to Wikipedia and other public fora of scientific information as part of their science outreach duties. In fact, this public outreach duty (“tredje uppgiften” in Swedish) is governed in Swedish law. In 2009, the universities in Sweden have been assigned to “collaborate with the society and inform about their operations, and act such that scientific results produced at the university benefits society” (my translation). It seems rational that Wikipedia editing would be part of that duty, as that is the place were many (most?) people find information online today. Consequently, it is only up to the universities to demand 30 minutes of Wikipedia editing per week/month from their employees. Note here that I am referring to paid editing.

Another way of increasing the economic appeal of writing Wikipedia articles would be to encourage funding agencies and foundations to demand Wikipedia articles or similar as part of project reports. This would require researchers to make their findings public in order to get further funding, a move that would greatly increase the importance of increasing the common wisdom treasure. However, I suspect that many funding agencies, as well as researchers would be reluctant to such a solution.

Lastly, as shown by the Rfam/RNA Biology/Wikipedia relationship, scientific publishing itself could be tied to Wikipedia editing. This process could be started by e.g. open access journals such as PLoS ONE, either by demanding short Wikipedia notes to get an article published, or by simply provide prioritised publishing of articles which also have an accompanying Wiki-article. As mentioned previously, these short Wikipedia notes would also go through a peer-review process along with the full article. By tying this to the contribution factor, further incitements could be provided to get scientific progress in the hands of the general public.

Now, all these ideas put a huge burden on already hard-working scientists. I realise that they cannot all be introduced simultaneously. Opening up publishing requires time and thought, and should be done in small steps. But doing so is in the interest of scientists, the general public and the funders, as well as politicians. Because in the long run it will be hard to argue that society should pay for science when scientists are reluctant to even provide the public with an understandable version of the results. Instead of digging such a hole for ourselves, we should adapt the reward, evaluation, funding and publishing systems in a way that they benefit both researchers and the society we often say we serve.

  1. Bateman and Logan. Time to underpin Wikipedia wisdom. Nature (2010) vol. 468 (7325) pp. 765
  2. Seglen. Why the impact factor of journals should not be used for evaluating research. BMJ (1997) vol. 314 (7079) pp. 498-502

I listened to a great talk by Alex Bateman (one of the guys behind Pfam and Rfam, as well as involved in HMMER development) at FEBS yesterday. In addition to talking about the problems of increasing sequence amounts, Alex also provided some reflections on co-operativity and knowledge-sharing – not only among fellow researchers, but also to a wider audience. The starting point of this discussion is Rfam, where the annotation of RNA families is entirely based on a community-driven wiki, tightly integrated with Wikipedia. This means that to make a change in the Rfam annotation, the same change is also made at the corresponding Wikipedia page for this RNA family. And what’s the use of this? Well, as Alex says, for most of the keywords in molecular biology (and I would guess in all of science), the top hit on Google will be a Wikipedia entry. If not, the Wikipedia entry will be in the top ten list of hits, if a good Wiki page exists. This means that Wikipedia is the primary source of scientific information for the general public, as well as many scientists. Wikipedia – not scientific journals.

The consequence of this is that to communicate your research subject, you should contribute to its Wikipedia page. In fact, Bateman argues, we have a responsibility as scientists to provide accurate and correct information to the public through the best sources available, which in most cases would be Wikipedia. To put this in perspective (and here I once again borrow Alex’ words), if somebody told you ten years ago that there would be one single internet site that everybody would visit to find scientific information, and where discussion and continuous improvement would be allowed, encouraged and performed, most people would have said that was too good to be true. But that’s what Wikipedia offers. It is time to get rid of the Wiki-sceptisism, and start improving it.

And so, what about the future of publishing? Bateman has worked hard to form an agreement with the journal RNA Biology to integrate the publishing into the process of adding to the easily accessible public information. To have an article on a new RNA family published under the journal’s RNA families track, the family must not only be submitted to the Rfam database, but the authors must also provide a Wikipedia formatted article, which undergo the same peer-review process as the journal article. This ensures high-quality Wikipedia material, as well as making new scientific discoveries public.

I don’t think there’s a long stretch to guess that in the future, more journals and/or funding agencies will take on similar approaches, as researchers and decision-makers discover the importance of correct, publicly available information. The scientific world is slowly moving towards being more open, also for non-scientists. This openness is of extremely high importance in these times of climate scepticism, GMO controversy, extinction of species, and nuclear power debate. For the public to make proper decisions and send a clear message to the politicians, scientists need to be much better at communicating the current state of knowledge, or what many people prefer to call “truth”.