Tag: Antibiotics

Yesterday was an intensive day for typesetters apparently, since they put two of my papers online on the same day. This second paper was published in Environment International, and focuses on predicting minimal selective concentrations for all antibiotics present in the EUCAST database (1).

Today (well, up until yesterday at least), we have virtually no knowledge of which environmental concentrations that can exert a selection pressure for antibiotic resistant bacteria. However, experimentally determining minimal selective concentrations (MSCs) in complex ecosystems would involve immense efforts if done for all antibiotics. Therefore, efforts to theoretically determine MSCs for different antibiotics have been suggested (2,3). In this paper we therefore estimate upper boundaries for selective concentrations for all antibiotics in the EUCAST database, based on the assumption that selective concentrations a priori must be lower than those completely inhibiting growth. Data on Minimal Inhibitory Concentrations (MICs) were obtained for 122 antibiotics and antibiotics combinations, the lowest observed MICs were identified for each of those across all tested species, and to compensate for limited species coverage, we adjusted the lowest MICs for the number of tested species. We finally assessed Predicted No Effect Concentrations (PNECs) for resistance selection using an assessment factor of 10 to account for the differences between MICs and MSCs. Since we found that the link between taxonomic similarity between species and lowest MIC was weak, we have not compensated for the taxonomic diversity that each antibiotic was tested against – only for limited number of species tested. In most cases, our PNECs for selection of resistance were below available PNECs for ecotoxicological effects retrieved from FASS. Also, concentrations predicted to be selective have, for some antibiotics, been detected in regular sewage treatment plants (4), and are greatly exceeded in environments polluted by pharmaceutical pollution (5-7), often with drastic consequences in terms of resistance gene enrichments (8-10). This is a central issue since in principle a transfer event of a novel resistance determinant from an environmental bacteria to an (opportunistic) human pathogen only need to occur once to become a clinical problem (11). Once established, the gene could then spread through human activities, such as trade and travel (7,13). Importantly, this paper:

• Provides upper boundaries for selective concentrations (MSCs) for 111 antibiotics
• Predicts no effect concentrations (PNECs) for resistance selection
• Can guide implementation of compound-specific emission limits based on the provided concentrations

The paper is available under open access here. We hope, and believe, that the data will be of great use in environmental risk assessments, in efforts by industries, regulatory agencies or purchasers of medicines to define acceptable environmental emissions of antibiotics, in the implementation of environmental monitoring programs, for directing mitigations, and for prioritizing future studies on environmental antibiotic resistance.

References:

1. Bengtsson-Palme J, Larsson DGJ: Concentrations of antibiotics predicted to select for resistant bacteria: Proposed limits for environmental regulation. Environment International, 86, 140-149 (2016). doi: 10.1016/j.envint.2015.10.015 [Paper link]
2. Ågerstrand M, Berg C, Björlenius B, Breitholtz M, Brunstrom B, Fick J, Gunnarsson L, Larsson DGJ, Sumpter JP, Tysklind M, Rudén C: Improving environmental risk assessment of human pharmaceuticals. Environmental Science and Technology (2015). doi:10.1021/acs.est.5b00302
3. Tello A, Austin B, Telfer TC: Selective pressure of antibiotic pollution on bacteria of importance to public health. Environmental Health Perspectives, 120, 1100–1106 (2012). doi:10.1289/ehp.1104650
4. Michael I, Rizzo L, McArdell CS, Manaia CM, Merlin C, Schwartz T, Dagot C, Fatta-Kassinos D: Urban wastewater treatment plants as hotspots for the release of antibiotics in the environment: a review. Water Research, 47, 957–995 (2013). doi:10.1016/j.watres.2012.11.027
5. Larsson DGJ, de Pedro C, Paxeus N: Effluent from drug manufactures contains extremely high levels of pharmaceuticals. Journal of Hazardous Materials, 148, 751–755 (2007). doi:10.1016/j.jhazmat.2007.07.008
6. Fick J, Söderström H, Lindberg RH, Phan C, Tysklind M, Larsson DGJ: Contamination of surface, ground, and drinking water from pharmaceutical production. Environmental Toxicology and Chemistry, 28, 2522–2527 (2009). doi:10.1897/09-073.1
7. Larsson DGJ: Pollution from drug manufacturing: review and perspectives. Philosophical Transactions of the Royal Society London, Series B Biological Sciences, 369 (2014). doi:10.1098/rstb.2013.0571
8. 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 [Paper link]
9. 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.
10. 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
11. Bengtsson-Palme J, Larsson DGJ: Antibiotic resistance genes in the environment: prioritizing risks. Nature Reviews Microbiology, 13, 369 (2015). doi: 10.1038/nrmicro3399-c1 [Paper link]
12. Bengtsson-Palme J, Angelin M, Huss M, Kjellqvist S, Kristiansson E, Palmgren H, Larsson DGJ, Johansson A: The human gut microbiome as a transporter of antibiotic resistance genes between continents. Antimicrobial Agents and Chemotherapy, 59, 10, 6551-6560 (2015). doi: 10.1128/AAC.00933-15 [Paper link]

Yesterday, a paper I co-authored with my colleagues Chandan Pal, Erik Kristiansson and Joakim Larsson on the co-occurences of resistance genes against antibiotics, biocides and metals in bacterial genomes and plasmids became published in BMC Genomics. In this paper (1) we utilize the publicly available, fully sequenced, genomes and plasmids in GenBank to investigate the co-occurence network of resistance genes, to better understand risks for co-selection for resistance against different types of compounds. In short, the findings of the paper are that:

• ARGs are associated with BMRG-carrying bacteria and the co-selection potential of biocides and metals is specific towards certain antibiotics
• Clinically important genera host the largest numbers of ARGs and BMRGs and those also have the highest co-selection potential
• Bacteria isolated from human and domestic animal origins have the highest co-selection potential
• Plasmids with co-selection potential tend to be conjugative and carry toxin-antitoxin systems
• Mercury and QACs are potential co-selectors of ARGs on plasmids, however BMRGs are common on chromosomes and could still have indirect co-selection potential
• 14 percent of bacteria and more than 70% of the plasmids completely lacked resistance genes

This analysis was possible thanks to the BacMet database of antibacterial biocide and metal resistance genes, published about two years ago (2). The visualization of the plasmid co-occurence network we ended up with can be seen below. Note the strong connection between the mercury resistance mer operon and the antibiotic resistance genes to the right.

On a side note, it is interesting to note that the underrepresentation of detoxification systems in marine environments we noted last year (3) still seems to hold for genomes (and particularly plasmids), supporting the genome streamlining hypothesis (4).

References:

1. Pal C, Bengtsson-Palme J, Kristiansson E, Larsson DGJ: Co-occurrence of resistance genes to antibiotics, biocides and metals reveals novel insights into their co-selection potential. BMC Genomics, 16, 964 (2015). doi: 10.1186/s12864-015-2153-5 [Paper link]
2. Pal C, Bengtsson-Palme J, Rensing C, Kristiansson E, Larsson DGJ: BacMet: Antibacterial Biocide and Metal Resistance Genes Database. Nucleic Acids Research, 42, D1, D737-D743 (2014). doi: 10.1093/nar/gkt1252 [Paper link]
3. Bengtsson-Palme J, Alm Rosenblad M, Molin M, Blomberg A: Metagenomics reveals that detoxification systems are underrepresented in marine bacterial communities. BMC Genomics, 15, 749 (2014). doi: 10.1186/1471-2164-15-749 [Paper link]
4. Giovannoni SJ, Cameron TJ, Temperton B: Implications of streamlining theory for microbial ecology. ISME Journal, 8, 1553-1565 (2014).

UPDATE: This post has been updated to reflect a valid comment that I messed up in the original post. Specific use of antibiotics as feed additives has been forbidden for years in the EU. The petition was about cutting the prophylactic use of antibiotics in animals, but that was very unclear from the original post. I thank my readers for pointing this unclarity out.

I’m going to do something unusual and ask you to sign a petition targeted at European Union ministers to support new EU laws to drastically cut the prophylactic use of antibiotics in agriculture as growth promoters. The problem is that if ministers don’t feel the public pressure to act, the laws may be delayed or not be implemented. Examples from Denmark, Sweden, Norway and the Netherlands show that it is possible to produce meat with little or no antibiotics, but since bacteria can travel across borders (1), we need to bring the rest of the world onboard, and the EU is good first step. Therefore I ask you to sign the Avaaz petition here.

1. Bengtsson-Palme J, Angelin M, Huss M, Kjellqvist S, Kristiansson E, Palmgren H, Larsson DGJ, Johansson A: The human gut microbiome as a transporter of antibiotic resistance genes between continents. Antimicrobial Agents and Chemotherapy, 59, 10, 6551-6560 (2015). doi: 10.1128/AAC.00933-15 [Paper link]

The paper we published in August on travelers carrying resistance genes with them in their gut microbiota has now been typeset and got proper volume and issue numbers assigned to it in Antimicrobial Agents and Chemotherapy. Take a look at it, I personally think it’s quite good-looking.

Also, if you understand Swedish, here is an interview with me broadcasted on Swedish Radio last month about this study and the consequences of it.

The new citation for the paper is:

• Bengtsson-Palme J, Angelin M, Huss M, Kjellqvist S, Kristiansson E, Palmgren H, Larsson DGJ, Johansson A: The human gut microbiome as a transporter of antibiotic resistance genes between continents. Antimicrobial Agents and Chemotherapy, 59, 10, 6551-6560 (2015). doi: 10.1128/AAC.00933-15 [Paper link]

There have been quite a lot of buzz this week around the travel paper we published earlier this month. Twitter aside, the findings of the paper has also been covered by a range of news outlets, both in Sweden and internationally. Today, I was on Swedish radio talking resistance problems for about ten minutes (listen here; in Swedish). Here’s a few takes on the story I gathered around the web:

Science Daily
Business Standard
Z News
Englemed Health News
Läkemedelsvärlden (in Swedish)
Sveriges Radio (in Swedish)
Göteborgs-Posten (in Swedish)

Earlier today, my most recent paper (1) became available online, describing resistance gene patterns in the gut microbiota of Swedes before and after travel to the Indian peninsula and central Africa. In this work, we have used metagenomic sequencing of the intestinal microbiome of Swedish students returning from exchange programs to show that the abundance of antibiotic resistance genes in several classes are increased after travel. This work reiterates the findings of several papers describing uptake of resistant bacteria (2-8) or resistance genes (9-11) after travel to destinations with worse resistance situation.

Our paper is important because it:

1. Addresses the abundance of a vast range of resistance genes (more than 300).
2. Finds evidence for that the overall relative abundance of antibiotic resistance genes increased after travel, without any intake of antibiotics.
3. Shows that the sensitivity of metagenomics was, despite very deep sequencing efforts, not sufficient to detect acquisition of the low-abundant (CTX-M) resistance genes responsible for observed ESBL phenotypes.
4. Reveals a “core resistome” of resistance genes that are more or less omnipresent, and remain relatively stable regardless of travel, while changes seem to occur in the more variable part of the resistome.
5. Hints at increased abundance of Proteobacteria after travel, although this increase could not specifically be linked to resistance gene increases.
6. Uses de novo metagenomic assembly to physically link resistance genes in the same sample, giving hints of co-resistance patterns in the gut microbiome.

The paper was a collaboration with Martin Angelin, Helena Palmgren and Anders Johansson at Umeå University, and was made possible by bioinformatics support from SciLifeLab in Stockholm. I highly recommend reading it as a complement to e.g. the Forslund et al. paper (12) describing country-specific antibiotic resistance patterns in the gut microbiota.

Taken together, this study offers a broadened perspective on how the antibiotic resistance potential of the human gut microbiome changes after travel, providing an independent complement to previous studies targeting a limited number of bacterial species or antibiotic resistance genes. Understanding how resistance genes travels the globe is hugely important, since resistance in principle only need to appear in a pathogen once; improper hygiene and travel may then spread novel resistance genes across continents rapidly (13,14).

References

1. Bengtsson-Palme J, Angelin M, Huss M, Kjellqvist S, Kristiansson E, Palmgren H, Larsson DGJ, Johansson A: The human gut microbiome as a transporter of antibiotic resistance genes between continents. Antimicrob Agents Chemother Accepted manuscript posted online (2015). doi: 10.1128/AAC.00933-15 [Paper link]
2. Gaarslev K, Stenderup J: Changes during travel in the composition and antibiotic resistance pattern of the intestinal Enterobacteriaceae flora: results from a study of mecillinam prophylaxis against travellers’ diarrhoea. Curr Med Res Opin 9:384–387 (1985).
3. Paltansing S, Vlot JA, Kraakman MEM, Mesman R, Bruijning ML, Bernards AT, Visser LG, Veldkamp KE: Extended-spectrum β-lactamase-producing enterobacteriaceae among travelers from the Netherlands. Emerging Infect. Dis. 19:1206–1213 (2013).
4. Ruppé E, Armand-Lefèvre L, Estellat C, El-Mniai A, Boussadia Y, Consigny PH, Girard PM, Vittecoq D, Bouchaud O, Pialoux G, Esposito-Farèse M, Coignard B, Lucet JC, Andremont A, Matheron S: Acquisition of carbapenemase-producing Enterobacteriaceae by healthy travellers to India, France, February 2012 to March 2013. Euro Surveill. 19 (2014).
5. Kennedy K, Collignon P: Colonisation with Escherichia coli resistant to “critically important” antibiotics: a high risk for international travellers. Eur J Clin Microbiol Infect Dis 29:1501–1506 (2010).
6. Tham J, Odenholt I, Walder M, Brolund A, Ahl J, Melander E: Extended-spectrum beta-lactamase-producing Escherichia coli in patients with travellers’ diarrhoea. Scand. J. Infect. Dis. 42:275–280 (2010).
7. Östholm-Balkhed Å, Tärnberg M, Nilsson M, Nilsson LE, Hanberger H, Hällgren A, Travel Study Group of Southeast Sweden: Travel-associated faecal colonization with ESBL-producing Enterobacteriaceae: incidence and risk factors. J Antimicrob Chemother 68:2144–2153 (2013).
8. Kantele A, Lääveri T, Mero S, Vilkman K, Pakkanen SH, Ollgren J, Antikainen J, Kirveskari J: Antimicrobials increase travelers’ risk of colonization by extended-spectrum betalactamase-producing enterobacteriaceae. Clin Infect Dis 60:837–846 (2015).
9. von Wintersdorff CJH, Penders J, Stobberingh EE, Oude Lashof AML, Hoebe CJPA, Savelkoul PHM, Wolffs PFG: High rates of antimicrobial drug resistance gene acquisition after international travel, The Netherlands. Emerging Infect. Dis. 20:649–657 (2014).
10. Tängdén T, Cars O, Melhus A, Löwdin E: Foreign travel is a major risk factor for colonization with Escherichia coli producing CTX-M-type extended-spectrum beta-lactamases: a prospective study with Swedish volunteers. Antimicrob Agents Chemother 54:3564–3568 (2010).
11. Dhanji H, Patel R, Wall R, Doumith M, Patel B, Hope R, Livermore DM, Woodford N: Variation in the genetic environments of bla(CTX-M-15) in Escherichia coli from the faeces of travellers returning to the United Kingdom. J Antimicrob Chemother 66:1005–1012 (2011).
12. Forslund K, Sunagawa S, Kultima JR, Mende DR, Arumugam M, Typas A, Bork P: Country-specific antibiotic use practices impact the human gut resistome. Genome Res 23:1163–1169 (2013).
13. Bengtsson-Palme J, Larsson DGJ: Antibiotic resistance genes in the environment: prioritizing risks. Nat Rev Microbiol 13:396 (2015).
14. Larsson DGJ: Antibiotics in the environment. Ups J Med Sci 119:108–112 (2014).

Late last year, an opinion paper by José Martínez, Teresa Coque and Fernando Baquero was published in Nature Reviews Microbiology (1). In this paper, the authors present a system – resistance readiness conditions (RESCon) – for ranking the risks associated with the detection of antibiotic resistance genes. They also outline the obstacles associated with determining risks presented by antibiotic resistance genes in environmental microbial communities in terms of their potential to transfer to human pathogens. Generally, I am very positive about this paper, which I think is a must-read for anyone who works with antibiotic resistance genes in metagenomes, regardless of it they stem from the human gut or the external environment.

There is, however, one very important aspect that struck me and many other members of our research group as curious: the proposed system assign antibiotic resistance genes already present on mobile genetic elements in human pathogens to the highest risk category (RESCon 1), while resistance genes encoding novel resistance mechanisms not yet been found on mobile elements in a pathogen are considered to be part of lower risk categories. We believe that this system will overestimate the risks associated with well-known resistance factors that are already circulating among human pathogens and under-appreciate the potentially disastrous consequences that the transfer of previously unknown resistance determinants from the environmental resistome could have (exemplified by the rapid clinical spread of the NDM-1 metallo-beta-lactamase gene (2,3)).

With this in mind me and Joakim Larsson wrote a response letter to Nature Reviews Microbiology that went online last monday (4), together with the authors’ reply to us (5). (I strongly suggest that you read the entire original paper (1) before you read the reply (5) to our response letter (4), since Martinez et al. changes the scope slightly from the original paper in their response letter, and these clarifications may (or may not) have been in response to our arguments.)

In our response, we also stress that the abundances of resistance genes, and not only their presence, should be accounted for when estimating risks (although that last point might have been slightly obscured due to the very low word limit). In other words, we think that identifying environmental hotspots for antibiotic resistance genes, where novel resistance genes could be selected for (6,7,8), is of great importance for mitigating public health risks related to environmental antibiotic resistance. Please read our full thoughts on the matter in Nature Reviews Microbiology.

Similar issues will be touched upon in my talk at the EDAR2015 conference later in May. Hope to see you there!

References

1. Martinez JL, Coque TM, Baquero F: What is a resistance gene? Ranking risk in resistomes. Nat Rev Microbiol 2015, 13:116–123.
2. Kumarasamy KK, et al.: Emergence of a new antibiotic resistance mechanism in India, Pakistan, and the UK: a molecular, biological, and epidemiological study. Lancet Infect Dis 2010, 10:597–602.
3. Walsh TR, Weeks J, Livermore DM, Toleman MA: Dissemination of NDM‐1 positive bacteria in the New Delhi environment and its implications for human health: an environmental point prevalence study. Lancet Infect Dis 2011, 11:355–362.
4. Bengtsson-Palme J, Larsson DGJ: Antibiotic resistance genes in the environment: prioritizing risks. Nat Rev Microbiol 2015, Advance online publication. doi:10.1038/nrmicro3399‐c1
5. Martinez JL, Coque TM, Baquero F: Prioritizing risks of antibiotic resistance genes in all metagenomes. Nat Rev Microbiol 2015, Advance online publication. doi:10.1038/nrmicro3399‐c2
6. Kristiansson E, et al.: Pyrosequencing of antibiotic‐contaminated river sediments reveals high levels of resistance and gene transfer elements. PLoS ONE 2011, 6:e17038.
7. 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. Front Microbiol 2014, 5:648.
8. Marathe NP, et al.: A treatment plant receiving waste water from multiple bulk drug manufacturers is a reservoir for highly multi‐drug resistant integron‐bearing bacteria. PLoS ONE 2013, 8:e77310.

I will be giving a talk at the Third International symposium on the environmental dimension of antibiotic resistance (EDAR2015) next month (five weeks from now. The talk is entitled “Turn up the signal – wipe out the noise: Gaining insights into antibiotic resistance of bacterial communities using metagenomic data“, and will deal with handling of metagenomic data in antibiotic resistance gene research. The talk will highlight the some particular pitfalls related to interpretation of data, and exemplify how flawed analysis practices can result in misleading conclusions regarding antibiotic resistance risks. I will particularly address how taxonomic composition influences the frequencies of resistance genes, the importance of knowledge of the functions of the genes in the databases used, and how normalization strategies influence the results. Furthermore, we will show how the context of resistance genes can allow inference of their potential to spread to human pathogens from environmental or commensal bacteria. All these aspects will be exemplified by data from our studies of environments subjected to pharmaceutical pollution in India, the effect of travel on the human resistome, and modern municipal wastewater treatment processes.

The talk will take place on Monday, May 18, 2015 at 13:20. The full scientific program for the conference can be found here. Registration for the conference is still possible, although not for the early-bird price. I look forward to see a lot of the people who will attend the conference, and hopefully also you!

It is nice to see that Indian media has picked up the story about antibiotic resistance genes in the heavily polluted Kazipally lake. In this case, it is the Deccan Chronicle who have been reporting on our findings and briefly interviewed Prof. Joakim Larsson about the study. The issue of pharmaceutical pollution of the environment in drug-producing countries is still rather under-reported and public perception of the problem might be rather low. Therefore, it makes me happy to see an Indian newspaper reporting on the issue. The scientific publication referred to can be found here.

In a recent paper in Nature, a completely new antibiotic – teixobactin – is described (1). The really cool thing about this antibiotic is that it was discovered in a screen of uncultured bacteria, grown using new technology that enable controlled growth of single colonies in situ. I really like this idea, and I think the prospect of a novel antibiotic using a previously unexploited mechanism is super-promising, particularly in the light of alarming resistance development in clinically important pathogens (2,3). What really annoys me about the paper is the claim (already in the abstract) that since “we did not obtain any mutants of Staphylococcus aureus or Mycobacterium tuberculosis resistant to teixobactin (…) the properties of this compound suggest a path towards developing antibiotics that are likely to avoid development of resistance.” To me, this sounds pretty much like a bogus statement; in essence telling me that we apparently have not learned anything from the 70 years of antibiotics usage and resistance development. After working with antibiotic resistance a couple of years, particularly from the environmental perspective, I have a very disturbing feeling that there is already resistance mechanisms against teixobactin waiting out in the wild (4,5). Pretending that lack of mutation-associated resistance development means that there could not be resistance development did not help vancomycin (6,7), and we now see VRE (Vancomycin Resistant Enterococcus) showing up as a major problem in clinics. The “avoid development of resistance” claim is downright irresponsible, and the cynic in me cannot help to think that NovoBiotic Pharmaceuticals (the affiliation of almost half of the authors) has a monetary finger in this jar. In the end, time will tell how “resistance-resilient” teixobactin is and how well we can handle the gift of a novel antibiotic.

1. Ling LL, Schneider T, Peoples AJ, Spoering AL, Engels I, Conlon BP, Mueller A, Schäberle TF, Hughes DE, Epstein S, Jones M, Lazarides L, Steadman VA, Cohen DR, Felix CR, Fetterman KA, Millett WP, Nitti AG, Zullo AM, Chen C, Lewis K: A new antibiotic kills pathogens without detectable resistance. Nature (2015). doi:10.1038/nature14098
2. 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, 57: 704–710 (2013).
3. French GL: The continuing crisis in antibiotic resistance. Int J Antimicrob Agents, 36 Suppl 3:S3–7 (2010).
4. 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).
5. Larsson DGJ: Antibiotics in the environment. Ups J Med Sci, 119: 108–112 (2014).
6. Wright GD: Mechanisms of resistance to antibiotics. Curr Opin Chem Biol, 7:563–569 (2003).
7. Werner G, Strommenger B, Witte W: Acquired vancomycin resistance in clinically relevant pathogens. Future Microbiol, 3: 547–562 (2008).