Fighting Antibiotic Resistance One Gene at a Time: Karl Hassan Discusses His Research

Richard Jacobs: This is Richard Jacobs with the Finding Genius Podcast.  I have Karl Hassan.  He’s a ARC Future Fellow.  He’s at the University of New Castle In Australia.  I thought there was New Castle in England but no, there’s probably New Castles in US and all over the world.  So, we’re going to talk about his research fellowship.  He’s working at Antimicrobial drug business, very important issue.  So Karl, thanks for coming.

Karl Hassan: Yes, thank you.  Thanks for the opportunity.

Richard Jacobs: If you would tell me about your work.

Karl Hassan: So, I guess broadly I’m interested in the origins and mechanisms of antimicrobial resistance in bacteria.  I’m primarily looking at hospital-associated pathogens.  So, these are the ones that cause opportunistic infections so invulnerable patients in hospitals.  These bacteria have done a really good job of adapting to the hospital niche and we normally consider the hospital niche to be quite sterile because we use disinfectants and antiseptics to clean the surfaces and then patients that are in hospitals are given antibiotics to prevent infections.  But these bacteria have developed resistance to a lot of the antibiotics that we use to prevent infections and they’re able to tolerate some of the antimicrobials that we use to clean surfaces and to prevent their transfer between patients and so they have become resistant to those.  And because they’ve become resistant to those, they’ve become more of a problem and some of these bacteria have become superbugs, so we call them superbugs because they’re resistant to multiple classes of different antibiotics.  And in the past, we would have considered new drugs to be developed by big pharma companies but they’ve sort of withdrawn from that because of lower profits and so that’s meant that we don’t have new drugs in the pipeline.  So, what we’re interested in is the ways that bacteria have become resistant to the compounds that we’ve got so that we can potentially tweak them or use them in different ways to kill these bacteria.

Richard Jacobs: A friend of mine works for the CDC and he was saying that the P-traps in sinks and toilets and things like that are places where the patient will urinate or stuff gets poured down the train and then, drugs will go down there either through body secretions or otherwise and stuff sits [inaudible – 0:02:26.9] witches brew of the most evil bacteria on earth and then they get aspirated out of the drains and infect people.  Is that a mechanism you see?

Karl Hassan: Yes, that’s definitely a way or an environment where bacteria could become resistant to antibodies.  So, we also use antibiotics in agriculture and elsewhere and so where the origins of the resistance come from are quite complicated and how they find their way into hospitals.  But yes, certainly in those sorts of environment, when you’ve got multiple drug-resistant bacteria together, they can exchange DNA and become more resistant to multiple compounds.  And then, if they find their way back up into patients, then there are problems.  So, these groups that are looking at those types of mechanisms and where …

Richard Jacobs: So, you’re studying this particularly in hospitals?

Karl Hassan: So, we study the bacteria that come from hospitals but yes, so …

Richard Jacobs: What are some of the worst ones that are the most resistant to all drugs right now?

Karl Hassan: So, the ones that we’re particularly interested in have a gram-negative bacteria, so these are things like Acinetobacter baumannii, Pseudomonas aeruginosa and Enterobacteriaceae.  So, these are a problem particularly because they’ve got two membranes so that makes them extremely resistant or tolerant intrinsically to a lot of different compounds, so the added a membrane that they have is covered with a dense layer of sugars and it’s very impermeable to most different classes of chemicals.  And so, they’re becoming more of a problem because into the future, these are the bacteria that are the hardest ones to develop new compounds for.

Richard Jacobs: They have two membranes and the membrane outside is so hardened, how do they transit plasmids or things to membranes?  Wouldn’t that be harder for them to do that?

Karl Hassan: Yes, they’ve got mechanisms for that.  So for small molecules, they’ve got pores and the pores are quite selective to the types of compounds that they want to bring into the cell.  So, because of the characteristics of the inside of the pore, they may only allow hydrophilic compounds to pass through so things like metabolites and things that they want to have inside the cell, which makes it really hard to design an antibiotic that can pass through those pores to get entry in-between their two membranes and then also have the characteristics needed to get across the inner membrane.  The inner membrane, one of my big areas of interest, is in flux so the inner membrane also has quite a number of efflux pump so these are proteins that sit in that inner membrane and basically any compound that shouldn’t be in the sell, they’re able to recognize and pump it out.

Richard Jacobs: Is there a space between the inner membrane and the outer membrane or is it so close and so tight that nothing could reside between the two?

Karl Hassan: Yes, there’s a space in there.  So, in that area, one of the major things that you find in there is a layer of what we call peptidoglycan, just kind of like a mesh which protects the cell.  But yes, it’s kind of a viscous environment with lots of proteins and small molecules.  What there are some efflux pumps that span both the inner and outer membranes so they’re able to move compounds from within the periplasm or within the inner membrane across the outer membrane because the outer membrane is so impermeable that it makes it really difficult for those compounds to get back inside the cell.

Richard Jacobs: How do you know if a given bacteria is resistant to antibiotics?  In a dish, do you put some antibiotic with it and does it enter the cell and gets pumped back out?  How do you know what’s going on?

Karl Hassan: Yes, that’s right.  So, we can tell by doing simple resistance type tests, whether or not they are resistant, and then we’ve got different approaches that we use to try and understand why it is that they’ve become resistant.  So, one of the simple things that we can do is comparative genomics so we can sequence the genomes of multiple different strains of the same species, which have different levels of resistance and then compare the genes that they have to see why it is that some of them have become resistant.  And we’ve got quite good databases now of genes that are known to be involved in resistance and so we can look for those types of sequences in the ones that are resistant compared to the ones that aren’t resistant to get an idea about why they’ve become resistant.

But something that we’re also interested in is intrinsic resistance.  So, not these resistance genes that are getting past around or mutations that are required when bacteria are in hospitals, which make them resistant but they core resistance mechanism, so a good example is the outer membrane in gram-negative bacteria.  And to try and identify which of those intrinsic resistance mechanisms is involved in resistance to different compounds, we need to use different approaches.  And so for that, we use a lot of functional genomics.  So, one example is transcriptomic, so we can look at the expression of all the genes in the genome and bacteria are very efficient so they don’t want to express genes and waste their energy unless they need to.  So, we can challenge bacteria with different antibiotics and see how they respond in terms of the genes that they’re expressing and often, what we’ll say is that the ones that are involved in resistance that allow them to tolerate those compounds induced or at least the transcripts are higher in those cells.  And so that points us in a direction of the genes that are involved in intrinsic resistance.

Richard Jacobs: How does resistance arise, does the bacteria takes into molecule and changes its DNA or changes its gene expression so that it hardens its exterior membrane or the activity changes or how does it manifest?

Karl Hassan: So, there are different mechanisms for that.  So, what you’re talking about is acquired resistance so the development of resistance over time.  So, one way is that bacteria can pass pieces of DNA between each other and those pieces of DNA may encode for an enzyme or an efflux pump, which is able to confer resistance to a particular compound, so the enzyme might degrade the compound when it’s expressed and that confers resistance.  Sometimes you see mutations appear in different parts of the genome so often at the target-site of antibiotics, so antibiotics usually target things that are essential.  So, the molecules involved in transcription, for example, or translation of sequences and so we can say mutations appear at the sites of — the target-sites of the antibiotics.  So, that’s how acquired resistance works.  But yes, bacteria, a lot of the efflux pumps and the genes involved in producing the outer membrane are intrinsic resistance mechanisms.

So, sometimes when we challenge a bacteria with a compound, an antibiotic or antimicrobial, we’ll see the induction of something quite novel that we’ve never seen before.  So, in a study that we did 5 or 6 years ago, now we challenged Acinetobacter baumannii with a biocide called Chlorhexidine so that is commonly used as an antiseptic in hospitals and it’s typically in soaps so it’s used to prevent the movement of bacteria between patients on the hands of the hospital.  And when we challenged Acinetobacter with that biocide, we found that there are only a few genes that are induced, some of them encoded for an efflux pump which we knew about but there was another gene that was quite highly induced by that compound, which was completely unknown so it was annotated as a hypothetic protein, which means it was a piece of DNA that looked like it encoded for a protein but we had no idea what it did.  So, we were quite interested in why that gene was induced by chlorhexidine and so we went on to take it out of Acinetobacter and put it into another bacteria that was susceptible, and in there, we were able to show that it gave resistance to chlorhexidine.  So that was an example of identifying a completely novel intrinsic resistance to determine in a hospital.

Richard Jacobs: How quickly does this happen when a bacteria is challenged, how quickly does it change itself up?

Karl Hassan: I don’t know if we know the answer to that specifically.  You can, in the lab, induce so in the hospital it may be different but in the lab, you can induce resistance for some compounds I guess with a few passages of the bacteria so if you grow them on increasing concentrations of antibiotic in the lab, after a few days, you can have mutants that sort of breakthrough and resistant to that antibiotic.  So, it’s something that we would do if we were developing a new compound try and look at how quickly the bacteria can become resistant to it.

Richard Jacobs: But when you’re challenging a bacteria, is it that bacteria itself that would change its gene expression or future generations of the ones that change their gene expression?

Karl Hassan: Yes.  So, in terms of changing gene expressions, so the genes that are induced by the antibiotics, sort of part of an adaptive resistance response I guess, so they are sort of tuned to be expressed in response to an antimicrobial.  But sometimes, yes, you get a mutation that fixes a high level of gene expression, which confers the higher level of resistance.  And so there, I guess we assume that those mutations are typically spontaneous within a population.  So, because you’ve got such a large population of bacteria, there’s a good chance that these mutations just randomly pop up in the right spot and then because you’re challenging them with a high concentration of an antimicrobial, you kill-off the ones or the ones that don’t have that mutation are a lot less fit than the ones that do, so the ones that have the mutation then grow up to become the dominant type of cell in that population.

Richard Jacobs: Is anyone doing like single bacteria antibiotic challenging to look and see what happens?

Karl Hassan: So, at the single-cell level?

Richard Jacobs: Yes.  Has anyone looked at that?

Karl Hassan: Yes, people are doing things at the single-cell level and looking at how specific parts of the bacteria respond.  Yes, it’s not sort of my field of expertise.

Richard Jacobs: What are you discovering in particular then?  What kind of useful information you’re finding?

Karl Hassan: Yes. So, the hypothetical protein that I mentioned, it was a new type of resistance gene.  We then decided that to really understand how it works, we needed to get some insight into its function.  So, we were able to express and purify the protein and we could do experiments on it to test how it works.  So, we could tell from its sequence that it encoded for a membrane protein so a protein that sits in the bacterial membrane and we could purify it from the membrane.  So, we knew that it was a membrane protein for sure and then, we could speculate because it was a membrane protein on its specific mechanism of activity.  So, there are a number of different ways that a membrane protein could confer resistance to an antimicrobial so it could change the composition of lipids in the membrane, which make it less permeable to antimicrobials or it could be a regulatory protein that signals the inside of the cell to change the expression of genes to make the cell more resistant.  Or as I mentioned earlier, it could be an efflux pump.

So, when we looked at the protein that we found — the gene that we found that was giving resistance to chlorhexidine, we then tested to see if it was giving resistance to other antimicrobials.  So, if it’s an efflux pump, often we do see conferring resistance to other antimicrobials but it seemed to be very specific to chlorhexidine.  That was kind of unusual because chlorhexidine is a completely synthetic biocide so it hasn’t been seen in nature and in the long-distant past but we could see that this protein was conserved among a lot of different bacteria so we knew that it had been in the genomes of these bacteria for a very long time.

So, we did some studies to see if it interacted with chlorhexidine and we found that it could bind directly to chlorhexidine and so that was sort of consistent with it potentially being an efflux pump.  So we did some SAs in bacterial cells and found that it could reduce the concentration of chlorhexidine in those bacterial cells and so that, again, was consistent with it being an efflux pump.  We also looked to see how broadly conserved it was and we’ve found that they were homologs related sequences and a lot of other gram-negative bacteria.  And we went on to look at the functions of 40 or 50 of those and found that some of them also conferred resistance to chlorhexidine but some of them were giving resistance to more than just chlorhexidine, so other biocides and we could do different types of transport experiments to show that they’re able to lower the concentration of those compounds in bacterial cells, which is, again, consistent with them being efflux pump.  So, through this, we discovered that basically it’s a new family of multidrug efflux pump, which is quite exciting because we haven’t seen one of those for 15 years or so.

Richard Jacobs: Is the new one looked at literally at efflux pump structurally and actuated and somehow either using to computer modeling or something to see how it functions?  What does that look like to you and what does that tell you?

Karl Hassan: So in bacteria, now there are six or seven families of efflux pumps, and most of those, we have high-resolution structures, so most of them have come about through crystallography type studies.  Some of them now are coming through high-powered electron microscopy.  So, it tells us that the different families do sort of function in different ways, so some of them sit just in the inner membrane of bacteria and they’re able to transport substrates in the cytoplasm into that space between the inner and outer membranes, which we call the periplasm.  And so they seem to sort of function typically in a fairly simplistic way.  So they have essentially located binding site and substrates are able to bind into that binding site and then there’s a conformational change, which is energized in different ways either by the movement of another molecule into the cell or by the hydrolases of ATP and then, it is then exposed to the periplasm and the substrate is released.  Other than others, so there is a family called the RND Superfamily, which is a particular problem in gram-negative bacteria because it gives really high levels of resistance to lots of different compounds.  They function in a different, so they is a much bigger, more complicated structure and they have three sets of proteins that work together to capture substrates from the periplasm or the inner membrane and sort of move them through the top of the protein and then through a channel across the outer membrane.  So, those structures have given us a lot of insight into the ways that these proteins are able to recognize and interact with a very broad range of different compounds and to move them across the membranes out of the cell.  But for the family that we’ve identified, yes, we don’t have any structural information at the moment.  So, it’s very difficult to sort of speculate on how it actually works to reduce concentrations of compounds in the cell.

Richard Jacobs: What’s your end goal?  Let’s say you understand how the bacteria of your choice are resisting chlorhexidine, for instance, then what?  What’s your endgame here?

Karl Hassan: So, I guess sort of more broadly thinking about efflux and cell permeability, what we would really like to do is to understand what types of compounds are able to come into the cell and stay in the cell and not be subject to efflux.  So, if we can understand the specificity of all of the different efflux pumps, so it’s interesting bacteria typically have at least five or more different efflux systems, which seem to have overlapping specificity when it comes to antimicrobials.  But if we can understand the collective specificity of those different efflux pumps as well as the types of compounds that are able to enter the cell, we can potentially identify the characteristics of what would be good future antimicrobials against gram-negatives.  So, I guess in terms of resistance, that’s the end-goal but yes, we can use efflux for a lot more than just that.  So, the family that I was describing earlier on, which gave resistance to chlorhexidine, we knew that chlorhexidine, because it is just a man-made biocide and that’s only been around for a 100 years or so that that protein probably had other functions, so we thought that it would have been maintained in these genomes for millions or billions of years to transport chlorhexidine because it hasn’t been around.

So, we are really interested in looking at what its physiological function was.  And this is something that we see with a lot of efflux pumps, they don’t just transport drugs, they’ve got a range of other functions and that’s partly because they can recognize so many different compounds.  So, we went looking for its native substrate and we found that it was able to recognize and transport a class of compounds called Diamines.  So, these are found in all different cell types and they’ve got a whole range of different functions.  So, in DNA and protein stability, potentially in signaling and their male so act as surfactants, which allow bacteria to move around.  So, there are reasons why bacteria want to pump them out and they become toxic at very high concentrations and they may also act in signaling as surfactants so they need to be moved out of the cell.  And so we’ve found these proteins were able to transpose out of the cell.

They’re an interesting group of compounds because they’ve also got some commercial value, so diamines are used, for example, in the production of nylon and at the moment, we get a precursor for making nylon from petroleum but some of the other diamines which are recognized by these efflux pumps have different properties that we can use to make different nylons and they’d also potentially make cleaning nylons because we’re not using petroleum-derived compounds.  So, there is potential to use these types of proteins as well as the systems that regulate them in biotic to try and improve the production of these types of compounds in bacteria so that’s another sort of end-game when it comes to these discoveries.

Richard Jacobs: Do bacteria tend to alter antibiotics before they spit them out or do they just tend to take them in and spit them out unchanged?

Karl Hassan: I guess it depends on the antibiotic.  So, some of the enzymes that they produce are hydrolytic enzymes or modification enzymes.  So, these are things that are typically passed around between bacteria especially in these hospital bacteria and they themselves can give resistance because when degrading the antibiotic or modify it, it can no longer bind to its target site and so the bacteria are no longer susceptible to them.  What happens to them after they’re modified or degraded, I guess we’re not completely sure.  There are some bacteria that can use them as a carbon source so they don’t spit them out, they then use them to grow, which is really scary because it shows that they are completely resistant to them.

Richard Jacobs: What transits most easily into a gram-negative bacteria, you know, as plasmids from other gram-negatives and if so, why not try to package things inside of plasmids so that they can trans it very easily into the bacteria and surprise them in a bad way?

Karl Hassan: Yes.  So, I guess they’ve got specialized mechanisms for taking up DNA, which a lot of research groups are actively studying.  And some bacteria are able to take in foreign DNA a lot more readily than others.  DNA itself I probably — it would be challenging to package up a small molecule into a [inaudible – 0:24:43.6] strand of DNA but there are groups that are working on delivery systems for antimicrobials with nanoparticles, which might work in a different way.  So, yes, targeting in that respect is a potential future approach.  It’s not really my field but yes, delivery of those into a patient may be more challenging than [inaudible – 0:25:08.5] potentially.

Richard Jacobs: I thought plasmids were like [inaudible – 0:25:11.8] in a way and they have their own membranes or they’re naked pieces of DNA.

Karl Hassan: Yes, they’re naked pieces of DNA.  And often, when plasmids are transferred between two bacterial cells, it’s via a pilus so a structure that moves the DNA specifically from one bacterial cell to another but some bacteria have natural competence so they’re able to just take up fragments of DNA from the environment but yes, they’re not encapsulated by anything.  There are some animal viruses encapsulated, there are viruses that target bacteria but typically, they’re just a protein structure surrounding nucleic acid, which gets injected but they are a potential future avenue of treatments using viruses that target bacteria or against bacteria, something that was actively researched quite a lot in the past, particularly in the former soviet states and something that’s coming back into fashion in terms of potential treatment against bacteria with the lack of new antibiotics.

Richard Jacobs: In the beginning, you said that there was not money in new antibiotics but how could that be?  There’s resistant bacteria and it’s killing people as no one cares and no money in there?

Karl Hassan: I guess it just comes down to the profits that big pharma companies would make.  So, antibiotics are different to other types of drugs because typically we don’t take them continuously.  So, if you have an infection, you’ll take a course of antibiotics, hopefully, that will clear the infection.  If it doesn’t, then maybe you’ll take a course of different antibiotics.  So they don’t get used as much as other compounds and if we do develop something that’s new, then the clinicians are less likely to prescribe it because they want to keep it in reserves so that the bacteria in hospitals and so on don’t become resistant.  So, companies could spend millions or billions of dollars developing a new compound and then not be able to sell much of it so I guess there are issues in that respect.

And then, by the time they start to make profits, the compounds come of patent and so then, they’ve invested a great deal of money for a very tiny gain.  So, we can understand why pharmaceutical companies are less likely to pursue antibiotics but it’s sort of pushed a lot of the discovery of new compounds to the academic level to researches at university.

Richard Jacobs: It sounds like a broken system.

Karl Hassan: Yes.  I think that there are potential changes that could be put in place and I think some countries are starting to do that type of thing so that things can be on patent for a longer period of time or fast-track in terms of the trials that are needed when there are situations that people really need those compounds.  But it’s something that people or researches in academia need to keep in mind.  There’s only so far that you can take the development of an antibiotic at a university level before it becomes too expensive.  So, when people are developing potential new antibiotics, they need to keep in mind that in the future, this is something that needs to be taken on by a pharmaceutical company and made patentable by them if we want them to take them on and do the expensive clinical trials and so on.

Richard Jacobs: What do you think is going to be possible with your research over the next couple of years?  Anything that is close to a breakthrough or it’s still a long way?

Karl Hassan: We are still continuing to do those sort of functional genomic type SAs and there are other hypothetical proteins that colleagues and I have identified, which could potentially be other new classes of resistance determinants but ultimately and hopefully at least for some of these bacteria, we can get a better idea about what types of compounds are able to penetrate into those cells and evade being effluxed by these many efflux systems.  And so those characteristics will potentially find their way into the next generation of antimicrobial.

Richard Jacobs: What’s the best way for people to find out more about your work?

Karl Hassan: They can go to my website at the University of New Castle and have a look, so on there, we sort of keeping an up-to-date list of publications and research activities.

Richard Jacobs: Very good, Karl.  Thanks for coming on the podcast.  I appreciate it.

Karl Hassan: Yes, thanks very much for the opportunity, Richard.

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