Tag: Antibiotics

If you’re looking for super-interesting jobs within bioinformatics, you don’t need to look any further. Instead, you should apply for a position at 1928 Diagnostics here in Gothenburg and join them in the fight against antibiotic resistant bacteria. The position is in the development team and the deadline for application is December 19. All the details can be found here.

Our paper describing the bacterial community of a polluted lake in India has now been typeset and appears in its final form in Frontiers in Microbiology. If I may say so, I think that the paper turned out to be very goodlooking and it is indeed nice to finally see it in print. The paper describes an unprecedented diversity and abundance of antibiotic resistance genes and genes enabling transfer of DNA between bacteria. We also describe a range of potential novel plasmids from the lake. Finally, the paper briefly describes a new approach to targeted assembly of metagenomic data — TriMetAss — which can be downloaded here.

Reference:
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

The first work in which I have employed metagenomics to investigate antibiotic resistance has been accepted in Frontiers in Microbiology, and is (at the time of writing) available as a provisional PDF. In the paper (1), which is co-authored by Fredrik Boulund, Jerker Fick, Erik Kristiansson and Joakim Larsson, we have used shotgun metagenomic sequencing of an Indian lake polluted by dumping of waste from pharmaceutical production. We used this data to describe the diversity of antibiotic resistance genes and the genetic context of those, to try to predict their genetic transferability. We found resistance genes against essentially every major class of antibiotics, as well as large abundances of genes responsible for mobilization of genetic material. Resistance genes were estimated to be 7000 times more abundant in the polluted lake than in a Swedish lake included for comparison, where only eight resistance genes were found. The abundances of resistance genes have previously only been matched by river sediment subject to pollution from pharmaceutical production (2). In addition, we describe twenty-six known and twenty-one putative novel plasmids from the Indian lake metagenome, indicating that there is a large potential for horizontal gene transfer through conjugation. Based on the wide range and high abundance of known resistance factors detected, we believe that it is plausible that novel resistance genes are also present in the lake. We conclude that environments polluted with waste from antibiotic manufacturing could be important reservoirs for mobile antibiotic resistance genes. This work further highlights previous findings that pharmaceutical production settings could provide sufficient selection pressure from antibiotics (3) to drive the development of multi-resistant bacteria (4,5), resistance which may ultimately end up in pathogenic species (6,7). The paper can be read in its entirety here.

References:

1. Bengtsson-Palme J, Boulund F, Fick J, Kristiansson E, Larsson DGJ: Shotgun metagenomics reveals a wide array of antibiotic resistance genes and mobile elements in a polluted lake in India. Frontiers in Microbiology, Volume 5, Issue 648 (2014). doi: 10.3389/fmicb.2014.00648
2. Kristiansson E, Fick J, Janzon A, Grabic R, Rutgersson C, Weijdegård B, Söderström H, Larsson DGJ: Pyrosequencing of antibiotic-contaminated river sediments reveals high levels of resistance and gene transfer elements. PLoS ONE, Volume 6, e17038 (2011). doi:10.1371/journal.pone.0017038.
3. Larsson DGJ, de Pedro C, Paxeus N: Effluent from drug manufactures contains extremely high levels of pharmaceuticals. J Hazard Mater, Volume 148, 751–755 (2007). doi:10.1016/j.jhazmat.2007.07.008
4. Marathe NP, Regina VR, Walujkar SA, Charan SS, Moore ERB, Larsson DGJ, Shouche YS: A Treatment Plant Receiving Waste Water from Multiple Bulk Drug Manufacturers Is a Reservoir for Highly Multi-Drug Resistant Integron-Bearing Bacteria. PLoS ONE, Volume 8, e77310 (2013). doi:10.1371/journal.pone.0077310
5. Johnning A, Moore ERB, Svensson-Stadler L, Shouche YS, Larsson DGJ, Kristiansson E: Acquired genetic mechanisms of a multiresistant bacterium isolated from a treatment plant receiving wastewater from antibiotic production. Appl Environ Microbiol, Volume 79, 7256–7263 (2013). doi:10.1128/AEM.02141-13
6. Pruden A, Larsson DGJ, Amézquita A, Collignon P, Brandt KK, Graham DW, Lazorchak JM, Suzuki S, Silley P, Snape JR., et al.: Management options for reducing the release of antibiotics and antibiotic resistance genes to the environment. Environ Health Perspect, Volume 121, 878–885 (2013). doi:10.1289/ehp.1206446
7. Finley RL, Collignon P, Larsson DGJ, McEwen SA, Li X-Z, Gaze WH, Reid-Smith R, Timinouni M, Graham DW, Topp E: The scourge of antibiotic resistance: the important role of the environment. Clin Infect Dis, Volume 57, 704–710 (2013). doi:10.1093/cid/cit355

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

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

A new year has begun, and it brings with it a few updates on the website. I have added a summary of the year 2013 from my perspective, and (as you may recognize) updated my picture on the front page. Briefly, this year will bring lots of exciting stuff. Personally, I am quite excited to finally be able to share the new version of Metaxa – Metaxa2 – which will be released to the public late this Winter (or early Spring). Additionally, I look forward to wrap up some manuscript on metagenomics and antibiotic resistance, which I have been working with for more than 2.5 years now. Also, we look forward to some super-intersting technology developments in DNA sequencing, with PacBio finally finding proper usage scenarios, Nano-pore sequencing around the corner, and super-multiplexing on the Illumina instruments. We’re in for a treat with DNA sequencing in 2014!

It seems like our paper on the recently launched database on resistance genes against antibacterial biocides and metals (BacMet) has gone online as an advance access paper in Nucleic Acids Research today. Chandan Pal – the first author of the paper, and one of my close colleagues as well as my roommate at work – has made a tremendous job taking the database from a list of genes and references, to a full-fledged browsable and searchable database with a really nice interface. I have contributed along the process, and wrote the lion’s share of the code for the BacMet-Scan tool that can be downloaded along with the database files.

BacMet is a curated source of bacterial resistance genes against antibacterial biocides and metals. All gene entries included have at least one experimentally confirmed resistance gene with references in scientific literature. However, we have also made a homology-based prediction of genes that are likely to share the same resistance function (the BacMet predicted dataset). We believe that the BacMet database will make it possible to better understand co- and cross-resistance of biocides and metals to antibiotics within bacterial genomes and in complex microbial communities from different environments.

The database can be easily accessed here: http://bacmet.biomedicine.gu.se, and use of the database in scientific work can cite the following paper, which recently appeared in Nucleic Acids Research:

Pal C, Bengtsson-Palme J, Rensing C, Kristiansson E, Larsson DGJ: BacMet: Antibacterial Biocide and Metal Resistance Genes Database. Nucleic Acids Research. Database issue, advance access. doi: 10.1093/nar/gkt1252 [Paper link]

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.

It seriously worries me that a number of indications recently have pointed to that the heavy use of antibiotics does not only drive antibiotic resistance development, but also the development towards more virulent and aggressive strains of pathogenic bacteria. First, the genome sequencing of the E. coli strain that caused the EHEC outbreak in Germany in May revealed not only antibiotic resistance genes, but also is also able to make Shiga toxin, which is causes the severe diarrhoea and kidney damage related to the haemolytic uremic syndrome (HUS). The genes encoding the Shiga toxin are not originally bacterial genes, but instead seem to originate from phages. When E. coli gets infected with a Shiga toxin-producing phage, it becomes a human pathogen [1]. David Acheson, managing director for food safety at consulting firm Leavitt Partners, says that exposure to antibiotics might be enhancing the spread of Shiga toxin-producing phage. Some antibiotics triggers what is referred to as the SOS response, which induces the phage to start replicating. The replication of the phage causes the bacteria to burst, releasing the phages, and with them the toxin [1].

Second, there is apparently an ongoing outbreak of scarlet fever in Hong Kong. Kwok-Yung Yuen, microbiologist at the University of Hong Kong, has analyzed the draft sequence of the genome, and suggests that the bacteria acquired greater virulence and drug resistance by picking up one or more genes from bacteria in the human oral and urogenital tracts. He believes that the overuse of antibiotics is driving the emergence of drug resistance in these bacteria [2].

Now, both of these cases are just indications, but if they are true that would be an alarming development, where the use of antibiotics promotes the spread not only of resistance genes, impairing our ability to treat bacterial infections, but also the development of far more virulent and aggressive strains. Combining increasing untreatability with increasing aggressiveness seems to me like the ultimate weapon against our relatively high standards of treatment of common infections. Good thing hand hygiene still seems to help [3].

References

1. Phage on the rampage (http://www.nature.com/news/2011/110609/full/news.2011.360.html), Published online 9 June 2011, Nature, doi:10.1038/news.2011.360
2. Mutated Bacteria Drives Scarlet Fever Outbreak (http://news.sciencemag.org/scienceinsider/2011/06/mutated-bacteria-drives-scarlet.html?etoc&elq=cd94aa347dca45b3a82f144b8213e82b), Published online 27 June 2011.
3. Luby SP, Halder AK, Huda T, Unicomb L, Johnston RB (2011) The Effect of Handwashing at Recommended Times with Water Alone and With Soap on Child Diarrhea in Rural Bangladesh: An Observational Study. PLoS Med 8(6): e1001052. doi:10.1371/journal.pmed.1001052 (http://www.plosmedicine.org/article/info%3Adoi%2F10.1371%2Fjournal.pmed.1001052)