Reply To: Dr. Tim Lu – Bacteriologist & EE (Excerpt& Video Available)

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    Question: The studies from the CDC state that 60-80% of chronic infections involve biofilms. What’s the connection between bacterial biofilms and chronic health conditions?

    Answer: There’s an increasing recognition that biofilms play a role in chronic diseases and the reason for that is that biofilms are very difficult to eradicate. They live on surfaces, they produce extracellular material that can defend themselves against human immune cells or antibiotics, and therefore they’re very difficult to remove. And once they form on a surface, they’re very difficult for your body or for drugs to kill them. Examples of this can include infections of bone, infections of your heart valves, infection of the urinary tract, in which bacteria can colonize these surfaces of the human body and live there for long periods of time.

    Question: Tell us a bit more about biofilms.

    Answer: So biofilms are a complex type of organizational structure that bacteria and these yeasts like to live in. I like to generally think of bacterial biofilms kind of like fruit Jell-O. That’s kind of the analogy I like to talk about, and the reason for that is, if you think about a fruit Jell-O, where you have the individual pieces of fruit, embedded in this gelatinous material, the biofilm cells, the bacterial cells, are like the fruit in the, inside the Jell-O and they produce materials, for example, polysaccharides, DNA, all those types of different materials that form this gelatinous layer that surrounds them and provides structure for them to live in. Depending on the species of the bacteria, if you’re talking about E. coli or staphylococcus species or pseudomonas species. The type of extracellular material they produce is different, but they all have one thing in common: they provide some kind of protection and provide some stabilization structure for the rest of the community.

    Question: How does a colony form, when does it form…what are the conditions necessary for a biofilm to form?

    Answer: The life cycle of a bacterial biofilm is pretty interesting. So it would usually involve some type of bacteria that can swim around we call “planktonic culture”, which is in liquid, and they’re not stuck to any surfaces. They swim around until they find a surface that they like, and then they stick to that surface, and they start growing on that surface and growing more and more complex structures as they grow. At a certain point, when the biofilm is grown to a certain size, it reaches its mature state. At that point, it can shed bacteria that kind of can swim to other locations. This happens in human cells, the human body, happens out in the environment, and the conditions that are necessary for biofilm formation are essentially like a surface that the bacteria would like to live on and the bacteria needs to be able to produce the right types of material so he can stick to those types of surfaces.

    Question: How do you think the biofilm colonies are hiding from the immune system and/or what makes a biofilm more resistant to interventional therapies?

    Answer: Biofilms are difficult to eradicate types of infections for a variety of different reasons. is just by the purely that they live on surfaces. So, if you think about it, if you have a bacteria floating around in a liquid and your immune cell comes by and tries to eat it, that’s very easy for the immune cell to reach around the bacteria and eat it, as opposed to if you’re trying to clear something off of a surface, there’s no easy way for your immune cells to kind of get around the infection. It can’t, it can’t really flank the type of infections. So that’s one kind of big problem in terms of trying to eradicate biofilms from any surface.

    The second reason is that when a biofilm forms, it tends to produce these extracellular materials that can either prevent the penetration of things like bacteriophages or immune cells deep into the biofilm.

    The third thing is that when biofilms form, a lot of these cells adopt what is called a persistence state, in which they kind of shut down, they’re not very metabolically active, and, as a result, a lot of the drugs that we use, like antibiotics, can’t really kill those cells. A lot of the antibiotics we use actually target actively dividing cells. So, if a cell is kind of just sitting around, not dividing, not very active, it’s very hard for antibiotics to kill these types of cells.

    I think that’s definitely a problem, since more research has shown that especially some of the antibiotics that are very active against killing bacteria can never sterilize an entire population. So let’s say you start off with a culture of a million bacteria, and you apply this very strong antibiotic. You may kill off the majority of that bacteria, but, for example, some of the studies we’ve done show that, let’s say you start off with a million bacteria, you may be able to kill 999,000 of them, but you still have about a thousand bacteria left, which are kind of immune to the antibiotics at this point. They can adopt a persistence state, and once you remove the antibiotics, they can start forming again and growing into a biofilm. So the presence of persister cells and the inability for us to really completely eradicate a biofilm leads to things like antibiotic resistance and future biofilm formation.

    Question: How many different microbes work together to create biofilm communities? E.g., gram negative, gram positive, yeasts…?

    Answer: I think your question about how different microbes work together to create biofilm communities is a very interesting one, and it’s something that the communities only now are really starting to get a grasp on. There are many theories, where how different microbes can work with each other and the limitation is that we don’t necessarily have great technologies by which we can study these. There are several mechanisms by which this might work. For example, bacteria can produce these molecules called quorum sensing molecules, and they produce these when the bacteria grow to a significant concentration, and it kind of signals to their friends, like a bacterial cell will tell it’s friends, like, “Oh, now that we’ve reached a certain concentration, maybe we should switch into a different state, like a biofilm state or a virulent state.” And it’s been found that, actually, different types of bacteria can communicate with each other by producing these quorum sensing molecules, and so quorum sensing is a big component of what people are trying to study now in terms of polymicrobial biofilms.

    In other situations, different microbes may have kind of more of a symbiotic relationship, where one microbe might produce a nutrient that the other one likes and vice versa, and therefore, they would like to form a, a biofilm where they’re in close proximity to each other, and they can both have increased survival because they live in a biofilm together.

    The third thing maybe that different microbes produce is different factors that can help protect each other from extracellular insults. For example, bacteria may produce a certain type of polysaccharide that another one doesn’t, and in these polymicrobial biofilms, they may produce different types of polysaccharides that add increased protection for the entire community as a whole. And the last thing that people like to talk about in terms of polymicrobial biofilms is that, because these different cells are in close proximity with each other. They’re in an ideal situation where one bacterial species can transfer genes to another one, and they can share genetic elements that can promote survival. One of these survival elements are antibiotic resistance genes, for example, and that can be traded between bacteria, and cause antibiotic resistance to spread through a community of different types of species.

    Gene transfer can occur between bacteria or between bacteria and non-bacterial species — studies have been done between bacteria — there’s a huge diversity of bacteria that have antibiotic resistance and even just studying that reservoir of antibiotic resistance genes is quite interesting already.

    Question: How are your T3 and T7 phages able to enter bacterial biofilms and is the hydrolysis of the biofilm adhesives the key part of that strategy? Is it akin to like chipping away the cement between bricks?

    Answer: That’s a great analogy. So the way our engineered bacteriophage technology works against biofilms is that they produce components, or these enzymes that can chew up the biofilm, extracellular matrix. So, as I mentioned before we like to think about these things as fruit Jell-O, where the, the fruit inside of the biofilms are the cells and the Jell-O is the extracellular components. And, if you think about it, if you’re trying to deliver an antibiotic or virus deep into the fruit Jell-O, it’s going to be very hard to penetrate through all the Jell-O to the bottom of the bowl. So, by producing enzymes that can chew up the gelatinous material you can allow these phages and antibiotics to penetrate deeper and deeper into the biofilms and eradicate much more of the biofilms than you’re able to do otherwise.

    Question: Regarding your future solutions: are you focusing on monomicrobial, or could it also be true for the polymicrobial films?

    Answer: So our technology, as currently tested, is focused on the monomicrobial biofilms, and this is for a variety of reasons. For regulatory reasons, it’s advantageous to go after monomicrobial biofilms when you’re trying to put a, push a new technology through with the FDA. It’s also easier to work with in the laboratory. However, we do believe that a lot of these technologies are applicable to polymicrobial biofilms, and the reason is as follows: all…the enzymes that we’re using actually target a variety of different types of bacterial biofilms. So, not only do they go after E. coli, but they go after other different types of bacteria as well, the same enzyme, and therefore we think that it should be able to go after polymicrobial biofilms as well, although it’s something that is a little bit further along in our research plan.

    Question: Can you differentiate between acute conditions, chronic conditions, systemic bacterial infections and where your future solutions may apply?

    Answer: So the technology that we’re developing can probably be used for acute conditions, chronic conditions, and in certain cases, systemic infections as well. Acute bacterial infections, such as the ones that cause sepsis, in which you have bacteria flowing through your bloodstream can be treated with bacteriophage, although it may not be the ideal technology to use at that point. In those acute infectious conditions, the bacteria usually has not had enough time to form a very thick biofilm, and therefore those bacteria generally can be killed by antibiotics, and these antibiotics can spread throughout your bloodstream very quickly, and can treat these types of infections.

    Question: Are there any things, any other things you wanted to mention today that we didn’t cover that are important for the audience to hear?

    Answer: Biofilms are actually intricately related to the problem of antibiotic-resistant bacteria, which is a topic people have heard about a lot more in the popular press. So antibiotic-resistant bacteria are essentially infections that are difficult to treat because these bacteria have evolved resistance to antibiotics. But awareness of the biofilm issue has really spread significantly throughout the medical community.

    Examples include MRSA infections, but there are a lot of other examples, e.g., gram-negative and gram-positive bacteria that have acquired genes that make them immune to a lot of the antibiotics we’ve developed. Now the issue is that, in the last 30 years, investment in developing new antibiotics has really decreased significantly, and, therefore clinicians are very restrained at this point in terms of what antibiotics are effective in terms of treating these types of infections.

    The link between biofilms and antibiotic-resistant bacteria is quite close; in that biofilms, because of the presence of persister cells and being able to trade genes between each other, can often have an increased level of antibiotic resistance. And so moving forward, the type of technologies we need to develop and the type of research that we need to do should really focus on both of these problems together: biofilms as one distinct entity, but also on antibiotic resistance and how they interplay with each other to cause human disease.