Resistance without genetics - persistance in bacterial populations

Most work on bacterial resistance to antibiotics tends to start with genetics. If a bacteria is able to survive a certain antibiotic, it is assumed that it has gained a gene from somewhere (and bacteria can get genes from almost anywhere) which allows it to survive. That makes sense after all...surely two bacteria with exactly the same genetic information should react identically to antibiotics?

No. Not all the time. There is an unfortunate habit of biochemists to get too wrapped up in the genetics to think about epigenetics (control of genetic expression by proteins), especially with 'simpler' organisms like bacteria. Persistence is the property of a totally identical population of bacteria to respond differently to antibiotics, despite having identical genomes they are phenotypically (phenotype = set of observable characteristics) different.

Persistent bacteria aren't only seen in response to antibiotic's either...they can be found for many types of stress, such as heat or starvation. They've been roughly split into two different types:

• Type I persisters: form in response to stress, usually at stationary phase (i.e after the initial burst of bacterial growth)
  
• Type II persisters: are seen forming throughout the bacterial lifecycle

Monitoring the growth rates of individual cells showed that even before treatment with antibiotics, many of the persister cells had reduced growth rates. The mechanism for this is not yet clear, a few genes that may be involved have been identified, but no reason for activating them (or repressing them in non-persisters), has yet been identified.

Although these sound dangerous at first glance, the medical implications of persisters are relatively limited (probably the reason why the mechanism has not been more thoroughly studied). As they have a slower growth rate, and as there are so few of them within an overall susceptible population, they can usually be cleared relatively easily by the immune system. There are, therefore, only three main areas where they are clinically relevant: immunosuppressed patients, pathogens that have adapted to the immune system, and in niches in the body that are less available to the immune system (such as within a biofilm).

The main ecological and evolutionary implications of this are that a colony of identical bacteria can 'survive' antibiotic attack by having a few of its members able to withstand that attack. Although they will grow slower under normal conditions, their ability to withstand stressful conditions means that they can essentially recover the whole colony, without the need for a major genetic change. This is true especially of diseases such as tuberculosis, where very few bacteria are needed to re-start an infection.

One thing that would be really interesting would be to study the behaviour of persisters within biofilms. Along with swarming and quorum sensing, biofilms are an example of pseudo-multicellular bacterial behaviour, circumstances under which having a small population of cells that can regenerate the whole colony would be very useful.

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Orit Gefen & Nathalie Q. Balaban (2009). The importance of being persistent: heterogeneity of bacterial populations under antibiotic stress FEMS Microbiology Reviews, 33 (4) : 10.1111/j.1574-6976.2008.00156.x

On the classification of blobs

The first forays into microscopy revealed a whole world of blobs, tiny microscopic organisms that were invisible to the naked eye. These went through a range of different names, from the 'animalcule' denomination given by Anton van Leeuwenheok (which he used to describe everything small he saw under the microscope, including his own sperm) through 'monera' (a more specific name for certain types of blobs, namely those that weren't eukaryotic) to 'prokaryotes', a name that still stands.

Prokeryote means, literally, 'no nucleus', and it's use allows the world of living organisms to be split up into two groups: Prokaryotes and Eukaryotes. Eukaryotes are things with a nucleus, a membrane covered partition to hold DNA, as well as many separate organelles existing within their cells, such as mitochondria, endoplasmic reticulum, the Golgi apparatus...

Prokaryotes are...uh...everything else.

Which means that the label 'prokaryote' was always waiting to fall apart. After all, they may just be blobs but there are a lot of them, and some are very different blobs. Around the 1970's people started noticing that there were a group of the prokaryotes that behaved differently, mainly through studies done by Carl Woese and George E. Fox who created classification tables based on the genetic sequences of ribosomal RNA (the part of the genome most likely to be conserved, this is often used for classification, especially of things in Deep Time). This showed that there was a distinct group of prokaryotes with a mostly separate evolutionary history (more on the mostly later) to the rest of the prokaryotes. They were originally named 'archaebacteria', and together with 'eubacteria' (true-bacteria) were put in the prokaryotes group. They were blobs without a nuclei, and that was where they belonged.

However, things started to get a bit more complicated the more people looked at archaebacteria. They weren't just a group of slightly odd bacteria, they were something else. Something different. Although their metabolic pathways are similar to bacteria, their methods of turning DNA into proteins more resembles eukaryotic processes. Their flagella (tentacle like structures used for movement) have a markedly different structure from bacterial flagella. Like bacteria, they reproduce asexually and (also like bacteria) they can share their DNA around, in fact they can also share there DNA with bacteria, which makes taxonomists tear their hair out. It's very difficult to classify something when it keeps giving its DNA away, and collecting bits from other sources.

It is proposed in the SGM journal (Society for General Microbiology-journal not available on line) that the term 'prokaryote' should be scrapped altogether. As well as being an incorrect label for a large group of organisms it also produces an incorrect evolutionary perspective. The use of the eukaryote/prokaryote terms suggests a very human based linear "One upon a time there were blobs with no nuclei and then they got nuclei and then they were better" sort of story. A more correct view is that of all three superkingdoms; bacteria, archaea and eukaryotes splitting away from each other. Eukaryotes safely packaging their DNA away, allowing a more complex system to build up, yet forfeiting the ability to share bits of DNA. The archaea and bacteria on the other hand, continued to share their genetic material, just became more selective about it as they diverged (hense the 'mostly' seperate history).

Or maybe not. It might be that the archaea/eubacteria formed a very selective group of blobs, which then split further when some developed a nucleus, while the others continued to share their DNA with the bacteria, picking up different metabolic secrets. It's hard to work out; especially given that similarities between the DNA of archaea and bacteria does not necessarily show their relatedness; it might be a gene that has remained conserved in both of them for millions of years, or it might just be one that was exchanged last week.

There a several arguments against removing the 'prokaryote' as a naming system but most of them boil down to the very multicellular-centric argument of: "but they're all just blobs!" The three superkingdoms of archaea, bacteria and eukaryote are a far more accurate, and scientifically and taxonomically correct way of looking at things than the prokaryote/eukaryote model.

My only complaint is that I spent ages in secondary school trying to learn how to spell 'prokaryote'... removing the name means I could have spent that time doing something far more useful...like building paper planes and reading 'Redwall'.

End of exams!

So exams finally finished! Going out with a bang for the final practical paper, which was evil to the point of being insane. It seemed to be designed to cover material we hadn't been taught, about half way through I seriously began to wonder whether I'd missed a few lecture courses somewhere.

Had there been a lecture on how to calculate glycolytic flux? Was there a chemistry course I'd somehow missed? Had flow cytometry been covered at some point without my knowledge?

But no. it turned out they did actually expect us to work out how to calculate glycolytic flux from first principles. In an hour. IN FINALS. AND apparently remember back to A-level chemistry.

*is dead, Jim*

Anyway now I have more time on my hands, I want to start properly getting into research blogging. And the truly interesting stuff should start next month, when I get back into the lab. =D

Revision...

...would be a lot easier if the lecturers gave better notes. Notes that didn't leave me trying to work out colour-coded protein domains printed in greyscale with four very small slides to a page surrounded by unhelpful notes which turn into random squiggles when I drift off.

It's the option lectures that are scaring me at the moment. First terms stuff I've kind of got a hold on but I'm a bit lost with the options stuff, because I wasn't revising it as I went along. The reason I wasn't revising it? Because I was simultaneously trying to do an 8am-6pm five-days-a-week lab work project with a 5000 word write-up worth (wait for it) a whole 10% of my mark.

I swear this department is trying to kill me. Why I've agreed to do another year of it I don't know. (Except that the lab work was FUN. Just mental. Fun and mental. Like me :p )

But as soon as I get out of here, I am definitely going to make a headlong dash for the microbiology department and stay there. They will probably work me just as hard and just as crazily, but at least the scenery will be different.

Either dull or revolutionary, no middle ground...

I decided to have a go at some proper 'research blogging' today; taking an article and turning it, figures and all, from highly scientific language into understandable English. But I got as far as looking at the current issue of Nature, when I saw this quote, in a short piece down the side (about whether scientists are dull or not):

"Because, it seems to me, most working scientists have either long since accepted that they are not of the ‘revolutionary’ type exemplified by greats such as Isaac Newton, Charles Darwin and Albert Einstein, or never strived to be."

I'm sorry, striving to be revolutionary? Darwin? The conservative family man who always vaguely reminds me of Dickens? Although if 'revolutionary' means 'a bit of a bastard' then Newton certainly fits the bill. Their ideas were revolutionary, true, at least in the world of science, but the author here seems to be using revolutionary as an antonym for dull, which in my mind does both words an injustice.

The rest of the piece also seems to suggest that there are two types of scientist: the 'revolutionary' who is vibrant, exciting and unhindered by paperwork, and the 'dull' who just wants to get on with a normal life, seeing research as work rather than vocation. The tone veers wildly between apologetic, defensive, and trying to allot blame. Paperwork and bureaucracy are mainly blamed for the dullness, because apparently none neither of them existed in sixteenth century Cambridge, or Second World War Germany. Apparently geniuses (because the revolutionaries are now geniuses, which should come as news to the Les Mis boys on the barricade) must have "the requisite levels of selfishness and creativity" which fits Newton, certainly, and James Watson as well, but falls short of being fair to Einstein and Darwin. They are also described as "the ‘clever crazy’ type that might belong in an institution" which kind of fits Einstein slightly (or at least fits his hair), Newton maybe, but again, does nothing for Darwin or Watson.

My problem here is that the author is dealing with a stereotype. Why aren't scientists all crazy and enthusiastic geniuses? Because they never were! Some were, true, but that doesn't mean everyone else was dull by comparison. Science is not limited to research drudgery working tirelessly to support and uphold the occasional flash of brilliance; rather the whole process is brilliant, occasionally flashing up bright in the public perception when it produces a particularly intelligent person (Einstein etc), or a particularly good idea (vaccination, antibiotics, etc). It is these flashes which get remembered, and worked into stories, and pointed out years later as stark events that existed on their own.

I resent the fact that I am meant to believe my work is dull but necessary just because I'm never going to reach the dizzy heights of selective fame. I also resent that I am told I should be dull but necessary. Maybe I should dye the front of my hair orange again...

You can read the full article here if your library or institution will let you in. Otherwise, here is a quick summary:

• There used to be great scientists, revolutionaries, geniuses
• Nowadays all is dull and boring
• Well obviously, because there's lots of paperwork and bureaucracy
• And you need some dull people, to support the ones who make the big discoveries
• Acutally it's better to be dull
  
• The revolutionaries are a bit odd anyway.

Underlying subtext: No I am not envious or unfulfilled, not at all, of course not, why should I be?

Emotions...I knew i was forgetting something...

Slightly baffling BBC Health headline today: "Emotional intelligence 'aids sex'"

Apparently woman with a high 'emotional intelligence' (defined rather shakily as being better able to monitor their own and others emotional feelings) enjoy it more. 2 035 female patients were given questionnaires about their experiences during sex and their emotional intelligence. Apparently there was enough correlation between the two for the BBC to conclude that a greater ability to feel, express, and enjoy emotional connections made sex better.

Well, who'da thunk!

*facepalm* this really should not be a headline.

And interestingly enough, there don't seem to be any similar studies done on men. Making this whole article, and indeed the study, border on the lines of the pointless and the obvious.

Ah well, it provided a brief (and because there is a part in all of us with the mentality of an immature 14-year old) slightly snigger-inducing break from revision. Membrane translocation can only hold my interest for so long.

Monoclonal antibodies

During my pathology course last year, monoclonal antibodies were one of those things I just couldn't 'get'. It was explained to me numerous times, by increasingly more irate and disappointed looking supervisors, but every time it was re-mentioned in lectures and supervisions I would sort of stare despairingly at whatever piece of paper was in front of me thinking 'what the hell are they again'.

"Something to do with mice, and antibodies, and making them human, or something" was usually the best I could do.

So when the subject appeared yet again during this years course, I decided to finally look it up properly and work out just what was going on.

Antibodies look like this:
The two variable regions recognise bind to antigen (parts of invading bacteria) leading to the invading bacteria being destroyed. Antibodies produced in the body are polyclonal, because each one has a different variable region and can target a different antigen (until a threat is realised in which case they massively overproduce the relavent antibody).

The idea of monoclonal antibody therapy is to produce a large number of essentially the same antibody, that can find and potentially destroy a specific target. The idea was to produce a kind of 'magic bullet' that went through the body picking out the specifically ill parts and removing them. And antibodies are very specific, and can be targeted to lots of different proteins.

The problem with producing them is that a single B cell (antibody-producing cell) will only last a few generations before dying. Not long enough to produce the large amounts of specifically-target antibody needed for therapy. The original solution to this problem was to use a technique known as hybridoma. Individual B cells that had been grown in mice and produced antibodies that destroyed whatever target the therapy was being designed to remove were fused with immortal myeloma cell lines. The B cell could then propagate for much longer, secreting monoclonal antibodies. The main problems with this technique were that is was slow and laborious and created problems for purifying the antigen.

The most modern technique I know of (although others are being developed) is called SLAM, which stands for Selected Lymphocyte Antibody Method. B cells are isolated from mice (or rabbits, other animals can potentially be used as well) and grown in little plastic wells until they start secreting antibodies. Single B cells are then isolated, and screened for activity. The relevant antibody genes are then cloned through PCR and expressed as recombinant antibodies. This technique is a lot faster and produces high affinity antibodies from a number of species.

Monoclonal antibodies are used in various drugs currently on the market. Lymphomas (cancerous B cells) can be treated with Zevalin (R) or Bexxar (R). Apparently on 3 February 2005, the New England Journal of Medicine reported that 59% of patients with a B-cell lymphoma were disease-free 5 years after a single treatment with Bexxar.

The thing is though, I'm being taught this as a biochemist student/researcher, not as a medical researcher. Which means that I have very little idea how useful, common, or applicable most of these techniques and products are. Academic researchers and medical researchers seem to live a world apart, something that hit me particularly hard during the conference. You could almost always tell, about half way through a talk, whether the speaker was a medical or academic researcher. There doesn't seem to be a whole lot of cross-talk between them either, which is a pity because academic research does often come up with the odd useful medical application, but of course they aren't in any position to implement it.

Tee-four and Colin

They're getting on so well together!
Tee-four (the phage on top) was a present from Genetic Inference
And Colin (the rather worried looking E. coli underneath) was actually a long-ago present from Paradigms as well.

Many apologies to Genetic Inference for taking so long to realise who tee-four was from. I've been neglecting the Internet in general lately to try and encourage me to revise more :)

[The bow-and-arrow on the wall behind is not mine]

Science and Yoga

I have started doing Yoga recently, to give me a bit of calming time during revision, and to get a work out which doesn't involve undue damage to my knees. This has brought about a distinct amount of light-hearted teasing from a Certain Special Someone, who as well as insisting that this means I'm turning into a middle-aged woman also asks me how I can cope with it not being 'scientific'. I'm meant to object to phrases like 'positive energy flows' and things.

Well I do object to them. A little. Our yoga instructor is a cheerful guy in about his thirties who said on the very first day that his science background was pretty much non-existent. Positive energy flows have been mentioned, as have muscles opening up and the mind drifting away etc.

But then I starting thinking about it. What he's saying is not strictly true, he knows that. It's not meant to be true. What it is is a model for how you're thinking and how your body is reacting. A model that fits within the thoughts and philosophies of yoga. There is no positive energy in my legs at any point, there's a lot of lactic acid at some points but that doesn't make any sense in the context of yoga. Like many parts of science, yoga takes the model that works best within the context of what it's doing and rolls with that.

I'm not claiming that yoga is scientific here. It isn't, it hasn't got the scientific method and evaluation behind it (it's more of a philosophy if it's anything, which means the aforementioned Certain Special Someone should have a little more respect for it :p ). But using chakras and energy points to describe what's happening is no worse than enzymologists using clunky atoms-as-large-balls models to work their models of catalysis.

[As an aside, enzymologists tend to be very realistic when it comes to thinking of science as a model. They know that there's very little chance of ever finding out exactly what's going on in the catalytic centre of an enzyme. They thought they were in with a chance, somewhere in the 70s, but then quantum entered biology and it all went a bit weird]

There is, however, a difference between 'working model' and 'bad science'. Claims that yoga does things like 'release toxins' or 'energise the DNA' or whatever are nonsense and bad science; Pseudo-scientific babble that could easily be described as downright lies. But being told to feel your breath being used to flow energy through you (or whatever, he's not yet gone that far but it's a nice phrase nevertheless) I would say isn't bad science. It's a way of describing a feeling, that allows you to grasp what you're trying to do. There's no scientific way of putting it. It's a model for a way of feeling, and it's the best model within the context of yoga.

Until you start taking it too seriously, obviously. But then, that's true for everything.

Yay Procrastination!

To reassure the people I know who read this blog, I am actually working quite hard. However I decided to take a bit of time out today to write out my own skippy's list for Lab Rat laboratory work. This is, in effect, the Other Dissertation, based around my project last term. All of them come from things done, or discussed by either me or my fellow student Lab Rat during the project:

Thing’s Lab Rat has learnt in the Lab

1) Bacteria are to be referred to as ‘bacteria’, ‘microbes’ or ‘bugs’ not ‘bloody-minded bastards’
2) Warning labels are to be taken seriously
3) The Bunsen burner is not ‘out to get you’
4) Neither is the autoclave
5) Flame sterilised items remain hot for a while after being taken out the flame
6) If you must sing, sing quietly
7) Stick figures are not allowed on reports
8) Neither are smiley faces or any kind of emoticon
9) The phrase “It’s OK, no one will notice” should NEVER be uttered
10) Your PI can tell the difference between bacteria and fungal contamination
11) The autoclave does not have a mind of its own, and should not be blamed for any contamination
12) Making Darth-Vader breathing sounds while working under the fume hood is not necessary
13) Bacteria are not to be described as evil, no matter how many rounds of mutation they’ve been through
14) Sliding down the corridor is not encouraged, especially when holding plates
15) Juggling of any laboratory equipment should not be attempted
16) There is no such thing as approximate accuracy
17) Repeating an experiment until it gives you the correct results is not proper scientific technique
18) The above rule always applies, no matter how close to the deadline you are
19) Your lab coat is not a trench coat
20) There is a limit to how much glassware can be balanced on top of a lab book
21) ‘Bench space’ should not be an oxymoron
22) COSHH stands for ‘control of substances hazardous to health’, attempts to pretend it stands for anything more obscene are in bad taste
23) Molten agar does not remove fingerprints
24) The UV light should not be used for tanning [just for the record, this was never tried, only half-jokingly discussed]
25) The bacteria are not your minions, and should not be referred to as such.
26) Maniacal laughter is not encouraged
27) Thanking God in three different languages is not necessary, no matter how wonderful your results turn out to be
28) The computers are to be used for analysing data, not reading webcomics
29) The above rule applies even if the webcomic in question mentions bacteria
30) Many things in the lab sometimes produce smells of burning. This is no cause for total panic
31) Neither plates, nor agar bottles have the capacity to move on their own
32) Lab reports need not be written in Iambic Pentameter
33) Mutant bacteria are given numbers, not ‘codenames’
34) X-23 is not a number
35) The words ‘gloopy’ ‘thingy’ ‘unfortunate’ ‘awesome’ and ‘combobulated’ should not be used in scientific reports
36) Plate is not a verb (as in ‘to plate’)
37) Bacteria are killed by autoclaving, not by repeated stabbing with a spreader, no matter how satisfying the latter may be
38) The difference between 10?g and 100?g is not ‘just another zero’
39) Laboratory antibiotics should not be used to heal personal illnesses
40) If the thought of doing something makes you giggle, that thing is probably prohibited within the laboratory

Number 36 came up surprisingly often in my dissertation. "The bacteria were plated onto an agar disk..." "Plate 30 microlitres of bacteria" etc. it just flows so naturally.

Anyway! Back to G proteins...