1. 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 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:
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:
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