Bacterial persisters tolerate antibiotics by not producing hydroxyl radicals

https://doi.org/10.1016/j.bbrc.2011.08.063Get rights and content

Abstract

In a phenomenon called persistence, small numbers of bacterial cells survive even after exposure to antibiotics. Recently, bactericidal antibiotics have been demonstrated to kill bacteria by increasing the levels of hydroxyl radicals inside cells. In the present study, we report a direct correlation between intracellular hydroxyl radical formation and bacterial persistence. By conducting flow cytometric analysis in a three-dimensional space, we resolved distinct bacterial populations in terms of intracellular hydroxyl radical levels, morphology and viability. We determined that, upon antibiotic treatment, a small sub-population of Escherichia coli survivors do not overproduce hydroxyl radicals and maintain normal morphology, whereas most bacterial cells were killed by accumulating hydroxyl radicals and displayed filamentous morphology. Our results suggest that bacterial persisters can be formed once they have transient defects in mediating reactions involved in the hydroxyl radical formation pathway. Thus, it is highly probable that persisters do not share a common mechanism but each persister cell respond to antibiotics in different ways, while they all commonly show lowered hydroxyl radical formation and enhanced tolerance to antibiotics.

Highlights

► There always remain persisters after treating a bacterial population with antibiotics. ► The persistence is no less tractable than resistance in many aspects. ► We analyzed persisters using FACS for various features. ► Persisters tolerated antibiotics by not producing hydroxyl radicals. ► Persisters do not seem to share a common mechanism but can be different from each other.

Introduction

Within a population of bacteria, a sub-population of multidrug-tolerant cells exists. These cells, termed persisters, are believed to be in a state of dormancy [1], which enables them to tolerate antibiotics. Upon reinoculation of cells that survived from antibiotics, the persisters give rise to new populations that have the same vulnerability to antibiotics as the ancestral population. Unlike antibiotic-resistant cells, the persisters are not genetically different from normal antibiotic-sensitive cells but are phenotypic variants of the wild-type cells [2], [3].

This non-inherited bacterial resistance to antibiotics, called persistence, can be considered as an insurance policy that permits survival of a sub-population from an antibiotics encounter [4]. Alternatively, persistence may be a social trait of bacteria that benefits other individuals [5]. Paradoxically, this phenomenon can be disastrous to humans because it disarms antibiotics which have been the strongest weapon that humans have developed to cure bacterial infection. Some well-known examples are tuberculosis, syphilis, typhoid fever and gastric ulcer. The pathogens linger in the host for long periods of time in spite of prolonged antibiotics treatment. More importantly, persisters are a potential source for the emergence of inheritable antibiotics resistance [6]. Thus, the problems posed by persistence are no less intractable than those by the resistance [2], [7], [8], [9], [10], [11].

Although persistence was first described ∼70 years ago, its mechanism still remains unknown. A major hurdle for studying persistence is in the fact that bacterial persisters are formed at a very low rate (i.e. ∼10−6) [7]. Moreover, the persisters, which are phenotypic variants of the normal population, have a transient nature. While it is still a mystery how persisters survive from an antibiotic encounter, several studies have suggested that toxin–antitoxin (TA) modules are important for persister formation. In the 1980s, the hip (high frequency of persisters) mutant was identified [12]. The hipA7 allelic strain produced 1000× more persisters than the wild-type strain. The hipBA operon was shown to act as a TA module, in which the HipA toxin is tightly regulated by the HipB antitoxin [13]. Lewis and colleagues proposed that various TA modules such as HipBA and RelBE could lead to multidrug tolerance on the basis of microarray analysis of the Escherichia coli transcriptome [14], [15]. Recently, a mechanism for HipA-mediated persistence and its neutralization by HipB was suggested based on the HipA and HipA–HipB–DNA crystal structures [16]. However, the DnaJ, PmrC and DskS proteins, which are unrelated to TA modules, changed persister frequency [17]. Moreover, several genes that do not show direct relevance to TA modules but display altered persister formation were identified from genetic screens [18], [19], [20]. Thus, persistence may be a result of the coincidence of many different events.

Kohanski et al. suggested an intriguing mechanism of antibiotics for killing bacteria [21]. They showed that three major bactericidal antibiotics stimulate hydroxyl radical formation in bacteria, and this toxic chemical contributes to the killing efficiency of lethal antibiotics. This process was accompanied by hyperactivation of NADH dehydrogenases and a depletion of NADH. Based on the results, they concluded that generation of hydroxyl radicals is a common mechanism of bacterial cell death caused by antibiotics. Hydroxyurea, which is an inhibitor of class I ribonucleotide reductase, also induced hydroxyl radical-mediated cell death in E. coli [22]. Based on these findings, we questioned whether a lack of hydroxyl radical generation inside a bacterial cell upon antibiotic treatment could lead to the cell becoming a persister. Consistent with this idea, our experiments using flow cytometer and fluorescence microscopy reveal that upon bactericidal antibiotic treatment, the majority of E. coli cells which are killed display morphological changes (i.e. filamentation) and intracellular accumulation of hydroxyl radicals, whereas persisters maintain normal morphology and do not overproduce hydroxyl radicals. Based on the result, we conclude that persisters do not share a common mechanism of antibiotic-tolerance because hydroxyl radical formation can be blocked in various ways.

Section snippets

Bacterial strains and chemicals

E. coli K12 BW25113 was obtained from the Korean Culture Center of Microorganisms (Seoul, Korea). All antibiotics were purchased from Sigma. The following concentrations of antibiotics were used: 10 μg/ml ampicillin, 250 ng/ml norfloxacin. 5 μM hydroxyphenyl fluorescein (HPF) and 1 μg/ml propidium iodide (PI) were used to analyze persister cells by flow cytometry and confocal microscope.

Determination of colony forming unit (cfu) in a liquid culture

Cells were pre-cultured in 10 ml LB overnight. The pre-culture was inoculated into fresh LB (50 ml) and cultured at

Results and discussion

We initially observed the formation of bacterial persisters. When E. coli cells were treated with ampicillin (Amp) or norfloxacin (Nor), they were rapidly killed: the colony forming unit (cfu) was reduced by a factor of ∼3 during the first 2 h of antibiotic treatment (Fig. 1A). However, due to the formation of persisters [2], [6], the cell death rate was reduced after 2 h of antibiotic treatment. Decimal reduction time (D), which is the time required to reduce cfu by a factor of 10 and is the

Acknowledgments

This research was supported by the Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education, Science and Technology (2011-0006268 and 2009-0058612) and by iPET (Korea Institute of Planning and Evaluation for Technology in Food, Agriculture, Forestry and Fisheries), Ministry for Food, Agriculture, Forestry and Fisheries, Republic of Korea.

References (22)

  • R. Jayaraman

    Bacterial persistence. some new insights into an old phenomenon

    Journal of Biosciences

    (2008)
  • Cited by (43)

    • Understanding effect of interaction of nanoparticles and antibiotics on bacteria survival under aquatic conditions: Knowns and unknowns

      2020, Environmental Research
      Citation Excerpt :

      However, persistent bacteria can tolerate ABs by not producing •OH radicals. This phenomenon was noticed in sub population of Escherichia coli which are able to survive and maintain normal morphology whereas most bacterial cells were killed by hyper-accumulation of •OH radicals and shown filamentous morphology (Kim et al., 2011). Keren et al. (2013) suggested that AB killing was unrelated to ROS production and was almost similar under aerobic and anaerobic environment for studied ABs (norfloxacin, ofloxacin, ampicillin and kanamycin).

    • General Mechanisms Leading to Persister Formation and Awakening

      2019, Trends in Genetics
      Citation Excerpt :

      It has been suggested that the induction of ROS production is a common mechanism by which bactericidal antibiotics kill their target cells [81], although this view is still a matter of scientific debate [82–84]. Nevertheless, detailed examination of isolated E. coli persisters revealed a lower level of hydroxyl radicals upon antibiotic treatment compared to their sensitive kin [85]. This suggests that persister cells either accumulate less ROS or harbor scavenger enzymes such as superoxide dismutase, catalases and alkyl hydroperoxide reductases that degrade ROS [86].

    • Synergistic and long-lasting antibacterial effect of antibiotic-loaded TiCaPCON-Ag films against pathogenic bacteria and fungi

      2018, Materials Science and Engineering C
      Citation Excerpt :

      Although the joint action of AgNPs and antibiotics was not well established, a number of possible mechanisms were proposed. Generation of hydroxyl radicals, a highly reactive oxygen species, was demonstrated to be a common mechanism of bacterial cell death caused by antibiotics [17,18]. AgNPs also have the ability to generate hydroxyl radicals [19].

    • Physiologically distinct subpopulations formed in Escherichia coli cultures in response to heat shock

      2018, Microbiological Research
      Citation Excerpt :

      Destabilized ferrous iron can react with hydrogen peroxide in a Fenton reaction leading to the generation of highly toxic hydroxyl radicals which damage DNA, lipids and proteins. Kim et al. (2011) proposed that persisters tolerate antibiotics by not producing hydroxyl radicals, because the reactions involved in the hydroxyl radical formation pathway, including destabilization of iron sulfur clusters and the Fenton reaction, do not operate in persisters (and likely in VBNC cells). This mechanism may also explain the observed LD50 tolerance to hydrogen peroxide.

    View all citing articles on Scopus
    View full text