Category: Thoughts

The Core Facilites at Sahlgrenska are looking for a skilled bioinformatician that can support research projects employing the Core Facilites’ services. The employee will e.g. deal with setting up analysis pipelines for next generation sequencing data. They (of course) want an experienced bioinformatician, who also knows programming (Java, C and/or C++, and scripting languages such as Perl or Python). It is also preferable if the applicant knows how to set up secure systems and manage work with the Unix/Linux terminal. More on the position can be found at GU’s web site. The application time closes on the 17th of September.

I know that this is not supposed to be a political page, but writing this up, I realized that there is no way I can keep my political views entirely out of this post. So just a quick warning, the following text contains political opinions and is a reflection of my views and believes rather than well supported facts.

So, Swedish minister for education Jan Björklund has announced the government’s plan to spend 3 billion SEK (~350 million EUR, ~450 million USD) on “elite” researchers over the next ten years. One main reason to do so is to strengthen Swedish research in competition with American universities, and to be able to recruit top researchers from other countries to Sweden. While I welcome the prospect of more money to research, I have to say I am very skeptical about the nature of how this money is distributed. First of all, giving more money to the researchers that have already succeeded (I guess this is how you would define elite researchers – if someone has a better idea, please tell both me and Jan Björklund), is not going to generate more innovative research – just more of the same (or similar) things to what these researchers already do. If the government is serious about that Swedish research has a lower-than-expected output (which is a questionable statement in itself), the best way of increasing that output would be to give more researchers the opportunity to put their ideas into action. Second, a huge problem for research in Sweden is that a lot of the scientists’ time is spent on doing other stuff – writing grant applications, administering courses, filling in forms etc. Therefore, one way of improving research would be to put more money into funding at the university administration level, so that researchers actually have time to do what they are supposed to do. I will now provide my own four-point program for how I think that Sweden should move forward to improve the output of science.

1. Researchers need more time
My first point is that researchers need more time to do what they are supposed to do – science. This means that they cannot be expected to apply for money from six different research foundations every year, just to receive a very small amount of money that will keep them from getting thrown out for another 8 months. The short-term contracts that are currently the norm in Sweden create a system where way too much time is spent on writing grant applications – the majority of which will not succeed. In addition, researchers are often expected to be their own secretary, as well as organizing courses (not only lecturing). To solve this we need:

• Longer contracts for scientists. A grant should be large enough to secure five years of salary, plus equipment costs. This allows for some time to actually get the science done, not just the time to write the next application.
• Grants that come with a guaranteed five-year extension of grants to projects that have fulfilled their goals in the first five years. This further secures longevity of researchers and their projects. Also, this allows for universities to actually employ scientists instead of the current system which is all about trying to work around the employment rules.
• More money to university administration. It is simple more cost efficient to have a secretary handling non-science related stuff in the department or group, as well as economic people handling the economy. The current system expects every researcher to be a jack of all trades – which efficiently reduces one to a master of none. More money to administration means more time spent on research.

2. Broad funding creates a foundation for success
Another problem is that if only a few projects are funded repeatedly, the success of Swedish research is very much bound to the success of these projects. While large-scale and high-cost projects are definitely needed, there is also a need to invest in a variety of projects. Many applied ideas have originated from very non-applied research, and the applied research need fundamental research to be done to be able to move forward. However, in the shortsighted governmental view of science, the output has to be almost immediate, which means that applied projects are much more likely to be funded. Thus, projects that could do fundamental discoveries, but are more complicated and take longer time will be down-prioritized by both researchers and universities. To further make situation worse, Björklund et al. have promised more money to universities that cut out non-productive research, with almost guarantees that any projects with a ten-year timeframe will not even be started.

If we are serious about making Swedish research successful, we need to do exactly the opposite. Fund a lot of different projects, both applied and fundamental, regardless of their short-term value. Because the ideas that are most likely to produce short-term results are probably also the ones that are the least innovative in the long-term. Consequently, we need to:

• Spend research funding on a variety of projects, both of fundamental and applied nature.
• Secure funding for “crazy” projects that span long periods of time, at least five to ten years.

3. If we don’t dare to fail, we will not have a chance to win
Finally, research funding must become better at taking risks. If we only bet our money on the most successful researchers, there is absolutely no chance for young scientists to get funded, unless of course they have been picked up by one of the right supervisors. This means that the same ideas get disseminated through the system over and over again, at the expense of more innovative ideas that could pop up in groups with less money to realize them. If these untested ideas in smaller groups get funded, some of them might undoubtedly fail to produce research of high societal value. But some of them will likely develop entirely new ideas, which in the long term might be much more fruitful than throwing money on the same groups over and over again. Suggestions:

• Spend research funding broadly and with an active risk-gain management strategy.
• Allow for fundamental research to investigate completely new concepts – even if they are previously untested, and regardless (or less dependent on) previous research output.
• Invest in infrastructure for innovative research – and do so fast. For example, the money spent on the sequencing facilities at Sci Life Lab in Stockholm is an excellent example of an infrastructure investment that gains a lot of researchers at different universities access to high-throughput sequencing, without each university having to invest in expensive sequencing platforms themselves. More such centers would both spur collaboration and allow for faster adoption of new technologies.

4. Competing with what we are best at
A mistake that is often done when trying to compete with those that are best in the class is to try to compete by doing the same things as the best players do. This makes it extremely hard to win a game against exactly those players, as they are likely more experienced, have more resources, and already has the attention to get the resources we compete for. Instead, one could try to play the Wayne Gretzky trick: to try to skate where the puck is heading, instead of where it is today. Another approach would be to invent a new arena for the puck to land in, where you have better control over the settings than your competitors (slightly similar to what Apple did when the iPod was released, and Microsoft couldn’t use Windows to leverage their mp3-player Zune).

For Sweden, this would mean that we should not throw some bucks at the best players at our universities and hope that they will be happy with this (comparably small) amount of money. Instead, we should give them circumstances to work under that are much better or appealing from other standpoints. This could be better job security, longer contracts, less administrative work, securer grants, more freedom to decide over ones time, and larger possibilities to combine work and family. Simply creating a better, securer and nicer environment to work in. However, Björklund’s suggestions go the very opposite way: researchers should compete to be part of the elite community, and if your not in that group, you’d get thrown out. Therefore, I suggest (with the risk of repeating myself) that we should compete by:

• Offering longer contracts and grants for scientists.
• Giving scientists opportunities to combine work and family life.
• Embracing all kinds of science, both fundamental and applied, both short-term and long-term.
• Allowing researchers to take risks, even if they fail.
• Giving universities enough funding to let scientists do the science and administrative personal do the administration.
• Funding large-scale collaborative infrastructure investments.
• Thinking of how to create an environment that is appealing for scientists, not only from an economic perspective.

A note on other important aspects of funding
Finally, I have now been focusing a lot on width as opposed to directed funding to an elite research squad. It is, however, apparent that we also need to allocate funding to bring in more women to the top positions in the academy. Likely, a system which favors elite groups will also favor male researchers, judging from how the Swedish Foundation for Strategic Research picks their bets for the future. Also, it is important that young researchers without strong track records gets funded, otherwise a lot of new and interesting ideas risk to be lost.

In the fourth point of my proposal, I suggest that Sweden should compete at what Sweden is good at, that is to view researchers as human beings, which are most likely to succeed in an environment where they can develop their ideas in a free and secure way. For me, it is surprising that a minister of education representing a liberal party wants to excess such control over what is good and bad research. Putting up a working social security system around science seems much more logical than throwing money at those who already have. Apparently I have forgotten that our current government is not interested in having a working social security system – their interest seem to lie in deconstructing the very same structures.

I am on my way to Copenhagen for the ISME14 conference that begins today. I’m myself quite excited about this event, and will present three posters (two as first author), and give a short talk on antibiotic resistance gene identification and metagenomics. My talk will be in the Bioinformatics in Microbial Ecology session on Thursday afternoon (at 13.30).

If you’d like to talk about Metaxa and Megraft, I will present an SSU-oriented poster in the Monday afternoon poster section (board number 267A). My antibiotic resistance gene poster will be presented on Thursday afternoon (board number 002A), and I really encourage everyone interested in metagenomics (especially metagenomic assembly) to come talk to me then! Finally, I am also partially responsible for a poster on periphyton metagenomics with Martin Eriksson as its main author. This poster is also presented on Monday, in the Microbial Dispersion and Biogeography session (board number 021A).

I hope to be able to make another post later tonight on what are the “essential” sessions for me on this conference. Hope to see you there soon!

As many of you probably know, I have gotten married, which of course is a huge step for me. In short, I think the main way it will affect my scientific life is that I am changing my surname. So from now on, I am going to publish under the name “Johan Bengtsson-Palme” instead of just “Johan Bengtsson”. Not that much of a change, but it could still be nice to know that Bengtsson-Palme J, and Bengtsson J might very well be the same person.

On a side note, we just recently got a paper accepted on guidelines for quality control of ITS barcode sequences. More on that to follow soon.

The guys at Pfam recently introduced a new database, called AntiFam, which will provide HMM profiles for some groups of sequences that seemingly formed larger protein families, although they were not actually real proteins. For example, rRNA sequences could contain putative ORFs, that seems to be conserved over broad lineages; with the only problem being that they are not translated into proteins in real life, as they are part of an rRNA [1].

With this initiative the Xfam team wants to “reduce the number of spurious proteins that make their way into the protein sequence databases.” I have run into this problem myself at some occasions with suspicious sequences in GenBank, and I highly encourage this development towards consistency and correctness in sequence databases. It is of extreme importance that databases remain reliable if we want bioinformatics to tell us anything about organismal or community functions. The Antifam database is a first step towards such a cleanup of the databases, and as such I would like to applaud Pfam for taking actions in this direction.

To my knowledge, GenBank are doing what they can with e.g. barcoding data (SSU, LSU, ITS sequences), but for bioinformatics and metagenomics (and even genomics) to remain viable, these initiatives needs to come quickly; and automated (but still very sensitive) tools for this needs to get our focus immediately. For example, Metaxa [2] could be used as a tool to clean up SSU sequences of misclassified origin. More such tools are needed, and a lot of work remains to be done in the area of keeping databases trustworthy in the age of large-scale sequencing.

References

1. Tripp, H. J., Hewson, I., Boyarsky, S., Stuart, J. M., & Zehr, J. P. (2011). Misannotations of rRNA can now generate 90% false positive protein matches in metatranscriptomic studies. Nucleic Acids Research, 39(20), 8792–8802. doi:10.1093/nar/gkr576
2. Bengtsson, J., Eriksson, K. M., Hartmann, M., Wang, Z., Shenoy, B. D., Grelet, G.-A., Abarenkov, K., et al. (2011). Metaxa: a software tool for automated detection and discrimination among ribosomal small subunit (12S/16S/18S) sequences of archaea, bacteria, eukaryotes, mitochondria, and chloroplasts in metagenomes and environmental sequencing datasets. Antonie van Leeuwenhoek, 100(3), 471–475. doi:10.1007/s10482-011-9598-6

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.

Michael Barton, Pierre Lindenbaum, and Rob Syme are currently running a survey on what it is like to be a bioinformatician today. The survey has a history since back in 2008, and I think everyone who’s doing bioinformatics should take it. It aims “to understand the field of bioinformatics by surveying the people whom work in it,” which I think is a nice objective for running a survey. It will be interesting to see what comes out of it. Take the survey, and read more about it at: http://bioinfsurvey.org/

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.

I just want to wish everybody a merry Christmas and a happy new year, from the sunny town of Stellenbosch in South Africa. I will have the pleasure of spending Christmas here this year. As some holiday reading I have provided a longer peace on metagenomics, and I hope to be able to provide a shorter one on resistance genes as well. Happy holidays!