Friday, July 11, 2014

4 Antibiotic Resistance and Persisters In Lyme disease

The other day, someone asked me what the difference was between bacteria which is antibiotic resistant and bacteria which are persister cells, thinking they were the same thing.

When one first hears about bacteria persisting, one often makes the assumption that must mean it's antibiotic resistant.

But there is a difference between bacteria which are antibiotic resistant and bacterial persister cells - which are antibiotic tolerant .

I'm going to provide a simple illustration (ragged as it is) to show what that difference is without getting too bogged down in terminology.

First, since many people are familiar with the term "antibiotic resistant" and have heard it in the news a lot when journalists discuss MRSA and other major bacterial infections, I'll start there, and explain what it means for bacteria to be antibiotic resistant...

What Is Antibiotic Resistance, Anyway?

When an antibiotic is used, susceptible bacteria are killed or inhibited by an antibiotic, resulting in a selective pressure for the survival of resistant strains of bacteria.

Some bacteria are naturally resistant to certain antibiotics - they just happen to have the genetic makeup and qualities which make them instantly resistant to certain antibiotics. But in other cases, bacteria develop a resistance to antibiotics through genetic mutation or acquisition of genetic material from another source.

Antibiotic resistance may take the form of a spontaneous or induced genetic mutation - that is, either a random mutation or one which arose due to changes in the bacteria's environment.

It can also occur when bacteria acquire resistance genes from other bacterial species by horizontal gene transfer via conjugation, transduction, or transformation - in other words, bacterial sex, viral sharing of DNA via bacteriophage, or acquisition of DNA from another external source.

Exposure to an antibiotic naturally selects for the survival of the organisms with the genes for resistance. A gene for antibiotic resistance may readily spread through a population of reproducing bacteria.

This is a quick and dirty diagram illustrating what antibiotic resistance looks like:




One thing to keep in mind in the illustration above is that non-resistant bacteria and drug or antibiotic resistant bacteria can exist at the same time in the same host. But eventually, all the non-resistant bacteria will die off and the antibiotic resistant bacteria will survive and reproduce, creating an entirely resistant population of bacteria.

The infection will continue to spread - unless the host's immune system is strong and can manage to kill off the antibiotic-resistant bacteria - or a different antibiotic can be found that kills them.

But some antibiotic resistant infections are so virulent and so successful, that neither antibiotics nor the host's immune system can overwhelm them.

This is catastrophic when it happens on a large scale. Right now, we are headed that way, as we are having a worldwide antibiotic resistance crisis: Too many people are contracting infections which we are having difficulty effectively treating with existing antibiotics - or in some cases we cannot treat them at all.

So, this explains what antibiotic resistant bacteria is: Bacteria which either naturally has a genetic makeup which makes it resistant to a specific antibiotic or bacteria which mutates (due to various factors) in such a way that it is more likely to survive the onslaught of antibiotics and produce a new generation of resistant bacteria.

How Are Persisters Or Persister Cells Different From Antibiotic Resistant Bacteria?

Persister cells are a small subset of a given bacterial population which are not produced by genetic mutation, nor are they resistant to a specific antibiotic by default.

Persister cells are a specific phenotype of the same bacteria. A phenotype results from the expression of an organism's genes as well as environmental factors and the interactions between the two.

When we specifically think about a phenotype in terms of people, we think of their physical characteristics - such as blue or brown eyes, curly or straight hair, and so on. Phenotypes are physical characteristics which are expressed, based on an organism's genes.

In terms of bacterial phenotypes, we can think more about size and shape, rod shaped or spiral cells, and so on. But we can also think in terms of tendencies towards dormancy, and that certain environmental factors can lead to bacteria expressing their innate tendency to go dormant.

One difference between antibiotic resistant mutants and persister cells is that, unlike mutants, cells regrown from such persistent bacteria actually remain sensitive to the antibiotic. But the entire population will not be as sensitive, and there will be a subpopulation which will be antibiotic tolerant.

Here is a simple illustration showing the difference between antibiotic resistant bacteria, and persister cells, which are antibiotic tolerant:


Image from Kenyon College's awesome MicrobeWiki.

The above illustration shows the key difference between antibiotic resistant bacteria and persister cells.

In the first row, the bacteria in the first dish are mostly susceptible to antibiotics, except a small number of bacteria which are resistant. Almost all bacteria die when exposed to antibiotics - leaving behind the resistant bacteria, which multiply and fill the dish. Throwing more of the same antibiotic at these bacteria will not kill them.

In the second row, we have bacteria which are also mostly susceptible to antibiotics, but a small number of bacteria are persisters. Almost all the bacteria die, and the persister cells become active and reproduce, creating a new population of bacteria of which some can still be susceptible to the same antibiotics and be killed off - but there will continue to be persister cells which could slowly multiply and survive more of the same antibiotic.

Persister cells have a phenotypic "switch" which can turn on at random or in response to environmental events. There are at least three main phenotypes of persister cells:

1) Those that reenter a normal growth phase and are quickly killed.

2) Type I persisters exit the stationary phase very slowly, in a matter of hours (if not longer) rather than minutes after getting nutritional requirements met.

3) Type II persisters occur via a spontaneous switch from a normal growth rate to a consistently slow growth rate - regardless of growth conditions and rarely switch back to the normal growth rate.

Anyway, overall, the mechanisms of persisters are poorly understood, and it's only in the past several years that interest has been ignited (or actually, reignited, as it was known some cells persisted after antibiotic use in the 1940's!) in persister cells and these mechanisms are being more closely investigated.

Antibiotic Resistance And Borrelia Burgdorferi - Not So Fast?

So what about the bacteria which causes Lyme disease, Borrelia burgdorferi? Is it antibiotic resistant? Has it shown signs of going the way of staph infections and tuberculosis, by becoming increasingly resistant to the antibiotics thrown at it?

No - at least not so far. In general, there is no evidence of acquired antibiotic resistance by Borrelia burgdorferi in response to treatment.

There is evidence, however, that some isolates have different preexisting susceptibilities to different antibiotics. Borreliae are known to be naturally resistant to aminoglycosides and quinolones, such as ciprofloxacin acid and ofloxacin. There have been Borrelia burgdorferi strains grown in laboratories and clinical isolates which show resistance to erythromycin. This particular resistance was first written about in depth in a 2002 study. Strain N40 is known to be resistant to erythromycin.

Erythromycin resistance is not an issue for most people who contract Lyme disease because erythromycin is a second line antibiotic which is not used to treat Lyme disease very often. It is important to be aware of it in case a patient has an allergy to the other antibiotics which are typically given and is offered erythromycin.

A study completed in 2010 demonstrated that Borrelia burgorferi is not eradicated by tigecycline, and in this study the issue of Borrelia burgdorferi potentially having persister cells and being antibiotic tolerant was raised.

It is possible that in the future Borrelia burgdorferi will show signs of acquired antibiotic resistance, and studies will reveal this to be the case. There is recent evidence that Borrelia burgdorferi has an efflux pump - also known as a membrane transporter protein - which could "pump" antibiotics out of cytoplasm as a mechanism of resistance. (More about efflux pumps here: http://en.wikipedia.org/wiki/Efflux_(microbiology))

Intriguingly, the researchers who wrote about these efflux pumps in Borrelia burgdorferi stated, "Existing evidence indicates that the possible heterogeneity of B. burgdorferi may enable certain isolates to evade antimicrobial therapy and may account for the subsequent relapses suffered by some patients," and cited three papers which support this statement.

At the time of publication up to the present, though, acquired resistance to specific antibiotics in specific isolates has yet to be confirmed.

However, new research has identified that Borrelia burgdorferi does have evidence of a persister cell population in vitro.

Borrelia burgdorferi And Persister Cells

Some researchers - such as Dr. Stephen Barthold, who completed the study mentioned above on the effectiveness of tigecycline to treat Lyme disease - have suspected for some time that Borrelia burgdorferi might have a persister cell population.

The issue of persistence in Lyme disease has become an oddly controversial topic, for which there is no reason - research will surely sort out what the nature of these wily spirochetes is one way or the other.

And there is a renewed interest in determining what the nature is of Borrelia burgdorferi spirochetes which have been known to survive initial antibiotic treatment in animal models, and could survive treatment in humans as well.

Recently, the CDC webcast a seminar via live streaming about government-funded studies touching on the persistence of Lyme disease spirochetes (this blog will discuss this seminar more in the near future).

But even more recently, two publications were announced which discuss Borrelia burgdorferi persister cells as well as which antibiotic compounds to which those persister cells may be at least partially if not totally susceptible.

The first publication was presented at the American Society for Microbiology conference this year (ASM2014) by Sharma et al, who are Dr. Kim Lewis' team which studies persister cells at Northeastern University. In their presentation, "Persister formation in Borrelia burgdorferi", the authors determined that 0.001% to 1% of Borrelia burgdorferi cells can survive lethal doses of various antibiotics in vitro.

If 0.001% to 1% of the cells can survive lethal doses of various antibiotics in vitro, then once you've killed off all but the persister cells, the original population will be replaced in vitro in a range of times and conditions as follows:

If only .001% of the spirochetes remain, and it takes 24 hours for each division, it will take 17 days for the bacteria to return to their original population size.

If only .001% of the spirochetes remain and it takes 12 hours for each division, it will take 8.5 days for the bacteria to return to their original population size.

If 1% of the spirochetes remain and it takes 24 hours for each division, it will take one week for the bacteria to return to their original population size.

If 1% of the spirochetes remain and it takes 12 hours for each division, it will take 3.5 days or so for the bacteria to return to their original population size.

All these are back-of-the-napkin calculations for what would happen to these bacterial persisters in vitro, provided they aren't environmentally challenged and have a stable medium; provided they don't lapse into dormancy. What exactly would happen in vivo, in the host, has yet to be clearly determined.

The second paper, by Feng et al, "Identification of novel activity against Borrelia burgdorferi persisters using an FDA approved drug library", with Dr. Ying Zhang as PI, evaluated the use of specific FDA-approved antibiotics which affected persister cells. A series of fluorescing protein studies were completed which gave visual confirmation of which of the 1524 compounds reviewed were more effective at killing persister cells. So far, daptomycin appeared to be at the top of the list for effectiveness in vitro. Additional studies in the future may be performed on animal models.

The executive summary:

Antibiotic resistant bacteria is resistant due to internal or induced genetic mutation of the bacteria, or by introduction of new genetic material to the bacteria.

Persister cells have a phenotypic switch, and can become dormant randomly or due to environmental stress. All the genes the bacteria needs to do this are already there; they are not mutants. Persister cells can be susceptible to antibiotics, but a subset will be tolerant to antibiotics - and that degree of tolerance depends on which phenotype they are.

Borrelia burgdorferi do not show signs so far of acquired antibiotic resistance. But the bacteria do have natural resistance to several antibiotics, and some specific resistance based on strain.

Borrelia burgdorferi have a population of persister cells, on which further studies are needed to determine their role in the infection process and how best to address them.




4 comments:

  1. Hi Camp Other, thank you for this easy to read explanation on persister cells and antibiotics resistance. I still have to re-read it - my brain is "lazy" these days…you know brain fog.

    But one question just of the cuff, if you don't mind. What do you think happens if you use an antibiotic repeatedly in a what seems a chronic infection, there is an improvement of symptoms, but the the infection(s) keep reoccurring (flare up of symptoms after a few days or weeks)? Is this now a sign of persister cells or an acquired antibiotic resistance, or both ? Best wishes Anushka

    ReplyDelete
  2. Hi Anuska, and welcome to Camp Other blog!

    I'm glad you got something out of this entry. I tried to make it easy and provide simple illustrations for examples.

    Regarding your question:

    If you use an antibiotic repeatedly and there IS a chronic infection and you experience improvement, then symptoms keep recurring, it can be for a variety of reasons:

    1) There could be an infection with persister cells in which some are being killed off because they are no longer dormant and in a sensitive state (sensitive to the particular antibiotics given).
    2) It could be the bacteria did acquire resistance. So far this hasn't been discovered in Borrelia burgdorferi as I've stated above - though it is naturally resistant to certain antibiotics to begin with. But so far, no one has found an example in research of it acquiring resistance in human patients.
    3) It could be that the infection has been eradicated and there is some immune dysregulation and inflammation going on and antibiotics have an impact on that dysregulation and inflammation.
    4) A combination of any of the above.

    I make these statements based on bacterial infections in general - not so much Borrelia burgdorferi in specific. It is still not 100% clear what is happening in patients with persisting symptoms (including myself) and why some respond better to additional antibiotics than others.

    My guess would be that it wouldn't be resistance for those who find some relief from taking antibiotics, based on what is known about B. burgdorferi. But there's a whole lot we need to learn about it.

    For an idea of how other bacteria can respond to different antibiotics at different times during the course of infection when it has persisters, you might want to check out this video about the Mitchinson Hypothesis regading tuberculosis:

    John McKinney (EPFL) Part 3: Phenotypic Heterogeneity and Antibiotic Tolerance - video [27 miinutes]

    I apologize that it is somewhat complicated, but it may shed light on how antibiotics can affect specific bacteria and how certain antibiotics are better treatment than others at different points in time.

    CO

    ReplyDelete
  3. Great article. I've read that daptomycin does not penetrate the blood brain barrier. Wouldn't this be problematic for treating a bacterial infection that is known to traverse the blood brain barrier?

    ReplyDelete
  4. Hi Curtis,

    Thanks - glad you enjoyed reading it.

    Daptomycin does penetrate the blood brain barrier, but it has minimal penetration cited anywhere from 2-3% up to 5-6% in the CNS. It's been effective in treating bacterial meningitis, but it's unknown how effective it would be for treating neuroborreliosis - particularly if it affects the brain directly.

    This is why I emphasize that the above studies are in vitro studies, too, and not in vivo. We don't yet know how effective these agents will be in animals - let alone people - for treating Borrelia infections. It's also possible that experiments will be conducted using a combination of antibiotics and antifungals, and there may be a synergy between them which facilities permeability of the blood-brain barrier and allows for greater saturation. I don't know, and more experiments would need to be conducted. I think this is the next step the researchers mentioned may be taking, too.

    CO

    ReplyDelete

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