Bacterial persisters tolerate antibiotics by not producing hydroxyl radicals Jun-Seob Kim a , Paul Heo a , Tae-Jun Yang a , Ki-Sung Lee a , Yong-Su Jin b , Sung-Koo Kim c , Dongwoo Shin d,⇑ , Dae-Hyuk Kweon a,⇑ a School of Life Science and Biotechnology and Center for Human Interface Nano Technology, Sungkyunkwan University, Suwon, Gyeonggi-do 440-746, South Korea b Dept. of Food Science and Human Nutrition, University of Illinois, Urbana, IL 61801, USA c Dept. of Biotechnology and Bioengineering, Pukyong National University, Busan 608-737, South Korea d Dept. of Molecular Cell Biology, Sungkyunkwan University School of Medicine, Suwon, Gyeonggi-do 440-746, South Korea article info Article history: Received 23 July 2011 Available online 22 August 2011 Keywords: Persistence Resistance Antibiotics Hydroxyl radical Flow cytometer abstract In a phenomenon called persistence, small numbers of bacterial cells survive even after exposure to anti- biotics. 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 differ- ent ways, while they all commonly show lowered hydroxyl radical formation and enhanced tolerance to antibiotics. Ó 2011 Elsevier Inc. All rights reserved. 1. 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 anti- biotics. 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 anti- biotic-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]. Alter- natively, persistence may be a social trait of bacteria that benefits other individuals [5]. Paradoxically, this phenomenon can be disas- trous 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 emer- gence of inheritable antibiotics resistance [6]. Thus, the problems posed by persistence are no less intractable than those by the resis- tance [2,7–11]. 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 iden- tified [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 0006-291X/$ - see front matter Ó 2011 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2011.08.063 ⇑ Corresponding authors. Fax: +82 31 299 6229 (D. Shin), +82 31 290 7870 (D.-H. Kweon). E-mail addresses: shind@skku.edu (D. Shin), dhkweon@skku.edu (D.-H. Kweon). Biochemical and Biophysical Research Communications 413 (2011) 105–110 Contents lists available at SciVerse ScienceDirect Biochemical and Biophysical Research Communications journal homepage: www.elsevier.com/locate/ybbrc