The magic of antibiotic tolerance

Bactericidal antibiotics are effective at killing proliferating bacteria as long as they don't carry mutated or acquired genes that encode resistance to the antibiotics. Unfortunately even antibiotic-sensitive bacteria can tolerate antibiotics under some circumstances. Bacteria that are in a nondividing "dormant" state often survive antibiotic exposure.  When the antibiotic is removed and growth resumes, the bacteria regain susceptibility to antibiotics.

At first glance antibiotic tolerance appears to be a passive process in which nondividing cells survive simply because the target of the antibiotic is inactive.  However, this is not correct.  Antibiotic tolerance requires an active response by the bacteria.  The nondividing bacteria that survive antibiotic treatment are called persisters.  Persisters may account for infections that are difficult to eradicate with antibiotics.

Persisters were first identified in 1944 by Joesph Bigger, who was testing the effectiveness of the new miracle drug penicillin on cultures of Staphylococcus.   However, interest in antibiotic tolerance waned as antibiotic resistance came to be a problem.  In the 1980s Harris Moyed revisited the issue of antibiotic tolerance, and his group harnessed the power of bacterial genetics to isolate mutants that exhibited abnormally high frequencies of persister formation and to map the mutations that caused the novel trait.

As Joseph Bigger discovered, very rare persisters can even be identified in growing cultures, around 1 in 100,000 bacteria.  How can these rare bacteria behave so differently from those surrounding them if antibiotic tolerance doesn't involve alterations to the bacterium's DNA?  The current thinking is that the persister state is triggered randomly in a small fraction of proliferating bacteria due to random fluctuations in the expression of a small number of persister genes.  In the rare bacterium (about 1 in 100,000), the persister genes will be expressed at a high enough level to induce the persister state and slow growth of the bacterium.

Certain environmental cues can also enhance the development of persisters.  For example, the fraction of persisters in a culture increases substantially as nutrients are depleted.  At stationary phase, when the bacteria stop increasing in number, at least 1% of the bacterial cells become antibiotic tolerant.

One of the active responses that stimulates persister cell formation is called the stringent response, which rapidly generates the unusual nucleotides ppGpp and pppGpp when bacteria are starving for nutrients.  Historically these derivatives of GDP and GTP were known as "magic spots" because they appeared as novel radioactive spots on thin-layer chromatograms when E. coli starved for amino acids were labeled with ³²P-phosphate. (p)ppGpp has wide-ranging effects on bacterial physiology.  The best-known activity of (p)ppGpp is its attachment, along with the protein DksA, to RNA polymerase during amino acid starvation, causing transcription of rRNA and tRNA genes to cease and transcription of amino acid biosynthetic operons to increase.  This make sense since there's no point in making more ribosomes until more amino acids are available to support protein synthesis and growth of the bacteria.

Magic spot!  Source

(p)ppGpp production is also necessary to generate the rare persisters in cultures of proliferating bacteria.  When the two genes coding for the enzymes that make (p)ppGpp, relA and spoT, are deleted, persister cells become even more rare in growing E. coli cultures.

A recent study published in the journal Science looked at the role of the stringent response in inducing antibiotic tolerance in biofilms, in which nondividing bacteria are embedded in a matrix secreted by the bacteria.  These studies were conducted with E. coli and Pseudomonas aeruginosa strains with deletion mutations in relA and spoT.  When the biofilms formed by the mutant and wild-type strains were treated with different antibiotics, the mutants turned out to be much more sensitive to the antibiotics even though the bacteria were not dividing (data for P. aeruginosa shown in bar graphs below).  Similar results were obtained when standard cultures of the P. aeruginosa mutant and wild-type strains were grown to stationary phase and then treated with antibiotics.

Figure 1E from Nguyen et al.  P. aeruginosa was allowed to form biofilms on polycarbonate membrane filters resting on agar culture medium.  The biofilms were incubated with different antibiotics or with no antibiotic (control).  To determine the number of bacteria surviving antibiotic treatment, the bacteria in the biofilms were dispersed by vigorous mixing and plated onto agar culture medium to determine cfu counts.  +SR, complementation of the ΔrelA ΔspoT mutant with intact copies of relA and spoT; *P ≤ 0.05, ***P ≤ 0.0005 versus wildtype.

Bactericidal antibiotics, regardless of their target, are now known to enhance production of reactive oxygen species (ROS) within bacteria.  If not detoxified by enzymes such as catalase and superoxide dismutase, ROS can fatally damage the bacteria by reacting with their DNA, protein, and lipids.  ROS is generated within bacteria during the course of their normal metabolic activities, even when antibiotics are not present.  When the authors measured the amounts of hydroxyl radical generated within untreated E. coli and P. aeruginosa biofilms, the ΔrelA ΔspoT mutants were burdened with higher levels of hydroxyl radicals than the wild-type strains. Why do the mutant strains have higher levels of hydroxyl radicals?  Further analysis of the bacteria in the biofilms showed that the ΔrelA ΔspoT mutants also had lower levels of catalase and superoxide dismutase activity.  It's possible that bactericidal antibiotics triggered production of lethal amounts of ROS that the mutants could not handle due to their insufficient production of catalase and superoxide dismutase, but this needs to be confirmed by further experimentation using bacterial strains with the superoxide dismutase and catalase genes knocked out.

The Science study also looked at infected laboratory mice treated with antibiotics.  Wild-type and ΔrelA ΔspoT P. aeruginosa strains were grown to stationary phase, and a lethal dose of the bacteria were injected into the abdominal cavity of mice.  Four hours later the animals were treated with the antibiotic ofloxacin.  The antibiotic was more effective at preventing lethal infection by the ΔrelA ΔspoT mutant than those caused by the wild-type strain.  Similarly, ofloxacin was more effective at reducing the number of bacteria in a biofilm chamber implanted underneath the mouse skin when the biofilm was composed of the ΔrelA ΔspoT mutant.

One must keep in mind that multiple pathways to persister cell formation have been identified (see figure below).  Note that for the animal experiments persisters were allowed to develop in the test tube prior to animal inoculation.  It is possible that the stringent response would not be involved at all if persisters were allowed to develop during infection instead.

Figure 4 from Lewis, 2010.  The redundant pathways to persister formation are shown.  FMN, flavin mononucleotide pool; pmf, proton motive force; TAs, toxin/antitoxin modules

Featured paper

Nguyen, D., Joshi-Datar, A., Lepine, F., Bauerle, E., Olakanmi, O., Beer, K., McKay, G., Siehnel, R., Schafhauser, J., Wang, Y., Britigan, B.E., & Singh, P.K. (2011). Active starvation responses mediate antibiotic tolerance in biofilms and nutrient-limited bacteria Science, 334 (6058), 982-986 DOI: 10.1126/science.1211037

Key references

Bigger, J.W. (1944). Treatment of staphylococcal infections with penicillin by intermittent sterilisation The Lancet, 244 (6320), 497-500 DOI: 10.1016/S0140-6736(00)74210-3

Moyed H.S., & Bertrand K.P. (1983). hipA, a newly recognized gene of Escherichia coli K-12 that affects frequency of persistence after inhibition of murein synthesis. Journal of bacteriology, 155 (2), 768-775 PMID: 6348026

Cashel, M., & Gallant, J. (1969). Two compounds implicated in the function of the RC gene of Escherichia coli Nature, 221 (5183), 838-841 DOI: 10.1038/221838a0

Paul, B., Barker, M.M., Ross, W., Schneider, D.A., Webb, C., Foster, J.W., & Gourse, R.L. (2004). DksA: a critical component of the transcription initiation machinery that potentiates the regulation of rRNA promoters by ppGpp and the initiating NTP. Cell, 118 (3), 311-322 DOI: 10.1016/j.cell.2004.07.009

Korch, S.B., Henderson, T.A., & Hill, T.M. (2003). Characterization of the hipA7 allele of Escherichia coli and evidence that high persistence is governed by (p)ppGpp synthesis Molecular Microbiology, 50 (4), 1199-1213 DOI: 10.1046/j.1365-2958.2003.03779.x

Kohanski, M.A., Dwyer, D.J., Hayete, B., Lawrence, C.A., & Collins, J.J. (2007). A common mechanism of cellular death induced by bactericidal antibiotics Cell, 130 (5), 797-810 DOI: 10.1016/j.cell.2007.06.049

Lewis, K. (2010). Persister cells Annual Review of Microbiology, 64 (1), 357-372 DOI: 10.1146/annurev.micro.112408.134306

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