Memoirs of a Defective Brain

Antibiotics & Agriculture Part 4: The Transfer of Antibiotic Resistance

2013-06-18T15:00:00.000+01:00

The patient was in dire condition. A forty year old woman from Michigan, she had suffered badly from diabetes, kidney failure and a number of complications related to those diseases. Two years after she had started dialysis, disaster struck. She developed painful foot ulcers, and an infection in her leg that was so severe that the whole leg had to be amputated. What was worse was that immediately after the operation, her amputation wound became infected, with Staphylococcus aureus. In her only stroke of luck that day, the Staphylococcus aureus was susceptible to antibiotics. But the next year, the foot ulcers were back, and she required even more amputations, as well as treatments to prevent the bacterial infections causing these ulcers from becoming fatal.
The catheter that linked her blood to the hospital's dialysis machine, the replacement for her riven kidneys, provided Methicillin-Resistant Staphylococcus aureus with easy entry into her blood. There was only one antibiotic that could stop this MRSA infection. Vancomycin was given to the patient while the physicians removed the infected catheter. In its place, the physicians used a number of temporary catheters, to ensure that the patient could still use the dialysis machine.
But a number of these catheters also became infected. When the physicians examined these catheters, they realised that against all odds, things had taken a turn for the worse. They discovered that the Staphylococcus aureus on this catheter had been joined by Vancomycin resistant Enterococci. Now the Staphylococcus aureus were resistant to Vancomycin too. They searched all of the possible options that could have lead to this situation, and it was the DNA evidence that revealed what had happened. The Vancomycin resistant Enterococci, commonly found in the community but rarely infectious, had given its resistance genes to MRSA.

This was the first of a series of outbreaks of VRSA that occurred in Michigan, and all of them had a similar theme. A person with an MRSA infection would spontaneously develop full blown resistance to vancomycin out of nowhere. The only commonality in all of these cases was the presence of vancomycin resistant Enterococci both before and during these cases. So how did vancomycin resistant Enterococci pass along their resistance to MRSA ?
The answer lies with DNA molecules known as plasmids.

These are rings of DNA which can carry genes between different bacteria. The exchange of plasmids between bacteria is a key driver of bacterial evolution, as it allows species to share genes between eachother. They enable bacteria to acquire new traits from other bacteria in the vicinity, which can allow them to adapt to their environment in new ways. In this case, the vancomycin resistance "trait" was carried on a plasmid in Enterococci, and this plasmid could be very easily transferred to Staphylococcus aureus. The high abundance of Enterococci with vancomycin resistance increased the probability of this occurring.

This constant transfer of plasmids between bacteria plays a key role in their evolution. It allows a bacterium entering a new environment to steal some useful genes from the bacteria that are already there, helping it adapt to that niche. This is what happened in the cases discussed above, and is one of the more insidious methods through which antibiotic resistance can spread.
As we've seen in the previous posts, the unregulated use (and in some cases regulated use) of antibiotics in agriculture leads to the evolution of new resistant strains of bacteria. These strains can exchange this resistance using plasmids. The transfer of these plasmids to human pathogens is a major threat to human health.

Making things worse is that some plasmids can carry multiple resistance genes, rendering a variety of different antibiotics useless. The problem with using antibiotics in agriculture comes primarily from increasing the net amount of these non-pathogenic bacteria with resistance genes.

In the above case study, we have seen that Enterococci can exchange its resistance with Staphylococcus aureus. But we only know about Enterococci because on rare occasions, they can cause disease in humans. We don;t keep a track of all of the bacteria that don't cause disease. These are the bacteria that live in our bodies, that help us digest food and maintain an immune system. we are constantly exchanging these bacteria with our environmental surroundings.
They live under the radar, and nobody notices when they develop antibiotic resistance. Since they never cause disease in humans, we never need to prescribe antibiotics against them. The only time they would encounter sustained levels of antibiotics is on a farm, where they are constantly infused into the feeds of their animal hosts. Here they can evolve new resistances, and when they get transferred to humans, can exchange their antibiotic resistances with the bacteria they find in their new niche.
It is difficult for us to tell what kind of resistances an invading pathogen could potentially pick up from these bacteria.
To use an analogy, these silent bacteria may act as weapons merchants, hoarding resistances until the can share them with one of our potential enemies.
One way for researchers to investigate this is to simply take a snapshot of bacteria within an area, and just test for the resistance genes. Instead of looking for the weapons merchants, they are focussing on checking for the weapons.
With this technique, the scientists directly checked for the presence of resistance genes in an environment. This is known as the “resistome”.
Recently a group of researchers took it upon themselves to catalogue the “resistome” of three different countries. They compared the types of resistances they found in different countries to the way antibiotics were used in each of them.
The types of antibiotics that bacteria were resistant to were slightly different in each of the three countries they investigated (USA, Spain and Denmark). The antibiotics to which bacteria were most commonly resistant were the ones that were approved for use in animals. Antibiotic resistances were lowest in the places that had the ban in place for the longest time.
This all indicates that the agricultural use of antibiotics has contributed to the creation of a number of antibiotic resistant bacteria, but increased the number of resistance genes in our environment available for other pathogens to become resistant.
The mountain of evidence is indisputable. There is no doubt that new strains of antibiotic resistant bacteria owe their genesis to the reckless use of the drugs on farms. But is it fair for farms to take on the full brunt of the blame for the fall of antibiotics ? Would we not have antibiotic resistant bacteria in our hospitals even if the farms had banned them ?
I'll be dealing with this question in the conclusion of this series next time.

To be continued.....

References

Chang S., Sievert D.M., Hageman J.C., Boulton M.L., Tenover F.C., Downes F.P., Shah S., Rudrik J.T., Pupp G.R. & Brown W.J. & Infection with vancomycin-resistant Staphylococcus aureus containing the vanA resistance gene., The New England journal of medicine, PMID: 12672861

Zhu W., Murray P.R., Huskins W.C., Jernigan J.A., McDonald L.C., Clark N.C., Anderson K.F., McDougal L.K., Hageman J.C. & Olsen-Rasmussen M. & (2010). Dissemination of an Enterococcus Inc18-Like vanA Plasmid Associated with Vancomycin-Resistant Staphylococcus aureus, Antimicrobial Agents and Chemotherapy, 54 (10) 4314-4320. DOI: 10.1128/AAC.00185-10

Forslund K., Sunagawa S., Kultima J.R., Mende D., Arumugam M., Typas A. & Bork P. (2013). Country-specific antibiotic use practices impact the human gut resistome., Genome research, PMID: 23568836

TMI Friday: Perfume, is there anything it can't do ?

2013-06-14T13:51:00.000+01:00

We've all been there (I assume). You've caught the eye of someone at a party, or some other social gathering, and you've felt that instant attraction. And before you know it you've both drank yourselves stupid, and blurrily stumble towards some form of sexual activity. You'll both probably regret it tomorrow, but there'll be a lot of things you'll regret tomorrow, such as those evil drinks with the umbrellas in them. As you lock together in a clumsy lustful fumble, you realise that the emergency condom you'd kept in your wallet for the past decade had finally crumbled to dust. What do you do ?

The year is 1984, Orwell's dystopia failed to materialise, but Prince was riding high in the charts and Ghostbusters was ruling the Box Office. A 20 year old woman arrived at the outpatient clinic the day after the night of passion. She and her partner had been faced with the conundrum I described above. with no condoms available, what could they do to satiate their mutual lust ?

Luckily , one of them had a perfume bottle, which they used for *ahem* stimulation. Unluckily, during the course of this stimulation, the perfume cap came loose from the rest of the perfume bottle, and became lodged in the woman's vagina. It was for this reason that the woman had to go to hospital. After taking an X-ray of the object, the physicians gave the patient some local anaesthesia, and pulled the offending object out with a speculum. Fortunately it turned out okay for the patient.

Can we just decide right now that bottles and genitalia don't go together ?

Chapman G.W. An unusual intravaginal foreign body., Journal of the National Medical Association, PMID: 6471118

Antibiotics & Agriculture Part 3: The Spread of Resistant Bacteria

2013-06-13T12:47:00.000+01:00

The application of antibiotics to livestock has provided a boon to the agricultural industry. Unfortunately an outbreak of Salmonella showed that this application could have some untoward side effects. The farmers and veterinarians not only failed to contain this outbreak of Salmonella, but botched the antibiotic treatment so thoroughly that a multi-drug resistant strain of this pathogen emerged and spread to humans.
Such was the outcry in response to this outbreak that the government set up the Swann report, which attempted to promote more responsible usage of antibiotics. Even though the 1964 outbreak was primarily a result of improper medication for farm animals, the use of antibiotic growth promoters emerged as a specific concern. One of the sole achievements of this report was to separate the antibiotics used in humans to those used in animals, with specific restrictions on the use of antibiotic growth promoters.

Other countries experienced similar issues. An investigation in the US found that between 1971-1983, the majority of outbreaks of Multi-drug resistant Salmonella stemmed from contact with either farms or animal products. These antibiotic resistant Salmonella proved to be more lethal than their antibiotic sensitive counterparts. In 1977 the FDA decided that it was no longer safe to use certain antibiotics as growth promoters. They tried to stop front-line antibiotics such as penicillin and tetracycline being used as agricultural growth promoters. But for reasons that are unknown, they never followed up on their declarations. It is likely that the FDA simply didn't have the resources or the public support to pass such a law.

In contrast, Northern Europe had begun to implement restrictions on the usage of antibiotics in livestock. Often these restrictions consisted of allowing only one set of antibiotics for the agricultural industry and one for the medical community. But this soon encountered a major setback.
Clinicians began to encounter Vancomycin resistant strains of Enterococci. Vancomycin is often the drug of last resort, and was supposedly tightly regulated so as to prevent resistance developing. These outbreaks often occurred in hospitals, but not always. When doctors examined patients to find out where this bacterium was coming from, they found something surprising. The source of these Vancomycin resistant Enterococci infections originated from the community. The doctors redoubled their efforts to work out the source of this infection. They checked farm animals, food from shops, sewage outflows, and any other possible place where Enterococci could hide. What they found surprised them. They found this bacterium in farm animals and food sources and the sewage outflow. They found that not only were these hospital outbreaks traceable to these community sources, but there was a veritable reservoir of vancomycin strains out there that had not yet reached the hospital. But this presented a puzzle.
Vancomycin was only available to hospitals. In accordance with laws, the farms in the area were using different antibiotics. So why were these bacteria in the community, who should never have even seen Vancomycin, suddenly becoming resistant to it ?
The truth is that the bacteria had not specifically developed a resistance to Vancomycin. They had developed a resistance to a drug named Avoparcin. The vancomycin resistance was just a lucky side effect of this. You may not have heard of Avoparcin. This is because it was never meant to be used in humans. It was one of the few antibiotics allowed to be used as a growth promoter. What no-one had foreseen was that it's structure was so similar to vancomycin that it would breed resistance to it. And as a result, one of the key antibiotics to stop hospital outbreaks was rendered useless against the Enterococci.

But why should we worry about these bacteria. Enterococci aren't much of a threat outside of the hospital, and even then they tend not to have multiple drug resistances. Whilst we can worry about Salmonella, we should remember that the best ways of treating Salmonella don't require antibiotics at all. So why should the spread of these antibiotic resistant bacteria be a worry for us ?

To Be Continued.....

References

Holmberg S., Wells J. & Cohen M. (1984). Animal-to-man transmission of antimicrobial-resistant Salmonella: investigations of U.S. outbreaks, 1971-1983, Science, 225 (4664) 833-835. DOI: 10.1126/science.6382605

Bates J., Jordens J.Z. & Griffiths D.T. (1994). Farm animals as a putative reservoir for vancomycin-resistant enterococcal infection in man, Journal of Antimicrobial Chemotherapy, 34 (4) 507-514. DOI: 10.1093/jac/34.4.507
O'Brien T. (2002). Emergence, Spread, and Environmental Effect of Antimicrobial Resistance: How Use of an Antimicrobial Anywhere Can Increase Resistance to Any Antimicrobial Anywhere Else, Clinical Infectious Diseases, 34 (s3) S78-S84. DOI: 10.1086/340244
http://docs.nrdc.org/health/files/hea_12032301a.pdf

#MicroTwJC : The Creation of a Superbug

2013-06-10T02:30:00.000+01:00

The year was 2004. The patient was a 6 month old baby girl. She was about to enter thoracic surgery, when the doctors found that she was harbouring methicillin resistant Staphylococcus aureus. Now, in most western hospitals, the origin of this bacterium would not be a mystery. But this was a hospital based in the Netherlands. The Dutch have a "search and destroy" mentality when it comes to dealing with superbugs, and have been very successful at keeping their hospitals free of MRSA. They wanted it to stay that way. They had to find the source of this MRSA, and put a stop to it. The hospital equipment was scrutinised for any traces of the bacterium. None could be found.
They eliminated the MRSA from the baby, and then sent her home with her parents. But when they followed up, the baby was once again colonised with MRSA. They went through the same process again and again, until they realised that the baby was continuously being re-infected with the bacterium from an unknown source. The doctors found that the babies parents were also carriers of MRSA. but where did they get the disease from ? If it wasn't coming from the hospital, then where was it coming from ?
It turned out that the family lived on a farm raising pigs. The pigs were tested. They were the source of the MRSA.
Other pigs on different farms in that area also carried this strain of MRSA. A number of other cases of farmers and vets catching MRSA off their pigs. They concluded that farmers were 760x more likely to get an MRSA infection than any other Dutch people. Further research revealed that 39% of pigs entering a slaughterhouse carried MRSA. Hospitals in close proximity to pig farms tended to see more patients with MRSA than hospitals that were far from pig farms. This MRSA appears to be different from the hospital associated MRSA's we are more familiar with. It is primarily carried by pigs, and was a leading cause of MRSA infection in the Netherlands.
So now we know that pigs can carry MRSA, it is time to ask an important question. How did they get MRSA ? How did this particular strain evolve ? These are the questions that this weeks #MicroTwJC paper aims to answer.

So how would you go about tracing the heritage of a bacterium ? It's not like we can use census records or old photo albums to track down their ancestors. But there are records that we can use to study the genealogy of a bacterium. Every single living thing on this planet keeps a record of their genealogy within their genetic code. All you need are the right tools and talent to read this code, and the ancestry of the bacterium should unfold before you.

Just like us, bacteria inherit their genetic code from their forebears. When a bacterium reproduces, it replicates it;s DNA , which is inherited by its two daughter cells. As the DNA is replicated, errors occasionally occur in the code, changing it from its ancestors. These errors are known as mutations, and can change the functions of genes. Most importantly, once these mutations creep into the code, they stay there. If the bacteria carrying a particular mutation survives to reproduce, it's descendants will still carry the mutation.
So if we see a group of bacteria with the same mutation, we can trace that quirk of the genetic code back to a common ancestor. This can tell us a lot about how bacteria are related.

This brings us to an important point. You can't look at one bacterium and divine the history of its genetic code from nothing. You always need to compare it with other bacteria. This is crucial. In order to build a family tree for this bacteria, we need to get a good look at its family. To this end, researchers sequenced the genomes of 63 livestock associated MRSA. The researchers needed to go further, so they also looked at other closely related bacteria, such as MSSA (methicillin sensitive Staphylococcus aureus. Also known as Staphylococcus aureus) and hospital MRSA's, which are the two most likely candidates from which this bacterium evolved. They looked at 40 and 43 genomes from each of these groups respectively. They also made sure to take samples from all over the world. This allowed them to peg the evolution of each of these strains geographically.
There are a number of possibilities that could explain how this particular livestock associated MRSA evolved.

MRSA Tourism: MRSA is common in hospitals, and most of the strains we know of evolved in the clinic. It could be that a human carrier of MRSA passed it on to a pig. The pigs would have been fed growth promoting antibiotics, which would select for MRSA, allowing it to be sustained within this population. In this case, the strain would show clear indicators that it had evolved from an MRSA strain, without showing any characteristics unique to MSSA strains.
The Origin of MRSA: Perhaps an MSSA strain jumped into livestock, and became resistant to agricultural antibiotics to become MRSA. It then leapt over into man where it eventually formed the basis for the whole MRSA population that currently infest our hospitals. In this case,Livestock MRSA would show mutations that are similar to the MSSA strains that also distinguish it from the MRSA strains.
MRSA Independant evolution: Perhaps Livestock associated MRSA is completely unrelated to the MRSA's we find in humans. It is like the above scenario, only this MRSA never made the jump into hospitals. Maybe In this case it would show no similarity to any of the MRSA strains tested, but definite similarity with the MSSA strains.
Convergent Evolution in Multiple Regions: Perhaps we are not just looking at one single pandemic strain of livestock associated MRSA. Perhaps MRSA evolved in livestock multiple times in different regions. In this case, we would see that different regions have different livestock associated MRSA strains, with different origins. This would be a situation in which one or more of the above pathways of evolution could have occurred in order to reach the same end point.

Once they had all of the MRSA and MSSA genomes sequenced, they had to analyse them. They tried MLST, a technique that I talked a fair bit about in a previous #MicroTwJC. The key thing about MLST is that it looks at seven genes and using their deletion/ alteration to measure the relatedness of strains. Conceptually it is sort of similar to the example of mutations I talked about above. But there is one quirk of bacterial evolution that I didn't mention, which is the main reason why MLST is necessary.

Bacteria can steal genes off eachother, and occasionally completely lose genes as well. If you are looking at the mutations in just one gene, you have to realise that the bacterium carrying that gene may not have evolved it itself, it may have stolen it off of another bacterium. Or it may have completely lost the gene, thus giving you no information at all. MLST avoids this problem by looking at multiple genes. These genes are spaced out along the bacteria's chromosome. If a bacteria decides to get rid of one section of its genome, it means that only one of the genes you are looking at is lost and you still have the other genes to study. In fact, this gene loss works in your favour. You can now tell how closely other bacteria are related by looking at the MLST pattern they have. If a group have similar gene patterns, except for one missing gene, then they are likely to be related. If one bacteria steals a gene off another, it creates a new lineage with a distinct gene pattern. These groupings tend to be names "eBURST" groups, after the kind of statistical analysis used to discover them.

Whilst MLST is good, it only tells you about the lives of seven genes. If you want to go into even more detail, you will need to look at the whole genome sequence. Instead of looking at just seven genes on the chromosome, we can look at all of them. But this comes with some degree of problems. Because there are some portions of the genome that are constantly mutating and changing at such a fast rate that there is a high probability they will generate the same sequences due to chance. You need to focus on relatively stable genes. But if you know what you're doing, this can be really useful. If there are specific genes you are interested in, like the ones which encode antibiotic resistance, or virulence, all of that information is stored in the genome sequence.

Whole genome sequencing also allows for an unparalleled look at the exchange of genes between different strains of bacteria. Bacteria exchange genes through a variety of factors, but one of the most important are through viruses called bacteriophages (phages for short). These are parasites that insert themselves within the genome of their hosts, tricking the host into using the genetic information they encode into making new viruses. When they splice their DNA out of the host genome, they can take a souvenir. In some cases, this can include a virulence gene, or an antibiotic resistance gene. These viruses can then become inactive for generations while their host goes forth and multiplies. On occasion, these viruses can become reactivated and spread their genes to a new host.

Over the years, a number of phages which benefit bacteria have evolved, and insinuated themselves in the genomes of bacteria. Whole genome sequencing allows for these phages to be identified, and we can go some way to tracking their journey across different strains of bacteria.

They used all of this information to build up a family tree of the different strains of livestock associated MRSA, which is shown below :

The left of this diagram shows the family, and on the right we see a table giving more details of each strain. Each block of colour indicates a specific group. The grey and orange blocks on the bottom are groups of MSSA that have been found to infect humans. The green colour indicates the live-stock associated MRSA. Each successive colour indicates a further subgroup for each lineage.
Those lines of numbers at the end of each tree are the names given to each strain. These strains are the family members that were used to work this tree out. We can learn further details about them if we look to the right side of this diagram. The first column indicates each strain's country of origin with a two letter code, Shown below.

AT, Austria; BE, Belgium; CA, Canada; CH, Switzerland; CN, China; DE, Germany; DK, Denmark; ES, Spain; FI, Finland; FR, France; GF, French Guiana; HU, Hungary; IT, Italy; NL, The Netherlands; PE, Peru; PL, Poland; PT, Portugal; SI, Slovenia; US, United States

The next column notes the host of these bacteria. H is for human, T is for Turkey, P is for Pig and B is for Cow.
The columns to the right of this are a bit more involved. The next column tells us about a gene called spa. spa encodes Staphylococcal Protein A, which is one of the proteins that has been used a lot in the past to classify different strains of staphylococci. It is usually produced in high amounts, making it easy to detect. It binds to antibodies, preventing it's host from using them against it*.
The next column indicates whether these strains carry a specific bacteriophage φSa3. This phage carries a few genes that help Staphylococcus aureus survive in humans. There are other types, but we don't really need to get into them for this figure. I could talk about φAvβ phage which allows Staphylococcus to colonise birds.
The next three columns indicate what types of antibiotic resistances each of these strains have. The Tet column indicates whether the bacteria are resistant to tetracycline or not. The MET column indicates whether the bacteria are resistant to methicillin or not. The final column SCCmec tells us about the kind of genes the Staphylococcus aureus carries in order to encode these resistances.

Conclusion
So now we can understand this graph, let us take a closer look at what it tells us.

It shows that all of these livestock associated MRSA's share a common ancestor
That ancestor came from the same family as the MSSA's studied here. This knocks out "MRSA tourism" theory of evolution. At some point, a human MSSA crossed over to a farm animal, which could have been a pig or a cow.
In the course of making this transition, these MSSA's lost the φSa3, which helps it infect humans better.
These bacteria also acquired a resistance to tetracycline. This shouldn't be too surprising, because tetracyclines were amongst the first antibiotics that were discovered to be growth promoters, and they are still used by many farms. Any bacteria attempting to survive in livestock would have to develop resistance to these antibiotics.
The evidence suggests that the crossover between humans and their animals occurred in the US. After this crossover, the Staphylococci branched into two lineages.
In one lineage, bacteria acquired the φAvβ phage, which allowed them to infect turkeys. This lineage remained methicillin sensitive, and has not been known to colonise humans. This strains descendants have not been found outside the US
The other lineage acquired Methicillin resistance through different SCCmec genes on multiple occasions. Furthermore, this strain had legs. It spawned descendants that would invade Canada and Europe.
The Strains that invaded Europe evolved and diversified, until you get to the top group in pink, which began to spread to humans in Denmark.

From this information, we can see that livestock associated MRSA evolved in the US, and then made the jump into Europe, where it was eventually discovered in the Netherlands.

Because of the common ancestor, we can rule out the "Convergent evolution in multiple regions" hypothesis.

So this leaves two hypotheses standing. The "Origins of MRSA" hypothesis would require that these livestock MRSA's spread to a hospital and cause a raft of human infections. This requires is a cluster of MRSA's within the green area of this graph that cause human infections. What we actually see is an occasional human infection occurring across a broad range of lineages that infect livestock. This suggests that the livestock MRSA's have not completely made the jump back into human infections.

This leaves one hypothesis left for the evolution of livestock associated MRSA- the one that is completely independent of the MRSA's we usually get in hospitals.

So why is it that livestock associated MRSA's were first discovered in the Netherlands, and not the US ? Have the Danish MRSA strains become better at infecting humans ? Or are livestock MRSA most commonly observed there because they conduct the most thorough searches for it ? Should we even be concerned about these strains of MRSA, especially seeing as they don't tend to infect humans ? Is the intensive agriculture required to raise cheap meat worth the potential danger of creating new types of antibiotic resistant bacteria, as demonstrated here ?
These are the questions that this paper raises, and how you answer it affects us all whether we like it or not. Perhaps you think that this is reason enough to ban all antibiotics in agriculture, and food budgets across the world tighten to cope with the rising cost of meat. Or you may decide that the threat from these bacteria in livestock is overblown, and stopping the creation of antibiotic resistant bacteria is not worth the extra costs.
But always remember that as consumers and active participants in democratic society**, you have the power to choose.
But remember, that the choice you make could determine whether a dangerous bacterium in the Netherlands attacks 6 month old baby.

References

Price L.B., Stegger M., Hasman H., Aziz M., Larsen J., Andersen P.S., Pearson T., Waters A.E., Foster J.T. & Schupp J. (2011). Staphylococcus aureus CC398: Host Adaptation and Emergence of Methicillin Resistance in Livestock, mBio, 3 (1) e00305-11-e00305-11. DOI: 10.1128/mBio.00305-11

van Rijen M.M.L., Van Keulen P.H. & Kluytmans J.A. Increase in a Dutch hospital of methicillin-resistant Staphylococcus aureus related to animal farming., Clinical infectious diseases : an official publication of the Infectious Diseases Society of America, PMID: 18171259

Voss A., Loeffen F., Bakker J., Klaassen C. & Wulf M. Methicillin-resistant Staphylococcus aureus in pig farming., Emerging infectious diseases, PMID: 16485492

I've been writing a five part series of blog posts about our relationship with Antibiotics and Animals. If you liked this post, you may want to check them out. You can check out parts 1, 2 and 3 . Parts four and five will be published on the next two days that begin with a T.

*If you ever try to perform a western blot, or any other process reliant on antibodies, on Staphylococcus aureus without accounting for this protein, you will run the risk of getting the following reaction.
** Unless you are one of my readers from China. However, as most of my Chinese friends assure me, democracy is over-rated, and the leaders only ever do what's best for the health of their nation.

TMI Friday: I'll tell you where you can stick that Bratz doll !

2013-06-07T12:00:00.000+01:00

I missed the whole "Bratz" craze, on account of not being a pre-teen girl at the time it happened. But I am aware that it existed, and that there were some out there who disapproved of it. I heard that there was something of a "Bratz" controversy, because the toys were "oversexualized" and presented an unrealistic body image, as opposed to Barbie dolls. Because Barbie doesn't present an unrealistic body image or is overly sexual, obviously.

One four year old child, who showed up to an emergency room with trouble in her genital area. After questioning the child, and her mother, the surgeons could not figure out what happened. So they took an X-ray, which showed a pair of legs, curiously without feet. This clued the physicians into the brand of doll in question. Bratz Dolls have interchangeable feet, to allow different shoe designs to be used with each doll and so girls can re-enact the final scene of Saw, minus Cary Elwes performance.

It turned out that this girl had stuck a Bratz Doll up her vagina for reasons that may never become clear. I still have no idea why I ate toilet paper when I was four, and probably never will. I hadn't even learned to spell "Logic" at that age, let alone know how to apply it. The theory was that she was mimicking her mother, whom she had seen inserting a tampon. Which is probably the most damning criticism of a Bratz doll as I've seen anywhere.

I mean if the accident occurred during the course of play, it would almost be understandable. It's like getting a Boba Fett action figure lodged in your partners rectum whilst re-enacting the Sarlacc scene from Return of the Jedi, as opposed to it being the closest available object scratch their haemarrhoids. One of those reasons implies that the toy was still fulfilling its function, the other indicates that it was just a convenient piece of plastic.

Someshwar J., Lutfi R. & Nield L.S. (2007). The Missing "Bratz" Doll, Pediatric Emergency Care, 23 (12) 897-898. DOI: 10.1097/pec.0b013e31815c9dd2

Antibiotics & Animals Part 2: The First Warnings

2013-06-06T12:21:00.001+01:00

In the previous post, we were wowed by the miraculous discovery that antibiotics could improve the growth and well being of farmed animals, such as pigs and baby chicks. The use of these growth promoters enabled farmers to save money on animal feed and improve the health of their animals. Soon, nearly 50% of all antibiotic sales went to the agricultural industry. Whilst there were some concerns over this unregulated use triggering the development of antibiotic resistant bacteria, without evidence these fell on deaf ears. This would soon change.

We begin this chapter of the story at the Enteric Reference laboratory. The job of this reference laboratory was to receive and catalogue samples of bacteria obtained from intestinal infections occurring around the country. It was during the 1960's that they began to receive samples from concerned farmers.
The environments on intensive farms of this era could best be described as overcrowded factories for disease. The farmers had noticed that calves were particularly prone to getting diarrhoeal infections. The bacteria causing these infections was Salmonella typhimurium, the bacterium responsible for human typhoid disease. This was not only a threat to the health of the herd, and those who interacted with them. Calves were dying. The Salmonella outbreaks needed to be brought under control. This is where it all started to go wrong.

There were two methods that were used to put a stop to Salmonella on these farms. The first method was to use high doses antibiotics to treat visibly sick cattle. The second method was to give lower doses of antibiotics to the rest of the visibly healthy herd, to prevent them getting ill. I say "visibly" because cows can carry Salmonella without showing any symptoms, so it is likely that plenty of the cows with Salmonella received the lower doses of antibiotic.
Unbeknownst to the veterinarians, they were creating the perfect environment for bacteria to develop resistance.
Antibiotic resistant strains began to make their first appearance in the beginning of 1963, when a strain developed resistance to sulfonamides and streptomycin. A year later these bacteria had become resistant to six more antibiotics.

Soon, this multi-resistant strain of Salmonella began to spread to humans. The Enteric reference laboratory received over 500 samples of this same bacterial strain, obtained from human infections. The antibiotics that would normally used in these situations turned out to be useless. This outbreak provided dramatic evidence of the hazards of utilising antibiotics in agriculture. The UK government was forced into action

In 1969, the Swann committee convened to change the way we used antibiotics, so that this kind of outbreak would never be repeated. They recommended that a quasi-non governmental organisation (Quango) be created, which would act to oversee the use of antibiotics for both humans and animals. It was there to increase transparency, to make sure that people knew what antibiotics were being used for, and how much they were used. It would bring together the usage of both veterinary and medical antibiotics under one authority. This co-ordination would enable scientists to better understand the threat of resistance in all of its facets.
Whilst the committee’s job was to regulate the use of antibiotics in both humans and animals, it ran into a number of problems. But the various different interest groups involved in antibiotics had no compulsion to co-operate. The committee had no real power to control the use of antibiotics, nor did it have any resources to investigate the impact of antibiotic overuse. Eventually it died a quiet death, having never quite lived up to the promise of its birth.

To be Continued Next ~~Tuesday~~... Thursday...

Anderson E.S. (1968). Drug Resistance in Salmonella Typhimurium and its Implications, BMJ, 3 (5614) 333-339. DOI: 10.1136/bmj.3.5614.333

(1981). Death of a quango., BMJ, 282 (6274) 1413-1414. DOI: 10.1136/bmj.282.6274.1413-a

http://www.guardian.co.uk/society/2006/mar/22/health.science

Antibiotics & Agriculture Part 1: The Discovery of Growth Promoters

2013-06-04T18:48:00.003+01:00

This story begins with Robert Stokstad, an agricultural scientist brought up on a Californian poultry farm . He had started his career fighting against malnutrition in chicks. He had found that a haemorraghic disease in chicks was in fact caused by malnutrition. He had followed this up by examining the diet of baby chicks, to work out which parts of the diet are the most essential, and which of those, if neglected could lead to disease. He was one of the first to discover that folic acid is an important component of nutrition in chicks, before people realised it’s importance for humans.

It was at Lederle pharmaceuticals, whilst working with Thomas Juke, that he made another significant discovery about the right things to feed baby chicks. He had found during his work that feeding chicks a diet of vegetables alone was not enough. In fact, many chicks would end up dying on this diet. If they were to survive, then some degree of animal protein was needed. Other people working in his field had found that adding a small amount of “sardine meal” to the mix helped this. But then in a later paper, those same researchers, Hammond and Titus, found that mixing in cow manure produced a similar effect. Yes, you read that right, there were people feeding chicks cow manure, and found that it was more healthy than feeding them a diet of just vegetables.

It was known at the time that vitamin B12 was a key factor needed for chicks to grow, and that often the vegetable diets given to these chicks did not have enough of it. So Stokstad and Juke fed the chicks different mixtures of foods, and looked at how well they grew afterwards. One of the foods they included was a bacterium, Streptomyces aureofaciens, which they grew up and dried out and added to the feeds of the chicks. This was to work out why the cow manure turned out to be such a great dietary supplement. Stokstad knew that manure is full of bacteria, and that bacteria could produce B12. So the reason that cow manure was good for chicks was that it was a source of B12.

But Stokstad was not the sort to rule anything out. He decided to compare the potency of Streptomyces aureofaciens against B12 purified from liver extract. He found that the purified liver extract improved the growth of the chicks, nearly doubling their final weight. But when he fed the chicks Streptomyces aureofaciens , he discovered that they grew far faster and bigger than the ones fed with just the liver extract. This growth spurt was about more than vitamin B12. Streptomyces aureofaciens was producing something else that was boosting the growth of these chicks. So what was this mysterious factor which made these chicks grow up so well ?

It was a compound known then as aureomycin, and it was amongst the first tetracycline antibiotics ever discovered. It was also one of the first antibiotic growth promoters. Other researchers were also beginning to discover the benefits of antibiotics in promoting the growth of animals. The use of antibiotics as feed additives caught on like wildfire.

One of the first to express their concerns over the growth of this industry was Robert Wrigglesworth, who in 1952 wrote a letter to the British Medical Journal

We have the prospect of more antibiotics being sold in the USA, as growth promoters for food in farm animals than are used for clinical medicine.

But at the time, these kinds of concerns were brushed aside, with some justification. So what if the bacteria that infect livestock become slightly resistant to antibiotics ? The bacteria that live within pigs and chicken don’t pose a problem to the health of people, because the only time that those aforementioned bacteria could possibly come into contact with us is after being thoroughly cooked. Right ?

To Be Continued.....

References

STOKSTAD E.L.R. & JUKES T.H. (1949). The multiple nature of the animal protein factor., The Journal of biological chemistry, PMID: 18135798

Shane B. & Carpenter K. (1997). E. L. Robert Stokstad, Journal of Nutrition, (127) 199-201. DOI:

Wigglesworth R. (1952). Value of Organic Manures, BMJ, 1 (4772) 1357-1358. DOI: 10.1136/bmj.1.4772.1357-c

TMI Friday:...And the mystery of the lost piercing

2013-05-31T12:00:00.000+01:00

So let's start this month of "TMI Fridays" focussing on women by talking about penis piercings !
Let us consider the Prince Albert. The legend goes that Queen Victoria's husband, Prince Albert, was rather generously endowed, and as a result required a ring piercing on the end of his member in order to prevent it slipping down his trouser leg. This was meant to explain why Queen Victoria forced him to make so many children with her despite the fact that she despised them. As a result of this urban legend, many young men have also adopted this piercing.

It is because of this kind of penis piercing that a 20-year old psychology student decided to admit herself into A&E in 2004, in spite of not having any visible symptoms.
Earlier that day she had been intimate with her boyfriend, who possessed a Prince Albert piercing on his manhood. However, during the course of their intimacy, they found that the genital piercing had been lost. Here we come to the central mystery. Where did it go ?

They assumed that it had been unfastened in the student's vagina, and this is why she went to A&E immediately. The Doctors examined her vagina, but it was perfectly healthy and there was absolutley no sign of the piercing. The couple insisted that it was somewhere in the vagina, and the physician performed an X-ray to appease them..
To his surprise, the piercing was found to be high up in the pelvis. So the attendant physician decided that it would be necessary to perform major surgery to remove the Prince Albert ring from her.
But then the couple were seen by another physician, who asked them an important questions about whether the couple had utilised any other orifices for intercourse. It turned out that they had performed oral sex in the course of their intimacy. This was the key to the mystery.

You see, whilst the pelvis of a lady contains their vagina, it also contains their digestive tract. The physicians realised that their initial examination of the patient's vagina was correct. The ring wasn't there. The ring had been accidentally swallowed during oral sex, and had ended up in the intestine. The reason it appeared on the X-ray was that it was working its way though the gut. The piercing would pass naturally through the gut without causing any problems. To the relief of the couple, no invasive surgery would be needed.
Another X-ray performed a week later confirmed that it had passed without incident, and was discharged.
This is another example about how being open and truthful with your doctors can prevent you needing invasive surgery, and other stories have shown what happens when people are too embarrassed to do this.

Das G., Rawal N. & Bolton L.M. (2005). The Case of the Missing “Prince Albert”, Obstetrics & Gynecology, 105 (Supplement) 1273-1275. DOI: 10.1097/01.AOG.0000160487.59892.d8

TMI Friday, Girls Month

2013-05-31T11:00:00.000+01:00

Regular readers of the TMI fridays on this blog may have noticed a worrying trend. I didn't notice it until I came across a post on twitter:

@funky49 LMAO - I love that stuff! All the posts were of men's antics...I've no doubt that women have a few Darwin Awards up their...sleeve.
— Mary(@SRQ2U) May 17, 2013

I didn't notice this at first, and I looked through my back catalogue to disabuse this notion, and realised how spot on this tweet was. At first, I thought that maybe there weren't any stories about women's antics out there. But I immediately knew this was wrong, because I had a couple of stories about women on my reject pile. That was when I realised. I've been sexist about the stories I've been telling.
I'll give an example.

I read a story about a man cheating on his wife, and how in retaliation, she slipped a wedding ring over his penis, causing a condition known as "penile" strangulation, and I found this story hilarious. When hearing a story of genital mutilation with the genders reversed, I suddenly felt very ill.

I realised that I had a mental block that could be summarised like this :

"Violence against women = Not funny"
So when I read a story that could be genuinely be funny about some poor girl injuring themselves whilst masturbating, I don't generally feel like discussing it. But this has to change. I've been missing out on a rich vein of weird and embarrassing stories because of my prejudices.
To redress the balance, for the next month, I will be posting "TMI Friday" stories focussing on stories women, because men don't have the monopoly on stupidity, even if it sometimes feels that way.

Can a bacterium help you lose weight ?

2013-05-27T14:18:00.000+01:00

Yes, it's called Cholera. There, story over. Nothing to see here.

All right, I'll try to leave the snark aside for this story. It's just that every time I look at a story extolling the virtues of a new weight loss treatment based only on data from mice, I get slightly sceptical. But let's take a look at this new piece of research published in PNAS this week and give the research a fair hearing.

The bacterium that is the focus of this research is Akkermansia mucinophila. This bacterium was discovered in 2004 in the guts of humans, where it was suspected to degrade gut mucus. It is one of the "friendly" commensal bacteria which live in the diverse ecosystem of your gut.
A question that has interested a number of researchers worldwide is whether the type of bacteria that inhabit your gut ecosystem affect your weight.
I have mentioned in one of my earlier posts about how researchers can study obesity, through the use of genetically modified mice which don't produce a hormone (called Leptin) which acts to signal to them when they have too much fat and tells them to stop eating. With this gene turned off, mice eat uncontrollably and become fat. Early studies showed that these "Super" fat mice had a different gut ecosystem to the normal mice. Interestingly, if you were to take the "gut ecosystem" of a fat mouse, and gave it to a thin mouse, the thin mouse would start to gain weight. Unfortunately, the researchers didn't show the reverse effect occurring. But it was a promising start.
In other work using humans, the gut ecosystem of a fat person has been shown to be different to the ecosystem of a thin person. In these studies, one bacterium tended to stand out. Akkermansia mucinophila, whilst a key component in the healthy digestive system, is rarely found in obese people.
What is it about this bacterium ?

The researchers found that Akkermansia had a lower population within the gut of Leptin deficient mice compared to healthy mice. I mentioned in the previous research that people looked at leptin deficient mice. Whilst these mice are fat, they aren't like most fat humans. Only ten people have ever been shown to have a genetic leptin deficiency. So these leptin deficient mice (called ob/ob) aren't like normal overweight humans.
A more relevant model would be to feed mice fattier food. The equivalent of moving a human from salads to hamburgers. So they moved mice from the normal standard diet to a high fat diet, which tends to make them gain weight. As a result, the numbers of Akkermansia in these mice was lower than mice fed on a healthy diet.
But what would happen if you tried to increase Akkermansia numbers within the gut of a mouse when you start to feed it high fat food ?

They mixed in a prebiotic compound with the high fat diet to nswer this question. The compound they used was Oligofructose. This compound is known to increase the amount of mucus producing cells in the gut. Thus, it increases the amount of mucus. Since Akkermansia eats mucus, increasing the amount of food available for it causes its population to increase in the gut.

The mice fed on the high fat diet with the prebiotic added did not gain as much weight as the mice on a diet without the prebiotic.
Further more, the researchers noted that there were less inflammatory signals from the intestines from mice fed on the prebiotic than mice which were not. Why were the researchers interested in this ?

They were interested, because of something called Metabolic Endotoxemia.
The story goes like this :

Fat cells play an important role in regulating the energy balance of the body, telling you when you have had too much to eat, or when you have had too little.
In a number of studies, Obese people have been shown to have muscle and fat cells that send out unhealthy levels of inflammatory signals.
The increased levels of fat in the blood can be helpful to the immune system during infection, but if the fat levels in the blood remain high for too long, they keep the immune system activated, which leads to chronic inflammation.
This inflammation leads to the cells of the immune system to send out signals to eachother. The signalling between cells that regulate the immune system and those that regulate your metabolism often interact with eachother in normal situations. But in this case, the signals interfere with eachtoher, leading to some cells to stop producing insulin, which leads to diabetes.

Metabolic Endotoxemia can lead to the cells responsible for regulating the energy balance of you body to become less effective. It not only ruins a persons ability to control their own blood sugar, it also affects their ability to regulate their body fat and energy expenditure.

So with that, we have to look a the next experiment the researchers did. They took groups of mice, and fed them concentrated cultures of Akkermansia every day for four weeks. Mice were either fed on a healthy diet, or the High fat diet.

They found that once again, mice fed with a high fat diet and Akkermansia gained less weight,and reduced the signs of inflammation caused by a rapid increase in weight. But how do we know whether this is the right kind of weight loss ? What if the mice are losing weight just because these treatments are making them ill ?

To work this out, they had to look at where exactly the mice are losing weight from. Were the fat stores being depleted, or were the mice losing muscle mass instead? So they used a neat piece of technology known as a body composition analyser. The body composition analyser is a tool to analyse the different chemical forms of fat and muscle within the body. They can put a live mouse in the machine and get a reading for the levels of fat in proportion to "lean tissue", and then take it out unharmed. So you can watch exactly what type of fat is accumulating within the mouse throughout the experiment. On all of these scales, the mice who were fed Akkermansia not only put on less fat, but had a body composition similar to healthy mice.

Unfortunately, I don't exactly know what a sick or starving mouse's body composition would look like to compare with these graphs. In fact, despite the fact that they allegedly took measurements every day, they only display the final time points, and seem to show the kind of data that could only be obtained from dissection rather than the use of a body composition analyser.

But fat in itself isn't the most damaging part of obesity. As I've indicated before, Metabolic Endotoxemia can lead to diabetes. They showed that inflammation can occur after this high fat diet. But did the mice show any signs of developing diabetes.

This is why they tested fasting hyperglycaemia. What is fasting hyperglycaemia, and why do we care about it ?
In normal people you see hyperglycaemia occurring immediately after they have eaten. If a person has not consumed any food for a significant amount of time and is still hyperglycaemic, then it indicates that they cannot control the levels of sugar in their blood. In essence, it is a key sign of diabetes.

So they starved mice on these diets, and then tested to see whether they were still hyperglycaemic after a set amount of time. Mice provided with a high fat diet tended to show fasting hyperglycaemia, but the introduction of Akkermansia changed this. It appeared to lower the amount of sugar in the blood of mice fed on the high fat diet, although not the the levels of healthy mice.

So how were the mice suddenly able to control the levels of glucose in their blood ?
It comes down to insulin. When the liver senses this hormone, it soaks up the excess glucose from the blood, and converts it to glycogen. When there is a drop in the blood glucose levels, the liver converts glycogen back into glucose. In unhealthy animals, the conversion of glycogen into glucose is less well regulated, and leads to more glucose being released into the blood.
They tested the mice to see how well they reacted to insulin. Mice that don't react at all tend to become diabetic. So it is a good sign that the inclusion of Akkermansia into a high fat diet tended to give the mice a better reaction to insulin than if mice were fed with a high fat diet only.
All of these bits of evidence seem to show that Akkermansia can somehow prevent weight gain and hamper the progression of diabetes in mice fed with fatty foods.

So the next part of this research is where is gets complicated. You see, the storage of fat in your body is a dynamic process. You have special fat storing cells known as adipocytes, which can differentiate and change and adapt to changes to the way you eat. The amounts of lipids they manufacture, just for the day to day uses in the body, change depending on diet. If you have a high fat diet, they don't manufacture as much fat. They also can oxidise the fats they store, and break them down. So the researchers took cells from the fat layers of the mice, and tested them to examine what they were doing.
The adipocytes of mice fed a high fat diet tend to differ from those fed a healthy diet. But when you add Akkermansia into the mix, in nearly all cases, it make the adipocytes behave as if they were in a healthy mouse, even if the mouse they are residing within is eating a high fat diet. So somehow, Akkermansia can controlling the way fat is stored within the body.

The researchers suspected it may have something to do with the way the intestine defends itself from infection. Some cells within the intestine can produce chemicals to kill off bacteria, known as Antimicrobial peptides. But they found no statistical differences between any of the treatment groups in terms of antimicrobial compounds.
They looked at the cells that produce these compounds, and found that they were more highly activated in mice fed on a healthy diet when exposed to Akkermansia. But Akkermansia produced no such effect in mice fed on a high fat diet.

There is a theory that the gut microbiota can regulate fat levels by communicating with the intestine via compounds known as endocannabinoids.

They examined the levels of three different endocannabinoids, 2-palmitoylglycerol (2-PG) 2-oleoylglycerol (2-OG) and 2-Arachidonoylglycerol (2-AG). These endocannabinoids are signalling compounds which can change the way the intestine absorbs certain compounds.
The levels of these compounds were much higher in the mice which were fed Akkermansia in groups fed on the healthy and the fat diet. This suggests that the Akkermansia is in communication with these cells in the intestine, and somehow changes the way they absorb molecules.

They then looked inside the gut itself. They wanted to see whether the Akkermansia affected the thickness of the mucus layer within the gut. This acts as a barrier to pathogens, and protects the living cells on the guts surface, whilst still allowing nutrients through. The increase in mucus thickness could impair the guts ability to absorb nutrients. This layer is thinner in mice fed on a high fat diet, but when those mice are instead given Akkermansia, the mucus layer becomes thicker.

So this draws us to the final set of experiments. They know that Akkermansia is needed to for all of these things to occur, but does it need to be alive ? This a chance for them to test how active Akkermansia is in regulating fat deposits. They repeated all of their previous tests, but this time with an additional control group. One group were fed a high fat diet, with dead Akkermansia as the supplement. The dead bacteria did nothing.

Paper Readability
I would heartily recommend you read this paper, because why should I be the only one the suffer ? The main problem with this paper is that has been horribly compressed to fit the space allocated to it by the journal, and that a lot of the juiciest results are in the supplementary section. It is because of this that it is really difficult to understand what the researchers did. The methods section has been chopped up between the supplementary section and the actual paper. They only mention the whole body fat analyser in passing, and its unclear whether any of the data they show comes from using that piece of equipment.
It is a complex paper, and it really needs more space than it was given.

Conclusions
There is a great story at the centre of this article.

Once upon a time there was a bacterium in your gut called Akkermansia mucinophila. It lives in the protective mucus lining of your gut, and feeds on it. It always made sure there was enough delicious mucs by telling the cells of your gut to keep making it. This made the mucus layer nice and thick, which was not just good for Akkermansia, it was good for you. That thick mucus layer stopped harmful bacterial components entering your body, and reduced the amount of fat entering your blood stream.
But then you decided to eat your own weight in milkshakes every day for nine years, and everything went downhill from there. The poor Akkermansia couldn't survive in these conditions, and eventually became extinct in your gut. The protective mucus layer in your gut that it had so diligently maintained, became thinner. Harmful bacterial components entered your blood stream, alongside big globs of fat, giving you metabolic endotoxemia. Your fat cells and you immune system couldn't cope, and went to war. The cells that produce insulin were killed in the crossfire. That is when you became diabetic, and it is why you died after one particularly grotesque Oreo milkshake binge. Witnesses say that your last words were "Totally worth it"

The researchers have shown that Akkermansia could reduce some of the negative outcomes. They have shown that in mice at least, it can reduce insulin sensitivity, and possibly slow down the progression to diabetes. It does this through maintaining the mucus layer in your gut wall for completely selfish reasons.
Hypothetically, this causes the gut to be less absorbent to fats to bacterial compounds.
But whilst this paper produces an interesting case that there is some communication occurring between the bacteria and the gut, it doesn't tell us anything about how this happens. We still don't know what the signals are between the bacteria and the gut wall, and even the immune system.

So, can Akkermansia help you lose weight ?

No. There is no evidence in this paper to suggest this. All of the tests looked at the reduction in weight gain. Changing the amount of weight you gain is quite different from weight loss. But Akkermansia is still an interesting example of how the bacteria that coexist within us act for both their own selfish reasons, and for our benefit.

Everard A., Belzer C., Geurts L., Ouwerkerk J.P., Druart C., Bindels L.B., Guiot Y., Derrien M., Muccioli G.G. & Delzenne N.M. & Cross-talk between Akkermansia muciniphila and intestinal epithelium controls diet-induced obesity, Proceedings of the National Academy of Sciences, DOI: 10.1073/pnas.1219451110

TMI Friday: Batteries should NOT be included

2013-05-24T12:24:00.000+01:00

There are some days when you have to ask yourself, just what is the deal with men. I don't mean our general demeanour, or the pretensions of superiority over other genders. No, I'm talking about the strange things that men decide to do when left alone for too long.The men who happily dangle their members inside bottles, or in reach of the spinning blades of a vacuum cleaner not thinking of the consequences.
With this in mind, we arrive at a case study by Bedi et al, where they describe the case of an elderly gentleman who was referred to them from a nursing home. He had been experiencing extreme pain during urination. A year previously, he had a similar problem. On that occasion, it was found that he had stuffed a pen lid inside his penis.
Now, one shouldn't make assumptions about people. Just because he had abused the elasticity of his urethra before was no reason to suspect that it had happened again. He could be experiencing difficulty urinating for a whole number of important medical reasons.
Then he pissed out a rusty Triple A battery.
And he still had trouble urinating ! This is when the nurses at the home realised that they needed to get help from doctors.
An X-ray revealed the source of the problem. Two batteries were still lodged in the man's urethra. Meanwhile one of the residents of the home for the elderly is wondering why the remote stopped working.
So how do you pull these batteries out ? The surgeons decided to use special endoscopic forceps to pull out the batteries. Endoscopic forceps are essentially long tubes with jaws on the end (for grasping things).
They had to insert these forceps up the urethra to grasp at these batteries.
Upon being presented with these batteries, the man admitted to jamming these batteries up his urethra four weeks earlier whilst masturbating. The train of thought which led him to put lead in his pencil may in fact be similar to all of the other male "thrill seekers" who decide to risk their genitalia for the sake of sexual gratification. But even after reading all of these articles, that trainwreck of thought is completely unknown to me.

Bedi N., El-Husseiny T., Buchholz N. & Masood J. (2010). 'Putting lead in your pencil': self-insertion of an unusual urethral foreign body for sexual gratification, JRSM Short Reports, 1 (2) 18-18. DOI: 10.1258/shorts.2010.010014

The Even Earlier Discovery of Antibiotic Resistance

2013-05-21T12:00:00.000+01:00

So about a month ago, I wrote about how amazing it was that penicillin resistance was discovered as early as 1940, two years before it went on general sale. But whilst researching that article, I realised that Sulphonamide drugs entered the market long before penicillin, with their discoverer, Gerhard Domagk, being nominated for a Nobel prize in 1939. He had been tasked by Bayer pharmaceuticals to test out a gargantuan number of dye molecules to see whether they could kill off bacteria, and in the process , stumbled across the first antibiotic.
You may recall from the previous instalment that Heinrich Hoerlein was the man to recruit Gerhard Domagk into Bayer. Heinrich Hoerlein was a talented chemist, who had specialised in developing dyes for wool. How did this dye maker end up working to create one of the most important pharmaceuticals that the world had seen up until that point ? Why was it that when Bayer decided to devise new treatments against bacterial disease, they focussed on the compounds used to colour clothes ?

To understand how this state of affairs occurred, we need to go back further in time to the 1800s, and look at bacteria. If we were to do this in this era, we would need to get a good microscope. Bacteria tend not to be visible to the naked eye. So let us look down our microscope, what would we see ?
We would probably see tiny transparent blobs. This could mean that we either have lots of bacteria, or are looking at some air bubbles. There are various legends of scientists proclaiming that they have found an entirely new type of bacteria, only to later realise that this bacteria was nothing more than a bubble of air in the wrong place at the wrong time.
This is where dyes become important. The odds are that you are wearing clothes which have undergone the dye process. A dye works by chemically binding to the surface of whatever you want coloured.
A number of scientists of that era began to work on dyes that bind to cells, allowing them to be more easily seen under a microscope. This allowed scientists to see that bacteria came in all sorts of shapes and sizes, and that different species were associated with different diseases.
One of the pioneers of this research was a man named Paul Ehrlich. His PhD had been dedicated to studying how aniline dyes, which had previously only been used for fabrics, could actually be used to colour cells. He also noticed that dyes he used would colour some cells differently to others. Some cells would take up a lot of the dye, whilst others would not and remain transparent. At the age of 24, using the new "staining" techniques, he had managed to discover a new type of cell, known as a Mast cell.
A lot of his scientific achievements could be tracked down to the one simple question he asked himself when he saw this effect: Why did some cells take up more dye than others?
He theorised that cells took up specific nutrients, and that receptors on the surface of these cells played a crucial role in this process. Different cell types have different receptors on their surface. some of these receptors allow dye molecules to enter the cell, and some do not. The reason that different cells "stained" differently was due to the different receptors on their surfaces.
He suggested that toxins on the surface of the bacteria bound the receptors on the surface of the host during infection. In response, the host cell would secrete these receptors to flood the toxins on the surface of the bacteria, thus neutralising them. He called these secreted receptors antibodies.
Whilst this is far from our modern understanding of antibodies, it was a crucial step in the right direction, and he would be credited as one of the founders of immunology as a result of it.
He also suspected that bacteria also had receptors which they used to ingest dye molecules. He noted that some dyes were taken up by bacterial cells, but not human cells. He suggested that this was because the dye molecules resembled nutrients that the bacteria eat. If he could manufacture the chemical structures of these dye molecules to include poison, then he could have a chemical that kills of bacterial cells, and leave human cells alone. He termed these chemicals "Magic Bullets".
In 1904 came his first breakthrough, with a compound, known as Trypan Red, due to it's colour, and sucess for treating mice infected with trypanosomes. Whilst this was useful as a proof of concept, Trypan Red only worked against the types of trypanosomes that infect mice, but not those which attack humans
It was while he was working on this problem that researchers at the Liverpool School of Tropical Hygiene, Anton Breinl and Harold Wolferstan Thomas, discovered that a compound known as Atoxyl, though to be non-toxic for humans, could kill off trypanosomes. From 1906, a number of expeditions to Africa took it with them to protect themselves from the Sleeping Sickness caused by these organisms. Robert Koch, one of the founders of microbiology, used it to treat patients on the shore of Lake Victoria. It became incredibly popular at the time.
Intrigued, Paul Ehrlich investigated this wonder drug, in addition to the other dye based drugs he was developing. During the course of his research, he noticed a worrying trend. After prolonged therapy with these drugs, the resistance of the trypanosomes to these chemicals increased, until they were completely resistant to the therapy. He coined the term "fastness" to describe this trait in bacteria. The fact that he had observed this "fastness" occurring in response to such a broad range of chemicals suggested to him that this was an inevitable event.
Ehrlich was unexpectedly energised by this discovery of resistant organisms. This was because of the finding that once an organism became resistant compound, it was also resistant to chemicals with the same shape and structure. This provided evidence for his fledgling theory of surface receptors which bind to specific chemicals based on their shape and structure.
But his discovery of resistance put him on a crash course with Robert Koch, who had not observed this effect, and thus disputed that it had ever occurred outside of the lab. The main differences were that Robert Koch used much higher doses of Atoxyl than Ehrlich. Either way, Atoxyl was fast falling from popularity. Patients treated with it would go blind due to its severe side effects. A study in 1910 would show that it merely halted progression of trypanosome disease, and that patients were no better off using it.
Ehrlichs lab was still screening drugs to fight off pathogens, and it came across compound 606, a derivative of atoxyl that not only had less severe side effects, but had proven utility against syphilis.
This was marketed as Salversan, and became an important drug in the fight against syphilis, and was used up until the 1940's, when penicillin replaced it.
The discovery of these drugs, and the apt demonstration that dye molecules could make good antibiotics cemented his place in history. One of his assistants, Wilhelm Roehl, would go on to head a research department at Bayer. It was he who would recruit a Dye chemist, Heinrich Hoerlein, to find the next big drug.
So whilst Ehrlichs theory of "magic bullets" would live on, and laid the foundations for the discoveries of Domagk and Fleming, what happened to his theories on antibiotic resistance ?
There were a number of weaknesses in Ehrlichs theories that a number of researchers called into question. Ehrlichs theories of antibiotics and antibiotic resistances were too simplistic. He posited that for each compound, there was a single path to resistance through the mutation of a single receptor. But we know that bacteria and other pathogens can adapt to antibiotics in multiple ways. This made it difficult to replicate his results, and even more difficult for him to explain why they didn't replicate. The rules he had set out for explaining antibiotic resistance did not always hold true. The disparity observed between his experiments of Atoxyl, and of Kochs experiments did not help his case.
This apparent wooliness would mask the threat of antibiotic resistance as a merely theoretical phenomenon. It would for the next 30 years be regarded as a curiosity that would never pose a threat to people.
Over a hundred years after Ehrlich's initial observation of antibiotic resistance, we have a slightly different perspective on his discovery than his contemporaries.

References

Gradmann C. (2011). Magic bullets and moving targets: antibiotic resistance and experimental chemotherapy, 1900-1940, Dynamis, 31 (2) 305-321. DOI: 10.4321/S0211-95362011000200003

Titford M. (2010). Paul Ehrlich: Histological Staining, Immunology, Chemotherapy, Laboratory Medicine, 41 (8) 497-498. DOI: 10.1309/LMHJS86N5ICBIBWM

Casanova J.M. (1992). Bacteria and their dyes: Hans Christian Joachim Gram, Historia de La Immunologia, 11 (4) DOI:

Ehrlich P. Address in Pathology, ON CHEMIOTHERAPY: Delivered before the Seventeenth International Congress of Medicine., British medical journal, PMID: 20766753

Kaufmann S.H.E. (2008). Immunology's foundation: the 100-year anniversary of the Nobel Prize to Paul Ehrlich and Elie Metchnikoff, Nature Immunology, 9 (7) 705-712. DOI: 10.1038/ni0708-705

The Earlier discovery of Antibiotic Resistance

2013-05-20T12:00:00.000+01:00

A couple of weeks ago, I wrote about how quickly penicillin resistance was discovered not long before it was distributed to the public, and how even Alexander Fleming noted his worries over penicillin resistance in the closing of his Nobel prize acceptance speech.

But even in the process of researching this article, I realised that I was merely scratching the surface. You see penicillin was not the first antibiotic discovered. If I want to talk about the first discovery of antibiotic resistance, then I will need to tell this story as well.

In 1932 in Germany, a scientist patented an incredibly important discovery, one that would eventually win him the Nobel prize.

Domagk had been working at Bayer pharmaceuticals at the time of his discovery. In the early 1920's, Bayer had begun to experimenting with different methods for treating bacterial diseases. The experiences of World War 1 had left many researchers with the desire to find ways of preventing deaths from wound infections. Domagk had served in World War 1, and had worked in a cholera hospital near the eastern front. He noted the seeming futility of treating patients with infections.

He came to the attention of Bayer pharmaceuticals after Professor Heinrich Hoerlein* had come across his thesis and decided to hire him. Hoerlein believed that dye molecules could be the key to solving bacterial infection.

The chemists at Bayer would synthesise new chemicals, and then send them to Domagk, and he would then test them on whether they could kill bacteria in vitro, or whether they could prevent mouse deaths from Streptococcus infection. Domagk managed to speed up this process to the point where he could test 30 new chemicals every week.

The chemist on the other end of this process was a man named Josef Klarer. He was the one rushing to make the chemicals for testing. He had tried a number of quinine derivatives, but had no luck. However, in 1932, things would change when he decided to make products based off of Azo Dye molecules. His first success came with Kl-695**, which Domagk found to protect mice during an infection, even though it didn't seem to kill the bacteria in the petri dish. But based off of this finding, Klarer modified Kl-695 again and again. Until it came to a red dye compound that was at the time named Kl-730.

Of course, even though this chemical had been proven in mice, it was as of yet unknown whether it would work in humans. But then Domagks daughter fell ill with a streptococcal disease, and desperate, he gave her a dose of the drug, curing her of the disease.

By 1935, Prontosil Red was being trialled internationally, with Leonard Colebrook, himself a frequent experimenter with antibiotics, demonstrating the effectiveness of Prontosil Red in treating pregnant women, albeit with the side effect of turning his patients bright red. Prontosil Red was the first Sulphanilamide drugs.

Such was the success of this drug that he was nominated for a Nobel Prize in 1939. However, at this time the Nazi's were running Germany. They held a dim view of the Nobel prizes due to the previous German to win a prize. Carl von Ossietzky was a pacifist, who exposed the Nazi's breaking of the treaty of Versailles by training an air corp, and won the Nobel peace prize for his opposition to the Nazi's. As a result of this, the Nazi's forbade any German from accepting Nobel prizes

So when Domagk won a Nobel prize, he was immediately thrown in jail for a week by the Gestapo. This was enough to convince him not to accept the Nobel prize until 1947, two years after Fleming.

By this time, Doctors were already discovering the limits of antibiotics. A.J. Cokkinis wrote in 1938

Inadequate dosage and too short a period not only fail to do any good but seem to lead to the development of acquired resistance on the part of the organism to the drug

Amongst the first to analyse these limitations were a group of researchers based at St Mary's, one of whom was Alexander Fleming**. They had discovered that bacteria could adapt to antibiotic concentrations. The same year, Connor Macleod, a researcher based in New York, investigated this in more detail. He discovered that gradually increasing the amount of antibiotics in broth could increase the numbers of resistant bacteria.

Sulfa drugs like Prontosil Red changed the way medicine worked, and laid down the foundations upon which modern medicine would arise. Unlike penicillin, Prontosil and the related sulphonamide and sulphanilamide drugs could be created entirely synthetically from available chemicals.

Bayer's technique for finding drugs could best be compared to throwing spaghetti against a wall until it sticks, testing random chemicals until they produced the effects they wanted. and people say that Alexander Fleming relied on luck ! Bayer appeared to be basing its company policy on it.

But the question remains as to why they decided to use dye compounds as antibiotics, how did they even know it could work. It's not like there was someone before them who discovered antibiotics even earlier...was there ?

To be continued tomorrow........

References
Wollheim Memorial- Phillip Heinrich Hoerlein
Bayer- Gerhard Domagk
Nobel Prize- Gerhard Domagk

Bentley R. (2009). Different roads to discovery; Prontosil (hence sulfa drugs) and penicillin (hence β-lactams), Journal of Industrial Microbiology & Biotechnology, 36 (6) 775-786. DOI: 10.1007/s10295-009-0553-8

Macleod C. & Daddi G. (1939). A ''Sulfapyridine-Fast'' Strain of Pneumococcus Type 1, Proceedings of the Society for Experimental Biology and Medicine, 41 69-71. DOI: 10.3181/00379727-41-10575P

Cokkinis A.J. (1938). SULPHONAMIDE CHEMOTHERAPY IN SURGICAL INFECTIONS--I, BMJ, 2 (4059) 845-847. DOI: 10.1136/bmj.2.4059.845

Gerhardt Domagk: The First Man to Triumph Over Infectious Diseases By Ekkehard Grundmann

* Heinrich Hoerlein would eventually rise up to the managing board of IG farben, which was the conglomerate which ran a number of companies, including Bayer. Originally, it was primarily a dye making company. But it's activities during World War 2 were infamous. It was the company that developed Zyklon B, in the time that Hoerlein served on its board, which is why he found himself at the Nuremberg trials alongside many of the other company directors. It didn't help that at least one of these directors had been conducting experiments at Auschwitz under the direction of the SS. These experiments involved inducing artificial infections deliberately, and then giving the test subject antibiotics to cure the disease. Heinrich Hoerlein was amongst a number of IG Farbens executives who tried to stop the supply of these chemicals once he had found out what the Nazis were doing with them. When this came to light, the charges were dropped, but the reputation of IG Farben never really recovered, and the conglomerate didn't last long after the war, although some of it's constituent companies are still around today.

** Unfortunately the original paper is locked in the vaults of the Lancet, and so I am forced to diminish his role in the discovery of Antibiotic resistance, because there is no way for me to find out exactly what he did.

TMI Friday: Taking it to third base ..literally

2013-05-17T12:32:00.000+01:00

The variety of foreign bodies in the rectum tests a surgeon's ingenuity to solve a myriad of geometric puzzles

So begins Major PT Mcdonald's 1976 paper, in which he has to deal with a patient with a somewhat unique problem.
The patient, a 49 year old baseball fan, who had serious trouble with his bowels ever since the Oakland A's won the world series in 1974. The doctors examined him, and noticed

" a firm, fixed, round object barely palpable which was lodged high in the rectum"

It was a baseball. To celebrate the Oakland A's victory, he had his sexual partner force the hardball up his rectum, where it got lodged. Unable to get any purchase on the surface of the ball, retrieval seemed impossible.
Thus it was left to the surgeon to figure out how to get the ball out. They drained the man's bladder using a catheter to take some of the pressure off the baseball. They tried to hook the ball, and drag it out as you would a particularly large fish. But this anal fishing expedition was for naught, as they only managed to rip some of the skin from the baseball.
They then decided that perhaps a better way of extracting the ball was through using obstetrics forceps. For those of you who don't know, these are generally used to deliver babies. So they pumped a little bit of air around the baseball, and tried to use the forceps to grab the ball.
It didn't work. They realised what the problem was. The baseball had travelled up through the pelvic arches, and after it had done so, it had become swollen with fluid, and become lodged in the pelvis.
It was a dire situation. The surgeon decided to cut into the man's abdomen to get access to the baseball. It was still stuck fast, and he needed to get some grip on the surface. So he skewered the baseball with a corkscrew, and tried to use it to pull it out. It still wasn't enough. So he got an assistant to stick their fingers up the patients arse from the other end whilst also pulling on the corkscrew, and with " a force enough to lift the patient off the table", popped the baseball out.

McDonald M.P.T. & Rosenthal C.D. (1977). An unusual foreign body in the rectum—A baseball report of a case, Diseases of the Colon & Rectum, 20 (1) 56-57. DOI: 10.1007/BF02587455

TMI Friday: Using a Bottle for a Throttle

2013-05-10T14:32:00.000+01:00

Today we once again must again take a look at men who take incredible risks in order to find new and grotesque methods of masturbation. You have been warned.

This week, the object of their fascination is.. the plastic bottle.

In the grand scheme of things, at least the plastic bottles don't have spinning blades inside them, so in theory, these individuals are better off than those who turn on the vacuum cleaner for stimulation.

The first case we shall be examining comes from 2004, when a 27 year old man in India was admitted to hospital with a peculiar problem. His penis was stuck in a hard plastic bottle. There apparently were no attempts at an excuse, just the simple explanation that he had attempted to use it for masturbation. They called in the hospital carpenter to cut away the bottle *very* carefully using an Iron cutting saw. After 15 minutes of struggle, the bottle was removed.

In 2009, a 77 year old man in Singapore was admitted into hospital with complaints of blood in his urine, and difficulty urinating. Initially he wasn't forthcoming about his case history, for reasons that will soon become clear. You see, one week previously he had pushed a 1.5 litre bottle over his genitals, and got stuck. Over the next 3 days, he managed to cut away most of the bottle. But he still couldn't remove the neck of the bottle, despite attempts at lubricating it with soap. The surgeons managed to pry off the bottle neck with scissors, and they managed to repair some of the damage, but he died 3 days after admission.

The third case we will look at came from 2010 , and also occurred in India. A 47 year old man came in 14 hours after attempting to masturbate himself with a plastic bottle. That in itself is not the hair raising part of this case. We are told that the bottle neck was placed in such a position that it was impossible to access with a normal cutting device. So what did they use ? A soldering iron. Think about that. They mitigated the heat somewhat through adding cold saline in order to regulate the temperature. Still, it's not exactly a pleasant thought.

The final case I'll be talking about involves a 58 year old man. His flatmate called an ambulance for him after recognising that he was behaving oddly. But he sent them away, claiming that he didn't have anything wrong with him. Two days later his flatmate found him dead. The autopsy revealed that his genitals had been constricted with a plastic bottleneck. This bottleneck had cut off the circulation to this region, and allowing parts of his genitals to begin decaying. This lead to bacteria entering the bloodstream, and eventually caused multiple organ failure.

So how could these people stick their members into such small openings, and then get stuck. In order to answer this, we must examine how the penis works. Essentially it is a balloon filled with blood. To become erect, arteries dilate in order to increase blood flow. However, if the veins that take the blood out of the penis are constricted, by say, a plastic bottleneck, then blood takes longer to escape, and so it swells up and gets stuck. This can actually be very dangerous. when the circulation is cut off, it can become gangrenous and in severe cases of penile strangulation, the only option is amputation. this is not even the worst case scenario, as we have seen, if this is not dealt with as soon as possible, then there is a risk of death.

Penile strangulation appears to have a higher body count than men who stick their appendages into the whirling blades of a vacuum cleaner !

References

Jain S., Gupta A., Singh T., Aggarwal N., Sharma S. & Jain S. (2004). Penile strangulation by a hard plastic bottle : A case report, Indian Journal of Surgery, 66 (3)

Ooi C.K., Goh H.K., Chong K.T. & Lim G.H. Penile strangulation: report of two unusual cases., Singapore medical journal, PMID: 19296009

Shamrao Kumbhar U., Dasharathimurumu & Bhargavpak (2011). Acute penile incarceration injury caused by a plastic bottle neck., International Journal of Biological & Medical Research, 2 (4)

Morentin B., Biritxinaga B. & Crespo L. (2011). Penile strangulation: report of a fatal case., The American journal of forensic medicine and pathology, PMID: 22101437

TMI Friday: An Unusual Rectal Injury

2013-05-03T12:00:00.000+01:00

The year was 1953, it was the fifth of November and a 24 year old man stumbled into Beckett Hospital complaining of abdominal pains. He told the doctors that it was a regular occurrence, that he had been plagued by abdominal pain for the past ten years. He told them that the evening before, he had noticed blood issuing from his bowels, and that he had vomited that morning.

As the doctor noting his horribly swollen and tender belly, he fainted. This made finding the source of the problem more difficult. The doctor checked for tumours, and ended up trying to perform a proctoscopy. This involved the insertion of an instrument known as a proctoscope up the anus in order to get a look at the inside of the rectum. However, the copious amount of blood and faecal matter belching from the anus made it impossible to see anything.

This called for more drastic measures. The patient was anaesthetised, and the abdomen was opened up to get some idea what was going on. The abdominal cavity was full of blood, that was likely caused by some form of internal bruising. In the absence of an obvious injury, the abdomen was closed up. The doctor took another examination of the rectum, which was easier now that the patient was sedated, and found the source of the bleeding, a three cm rip in the rectum. With this established, surgery was performed to heal the wound, and to reverse the damage. But an important question still remained.

How did this injury occur ? Clearly, the patient had not told the whole story about what had happened to him. The three centimetre rip was clearly caused by a traumatic injury. The patient admitted this, and then gave them another explanation for what happened.

It was the fifth of November, Bonfire night, where the English stage fireworks displays to celebrate the foiling of the gunpowder plot. The man said that he had bent over at the wrong moment, and a carefully aimed firework had shot up his anus.

But this story didn't make sense. There would be damage to the anal sphincter and the butt cheeks had this been the case. When confronted with this evidence, the man told them a third story. I shall now quote directly from the article.

For domestic reasons he had become unhappy and morose, and on the evening of November 4 he decided to explode a firework up his seat. He accordingly fashioned a narrow tube, using cartridge paper, and with the aid of a pencil introduced one end of this tube, approximately 6 inches (15cm.) in length, into his rectum. He then placed a lighted firework into the end of the tube projecting out of his anus...

This story still left the question as to where the firework went, as no fragments were found. and there was a distinct lack of singeing. It is possible that the sheer volume of effluence issuing from the anus could have washed out the bits of firework. The patient maintained their story under psychiatric evaluation, so we must assume that this was the true story.There is no escaping the image of a man not only lighting a firework up his own anus, but then hitching up his pants and waiting until the next day to actually go to hospital.
The frustrating thing about this tale is that the patient went to some length to deceive the doctors with a fake medical history. Had he told the doctors the truth, he would have been put under anaesthesia, and have been treated more rapidly. That is one of the lessons we can draw from this study, aside from the obvious one.

Butters A.G. (1955). Unusual Rectal Injury, The British Medical Journal, 2 (4939) 602-603. DOI: 10.1136/bmj.2.4939.602

TMI Friday: Vacuum Cleaners Suck

2013-04-26T12:00:00.000+01:00

When you look at a vacuum cleaner, what do you see ? A tool perhaps, to help you deal with the crisps trodden into the carpet from a party the night before. A way to keep armies of dust from taking over your house and to frighten your pet dog.

But there are some people (Who am I kidding, It's men.) who look at vacuum cleaners in a different way. Who observe the coquettish expression on a Henry vacuum cleaner and contemplate a universe of intimacy. Not much is known about these people. Are they a real sub-culture ? Do they compare notes on Rule 34 ?

What we do know is that these individuals tend to end up in an emergency room, crossing their legs out of embarrassment and to stem the flow of blood.

Dr Ralph Benson, in his article "Vacuum Cleaner Injury: A Common Urologic Problem ?", he examines five cases where men had attempted intimacy with these household implements... and suffered the consequences. All of these cases occurred when men attempted to use vacuum cleaners as masturbatory aids. I don't want to get into the details of the injuries, but the words "laceration" and "penis" should give you an idea of what happened. If you don't know what the terms "avulsion" and "degloving" mean, then don't worry, the paper comes with helpful photographs. Why not download the paper and look at them ?

Dr Benson notes in the paper that the cases he describes are not isolated incidents. Reports of vacuum cleaner injury go right back to the 1960's. But the interesting thing that Dr Benson notes is that even though he lived in a relatively small town, he encountered a cluster of these vacuum cleaner cases. In the absence of a tangible connection between these people, he came to a somewhat incredible conclusion.

His conclusion was that this was not an isolated cluster of vacuum abusers. Instead , he suggests that vacuum cleaner abuse may be far more common than is generally recognised. He notes that often, people will lie about the causes for the injury, and will only reveal the truth if the doctor subjects them to more intense questioning. He proposes that in busier emergency rooms, medics don't have the time or the inclination to probe into these lies, and just get on with repairing the injury.

Nevertheless, there is an important lesson in here, that I never would have thought needed to be iterated, but nonetheless here it is- Do not stick any part of your body into a hole that contains whirling blades.

C. Benson R. (1985). Vacuum cleaner injury to penis: A common urologic problem?, Urology, 25 (1) 41-44. DOI: 10.1016/0090-4295(85)90561-8

TMI Friday: A Vexacious Consequence of a Vasectomy

2013-04-19T12:00:00.000+01:00

It was an emergency. The patient was 51 years of age, running a high fever, and pain and swelling in a particularly sensitive area, in which an operation had been performed a week previous. Gentlemen of delicate dispositions may wish to avoid reading further, for that operation was a vasectomy.

The purpose of a vasectomy is contraception, to make sure that a man cannot impregnate a woman with his sperm. A vasectomy works through preventing sperm from escaping from your testicles, where they are manufactured. It does this by cutting the vas deferens, the tube through which the sperm travel out of the testicles. This procedure has become relatively advanced in recent years.

The gentleman in question had a "No scalpel" incision vasectomy. This has a number of benefits , not least that it doesn't involve a scalpel being wielded near to a "gentleman's dangling region". It its quicker, leaves a tiny operation scar, which means less bleeding pain and infection, and more importantly, a quicker return to sexual activity.

The 51 year old gentleman however had clearly acquired some form of infection after the operation. Infection after a vasectomy is generally uncommon. They then found the identity of the bacterium causing this infection. It was Streptococcus pyogenes, the bacterium that commonly causes sore throats. Long time readers of this blog probably have some idea of where this is going...

His wife had been looking after their children who were suffering from sore throats. And unbeknownst to anyone, the Streptococcus pyogenes had been passed to her, and was settling on her tonsils. The night before the fateful emergency visit, she and her husband had an intimate moment. During this process, the bacteria on the wife's tonsils somehow ended up on the husbands genitals. The paper describing this clinical case describes the infectious process that followed:

It is reasonable to assume that the vasectomy incision was only superﬁcially healed, and therefore, violated and impregnated during the “trauma” of oral intercourse.

This is one of those cases where a series of events coincide, which results in a bizarre disease complication.

That was my TMI Friday, I hope you endured it as well as I did.

Ramaswamy K. &; Kaminetsky J. (2011). Unique Infective Complication after Routine Vasectomy: A Case Report, The Journal of Sexual Medicine, 8 (9) 2655-2658. DOI: 10.1111/j.1743-6109.2011.02345.x

TMI Friday: Taking a Bite out of Love

2013-04-12T12:00:00.000+01:00

Love isn't commonly encountered within the medical literature. The romantic lives of two people in love is a subject that rarely requires the attention of a doctor.

But occasionally in the violent throes of a passionate embrace, there is an emphasis on the violence. With this in mind, let us consider the Hickey.

Once when I was in school, I met up with my friend and the first thing I said to him was "What the f**k happened to your neck ? Did you get attacked ?" at which point I realised the girl next to him started to giggle.
I later learned that during the violent throes of passion, that occasional nibble may occur, leaving a bruise, or just a red mark. But on occasion, some couples go a little bit too far. This is where medical professionals get involved.
In a set of case reports published by the British Journal of Surgery in 1990, 7 cases of what are described as "Traumatic" love bites are reported. I shall summarise them below

Patient 1, a 35 year old man, came into hospital complaining of a hard lump in his shoulder that had been bothering him for long time. It was a worrying lump, and the doctors initially suggested that it was some sort of cyst. When they removed the cyst, they were dismayed to find... a plastic tooth. It turned out that he had engaged in intimate relations with a lady who was not only dressed as a vampire, but possessed an incredible commitment to the lifestyle.
Patient 2 arrived at the hospital with an abscess in his neck that was swollen with bacteria, caused by a particularly violent love bite that had happened 3 weeks previously
Patient 3 arrived at the emergency room, bleeding from the jugular vein that was caused by deep bite marks that had been inflicted 2 hours earlier under undisclosed circumstances.
Patient 4, a 26 year old woman had been suffering from cellulitis in the neck for about 3 days before she went to hospital, and later confessed it was due to "ferocious love biting" by her boyfriend, and in response, the surgeons gave her a course of antibiotics and also a tetanus shot.
Patient 5 appeared to accept some form of responsibility for the infected wound on his neck, as the wounds were inflicted after he had returned from a long holiday by his "frustrated" girlfriend.
I feel sorry for Patient 6, who had to cut off her honeymoon early after her drunken husband accidentally bit off her left nipple. The doctors don't mention the fate of this relationship, but I would be very surprised if "Divorce" was not a key feature of it.
Patient 7 suggested that the primary reason for the infected injury in the left breast was due to the short stature of her paramour.

Only in two of these cases is the injury in itself severe enough to merit an immediate visit to hospital. The majority of the problems caused by these human bites come from infection. The human mouth is generally full to the brim with bacteria, that could potentially become hazardous if introduced into a wound.

When one attempts a lovebite, always remember to take a sensible nibble, if you end up with a mouthful of blood then you are probably doing it wrong. Unless you are a vampire, in which case, check that you still have all of your teeth at the end of it.

Al Fallouji M. (1990). Traumatic love bites, British Journal of Surgery, 77 (1) 100-101. DOI: 10.1002/bjs.1800770134

The Early Emergence of Antibiotic Resistance

2013-04-07T16:15:00.001+01:00

The development of resistance to the antibiotics is a phenomenon of great theoretic interest to a bacteriologist, and it may some day become a matter of major concern to the clinician.

This is the opening line in C. Phillip Miller's paper on the development of resistance to antibiotics, which he published in 1947. Penicillin had only just been in production for five years. It was saving countless lives. It was emerging as a miracle drug. But even in this relatively optimistic era, a number of scientists were getting a taste of things to come.

Alexander Fleming, the discoverer of penicillin, was well aware that it does not kill all bacteria. Whilst it was effective against bacteria like Staphylococcus aureus, he found that E. coli (it was called B.coli at this stage in history) was not killed by penicillin. These bacteria appeared to be naturally resistant to penicillin. The question of what caused this resistance in some species of bacteria was taken up ten years later, by Edward Abraham and Ernest Chain. In 1940 they took cultures of E.coli and then broke them down into a mush. They filtered penicillin through this mush, and then tested whether it still killed Staphylococcus aureus. It turned out that the penicillin no longer worked, and they inferred that E.coli had some form of enzyme that broke down penicillin.

But this paper only talked about E.coli’s natural ability to break down penicillin. Surely this wouldn’t be a problem. This just means that penicillin won’t be useful against infections caused by E.coli. It’s not as if it would ever be a problem for fighting bacteria which they knew to be sensitive to penicillin, like Staphylococcus aureus ? Right ?

In 1942** it became clear that Staphylococcus aureus could become resistant to penicillin. Charles Rammelkamp and Thelma Maxon were trying out penicillin therapy for human infections. They noticed that penicillin therapy was not always effective for treating patients infected with Staphylococcus aureus. Some of the Staphylococcus isolated from these patients were now resistant to penicillin.

They then took some strains of Staphylococcus aureus that they knew could be killed by penicillin. The exposed these bacteria to increasing concentrations of penicillin, and over the course of 50 days they managed to dramatically increase the resistance of these bacteria to penicillin. The reason this had not been spotted before was because it had taken so long to develop. In the years after, similar methods were used to show that other bacteria could also develop resistance to penicillin.

By the time Alexander Fleming gave his Nobel Prize lecture in 1945, he was very much aware of the threat that antibiotic resistance posed. At the end of his lecture, he issued a warning about the perils of underdosing penicillin, and how it could eventually lead to the proliferation of penicillin resistance.

The time may come when penicillin can be bought by anyone in the shops. Then there is the danger that the ignorant man may easily underdose himself and by exposing his microbes to non-lethal quantities of the drug make them resistant.

In this speech, he also proposed his solution to this problem. He observed that since penicillin wasn’t toxic, there was no such thing as an overdose.

C. Phillip Miller’s paper further expands on this theory. He compared the development of resistance to Penicillin and Streptomycin. Other works had shown that antibiotic resistance had developed when low doses of penicillin were used to treat animal infection. Miller however disputed that this was relevant to the human situation, as penicillin was usually given in high doses to produce a “margin of safety”. He said that most bacteria develop resistance so slowly that infections are brought under control before a detectable degree of resistance has begun to build up. In his work, he noted how comparatively slowly organisms developed resistance to penicillin, and thus predicted the rise in Streptomycin resistant strains of bacteria, but not penicillin resistant strains. At this point in time, penicillin resistance could be dismissed as a rare event.

By 1957, the emergence of Staphylococcus aureus strain 52/42B/81, which possessed resistance to penicillin, streptomycin and tetracyclines, put paid to the idea that the threat from antibiotic resistance was only theoretical. When we talk about the emergence of antibiotic resistant strains, it must be remembered that they have been around for nearly as long as antibiotics themselves, and even the earliest pioneers could foresee the danger they presented.

References

Miller C.P. (1947). Development of Bacterial Resistance to Antibiotics, The Journal of the American Medical Association, 135 (12) 749. DOI: 10.1001/jama.1947.02890120003002

Abraham E.P. & Chain E. (1940). An Enzyme from Bacteria able to Destroy Penicillin, Nature, 146 (3713) 837-837. DOI: 10.1038/146837a0

Rammelkamp C. (1942). Resistance of Staphylococcus aureus to the Action of Penicillin, Proceedings of the Society for Experimental biology and Medicine, DOI: 10.3181/00379727-51-13986

Alexander Fleming's Nobel Prize Lecture (1945)
http://www.nobelprize.org/nobel_prizes/medicine/laureates/1945/fleming-lecture.html

Finland M. (1979). Emergence of antibiotic resistance in hospitals, 1935-1975, Reviews of infectious diseases, PMID: 45521

Footnotes

*They called resistant bacteria “antibiotic-fast”, for linguistic reasons that are unclear to me.

** The earliest paper that I could find which demonstrates penicillin resistance was from 1942. In the paper the authors say that penicillin resistance had been described before in 1941, and then cites those papers…. incorrectly. The first paper cited only looked at resistant to sulphonamide antibiotics. The second paper they cite was by howard florey, and in that paper there is no reference to either resistant (or “fast” as the term would be) Staphylococcus aureus. So somewhere out there there may be a paper in 1941 that shows the development of antibiotic resistance in Staphylococcus aureus, but it has been expunged from the record for completely unknown reasons.

#Microtwjc: The Evolution of Virulence

2013-03-31T18:37:00.000+01:00

A long time ago, a bacterium noticed the odd behaviour of its cousins. It had noticed that they had formed a group, and were spending a lot of time together. An unsettling amount of time together. The bacterium's friends told it to not worry. It is perfectly fine for related bacteria to stick together, to live in colonies of individuals.

The bacterium told its friends how its cousins behaviour was different. The cousins had forfeited the life of independence, and had reduced themselves to mere parts of a greater whole, a multicellular organism. it was disgusting, it was socialism. It had to be stopped.

Bit by bit, it rallied other bacteria to it's cause, and together they came up with a plan. They would become virulent. They would evolve the resources to fight their multicellular competitors, invade their cells and feast on their nutrients. So they went to Professor Oak, who happened to have just the right thing for them...

Okay, maybe my "Pokemon Virus Vendetta" theory of bacterial evolution is a bit off the mark. In my defence, a microbiologist looking at bacterial evolution doesn't have any fossils to help them. And even if they did, they won't really tell them anything other than the shape of the bacteria, which has no real bearing on whether a bacterium is virulent or not.

Evolutionary bacteriology is primarily based on using the bacteria's genetic code. We can look at bacteria that inhabit different environments, and look at the similarities and differences between them and formulate ideas about bacterial evolution.

So there are questions as to how bacteria evolved to live inside animals, and more importantly, how they learned how to survive the immune system. One of the great threats to bacteria are phagocytes, which roam the body gobbling up bacteria and digesting them. Yet some bacteria have not only evolved ways to prevent themselves being eaten, but to survive, even thrive, within phagocytes, such as Mycobacterium tuberculosis. One of the first steps of infection with this pathogen actually requires a phagocyte to consume it, and then it grows and proliferates within this phagocyte in order to eventually cause disease.

M. tuberculosis possesses many genes which help its survival within the host, and the set which is studied in this paper is known as "Mammalian Cell Entry" genes, so called because when E.coli were given these genes, it enabled them to invade mammalian cells*. In M. tuberculosis it is thought to in some way control its survival within phagocytes.

So when the "Mammalian Cell Entry" genes were found in a soil microbe, Streptomyces coelicolor , a question is raised. Why would S. coelicolor need these genes ? The answer may tell us something interesting about how bacteria evolved to attack us and cause disease.

Does Streptomyces actually use these genes ?

Just because it's in your genome doesn't mean you use it. If the genes have no function in S. coelicolor then we can only speculate on what function their ancestors had for these genes. So they measured the presence of mRNA within these cells at the different stages of Streptomyces growth, and checked its presence on different media.

This is shown on Figure 2 A, with the white bands indicating the presence of mRNA.

hrdB is a house keeping gene which is always active, and mceA is one of the "mammalian cell entry" genes. mtrA controls whether mceA is switched on or not.

So from figure 2A, we can see that mceA is switched on when grown in YEME medium, but not in MS medium, and mtrA is switched on in this medium. This suggested that the medium's effect on mceA works independently of mtrA.

In Figure 2 B, we are shown what happens if S.coelicolor grown in cholesterol compared to YEME. A mutant with no functional mtrA was also grown as well. This shows that mceA is not active when cholesterol is present within the medium, and it is not present when mtrA is removed from the medium as well. This indicates that mtrA is needed for the expression of mceA, except when the bacterium is grown in MS medium, in which case not even mtrA will not prevent mceA being repressed.

So at least we know that the mammalian cell entry genes are doing something in Streptomyces, but what are they doing ?

How well does Streptomyces do without the Mammalian Cell Entry (mce) genes?

One way of working out what a gene does is getting rid of it, and watching what the bacterium now cannot do without those genes.

Figure 3 looks at a number of things that change when Mammalian Cell Entry (mce) genes are removed.

They took a lawn of streptomyces, and then they added a drop of SDS into the middle of it. SDS is a surfactant, that attacks bacterial cell membranes kills off the streptomyces, which produces a dark pigment as if to protest***. As you can see from the image, the darker patches are larger when 20% SDS is applied compared to the 10%.

The mce mutation appears to give the two colonies we are shown an edge when growing with lysozyme, a molecule which acts to break down cell walls. Whilst the dark patches are still there, you can see some bits of white poking through, showing that some of the cells have survived, although they are still injured***

Electron microscopy revealed that mce mutants have more "wrinkled" cells, that were on average shorter than the normal cells from a sample of a hundred cells.

So what is the significance of these effects?

What happens if we feed our bacteria to an amoeba ?

An Amoeba is the natural predator of bacteria like S. coelicolor in its natural habitat within the soil. So what happens when we feed these bacteria to an amoeba, and what effect does the mutant have on this ?

In Figure 4, this is what they do.

It turns out that the mce mutant kills the amoeba. If you look under a microscope in 1 A , the bacteria germinate and grow within the amoeba after they are eaten, and kill the amoeba in 24 hours.

In the next experiment, they made a "lawn" of S. coelicolor grow on the surface of an agar plate, before adding amoeba to the centre of the plate. When the bacteria are eaten, clear patches form on the surface of the plate. So we can see that the deletion of mce, and the Mtr gene that promotes its growth prevents the bacteria getting eaten. If you try to correct the mutation by adding a plasmid (pLS006) with functioning mce, then you find that the bacteria once again get eaten by the amoebae.

So mce expression enables bacteria to be eaten by amoebae, and when its gone, the amobae stop being a threat.

So if all mce does is allow bacteria to get eaten, then what is it's point ?

What effect does mce mutation have on root colonisation ?

S. coelicolor lives on roots, and its possible that mce plays a part in root colonisation. And it turns out that when mce is deleted, bad things happen to the plants it colonises, which is shown in 5A. On the microscopic pictures we are shown, we can see that there are less bacteria on the roots when the mce is knocked out.

In 5B, we can see that there are less of the mce mutant on these plants compared to S. coelicolor with functioning mce.

So now we must consider what this data has told us, and then what we can deduce from it.

Summary

This paper characterises the function of a gene system in S. coelicolor which is somewhat related to an important gene in Mycobacterium tuberculosis. So what have we found out about this gene in this paper ?

When mce is active :

It decreases resistance to Sodium dodecyl sulphate (SDS)***
It does not prevent resistance to lysozyme.
It makes nice and healthy looking cells.
It does not prevent amoebae eating it
It allows growth of the bacteria on roots

When mce is inactive:

It increases resistance to SDS***
In increases resistance to lysozyme, which is lucky because lysozyme is an important digestive enzyme used by amoebae to eat their prey.
It also kills off amoebae when they attempt to eat S. coelicolor.
It prevents plants growing very well if they get into the roots
It also doesn't allow S. coelicolor to grow very well either on the surface of roots.

What does this tell us about the evolution of virulence ?

On its own, this data only really tells us about S. coelicolor, and what the mce gene does in this bacterium. But when we compare it to the action of the mce in M. tuberculosis, we can start thinking about the different ways these genes have evolved to match the life cycles of their respective bacteria.

There are multiple mce's in M.tuberculosis which have slightly different functions, but mostly they play roles in surviving within macrophages, primarily in reducing the expression of cytokines by macrophages after they've been infected with the bacterium, and its general survival. It is very difficult to compare the functions of the mce's in Mycobacterium to Streptomyces because of this.

But this isn't the point of the paper. They say in the paper that Streptomyces split off from the ancestor of Mycobacteria at around 440 million years ago**. Based on this, we assume that the common ancestor of these two bacteria probably expressed some variant of the mce gene. So based on this, we can try to deduce what this ancient bacteria from the Silurian era used these genes for. This was around the time that plants were colonising the land. The authors suggest that these genes evolved to allow soil bacteria to colonize the surface of plants, and allow it to control when it expresses the genes that allow it to survive being eaten by amoebae.

In many ways, phagocytes behave like amoebae. Like amoebae, phagocytes use lysozymes to digest their prey. So soil microbes which have methods of resisting amoebae come ready made with methods of resisting phagocytes.

So the idea is that a long long time ago, these soil microbes were minding their own business, when they end up in the body of a mammal. The mammals immune system immediately recognises them as foreign, and sends phagocytes to destroy them. The bacteria, assuming that they are being eaten by amoebae, shut down their mce system to resist being eaten, and end up causing a disease in the mammal.

Do you see the irony here ?

The presence of these macrophages actually make a host more likely to get a disease after ingesting these microbes.

Why does any of this matter ?

So let's imagine a world where we fully eradicate tuberculosis, and other diseases. The primary threat of disease comes from the fat tail of emerging pathogens which can now exploit the empty niches left by the other bacteria. What was originally just one bacterium causing a disease is now a hundred bacteria causing a disease.

Understanding the evolution of virulence allows us to get an idea of where possible threats can come from.

There are Mycobacteria, Legionella and Chlamydia like bacteria which currently reside in our environment and attack amoebae, which can cause pneumonia in humans if they end up in the lung. If we understand the evolutionary process which allows soil bacterium to cause disease in humans, perhaps we can devise strategies to prevent it from happening.

This paper was up for discussion by the microbiology twitter journal club this Tuesday (2nd April) at 8pm BST.

The transcript of that discussion can be found here

Reference

Clark L.C., Seipke R.F., Prieto P., Willemse J., van Wezel G.P., Hutchings M.I. & Hoskisson P.A. (2013). Mammalian cell entry genes in Streptomyces may provide clues to the evolution of bacterial virulence., Scientific reports, PMID: 23346366

*Now I should note here that it is unclear at this time how the "Mammalian Cell Entry" system actually works, and whether it actually serves any function in helping mammalian cell entry, or whether it simply allows bacteria to survive better after naturally being eaten by cells.

**I couldn't find the reference for this observation. Well, they do give a reference for fungal and plant evolution occurring around 400 million years ago, but streptomyces is a bacterium, and I'm not sure how this would apply.

*** In the original article, I assumed that the black patches were the colonies themselves, rather than zones of dead bacteria, when in actuality, they represent dead bacteria, and I thought that the SDS had the reverse effect to what it really does. Had I known that S. coelicolor painted itself black upon death, I would have drawn a picture of it dressed as a goth. Hat tip to @clonemanager and for explaining this to me.

The Ten things I can do now I have a PhD

2013-03-27T21:05:00.000Z

Ten Things I can now do with a PhD

I can introduce myself as the "lu-uurve" doctor to members of the opposite gender.
I can finally publish a book about the "Lint" diet , and have it fester on bookshelves around the country as its devotees staunchly defend it even as they succumb to rickets, scurvy and starvation.
I can start an advice column, in which I couch my advice in impenetrable jargon to conceal its vacuity.
I can drop my PhD into conversations, knowing that it makes me knowledgeable on all subjects, as opposed to the one I actually specialised in.
I can now end any argument by taking a tally of people in the room with PhDs, and those without, and then cement my victory by blowing raspberry lasting not less than 3 minutes.
I can buy a pair of glasses for the sole purpose of peering over them when talking to people. And occasionally ripping them off dramatically to let everyone know that shit just got real.
Whenever someone utters the words "Doctor Who?", I can shout my own name really loudly and pointing at myself. Raucous applause will no doubt follow.
I can now defuse "Doctor Doctor" jokes by simply saying "Yes, what is it?". This will always be funnier than the actual joke.
I can line up the photos from my BSc, MSc and PhD graduations, put attack statistics under them and pretend that I am a extremely rare and esoteric pokemon, and that these were my evolutions.
I can don a cape and mask to begin a career in super-villainy. It's the profession with the best hiring prospects in this economy.

The Final Hurdle

2013-03-25T21:08:00.002Z

I'd pretty much done everything at this point. Through struggle and sweat and 263 pages of thesis, I found myself edging closer to the finish line.
In theory, anyone can write a long thousand page tract on a topic without applying observation, logic, creativity or even basic grammar to it.
The manuscript I wrote needed to be assessed. But herein lies the problem.
A PhD, unlike many other qualifications, is granted based on the synthesis of new knowledge. The problem of this becomes apparent when it comes to examining a PhD. I'll compare it with learning in a school.
The learning that occurs in schools occurs in the classroom, by a teacher who at least know the standardised answers on their test sheets, if not the subject itself. As a result, examining students knowledge of these subjects is the simple task of comparing their answers to the standardised tests.
A PhD is very different. A student does not do most of their learning in lecture rooms regurgitating the knowledge that is fed to them. A student must use the tools, and the advice of experts (including , but not exclusively their supervisor(s)) in order to gain new knowledge or insight about a subject. In the case of the sciences, the teacher is nature. This becomes problematic when examining PhD, because Nature doesn't have a mark scheme. If it did, it would be behind an infinitely expensive paywall.
This is where the Viva Voce comes in. The exact details for the examination differ between countries, but the basic jist is that it requires the thesis to be assessed by a group of experts in that field, who are able to assess the work, and discuss the new findings of the thesis with the candidate.
A number of people in my lab told me that I would be fine, that it would amount to a nice conversation about my thesis by the only people* who would actually read it. I guess I should have found this reassuring. An experienced tight rope walker may tell you that a hundred metre walk on spider silk over hot lava was simple enough when they did it, but does that mean you'll have no trouble on your first go ?
As it turned out, this was pretty much the case. Aside from one moment where we got into a somewhat philosophical argument about the possible detriments of eradicating an infectious disease, a few moments where I talked myself into a corner, it went well.
In the end, I passed !

*not including those involved with its production

Elementary; The Science of the Perfect Murder

2013-03-11T14:11:00.001Z

It was television that inspired me to devise the perfect murder. Not for the usual reasons relating to scientific inaccuracy. This isn’t about watching a CSI unbalance a centrifuge, contaminate a sample or holding a pippette backwards. Small errors like this just don’t bother me anymore. I long ago accepted that most television shows are set in a science fiction fantasy world, where science only works when the gods of plot convenience allow it. It was in this frame of mind of that I watched a recent episode of Elementary (S1e17 “Possibility Two”).

For those of you who aren’t familiar with it, “Elementary” is a procedural cop drama with the twist that it is also a Sherlock Holmes pastiche. It follows the detective (played by Johnny Lee Miller) and Joan Watson (played by Lucy Liu) as they solve crime in modern day New York. Johnny Lee Millers interpretation of Holmes can be described by shouting “QUIRKY” and doing the jazz hands so hard that they violently detach and fly across the room. This is a show that proudly flies the flag and wears the matching underpants of silliness. The very definition of TV to turn your brain off and enjoy. Unfortunately, my brain’s off switch is always slightly faulty and against all odds, this episode did make me think. I am going to spend the rest of this post exploring these thoughts, so I should warn you that this article contains major spoilers.

This episode kicks off with millionaire Gerald Lydon attempting to get Holmes to reveal that he has hereditary Cerebral Amyloid Angiopathy (CAA). The twist is that he believes that someone gave him this hereditary genetic disease deliberately. To Sherlock, the answer to this particular puzzle is as simple as cluedo. The culprits were Lydon’s parents in the bedroom using the *ahem*. But they have to be ruled out as suspects, because they didn’t have CAA (and it is a dominant genetic trait).

And then Sherlock asks “Have you considered that you may not know your true parentage ?”, and they spend the next forty minutes of the episode tracing Lydon’s real father whilst discussing the degree to which parents are responsible for the genetic inheritance they bestow on their children and the episode finishes with Sherlock explaining the mystery to his dementia ridden client using sock puppets. Just kidding, this wouldn't be a murder mystery procedural drama without an actual murder mystery.

Someone did find a way to give Gerald Lydon CAA, and they did it by using science. In the process of this episode, they frequently invoke fictional geneticists that say “this is totally possible” in a naked attempt to justify the absurdity at the heart of the episode. Sherlock’s investigations lead him to a slick biotech company where their one jobbing scientist to explain the basis of CAA.
CAA is essentially caused by a an excess build up of beta amyloid protein in capillaries that carry blood within the brain. as these build up, blood is prevented from going where it’s needed, leading to brain cells dying off. As the scientist explains, the hereditary forms can occur through a mutation in the APP gene. This gene encodes the Amyloid precursor protein, which forms the harmful Beta Amyloid that is the cause of CAA. Later in the episode, that scientist sends the Sherlock a chemical structure, with a note that says something akin to “This is the murder weapon”.
.

Unfortunately, I’m not an expert on chemistry so I don’t know the identity of this chemical*. In this episode, it acts as a mutagen which specifically interacts with the APP gene to cause the build up of amyloid beta. How it does this is not explained, and I think I am safe in saying that this compound wouldn't actually work in real life**. But just because this compound is unlikely to give someone CAA, it doesn’t mean that it isn’t possible.
For the past 10 years, people have been developing gene therapies to cure hereditary diseases through manipulating the human genome. But what if we re-purposed the tools used for gene therapy into a murder weapon, in order to cause a disease rather than to cure it ? What if we put ourselves in the shoes of the murderer of this episode. How would we go about giving someone CAA for real ?
Let’s get back to the APP gene, which encodes the Amyloid precursor protein. We want to alter this gene so that it ends up forming as much Beta amyloid protein as possible. The APP gene, through a clever trick using short RNAs, actually codes for a number of different APP’s, only a few of which can be broken down to form Beta amyloid. The gene itself is structured into segments (which are called “exons”) which are transcribed depending on the presence or absence of microRNA’s.
It is known that the production of Beta amyloid is related to the presence of one particular section of the APP gene, known as the “Kunitz protein inhibitor” (KPI) is associated with the build up of beta amyloid [1,2]. However, this section of the gene often excluded from APP transcripts by a microRNA known as MiR-124 [3].

When the DNA code is being translated, this MiR-124 binds to the KPI section of the gene, and excludes it from the APP. If we stop MiR-124 from doing this, we increase the amount of APP with the KPI section in it, and in theory this could increase the production of beta amyloid, which would give an individual CAA. How do we do this ?
We can do it by using some nifty chemicals known as Morpholinos. They mimic RNA, except they don’t get broken down as easily, and they can be attached to other chemicals to make sure that they enter cells. We could design a morpholino to mimic the action of MiR-124 if our intention was to prevent someone from getting a build up of Beta amyloid in their brain. But that isn’t what we want to do. We want to stop MiR-124. So we can design a morpholino with a sequence that binds to MiR-124. This would effectively halt its function, and allow for more beta amyloid to build up.
But how do we give this lethal morpholino to a patient without them noticing ?
The murderer from Elementary fortunately solved this one for us. We are told that the murderer targeted three millionaires, and gave the drug intravenously whilst they were staying as patients in hospital. Delivering a drug intravenously allows it to go straight to the blood. Blood goes everywhere in the body, including the brain. While these patients are being infused with the anti-MiR-124 morpholino, Beta amyloid builds up, eventually to the levels needed for them to develop CAA ***.
Now we need to know how much Morpholino to give our murder victims. The main challenge here is the blood brain barrier, which prevents many drugs, including morpholinos, entering the brain. We are going to try to overcome this by simply putting in such a high dose that it overcomes the blood brain barrier.
In studies in rats, researchers have administered a dosage of approximately 300pmol of morpholino per rat brain [1]. Let’s imagine that to get the same effect in humans, all we need to do is increase the dose based on weight****. A rat brain weighs around 2 grams, and a human brain weighs around 1400g.

Anti-MiR-124 dose/Rat Brain= 300pmol/2 g
Human brain= x/1400g
Therefore the dose = 150 x 1400g

210,000pmol of morpholino needs to enter the human brain in order to have the desired effect. I could only find one study that makes quantitative measurements of morpholino concentrations in the brain after an intravenous dose, and it suggests that the dosage of morpholino can be reduced by around a thousand fold [5] *****. So we’re going to have to up that dose to 210,000,000 pmol, per person.
At this point, one may wonder why anybody would go to this much trouble to kill some rich philanthropists in such a convoluted way ? The answer is as demented as it is ludicrous. The killer is a scientist suffering from CAA. In order to cure himself, he needs more funding. So he decided that the best way to go about this was to deliberately give rich people CAA, which would compel them to donate money to him in order to fight the disease. I will demonstrate the ludicrousness of this plan through the use of simple arithmetic.
10,000,000 pmol of a morpholino costs at least $9,000 dollars (based on numbers from Gene-tools.com). So the total cost of giving one dose of the drug to reach their brain would be around $189,000 dollars. I am going to assume that they were given the drug throughout their stay in hospital. All of these individuals are over the age of fifty, and the average hospital stay for that age group in the United States is 5-6 days. So at most, it would cost $ 1,134,000 to poison one person.
During the course of this episode, not one, but three people have been poisoned successfully in this way, bringing the total budget of this endeavour to $3,402,000. This assumes that it all went according to plan (which science so often doesn’t) and that the murderer only produced enough chemical for three people. Let’s not forget that the technology for morpholinos already exists . If the murderer wanted to develop some other method to cause this disease, they would have to start from scratch, requiring even more money. This highlights the central contradiction within the plot.
As the scientists often say during the episode, with unlimited money and resources a scientist could develop the technology to give someone CAA. As I have demonstrated, the killer had plenty of money around to throw at this project. Yet the whole reason behind this convoluted murder scheme is to allow the perpetrator to acquire money. It’s almost like the murderer has some sort of degenerative brain disease.
In thinking through this murder plot, I am forced to consider the many barriers to actually pursuing it in real life, which I have boiled down to three points

To induce a genetic disease, we need to know a lot about it. I kept coming up against this problem with CAA. It’s not that I couldn’t find a cause, but that there were so many different genes that could play a role that I couldn’t point to any of them being the root cause.
Our technology isn’t there yet. There are a lot of promising developments in gene therapy that are just over the horizon. I’ve spent most of the this post talking about morpholinos, but there are other treatments in the works. Viruses that have been re-purposed to deliver genes are now commonplace in some labs, and advances in nanotechnology may allow for promising developments in the future. Very few of these have ended up in successful human trials, and they are still being tweaked to make them more effective.
Resources. The cost of using this technology immediately puts it out of the hands of a casual amateur. And if you are a professional scientist, you will eventually be called in to explain the half million dollar hole in your lab finances that is designated “convoluted murder plot”.

Our knowledge of genetic diseases is expanding as are the gene therapies that are being developed to combat them. The “Genetic disease” method I have described here may be plausible at some point in the future. But I have to note that the three points I have described are also barriers to curing genetic diseases.
When we do get to the point where our knowledge and technology is advanced enough to manipulate a person’s genome so subtly that we can give them a genetic disease, we will also be at the point where these advancements will also allow for the treatment, prevention and possibly even a cure for it.
It is somewhat amazing that a run-of-the-mill procedural drama catalysed this thought experiment. But sometimes inspiration can come from the unlikeliest places. Sherlock sums it up the best in the last line of the episode.

“A good detective knows that every task, every interaction no matter how seemingly banal, has the potential to contain multitudes.”

References

[1] Matsui T., Ingelsson M., Fukumoto H., Ramasamy K., Kowa H., Frosch M., Irizarry M. &; Hyman B. (2007). Expression of APP pathway mRNAs and proteins in Alzheimer’s disease, Brain Research, 1161 (3) 116-123. DOI: dx.doi.org/10.1016/j.brainres.2007.05.050
[2] Tharp W.G., Lee Y.H., Greene S.M., Vincellete E., Beach T.G. & Pratley R.E. (2012). Measurement of altered AβPP isoform expression in frontal cortex of patients with Alzheimer's disease by absolute quantification real-time PCR., Journal of Alzheimer's disease, 29 (1) 449-457. DOI: dx.doi.org/10.3233/JAD-2011-111337
[3] Smith P., Al Hashimi A., Girard J., Delay C. & Hébert S.S. (2011). In vivo regulation of amyloid precursor protein neuronal splicing by microRNAs, Journal of Neurochemistry, 116 (2) 240-247. DOI: 10.1111/j.1471-4159.2010.07097.x
[4] Fujikawa T., Tamura K., Kawase T., Mori Y., Sakai R., Sakawa K., Yamaguch A., Ogata M., Soya H. & Nakashima K.; (2005). Prolactin Receptor Knockdown in the Rat Paraventricular Nucleus by a Morpholino-Antisense Oligonucleotide Causes Hypocalcemia and Stress Gastric Erosion, Endocrinology, 146 (8) 3471-3480. DOI: 10.1210/en.2004-1528
[5] Stein D.A., Huang C.Y.H., Silengo S., Amantana A., Crumley S., Blouch R.E., Iversen P.L. & Kinney R.M. (2008). Treatment of AG129 mice with antisense morpholino oligomers increases survival time following challenge with dengue 2 virus, Journal of Antimicrobial Chemotherapy, 62 (3) 555-565. DOI: 10.1093/jac/dkn221

Footnotes

*It looks like a unsaturated aromatic ring linked via three keto groups to branched carbon chains that end in either a napthalene group or an adenine, and these speculations are purely based on the diligent use of google image search.
** If I am wrong about this compound being ineffective, then not only does this cement my inexperience as a chemist, but also worryingly suggests that not only is one of the scriptwriters for the show devising bioweapons, but they are broadcasting them on air for the whole world to see. That’s a scary thought. What will we see next, plans for an Antimatter bomb in the background of Sherlock’s study ?
*** You’ll need to expose the victims to the drug for a long time for it to be effective, as people who are born with the mutation we are attempting to replicate often take an entire lifetime for symptoms to become visible. And this is not to mention that the link between the presence of KPI and high levels of amyloid beta have only really been shown to have a correlational relationship, so this entire method of inducing it could be completely wrong. But let us assume that it works as a method of murder.
**** The actual art of comparing the biological reactions of all the different creatures within the animal kingdom (known as Allometry) is a massive subject which touches on evolution as well as medical science, and boiling it down to a mathematics exercise misses out a lot of its more subtler details.
***** I experienced severe math fail when reading reference [5]. They administered the drug at 10mg/kg to a mouse. In 200 micrograms of morpholino compound was given to a mouse, equating to approximately 0.26 micromols. This is assuming a mouse weighing 20 g, that a 24 nucleotide morpholino has a similar molecular weight to an RNA oligo of approximately ~ 7500 m.w. They later measured the presence of morpholino in the brain, and found that it hovered at around 0.02 micromols, which would make it look like it goes to the brain very well. However, in this same experiment they detected 9.34 micromols in the liver, which would suggest either that the morpholinos are replicating inside the mouse, or that my math went wrong somewhere. It is almost certainly the second option, because Figure 6 of that particular paper shows quite clearly that barely any of the morpholino reaches the brain, and my thousand fold guess comes from looking at the graph, and also comparing the amount of morpholino in the brain compared to other organs in table 2 of the paper.

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I've written a shorter version of this article on Kinja (http://faz-alam.kinja.com/). to make it clear, it's that article where I'm self plagiarising, as I wrote it after this one.

Taking DRACOnian measures against viruses

2013-02-09T16:45:00.001Z

When Alexander Fleming shamelessly took credit for a drug produced by the penicillium bread mould, our fight against bacteria completely changed. Even now, bacterial diseases are nowhere near the threat they were before that discovery. But today, we are seemingly having to put up with emerging viruses. Every year, we have to put up with entirely new strains of flu. There is nothing we can do about the viruses that cause the common cold. There are various antivirals out there, but they are specific for specific diseases, like flu, or HIV.
Generally, it’s been fairly difficult to make anti-viral drugs. Often, a lot of the problem comes from the way viruses work. When they are outside the cell, they don’t have any active metabolic processes that can be targeted by drugs. And when they are inside the host cells, the metabolic processes they’re mostly using are ours. So it’s very hard to target the viruses without attacking the host. Compared to bacteria, viruses present a very small target.
Could a paper published in PLOS One called “Broad Spectrum Anti-viral Therapeutics” represent a penicillin moment for viruses ? A trailblazer that can transform our relationship with viruses ? To work this out, let’s take a good long look at what these anti-viral therapeutics are, and what they do. To do that, we have to talk about DRACO.

Are we talking about the DRACO from Harry Potter, or the dragon voiced by Sean Connery in Dragonheart ?

Whilst I compliment you on your knowledge of pop culture, you are wrong on both counts. DRACO is a handy acronym that stands for Double-stranded Ribonucleic acid Activated Caspase Oligomizer. DRACO is the drug that is being purported to extinguish all of those nasty viruses. To understand how it works, we need to take a look at how some of our most pernicious viruses work.
You may have heard about DNA, and how it’s genetic code is a blueprint for life. Your genome is encoded in your DNA. The nucleus of your cell holds all of the DNA, and acts like a library for your genetic code. When your cell needs a specific genetic code to make a certain protein, the nucleus makes an RNA copy of the appropriate gene and sends it out into the rest of the cell, where it can be used to construct a protein.
The goal of a virus is to enter a cell, and to hijack this process to make more viruses. Usually at some point, the virus will attempt to substitute it’s own genetic code for that of the host cell, tricking the host cell to make more viruses ^[1].
However, some viruses can store their genetic code using RNA only. Whilst RNA is less stable than the DNA, it means that these viruses can go straight to the cell machinery that translate RNA into protein, and get ahead with making virus based proteins.

Influenza, Hanta Virus , Dengue Fever, Rhinovirus, Lassa fever virus, and reoviruses all use RNA as the basis of their genomes, and at some point during their replication, form double stranded RNA. This last bit is important, because double stranded RNA is not usually found in human cells, and when they are found, they don’t last for very long^[3]. So essentially the creation of double stranded RNA has been the achilles heel for many viruses.
So naturally, humans (and other animals) have evolved ways to detect the presence of double stranded RNA. I’ll give one example, a protein known as TLR-3^[4].

TLR-3 detects dsRNA, and then it signals to other proteins within the cell, so that it can do a number of things that the virus won’t like. For instance, it leads the cell to produce interferon, which acts as a distress call to summon the immune system. The most drastic reaction to the detection of viruses is the activation of caspases, which are the enzymatic equivalent of a self destruct button.

However viruses can evolve very quickly, and have matched pace with us in this evolutionary arms race. Viruses have evolved a number of tricks to get past our natural defences against them. While they can’t evolve ways out of being detected by RNA binding proteins, they have evolved ways to short circuit the signalling cascade that can occur after their initial detection. Let’s say that TLR-3 binds dsRNA, it tells another protein, and then a virus protein get’s in the way, and the message is lost.

So the idea underlying DRACO is taking the dsRNA detecting ability of enzymes like TLR-3, and shortcutting all of the signalling pathways and go straight to pushing the self destruct button Calm down, it’s not as bad as it sounds.

Your body naturally kills of virus infected cells. a cell infected with virus is not your friend any more it’s a factory for the enemy, churning out viruses to infect other cells. The normal immune response against viruses often involves killing off these cells, so don’t worry too much about them.

How does one make DRACOs ?

Now we’re getting into the actual paper, and away from the background. So how did this research group go about making DRACOs ?
They looked at a whole raft of different RNA binding proteins and looked at the parts of them which directly bind to the RNA. And then they looked at caspase proteins, and worked out which parts of those tend to cause human (or mouse cells) to self destruct. and they took those bits, and stuck them end to end.

Figure 1.A. of the paper shows the basic blueprint for these proteins. They also deliberately made versions of these DRACOs that were broken, to use as controls, just in case randomly giving cells proteins will protect them for no good reason. Figure 1.B. shows a western blot to prove that they created these proteins.

That’s great ! Do they work ?

So now that these DRACOs have been “designed” and created, the question is whether they work. So the first thing that we need to look at is whether these proteins actually get into cells. If they can’t do that, then there isn’t much hope that they can be effective.
The thing is, cells don’t just take up every protein that they get into contact with willy nilly. In order to gain passage into the cell, these proteins must have special tags on the end of them. In this case, they tried out DRACOs with PTD and TAT tags. They then added them to a culture with cells derived from humans (HeLa cells). They then extracted the cells from this,and tested whether the DRACOs had managed to get into the cells.

The western blot in Figure 2 A shows that DRACOs managed to get into the cells when they had the PTD or TAT tag added to them, but not without it. Furthermore, in Figure 2 B, they added the DRACOs to a culture of cells, and took out samples at specific time points to test whether the DRACOs had entered. Judging by the image, the DRACOs were taken up as early as 10^[5]-15 minutes after administration. Figure 2C shows that the DRACOs were retained by these cells for about 6-7^[5] days after they were first applied.
So now we know it actually gets into the cells, the question is whether they can detect the presence of double stranded RNA (dsRNA), and furthermore whether they can cause cells to “self destruct” when they are present. So they took some human cells, and genetically modified them to produce dsRNA. If the DRACO’s worked , then they would immediately kill off these cells.

How do you work out whether these cells have caspases becoming active ?

To work out whether the cells were pushing their self destruct button by activating caspases, the scientists here did some clever cell manipulation. They added the gene for luciferase to the cells. Luciferases are enzymes that produce light when they grab onto specific chemicals called luciferins.
To these cells, they introduced a protein which mimics the kind of proteins that caspases usually act upon when they are activated, with one difference. Tied up within the structure of these proteins is Luciferin. So when the caspases are activated, they bind this substrate, the process causes luciferin to be released into the cell. This is then found by the luciferase enzymes which then cause light to be produced. So the researchers could work out how active the caspases are in these cells just by looking at how much light they are making.
So they gave some of these cells caspase inhibitors. If DRACO’s were naturally lethal to these cells, the presence of these inhibitors would make no difference to whether the cells would self destruct (a process known as apoptosis).

In Figure 3, they added the DRACO’s to these cells, either with, or without the inhibitor. They also included a product which makes cells which have just self destructed glow in the dark. The first four sections on the graph are simply controls, to pick up the background levels of cell death. Since the main function of caspases is to cause cell death to occur, you can guess what would happen if we were to add caspase inhibitors to a normal set of cells. The blue and red bars are both lower than the green bar , because they have the caspase inhibitors added. The next three sections show what happens when DRACO’s are added to the mix, and they show that they kill off a lot of cells. And importantly, you can tell that it’s performed using caspases, because in the presence of inhibitor, the cells do not die as much. In fact, the levels of death seen is more or less the same as the other controls with inhibitors.
Ah yes, but this doesn’t necessarily prove anything ! The DRACO’s were created using e.coli cells, and it could be possible that when extracting these cells, some other nasty substances were pulled out that could account for this effect. So in the next set of data, they add some of the e.coli extract, and show that actually it doesn’t have any effect, when compared with controls.
But you forget, cells naturally try to off themselves when confronted with dsRNA ! how do we know that the supposed effect of the DRACO isn’t caused by that ? Because they then tested the cells with added dsRNA. And while the cells did indeed die off more than controls, it was still much lower than when the DRACO’s were added. They also added a compound known as camptothecin to deliberately trigger the self destruct in these cells.
So at the end of this last figure, we know that DRACO’s do what they say on the tin. When DsRNA is present, it activates Caspases and causes infected cells to Off themselves. But we haven’t yet tested them with real life actual viruses.
Whilst in the last experiment, we were looking at caspases only, in the next one we want to know whether it actually improves cell survival.
The theory behind DRACO’s is that the first cells to get infected should be the last cells to get infected, and die off before they can spread virus to the rest of the cells in the culture.

In Figure 4 A, they use rhinovirus, one of the viruses that cause the common cold, and add it to Normal Human Lung Fibroblasts, which are a cell type usually found in the lung. They are the normal target for rhinovirus. So they added the rhinovirus to the culture of lung cells, to see whether the presence of DRACO would save them. The first four sections show the controls, showing that no one part of the DRACO extract protects the cells on their own. Within 12 days post infection, all of the cells are dead.
The last two sections are the most interesting part of this graph. The last section shows that when the complete , fully functional DRACO is added to these cells, they are protected against rhinovirus infection.
Okay, that’s good. I should note that for this experiment, the DRACO’s were already in the cell culture when the viruses attacked. But what happens if you got rid of the DRACO? Could the virus bounce back, only being momentarily delayed ? How long do these cells need to be given DRACO to stop the infection?
Figure 4B goes some way to attempting to answer this question. A number of different types of DRACO were added to cell cultures, which were then exposed to the rhinovirus. After three days of marinating in DRACO filled medium^[6], the some cells were removed and left in plain old normal medium. Whether or not media were changed, the effects were still the same after seven days. This shows that whatever DRACO is doing, it’s happening within three days of infection.
In Figure 4C, we drive down further into this question. If you remember earlier, that DRACO’s can stick around in cells for around 6 days? So what happens if we treat cells with DRACO, and add in rhinovirus after 6 days ? it turns out that the cells survive. But the question is, can DRACO work if it is given after infection ? The last 3 sections of 4C show that it can work for up to 3 days after the initial infection.
So they next tested a whole raft of different types of DRACO’s (Figure 5 A) to see their efficacy against rhinovirus infection. In the previous studies, they showed that DRACOs can allow increased cell survival. But they haven’t yet shown a reduction in the levels of virus. Figure 5B solves this. Cultures of human lung cells were tested four days after infection to see detect the whether any rhinovirus was present. Turns out, the cultures with DRACO’s did not have any viruses, whereas the controls did.
The next experiment was a basic dose response, which asked the question ; What concentration of DRACO will save all of the cells in a culture ?
So they took a set of cell cultures, and added different concentrations of DRACO to them. They then infected each of these cultures with different species of virus. They used Rhinovirus, murine encephomyelitis and intriguingly murine adenovirus. At concentrations of 0.1 nM, no protective effects were found. The DRACOs seemed to be effective against all the viruses at around 10nM. As with all experiments that involve a line graph and phenomena that could possibly be described by an equation , I wonder where the regression’s at ? But I’ll talk more about that later.
But the interesting thing here is that for some reason the DRACO is effective against adenoviruses. This is interesting because adenoviruses do not have a genome of RNA- they have a genome of DNA, and are not noted for using dsRNA at any point during their life cycle.
In figure 6, they tested whether DRACOs were as effective against adenovirus as they were against rhinoviruses. DRACOs were effective against rhinovirus seemed to be even more effective against adenovirus, with it conferring 100% protection even if applied 3 days after infection. And a whole raft of DRACOs were effective against the adenoviruses. Similar tests were applied to the murine encephomyelitis, amapari, dengue and guama viruses, which are each viruses from different families, and all of them form dsRNA at some point in their replication cycle. I could go into more detail about all of the viruses they’ve tested, but suffice to say that DRACOs look like they do what they say on the tin.
But a drug needs to do more than just stop viruses in a cell culture. A cell culture is basically just a culture of one cell type floating in fluid. The interior of an organism has connective tissue, a circulatory system and a whole variety of cell types all interacting with eachother in a complex structured mass which can’t be replicated in a cell culture. If this drug is to be effective, then it needs to be able to be absorbed into the body, and survive long enough to reach the same cells that the virus intends to infect. Since we’re talking about this drug fighting against different types of virus, then it’ll have to go into different places in the body. If it needs to fight against hepatitis C, then it’ll need to get to the liver. For rhinovirus, the lungs, and for something really horrible like hantavirus, it pretty much needs to get to the entire circulation.
So they need to know how fast this drug can pass through the circulatory system. Drugs have two routes out of the circulation system after they enter. Route 1 is via the kidneys, which act to filter out waste and toxic products from the blood. Route 2 is via the liver, which generally drains blood coming from the gut, and is the main place in the body where drugs accumulate and are detoxified. So when drugs end up in these areas, a fair bet is that they’ve gone through the circulation.

In figure 9 A, they inject the drug into the circulation of a mouse, and then tested out specific organs at specific times to see how long it took for the drug to appear in these organs. within 2 hours, the DRACOs appear in the lung and the kidneys. It starts to appear in the liver not long after, and it stays there for over a day.
So the question arises- can this drug actually work when given to a real live whole organism ? To assess this, they performed a survival experiment (Figure 9 B). They gave two groups of mice different types of DRACO, and one group no treatment at all. The treatment was given the day before, and for three days after infection with H1N1 flu virus. 13 days after infection, most of the mice that didn’t receive the DRACO were dead, whilst most of the mice who received the drug survived. Some mice had their lungs extracted, and the numbers of viruses present in their lungs were assessed. The mice treated with the DRACOs had much fewer viruses within them than the untreated mice. In Figure 9C , a repeat of this experiment was performed , only using different DRACOs, and smaller animal numbers.
But wait ! Let’s take another look at this infection model. The mice received intraperitoneal injections of DRACO of 200 microlitres. Now this may not be the most practical methods of application for use in people, it’s sort of (but not really) the equivalent of an iv drip. But perhaps if the DRACO was administered through a different method, such as via the lungs, it may be either more or less effective.

True to this, Figure 10 A shows the distribution of different types of DRACO to the lungs, after they were administered to the lung. The DRACO that remained in the lung the longest was also the one which was the most protective for mice infected with influenza.
So those were the experiments, but the question is, what can we take away from this research.

Summary

Cons

Error Bars: I know always tend to bang on about this when I review papers, but it’s really important that when you put error bars on a graph, that you explain what the hell they are. Now, to be fair, this isn’t a problem for all the figures, as some do note that the error bars show standard error of the mean. But when I see a graph with error bars, but doesn’t tell you what they mean, I often assume the worst, that they’ve let excel do it’s automatic error bar thing, or they are too embarrassed to show you the actual standard deviation.
Statistics: This paper was relatively thin on the statistics. Often people tend to think that the only point of stats is to get a small p-value to “prove” that their pet theory is true. But it’s so much more than that ! It’s the best way to really get to grips with the shape of your data without your preconceptions getting in the way. The statistics they use are so basically to convert the numbers of dead/live cells into “percentage viabilities”. But they don’t seem to use any statistical analysis. I made a comment earlier about how they could have fitted their dose response data to an equation using regression, as it does look like a traditional hill slope. With that sort of information, you can actually get some interesting information about the similarities/ differences between the viruses. Without the stats, I have to make a guess that the variations in the cell viability are not large enough to account for these differences.
dsCARE. In 2009, a group based in boston/ china also created anti-viral based dsRNA that had a similar design to the DRACOs described here , except there they were called dsRNA dependant caspase recruiters. There are only a few differences with this paper- they use microscopy to judge whether viruses are there or not, as well as cell viability, and use direct counts for virus.They even discovered adenoviruses vulnerability to dsRNA based treatment. But they don’t look at the drug distribution, a much broader set of viruses and cell types, show no animal work, and didn’t get the patents. Read it here.

Pros

Breadth: They have shown quite effectively (and exhaustively) that DRACOs can prevent an infection propagating throughout a culture of cells. Now, I should note that without statistics, I am giving them the benefit of the doubt on this. But I think that the evidence they present is really compelling, and grounds for optimism. I mean the great thing about this paper is the sheer breadth of it. I may have name checked an earlier paper with a similar concept, but personally I like this one a fair bit more. This is the kind of paper that simply couldn’t have been published anywhere else, I mean there are 22 f***ing figures. That’s a huge amount of data. I mean, I’ve seen papers jump to far greater conclusions with less than a quarter of what this group is presenting.

So should we get excited ? Well a little bit. You can allow yourself a little chuckle perhaps. This is good news in the fight against viruses. They may not have been the first to come up and test the concept for this drug, but they have done a lot to build a case for it being broad spectrum, and for the possibility of it being useful for treating viral diseases. Even though the outlook is promising, this treatment is still in it’s early stages. The proteins may need to be altered, improved. They may even be ditched altogether and the genes encoding the drugs may be given. The treatment that will eventually result from these findings may look nothing like what was used here. It may look promising all the way to human tests, and then something could go wrong then.
This potentially could be a penicillin moment for viruses. But it’s too soon to tell. Such historic moments can only ever be judged with hindsight. I only hope that when I’m judging this moment in the future, it’ll be a future in which viruses have been knocked back as badly as bacteria once were.

Footnotes

[1] I should note that whilst this is the way that many viruses work, there are some exceptions. Mimiviruses, which generally are found in the sea infecting amoebas, may work in a completely different way that is mostly unknown at this moment.
[2] This footnote has no point to it. There is no reference in the text. It exists for its own sake, at the expense of the rest of the article.
[3] One exception is small interfering RNA. small interfering RNA’s do form double stranded RNA, which is recognised and rapidly broken down by the cell. The fact that double stranded RNA is broken down rapidly by the cell is why siRNA’s are so useful for a cell. For the first time, I can say that is the exception that proves the rule.
[4] TLR-3 stands for “Toll-Like receptor 3” because it was similar to a protein called Toll. and there were three more discovered before it that also looked like Toll. Yeah, it doesn’t really have anything to do with it’s function, but most proteins have names that occur by accident, and have nothing to do with their function.
[5] you may really need to squint your eyes to see the protein band here. This may be an instance of what my supervisor would refer to as “visible by photoshop”
[6] Feel free to use this phrase in your harry potter slash fic

Rider T.H., Zook C.E., Boettcher T.L., Wick S.T., Pancoast J.S., Zusman B.D. & Sambhara S. (2011). Broad-Spectrum Antiviral Therapeutics, PLoS ONE, 6 (7) e22572. DOI: 10.1371/journal.pone.0022572.t002