Flu researchers caught red-handed with plagiarism

Being caught red-handed is never a good thing. Best is of course not to cheat (or strangle your kids)

By Jonas Waldenström

Two weeks ago, Professor Randy Shekman, one of this year’s Nobel laureates, made the headlines when he accused the top journals Nature, Science and Cell to be ‘damaging to science’. A single publication in these journals can boost a scientist’s career, increasing the chances to get grants, positions, and tenure. But with great prospects of fame, the risk for ‘cutting of corners’ increases. Shekman wrote in a column in the Guardian that these journals thereby ‘contribute to the escalating number of papers that are retracted as flawed or fraudulent’.

Although the top journals are over-represented in terms of retracted papers, you still get them in many other journals, too. Sometimes it is just due to a human error, and the authors retract their paper when they found out that the data, or analysis, was wrong. But, there are also plenty of cases where it is the journal that retracts the paper, after having investigated an accusation of fraud. Even though we like to think of scientist as the pillars of wisdom, there is a bit of rot here and there. And it is not only manipulated data – another common form is plagiarism, with text being copied from other articles without proper citation.

Yesterday, Alex Bond in Canada sent me a tweet looking like this:

Retraction Watch  – the highway patrol of science – had reported a retracted paper in Virologica Sinica (Springer), pulled back by the journal due to plagiarism. In fact, plagiarism of a paper that I had co-authored! The retraction notice said that:

“This article has been retracted at the request of the Editor-in-Chief, as it contains large portions of text that have been duplicated from “Phylogenetic analysis of the non-structural (NS) gene of influenza A viruses isolated from mallards in Northern Europe in 2005” published in Virology Journal (2008) 5:147, without sufficient attribution being given to the article. Despite the data / conclusion being original in the paper, this is violating the journal policy.”

I had not read the paper from Virologica Sinica, and wasn’t aware of neither the paper’s existence, nor the plagiarism before yesterday. On Retraction Watch’s blog they show examples from the abstract and other sections that are clear copy-and-paste jobs.

But why? How hard can it be to write your own abstract? Why did the eleven authors on the paper chose to borrow text, instead of writing their own? Perhaps it was a language problem, as all authors belong to institutes in Kazaksthan and Russia? Or was there a single bad egg who cut the corners, and the remaining authors were not involved sufficiently in the writing of the article? I guess we will never know for sure, but one thing I immediately noted when reading the published version of the retracted paper is the frequent occurrences of grammatical errors.

The moral standards in science are set high, and rightly so. You shall be objective, you shall justify your claims, and you shall not cheat. Simple as that. The paper on non-structural gene variation in influenza virus collected in Kazakhstan is no longer a part of the scientific literature. Kudos to Retraction Watch for highlighting the retraction of this and other papers!

If you now want to read the original paper you will find it here:

Siamak Zohari, Péter Gyarmati, Anneli Ejdersund, Ulla Berglöf, Peter Thorén, Maria Ehrenberg, György Czifra, Sándor Belák, Jonas Waldenström, Björn Olsen, Mikael Berg. Phylogenetic analysis of the non-structural (NS) gene of influenza A viruses isolated from mallards in Northern Europe in 2005. VIROLOGY JOURNAL, 2008 5:147.

And the retracted paper (is/was) here:

Andrey Bogoyavlenskiy, Vladimir Berezin, Alexey Prilipov, Ilya Korotetskiy, Irina Zaitseva, Aydyn Kydyrmanov, Kobey Karamedin, Nailya Ishmukhametova, Saule Asanova, Marat Sayatov, Kainar Zhumatov. Retraction Note: Phylogenetic Analysis of the Non-structural (NS) Gene of Influenza A Viruses Isolated in Kazakhstan in 2002–2009[J]. VIROLOGICA SINICA, 2013, 28(6): 373-373.

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#MyGenderGap – my history of inequality in numbers

 

By Jonas Waldenström

Last week Nature published a news story on the gender gap in science. That is, the ever-growing gap between women and men as their careers develop. Typically, women are in majority in undergraduate studies, are more or less equally represented at the PhD level, but plummet at the postdoc, tenure track, and tenure levels. As a consequence the number of female professors is low at most universities. As an example, in the comparatively gender friendly Sweden only roughly 20% of professors are women. Clearly something is rotten in the state of research.

We all know this, it is nothing new. However, knowing it is wrong at a systematic level is not the same as being aware of your own part in replicating it. That’s where change is needed. Alex Bond, an ecologist and blogger in Canada, did this. He took the larger question down to the personal level and asked people to calculate their own gender gaps and tweet it on Twitter under the hash tag #MyGenderGap. This is a great initiative and I suspect he will write an informed and eloquent blog post about it soon (as he usually does – great blog, please visit it). I calculated mine too, and tweeted it. But as twitter-tweets are limited to 140 characters I thought that I should devote some space here in the blog to present my own data in a bit more depth. And it doesn’t look very good…

First of all, my own supervising career mimics the field as a whole. I have supervised 12 undergraduate students at honor’s and MSc levels, 9 women and 3 men. I supervise 4 PhD students, plus one that graduated last year, in total 3 women and 2 men. I have three postdocs in the lab, 1 woman and 2 men. On top of that I have acted as co-supervisor for an additional 9 PhD-students, 4 woman and 5 men. Over all not too bad, but the trend with a stepwise reduction in women with each step up the academic ladder is obvious.

But it is when you look at the publications that things become really clear. I went through the 102 peer-reviewed publications that I have authored/co-authored and for each publication I counted the number of men and women co-authors, and whether the female scientist was my own PhD student and if the leading PI was woman or man. The table looks like this:

Year

Men in pub

Women in pub

Women PhD students

Ratio F/M

# publications

2000

1

1

2001

2

2

2002

12

6

0.50

4

2003

12

5

0.42

5

2004

19

5

0.26

7

2005

25

8

0.32

8

2006

22

1

0.05

5

2007

98

14

2

0.14

13

2008

45

17

3

0.38

9

2009

35

10

6

0.29

7

2010

44

23

10

0.52

10

2011

45

13

3

0.29

10

2012

56

16

3

0.29

11

2013

47

21

11

0.45

10

463

139

38

0.30

102

Out of 602 co-author across 102 publications (note that the same co-author often is found on the author line-up of several articles), only in 139 occasions the co-author was woman, and in many cases my own PhD student. This gives a gender gap statistic (the simple division of #F/#M) of 0.30. A totally equal proportion would be 1.0. Thus, my stats are not very flattering. And as regards the long-term trend the line is depressingly flat. Why? One reason that I can see is that I have established a core of collaborators that I tend to publish with. Most of them are men. Most of them are in turn PIs in their labs, thus it reinforces the system.

My Gender Gap in publications across time. I took out the data from 2000 and 2001, since only few publications were published.

My Gender Gap in publications across time. I took out the data from 2000 and 2001, since only few publications were published.

What could be done about it? That’s a great question; one which I don’t have a good answer to. In my mind I haven’t chosen collaborators because of their gender, but of common research interests. But seeing your own stats staring you in the face is the first step in a thought-process that may lead to actual change.

We should remember that the university system is strange. It has a high intrinsic growth rate [the cohorts upon cohorts of PhD-students that start each year], but a very stable carrying capacity [the old folks sits on their position for life and most departments don’t grow]. Thus, if you have succeeded in science and is a PI, the highest likelihood is that (on average) only one of your students you teach during your career will make it in academia. Which gender will that person have?

Now go compute your own gender gap.

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Looking back on 2013 (part I): A dozen publications, some zombies, but no pandemics

By Jonas Waldenström

Post-apocalyptic dawn will have to wait some time more

Post-apocalyptic dawn will have to wait some time more

A year goes by so fast, and soon it is time to close the book on 2013. One thing we can conclude, at least, is that there was no apocalypse, and no end-of-humanity pandemic. However, there have been some worrying notes on new emerging pathogens in 2013. On top of the list of concern we find the MERS coronavirus in the Middle East, and the H7N9 low-pathogenic influenza virus in China. Neither of them has caused many human casualties, nor are they common or widespread. No, it is not what they do, but what they potentially could do that worries the disease world. The MERS virus is related to SARS – a deadly viral pathogen that in 1997 jumped from bats, to civets, and further to humans, and which was on the brink of causing a pandemic before it was fortunately contained and stopped. The other bad guy, the H7N9 influenza virus, carries novel antigenic properties to which the human population lacks immunity; thus, if it becomes adapted to spread between people (and not as today, between infected poultry and humans) it could turn into pandemic flu. Both viruses face strong guilt by association, you can say. These pathogens, and others, are like butterflies, fluttering in and out of detection. Worrisome echoes on the radar screens at WHO and CDC. They are also good examples to why field biology is needed in medicine: we need to track reservoirs of diseases, new and old, and we need to understand how diseases evolve. And that’s exactly what we try to do in ZEE. (In sale pitch jargon: we are the good guys!)

So what happened in ZEE during 2013? In this and other posts we will give you a hint of what we did, and how things went.

Publications. 2013 has been a productive year for the ZEE group! More than a dozen publications were published from Linnaeus University (plus a bunch from Uppsala). Most of these are available freely and you can reach them by following the links below. This year was also the year when this blog was launched! A motto we have is to provide popular accounts on the science we do. Thus, for some of the publications there is a link to a blog post in the list below. Read them – lots of fun!

  1. Tolf, C., Wille, M., Haidar, A-K., Avril, A., Zohari, S. & Waldenström, J. Prevalence of avian paramyxovirus type 1 in Mallards during autumn migration in the western Baltic Sea region. Virology Journal 10: 285  [Ebola, Chikungunya and Newcastle – of places, names and Mallard viruses] http://bit.ly/IToOVG
  1. Gillman, A., Muradrasoli, S., Söderström, H., Nordh, J., Bröjer, C., Lindberg, R.H., Latorre-Margalef, N., Waldenström, J., Olsen, B. & Järhult, J. 2013. Resistance mutation R292K is induced in influenza A(H6N2) virus by exposure of infected Mallards to low levels of oseltamivir. PLoS ONE 8(8): e71230. [This flu, that flu, and Tamiflu®] http://bit.ly/1gw2roa
  1. Safi, K., Kranstauber, B., Weinzierl, R., Griffin, L., Rees, E., Cabot, D., Cruz, S., Proaño, C., Takekawa, J. Y., Waldenström, J., Bengtsson, D., Kays, R., Wikelski, M. & Bohrer, G. Flying with the wind: scale dependency of speed and direction measurements in modelling wind support in avian flight. Movement Ecology 1: 4. [As the Mallard flies] http://bit.ly/19mFVtC
  1. Wille, M., Tolf, C., Avril, A., Latorre-Margalef, N., Bengtsson, D., Wallerström, S., Olsen, B. & Waldenström, J. 2013. Frequency and direction of reassortment in natural influenza A virus infection in a reservoir host. Virology 443: 150-160. [How do you do, the things that you do, Mr Flu?] http://bit.ly/18JpZkp
  1. Latorre-Margalef, N., Grosbois, V., Wahlgren, J., Munster, V.J., Tolf, C., Fouchier, R.A.M., Osterhaus, A.D.M.E., Olsen, B. & Waldenström, J. Heterosubtypic immunity to influenza A virus infections in Mallards may explain existence of multiple virus subtypes. PLoS Pathogens 9(6):  e1003443. [Why are there so many flu viruses?] http://bit.ly/IUiwoM
  1. van Toor, M. L., Hedenström, A., Waldenström, J., Fiedler, W., Holland, R.A., Thorup, K. & Wikelski, M. Flexibility of continental navigation and migration in European mallards. PLoS ONE 8(8): e72629. [Perdeck revisited – or how does a Mallard know its way?] http://bit.ly/1904O0h
  1. Tolf, C., Latorre-Margalef, N., Wille, M., Bengtsson, D., Gunnarsson, G., Grosbois, V., Hasselquist, D., Olsen, B., Elmberg, J. & Waldenström, J. 2013. Individual variation in influenza A virus infection histories and long-term immune responses in Mallards. PLoS ONE 8(4): e61201. [Disease is a property of the individual] http://bit.ly/1ebcGOz
  1. Hellgren, O., Wood, M. J., Waldenström, J., Hasselquist, D., Ottosson, U., Stervander, M. & Bensch, S. 2013. Circannual variation in blood parasitism in a sub-Saharan migrant passerine bird, the garden warbler. Journal of Evolutionary Biology 26: 1047-1059.
  1. Griekspoor, P., Colles, F.M., McCarthy, N.D., Hansbro, P.M., Ashhurst-Smith, C., Olsen, B., Hasselquist, D., Maiden, M.C.J. & Waldenström, J. 2013. Marked host specificity and lack of phylogeographic population structure of Campylobacter jejuni in wild birds. Molecular Ecology 22: 1463-1472. [Of chickens, wild birds and men – host specificity in Campylobacter jejuni] http://bit.ly/1bCcFeu
  1. Griekspoor, P., Olofsson, J., Axelsson-Olsson, D., Waldenström, J. & Olsen, B. 2013. Multilocus Sequence Typing and FlaA sequencing reveal the genetic stability of Campylobacter jejuni enrichment during coculture with Acanthamoeba polyphaga. Applied and Environmental Microbiology 79: 2477-2479.
  1. Hernandez, J., Johansson, A., Stedt, J., Bengtsson, S., Porczak, A., Granholm, S., Gonzalez-Acuna, D., Olsen, B., Bonnedahl, J. & Drobni, M. 2013. Characterization and comparison of Extended-Spectrum β-Lactamase (ESBL) resistance genotypes and population structure of Escherichia coli isolated from Franklin’s gulls (Leucophaeus pipixcan) and humans in Chile. PLoS ONE 8(9): e76150. [Travel the world – can antibiotic resistant bacteria hitchhike with migratory birds?] http://bit.ly/19mF2kV
  1. Olofsson, J., Axelsson-Olsson, D., Brudin, L., Olsen, B. & Ellström, P. 2013. Campylobacter jejuni actively invades the amoeba Acanthamoeba polyphaga and survives within non-digestive vacuoles. PLoS ONE 8(11): e78873. [doi:10.1371/journal.pone.0078873]. [Good morning Mr Amoeba, may I come in?] http://bit.ly/1cqPybs
We study ducks, and they study us.

We study ducks, and they study us.

Staff and students. A year is also a quarter of a PhD time span, and half of a master student’s time. This means that there are many comings and goings in a research group over time. This year, one PhD left the nest and graduated, and four are due in 2014. No new PhD student started, but there were three babies born, thereby boosting the current ZEE children count to more than 10, enough for a football team!

Two former PhD students got a flying start: Dr Neus Latorre-Margalef is on a postdoc in Athens, Georgia, funded from the Swedish Research Council (VR), and Dr Josef Järhult in Uppsala got a huge researcher grant from VR to build up his own group! Fantastic news!

After finishing her MSc last year, Anna Schager got a PhD position in Italy in the spring. Olivia Borg and Anu Helin made their honors’ degree in the lab and then moved to Uppsala for MSc studies, while Johanna Carlbrand and Andras Turai stayed on with MScs at Linnaeus University.

With no real apocalypse in sight, 2013 instead became the zombie year, a trend that culminated with the WWZ movie. If you want to prepare for the coming zombie apocalypse, the author and blogger Colin M. Drysdale has a range of tips for you, including how to make projectile weapons with toilet brushes. You never know, such a skill may come in handy one day when the undead are going for your entrails. Next week we will get back on the-end-of-year-theme and present the best and the worst links/papers/topics of 2013! Cheers!

‘MOOC it, MOOC it’? – My impressions of an online Coursera course in Epidemics.

By Jonas Waldenström

I just finished a course in epidemics at Penn State University. But without setting my foot on campus, or, in fact USA! And guess what, I didn’t miss a single lecture! Sounds weird? Thing is, this was not an ordinary course; this was a MOOC – the latest development in distance learning. And it was great! But more on that later.

We live in an ongoing digital and technical revolution – just compare everyday life today with how it was ten, or twenty years ago. The change is not linear; rather it is a series of punctuated shifts, where each new wonder gadget push the field to a new plateau. For instance, back in 1999 when I worked in northern Nigeria it was virtually impossible to phone home. In the bigger cities you could pay for a call via landlines, but in the area where we worked the closest phone was 10 hours away, across the sand dunes. A few years later mobile phones were everywhere, and Internet cafes were popping up on the posh streets. In fact, these days, you often have better connection between Nigeria and Sweden, than between Kalmar and Öland…

The hallmark of an obsolete technique

The hallmark of an obsolete technique

Another example, although more linear, is the ever-faster computer processors, or the speed at which you can sequence DNA. When I started my PhD, the art of drawing figures by hand, in ink, for publications was still remembered. And during my first year I was extremely happy if I managed to run 10 sequences (of roughly 500 bp each) over night on the capillary sequencer! Now you can print a gun in 3D, or sequence a genome over night. At least a microbial genome, and a very simple gun. But still, who would have believed this 20 year ago?

Education, however, has evolved at a slower pace, even after the onset of computers. Yes, some guy invented PowerPoint in 1987 and sold it to Microsoft, thereby turning a bad lecture into a worse, but more colorful lecture. (Actually, the ‘internets’ tell me they were two guys: Robert Gaskins and Dennis Austin, and that they both think their creation has sometimes turned into an abomination.) Otherwise the key concepts of a lecture have remained the same for many centuries. Teaching is primarily occurring here and now, in a room of varying size at a campus. Teacher goes in, does his/her thing and hopefully the students become enthusiastic, interact and learn. A familiar process for all of us, unless you drop out of school at the age of seven. A good lecture can change your purpose of life. On the other hand, a bad lecture can be akin to torture and be remembered for far too long.

The future of education as seen in 1905

The future of education as seen in 1905

The question now is whether this is about to change? At least, for the first time it seems as technology is there, or nearly there, to allow things to be done differently. And this is where the MOOC – the Massive Online Open Courses – comes in to play. Education for the masses, free and online. Some believe this way of teaching will make education democratic and open it for everyone, not only the fortunate who by smartness or rich parents study at the prestigious universities. Via a connection to the Internet it is now possible to enroll in courses on a vast number of subjects. A smorgasbord of education. Through sites as Coursera, the disparate courses can be collected into a degree; tailored and individual-based degrees.

So what about the epidemics MOOC? Truly, it was a magnificent course! The team of teachers was like a NHL team of only seriously talented scientists: Dr. Marcel Salathé, Dr. Ottar N. Bjornstad, Dr. Rachel A. Smith, Dr. Mary L. Poss, Dr. David P. Hughes, Dr. Peter Hudson, Dr. Matthew Ferrari, and Dr. Andrew Read. And it was indeed a massive course, even for a MOOC! One figure that was mentioned was 27,000 enrolled students! One year I gave my course, with a rather similar content, to 6 students – that tells you of the potential outreach a well-managed online course can have compared to a campus course at a small university.

A screen shot of Dr Andrew Read in action.

A screen shot of Dr Andrew Read in action.

A MOOC, or any type of distance learning, is as good as the teachers are, and the time and resources put in by the university. The Epidemics MOOC was certainly an extremely good example, where it was evident that they wanted to do something more than the ordinary, set a new standard even. However, even if the course was beautifully made and stuffed with interesting lectures, I reckon that there must have been a massive drop out of students. Not because the course was too advanced, but because it is so easy to procrastinate things to a point where you have several weeks of lectures to digest. A human fallacy, I’d say. Also, I guess that a significant fractions of students, and perhaps over-represented among those that finished it, were folks like me. Folks that already have a degree, or even work with the subject. But that doesn’t matter too much after all – if only 10% of the students made it all the way, it still means that a staggering 2,700 people now are acquainted with the basics of epidemiology!

In case Marcel Salathé (or any of the other teachers) reads this little piece, I’d like to end with some reflections of the course:

You understood your audience. Most people that are enrolled in distance education are already busy. If you have a day job you don’t have all the time in the world. You want things to be condensed, edited and thoroughly thought through. In my case I watched the videos when my youngest daughter was napping – which isn’t forever, I can tell you. Instead of long lectures there were several short (5 – 9 min each); short enough to maintain focus, and long enough to say something important. Well done!

We could see you! I love the fact that the videos were not uploaded powerpoints with voice-over. These were actual lectures recorded in studio, where the lecturer looked into the camera. Instead of the ppt, the main notes were highlighted via cartoons appearing together with the talker. That’s smart!

I could rewind, skip, press forward, and even adjust the speed of the talks. The possibility to watch lectures when you want to is great, of course. And that you can download them and watch when you are offline too. I also liked the speed button. The Andrew Read NZ dialect required a 0.75 speed setting, while Ottar Bjornstad and Mary Poss were best enjoyed at a 1.25 pace. Splendid idea!

Ask us anything. The idea of picking questions from the study forums and have group discussion among the faculty was an excellent idea. And a possibility to pick up recent events like flu and MERS. I liked this, good move.

The heterogeneity of students was a problem, at least for discussions. A central idea is that the platform should stimulate discussion via online forums. For this course it was expected that the student engaged in at least 10 posts to earn credits. However, with 27,000 students enrolled the level of comments was often more Facebookish than intellectual. Perhaps it would have been good to add a few explanatory lectures (like a week zero) describing the very basics (such as what a virus really is).

With that said, I really enjoyed the course! Whether the MOOCs will change education as we know it is still an open question though. We will see what the future brings! Or to paraphrase Reel 2 Real: MOOC it, MOOC it!

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Four partially successful ways of supervising your academic troupe

Is that a student/postdoc waiting for his/hers supervisor?

Is that a student/postdoc waiting for his/hers supervisor?

By Jonas Waldenström

Science is the business that never goes to sleep. Even at our little university in the backwaters of SE Sweden there are some office windows that shine through the darkest November hours. Those rooms most often house a PhD student or a postdoc. Actually, in the case of our department, most often one of my PhD students or postdocs.

Of course, working during nights and weekends can be good. At least in the short run. You get things done, you can drink as much free coffee as you like, and you are not interrupted by colleagues expecting you to do stuff for them. Perhaps most importantly: you are not interrupted by your supervisor. In the long run, though, we need our free time to recuperate.

This autumn has been very busy for me. However, in my case it has been less due to science, and more to the domestic realm of life. You see, I have been on parental leave from August to December. It has been a wonderful time, even though the word leave has very little resemblance to vacation and relaxing. Parental leave is a full time job – a very rewarding job, but demanding.

My domestic life. Not.

My domestic life. Not.

But it is hard to completely step away from science when you have a research group to run. A PhD student cannot be put on pause, just because you are away. Manuscripts are still produced, revisions are accepted or rejected, deadlines for grant proposals approaches mercilessly, and people in admin want your opinion on this, that and everything. This is the academic’s dilemma, much harsher for women than for men, as women on average take longer leaves than men. In my case I have actually worked one day, and lately two days a week during the fall. This means I have been able to do some of the things I was supposed to do, but far from all the stuff I had intended to.

This is the third time I have been home with kids. When the time now is approaching for my return to the office I can look back at four ways things have been handled this fall:

That is a valid question!

That is a valid question!

Absence breeds creativity. Even though I haven’t been around the team has pulled off some incredible research stunts both in the lab and in the field. One infection experiment was carried out at the National Veterinary Institute, and two intensive studies were conducted at the bird observatory. All ran smoothly without me showing neither my hide, nor my hair. In my absence, people collaborated, found solutions and carried on. And if the shit had hit the fan – they could always have blamed me! (Which luckily it didn’t).

 

You cannot clone yourself, but you can get someone as a stand-in.

You cannot clone yourself, but you can get someone as a stand-in.

Find your doppelganger. Just as in the movies, it is nice with a stand-in (or several, actually). Someone who can take necessary charges of things. You know who you are – you have done great! Many thanks!

 

From Frederick William Fariholt's book Tobacco, its history and associations. From WikiMedia.

From Frederick William Fariholt’s book Tobacco, its history and associations. From WikiMedia.

Keep supervision short and worthwhile. One especially important tactic has been ultra-fast supervision. Or as long as it takes to smoke a cigarette outdoors on a Swedish November day. Without a jacket. Supervision on speed!

 

This is actually a thing you could buy http://www.thecheapplace.com/Ignorance-is-Bliss-Patch

Ignorance is bliss. Finally, accepting that you can’t do all is the best thing. For everyone’s sake. Be home. Enjoy. And, of course, blog about it 🙂

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Good morning Mr. Amoeba, may I come in? – Or how protozoa act as Trojan horses for Campylobacter

By Jonas Waldenström

The world of microbes is measured on a different scale from the world of humans. A single drop of seawater can contain more microbial life than there are inhabitants in Sweden. And the microbes are everywhere: in the sea, in the air, in the soil, even deep in the bedrock, or in the abysses of the ocean. The smallest and most numerous inhabitants of the microbial world are viruses; so tiny we need advanced electron microscopy to see them. They are obligate parasites, high jacking the cellular machinery of other organisms for replication. The prokaryotes – bacteria and archea – come next, in an abundance of shapes and forms. In many environments bacteria constitute the bulk of the microbe world in terms of mass. Next step up on the size ladder are the protozoa: a taxonomically diverse collection of single-celled eukaryotes that include amoebae, rotifers, foraminifers and many, many more bizarre creatures. The roles they play differ depending on species and circumstances. For instance, in marine systems protozoa drive photosynthesis, producing carbohydrates from sunlight. They can also be the bad guys, the trolls and ogres of the microbe world feeding on the smaller members.

Single-celled eukaryotes were lumped together as ‘Protozoa’ in the early ‘five kingdoms of life’. In the genomic age we now know that protozoa is not a valid taxonomic group, as the members in several cases are paraphyletic. But out of conveiniance the term is still used. This particular protozoa is an Ammonia tepidia, a benthic foraminifer (image by Scott Fay, UC Berkeley, via Wikimedia Commons).

Single-celled eukaryotes were lumped together as ‘Protozoa’ in the early ‘five kingdoms of life’. In the genomic age we now know that protozoa is not a valid taxonomic group, as the members in several cases are paraphyletic. But out of conveiniance the term is still used. This particular protozoa is an Ammonia tepidia, a benthic foraminifer (image by Scott Fay, UC Berkeley, via Wikimedia Commons).

Single-celled life has ruled this planet for three billion years. They are also ruling us, at the global scale by carbon cycling and atmospheric gas exchange, down to the individual scale by living in and on our bodies, either friendly as commensals, or aggressively as pathogens. In your gut there are billions of bacteria processing the food while taking tidbits for themselves. Some of these bugs work for the common good including the host (– that’s you!), some are just there on a quick visit, and some are interfering with the other microbes or you. Recent research show how intricate the microbiome of the gut is, and how important the composition of it is for our health. The ever-decreasing costs for DNA-sequencing have sparked a new branch of microbiology research. Some believe we are the doorsteps of a revolution in medicine, while others think it is snake oil.

What all can agree on, at least, is that the potential for interactions between microorganisms is nearly endless, but that we need to start addressing them to get a hold on disease epidemiology. A bug we have worked a lot with over the years is Campylobacter jejuni. This bug, also known as the ‘chicken bug’, is a major human pathogen. It rarely kills, but it affects many, many people. A common figure says that 1% of the human population in the US is infected with C. jejuni per year. Yes, per year! If you ever have had campylobacteriosis you are likely to remember it. Profuse diarrhea, vomiting and stomach pains are unpleasant, but common symptoms. Folks get sick, stay home from work or school, and some may need to seek health care. A few percent become hospitalized, and a fraction develops sequelae including paralytic symptoms. Recent research suggests that the exposure is higher than the 1% figure. Perhaps as high as 8% of the population per year, but that many infections don’t give symptoms.

Anatomy of an amoeba (image by Pearson Scott Foresman, via Wikimedia Commons)

Anatomy of an amoeba (image by Pearson Scott Foresman, via Wikimedia Commons)

Soon ten years ago, Dr Diana Axelsson-Olsson, then a graduate student, was working with C. jejuni and amoebae in the microbiology lab at Kalmar County Hospital. The aim of the project was to investigate if campylobacters interacted with amoebae, and if that was of importance for how the bacteria fared in the environment. In those days (and to a large extent still) the focus in our lab was wild bird campylobacters, and the environment represented a ‘black box’ in the epidemiology. Diana showed that if C. jejuni were exposed to amoebae the bacteria quickly ended up inside the amoebae. In the microscope they could be seen swimming around in amoebae vacuoles. More importantly, she found that raising the temperature to 37°C the bacterial cells started to multiply inside the amoebae. In fact, they grow so quick and so violently that they soon ruptured the amoebae and spilled out into the surrounding solution!

Perhaps this is a good place to pause for a while, and tell you something about the biology of campylobacters. Doing so make it easier for you to understand the importance of this finding. C. jejuni is an obligate gastrointestinal bacterium that only replicate at temperatures of 37-42°C, in essence the body temperature range of its host animals. It is also a rather sensitive bug, that doesn’t do well in aerobic conditions, doesn’t stand UV-radiation (sunshine, that is) or desiccation very well. Compared to Salmonella it is a real softie. Despite this, C. jejuni is one of the most common pathogens, equipped with a large host range of wild birds, domestic birds and mammals, and humans. Some would say, even, that campylobacters are ubiquitous. This duality of being sensitive and ubiquitous has been a longstanding conundrum in C. jejuni research.

Thus, in light of this, the campy-within-the-amoeba was the start of something new. A potential pathway for the bacterium to survive outside its animal host. Later studies have shown that it is not only amoebae of the genus Acanthamoeba (the ones the research started with) that take up campylobacters, rather this ability seems widespread among protozoa. So, if campylobacters from an infected host end up in an aquatic environment – and we are not talking oceans here, but rather puddles – there is likelihood they will meet an amoeba. When that happens, the bacteria somehow, either actively or passively, are taken up by the amoeba and become internalized. The life within the vacuole seems fairly cozy, in fact it has been shown that once internalized the bugs can survive and be able to become resuscitated many weeks, even months, later. And, of course, the raise of temperature to 37°C is expected to take place when an animal drinks the water with the bug-infested amoebae, leading to exponential growth of the bacteria, rupture of the poor amoebae and fait-a-complit infection of the new host! A Trojan horse epidemiological pathway!

The Spartans are up for a surprise (Giovanni Domenico Tiepolo, via Wikimedia Commons)

The Spartans are up for a surprise (Giovanni Domenico Tiepolo, via Wikimedia Commons)

Such pathways are known from other systems, perhaps most famously from Vibrio cholera, the bacterium causing human cholera. Vibrio bacteria are transmitted via contaminated water, and it has been shown that bacteria that invade protozoa start to express genes that make them more virulent in humans.

An open-ended question for the Campylobacter vs. Amoeba story has been whether it is an active or passive event from the bacterium’s side. Amoebae normally eat bacteria for breakfast, lunch and dinner, stretching out their long pseopodes to grab things in their environment. Food is ingested into phagosomes that turn into lysosomes and the contents get degraded. How then do campylobacters survive this? Jenny Olofsson in our lab has devoted her PhD on this and related questions, and a few weeks ago one of her studies was published in PLOS ONE.

Viable and heat killed C. jejuni are taken up into different types of A. polyphaga vacuoles. (A) Live/Dead stained viable (green) C. jejuni, confined to tight vacuoles. (B) Live/Dead stained heat killed (red) C. jejuni residing in giant spacious vacuoles. (C) Vaculoes with CTC stained viable (red) C. jejuni do not co-localize with Alexa fluor-488 labeled dextran filled vacuoles (green). (D) Vaculoes with CTC stained heat killed (red) C. jejuni have taken up Alexa fluor-488 labeled dextran (green). (E) In contrast to non digestive vacuoles, giant digestive vacuoles contained smaller vesicles (arrow). Picture D and E are from the same amoeba. doi:10.1371/journal.pone.0078873.g004

Viable and heat killed C. jejuni are taken up into different types of A. polyphaga vacuoles.
(A) Live/Dead stained viable (green) C. jejuni, confined to tight vacuoles. (B) Live/Dead stained heat killed (red) C. jejuni residing in giant spacious vacuoles. (C) Vaculoes with CTC stained viable (red) C. jejuni do not co-localize with Alexa fluor-488 labeled dextran filled vacuoles (green). (D) Vaculoes with CTC stained heat killed (red) C. jejuni have taken up Alexa fluor-488 labeled dextran (green). (E) In contrast to non digestive vacuoles, giant digestive vacuoles contained smaller vesicles (arrow). Picture D and E are from the same amoeba.
doi:10.1371/journal.pone.0078873.g004

By comparing live and dead campylobacters and the speed of which they were taken up by amoebae, Jenny and colleagues could show that it is a process that is induced actively by the bacterium. To start with, viable bacteria associated with a substantially higher proportion of Acanthamoeba trophozoites than heat-killed bacteria. The speed of internalization, the total number of internalized bacteria as well as the intracellular localization of internalized C. jejuni were also dramatically influenced by bacterial viability. Living C. jejuni ended up in small vacuoles that were tightly surrounding the bacteria, while heat-killed C. jejuni were observed in larger, spacious vacuoles. Using fluorescently labeled dextran, it was shown that the latter vacuoles were part of the normal degradative pathway – in other words, chewed, swallowed down and digested. Somehow the live C. jejuni cells manage to escape the amoeba metabolic machinery, perhaps by stopping the fusion of the phagosome with the lysosome. The next step would be to identify the genes responsible for theses processes and characterize the molecular mechanisms involved.

Clearly, the usage of amoebae and other protozoa has many advantages for pathogenic bacteria that are poor in surviving in the environment. By infecting amoebae they can take advantage of the intracellular environment and hope that the protozoan cell lives long enough to become swallowed by a suitable host animal. Each and every water body is in essence filled with a cavalry of potential mini-Trojan horses.

Link to the articles:

Olofsson J, Axelsson-Olsson D, Brudin L, Olsen B, Ellström P (2013) Campylobacter jejuni Actively Invades the Amoeba Acanthamoeba polyphaga and Survives within Non Digestive Vacuoles. PLoS ONE 8(11): e78873. doi:10.1371/journal.pone.0078873

Axelsson-Olsson D, Waldenström J, Broman T, Olsen B, Holmberg M (2005) Protozoan Acanthamoeba polyphaga as a Potential Reservoir for Campylobacter jejuni. Appl. Environ. Microbiol. 71: 987-992. doi:10.1128/AEM.71.2.987-992.2005

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