Antibiotic Persistence and Resistance

by Molly Sargen

Antibiotics are drugs that kill or inhibit the growth of microbes, including bacteria and fungi. These drugs work by blocking essential processes like protein production, DNA replication, and cell division. After Alexander Fleming’s serendipitous discovery of Penicillin, antibiotics became a central feature of medical care. Today, antibiotics are used to treat a wide variety of infections and prevent new infections during invasive medical procedures. However, the efficacy of many antibiotics is waning, leading to failed courses of antibiotic treatment that may result in chronic infections and increased risk to patients who are immunocompromised and/or undergoing invasive medical procedures. 

Because of the central role of antibiotics in healthcare, waning antibiotic efficacy poses a major threat to modern medicine. There are many factors contributing to reduced antibiotic efficacy, but some of the most important are the ways in which microbes evade antibiotics, including resistance and persistence.   Antibiotic resistance has gained increasing attention from the CDC and WHO; antibiotic persistence is less commonly discussed, but equally important.

Resistance

Antibiotic resistance is when microbes overcome the effects of antibiotics through a genetic change. This might involve acquiring new genes that enable the microbe to break down the antibiotic or mutations that change the shape of a protein and prevent the antibiotic from binding.  Thus, resistant microbes are able to grow even in the presence of the drug. Since resistant microbes outcompete susceptible microbes and can pass on the genetic information encoding resistance, antibiotic resistance spreads rapidly and is currently a global crisis. According to the CDC, in the US alone, there are nearly 2.8 million antibiotic-resistant infections each year, leading to nearly 35,000 deaths. Antibiotic resistance and the antibiotic resistance crisis are further described in previous SITN work (see the ‘For More Information’ section below).

Figure 1: Resistant bacteria are able to grow during antibiotic treatment. Non-resistant bacteria die and resistant bacteria take over the population. Resistance is due to a genetic change, so all of the bacteria in the resulting population are resistant.

Persistence

Antibiotic resistance is not the only mechanism through which microbes can survive antibiotic treatment; a lesser-known phenomenon through which microbes fail to succumb to antibiotics is antibiotic persistence. Antibiotic persistence is when a subpopulation of microbes escapes the effects of antibiotics by transiently stopping growth. Typically, this means that persisters stop cell division and thereby the processes involved in preparation for division.  Since antibiotics target these same processes, when microbes stop growing, the processes antibiotics target may become inessential.  Consequently, the antibiotics fail to kill the growth-arrested bacteria. These non-growing bacteria that survive antibiotics are called persisters. When the antibiotic is removed, persisters can resume growth.

Figure 2: Persisters survive antibiotic treatment. Persisters are able to resume growth in the absence of antibiotics. The resulting population looks exactly like the original population, since persisters are genetically identical to non-persisters.

Persisters typically make up only a small fraction of a microbial population (Figure 3). Scientists quantify the amount of persisters by measuring the number of bacteria that survive an antibiotic treatment. When the survival of bacteria is plotted vs. the duration of antibiotic treatment, there is an inflection point at which the rate of death declines (Figure 3A). The bacteria that are still surviving at this time are persisters. The proportion of persisters varies for different bacteria and with different environmental conditions.  For example, when the conditions are optimal for growth, such as in a nutrient rich medium, the proportion of persisters is extremely small for most bacterial species. Stressful conditions such as starvation or low (acidic) pH can increase the abundance of persisters (Figure 3B). One especially stressful condition that some infectious bacteria face is survival inside macrophages. Macrophages are immune cells that defend the body by engulfing bacteria and trying to destroy them. Certain bacteria such as Salmonella enterica (which causes Salmonellosis) and Mycobacterium tuberculosis (which causes Tuberculosis) are known to form persisters when they encounter macrophages during an infection.

Figure 3: Persisters are a small fraction of the population. A) The fraction of persisters is quantified in an antibiotic survival assay. B) The proportion of a population that is persistent changes in different conditions. Stressful conditions can increase the proportion of persisters. Figure 3 illustrates results similar to Helaine et al. Science 2014, but does not report actual data.

Persisters during infections can contribute to antibiotic treatment failure. Put simply, persisters may survive the antibiotics and cause relapse of the infection. Since persisters are such a small fraction of a microbial population (Figure 2), they may be undetectable at the end of antibiotic treatment; consequently, it may appear that the infection is cleared. However, as described above, when the antibiotic treatment is stopped, persisters can resume growing and re-establish the infection, leading to relapse. This is a problem, not only because of the health risk for the infected patient, but also because the relapsed infection will likely require a second course of antibiotic treatment. In this way, persisters can also contribute to the development of antibiotic resistance. It is well established that the more antibiotics are used, the more resistance will develop.

Open Questions About Antibiotic Persistence

While mechanisms of antibiotic resistance are fairly well-defined, mechanisms of antibiotic persistence are not well understood. Researchers are working to understand how bacteria become persisters, how persisters begin to grow again, and what is required for persister survival (Figure 4). By better understanding persisters, scientists aim to develop ways to prevent persister formation and regrowth, as well as ways to eliminate persisters.

Figure 4: Open questions about persistence. Scientists want to understand how persisters form and how persisters regrow.

First, researchers want to understand what pushes some bacteria into a non-growing persister state while others continue to grow. As described above, stressful conditions can increase the fraction of a population that are persisters, but still many bacteria fail to become persisters, suggesting that heterogeneous states from cell to cell are involved. In other words, stress can “trigger” persistence, but additional factors are involved. Differences in gene expression in individual cells may drive persistence, and individual cells might experience unique microenvironments and thus activate different responses.  A specific combination of responses could push some cells to become persisters. Direct causes of persistence have been so elusive that some scientists think persistence may even be a stochastic, or randomly determined, occurrence. Scientists have shown that bacteria in a uniform environment seem to randomly produce different amounts of a control protein. Likewise, heterogeneous production of multiple proteins in individual bacteria could be linked to persistence.

Additionally, researchers are working to understand how persisters begin to grow again. It is unclear if persisters can sense the removal of the antibiotic. Some research suggests that persisters can sense specific nutrients prior to regrowth. Alternatively, many researchers argue that exiting the persister state may be as random as entering the persister state. Ultimately, understanding persisters will allow scientists to identify ways to target persisters. For instance, persisters of Salmonella require DNA repair mechanisms to resume growth, so blocking such repair mechanisms could prevent them from resuming growth. 

Many questions remain about the mechanisms of persistence, but it is nonetheless clear that persistence affects antibiotic efficacy. In the short term, this affects infection relapse. In the long term, persistence may contribute to the development of antibiotic resistance and the antibiotic resistance crisis. Understanding persistence will be critical for developing practices for antibiotics and maintaining antibiotic efficacy for the future.

Molly Sargen is a third year PhD Student in the Biological and Biomedical Sciences Program at Harvard Medical School.

Cover image by qimono from pixabay

For more information: 

Antibiotic Resistance Content from SITN

  • For more details about antibiotic resistance, check out this article from SITN. 
  • Watch seminars from the SITN archives about antibiotic resistance (2017, 2020). 
  • Learn how antibiotic resistance has been impacted by COVID-19 in this article. 

External Information 

Primary Research about Antibiotic Persistence

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