Monday, January 25, 2016

MCR-1 gene: Update


I have previously blogged about colistin resistance factor in a plasmid that has had the world turn attention (Read earlier posts here and here). This discovery signalled that we are loosing out on antibiotics. Colistin is one of the last resorts against pan drug resistant gram negative bacteria. Resistance to this drug would be essentially a grave problem. It is generally agreed that Colistin is not the only drug, there are a couple of other options in case of such infections but the arsenal is very limited.

Ever since the first identification report was published, most of the countries have looked for the gene sequence in their strains and databases and there has been aggressive reporting of the findings. This is in part, a global effort. I need to stress here that the media reporting these as first instance is totally wrong. The gene has been described earlier in chromosomes of resistant organisms. The highlight of this issue is, now it is a plasmid which makes it more mobile. In other words, unlike the earlier known version, this is moving through different strains.

Fig 1, is a representation of the discovery of MCR-1 in bacterial isolates. The samples include from anything that can be imagined- human sample, commercially available meat, animals etc. 

Fig 1: Map of MCR-1 gene detected. Data as on 15 January 2016. Source
It has been established that this rise in antibiotic resistance is due to the heavy use of antibiotics in livestock. Interestingly, despite several earlier warnings the antibiotic sale has shot up by about 23% over the last 5 years, a majority of which is going into the livestock use.

It remains to be seen what is the true extent of spread of MCR-1. However, experts agree that it has already spread and some suggest that with surveillance and molecular evidence we maybe able to see more regions.

Friday, January 22, 2016

Who wins by count: Man Vs Microbiome


So often I have heard of talks and references stating that the total number of microbial cells in our body exceeds our own cell count by a ratio of 1:10. The original estimate was published in 1972 by Thomas Luckey. I myself like other microbiologists have quoted this estimate in a variety of my communication and classes. But the recent buzz has had me thinking. The new study claims that this is a myth and the real numbers are not anywhere near the estimated figure.

Table 1: Bacterial count estimates. Source
Microbiome was a big deal a few years ago. However, with better Sequencing and bioinformatic capabilities microbiome studies are becoming more common. The human microbiome project (HMP) has currently achieved to catalogue a huge amount of data. The aim of HMP was to generate resources that would enable the comprehensive characterisation of the human microbiome and analysis of its role in human health and disease. The current literature is pretty much aware of the average set of organisms that constitutes the microbiome.

Fig 1: Counting human cells
The original study was based on the estimates from stool samples. The new study by Milo etal, suggests that this is probably an overestimate. The new estimate is approximately 1.3:1, with an error factor of about 2 degrees. Yet that is way beyond the original estimate. The paper mined earlier data from multiple studies and put up the probable bacterial count from different body areas. See Table 1. My first set of question are based on the method of estimate. When I talk about the microbiome, I always want to think about all the microbial forms. I want to include fungus and viruses as well. It appears to me that this is obviously missing in the estimates. But even if I consider that there are at least a 100 phage for every bacteria, the estimate still doesn't shoot up to the classic 1:10 counts.

The study has provided a better number for quoting. But what is still an amazement, even a conserved estimate would be somewhere in a 1:1 ratio. That means there is at least one microbe for every cell the humans have. Needless to say, with defecation, bathing and other routine activities the ratio will change for a temporary period of time. Simplified into one line, “The numbers are similar enough that each defecation event may flip the ratio to favour human cells over bacteria”. Peer Bork comments, "It is good that we all now have a better estimate to quote. But I don’t think it will actually have any biological significance".
Ron Sender, Shai Fuchs & Ron Milo (2016). Revised estimates for the number of human and bacteria cells in the body bioRxiv DOI: 10.1101/036103

Abbott, A. (2016). Scientists bust myth that our bodies have more bacteria than human cells Nature DOI: 10.1038/nature.2016.19136

Wednesday, January 13, 2016

Lab series# 9- Pulsed field gel electrophoresis


Electrophoresis is a term that everyone has heard about. For people doing science in a wet lab setup, electrophoresis is kind of a usual work set up. There is a large number of variations of this technique, depending on the objective to be achieved. Gel electrophoresis is the one classic example. I haven't personally worked with a PFGE. Since I had to give a seminar on the basics of this topic, I did a little bit of literature reading. Here is the summary.

Photo 1: SEM  image of
agarose polymer. Source
Gel electrophoresis has the fame of being easy to work with and reliable enough for separation of biomolecules. Gel electrophoresis is used for DNA, RNA and protein separation. The way this system works is based on a mass is to charge ratio. In a set agarose gel, there are pores. The size of the pore depends on the concentration of the gel. Though the distribution of pore size is not entirely uniform on an average, higher the gel concentration, smaller the pore size. Gel electrophoresis works something like this. A charged molecule will move towards the opposite charge. The movement is restricted by the size of molecule since the molecule has to penetrate through the pore. For example, DNA has a negative charge due to Phosphate groups in its structure and thus will move towards the positive charge. However, smaller DNA fragments, say about 300 bp in size will move faster than the 600 bp size DNA molecule. The accumulation of similar DNA molecules in a region allows it to be visualised using DNA tracking dye such SYBR Green. This is what we call as "DNA bands".

But there is a drawback to this "Conventional" technique. Larger the DNA fragment you have to analyse the more you need to loosen up the pore for DNA to move. This means, a more diluted gel needs to be made. There is a limit to how much diluted gel you can make and hence an inherent limit to the size of DNA that can be resolved. It has been calculated that the conventional gel electrophoresis can effectively separate DNA fragments up to ~20 kb, though practical resolvable limit is probably much less than this value. Any DNA fragment larger than this limit will stay at the same place regardless of DNA size, since the size is too large for it to pass through the gel pores. This is where the technique of PFGE comes in handy. PFGE methodology is useful when the analytical DNA sample is expected be of larger size (in Kbp lengths).

Fig 1: Schematic representation of PFGE work flow.
So how does the PFGE basically work? PFGE was first demonstrated by Schwartz and Cantor (Link). The idea was based on a earlier observation that DNA molecules elongate upon application of an electric field and return to an un-elongated state upon removal of the electric field, the relaxation rate being dependent on the size of the DNA. Larger pieces of DNA will be slower to realign while smaller pieces will be quicker. Thus over a long run with consistent change of electric fields the larger DNA samples will migrate in the gel yielding a pattern which can be analysed.

Preparation of DNA sample for PFGE is slightly different from the conventional methodology. This is because handling large piece of DNA tends to be damaged easily. For bacterial typing the most recommended method is to use a fresh culture and embed the bacteria into a gel. This is known as the plug. Currently plug holds are commercially available. The cell lysis and restriction enzyme treatment is done on the plug. This plug is the loaded into the PFGE. A schematic representation of the PFGE- process is shown in Fig 1.

PFGE has multiple parameters that can be varied to achieve optimised results. Of all the variable parameters- Pulse time and reorientation angle appears to be the most crucial.

Table 1: Pulse time recommended for DNA fragments.
Pulse time (also known as switch time or switch interval) is defined as time interval for which a given set of electric field is active. The larger the expected size of fractionated molecules, the less frequently the switching of electric fields should be performed. However, In a given pulse time only molecules of a particular size class occur under optimal conditions for separation and may be effectively fractionated. This means that the over a range of DNA size different pulse time needs to be used. The change of pulse time during electrophoresis is called “Ramping”. This parameter may be increased throughout a run either constantly or discontinuously over a range of discrete values. Continuous increase of pulse time from minimum to maximum of specified values during a given time interval is called “Interpolation”. An alternative variant, the stepwise increase in pulse time, is called “Stepping”.

The acute angle between the two alternating electric fields is called as reorientation angle. In certain instruments this is not variable and fixed at 120°. In some instruments this can be varied as per requirements. Reorientation angle does not produce significant effect in DNA molecules with less than 1 Mbp size. The mobility of larger molecules increases with decrease of the angle; But it has been the common experience by users that the band resolution is significantly reduced with decreasing angle. Lesser than 90° is not recommended. For a start 110° is used as optimal in most cases.

Table 2: Variants of PFGE.
PFGE is a prototype version. The principle of this technique has been used in different instruments. Some of the most well known include- FIGE, TAFE, CHEF and RGE. See Table 2 for details.

CHEF is probably the most commonly used apparatus. The CHEF apparatus has twenty-four point electrodes equally spaced around the hexagonal contour and voltage is set at all points. It employs the principles of contour-clamped electrophoresis to generate homogeneous electrical fields by holding the electrodes at intermediate potentials, thus generating the homogeneous electric fields necessary for straight, distortion-free lanes. CHEF uses a fixed reorientation angle of 120° with gradiations of electropotential radiating from the positive to the negative pores. This allows resolution of DNA fragments up to 7 Mbp.

PFGE technique is currently considered as a gold standard for epidemiology of bacterial infection outbreaks. The resolution provided is quite high in comparison to other methods such as Ribo-typing or MLST. However, there is a lack of consensus on protocol for a variety of species and for some bacteria PFGE cannot be performed. Inter-laboratory variations have been another issue. Experts agree that PFGE gives a phylogenetic map but it should be considered as a relative map rather than absolute. According to me the only technique that is better is Whole genome sequencing.
Sambrook J, & Russell DW (2006). Pulsed-field Gel Electrophoresis via Transverse Alternating Field Electrophoresis Gels. CSH protocols, 2006 (1) PMID: 22485453

Sunday, January 10, 2016

Zika Virus


The best part of 2016 as of yet, is that the Ebola epidemic which is more than a year long, the worst among all previous Ebola spread looks like is over and currently not reporting any new cases. If the situation remains stable, WHO will declare the epidemic to be officially over in another couple of weeks. The sad part, blood-based Ebola therapy trial results is not promising. And the worst part is, there appears to be an increasing number of cases of a rare virus infection- Zika Virus.

Fig 1: Documented ZIKV outbreaks.
Zika Virus (ZIKV) is a member of mosquito borne- Flavivirus group. Structurally it is similar to other Flavivirus members. The vision has an icosahedral-like symmetry, containing nucleocapsid surrounded by a host-membrane derived lipid bilayer studded with envelope proteins E and M. First discovered in an experimental rhesus monkey which was caged in Zika forest in 1947 for research on yellow fever. Subsequently the agent was isolated and characterised in 1952 and named as Zika virus. The first human case was described from a Nigerian patient in 1954. Interestingly, this virus has been reported in extremely rare numbers upto 2007.

Fig 2: States with laboratory-confirmed cases ZIKV
Brazil 2015. Data as on 23 November 2015.
There has been a total of 3 outbreaks prior to the current suspected epidemic. The first documented outbreak was reported from Yap Island in 2007 (April to June 2007). There were a total of 185 cases of suspected Zika virus disease. Of these, 49 (26%) were confirmed and 59 (32%) were probable cases. The second was an outbreak in Tahiti. I have not been able to find the accurate number of cases, but literature published has estimated 8273 suspected cases. In 2014 two places, Cook Island and New Caledonia showed up in the map. The Cook island outbreak was over quickly with 932 suspected and 50 confirmed cases. New Caledonia had 1400 confirmed cases of which 35 was imported cases. Fig 1, shows a representation of documented ZIKV outbreaks. By May 2015 Brazil reported its first case of Zika and by December 2015 the virus had been demonstrated in several countries of Central and South America (See Fig 1). The outbreak continues to expand in terms of number of cases.

Photo 1: A child with Microcephaly,
mother diagnosed with ZIKV.
Microcephaly is a congenital defect represented by abnormally small growth of head, with incomplete brain development. In African region 2013, 167 cases and in 2014 147 cases of microcephaly was recorded. In 2015 nearly 3000 cases has been reported. The headlines over the media is that Zika virus has a role in microcephaly. Of course, as of yet there is no formal proof yet. But it appears that ZIKV genetic tests were positive in at least a good number of cases with children who are born of microcephaly. Further a CNS disorder- Guillain-BarrĂ© syndrome (GBS) has been previously shown to have some association with ZIKV infection. Infant 74 patients who had Zika symptoms, later developed neurological or autoimmune syndromes - out of them, 42 were diagnosed as GBS.

There are a couple of things to note here. ZIKV is similar to dengue and antibodies are cross reactive thus making ELISA tests sometimes confusing. Currently, only genetic test is diagnostic. It should also be noted that currently ZIKV infection is in quite good number and hence it is statistically hard to tell the association. But the sudden 20 fold increase in cases is something that needs to be looked into. There is also a good chance that this virus is slowly migrating.

At this point many experts have commented that it is too early to suggest that ZIKV is behind the small brains.
Duffy MR, Chen TH, Hancock WT, Powers AM, Kool JL, Lanciotti RS, Pretrick M, Marfel M, Holzbauer S, Dubray C, Guillaumot L, Griggs A, Bel M, Lambert AJ, Laven J, Kosoy O, Panella A, Biggerstaff BJ, Fischer M, & Hayes EB (2009). Zika virus outbreak on Yap Island, Federated States of Micronesia. The New England journal of medicine, 360 (24), 2536-43 PMID: 19516034

Roth A, Mercier A, Lepers C, Hoy D, Duituturaga S, Benyon E, Guillaumot L, & Souares Y (2014). Concurrent outbreaks of dengue, chikungunya and Zika virus infections - an unprecedented epidemic wave of mosquito-borne viruses in the Pacific 2012-2014. Euro surveillance, 19 (41) PMID: 25345518

Campos GS, Bandeira AC, & Sardi SI (2015). Zika Virus Outbreak, Bahia, Brazil. Emerging infectious diseases, 21 (10), 1885-6 PMID: 26401719

Tuesday, January 05, 2016

Blogger's Desk #9: For Science Blogger's


Wish everyone, a Happy New Year. Extending a big thanks to all the people who mailed me good wishes and patiently waiting for the "blog break", to be over. I will soon update all the blog stats for the the past 4 years in the blog statistics page. Coming to the science part of the blog, 2015 has been the best blogging year for me so far, as the stats will show. Recently some people have expressed interest in science blogging (Guess that is their new year resoultion) and have asked me if I would give them a some writing tips.

I have been running this blog now for 4 years. I have been thinking this through for a sufficiently longtime. I also read a lot of other blogs (Not just microbiology). From Indian context, I have found a very small percentage of blogs that actually talks science, and still lower- Microbiology. There are too many blogs that I could find that has not been updated for ages, and some even have as less as 4-6 posts in total and then author forgot that he had a page. It is worth a mention that there are several blogs that are updated regularly and has great content.

Personally, blogging has been a good experience for me, since it has allowed me to connect to a lot of people and also I get to think and ink about a lot of stuff. If you run a google search for blogging tips you will get zillions of posts and some have really good tips. But go through them and you will find that some of them are not applicable to science blogging. Though each blogger has a personal preference on how he/she will craft the piece there are some general things that I want to throw out- especially for science bloggers. If you think they are tips that's great. But let me put it as experience.

1. Science blog readers are a restricted population

In contrast with blogging fields such as fashion, cooking, sports, news, gaming etc the number of people browsing though a science blog is limited. People often arrive at your blog through a search hit and are there to read specifically that post. So the number of page hits (or what you call as traffic) will be less. Don't bother when your page hits is not climbing very fast.

2. Write a post, follow it up.

Science is ever evolving. In a field like microbiology, theories keep changing. So your post may be outdated in sometime. You have to follow up with your earlier posts. When you do so, include back-links to your posts. It not only lets reader get a deeper look, it also keeps the reader moving within your site. It has been my experience that, people often write a mail to you, and often don't drop a comment on the page. If you have sufficiently lot of comments its preferable to write about it in whole as a fresh post. Commenting on the same post, for pages and pages is not comfortable.

3. Reference and cross check your facts

This is one thing that is lacking severely in a lot of science blogs. It is very frustrating to a scientific reader to agree with your facts, when they disagree and also to have a deeper look if the posts are not referenced. Journal references and other online materials from which you borrowed the idea needs to be told. This also makes what you talk about, more authentic and credible. It also helps in doing a fact check of what you talk about, in case you missed something important. Try putting in reasonable number of links in the text to the source of data or ideas borrowed. This has a couple of advantages. Most importantly, you let your readers know that you don't plagiarise and respect original content of others. It also lets people go back without having to google for source when they want more information on the particular point.

4. Mix it up

The same thing being talked about for hundreds of post, start making the things monotonous and journalish. Blogging should be free sounding. Get someone else to write occasionally on your page. Guest posting is something that you don't ignore. If you get a chance, take it with both hands. Have a couple of varieties. If you know of someone who would be better suited to write about a topic (Such as personal experience) get that person to guest post. This is something that I learned from Small things considered. Lot of varieties makes people look for more and interested for a long time.

5. Your blog is your space. That's why it is called as "Blog space"

Take charge of what you talk about. Throw in stuff what you think is important. Important is rule of KISS (Keep it simple stupid). A page with lot of links and networks, too much graphics, unwarranted widgets will ruin what you want to say. As you write your work evolves and let it do so. Don't try to force yourself into. Block spam comments. One more way of avoiding unwanted pop-ups is don't let page have ads. Blogging should be for personal let-out. Not money making site. However, if you have a absolutely professional page which requires you to have some money in its maintenance, ads may be allowed in a controlled fashion. Declaration: I have not placed any ads in my page.

6. Let the world know you are there

This is one thing that every blogger needs to do. You have to blow your own trumpet and make regular announcements. Post links on Facebook, Science groups. If there is some discussion about a topic, put a link there and let them know you have some perspective on it. If some one has talked about your effort put in that too. Sometime self advertising is great (But don't spam). After all somebody needs to read what you wrote, and that somebody needs to know that you have written about it.

7. Keep yourself informed of what you are writing

This is a kind of extension of 2nd point. No person is an expert at everything. But we often make the mistake of laying hands in an unknown area when not warranted (Should admit I too have done these blunders). When there is something that you want write about, of which you don't have a good knowledge about, first learn it and teach it to yourself. If you could make a graphical abstract for the whole concept that had be excellent. Keeping yourself informed has a lot to do with how much you learn. Podcasts, news pages are some places where you could always look at for regular updates. Here is a list of Podcasts that I listen to regularly related to science. You would have your own list, based on your interest. If you regularly follow my posts, you will see that many of my references come from following.
  1. This week in Microbiology
  2. This week in Virology
  3. This week in Parasitology
  4. BBC Science hour
  5. Science AAAS
  6. Nature Podcast
  7. NeuroPod
  8. MedScape Infectious diseases podcast
  9. Emerging Infectious diseases
  10. Brain Science Podcast
In addition, I subscribe to following
  1. Asian Scientist
  2. Medical News Today
  3. Nature News
  4. Neuroscience News
  5. Scientific American
  6. Science Daily
  7. The Scientist
  8. Small things considered
  9. Coffee Table Science
For learning about research from the people who do it, I check the following
  1. Howard Hughes Medical Institute
  2. iBio seminars
  3. ICGEB Seminars
*There is of course Youtube and Coursera (MOOC Courses), for learning anything that you want to.

8. Don't bother too much about blog ranks.

Most of the blog ranking system to which you signed in, are algorithm based. It lets you have a superficial picture of your reach. There are several blog index sites which ranks your blog. This gives some visibility and serves as a blog search engine. You could try this. If you update your blog everyday, your rank is bound to increase by some percentage. But I'm not sure of how many people read it.

9. Does domain matter?

I'm not sure and many blogger's debate on this point. Having a blogspot or wordpress sub domain maynot be very attractive but unless contents are delivering it shouldn't matter. On the other side, a sub-domain is not considered as authentic. Well, you get to decide what serves best for you.

10. Enjoy the process

The ultimate of all. The day you feel, the process is not worth it or you want a break just do it. Blogging is mostly a free service. Nobody is going to penalize you for not posting a stuff. And perhaps a break may also help people to catch up on past. And take the pleasure of boasting about it. Not much people write about science. You are one of them.