Deep in the throes of infection, bacterial footsoldiers wage war against human cells, invading the tissues of the body. They multiply, stealing nutrients from their host and secreting offensive toxins. The host immune system rages in response: phagocytic cells swallow bacteria whole, the body’s temperature rises, and tissues become inflamed. The immune response is so effective that it will often kill the host in the process of eliminating an infection, causing sepsis, shock, and organ failure.

Medicine met this challenge with penicillin, the first drug to target and kill pathogenic (disease-causing) bacteria inside of a human host. Penicillin works by binding and inactivating key proteins needed for synthesis of the bacterial cell wall. This causes the swiftly-multiplying bacteria to lyse, or “burst,” and die. Human cells lack a cell wall, so the drug can target a pathogen without toxic effects on the infected host.

The concept of antibiotics is relatively straightforward: identifying a target (usually a protein) involved in a necessary bacterial function, then administering a drug which halts these functions. This molecular target should only be present in the bacteria; otherwise, human cells are at risk. In the plethora of metabolic processes undertaken by bacteria, there are many components that can serve as drug targets. For years after the discovery of penicillin, antibiotics entered a golden era of sorts. More and more drug classes were discovered, and they were increasingly effective in eliminating infections. Inhibitors of protein synthesis, DNA replication, cell wall formation, and other metabolic processes entered the clinic and stayed there.

Around the end of the 20th century, the discovery of new antibiotics slowed. Rising to counter this was a silent pandemic, slowly taking the world by storm. Antibiotic resistance had begun to develop.

The misclassification of antibiotics as “magic drugs” expanded their use to agricultural settings and non-bacterial medical applications like viral infections. Bacteria—because they divide so quickly—mutate frequently. When antibiotics aren’t administered at a high enough concentration to eradicate entire bacterial populations, these organisms adapt to the stressful environment. Selective pressure ensures the survival of helpful mutations that render the antibiotic ineffective. These mutant cells are called resistors and comprise 1/6th of all bacterial infections confirmed in medical laboratories.

Resistors are characterized by an immense ability to adapt. This ability is enhanced by improper antibiotic dosing and poor patient compliance: administering too little antibiotic or not finishing those nasty-tasting pills once symptoms begin to improve. This kills some bacteria and allows the survivors to re-populate and spread their beneficial mutations against antibiotics. Bacterial infections are once again becoming the formidable threat they were prior to penicillin.

There is a second, lesser-known component to the resistance problem. Persisters are bacterial cells distinctly different from resistors but similarly unaffected by antibiotics. The persister phenomenon was first conceptualized in the 1940’s by Dr. Joseph Bigger , who identified this sub-population of cells as a troublesome component of bacterial infections. Where resistant cells have developed some active mechanism against the drugs, persister cells are bacteria that enter a period of dormancy, either randomly or due to the presence of an environmental stressor like nitrogen depletion or an antibiotic drug.

The dormant period of persisters is similar to the bacterial growth stage known as the stationary phase, where metabolic processes slow and cell division halts. This results in bacterial tolerance to drugs, where they mitigate the effects of an antibiotic drug not by actively adapting to it, but by halting the processes the drug targets. Important to note is that resistance is heritable, while persistence is not. This is because resistance results from a genetic alteration that can be passed on, while persister cells do not experience a permanent change to their DNA.

The key to persistence is the ability to “re-awaken” from this period of dormancy. In the presence of a stressor, they remain unaffected, but the moment the stressing factor is removed, persisters can resume normal metabolic activity. This allows the bacteria to repopulate and jumpstart the infection process anew in a phenomenon known as recalcitrance. Recalcitrance is a significant contributor to chronic and recurring infections like UTIs, tuberculosis, and cystic fibrosis.

The Lewis Lab at Northeastern University studies persister cells responsible for tolerance to antibiotics. So far, the lab has identified three significant mechanisms of persister formation among various bacterial species: toxin-antitoxin modules in Escherichia coli (E. coli) and nitrogen depletion as well as random fluctuations in ATP levels in both E. coli and Staphylococcus aureus (S. aureus).

Toxin-antitoxin modules are two-component systems employed by many bacteria, and they provide a unique self defense mechanism. A toxin, such as tisB in E. coli , is released to protect the cell from a threat, briefly halting cell processes, then is later neutralized with an antitoxin molecule. Antitoxins are highly unstable and susceptible to degradation, meaning many E. coli can remain in this state of metabolic pause without neutralizing the toxins. Further, when the toxin halts bacterial growth, it removes the targets for an antibiotic, and again, persisters are born. Notably, toxin-antitoxin modules have not been found to play a role in the formation of S. aureus persisters.

Persisters can also form when bacterial cells lack nitrogen. Nitrogen is a necessary component for a variety of bacterial metabolic processes, including the synthesis of DNA, proteins, and other vital macromolecules. However, it is also a limited environmental resource for cells, and as such, many bacterial species compete for available nitrogen. E. coli and S. aureus, among many other bacterial species, have adapted a stress response that they can employ in the absence of nitrogen (“nitrogen starvation”), and this stress response is believed to increase the proportion of cells that become persisters. This occurs through a cascade of intracellular signals, which slows metabolic activity and results in persister formation.

Finally, the lab has observed that low ATP is associated with an increased formation of persister cells. Bacterial cells often randomly (“stochastically”) enter the stationary phase, where intracellular ATP decreases . This low-energy state slows metabolic activity, decreasing the activity of antibiotic targets and producing a tolerant population (persisters).

The Lewis Lab also aims to identify potential solutions to the problem of persistence by supporting natural drug discovery efforts to address both resistance and persistence. In 2013, Principal Investigator Kim Lewis discovered that an old molecule called Acyldepsipeptide (ADEP) could eliminate persister cells both in vitro and in vivo (mouse infection models). ADEP targets a protease called ClpP, hijacking it to degrade mature proteins in the cell. Normally, ClpP only degrades misfolded or damaged proteins, so this corruption is devastating for bacteria.

Beyond small molecule discovery efforts, the Lewis Lab has also pursued more creative endeavors such as pulse-dosing and metabolic stimulation. Pulse-dosing (another contribution of Dr. Bigger) involves treating an infection with multiple rounds of antibiotics, killing off the active population and leaving persisters behind. After each treatment, the antibiotic is cleared from the system and persisters are allowed to re-awaken in the absence of a stressful environment, and are then hit again with the drug. This is repeated until, in theory, all persisters are eradicated. Metabolic stimulation involves supplementing one step in the bacterial citric acid cycle in an effort to restore metabolic function, followed by antibiotic treatment to kill newly-awakened cells.

Resistance dominates public perception of the crisis of antibiotic inefficacy, but the Lewis lab has demonstrated the clear threat posed by tolerant persister cells. It isn’t quite clear what the future of persister cells holds, but without the research of microbiologists around the globe, we may continue to observe an increase in the frequency, duration, and severity of bacterial infections. What is obvious is that the solution will require scientific efforts as persistent as these little cells themselves.