Posted 5:03 PM by Paul

Biofilms, Quorum Quenching, and Antibiotic Resistance
It's been a rough couple of weeks (in a good way), but here's another installment of our saga. As we learned last week bacteria often form Biofilms, aggregates on surfaces that can cause lots of trouble. I didn't get too much into the details of how these aggregations form, but I a couple of points; 1) Biofilms play major roles in lots of things we aren't to happy about, and 2) Biofilm formation is controlled by chemical signals that bacteria produce. I mentioned bacteria forming biofilms as part of pathogenesis. Another bit of bad new is that Biofilms are more resistant to antibiotics than their planktonic (free floating) counterparts.

The ways in which bacteria cause us problems are manifold. For example, when someone goes into the hospital, they often come out with an infection they didn't have before. One of the very common ways this occurs is when bacteria grow in a biofilm on one of the various invasive catheters used commonly in medicine. These include urinary catheters (UTIs are very common, although usually not fatal) and lines into the bloodstream (much more dangerous). This problem is exacerbated by the well known fact that bacteria that colonize hospital workers are much more likely to be resistant to antibiotics (an aside; when I was a grad student, we did this experiment with the med students I was teaching. The ones who were currently working in the hospital had bacteria on their skin with higher levels of resistance to common antibiotics). This is even more problematic when we recall what I mentioned one paragraph ago, that biofilm bacteria are even more resistant.

So this leaves us with the following scenario; patient comes to hospital, gets an infection from a catheter, gets treated with antibiotic, then another, then another, till they eventually get vancomycin and get better. So now all their associated bacteria have been exposed to a very strong antibiotic. At a population level, this can lead to the emergence of vancomycin resistance in bacteria (like MRSA) that are resistant to virtually everything else.

Can this get worse? Oh, yes! Bacteria in biofilms (like the ones commonly causing these infections) are not terribly picky about their neighbors. Single species biofilms are cultivated in laboratories, multiple species biofilms are found in nature (such as dental plaque). So when a multiple species biofilm occurs, DNA transfer can occur. And does it? Hoo boy, does it ever!

So, to recap; bacteria in biofilms cause diseases, especially in hospitals, where antibiotic resistance is a bigger problem. Oh yeah, and biofilm bacteria are even more resistant.

So how to deal with this?
Well, in the last few years (about 5, give or take), a lot of investigators (including me, briefly) got interested in the idea of Quorum Quenching. The basic idea goes like this. A QS circuit involves a signal that is detected by a protein on the surface or in the cytoplasm (a receptor). The receptor either directly influences gene expression (a regulatory protein, or response regulator) or signals to a downstream protein that does this (Bonus points to people who recognize the similarity to hormone systems). So interfering with this would presumably prevent expression of genes that are regulated by quorum sensing (virulence factors and biofilm formation factors, to name a few). Two basic approaches have been attempted, with some success in vitro and in planta. Competitive inhibition with molecules (furanones) that bind to the receptors (perhaps you have heard of the analogous notion of estrogen-like molecules contaminating water and interfering with hormonal cycles of animals and humans), and enzymatic degradation of the signal (I worked on this type of thing). The basic notion is that if we can prevent biofilm formation, we can reduce the levels of biofilm related disease, and therefore 1) make people's lives better, and 2) reduce the use of antibiotics. So, how's it going? Well, I'm out of the business, so I can't really answer that. It's promising, and it will be very likely to work in industrial and agricultural settings, but in medicine, we'll see.

Posted 8:21 PM by Paul

Quorum Sensing, Biofilms, Bacteria as multi-cellular aggregates

When you were taught microbiology (as every last one of you should have been), you were probably shown a microbial cell structure like this one from a standard textbook. The parts are all there; DNA, membrane, cell wall, flagella, ribosomes, etc. If your micro class was pretty good, you learned about what all the parts do, then you went on to learn how bacteria get energy and carbon, as well as their other nutrition. You were probably told, in perhaps not so many words, that bacteria are just really simple versions of our cells, stripped down and optimized for their niche in the world. If your class was good (again!), you might even have learned about some of those niches besides us. However, if it was typical, you learned about diseases for most of the rest of the course.

Now, learning about infectious disease is very important. As a grad student in med micro, I made a lot of effort to learn the structures and functions of all sorts of toxins, including my favorites, the Superantigens from Staphylococcus aureus. Staphylococcus aureus makes a bewildering array of virulence factors, including things on the surface that protect it from the immune system, and lots of toxins (see above review paper). The order in which these various products are made, and which ones are being made, makes a huge difference in what disease S. aureus causes, whether it is a boil or toxic shock syndrome, or more recently, necrotizing fasciitis (By the way, I've found that as interesting as I think this is, gross pictures are worth at least a thousand words, so click through, if you have a strong stomach!). When I learned about this system, I found out a peculiar thing about bacteria. A million or a billion bacterial cells is not just the same cell multiplied a million or a billion times over. Bacterial cells change and develop over time, especially in the aggregate. In Staph (including the very, very relevant Methicillin Resistant S. aureus), there is a complex system for controlling cell behavior based on the population size and density...especially the density. The system (largely figured out by a very big group of researchers centering around Richard Novick at NYU) involves a peptide ( a short string of amino acids) secreted by the bacteria as they grow. Through a fairly complex mechanism, this peptide is bound by a protein on the surface of the cell, which activates a regulatory RNA molecule (called rnaIII). This regulatory RNA molecule (a relatively rare thing in microbial genetics) is a global regulator of gene expression. What does this mean? It means that rather than a single gene encoding a single protein that does something, this RNA molecule turns on and off a whole bunch of genes, and and that massive change in what genes are being expressed is a major factor in what disease results (the other factors are what toxin genes are present, and environmental factors such as pH, oxygen level, and CO2).

The astute reader will wonder what any of this has to do with the title of this post, which is about bacteria as multi-cellular aggregates. Well, see, the Staph stuff is the tease, to get you interested. Now here comes the science. The words I used in that paragraph; population, density, global regulator, are all words used to describe a system called a quorum sensing system. This means that the bacteria act one way when they are sparse, and as their density grows, they act another way. The canonical system was identified as a symbiosis, rather than a pathogenesis, between the marine bacterium Vibrio fischeri and the squid Euprymna scolopes. It turns out, after a great deal of fine work by too many people to name, that this system revolves around the production of a chemical called an acyl-homoserine lactone (don't worry about it!). Like the peptide I mentioned before, this is secreted by the bacteria as they grow, and it binds to a receptor (this time in the cell, because these can pass through membranes (maybe)), triggering responses in genes regulated by the system. In the V. fischeri / E. scolopes interaction, this results in bioluminescence (Finding Nemo, anyone?). In the squid symbiosis, bioluminescence is thought to be a defense against predators, because the light emitted matches moonlight in the visible spectrum, thereby preventing the squid from casting a shadow as seen from below by predators.

The astute reader who has made it this far will wonder "what does this have to do with multi-cellular aggregates, or biofilms?" After all, I wrote above about bacteria inside a squid, or in a person causing disease. But they are inside something, not on something, right? Well, your wrong, now pay attention! (kidding!) In fact, it turns out that microbiologists have pretty well been studying the wrong stuff for about the last 100 years, in some ways (we aren't going to take back the Nobel Prizes, or anything!) Most bacteria, in most environments, most of the time, live attached to something, and aggregated with other bacteria. It is a rare thing for a bacterium outside a lab to be floating in a fluid containing free nutrients. This attached lifestyle is called a biofilm. In the last 15-20 years, it has become a major field of study. I even wrote a grant about it! (yeah, I'm pretty proud of myself, why do you ask?) So what does this biofilm thing have to do with S. aureus disease? A lot, actually! It turns out that many diseases in eukaryotes, caused by bacteria, are caused by them when they form a film on a biotic surface. The biofilm is often a critical stage in the infection. Many hospital infections are entirely based on the formation of biofilms on medical devices or tissues.

It turns out (surprise, surprise) that many bacteria have complex systems for controlling the formation of these biofilms, with some significant commonalities. In many gram-negative bacteria (like Pseudomonas aeruginosa, bacteria that can cause wound infections and lung infections in cystic fibrosis) the signals that control expression of virulence genes and biofilm formation genes are one and the same, acyl-homoserine lactones. In gram-positive bacteria (like the aforementioned S. aureus) peptide signals are used. These signals in gram positives are important in transfer of DNA between bacteria, uptake of DNA from the environment, biofilm formation, and virulence factor production (at least).

I better stop here, but it is a huge, complex story. Just understanding one of these systems can take a lifetime of well-funded research (I hope!). But that last bit there is particularly interesting. The role of horizontal or lateral gene transfer in bacteria; how is this connected to quorum sensing, biofilm formation, and virulence (as well as other things)? That sounds like a topic for next time!