Persistent bacteria could aggravate the problem

Antibiotic resistance is one reason why antibiotic treatment sometimes fails, another reason is antibiotic persistence.

New insights into this phenomenon were presented at the EMBO workshop "Bacterial Persistance and Antimicrobial Therapy" in Ascona on 10 - 14 June 2018. Among the participants were biological physicist Nathalie Q. Balaban and microbiologist Sophie Helaine. In an interview, they talk about the causes and implications of persistence and possible links to antibiotic resistance.

Prof. Balaban, Dr. Helaine, it is generally known that bacteria can become resistant to antibiotics. But what does persistence mean?

N. Balaban: Persistence is the ability of bacteria to survive antibiotic treatment without being resistant. It usually doesn’t happen in the whole population but only in a subpopulation of bacteria.

S. Helaine: The best way for bacteria to do this is to arrest growth. It is well known that non-growing bacteria are less susceptible to antibiotics than actively dividing bacteria.

So the bacteria enter a kind of hibernation state and that is why they are not susceptible to antibiotics anymore?

N. Balaban: Yes, you could put it that way.

Bacterial resistance often only protects bacteria against specific antibiotics or antibiotic classes. Does the same principle apply to persistence?

N. Balaban: Typically, a reduced metabolism or reduced growth provides these dormant forms with protection against more than one antibiotic. If you change from one antibiotic class to another you can see that they are still persisting.

What happens to these dormant bacteria once the antibiotic treatment is over?

S. Helaine: Once the antibiotics are no longer in the picture, some of these bacteria will wake up and will be free to grow. It is possible that this is what happens when relapse of infection is occurring.

Have such persisters already been detected in a clinical environment or only in laboratories?

N. Balaban: There are several tests done in hospitals that can identify resistance. The problem with persisters is that they come out in these tests as if they are completely normal bacteria that are not resistant. You would need to do different tests to see if they are persistent.

So persistent bacteria have not been found in patients so far?s/strong>

N. Balaban: We find them when we perform specific tests. If you isolate bacteria from patients and test them in the petri dish, you do see that some bacteria strains have the persistent phenotype. But it is still not clear how important this is for real infections. However, there are persistent phenotypes in certain infections and it seems that they are increasingly found as a disease progresses.

Some people believe that persistent bacteria might develop into a major health problem soon. Do you agree?

S. Helaine: Well, it is a fact that many infections are hard to treat even in the absence of antibiotic resistance. If you have an infection which keeps relapsing because persistent bacteria are surviving, you will need multiple antibiotic treatments to clear the same infection. In times when we need to reduce antibiotic usage, this is a problem. Also, Nathalie [Balaban] has shown that the state of persistence can help the emergence of antibiotic resistance. So persistent bacteria surviving multiple rounds of antibiotic treatment could lead to an aggravation of the antibiotic resistance problem.

For what kind of diseases have persistent bacteria been found so far?

N. Balaban: Currently we have direct evidence for a link between infection and persistence in one infection that affects patients with cystic fibrosis, an incurable hereditary disease. These patients have to fight chronic lung infections caused by the bacterium Pseudomonas aeruginosa that often lead to an early death. As the disease progresses there are more strains of a persistent phenotype found in the lungs. Bacteria isolated from patients in Kim Lewis’ lab [at the Northeastern University in Boston] were also found to carry mutations that are linked to persistence. There are some other infections that are not cured by antibiotics although the bacteria are not resistant, for example urinary tract infections or infections of the blood. But whether this is really because of the predominance of persistence is not clear yet.

Dr Helaine, in your lab you are working with Salmonella. Can these bacteria also develop persistence?

S. Helaine: Salmonella mostly give rise to two types of disease. One is gastroenteritis, which does not tend to relapse. But depending on the species of salmonella or depending on the immunocompetence of the patient, salmonella infections can become systemic and give rise to diseases like typhoid fever. These patients have a high rate of clinical relapse, possibly because of persisters.

Is it known which kind of mechanisms induce a dormant state in bacteria like Salmonella and thus lead to persistence?

S. Helaine: There is no consensus yet on a general mechanism that is at work in all conditions and for the whole spectrum of species. It seems that there are many parallel ways of becoming persistent and quite a few different mechanisms have been identified. In Salmonella it is definitely stress that leads to persistence. When entering the host, Salmonella encounters host macrophages, cells of the immune system that can identify and ingest pathogens. This environment triggers a stress response in the bacteria and leads to growth arrest. Once growth-arrested these bacteria are virtually impossible to kill.

N. Balaban: In general, we see that in order to get persistence you usually require a stress signal to induce a dormant state. Other stress factors, besides macrophages, can also induce persistence, such as starvation or an acid environment, as we have heard at the workshop. In vitro it was shown that a subset of persistence is induced by antibiotics.

Are there any ideas how persistence could be fought – obviously antibiotics do not work?

N. Balaban: There are already compounds that have been shown to have very strong anti-persistent properties. One is a compound that induces bacteria to chew on their own resources even when they are not growing. There is another line of studies that takes persistent cells out of their dormancy with the goal to make them susceptible to antibiotics again. Actually, there are also persistent bacteria that are not persistent to all antibiotics. So there are studies that try different combinations of existing antibiotics.

S. Helaine: We have focussed a lot on trying to understand how persisters are formed. But at the moment I don’t see that it is very realistic to try to prevent persister formation. However, we are trying to understand what persisters need to survive during an infection. It might show us ways to impair their long-term survival and get rid of them.

Should clinicians be aware of persistence as an emerging problem? Or should they focus on fighting resistance?

N. Balaban: I think it is too early to tell them that persistence is really important in a clinical environment. But it is also definitely too early to say that it is not important. The field is currently moving towards a better understanding of the link between resistance and persistence. But this will require a lot more work.

Short Bios

Nathalie Q. Balaban is Professor for Biological Physics at the Hebrew University of Jerusalem. Among other projects she studies the link between dormancy, persistence and resistance evolution in bacteria, for which she received ERC [European Research Council] support. She has also developed quantitative assays for the detection of persisters.

Dr. Sophie Helaine is Senior Lecturer at the Centre for Molecular Bacteriology and Infection at Imperial College London. She started her lab in 2014 with the support from the Medical Research Council (UK) to investigate the mechanisms of persistence in Salmonella during infection. In February 2018 she received an ERC Starting Grant to explore how persisters resuscitate.

The EMBO workshop "Bacterial Persistence and Antimicrobial Therapy" was organized by Christoph Dehio (President NRP 72), Dirk Bumann and Urs Jenal (both researchers in NRP 72). In NRP 72, Urs Jenal (Biozentrum of the University of Basel) in collaboration with the University Hospital of Basel explores the creation of persistent Pseudomonas aeruginos bacteria in cystic fibrosis patients.

In the context of the EMBO workshop, a public event on antibiotic resistance took place. Professor Jean-Claude Piffaretti, director of Interlifescience, presented the most important developments and answered questions from the audience. The event was organised by the Biozentrum of the University of Basel in collaboration with NRP 72 and Congressi Stefano Franscini/ETH Zurich.