Resuscitation can be delayed using the QS inhibitor Cinnamaldehyde Quorum-Sensing Mediates the Resuscitation of Viable but Nonculturable Dormant Cells and Can Be Targeted to Manipulate Dormancy Mesrop Ayrapetyan, Tiffany C. Williams, and James D. Oliver University of North Carolina at Charlotte, Department of Biological Sciences Background: The antibiotic resistance crisis has become a worldwide threat and is leading us into a ‘post-antibiotic era’ where common infections may lead to life-threatening diseases. The pervasive use of antibiotics has contributed to this problem since the first reported appearance of antibiotic resistance in 1940. However, a less conspicuous and possibly even more widespread phenomenon leading to antibiotic failure is bacterial dormancy. In nature, microorganisms are threatened by environmental instability and must employ strategies that allow tolerance of harmful conditions. To cope, many bacteria enter a transient state of dormancy that is characterized by a decrease in growth and metabolic function. Dormant subpopulations can later resuscitate when the stress is alleviated. Although this bet-hedging strategy has likely evolved for survival in uncertain environments, it is increasingly evident that dormancy plays a significant role in disease. Entry into dormancy grants pathogens the ability to tolerate antibiotics, which has been linked to recurrent infections caused by several bacteria. In addition, dormant cells are undetected by culture-based methods and thus pose a significant public health hazard. The finding that dormant cells can resuscitate within a host and retain their virulence properties further corroborates this threat. Are quorum-sensing molecules produced during VBNC resuscitation? Methods: Results: Can the VBNC state be manipulated by targeting quorum-sensing? Methods: Results: Induced VBNC in V. vulnificus by incubating at 4°C for ~1 week until cells lost culturability Measured the production of QS molecules during resuscitation: Measured QS production using the V. harveyi bioluminescent reporter strain Measured culturability in parallel Induced VBNC in V. vulnificus by incubating at 4°C for ~1 week until cells lost culturability Assessed ability to manipulate resuscitation dynamics of VBNC cells by applying various treatments: Cell-free culture supernatants (CFS) from QS producing bacteria Synthetic QS molecules CFS from a mutant strain unable to produce QS molecules QS inhibitor (cinnamaldehyde) Increasing [QS] V. harveyi reporter bioluminescence is proportional to QS concentration Introduction: Bacterial dormancy Start of temperature upshift QS is detectable within 1hr of temp upshift QS production increases even before cells resuscitate Initial evidence of resuscitation Full resuscitation has occurred Many pathogenic bacteria enter a dormant state in response to environmental stress Cells remain viable (Figure 1) Intact cell membrane, continued gene expression and metabolic activity Metabolic activity is suppressed Fail to grow on routine laboratory media (Figure 2) Culturability versus viability of VBNC cells Culturable bacteria Viable bacteria Some pathogens that enter VBNC: Burkholderia spp. Campylobacter spp. Enterobacter spp. Enterococcus spp. Escherichia coli Helicobacter pylori Listeria monocytogenes Mycobacterium tuberculosis Pseudomonas spp. Salmonella spp. Shigella spp. Vibrio spp. Live cell Dead cell Resuscitation can be induced through the addition of CFS collected from AI-2 producing bacteria or by the addition of synthetic AI-2 Figure 2. As cells enter into the VBNC state as a result of stress the number of culturable bacteria drop below the levels of detection however a portion of the population retains viability. From: Oliver, J.D. (2010) Recent findings on the viable but nonculturable state in pathogenic bacteria. FEMS Micro. Rev. 34: 415-425 Conditions that induce VBNC: Starvation High/Low pH High/Low Temperature Oxidative Stress Chlorination Food Preservatives Oxygen limitation Copper Low Salinity QS molecules are produced by VBNC populations resulting in resuscitation In vitro VBNC dynamics in Vibrio vulnificus Is quorum sensing required for resuscitation to occur? Methods: Results: 4oC RT Figure 1. Fluorescence viability assays are used to distinguish between viable cells (green) and dead cells (red). Induced VBNC in V. vulnificus luxS mutant (unable to make AI-2) by incubating at 4°C for ~1 week until cells lost culturability, then evaluated ability to resuscitate Bacteria are typically less virulent in the VBNC state but are very resistant to lethal stressors (e.g. antibiotic treatment) VBNC cells are not detectable on standard laboratory media Cells can resuscitate from this dormant state (Figure 3) Regain metabolic activity and virulence properties Little in known regarding the molecular mechanisms that trigger resuscitation from the VBNC state Resuscitation can be delayed using the QS inhibitor Cinnamaldehyde Conclusions: Figure 3. The VBNC state is induced in V. vulnificus using cold temperature incubation. As cells are incubated at 4°C culturability declines until the entire population becomes VBNC. Temperature upshift allows for resuscitation in which cells regain metabolic activity and culturability. From: Nilsson, L., J.D. Oliver, and S. Kjelleberg. (1991) J. Bacterial. 173:5054-5059. Quorum-sensing is vital for the resuscitation of VBNC cells Considering that QS is a widespread bacterial communication system this mechanism may be utilized by other pathogens Dormancy dynamics can be artificially manipulated through the addition of QS molecules or inhibitors Ongoing research is aimed at manipulating quorum sensing dynamics to resuscitate dormant bacteria, to allow for more effective killing using current antimicrobials Introduction: Bacterial communication QS molecules Lux Q phosphatase Lux P Lux U Lux O Lux R LuxS Derepress LuxR A B C P Virulence and other genes Quorum sensing (QS) Widespread communication system in bacteria Coordinates group behaviors QS signaling results in global changes in gene expression Orchestrates virulence and adaptation to stressful conditions ΔluxS mutant was unable to resuscitate, unless it was supplemented with exogenous AI-2 molecules Acknowledgements: We would like to thank: Bonnie Bassler at Princeton University for kindly providing the V. harveyi bioluminescent reporter strain Matthew Parrow at UNC Charlotte for kindly providing the synthetic QS molecule The USDA and Duke University Marine Laboratory for funding this project We propose that quorum sensing is the signal that mediates the awakening of dormant populations Generalized schematic of QS phosphorelay system in bacteria