The great escape

A hallmark of many chronic illnesses caused by pathogenic bacteria is the recalcitrance to treatment with antibiotics, even in the face of laboratory tests showing the causative agents to be sensitive to drugs. Recent studies have begun to uncover a secret weapon in the bacterial arsenal - ‘persister’ cells.

Certain infectious diseases caused by pathogenic bacteria are typically chronic in nature. Deadly examples include tuberculosis, caused by Mycobacterium tuberculosis, and cystic fibrosis-associated lung infections, primarily caused by Pseudomonas aeruginosa. A hallmark of this type of illness is the recalcitrance to treatment with antibiotics, even in the face of laboratory tests showing the causative agents to be sensitive to drugs. Recent studies have attributed this treatment failure to the presence of a small, transiently multidrug-tolerant subpopulation of cells, so-called ‘persister’ cells.

In recent years, bacteria have often hit the headlines upon emergence of yet another pathogenic strain that has become resistant to most commonly used antibiotics, a property known as multidrug resistance. The spread of such bugs among the general population is indeed a major concern – acute infections by bacterial pathogens are mostly easily treatable with antibiotics, but only as long as the germs are susceptible. In the case of chronic infections, however, matters are not that simple. Antibiotic treatment is often unsuccessful despite laboratory tests indicating that the bacteria causing the infection ought to be sensitive to administered drugs. Recent evidence suggests that this discrepancy is likely due to so-far overlooked shortcomings of current testing methods. Indeed, antibiotic susceptibility testing typically involves looking for observable growth (or a lack thereof) of a particular isolate in the presence of a certain antibiotic, corresponding to growth inhibition of roughly 99% of the population.

However, it has long been known that a minority of the population can withstand the lethal effects of antibiotics1. A given strain will consistently produce a fraction of such ‘persister’ cells – individuals that are temporarily hyper-resistant to all antibiotics at once, with a frequency that can lie anywhere between 0.001% and 1%. Persisters are able to survive normally lethal levels of antibiotics without being genetically resistant to the drug. Consequently, reinoculation of these persisters in fresh growth medium will again produce a susceptible population with a similar level of persisters. Our apparent inability to effectively kill persister cells and their propensity to come to life again after the antibiotic threat has passed, has prompted comparisons to living dead or zombie bacteria. However, experimental evidence indicates that persister cells more likely represent a sleep-like state of being, as if in hibernation to escape the harsh spell of antibiotics.

Two major discoveries in the last decade have greatly fuelled interest in the scientific study of persistence. First, there was the observation that persisters are present at significant levels in biofilms2 – groups of microbes that stick together on surfaces and are associated with numerous chronic diseases through their resilient presence in the human body, e.g. in cystic fibrosis lungs, on heart valves and on indwelling medical devices such as catheters and implants. Persisters were encountered in biofilms to such an extent that it has been suggested that they are the long-looked for but elusive factor explaining antibiotics tolerance of biofilms. Eradication of biofilms might therefore require getting rid of persisters.

The second breakthrough consisted of finding experimental evidence that supports a role for persistence in the recalcitrance of chronic infections to antibiotic treatment, a role that was proposed upon encountering persisters in large numbers in biofilms. Sure enough, analysis of longitudinal isolates of Pseudomonas aeruginosa collected from the lungs of a single cystic fibrosis patient over the course of many years identified a sharp increase in persistence in late isolates3. Similar results were obtained by analysing isolates from other cystic fibrosis patients, suggesting that high persistence is advantageous in vivo to bacteria causing chronic infections and that persisters therefore merit attention in efforts to battle infectious diseases.

Currently, there are no drugs that specifically target persister cells. This can in part be explained by the fact that for a long time, the existence of persisters has been ignored by the research community. In addition, big pharma screening efforts aimed at identifying new antibiotics have focused, like clinical susceptibility testing, on the 99% or so weaker cells rather than on the hardy but hard to study persisters. Conceptually, an anti-persister drug could be envisaged to either prevent de novo formation of persisters, or better still to sensitise or kill pre-existing persister cells. It is likely that new methodologies will need to be developed in order to perform large-scale anti-persistence screenings, and the lack of a proven ‘golden standard’ will only serve to complicate matters. Furthermore, it is unclear as to what extent a drug should decrease persistence in order to have an impact on clinical outcome. Perhaps more importantly, no animal model has yet been developed to study persistence, and consequently the possible benefits that novel treatments might have, in vivo.

Any drug that is to target pre-existing persister cells will inevitably need to be administered concurrently with conventional antibiotics. However, recent reports have indicated that such a strategy could carry within it the seeds of its own demise. Work by Dörr and co-workers has shown that at least a part of the persisters in a population is formed only in the presence of antibiotics4. Conversely, De Groote and colleagues demonstrated a link between stable, genetically acquired antibiotic resistance mechanisms and transient, non-genetic persistence5. Since any antibiotic treatment inherently selects for resistant individuals, there is a real danger to also select for altered levels of persistence. While both studies focused on particular antibiotics and results therefore cannot yet be generalised, it is evident that the road ahead may well spring still more unpleasant surprises.

Persistence has been observed in numerous bacteria and also in a few fungal pathogens, suggesting a uniquely microbial affair. However, some features of persisters are eerily reminiscent of higher eukaryotes. Cancer cells often exhibit a heterogeneous response to drug treatment in which a minority of cells is unaffected, sometimes referred to as ‘fractional killing’ and requiring multiple rounds of chemotherapy. Both animals and plants hedge their bets by producing various forms of dormant stages, e.g. seeds, spores and resting eggs. Like persisters, these are typically less sensitive to adverse conditions and are programmed to resuscitate when chances of survival have improved. Future research will reveal whether these similarities are merely superficial or whether underlying mechanisms are also shared, and whether these insights will help us in fighting persisters.

Chronic infectious diseases are a major challenge in global health care. The spread of multidrug-resistant strains and the lack of novel antibiotics for the foreseeable future mean that this is likely to remain the case for some time to come. The problem of antibiotics resistance is compounded by the presence of persister cells, which – although formed in small numbers and only transiently so – are virtually impossible to kill with conventional drugs. Their importance in treatment failure is increasingly being recognised, and will no doubt bring them to the attention of a growing body of academic researchers and pharmaceutical companies alike. Time will tell whether their combined efforts are enough to overcome persistence.