->Jim Caryl

The idea of an enemy sleeper agent is a central plot device in many a spy novel or movie, and certainly the idea of going to ground behind enemy lines is not unheard of in many theatres of conflict. The idea in all cases is to remain undetected until re-activated to cause harm behind enemy defences.

The trick to identifying if there are latent sleepers operating is to try and re-activate them and get them to reveal themselves. Cue any number of spy stories about false radio signals or targets to lure sleepers into the open.

Curiously, it is a similar strategy that is being used in novel treatments for infections from two disparate areas of chemotherapy, one in the treatment of HIV and the other the treatment of persistent bacterial infections.

In HIV, one of the issues is that the virus can become dormant, they cease to replicate, and remain for all intents and purposes hidden and inactivated in a small pool of resting CD4+ T-cells. The problem with this is that the anti-retroviral drugs we have work by interfering with replication, thus if viruses aren't actively replicating, they're not exposed to the drug.

Likewise, in chronic bacterial infections there exists a phenomenon called 'persistence'. In a bacterial population a sub-population of 'persister' cells continue to exist in a stationary or growth-retarded state, regardless of the growth state of the population as a whole. Whilst there are ongoing arguments in the field about what constitutes 'persistence' and the genetic or environmental factors that result in such dormancy, it is none the less the case that most antibiotics have been developed to attack actively growing cells. In the absence of growth, the bacteria are simply not effected by the antibiotic.

(Image source: VGTI, Florida)

In both HIV and chronic bacterial infections, removal of the chemotherapy can result in a significant flare-up of the infection as a result of re-activation of the dormant virus/bacteria.

So how do we address these issues? Well, an approach adopted my many HIV/antibiotic research labs is to screen such latent cells/persisters against libraries of different types and shapes of drug molecules. The aim is to identify drug molecules that re-activate the cells, thus forcing the 'sleepers' back out into the open where they can be targeted by chemotherapy.

In HIV research circles there has been some work developing two potential approaches. Interleukin-7, a naturally occurring signalling molecule, has shown some signs it can re-activate dormant cells, and thus HIV. The other approach is looking at several histone deacetylase inhibitors, which can reactivate HIV directly. However, one of several major issues has been that it isn't much good flushing out the sleepers if there are still parts of the body where infected tissues can avoid exposure to such drugs; we'd need to know the extent of such reservoir tissues and how to target them.

In the case of treating bacterial persisters, a recent study demonstrates exactly the sort of prudent, rational approach that can be taken to addressing this issue. The study from the lab of Prof. Jim Collins at Boston University, focussed on targeting both E. coli and S. aureus persister cells with an aminoglycoside antibiotic called gentamicin. Aminoglycosides are a group of drug molecules that interfere with protein synthesis, an essential living process, but they can only work if they can actually get into the cell. It's well known that the process of aminogylcoside uptake by the cell is energy-driven, however, being metabolically dormant the persister cells have insufficient energy for drug uptake and so gentamicin has only weak activity.

Prof. Collins team found that treating persister cells with certain sugars (glucose, fructose, and mannitol) induced rapid-killing when gentamicin was present, and so seemingly the idea of metabolically stimulating such dormant cells hold true. The addition of sugars doesn't exactly turn these cells into native active cells, because this treatment only works to enable aminoglycoside-mediated killing. Other classes of antibiotics still had no effect. Sugars such as glucose, fructose and mannitol enter a cell's metabolism at the head of a process that generates high-energy molecules. Whilst this energisation is enough to enable aminoglycoside uptake, it's not enough to get all the cellular processes ticking over, hence the lack of reaction to antibiotic drug classes that target other cellular processes.

What is interesting is that the process works for both Gram-negative bacteria (such as E. coli) and Gram-positive bacteria ( S. aureus ), both representing a broad divide in the bacterial world, though in S. aureus only the fructose, rather than glucose and mannitol, resulted in rapid killing by gentamicin.

In our spy novel we might say that the enemy sleeper agent is being lured out into the open by a sugar coated package, the contents of which spell the agent's death. But certainly approaches such as these can only benefit the increasingly complex requirements of antibacterial chemotherapy that are required to treat persistent infections.

Ref.

Allison KR, Brynildsen MP and Collins JJ. (2011) Metabolite-enabled eradication of bacterial persisters by aminoglycosides. Nature 473: 216-220. doi: 10.1038/nature10069
[ pdf ]

I was recently called by an editor at NewScientist asking for some background on the field of fitness in bacteria, and particularly the issue of multi-resistant bacteria persisting in the environment (or clinic) in the absence of antibiotic selection. The reason for the question arose due to the upcoming publishing of an interesting paper in PLoS Genetics:

Silva RF, Mendonça SCM, Carvalho LM, Reis AM, Gordo I, et al. 2011 Pervasive Sign Epistasis between Conjugative Plasmids and Drug-Resistance Chromosomal Mutations. PLoS Genet 7(7): e1002181. doi: 10.1371/journal.pgen.1002181

Following this, I have been quoted in a NewScientist Health News item, and as I don't feel my response is quite in the context I gave it, I thought I would give a more detailed account. I spoke to NewScientist last Wednesday (the paper in question wasn't due to be published until the following day), but I was told that in the study the authors had observed that antibiotic resistance can have a positive effect on bacterial fitness, even in the absence of selection. I was asked whether this was a surprise to me, and more generally about research in bacterial fitness. What I perhaps should have done was ask specifically whether the paper was still embargoed and whether I could have more particulars of the study, because I could not have anticipated the particular nature of the study in question.

The paper describes an observation that the fitness costs of chromosomal mutations conferring resistance to antibiotics, whilst initially deleterious, can become beneficial through the acquisition of a transferable antibiotic resistant plasmid. The outcome of this might be cause for concern given the widespread occurrence of conjugative resistance plasmids in clinical strains; the idea that these may be stabilising otherwise costly resistance mutations is worrying.

We know from many studies that some mutations that give rise to antibiotic resistance come at a cost to bacteria. A bacterium may for example overcome an antibiotic by having a mutation in the key protein that the antibiotic targets, but the mutation may also make that protein less capable at its day-to-day function, and this presents a fitness cost to the cell. Obviously while there is antibiotic around, such mutations bring a strong fitness advantage, but when the antibiotic is no longer around, and these cells are forced to compete with their antibiotic-sensitive brethren, they can become a fitness cost.

The phenomenon in which the effect of one set of genes (say those encoded on a conjugative plasmid) may have a synergistic effect on another set of genes (i.e. those on the chromosome) that results in a more beneficial outcome is called 'positive epistasis'. Whilst I'm familiar with this phenomenon more generally in bacterial metabolism, as it has been documented by the inimitable Richard Lenski over the years, I would be hard pressed to cite examples where two negative fitness costs (one chromosomal, and one plasmid-borne) might cancel each other out, or indeed even result in a beneficial fitness advantage that is more than the sum of its constituent costs.

However, as I often quote, Crichton's Jurassic Park character Dr Ian Malcolm would say, 'Life finds a way..." Indeed, bacteria that encounter a fitness cost may under go 'mitigating' mutations that alleviate the fitness costs of the original mutation, restoring their fitness to that of their non-resistant brethren. This way the bacteria get to remain resistant at no extra cost, kind of like having your cake, and eating it too. It was this much that I described in my conversation with NewScientist. I have previously described the means by which bacteria may mitigate fitness costs (second half of post).

The study in question is more observational than mechanistic, so we are not clear why it is that acquisition of a conjugative resistance plasmid can be beneficial to cells already encoding chromosomal resistance to an antibiotic. This will likely have to await further investigation, however, the question inevitably turns to how to address this issue. As I mentioned to NewScientist, one aspect of my research is to identify those antibiotics against which resistance imposes the greatest fitness costs, the idea being that when resistance occurs, it may persist for less time once the use of that antibiotic is curtailed. However, this is not a one-stop solution, it would form one of many small changes in practise to prevent antibiotic persistence.

I don't disagree with Dionisio's idea of targeting the plasmids, and looking for ways to inhibit plasmid conjugation and replication, thus addressing one of the major causes of antibiotic resistance spread. Indeed, such approaches have been reviewed previously (Williams & Hergenrother, 2008, Exposing plasmids as the Achilles' heel of drug-resistant bacteria. Current Opinion in Chemical Biology 12: 389-399 doi: 10.1016/j.cbpa.2008.06.015), and some interesting chemical agents found.

In fact, I wonder if the presence of conjugative plasmids reduce the fitness costs of chromosomal mutations to the extent that the typical mitigating mutations seen to stabilise other chromsomal mutations don't occur. This at least presents the opportunity that a combined attack on the plasmid and the cell may be successful in eradicating particular resistance determinants before they become independently stable.

I'm glad that NewScientist took an interest in this paper as I think we need to keep the issue of antibiotic resistance, and what we learn about the associated problems and the solutions, in the public eye. I'm also open for comments on this area, but perhaps next time I will clarify what the interviewer has understood from my responses.

AS I think about how I'm going to get to Science Online London 2011 in September, when for half of it I am supposed to be at the Staph GBI conference in Edinburgh, I lament not being able to go to the Science Online unconference 'Fringe-Frivolous' event that usually takes place the night before.

Did I ever tell you how at last year's Fringe-Frivolous, as I drank bottles of cool beer and chatted to more interesting people you could shake a stick at, I found myself waiting in a queue for a BBQ burger with Peter Jenner?

For those of you born on Mars, Peter Jenner was Pink Floyd's first manager, and managed T.Rex, The Clash, Ian Drury, Robyn Hitchcock and Billy Bragg. Pete also has a lot, A LOT, to say about Digital Rights Management (DRM), which he says - and I quote - are f**ked .

So as we stood there, he talked about DRMs, and I talked about bacterial resistance to antibiotics, and you know what we both realised? We were pretty much talking about the same thing. DRMs, we agreed, are a bit like antibiotics. You can throw them out there to kill music theft/bacteria, but both won't work indefinitely, and they both generate resistance.

Hang on, isn't DRM supposed to protect music? Well, no, because it doesn't work. It merely forces music lovers to go exploring music on their own terms, and away from DRM, by whatever means possible. People want to listen to music, and will be damned if they'll let a DRM get in the way of that. Bacteria aren't afforded so much choice, but by dint of evolution, they are afforded the genetic prospect of resisting and find their own routes to tackle antibiotics.

The problems are worse when the you use too-low concentrations of antibiotics, or use them indiscriminately for the wrong conditions; both can not only generate more resistance, but a good deal of virulence (akin to giving the bacteria sharper claws and teeth). Weak or indiscriminate DRMs force people to by-pass the traditional money stream in the music industry and find free music elsewhere, widely disseminated in the population, much like antibiotic resistance.

I don't think we came to any firm conclusion as to how to tackle the problem of resistance. Pete said of DRMs that they will likely be replaced with blanket licensing, which allows people to freely exchange music, for a couple of pounds a month. Would that antibiotic resistance be tackled in such a manner; I guess the only comparable system would be the use of a wide-scale inhibitor of horizontal-gene transfer (the mechanism by much a lot of antibiotic resistance is spread), taken by a large enough group of people. This puts no selective pressure on bacteria, after all, HGT is only beneficial for the bacteria after the fact.

Perhaps both suggestions are naive, and we can only come up with a better approach, or set of approaches, once we better understand the behaviour of music-lovers/bacteria. What we can at least say, is that events like Science Online London, and the unconferences that precede them, are a melting pot of minds, and the ultimate forms of social networking.

TO SAY I've been busy recently would be an understatement, and none more so than the last two weeks where I have been taking part in a science education and engagement programme funded by the Wellcome Trust called I'm a Scientist, get me out of here (IAS).

So as I take a moment to close my calendar, which has been FULL every day, and move back into a week where I wing my experiments based on whether they work or not, I thought I'd take a minute to say something about my experience.

As I've been explaining (exhaustively) to my colleagues for the past two weeks, I was one of a hundred or so scientists taking part, with groups of five scientists allocated to 'zones' covering different scientific disciplines (more or less); I was in the 'Genes Zone'. Each group was given seven schools, and the schools booked chatroom sessions with the scientists in that zone, at any time within the school day. In addition to chatroom sessions, we were also inundated with submitted questions to answer in our own time, some targeted directly to me, and other to all of the scientists in the zone. The organisers recommended that we would need to commit at least two hours a day, which is to say, if you want the kids to vote for you, you've got to put some effort in! This is, at the end of the day, a bit of a science popularity contest. The winning scientists get £500 to put towards a science communication project, which was a strong feature of discussion in the chatroom sessions.

So how did I get into this?

I first heard of IAS via former-fellow NatNet blogger Stephen Curry, who won his zone in one of last year's events. Then at Science Online London 2010, I went to a session by Sophia Collins the event organiser of IAS, which gave me my first experience of a live-chat (with scientists participating in the Australian version). However, that live-chat was full of correctly punctuated sentences, good grammar and correct spelling.

This is not how the actual chat sessions go, they are frenetic, highly charged and typing naturally has to suffer (I'm still recovering now!); it's quite a rush, and there's really no time for Google. The kids see science as a subject, rather than a huge array of different disciplines, so despite being in the Genes Zone, we could expect questions on the age of the universe, the big bang theory and whether the world is going to end in 2012 was common place. If you didn't know an answer, you just needed to say so, because 'scientists' don't know everything. Fortunately I grew up as an insufferable astronomy geek with Carl Sagan and Richard Feynman books permanent residents next to my bed, so nothing I couldn't really handle.

So I entered the competition, and scientists were selected by pupils, teachers and a representative of the Wellcome Trust on the basis of:

A once sentence strap-line describing their work: "I run a fitness gym for bacteria, the 'Gene Gym', to see whether being resistant to antibiotics actually makes bacteria unhealthy."

A sentence describing the benefit to young people of IAS: "It gives first hand experience that you don't need to 'have all the answers', you just need the skills to go about 'finding any answer'."

Having been selected, and confirming I still wanted to do it, I received a welcome orientation pack in the post, and then sat back and waited for all the questions to start.

We got some great questions, but the most challenging and non-sequitur questions were generally in the chatroom sessions; it's a shame they couldn't be archived. However, of the submitted questions I liked some of these questions from my zone:

What is the meaning of life? (starting with the easy ones)

Do scientists get the girls? (A question I mis-understood on my first read)

How can chopping off a male dogs balls stop it from peeing as much?

What scientist do you think has contributed most to genetics?

There were some pretty oddball questions:

What did the Romans eat?

Hey Jimmy. What would you have as a more preferred pet? a) jelly fish b) spitting lama c) albus casius barbra percyvale woolfred vile dumble door d) lord voldemort e) tom mevarlo riddle f) ronald billious weasly g) a ppink unicorn rairy beast

Some questions were really well articulated:

You said that your 'Gene Gym' helps you to see whether being antibiotic resistant actually makes bacteria unhealthy. . Firstly, what inspired you/gave you the idea of calling it that? Could you please explain the gym concept of it all? Secondly, I do know that there are bacteria which don't do any harm, but could you please name a few and their functions?

We weren't limited to our own zone either, students and scientists could freely comment on answers in other zones. This was a great opportunity to engage with the students again, and get answers to questions you'd asked them in your answer.

In the second week evictions started. Each day students cast their votes and the scientist with the fewest was evicted. Harsh. The first scientists left on Tuesday, and two more on Wednesday and Thursday, leaving just two of us on the final Friday (and yes, I was one of those final two).

When Friday came I had two chat sessions booked in. The first was relatively quiet, due to technical problems, but the one on the afternoon was MAD - the winner was to be announced during the course of the chat and the atmosphere in the chatroom was exhilarating, the kids were clearly very excited (and the scientists hands were melting with the speed of typing).

After a long chatroom drum-roll, the news was in: I WON the Gene Zone! Thanks to all those pupils who voted for me, it made may day!

As for the winnings, I'm hoping to put these towards running some experiments with the two schools who put a great effort into participating with IAS, and whom I had regular chats with. I'll look forward to going in and talking more about my work, finding out their perceptions of science, answering more questions and running a practical microbiology experiment with them.

I'll be in London this Thursday (30th June) for an after event reception at the Wellcome Trust, so look forward to meeting some of my fellow scientists there.

Another account of my experience can be found on a guest post at NewScientist's Big Wide World blog.

YOU may have missed the fact that today was a World Health Day devoted to antibiotics; if you hadn't, then it is, or at least was. In any case, it's more or less over now and the issue can sink into the din of background noise.

As Frank Swain put it in in his well researched, and typically pithy, Guardian article today:

Health experts have been ringing the alarm over antimicrobial resistance for so long that it seems to have become part of our collective background noise, like the endless rasp of waves on the shore. And like stupid tourists, we sleep in the sun while the tide comes in.

I have to say that a little pithiness is warranted, because if we find ourselves still in this situation in 2021, I'm going to be either, a) A disgruntled cash-strapped senior lecturer / reader / professor with a serious Cassandra complex; b) long since departed from research due to lack of funding; or c) dead, or missing a limb, due to an untreatable bacterial infection, or grieving over the same in a loved one.

I've written previously about some of the reasons we don't have new drugs, and we can keep re-stating these issues til the cows come home, but it doesn't mean anything will actually change. The broad response of governments following the ReAct meeting in Stockholm last year was more words, then an eerie silence. Similarly, in a meeting of the British Society for Antimicrobial Chemotherapy (BSAC), bylined 'The Urgent Need', more words were said amongst people who already familiar with those words, following which there was also been an eerie silence.

There is a lot of pussy-footing around, and needless to say that governments need to put money where their inordinately large mouth is and start paving the way for incentives for drug discovery. In the meanwhile, what can we lowly researchers do? Well, we can actually get heavily involved in drug discovery ourselves, and on this subject I have several of my own pithy points to make:

1. Natural compounds are a way forward. As I have said before what are needed are truly novel chemicals; bat-shit crazy, funky, out of the box new chemicals developed by willing, well-funded and eager researchers. There are drugs out there that are irrational, don't conform to Lipinski's rule of five, are probably a little precocious, but amongst them could be gems. Our lab is now actively engaged in this process, and this area will be further aided by metagenomic approaches, which allow us to mine for genes of a far greater number of organisms than we could ever culture in a laboratory. I liken it to Googling for a search term, rather than buying a book and searching that for any given term. It is, however, still worth developing new culture techniques to grow previously unculturable bacteria, because there may be useful chemical products that they produce - perhaps when grown in the presence of its community members - that we can't really identify via metagenomic approaches. I'll be writing more about the specifics of these approaches in coming posts.

2. For well over a decade people have lamented that fact that all the antibiotics we have are targeted at a handful of bacterial physiological processes, i.e. interfering with DNA, proteins, a couple of metabolic pathways, or the components of the wall that bacterial cells use to keep their insides from becoming their outsides. Thus, a large number of labs invest much time and effort in identifying new physiological targets for drugs. This is all very well, and very academic, but my retort has always been, 'What drugs?' This is the crux of the matter; it's all very well having new targets, but if you have no drugs, what good is that? What we need are drugs, and what we already have are a set of awesomely good and pretty well characterised targets. So let's just go fill the arsenal full of drugs that hit those targets we have shall we, because by my clock we've got 15 years even if we know the target and find a new drug tomorrow; if you want to add a new target on to that, you may as well add another 5-10 years to that tally.

3. The 'Analogue Age', so defined by Prof Tony Coates as the age when the original set of natural compounds were modified slightly (to make an analogue) to overcome bacterial resistance to them, may yet be useful for the refinement of some older antibiotics. Chemical technology has advanced apace, and there may be scope to take very old antibiotics such as colistin, which I'm assuming was one of those drugs Frank referred to as, 'our most clumsy, brutal antibiotics', and reduce their toxicity. Likewise, there are moves afoot to consider ways of taking some of the successful anti-MRSA drugs and re-tasking them to Gram-negative bacteria by developing ways to get them past the intrinsic (i.e naturally occurring, rather than acquired) resistance Gram-negatives have to many drugs.

4. The current model for drug discovery is towards drugs that interfere with actively growing bacteria, however, bacteria aren't always actively growing. I've written before about how being in a different growth-phase can render a bacterial cell resistant to antibiotics. This can lead to repeated flare-ups of the infection until, eventually, true genetic resistance evolves that allows the bacteria to survive, and continue growing in the presence of the antibiotic. Thus there is the proposal that as part of enhanced efforts in drug discovery, that a platform for developing drugs at slow- or non-growing bacteria be practised.

4. The most common effect we find when testing a new chemical agent is that it it disrupts the bacterial membrane that envelopes the cell, a very important component of any cell. Whilst this can be a good thing to see in bacteria, such agents often also do the same in human cells. The lesson here is that it's not hard to find drugs that kill bacteria, the trick is to find drugs that kill bacteria, and not us. Thus, how useful are continued announcements of new drug discoveries where the membrane-damaging activity of the drug against human cells (which, as I say, isn't good a good thing) has yet to be established? To researchers in the field, unless you've demonstrated that your compound isn't cytotoxic to human cells, irrespective of how awesomely good it is at killing bacteria, perhaps delay the press release? On the flip side, drugs that are no good for our needs, due to their toxicity, can be quite interesting to cancer researchers who may then look to see whether the toxicity is more pronounced on fast-growing cancer cells and thus become viable chemotherapy leads for them.

5. There's always someone who, at this point, mentions phage-therapy, and how there is a big conspiracy in the west to not work on the technology. Phage therapy is a nice idea in theory, but in practice it has many flaws. The Russians aren't stupid, if phage therapy could have been made to work effectively, they would have done so; the reason they worked with phage in the first place was because they didn't have access to antibiotics. The fact that we went with antibiotics, rather than phage, following their discovery is testament to their effectiveness. A few choice anecdotes of successful phage-treatment does not mean it is a wholly viable technology; there has yet to be a full double-blind, placebo controlled clinical trial of systemically delivered phage therapy for the treatment of systemic infection. Phage may, however, have some utility as additives to topical wound dressings, much like silver impregnation is currently employed, and this may improve wound healing.

6. Antimicrobial peptides. Again, nice idea, relatively easy to work with and derivatise. However, much of the utility of antimicrobial peptides is built on the premise that little or no resistance to them has been seen. This simply isn't true, and given that antimicrobial agents are an important component of our own innate immune system, the idea of genes conferring cross-resistance to our own antimicrobial peptides worries me; this will be the subject of my next post.

So says Brooks Hatlen in the Shawshank Redemption, and this is never more true than in research science. Ask any professor over 50 yrs old what they achieved in their thesis; some may have sequenced a few base-pairs of DNA, others perhaps purified a single protein or a virus. I don't mean to disparage their achievements by any means, for these were the cutting edge of physical achievements with the technology of the day. However, such things can be achieved within a month of starting a PhD these days.

One thing that I actually quite enjoyed when I first started working in a lab was having periods of time, almost always of one hour, when I could take time out to read, plan the next step of an experiment or chat with colleagues, without any guilt. A typical day may have begun with a flurry of activity, setting up a gel to separate fragments of DNA or proteins, or setting a culture of bacteria to grow. Then would come 'the wait', the much sought after eye of the storm; this was part of the process of doing science, and there was little I could do to speed things up.

Fast forward a decade and all these little one hour time-outs are effectively gone, at least if you want to keep up. Now is the age of the 'kit', the packaging of methodologies that even a monkey could use. Let me illustrate this a little more descriptively with examples of standard laboratory cloning from my old lab books:

Restriction digest: use of a particular class of enzymes that cut DNA into specific fragments.
Time taken: (1999) 1 hour; (2011): 5 min.

Gel electrophoresis: use of an electric current to separate fragments of DNA (or proteins) on the basis of their charge.
Time taken: (1999) 1 hour; (2011): 15 min.

Ligation: use of a particular class of enzymes that join together fragments of DNA.
Time taken: (1999) 1 hour - 14 hours; (2011): 5 min.

Transformation: the process by which you introduce DNA into (in my case) bacterial cells.
_Time taken _: (1999) 1 hour 32 min; (2011): 5 min.

Cell growth: after 'transforming' cells, you allow the cells to grow on a nutrient rich growth medium until you see colonies. You then take a colony and grow this in a liquid growth medium until you have enough cells to play with.
Time taken (_E. coli_): (1999): 16 hours, then another 16 hours; (2011): 6.5 hours, then 4 hours.

This means that assuming I have all the materials I need, and all goes according to plan, I can clone a gene within a long working day, rather than the three days that was my recod in 1999. I have to say, I am still sceptical of enzymatic reactions that are done in 5 minutes, even though they work. This is perhaps quite human, we have a certain temporal encoding in our brain that processes are expected to take a certain amount of time, as if we can imagine the little enzymes chugging away like a steam engine; of course biochemistry in living cells takes place at unbelievable speeds.

I appreciate that even in 1999 there were ways of speeding up the above steps, especially if you were willing to take a loss of efficiency, or your lab was flush with enough cash to get early versions of the new molecular biological tools available, but the main thrust of my rant/lament is, where have my one hour slots gone?

I now have little excuse to ever step away from the bench, and I have to actively force myself from the bench if I want to analyse some results, or plan the next step, or dare I say - WRITE. The idea of the preparative process of lab-based science going from 90% of my day to about 10% of my day leaves me with cold sweats.

Sometimes I do feel like Brooks Hatlen, with young hot-shots zooming by, getting protein crystals and structures in months with their high-faluting high-throughput work-flows, where I was unable to in years.

I'm not bitter though, I love the new technologies, but they leave me more tired than the old ones!

N.B. This post was sponsored by a piece of older technology that STILL requires a one hour incubation.

In a convoluted intertwining of procrastination, stalling for time on several posts that I have been meaning to finish (but haven't, though I will) and blatant self-plagiarism, I'm re-posting a post I wrote on my old blog this time last year.

However, in an attempt to include something of at least momentary interest for readers, I'll preface it with two fantastically insightful articles on the subject of writer's block, with which I am currently suffering.

The first is a classic, and frankly I'll be surprised if you haven't read it, 'The unsuccessful self-treatment of a case of "writer's block".' Published in the Journal of Applied Behaviour Analysis (1973), the author, Dennis Upper, has this to say on the subject of writer's block:

As if such rousing and laudable prose on the subject couldn't be bettered, five gentlemen tenured at universities spanning the globe, wrote a deft follow-up article; published in the same journal in 2007, here the authors present, 'A Multisite Cross-Cultural Replication of Upper's (1974) Unsuccessful Self-Treatment of Writer's Block'. Lest you have concerns regarding the technical content of the paper, I have summarised it here:

Now, an ode to my block, on getting work past 'my' editor...

"IN this blogging age, we are all writers and self-publishers, bypassing the need of a publishing house to pit our meagre words to the scrutiny of all. Yet being the publishers we are, we must recognise that as in all publishing there are also editors.

Of course, each blogger is their own editor, though some may not reflect on this fact as they wilfully abandon great tomes of partially masticated drivel into the blogosphere. Others understand that the passionate cause for which they write, their gleeful discourse of some worthy note, must be married carefully with well nurtured words. These words, which are hunted down and tamed temporarily, often fidgeting and squirming in the embrace of their neighbours, must all stand before the judicious honing and pruning of the editor's critical vision, lest an anarchic cankerous sentence betray your message.

I work in a field in which my work is submitted to academic editors, and similarly I edit the work of others. However, my cruellest and most harsh editorial scrutiny is saved for my own work, and herein lies the problem.

I am my own worst enemy; I find myself, as a writer and publisher, in a battle of wills with my own editor - me. My editor seems to be overly concerned with this publisher's impending need to secure an alternative means of funding, and thus for the last month has accepted only written work for applications to achieve that aim.

This blog, this means of communicating my interests and what would otherwise be my tacit discoveries, may as well be Nature or the New Scientist for all the luck I'm having getting pieces past my editor.

In 2010 2011 I will start afresh, be less self-critical, and will try to post some of the 20 draft posts I've left to languish and die an ignominious death on the editor's desk."

Science content will commence very soon; the author is currently too busy reading real manuscripts and being hounded by automated mailing systems of various journals demanding return of said manuscripts. The author only wishes that more articles could exercise the brevity of those cited above.

Well gosh, I take a leave of absence to establish my Gene Gym (now established) and there seems to be a bit of a brouhaha with the departure of some wonderful people from NatNet. I wish them all the well with their new residence, Occam's Typewriter, and hope sincerely that teething troubles do not stunt their involving commentary on life and the universe; though it must be said that Occam himself would have found a typewriter most perplexing given his pre-dating the technology by some 600 years.

I will be staying put on NatNet of course, after all, I've just shifted a great deal of heavy equipment in (overly long blog posts), so it seems a shame to depart before saying a lot more about bugs and drugs in due course. You should really see the lard-arse on one of my MRSA strains, totally useless couch potato; if only his Special Forces cousins weren't so mean in their own environment.

Now, the title of this post has nothing to do with blogging networks (or bacterial fitness actually), but rather social networks closer to home, the confederacies of postdocs. I work in a faculty with a strange and varied history. I arrived here ahem-years ago to work in what was then the 'Division of Microbiology', which was later subsumed into the School of Biochemistry and Molecular Biology, later the School of Biochemistry and Microbiology. The other schools in the faculty included Biology, Biomedical Sciences and Exercise Sciences. Now the old schools are no more and we are a faculty of institutes, bringing together disparate labs formerly of the distinct schools into more specific research areas.

Throughout this process, I've always found it remarkable how well the individual small social networks of researchers have managed to resist the chopping and dicing of the faculty and remain as cliquey as ever. This is why, when recently invited to a faculty get together, it was with some trepidation that I finished work (all be it 90 mins after the 5 pm advertised time - seriously, do many postdocs actually finish at 5 pm?) and wandered on my lonesome to the pub for festivities.

What I was greeted with was about 26 postdocs, split into four tables, each of which were thoroughly uninvolved with each other, but for the efforts of one gallant soul buzzing between them. Needless to say this is an underwhelming turn out for a particularly large faculty. There is a basic rule of such get-togethers - mix things up a little. Case in point: at a recent conference I attended, everyone was assigned to random tables for eating so that we don't do what ALL labs do, which is to stick together; and do you know what, it was excellent, I had some fascinating conversations with people whom (I will admit) I would never have chosen to talk to.

As I figured 90 mins was quite enough time to have mixed things up, I didn't stick around long as I lacked the enthusiasm to break down the clique barriers. Those of you who have met me know I'm not afraid to blabber away on most subjects under the sun, nor to wander into a big city and start talking to strangers, but this is generally on the understanding that there is a critical mass of other people wanting to do likewise.

Perhaps I shouldn't find it amazing that after ten years working here the core culture of this place hasn't changed, and yet why should it? The splicing and dicing of academic departments is entirely a bureaucratic exercise, so why should I be surprised that people carry on doing what they've always done, which invariably results in everyone getting what they always got - not a great deal of social cohesion.

It always puts my back up somewhat, because if groups of scientists aren't talking to each other - and they speak the same language - then what hope has the public, but for the efforts of a philanthropic few?

I have been working to create a new strain of S. aureus with a few interesting properties, however, the little cooling fan in my brain is about to spin off its hinges, my CPU is running at 95%, and I'm beginning to admit to myself that I may not be able to pull this off.

There are of course alternatives to the particular approach I'm taking, there always are, but this route would have been SKILL. I have been playing an ambitious evolutionary chess game with some fancy molecular trickery, but as with all rather ambitious plans, this one was dependent on two strands of experimentation coming together at the same time. Like all the best laid plans, this then turned into an impossible mess and I have been trying to clone my way out of a dirty great staphylococcal-lined hole ever since.

Oh genes, genes, why do you DEFY ME!? Don't you know what's good for you? Please don't kill the intermediary host before I get the chance to put you into your intended host; I've made a nice little space for you, you'll like it, I promise, but once there you will also be ENTIRELY UNDER MY CONTROL.

Gym report card:

Name: Staphylococcus
Surname: aureus
Performance: Could do better.
Grade: D-

Normal service resumes soon now that the thrum (please let this be a word) of teaching and supervision that comes with each new academic year now idles to a distant, but still slightly annoying, hum.

Last week I had a little moan about some rather common, annoying, but not insurmountable sticking points in my type of research. As I mentioned, they concern the construction of plasmids, the rings of DNA that molecular biologists often use as tools to introduce new genetic material into a bacterium, many of which often come with instructions, but occasionally without a thorough list of ingredients. For me, they're one of the vehicles for introducing fitness challenges to my bacteria; think of them as barbells in the gym, but without the weights added yet. I need to standardise my barbells, so I can measure the fitness cost of each 'weight' (i.e. genes that have a fitness cost) I add.

Here's a typical schematic of one lab's plasmid construction. I should add that these guys got it dead right; this particular set of plasmids come from the lab of Nev Firth and Ron Skurray in Sydney, and being like-minded chaps, they provide comprehensive instructions and full sequences! My point though, is that many others don't.

In this image you can see some fairly typical components you find plasmid tools: Cm and Ap are antibiotic resistance genes, so this makes the bacterium containing this plasmid resistant to ampicillin and chloramphenicol. ColE1 ori and rep are used as the start point for the plasmid to replicate itself; in this case it can do it in two organisms, E. coli and Staph. aureus (we call such a plasmid a shuttle vector, as it allows us to transfer material between two rather different bacteria). These also have something called par, which is basically involved in ensuring that when the bacterial cell divides, that a copy of the plasmid ends up in each cell.

Yes, I know what you're thinking. It takes me some working to get through this schematic too, and I've been doing this for a decade.

Imagine you want to use the final products, i.e. one of the two plasmids at the bottom; you might want to take one of those plasmids and perform a similar jigsaw puzzle yourself, adding in a few other DNA pieces from other plasmids you have. Then imagine if you didn't have the sequence data to hand (it happens). Instead of sitting down with a coffee and piece of paper for an hour (or three) to plan your jigsaw puzzle, you'd have to track down each constituent fragment of DNA, see if there were any sequence data for it, and then use a computer program (though once we did it by hand) to go through the steps of the various 'cutting and pasting' steps until you have what you hope is the complete sequence of the plasmid. You can test this by using the sequence to tell you where particular DNA-cutting enzymes should cut, and then test that in the lab to see if the pieces of DNA that are left are the size you expected them to be.

Of course, one of the most important things about having DNA sequence data is that you can then check them for various genetic codes that may cause problems with your experiment. I could labour my barbell metaphor into the ground, but I'll spare you, needless to say various seemingly benign sequences can cause instability in plasmids if you're not careful. This is not something you want to discover after you've just cloned 40 individual genes into the plasmid.

So welcome to my molecular jigsaws, but we'll leave this for now and in my next post move on to some interesting recent findings.

As I've stupidly taken on one too many manuscripts to peer-review, as well as currently managing the arrival of some 500 new residents at the halls of residence where, for my sins, I happen to be the warden. Normal services will resume soon, but until then I offer only this rant this week. .

I imagine that historians find nothing more vexing than when they need to refer to primary written research material, only to find half of it is missing. This means they have to search around for other sources to fill in the gaps, before they can actually do the work that they wanted to do based on the original text.

I'm sure this can be fun for most, like a jigsaw puzzle; but sometimes you need the information, all of it, now. I find myself in the same situation again, faced with this pet peeve old foe. In bacterial genetics, DNA sequences are king. There was a valiant effort, as early as the early 80's, to get sequence data for various genes, but the prohibitive cost and limiting technology often meant that there were large gaps.

Over the past two decades sequencing technology has advanced exponentially, such that we can now sequence the genomes of complex organisms in less than a week, and at a fraction of a cost of only five years ago.

So why is it that I am continually faced with gaps in my source material, where no sequence data are available? I could understand if these were esoteric DNA sequences in obscure organisms, but the sequences I'm after are are those of plasmids that have been worked with for many years. Plasmids are naturally occurring rings of DNA that can encode multiple genes that supplement the suite of genes encoded by a bacterium's own genome. They're also indispensable tools for molecular biologists that allow us to monkey around with the physiology and chromosomes of bacteria.

There are commercially available plasmids that are fully characterised and sequenced, but many labs have their own plasmids, constructed over the years and passed from lab to lab with lots of bits added and taken away. Tracking down the blueprints of these plasmids is a puzzle that can be spread over 15 years of scientific articles.

The easy way to solve the puzzle is to send the plasmid to be re-sequenced, but sometimes they're really rather large, and sometimes you need the sequence NOW. I'm currently 'gene building', curating a suite of different resistance genes in different permutations, all of which come from different genetic backgrounds and are going into different plasmids. These are some of the genes I'll be using to exercise my bacteria in the gene gym. In order to isolate them all, I need to know the sequences so I can precisely engineer them.

This was always a perennial headache when I was doing my PhD and first postdoc in this area, one which I forgot about for a while when I stepped out in to the glittering world of bionanotechnology, where all sequences were known. Yet, here I am again piecing together the sequences of plasmids from old plasmids, and short scraps of sequences from databases, that someone, somewhere, really should have compiled and deposited in a public DNA database already. I guess I'm just being impatient as I'm keen to get these bacteria into my gym, but it's also a bit slack that many researchers make use of such tools for years without ever sequencing anything more than the genes they work on, and not the genetic background.

Killing avoidance strategies

A couple of recent research papers remind me that I promised to talk a little about a phenomenon by which bacteria can avoid being killed by antibiotics, without actually being resistant in the classical sense, i.e. they can't actually grow in elevated concentrations of the antibiotics they survive, and those cells that do survive give rise to populations that are no more, or less, likely to survive next time.

The first paper comes from the lab of Prof. Tony Coates at the Centre for Infection at St. George's, University of London. Prof. Coates has for a long time been heavily involved in research into the treatment of latent and persistent infections, most notably T.B./tuberculosis. His research team (as indeed are mine) are trying to understand why some antibiotics that kill actively growing bacteria of a particular species have no effect on cells of the same species that aren't actively growing; almost akin to the bit in Jurassic Park where T. rex kills the lawyer who's running, but wouldn't have done had the lawyer stood still ^§.

One of the reasons for this is that, historically, most drug discovery has been focussed on targeting actively growing cells, but what we are increasingly finding is that persistent infections can be mediated by a recalcitrant population of slow-growing or non-growing cells.

Whilst the idea of targeting non-growing bacteria is not a wholly new idea (you can find a review on the subject by Prof. Coates in my 'Further reading'), it does seem that together with the report's first author, Dr Yanmin Hu, their spin-out company (Helperby Therapeutics) has developed a platform and proof of principle drug that is now in trials, demonstrating the utility of such an approach. They have identified an antibiotic compound that has potent anti-Staphylococcal activity, but importantly, acts specifically against non-multiplying cells.

In a second paper, brought to my attention by Ed Yong, the Collins lab in Boston has identified that a sub-population of super-resistant bacteria act in a charitable manner to other members of the colony that are less resistant. Whilst the super-resistant cells could satisfy their own selfishness by merely allowing all their less-resistant siblings to die out, the bacteria in this case have a mutation that maintains their production of indole (a signalling molecule) when normally its production would be shut down on exposure to the antibiotic. When released by the cell, indole stimulates non-resistant cells to enter a state of phenotypic resistance or 'antibiotic survival', even though continued-production of indole incurs a fitness cost.

Why might they do this?

Well, for one of the reasons that is very much the subject of my blog, bacterial fitness. As I have mentioned before, antibiotic resistance can have a fitness cost, which means that cells committing themselves to this 'path of resistance' may find themselves at a disadvantage come the time when the antibiotic is no longer around. The subject of my research is to document the various ways in which antibiotic resistant Staph. aureus mitigate these fitness costs so that they get to remain resistant and just as competitive as they ever were in the absence of antibiotic. It seems that in the case of the Collins' lab's charitable bacteria, they may mitigate the fitness cost of antibiotic resistance at a population level by maintaining the presence of non-resistant cells that can come to the fore once the antibiotic is removed.

"These few drug-resistant mutants, by enhancing the survival capacity of the overall population in stressful environments, may also help to preserve the potential for the population to return to its genetic origins should the stress prove transient. Efforts to monitor and combat antibiotic resistance are complicated by these bet-hedging survival strategies and other forms of bacterial cooperation."

So what I want to do is briefly introduce the types of 'antibiotic survival' strategies seen in bacteria. It goes without saying that future drug discovery that targets the molecular/physiological underpinning for these strategies ( once we've identified what these are! ) will be important for the clinical management of infection.

Resistance or 'killing avoidance'?

I've discussed in a previous post what I might describe as mechanisms of antibiotic resistance, i.e. producing a enzyme that modifies or chews up the antibiotic; or changing the component of the cell so that the antibiotic targeted to that component no longer has any effect, or pumping the antibiotic out of the cell before it does any damage.

It was recognised early on, in the heyday of antibiotics, that penicillin could kill most bacteria in a culture, but could not sterilise a culture. This has been observed with numerous antibiotic compounds, thus at a practical level you cannot achieve a 100% kill with antibiotics. Now this isn't generally a problem for a healthy individual, as it is at this point the immune system takes over and clears away the remaining cells. However, many people receiving antibiotics aren't well, they may be immuno-compromised, or suffering from a deep-seated infection. The persistence of a bacterial infection becomes a perfect breeding ground for classical antibiotic resistance, with each resurgence of the infection from surviving cells increasing the probability that resistance may evolve; and thus is thought to play a significant role in the failure of antibacterial treatment.

1. INDIFFERENCE. Bacteria can avoid being killed by being in a stationary phase (non-growing or metabolically inactive). This is actually the default repose of bacteria in the environment, only submitting to bursts of growth in the presence of nutrients. Most current (and old) antibiotics are specific to the particular cell components and processes of actively growing cells, there is no reason to expect that such antibiotics would have any killing effect on cells not engaging in these processes.

2. TOLERANCE. Those antibiotics that do kill bacteria don't necessarily do so directly; they initiate a series of events, a cascade of physiological responses, which ultimately result in cell death. Unlike indifference, tolerance is not linked to the growth/metabolic state of the bacteria, but instead result from genetic changes that uncouple the killing activity of the drug from its inhibitory activity. In the clinic, tolerance seems to be specific to certain bacteria, and even then only in response to particualr antibiotics targeting the bacteria cell-wall.

3. PERSISTENCE. In a bacterial population there exists a sub-population of 'persister', cells that regardless of the growth state of the population as a whole, continue to exist in a stationary or growth-retarded state. It may be that persisters avoid antibiotic killing in the same way that indifferent bacteria do, but whilst there are some antibiotics that can kill indifferent cells, they don't kill persisters; this suggests that something different is going on in these cells, and there is increasing evidence to suggest that there are defined genetic differences implicated in persistence, including changes within the stress-response pathways, but what these are (and what they do exactly) remains to be seen.

4. BIOFILMS. Finally, and most stubbornly, there is the issue of biofilms. Biofilms are like a condominium (or halls of residence) of bacteria, a structured environment where the bugs are surrounded by a gelatinous matrix of sugar chains and many other macromolecules. They are involved in some 80% of human infections and represent a major cause of antibiotic treatment failure. Within the matrix the bacteria avoid antibiotic killing through indifference and persistence, thought to be brought on by the low oxygen and low nutrient environment; the matrix also provides some protection from certain classes of antibiotics, as well as the immune system. Even if a large number of matrix surface cells are killed off, the matrix structure survives and can be re-populated by the surviving cells. For some bacteria the biofilm environment stimulates them to massively increase their rate of mutation, which can increase the rate at which antibiotic resistance can evolve.

So what do we do?

Well again it comes down to idealism versus pragmatism. The current system of drug discovery is fraught and inefficient enough without an additional burden of esoteric and poorly understood mechanisms of bacterial antibiotic survival. I do think there is some merit in drug discovery targeted at non-growing indifferent bacteria, this is particularly important in the treatment of T.B. The problem is going to be that many of these killing avoidance strategies differ between pathogens and between the particular environment in which they're found, and also that in the absence of any ongoing preventative treatment, such as potential vaccines, by the time an infection manifests itself the antibiotic survival systems are likely to already be in place.

Other than indifference, biofilms are a system worth addressing in the immediate term. We have amassed a huge amount of data on biofilms, and demonstrated that they are of great clinical importance, thus efforts should be made to increase the number of biofilm busting compounds we have available.

Many people are familiar with antibiotic resistance, but I'm interested to hear (especially from other biologists) how much people knew about such antibiotic survival strategies. Also, as ever, please feel free to ask questions at any level. This (rather long) post barely touches the surface of this subject, there's plenty more to be said!

^§ The theory that T. rex would only 'see' moving objects is probably a little outdated.

Further reading

As always I will try to find open access material where available, and will update those references that aren't as and when they do.

Hu et al. (2010) A New Approach for the Discovery of Antibiotics by Targeting Non-Multiplying Bacteria: A Novel Topical Antibiotic for Staphylococcal Infections. PLoS ONE 5: e11818.
[ Open access ]

Coates, A. et al. (2002) The future challenge facing the development of new antimicrobial drugs. Nature Reviews Drug Discovery 1: 895-910.
[ Free pdf ]

Lee, H. et al. (2010) Bacterial charity work leads to population-wide resistance. Nature 467, 82-85.
[Sorry, article behind a paywall] - You can read Ed Yong's post on it though.

Levin, B.R. and Rozen, D.E. (2006) Non-inhertied antibiotic resistance. Nat Rev Microbiol 4: 556-562.
[ free pdf ]

- A very useful grounding to the subject of phenotypic resistance, as it was understood back in 2006.

Lewis, K. (2010) Persister cells. Annu. Rev. Microbiol. 64: 357-72.
[Sorry, another paywall paper ]

- Good review of bacterial persistence.

This Friday I will be getting up horrendously early in the morning to catch a train to London. Here I'll be meeting the many wonderful and varied people who are attending Science Online London 2010, this time at the British Museum Library. I went along to last year's event in the Royal Institution, and thoroughly enjoyed it. It gave me an opportunity to put a face (and an accent) to most of the bloggers I'd been reading for some time, and meet a huge number of new faces. Also like last year I be at the wonderfully informal FringeFrivolous rooftop debate.

For my own part, I have always been enthusiastic about communicating science. As to whether I succeed at this (or not) in my writing I have absolutely no idea, but then again, as I am not a professional science communicator, it never quite seemed to matter. However, I do love to talk about science more. On the conference circuit I fair pretty well in giving talks, but outside of academia I have bent the ear of many a friend, and enjoyed the rare opportunity to hold the rapt attention of roomfuls of school children, or humanist societies. These make me feel great!

However, given the thematic nature of this blog and my mission being to keep the issue of bugs and drugs in the public domain, it's time for me to put a more concerted effort into up-skilling on communicating 'as a scientist, to the public' (whether I get paid for it or not). Of course, we're fortunate to have the likes of Maryn McKenna (the journalist and blogger behind 'Superbug', the book and blog) who hits the subject with her inimitable journalistic verve, but I can't say I've encountered many scientists in the field giving their perspective.

I began this blog off the back of the NDM-1 story because whilst representative of an ongoing serious issue of multidrug resistant bacteria, it comes at a crucial time in the budgetary planning of future science funding. The recent media furore focussed on the lack of antibiotics (in between over-hyped commentary on cheap-ops in India), but failed to mention any of the reasons why most big industry doesn't want to touch antibiotics with a barge pole (economic in-viability), or any of the academic involvement in resolving this problem (needless to say that it is the efforts of academic labs that we even know we have a problem!)

So what of it? Between 1995 - 2001, the team at one of the remaining biggest players, GlaxoSmithKline, spent $14m for each drug 'lead' identified over a period of 7 years (they identified 5 'leads' in that time); remembering of course that a 'lead' may well never make it through clinical trials. One of the potential quick-fix options was screening of the pharmacopoeia, the pharmaceutical back-catalogue of drugs developed for everything from bone-loss to cardiac arrhythmia, for new leads. Companies have spent millions assaying such compounds, and derivatives thereof, to see if they can also hit a bacterial target. However, they haven't yielded the kind of hits that were hoped. One of the reasons cited for this is that whilst the majority of therapeutic drugs obey Lipinski's 'rule of five' (a set of molecular properties that define a good therapeutic drug), antibiotics don't follow these rules.

Image from Payne et al. (2007) Drugs for bad bugs: confronting the challenges of antibacterial discovery. Nature Reviews Drug Discovery 6: 29-40 [ link to pdf ]

Thus what are needed are truly novel chemicals; bat-shit crazy, funky, out of the box new chemicals developed by willing, well-founded and eager chemists.....but where are the chemists? Hell, where are the chemistry departments?

You might wonder that such problems are incumbent in all drug discovery, but then most 'lifestyle' drugs, the preferred pot of gold for big industry, have a relatively long shelf life. Not so antibiotics: most infections don't last long, thus people don't need many packets; physicians are told (correctly) to reserve their use only for bacterial infections (not colds/flu); moves are afoot to ban/scale back agricultural use = not much return for their investment. Then, just when the chips are down, resistance to your new drug is observed in the clinic and the death knell of the drug's usable life is sounded.

Physicians and industry also want to treat a 'disease', not a multiplicity of different infections; but how do you treat a disease like pneumonia with a single antibiotic? Pneumonia can be caused by any of eight completely different bacteria (or multiples thereof), some species of which have as much in common with each other as a human does with a paramecium [ pdf ]. There is a fundamental disconnect between the popular models of drug development and the issues at hand with antibiotic development.

Of course, if industry sees no economic viability in antibiotic drug development, we need to sweeten the deal; one way to do this is offer public money. I hear you gasp, but think about it, this is a global issue and treatment programmes for HIV/AIDS, TB and malaria are the great success stories of what happens when public support, political clout, industrial partnership and a huge pot of cash collide. Of course, in these days of pharma-bad / dolphins-good, no-one is going to throw any public money at industry. One of the best accommodations is for partnerships between industry and academia. These are already a mainstay in microbiology (because if we relied on research council money we certainly wouldn't still be here!), but it actually relies on the academic partner still being here.

Industrial partners can out-source a great deal of the legwork to academic partners for relatively low cost, increasing the network of players scanning for new drugs, informing new directions and finding. However, I can already run off a list of ten names of highly trained researchers in this field who have left the field over the past 8 years. At this time we're facing another budgetary crisis in science funding and we must, at all costs, prevent a further loss of both industrial and academic research capacity involved in antibiotic research.

The public needs to be aware of the strong part academic research will have to play in developing new courses of action in the advent of new 'superbugs'. For all the hope I have that there are academic solutions to better strategies of antibiotic use, anticipating and preventing resistance, better surveillance, and identifying new drug targets in bacteria (or better approaches to known targets), these are all rather like architect's plans when what we need right now are some bricks in the oven.

Look forward to seeing everyone at Science Online 2010

There was once an idea that we could address the problem of antibiotic resistant bacterial strains by simply removing the ailing antibiotic from clinical use. In the absence of selective pressure it was thought that the evolutionary traits that make a strain resistant to an antibiotic would actually put the strain at a competitive disadvantage compared with a strain that doesn't have such antibiotic resistance; the hope being that one day you could then go back to using the old antibiotic again. The basis of this argument is, at least superficially, quite sound:

1. As mentioned in my last post, one way bacteria can become resistant is via mutation of the cellular component that is attacked by the antibiotic; these components usually being those involved in DNA structure/function, protein synthesis or maintenance of the cell wall. By changing their shape slightly, these components stop themselves being a target. However, many components of the cell are the shape they are for good reason, it's because they have an essential day-job to do. By changing shape, they may be sacrificing their ability to do their day-job as well. So whilst they survive the antibiotic attack, in the absence of the antibiotic they don't do as well as their ancestral forms that weren't resistant to the antibiotic.

2. Another mechanism of resistance is to acquire a fully fledged 'resistance determinant', a gene (or suite of genes) encoded on some form of moveable DNA. However, this can be likened to being given a heavy backpack/rucksack to wear. Sure, you can keep extra things in it, including a stout stick for beating antibiotics to death, but it can be something of a hindrance once that stout stick has no more antibiotics to whack.

I should point out that (in the first example above) it appears that a cell produces just the right mutation in order to resist the antibiotic, which suggests that it is the antibiotic inducing this mutation. Whilst this may occasionally be true, what in fact is more likely is that these mutations already exist in the population, even if just a couple of cells have them. With the introduction of an antibiotic, all those without the mutation are killed, leaving only those with the mutation to re-populate. So when someone like me comes along and breaks open the bugs to see what made them resistant, the majority have this mutation. Likewise with the second example, only a few cells may have received a horizontally transferred piece of DNA encoding resistance, but as these cells are generally the ones that survive, this is what we see when we look more closely.

In any case, it seems that being resistant to an antibiotic may not be the free-ride people expect it to be. This sounds promising, suggesting that we could just ditch the antibiotic and wait for the bacteria to become sensitive to it again, which was where I began.

However, some years ago evidence started appearing that suggests reducing the use of a given antibiotic doesn't necessarily result in a loss of resistance to that antibiotic. Although in some circumstances the number of infections caused by a particular antibiotic-resistant pathogenic bacteria (in a hospital environment) are fewer upon switching to a different class of antibiotic (an approach not wholly without merit), it is not a universally the case, and is also reliant on good infection control practises in the hospital.

What we may be seeing is that, to a greater or lesser extent, the success of antibiotic cycling is dependent upon the degree of fitness cost associated with any given antibiotic resistance determinant. This suggests that (idealistically) when looking at antibiotic drug development, any antibiotics that incur a strong fitness cost on any bacterial resistance are those that will enjoy greater clinical longevity. I say 'idealistically' as the current paucity of available drugs means we can't be actually afford to be choosy, but knowledge of the fitness cost either way can allow us to anticipate a greater or lesser likelihood of drug resistance occurring in some drugs, and thus employing them strategically.

So what about these fitness costs...

When we start looking at the fitness costs in bacteria, we find that the growth environment (whether it be flask in the lab, a wound, or growth within a biofilm on medical equipment) has a profound effect on both the degree of fitness cost and the manner in which bacteria compensate for these fitness costs. It's therefore important to look at the response of bacterial resistance in different environments.

In the lab we can routinely generate highly resistant populations, but this usually comes with a high fitness cost, and so rapidly reverts to an antibiotic sensitive form when we remove the antibiotic. However, in strains associated with clinical epidemics, we see that such strains are resistant, but crucially with little or no fitness cost. In some cases these these resistant bacteria may even be fitter than their ancestral forms, even in the absence of the antibiotic. This was found to be the case with with a mutation conferring resistance to ciprofloxacin (a fluoroquinolone) in the pathogen Campylobacter jejuni ; in this situation the rapid emergence of fluoroquinolone resistance may actually be a result of enhanced fitness associated with the resistance.

When resistance has a cost, bacteria can indeed compensate with additional genetic 'nips and tucks', usually without the loss of resistance. So what sort of 'nips and tucks' might a bacterial population undergo in order to maintain a battery of costly genes, but which may provide an ongoing advantage? Well, this is the subject of much ongoing research, in fact, it's the subject of my research. Several mechanisms may include:

• In the absence of selective pressure, costly genes are simply 'silenced' - a molecular mechanism that prevents a gene from being 'switched on'. There are numerous mechanisms for this, but in horizontal acquired DNA fully fledged resistance determinants may arrive with proteins that actually regulate the determinant. In my own organism, Staphylococcus aureus (specifically MRSA), the fitness cost of vanA-mediated vancomycin resistance is very high when it is active (but of course such cells still have the advantage over sensitive cells when there is still vancomycin around), but once the vancomycin diminishes the resistance is effectively switched off, markedly reducing the fitness cost.

• Another mechanism is to mutate the target again, thus where the first mutation changed the shape to make it resistant to the antibiotic - at a cost of reduced ability to do its day-job - a subsequent mutation elsewhere may restore the day-job activity whilst still retaining the resistance.

• Related to the above mechanism, when a gene mutation makes a protein product that functions less well, the cell may accommodate by increasing the amount of that protein by duplicating the number of gene copies. This overcomes the apparent lack of activity, whilst also increasing the number of copies of a gene that are now subject to random mutation, until one such mutation improves the fitness of that gene. The same is true of genes present on plasmids, which can exist in multiple copies in a cell.

• Sometimes antibiotic resistance determines are acquired along with a suite of other genes that encode for other useful functions, so called gene linkage. Whilst the resistance component itself may constitute a fitness cost, the enhanced fitness associated with the other genes mitigates the fitness cost, permitting the resistance gene to remain.

These are just a few examples of compensatory mechanisms, the means by which bacteria get to have their cake and eat it too. Understanding both the costs of antibiotic resistance to bacteria, and the means by which they mitigate such costs, can provide important clues as to how we should manage antibiotic chemotherapy. It can help us form a basis to anticipate the occurrence and stability of resistance by any given bacteria, in any given growth environment. Also, by identifying high fitness costs in particualr growth environments, this may reveal behavioural or physiological weaknesses of bacteria that could be additional drug targets.

Thus, the sad fact is that reservoirs of drug resistance determinants may remain in clinical populations and become an integral, rather than an occasional, feature of many bacteria species.

Further reading:

European Commission Heath Research: PAR - Predicting antibiotic resistance. - This is the research programme within which I work. I will write more about this in due course.
[ Webpage: PAR ]

Andersson D.I. and Hughes, D. (2010) Antibiotic resistance and its cost: is it possible to reverse resistance? Nature Reviews Microbiology 8: 260-271
[ Sorry, full text is behind a paywall ] - a key review in this area from the co-ordinator of the above research programme.

Andersson, D.I. (2006) The biological cost of mutational antibiotic resistance: any practical conclusions? Current Opinion in Microbiology 9: 461-465
[ Sorry, full text behind a paywall ]

Brown, E.M. and Nathwani, D. (2005) Antibiotic cycling or rotation: a systematic review of the evidence of efficacy. Journal of antimicrobial chemotherapy 55: 6-9
[ Free full text] - meta-analysis of several other studies on antibiotic cycling.

Text books commonly state that in the natural environment antibiotics are a means by which bacteria (and yeasts) reduce competition for resources, by creating a 'zone of inhibition' around themselves; kind of like unleashing a smelly fart to stop people sitting too closely. However, antibiotics can also be seen as part a more complex system of cell to cell communication/signalling in microbial communities, in fact, they can also be food. When used at the concentrations we employ therapeutically, they can either stop bacterial growth, or kill outright. Just because they can have this effect, doesn't mean that this is what they evolved to do; 'antibiotic' is simply the name we give to the few (of many) small organic molecules produced by bacteria that happen to have an effect on a particular group of bacteria against which it (along with many other molecules) was screened.

When present at low concentrations, small organic molecules (including antibiotics) have been found to produce a whole range of metabolic activity in neighbouring cells, stimulating surrounding cells to change their behaviour, increase their mutation frequency, or increase the transfer of mobile pieces of DNA; thus at many levels, using antibiotics incorrectly stimulates exactly what we don't want to happen - it gives bacteria a kick up the arse, gets them talking and sharing information, including resistance to one or multiple antibiotics. The resistance can therefore be seen as an adaptation of a mechanism by which a bacterial cell can control its exposure to these small molecules, including antibiotics.

I mention this because it is a perspective that makes us think a little differently about the problem we're trying to solve. Without an understanding of the subtleties in the war of drugs and bugs, we're not going to get far. So for this post I just want to do a quick foray into the bugs and drugs background and mention something about the cocktail of conditions that we need to watch out for.

Antibiotics, and resistance to them, is not a new thing...

For a billion plus years there have been penicillin-like molecules out there, and for an equal amount of time there have been mechanisms to resist/control the effect of the same antibiotics. Numerous studies have shown that even a cursory screening of garden soil reveals a plethora of resistance determinants (genes or mutations that confer resistance), many of which may have not been (or may never be) documented in a clinical setting.

It's probably useful, before I go any further, to distinguish between several forms of antibiotic resistance as generally speaking bacteria can become resistant to antibiotics in several ways, which are not altogether mutually exclusive:

i. They can evolve an enzyme (usually co-opted from a pre-existing family of enzymes present within the original cell where the gene appeared), these either modify or cut up the antibiotic molecules;

ii. Another form of resistance occurs when component of the of the cell that the antibiotic normally attacks becomes mutated, these can often result from one or several mutations of the gene encoding that component;

iii. Some bacterial cells can exist in a form that is impenetrable to antibiotics, by surrounding themselves in a sticky goo (biofilm); or they can pump out (efflux) the antibiotics from the cell just as fast as they enter.

iv. As I will discuss in a coming post, some members of a bacterial population are simply not perturbed by antibiotics, yet not via a genetic or heritable mechanism, although one form of such 'killing avoidance' is the formation of biofilm, as in (iii).

Just to confuse matters, in some cases the resistance described in (ii) and (iii) can also be spread. There are examples of genes encoding pumps that can be transferred from one bacterium to another. Furthermore, mutated genes that are immune to the effect of an antibiotic, as described by (ii), can also be spread. One example from my own field is resistance to an antibiotic called mupirocin in Staphylococci spp. Mupirocin, which can be useful for treating some MRSA infections, attacks a crucial enzyme that makes a type of RNA needed to make proteins. Bacteria could become resistant if they mutate the specific enzyme that the antibiotic disrupts, but they can also acquire an alternative copy of this mutant enzyme from another cell. Many of the enzymes from (i) may have started life on a chromosome long ago, but have since taken up residence on segments of DNA that can be moved around, which I will come on to now.

The worrying cocktail...

So resistance determinants of many flavours are out there, but several factors need to be in play before we start worrying about finding them in an untreatable infection.

1. There needs to be ecological contact, a shared contact between the source of a gene encoding resistance, and the pathogen. Often there is no such link, but there is one place at least for which such contact is primer territory, sewage outlets. Here human pathogens can share an environment with environmental reservoirs (free-living environmental bacteria) of natural antibiotic resistance, or indeed, not so natural antibiotic resistance (where resistance has evolved due to improper use of antibiotics in agriculture). However, just because bacteria share the same space, doesn't mean they will share genetic information. This is the next factor.

2. Promiscuous mobile genetic elements. In addition to the chromosomal DNA, which is the large molecule of DNA in every cell that encodes the blueprints of that organism, there are also small(er) pieces of DNA that can move around. Sometimes they just move around within the chromosome - snipping themselves out and then inserting themselves somewhere else. Other times they can be autonomous, i.e. in control of their own destiny and taking care of their own replication. Some of these DNAs can move themselves from one organism to another; some can move themselves and other small bits of DNA that happen to be around. In this way, a fully evolved mechanism of resistance to a particualr antibiotic can be inherited, and established, within a bacterial generation (a matter of hours).

This is called horizontal gene transfer, as opposed to vertical gene transfer, which is how your parents passed their DNA to you, or when a cell divides to produce daughter cells. Horizontal gene transfer in humans might resemble you placing your hand on your cousin's shoulder and inheriting their hair colour (though presumably this isn't going to improve your chances of survival). Of course, over evolutionary history we have been subject to horizontal gene transfer, with numerous human genes being derived from viral genes.

3. Multidrug resistance. Mupirocin resistance, which I described above, is limited both in terms of what other bacteria it can spread to, and is also unlikely to confer cross-resistance to related antibiotics. However, some resistance determinants, such as NDM-1, confer resistance that can be readily employed by other bacteria, and confer resistance to numerous antibiotics.

Thus what the NDM-1 report (raised in my last post) describes is a heady cocktail ripe for troubled times:

Ecological contact + promiscuous mobile genetic elements + multidrug resistance = not good. These tick all the boxes for a situation that should be carefully monitored, as you would any invading pest species in a zoological sense.

NDM-1 is of course not the first determinant to raise concerns in this manner; within my own neighbourhood of bacteria (the Staphylococci), a resistance determinant identified in 2008 called 'cfr' was found to mediate resistance to several different classes of antibiotics, including Linezolid - a purely synthetic and important anti-MRSA antibiotic. It also be found on a plasmid, one of those potentially movable bits of DNA I mentioned, thus we might anticipate its propensity for spreading, though how much time this will take is anyone's guess. I'll discuss this further as part of a later post on surveillance of antibiotic resistance.

The future...

In the early days of antimicrobial chemotherapy we devoted a lot of time to identifying compounds that work, with often very little understanding of actually how they work. Over time we identified the (comparatively few) principle cell components that antibiotics attack (structure/function of DNA, protein synthesis, cell wall/membrane integrity; and at least one metabolic pathway), but even so there are still numerous compounds in use for which we don't have a clear molecular mechanism for their action, even if we know that they broadly interfere with.

A later development was an understanding of how bacterial resistance mechanisms work, their broad functioning being as I described earlier, but again there is much work to be done to understand the molecular mechanism behind it. Mechanisms of action and mechanisms of resistance are both areas focussed on in the lab where I work.

There is a great need to develop new antibiotics, of that there is no doubt. Part of the frustration in the the field of antibiotic chemotherapy is that we don't have a huge array of drugs to play with, and this severely curtails our options. I don't doubt that somewhere out there, there are chemical compounds that can do the job, some may even be sat on shelves as undeveloped test compounds because we don't have a the developmental infrastructure that makes their bringing to market (economically) worthwhile; others, are currently being held up by technical licensing issues and legal contests between discoverers and developers, such as with oritavancin , which showed potent activity against Staphylococcus aureus in biofilms.

We already employ strategies to improve the lifespan of antibiotics, using them as cocktails with other antibiotics, monitoring and isolating patients with resistant bacteria (I'll discuss these in later posts). However these are complicated because antibiotic chemotherapy is complicate, rife with side-effects and strict dosing regimes, and can be quite expensive. For such reasons, these strategies are not universally practised (worldwide) for socio-economic reasons, and such strategies in any case tend to be reactive (being employed only in serious cases, or when complications arise) rather than pro-active. If we're not working to the same strategic plan worldwide, then we will fall foul of the formula I've just discussed: Ecological contact, promiscuous genetic elements and multidrug resistance.

Without continuing investment in research to understand each of these processes, trying to solve the problem of antibiotic resistance will be as hard as trying to play Subbuteo wearing boxing gloves.

I will of course continue to expand on these themes in coming posts, but I'm sure you're all itching to know what this all has to do with bacterial fitness, and gene gyms; well next time...

Further reading:

I thought I'd name and fame some of the big names working in the field whose research I will be discussing, and point you in the direction of material that isn't behind a paywall (where possible).

Yim et al. (2007) Antibiotics as signalling molecules. Phil. Trans. R. Soc. B (2007) 362, 1195-1200 [ link to pdf ]

- Julian Davies is a mine of information about antibiotics and developed the thesis of antibiotics as signalling molecules.

Wright, G.D. (2007) The antibiotic resistome: the nexus of chemical and genetic diversity. Nature Reviews Microbiology 5: 175-186 [ link to pdf ]

- Gerry Wright has written some fascinating articles on the evolution of antibiotic resistance. You can listen to him giving an interview on U.S. National Public Radio (NPR) following the discovery that rather than being killed by antibiotics, some soil bacteria actually eat them.

Payne et al. (2007) Drugs for bad bugs: confronting the challenges of antibacterial discovery. Nature Reviews Drug Discovery 6: 29-40 [ link to pdf ]

- From the Infectious Diseases Centre of Excellence for Drug Discovery at GlaxoSmithKline, this is a sobering read on the learning outcomes from seven years of focussed, high-throughput antibiotic drug discovery, detailing the scientific and technical challenges to drug discovery.

I have been remiss, once again taking a protracted break from this blog. I was essentially forced, by economic factors beyond my control, into taking quarter of this year off work. I used this time to re-assess, travel, reflect and generally think about myself and my own directions; time I’ve not taken since 1999, which was careless of me. Whether I can consider myself rested enough within three months is neither here nor there, for I am back, or at least I will be back, but in a somewhat modified form.

One of the big problems with maintaining a science blog where I wander through a range of subject matter is that it does rather take my eyes away from my day job, and you only need read a few science blogs to realise the the day job of a jobbing postdoc is rather full of teaching, supervision, research, reading, writing and banging ones head against a wall. Thus, whilst I was increasingly thankful for this distraction in my last research position, I have recently joined a new research team who are doing some interesting and meaningful work; I will be taking the advice of Daniel McArthur of Genetic Future who at Science Online London 2009 said that one way to succeed at both blogging and your day job is to fuse the two. To that end, this is what I will be doing, obviously not giving the game of our own research away, that would be silly, but instead talking about the field in general.

So what field? Well, my new position sees me returning to my roots; I started out in Staphylococcal molecular genetics a decade ago and spent my most formative doctoral training years working in a rather academic aspect of horizontal gene transfer. It was then, and still is (though not for long) a highly under-studied area of research that couldn’t possibly be more important with what we are coming to understand about the nature of antibiotic resistance, evolutionary microbiology and the human microbiome. I am now working in an area that brings together several recurring themes of interest for me: evolution, horizontal gene transfer, bacteria (Staphylococcus aureus to be precise) and antibiotics research, specifically the evolution of bacterial fitness.

This is exciting times for me, and I can’t tell you how many fantastic papers I’ve already discovered from my period of absence in this field; people have been busy, and I intend to talk about it. However, much of what I have to say won’t necessary be in this blog, for I will be moving. This blog will remain of course, as part of my efforts to regale you with edifying titbits.

As for where I will be moving to, well I now have a blog over at the Nature Network, called ‘The Gene Gym‘.

I will not be closing this blog down, it will be a repository for all those science stories and titbits that don’t fit in with my Gene Gym remit.

Thanks for reading.

“FROM the perspective of a bacterium, higher eukaryotes are oversexed, unadventurous and reproduce in an inconvenient way.” So says Pål Johnsen and Bruce Levin in their commentary of today’s article for discussion, and nary a truer word said. Of course, one may state that inconvenient as reproduction may be, bacteria clearly have no sense of fun.

There was once an idea that we could address the problem of antibiotic resistant bacterial strains by removing the ailing antibiotic from clinical use. In the absence of selective pressure it was thought that the evolutionary traits that enable the strain to resist the antibiotic would actually put the strain at a competitive disadvantage compared with a strain that doesn’t have such antibiotic resistance. The proposed cause of this? Fitness costs – these are imposed by a resource-expensive set of mutations, or carriage of alien DNA, that make the resistant strain compete less well once its non-resistant brethren are no longer being killed off by the antibiotic.

However, some years ago now experimental evidence suggested that this is not always the case; it may in fact be often not the case. It is worth mentioning at this point that it has been shown that in some circumstances ^(alt) the number of infections caused by a particular antibiotic-resistant pathogenic bacterium have become fewer on reduction (or removal) of the antibiotic to which that strain is resistant, but to assume this would be the case with all strains/antibiotics is naïve.

It is true, with few exceptions, that initially both plasmid and chromosomally encoded resistances result in fitness losses. However, when resistance has a cost it is possible for compensatory mutations in a cell to ameliorate these costs, usually without the loss of resistance. The type of compensatory mutations that mitigate the fitness cost of acquiring antibiotic resistance, or any other incoming DNA that encodes potentially useful genes, will depend very much upon the environment in which the bacteria finds itself. These include the availability of resources, i.e. the growth environment of the bacteria, the environment of the genes (mobile or chromosomal), or whether the genes are being selected for by an external factor, such as the presence of antibiotics in the case of resistance genes.

So what sort of ‘nips and tucks’ might a bacterial population undergo in order to maintain a battery of costly genes, but that may provide an ongoing advantage? Well, this is the subject of much ongoing research; one example indicated that, in the absence of selective pressure, costly genes are simply silenced – a molecular mechanism often found in higher organisms that prevents a gene from being ‘switched on’. Thus a reservoir of drug resistance determinants may remain in populations that have compensated for their presence, remaining ‘inactive’ until a selective pressure removes the silencing.

A recent study by Peter Lind (et al.), a grad student working in the lab of Dan Andersson at Uppsala, Sweden, addresses a particularly pertinent question of compensatory mutations: those associated with genes acquired by horizontal gene transfer (HGT). HGT bypasses the slow and haphazard process of evolution (via random mutation, selection and recombination) by offering an opportunity for bacteria to receive fully fledged genes encoding pathogenicity factors (genes that make bacteria better at causing disease) as well as genes that encode resistances to disinfectants and/or antibiotics, amongst others. There is no doubt that such incoming DNA may pose significant fitness costs, so Lind et. al. set out to quantify the nature of compensatory mutations on such incoming DNA.

To do this they replaced three core genes encoding ribosomal proteins (key components of the protein-making factories of the cell) in Salmonella typhimurium with orthologues (genes performing a similar function) from various other microbes; some closely-related, and some very much not. The aim was to replicate receipt of DNA by HGT processes that can occur naturally in the environment, but here they used genes that form conserved parts of multi-protein complexes that perform a central role in the cells. These genes actually represent rare targets for HGT, but in these experiments the aim was to force the cells to make compensatory mutations to stabilize them (even if they’re poor substitutes) just to survive. Also, by studying proteins that are implicitly involved in the growth of the cells, they are able to link the stabilisation of these genes to the bacterial growth rate, the latter being a relatively easy property to measure experimentally.

So what of compensatory mutations? Lind points out, ‘as with any mutation, most transferred genes are likely to be neutral or deleterious in an alien cell, and over time become mutationally inactivated or lost.’ Alternatively, even if performing weakly, the gene may persist and its function can be fine-tuned by compensatory mutations to reduce fitness costs and improve gene function. This fine-tuning may take a number of forms: it may be achieved by multiple DNA basepair mutations, each resulting in slight improvement; or alternatively a phenomenon called ‘gene amplification’, which is relatively common, may result in a large number of extra copies of the gene.

This would have two outcomes:

a) It would compensate for the protein not doing its job very well, by making more of it; and,

b) provide a large number of extra copies of the DNA, each of which will be subject to mutation, so increasing the rate at which the gene can be ultimately improved.

In Lind’s (et al.) experiments, the Salmonella populations bearing costly genes were subjected to cycles of serial passage, where a small culture of bacteria is grown for 24 h, then diluted 1000-fold into fresh growth medium. Each strain producing the alien ribosomal proteins grew significantly more slowly than the aboriginal population, with the most distantly related proteins exacting the greatest fitness cost, as one might expect. At points between 40 – 250 generations (a bacterial generation being the time taken to double the population), the initial cost of carrying the foreign genes was mitigated. Populations of these improved growers were subjected to DNA sequencing to identify changes to the DNA sequence, as well as to techniques that test the relative abundance of a particular gene and corresponding protein.

What the researchers found is that compensation for fitness costs was achieved by increasing the amount of ribosomal protein several-fold, which was achieved by the cells duplicating the region of DNA encoding the alien genes by 100-fold. Thus Lind et al. show that by increasing the dosage of suboptimal genes, and thus proteins, gene amplifications can compensate for the fitness cost of carrying alien genes; and as mentioned earlier, may be subject to increased mutational frequency that can further refine their activity.

In this study the researcher focussed on how compensatory mutations affected a specific set of alien ribosomal genes. However, there are additional secondary genetic ‘nips and tucks’ that can happen elsewhere on the chromosome, within other genes or regulatory processes, that can compensate for fitness costs imposed by newly introduced genes. For the above set of experiments such mutations were controlled by pre-adapting the Salmonella strains in the experimental (growth) environment, prior to introduction of the alien genes. Echoing Pål Johnsen and Bruce Levin in their commentary, I think it will be particularly interesting to follow up this work with studies on how bacterial populations confront the costs associated with truly novel genes that have no corresponding genes in the host, e.g. those encoding antibiotic resistance.

____________________

THIS is the first of three posts about bacterial antibiotic resistance, given that I will hopefully be returning to research in this area. In the above post I essentially pointed out that bacteria can have their cake, and eat it too. In my next post I will talk about an alternative strategy that could be taken to prevent the spread of antibiotic resistance, and in a final post I will talk about how bacteria can (and do) avoid being killed by antibiotics without encoding a defined resistance mechanism in the conventional sense.

—

Lind, P., Tobin, C., Berg, O., Kurland, C., & Andersson, D. (2010). Compensatory gene amplification restores fitness after inter-species gene replacements Molecular Microbiology, 75 (5), 1078-1089 DOI: 10.1111/j.1365-2958.2009.07030.x

On Monday 19^th April a fantastic resource was re-launched. Sporting a shiny new website and a community of eager biologists, Ask a Biologist (AAB) is a site you should visit if you have any burning questions about biology. I have supported such sites for many years, originally as part of MadSci.org, but I am pleased to now give my time to AAB. Of course, biological sciences covers a huge range of disciplines, and as such the scientists who volunteer their time and experience to answer your questions provide expertise from a broad range of backgrounds, from medical sciences, microbiology, molecular biology, to ecology, marine biology, palaeontology and several specialist areas of zoology.

Ever wondered whether bacteria think?
Why a compost heap gives off steam?
Have you taken a picture of an insect you would like to have identified?
Are you confused about what evolution is all about?
Want to know how to become a biologist?

These questions, and many more, are typical of those we get asked. Often you will receive more than one answer to a particular question, especially if it is a complex question. This reflects the nature of science and the perspectives that different scientists can bring to a solve a problem. If there is no clear answer to your question, perhaps because the answer is not yet known, then we will point this out, and provide an answer based on our informed opinion.

When you arrive at AAB, you can search the site with your question to find out whether it has been asked, and answered, before. If not, then ask away and a biologist will get back to you very soon. One note however, we don’t do student’s homework assignments, and we’re pretty good at spotting homework questions, being teachers, lecturers and life-long students ourselves. If you have been told to go and research something, then asking a professional biologist for the answer really doesn’t count as research; it is in the process of finding things out that we learn more about a subject.

Ask a Biologist, tell your friends.

IN a strange series of events, today the Dutch firm Impossible BV announced that they have released a new B&W polaroid film that will fit traditional Polaroid cameras. This is great news, and they will be following up with standard colour Polaroid film soon.

It was exactly a year ago today that I first heard the news that Polaroid were ceasing producting of Polaroid film, and I set out to both acquire an old Polaroid camera, and stock up on film. I have two packets left, both of which are now out of date (which may yield some interesting results). I’ll be taking them on my travels next month.

After 17 months of research and development, The Impossible Project announced that it succeeded in its task of re-producing a new analog Instant Film for traditional Polaroid cameras. Containing more than 30 newly developed components, Impossible today introduced a new, monochrome Instant Film – the PX 100 and PX 600 Silver Shade – and is therewith saving millions of perfectly functioning Polaroid cameras from becoming obsolete. [pdf - 556 kb]

I’ve had photography on the mind today, having taken the day off to get the beginnings of my photographic portfolio up online (also available via the ‘Gallery’ tab above), and working on a new series of textured images. These images were from a fantastic visit to Skye, also this time last year, where I’d taken a macro shot of some Gabbro rock, incidentally my favourite igneous rock (because I have a favourite igneous rock), to use as a texture. This provides an additional link between the landscape images and the geology of the area. A nice fusion I think.

I will be selling these images at a very reasonable price once I have a convenient PayPal button set up, and have trialled the giclée printing I need for some of them.

Blog update:

This blog has been a little low on posts recently, which is bizarre as I’m staring at 12 browser tabs containing some fantastic papers – I will write some of these up in a research highlights format in the next day or so. Alas, as this is my last week of research work on this contract, I’ve been too busy wrapping things up in the lab.

I will (hopefully) be returning to do some research starting in May, on the evolution of bacterial fitness, but until then I’m also planning a lot of travelling, visiting old friends around Europe: I’ll be off to Czech Rep. (Prague, Karlovy Vary), Germany (Dresden), Iceland (Reykjavik), Belgium (Brussels, Gent, Geel) and Italy (Brescia), before finishing up at my annual Old Boys reunion with University of Wales friends in Snowdonia and on the beaches of Anglesey.

I’ll be posting pictures from the road – most likely on my photoblog over at The Overflow (also available in a tab above).

Hopefully I’ve given you a bit of procrastination fodder, but until I return to my regular science slot, add me to your RSS feed and you’ll see when I periodically resurface from my peripatetic sojourns.

Now for some embarrassing photos…

Reposted from 22nd March 2009…

POLAROID Corp will be ceasing to manufacture Polaroid film by the end of the year, and stopped making its commercial instant cameras a year ago. These were iconic cameras, and I have fond memories of the really bad photographs that they took.

Ok, ok, Polaroids aren’t so bad, in fact they’re kind of kitch and quite cool; but they should be bad, with their lens aberrations, wacko colour rendering, and emulsion streaks. No matter; in an era where the most stunning photographic reproduction of the real world is possible, the abstract, aberrant, wacko colours of Polaroidography deserves its nostalgic nod, much in the same that Lomography deserves its dues.

So I’ve gone and bought a Polaroid 636 CloseUp camera, not an expensive vintage as it’s only 13 years old, which I intend to take on my travels this year, until the (rather expensive) film expires.

Of course, if you’re willing to be accused of not being a traditionalist about it, it is possible to achieve the same effect using some fancy Photoshopping. Here I’m using the fantastic Polaroid Generator photoshop (Action) macro by rawimage.

The above pictures were photoshopped using the Time Zero render (Time Zero was a type of medium-speed general use film for the classic Polaroid SX-70).

One of the reasons I love these type of images is because the family photo album was always full of similarly poor photographs, but they captured the days, and my childhood, perfectly. My parents also lived out in Zanzibar, Tanzania, in the early 70s, and I was always fascinated by the grainy, stained old polaroid-type images of white sand and blue sea.

The above are originals, inserted into the neater photoframe of the polaroid generator. I’ll post some more in due course.

THERE you are, stood in a green grocers; you’re poring over your favourite variety of apple, but suddenly you catch the scent of something heavenly, a smell not unlike the apple you have in your hand, only better somehow. You abandon your apple and follow the scent to the next aisle where you find more apples, yet seemingly of the same variety. They smell rich, verdant, superior to the others. You pick one up and are compelled to take a bite; on doing so you realise something – it’s pretty bloody awful. You put down the unpalatable apple and move on to alternative apples.

I could be describing a situation reminiscent of our selectively bred, spotless, brightly coloured, sweet smelling fruits that line our supermarket shelves, but which in fact taste like a pallid, watery, tasteless facsimile of the original spots-and-all varieties. In this situation we are being manipulated by the supermarkets, but in nature it may be viruses doing the manipulating.

Viruses are parasites, making use of infected host cells to replicate more virus. Of course, it isn’t enough just to replicate, viruses also need to spread to new cells, and new hosts. Plant viruses are often carried from plant to plant by insects; the insects become known in this context as ‘vectors’. The study of the biology of insect vectors is, as you may imagine, fundamentally important to understanding the transmission of a whole range of parasites (viral, bacterial and protozoan) between plants, or between humans and animals. Of particular interest is how parasites, such as viruses, manipulate their insect vectors by altering the physical properties of the host they infect.

A Penn State based group, headed by Mark Mescher, have been using Cucumber Mosaic Virus (CMV), a known generalist plant pathogen, to study the effect it has on the interaction between cultivated squash plants and aphids (sap sucking bugs). The results of this study are reported by Kerry Mauck et al. in a recent paper.

They show that CMV-infected plants have elevated volatile (readily dispersing in air) emissions that attract aphid vectors. This in itself is not a revelation;  the authors cite two well documented examples of this phenomenon, from Potato leaf roll virus (PLRV) and Barley yellow dwarf virus (BYDV), where infected plants release volatiles that attract aphids. However, these other viruses employ a different method of transmission to CMV, and the main thrust of this paper is to identify how the mode of transmission modifies the host-insect interaction.

Viruses carried by insect vectors fall into two broad camps:

Persistent – in which virus from an infected host enters an insect vector, but needs a certain incubation period within the insect, usually allowing it to travel through the body cavity and locate itself in the salivary glands of the insect. In this position, the virus can ‘persistently’ infect new hosts each time the insect feeds. This mechanism takes time, with the insects required to feed for an extended period before the viruses can establish themselves in the insect vector.

Non-persistent – in which virus from the infected host literally sticks to the mouth parts of the feeding insect. This is a precariously exposed position for a virus where its transmission relies on the insect vector dispersing, within minutes, to a new host and introducing the virus to it.

Whilst there is a growing number of studies on the disease ecology of persistent viruses (including PLRV and BYDV above), comparatively little is known about that of non-persistent viruses (such as CMV study). Non-persistent viruses also represent a majority of plant viral pathogens, and thus have economic and agricultural significance.

With the persistent viruses (PLRV and BYDV), the infected plants are not only more attractive to aphids, they’re also more nutritious; the viruses actually improve the quality of the plant hosts. This fits their persistent method of transmission, encouraging the aphids to feed for much longer until the virus establishes a sufficient load in the aphids. The aphid population thrives and only begins to spread when the population is too large to be supported on one plant.

In Mauck et al.‘s study, they found that squash plants infected with the non-persistent virus CMV were actually poor hosts for aphids. CMV-infected plants sustain a lower population of aphids than healthy plants, and the winged variety of one of the aphid species they tested rarely colonised infected plants.

This too makes sense in the context of the non-persistent nature of CMV. The virus needs to be transferred rapidly from host to host, thus the aphids are attracted by the released volatiles that promise a good meal on a healthy plant; however, upon arrival, and after a quick probe – which is enough to pick up some viral passengers – they discover a rather unpalatable sickly plant from which they readily depart.

The researcher’s work therefore supports the hypothesis that the mechanism of virus transmission is a major factor shaping the evolution of pathogen-induced changes in the host plant. The authors state that ‘such studies also will facilitate the development of pathogen-management techniques that target vector transmission’. This is in recognition that insect pests and plant pathogens cannot be managed in isolation; such problems are best managed with an understanding of the complex evolutionary interactions between pathogen, host and vector.

To my mind it is interesting to speculate what the two modes of transmission mean for plant mortality. Whilst the viruses all attack the plant cells in slightly different ways, eventually leading to damage or death of the plant, the issue of aphid colonisation cannot be ignored. Aphids cause considerable plant damage themselves, and plants infected with persistent viruses support large populations of aphids compared with plants infected with non-persistent viruses. To what extent does the reduced aphid population – which is itself low enough that a single predator could gobble-up the who population – result in a prolonged stay of execution? To what extent does acquiring a persistent virus result in catastrophic damage by a huge aphid population?

As a further note of interest, the authors cite several interesting examples of insect attraction via volatile cues from infected hosts:

• Sandflies (a blood sucking fly) are more attracted to hamsters that are infected with Leishmania, the protozoan parasite they’re known for spreading, than uninfected hamsters.
• It is thought that Kenyan children who have the transmissible form of the malaria parasite, P. fulciparum, attract more mosquitoes than those who are uninfected, or have the non-transmissible stages of the parasite.
• Elm trees infected with the fungal pathogen that causes Dutch Elm Disease is more attractive to bark beetles that uninfected Elms.

I, for one, would like to know why it is that Scottish midges love my blood so much more than anyone with I travel in Scotland with; not that I’m suggesting that there is a virus involved, but there may be a volatile chemical cue?

—

Mauck, K., De Moraes, C., & Mescher, M. (2010). Deceptive chemical signals induced by a plant virus attract insect vectors to inferior hosts Proceedings of the National Academy of Sciences, 107 (8), 3600-3605 DOI: 10.1073/pnas.0907191107

UPDATE: This post is also available in Estonian – ‘Muru ei ole alati rohelisem…‘, translated by Anna Galovich

The following is an excerpt about the current interplay between science and the media, taken from an article in this week’s Nature by Colin Macilwain:

…thanks to the massive growth in public relations and to online media’s insatiable appetite for ‘content’, journalism in science, as in other spheres, has evolved into an ugly machine — called ‘churnalism’ by media-watcher Nick Davies and others. This machine delivers inexpensive and safe content, masquerading as news, to an increasingly underwhelmed public.

The machine prospers because it serves the short-term interests of its participants. Editors get coherent and up-to-date copy. Writers get bylines. Researchers, universities and funding agencies get clips that show that their work has had ‘impact’. And readers get snippets, such as how red or white wine makes you live longer or less long, to chat about at the water-cooler.

None of these groups is benefiting strategically from the arrangement. Science is being misrepresented as a cacophony of sometimes divergent but nonetheless definitive ‘findings’, each warmly accepted by colleagues, on the record, as deeply significant. The public learns nothing about the actual cut and thrust of the scientific process, and as a result is beginning to adopt a weary cynicism that can only rebound on science in the long run.

The thing I dislike about the issue of ‘churnaled’ factoids by some ‘stamp-collecting’ journalists (and uninventive bloggers) is their complete lack of apparent criticism, a sentiment echoed in the article. Such reports come across as cloyingly positive, but then this is to be expected of PR outputs from universities, societies and research institutes who obviously gloss over the likely criticism that the work encounters in peer-review. I’m not exactly looking for open detractors, or input from ‘fringe’ scientists who think the work is a load of baloney (in the measure of undue balance that an old school journalist may take), but I would like to hear about the context, limitations and realistic direction of the work being discussed. In short, a fuller, more rounded investigative piece, with input (and synthesis) from someone besides the original PR office.

I am a scientist, and I love reading about science, but I admit to a weary disinterest occasionally when faced with yet another science news snippet. Fortunately, unlike the general public, I have access to original published article; better still I have access to the letters and comments written to the journal regarding a specific article, and to sometimes in depth editorials or previews accompanying a milestone article. Faculty of 1000 is also an excellent source of expert opinions from distinguished scientists in the field, the sort of opinions that could be presented in more thoroughly investigative media reporting. In the absence of the above, I would recommend a good science blog write-up any day, or perhaps an article in NewScientist or The Scientist.

I’m tired of seeing university press-releases regurgitated in every form possible, mostly by syndication sites with no human intermediary, such that saturation (and annoyance) point is reached within hours of such a release. To re-iterate, more investigative reporting is required.

As Macilwain’s article finishes:

Andy Williams, Cardiff University’s journalism school, survey of science writers and editors identified widespread misgivings about growing workloads associated with multimedia reporting, the rise of public relations, ‘pack’ journalism (in which reporters are obliged to cover a story because their competitors will) and the lack of time for original research on stories.

It is hard, given the parlous financial state of newspapers and broadcasters, and the continued onslaught of the public-relations industry, to see what will reverse these alarming trends. One possible approach would be the unilateral abandonment, by writers and editors on influential publications, of the embargo system and the pack mentality that goes with it. Another would be far more willing and constructive engagement by scientists themselves in the public airing of the strengths, weaknesses and missteps that characterize scientific progress.

Thoughts?

A FEW years ago a Boston University team, headed by Jim Collins, published findings that suggested the means by which bactericidal antibiotics result in cell death (irrespective of the initial cellular target of the drug) was by stimulating the production of hydroxyl radicals, which a reactive oxygen species ¹. The hydroxyl radical is known to cause significant damage to cellular DNA, proteins and cell wall, leading to cell death.

Their 2007 study ¹ was initially met with raised eyebrows in some quarters, coming in for some criticism for having a few gaps, namely whether the role of the hydroxyl radical was even pertinent in real world infections settings, which are often in the low-oxygen environment of biofilms ². There was also some question of whether it was adequately demonstrated that the oxidative stress was a source or the result of cell damage. However, subsequent studies reported by Kohanski, as well as other labs, have described a more defined link between a bactericidal drug and resulting hydroxyl radical formation ³.

In the latest edition of Molecular Cell, a new article from Mike Kohanski, Mark DePristo and Jim Collins reports that prolonged exposure to sub-lethal concentrations of antibiotics can induce multiple drug resistance in E. coli and Staphylococcus aureus strains that are initially drug sensitive ⁴. E. coli strains were tested with sub-lethal levels of  three major classes of bactericidal antibiotics (quinolone, B-lactam and aminoglycoside), and were found to significantly increase the mutation rate, confirming their expectations.

After growing cells in the presence of low concentrations of antibiotic for five days (either norfloxacin, ampicillin or kanamycin – each representing an antibiotic with a different primary cellular target), they periodically tested samples for their resistance to a range of the same, and different classes, of antibiotics. The aim was to determine whether treatment with one antibiotic could confer cross-resistance resistance to other antibiotics.

Whilst antibiotic resistance mechanisms against one drug can often confer resistance to different molecule of the same class, development of resistance to a drug molecule in a different class is less likely, and would be an important finding.

The figure (left) produced for the accompanying preview article, by Ben Kaufmann and Deborah Hung ⁵, summarises the findings. The researchers found that, true enough, growth in one of the antibiotics – most notably ampicillin – resulted in  cross-resistance to a number of the other antibiotics. In contrast, when grown in the absence of any antibiotics, only spontaneous mutants could be expected when then exposed to high levels of antibiotic. Similar findings were observed with an S. aureus strain and a clinical E. coli strain, so were not just an artefact of laboratory E. coli strains, which can be poor models for in situ clinical infection.

Interestingly, in some cases the ampicillin-treated isolates resistant to another class of drug actually remained sensitive to ampicillin, indicating that sub-lethal antibiotic exposure results in a random mutation process, rather than a selective one.

Those cells newly resistant to ampicillin did not revert if grown for several days in the absence of the antibiotic, so represent a stable mutation, rather than transient adaptation, to growth in the presence of ampicillin. The positions of the mutations conferring cross-resistance were found in the genes encoding drug targets, as expected, rather than by mutation of a general efflux pump capable of pumping out multiple drugs. However, they did identify one isolate with a mutation in the acrAB gene, a multi-drug efflux pump that is linked with reactive oxygen species responses, and they propose that the mutation may contribute to multidrug resistance; they haven’t discussed this further in this paper.

The premise is that the development of the above mutants is mediated by the hydroxyl radical causing damage to the cell DNA. If the same experiments were performed in the absence of oxygen, it would be expected that no resistance to antibiotic would be observed, which was indeed the case. Whether or not it is the hydroxyl radical that directly mediates the mutation to the genes encoding the targets of the antibiotics is yet to be demonstrated unequivocally. It may be that DNA damaged by  hydroxyl radicals initiates other cellular processes, such as the bacterial SOS response, which is known to repair DNA at the cost of introducing mutations and recombination.

In such a case, the proposed mechanism may act as an alternative means of generating the necessary genetic variation needed to dig themselves out of a tight spot. Indeed, in bacterial populations there may also be naturally occurring mutator cells that can occupy anywhere between 0.5 – 30% of the cell population.  These cells are usually defective in DNA repair, and may undergo a 1000-fold increased rate of mutation compared with the background population ⁷.

The broader implications of the study are pertinent as current recommendation for antimicrobial chemotherapy is, ideally, to use a multi-antibiotic cocktail to mitigate the evolution of resistance that can result from monotherapies; though whether or not this is widely practised is another matter. Furthermore, the incubation of these strains in sub-lethal concentrations of antibiotic is relevant in clinical practice where bacteria can commonly experience such sub-lethal drug concentrations. The dosing regime for antibiotics means that the concentration of the drug will periodically drop to a low level for short periods, or longer if there is poor patient compliance with the regime instructions. Furthermore, some areas of the body are poorly supplied with drug, receiving less than the plasma concentrations, e.g. skin, joints and prostate. Such conditions may permit the evolution of cross-resistance as described in this work.

There remains a great deal of work to do in this area however. First and foremost, it will be important to determine the clinical relevance of the hydroxyl radical mechanism in situ. One of the drugs the researchers tested, kanamycin, is an aminoglycoside;  another study has previously shown that the sub-lethal levels of another aminoglycoside, tobramycin, induces biofilm formation ⁶. For both Gram-negative and Gram-positive bacteria, subinhibitory antibiotic treatment can stimulate production of exopolysaccharides necessary for biofilm formation – and the significance of biofilms is such that it protects cells from lethal levels of antibiotics, but the low oxygen environment may make hydroxyl radicals less significant.

Furthermore, the phase of growth will be important. Does the suggested mechanism work as well on stationary cells as it does on actively growing cells. It is well known that bacteria can become ‘indifferent’ to bactericidal drugs, simply because current drugs are designed to target processes that are active only in growing cells, rather than non-growing cells. This, in fact, will be the subject of a later post.

Research on antimicrobials since the 1960′s has focussed on identifying the mechanisms by which bacteria can physically modify a drug’s structure, disrupt the interaction of drug and target, or alter the activity of transport machinery that keeps the drug away from its target. An increased research focus to characterise the downstream physiological responses of bacteria to sub-lethal, as well as lethal, levels of antibiotics may help provide new impetus to inform future drug discovery ⁸.

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1. Kohanski et al. (2007) A Common Mechanism of Cellular Death Induced by Bactericidal Antibiotics. Cell 130: 797-810. DOI: 10.1016/j.cell.2007.06.049.

2. Hassett & Imlay (2007) Bactericidal Antibiotics and Oxidative Stress: A Radical Proposal. ACS Chem. Biol. 2: 708–710. DOI: 10.1021/cb700232k.

3. Kohanski et al. (2008) Mistranslation of Membrane Proteins and Two-Component System Activation Trigger Antibiotic-Mediated Cell Death. Cell 135: 679-690. DOI: 10.1016/j.cell.2008.09.038.

*4. Kohanski, M., DePristo, M., & Collins, J. (2010). Sublethal Antibiotic Treatment Leads to Multidrug Resistance via Radical-Induced Mutagenesis Molecular Cell, 37 (3), 311-320 DOI: 10.1016/j.molcel.2010.01.003

5. Kaufmann & Hung (2010) The Fast Track to Multidrug Resistance. Molecular Cell 37: 297-298. DOI: 10.1016/j.molcel.2010.01.027.

6. Hoffman et al. (2005) Aminoglycoside antibiotics induce bacterial biofilm formation. Nature 436: 1171-1175. DOI: 10.1038/nature03912.

7. Chopra et al. (2003). The role of mutators in the emergence of antibiotic-resistant bacteria. Drug Resistance Updates 6: 137-145. DOI: 10.1016/S1368-7646(03)00041-4.

8.Dwyer et al. (2009) Role of reactive oxygen species in antibiotic action and resistance. Current Opinion in Microbiology 12: 482-489. DOI: 10.1016/j.mib.2009.06.018.

IN evolutionary theory there is a phenomenon known as canalisation, a process in which the phenotype (i.e. the outward physical appearance of an organism) remains unchanged, despite the influence of genetic or environmental interference.  It suggests that a mechanism exists to buffer the phenotype from such changes, which may ultimately explain why species can remain mostly unchanged for millions of years.

If canalisation results in an unchanging phenotype, and the buffering results in the build up of genetic variation (without affecting the phenotype), a corollary of this is a situation where all this accumulated genetic variation may be released by an event that overcomes the buffering capacity of canalisation, this is called evolutionary capacitance. By such means, fuel (in the form of variation) is provided for evolution by natural selection, which potentially accelerates evolution.

The idea of capacitance was first suggested by Rutherford and Lindquist ¹ following experiments on a protein called heat shock protein 90 (Hsp90) in fruitflies. Generally speaking, heat shock proteins assist in the maintenance and correct folding of cellular proteins, especially when under temperature stress; Hsp90 plays a particular role in maintaining the unstable signalling proteins that act as key regulators of growth and development.

They suggested that in nature, a stressing event such as high or low temperatures may overcome the protective buffering effect that Hsp90 has on maintaining these key regulators. As Hsp90 becomes diverted from its usual role due to an increase of stress-damaged proteins in the cell, those cell signalling proteins it normally maintains are free to adopt a range of altered behaviours, interfering with the development of the organism. The result is morphological variants upon which natural selection can act. Indeed, they observed as much, with chemically and environmentally compromised Hsp90 resulting in flies with abnormal wings, legs or eyes; a remarkable number and variety of phenotypes were observed.

In experimental tests Rutherford and Lindquist demonstrated that the capacity for such remarkable variation was pre-existing, i.e. it was present genetically prior to the stressing event, but had been silenced. Evolutionary capacitance may therefore provide a mechanism of adaptive evolution in which a population under stress may release previously silent variation, resulting in the appearance of certain individuals with more desirable traits in that changed environment. When such revealed traits are selected for they can become fixed and independently of the buffering action of Hsp90.

This week, in a letter to Nature, Valeria Specchia et al.² report some fascinating evidence that indicates that beyond merely acting as a gate-keeper to unleash variation, mutations of Hsp90 that compromise its functionality result in new, rather than pre-exisiting, variation. They observed that mutations in Hsp90 affect the production of piRNAs, which are small RNA molecules that are involved in the silencing of genes, particularly those involved in development, i.e. sex cells like eggs and sperm, and all the cell types that give rise to these cells. These piRNAs are also responsible for repressing genetic elements called transposons.

To understand why this is important, it helps to know something about transposons. Transposable elements, initially known as ‘jumping genes’, were first identified by Nobel laureate Barbara McClintock in maize kernals. They are essentially segments of DNA that can cut themselves out of one location, and either remove themselves to another location, or copy themselves to another location. Whilst not entirely a random process, a transposon may well jump into an active gene and disturb its normal operation; in the figure shown you can see phenotypic variance in pigmentation as a result of transposable elements jumping into, and out of, different genes responsible for pigmentation.

“Because active transposons are mutagenic, these data suggest that the phenotypic variation observed in Hsp90 mutants could be due to de novo mutations produced by activated transposons.”

Specchia et al. show that mutation of Hsp90 is directly coupled with an increase of activated transposons jumping around to other regions of DNA, and that the phenotypic mutations they saw in their Hsp90-mutant fruitflies were due to mutations produced by these activated transposons. Rather than complement the mechanism proposed by Rutherford and Lindquist – that of releasing pre-exisiting variation – Specchia et al. may have identified the source of the remarkable variation seen in earlier work by demonstrating new mutation/variability caused by the release of transposons, which have a known mutagenic effect.

Specchia et al. seemed somewhat dismissive of the evolutionary capacity hypothesis itself, stating only that the mechanisms they have described ‘also potentially provides another molecular interpretation with respect to the vague capacitor concept.’

Vague. Perhaps; to robustly demonstrate evolutionary capacitance, it will be necessary to have more evidence that overcoming the canalising mechanism accelerates evolution to a new optimal phenotype, and that the process releases pre-existing variation in the process. I don’t think anyone is dismissing the earlier work of Rutherford and Lindquist; subsequent work in Lindquist’s lab further demonstrates an Hsp90-mediated mechanism for the evolutionof new traits in the form of drug resistance in fungi ³, however in this case, rather than buffering the effects ofnew mutations, it allows them to have immediate phenotypic consequences; fairly important when trying to resist a drug that might kill you.

What such studies do show is a highly complex system of phenotypic sculpting, and numerous mechanism via which even a single protein can go about promoting the emergence of new traits, when required.

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1. Rutherford, S.L. & Lindquist, S. (1998) Hsp90 as a capacitor for morphological evolution. Nature 396: 336-342 DOI: 10.1038/24550

2. Specchia, V., Piacentini, L., Tritto, P., Fanti, L., D’Alessandro, R., Palumbo, G., Pimpinelli, S., & Bozzetti, M. (2010). Hsp90 prevents phenotypic variation by suppressing the mutagenic activity of transposons Nature, 463 (7281), 662-665 DOI: 10.1038/nature08739

3. Cowen, L.E. & Lindquist, S. (2005) Hsp90 Potentiates the Rapid Evolution of New Traits: Drug Resistance in Diverse Fungi. Science 309 (5744): 2185-2189 DOI: 10.1126/science.1118370.

FURTHER to my recent post on why people don’t accept evidence, it turns out that an editorial ¹ and an opinion ² piece in this week’s Nature, the latter unfortunately behind a pay-wall, actually focus on just this issue. The editorial states:

“Empirical evidence shows that people tend to react to reports on issues such as climate change according to their personal values (see page 296). Those who favour individualism over egalitarianism are more likely to reject evidence of climate change and calls to restrict emissions. And the messenger matters perhaps just as much as the message. People have more trust in experts — and scientists — when they sense that the speaker shares their values.”

So people tend to accept the evidence that supports their personal proclivities, and in fact interpret evidence in a manner than does so, thus people tend to persist in cherished beliefs and views even when confronted with contradictory evidence. This of course is something probably appreciated by most of us. Dan Kahan, in his opinion piece, points out:

“People endorse whichever position reinforces their connection to others with whom they share important commitments. As a result, public debate about science is strikingly polarized. The same groups who disagree on ‘cultural issues’ — abortion, same-sex marriage and school prayer — also disagree on whether climate change is real and on whether underground disposal of nuclear waste is safe.”

Another factor that weighs heavily in the public perception, and acceptance, of facts is the messenger. Owing to the fact that most people are ill-equipped to evaluate the raw data from scientific studies, they rely on the position of credible experts; it seems that those experts laypersons see as credible are those perceived to share the same values.

Research into the mental processes involved in such public perception is, Dan tells us, being conducted by Donald Braman at George Washington University Law School in Washington DC, Geoffrey Cohen at Stanford University in Palo Alto, California, John Gastil at the University of Washington in Seattle, Paul Slovic at the University of Oregon in Eugene and Dan Kahan, the Elizabeth K. Dollard professor of law at Yale Law School. These processes are collectively referred to as ‘cultural cognition’.

So what is cultural cognition? Kahan describes it as, ‘the influence of group values (ones relating to equality and authority, individualism and community) on risk perceptions and related beliefs.’ I would imagine that peer-pressure represents one example within a spectrum of influences in cultural cognition.

Two techniques are forth-coming as means to mitigate public polarization on scientific evidence:

1. Ensure that facts are presented in a manner that ‘affirms, rather than threatens people’s values.’ This in essence means that science communication should not just be targeted according to the level of scientific literacy of the target audience, but that it also be targeted in a manner that presents the information in the most favourable way to respective ‘groups’, whether they be individualistic or egalitarian. Dan Kahan:

“…people with individualistic values resist scientific evidence that climate change is a serious threat because they have come to assume that industry-constraining carbon-emission limits are the main solution. They would probably look at the evidence more favourably, however, if made aware that the possible responses to climate change include nuclear power and geoengineering, enterprises that to them symbolize human resourcefulness. Similarly, people with an egalitarian outlooks are less likely to reflexively dismiss evidence of the safety of nanotechnology if they are made aware of the part that nanotechnology might play in environmental protection, and not just its usefulness in the manufacture of consumer goods.”

It is hard to envisage how such targeted differentiation in the means of communicating scientific information would work in a practical sense; however, one would imagine that the medium in which science is communicated, whether this be a particular newspaper, magazine, society, TV channel, or political party policy research documents, will tend to have a majority demographic that reflects a particular group to whom the information can be tailored.

This sounds a little contrived, but Dan Kahan says:

‘Unlike commercial advertising, however, the goal of these techniques is not to induce public acceptance of any particular conclusion, but rather to create an environment for the public’s open-minded, unbiased consideration of the best available scientific information.’

2. A second technique is to ensure the message is conveyed, and vouched for, by a diverse set of experts from different backgrounds and different values. The editorial points out that:

‘…scientists should be careful not to disparage those on the other side of a debate: a respectful tone makes it easier for people to change their minds if they share something in common with that other side.’

Kahan’s opinion is that as straightforward as these recommendations might seem, science communicators routinely flout them. There is the tendency to think that by putting good, sound scientific information into the public space, this is sufficient to win over opinion and push out the competitors. However, despite being well intentioned, it is at best a naive approach; people will make decisions using many more factors than just facts. As Kahan puts it, ‘[when the] truth carries implications that threaten people’s cultural values, then holding their heads underwater is likely to harden their resistance and increase their willingness to support alternative arguments, no matter how lacking in evidence.’

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1. Editorial (2010) Climate of suspicion. Nature 463: 269. http://dx.doi.org/10.1038/463269a.
2. Kahan, D. (2010) Fixing the communications failure. Nature 463: 296-297. http://dx.doi.org/10.1038/463296a.

FOLLOWING on from my post yesterday regarding people’s concept, or lack thereof, of evidence, it was suggested that it would be an interesting thought experiment for those of us who are willing to offer criticism on a subject to put ourselves on the receiving end. I think it’s a good idea to find something that each of us holds dear or true, and see if we can challenge ourselves to imagine how we’d feel if someone argued against that view. By understanding this, perhaps we can better approach our means of approaching such as subject with someone for whom such criticism would represent a paradigm shift.

As I managed to shake silly beliefs such as ghosts and ley-lines as a child, the only examples I have as a thinking adult are with particular scientific hypotheses that I’ve subscribed to, but subsequently had to ditch. This is the general method of science, and in my own research there have been any number of hypotheses I’ve formed and subsequently disproved on the basis of new evidence.

However, there have also been explanations for some natural phenomena that pre-date my research career, and to which I subscribed whole-heartedly. One example dates from my time as a first-year undergraduate studying marine biology. I had a particular interest in marine invertebrates and once attended a lecture by Donald Williamson, who was the major proponent of a larval evolution hypothesis, and recently came to light as being accused of ‘fringe science’ and getting a paper in the Proceedings of the National Academy of Sciences (PNAS) under the radar; thus also highlighting the pitfalls of the ‘I’ve got a mate in the club’ attitude to publishing.

Essentially Williamson felt that the immature forms (larvae) of many such invertebrates can be thought of as distinct organisms from the adult form, which are often comprehensively different both physically and physiologically; think caterpillar to butterfly, or blobby polyp jellyfish to its adult ‘medusa’ form.

Williamson felt that these different forms arose through hybridization — the fusing of two genomes (of two distinct organisms), one of which is now expressed early in an animal’s life, and the other late.

You can read an Sci. Am. article about it here.

I have to say, I absolutely LOVED this hypothesis, it was very exciting and I lapped it up with the typical fervour of an undergraduate.

Trouble is, since then it has been rebuked often and has not been substantiated by the experiments that were performed to test the hypothesis. I was quite recalcitrant about such rebukes up until the most recent PNAS rebuke that I’ve just linked to.

You can read rebukes to the Sci. Am. article here.

Changing my view about this hypothesis was hard, and a little embarrassing given I so animatedly communicated it to all my friends until I learnt it didn’t have strong grounding.

This is very true of many areas in which we are not experts, whether you are a scientist or not, and the fact is that we do tend to confer a great deal of trust in some individuals depending on their position. I would add that Donald Williamson was not ‘wrong’ to form this hypothesis at that time; scientific knowledge is by its very nature transitory, but once it has been tested, and alternatives developed, then we should seek to move on.

I could have easily ignored the evidence that Williamson’ hypothesis did not hold up to, and continued telling people an interesting and captivating story about why adult and juvenile forms of invertebrates are so different, but I didn’t. There’s still a part of me that thinks that there may still be something in it, which is why I can relate – to a point – with those people facing their first reality-check with regards some pseudoscience that they’ve hitherto believed in.

Donald Williamson is now retired and still stands by his hypothesis.

I don’t.