When nutrients are scarce, many bacteria slow down their metabolism and pause replication. In such a physiological state, starved cells have higher chances of survival while it also protects them from other forms of stress. The study “Antibiotic tolerance in environmentally stressed Bacillus subtilis: physical barriers and induction of a viable but non-culturable state” published in microLife explores the stress adaption of the soil bacterium Bacillus subtilis. Luiza Morawska and Oscar Kuipers explain for the #FEMSmicroBlog how this quiescent state can trigger antibiotic resistance in bacteria causing a high risk for public health. #FascinatingMicrobes
Bacteria entering dormant-like states
In natural environments, populations of bacterial communities can adapt to environmental stress conditions by entering low metabolic, non-replicating states. These include truly dormant endospores, persister and viable but non-culturable (VBNC) cells.
Each of these phenotypes can help a bacterial community endure harsh environmental conditions while awaiting better times. Also, bacteria in such a non-replicating state are known to be tolerant to lethal doses of antibiotics, making them difficult to eradicate.
Dormant-like physiological states help bacteria endure harsh environmental conditions while awaiting better times.
Generally, both persister and VBNC cells are stochastically present in bacterial populations and they share common features. Most importantly, bacteria in either state reside in a non-dividing, low metabolic mode, withstanding lethal stress conditions. Bacteria can also enter either of these physiological states in response to environmental stress.
Once the stressor is removed, both persisters and VBNC cells have the metabolic capacity to revive and grow on appropriate media. Interestingly, persisters can rapidly thrive again on growth-promoting media, while VBNC bacteria require prolonged treatment or an appropriate signal to awake. This is why VBNC bacteria are often overlooked when estimating the number of viable cells in environmental or clinical samples based on colony-forming unit counts.
Fast environmental changes lock bacteria into dormant-like states
Ever since the VBNC state in bacteria was discovered, the inducing conditions and mechanisms that drive the entry into VBNC were of prime interest. The article: “Antibiotic tolerance in environmentally stressed Bacillus subtilis: physical barriers and induction of a viable but non-culturable state” published in microLife showcases how the spore-forming Gram-positive model organism Bacillus subtilis enters the VBNC state.
In its natural environment, the soil, B. subtilis frequently encounters osmotic stress, mainly as interchanging periods of rain and drought. Thus, to survive, B. subtilis cells must adjust their physiology and metabolism, which often results in slower growth rates.
This study investigates how rapid changes in the environment’s osmolarity and simultaneous treatment with aminoglycosides modulate the bacterium’s membrane potential and metabolic activity. The subsequent adaptive mechanism locks B. subtilis cells into a non-dividing but still viable state.
With the use of a microfluidics system and fluorescent microscopy, the work shows that VBNC bacteria display a prolonged membrane hyperpolarization, lower levels of reactive oxygen species and lower metabolic activity. This phenotype was even observed 20 hours after antibiotic treatment indicating that these cells can survive prolonged periods of lethal doses of antibiotics but cannot regrow directly under typical growth-promoting conditions. The eradication of VBNC bacteria is, therefore, more challenging.
Bacterial dormancy is versatile
This study demonstrates for the first time that besides spore formation and switching to oligotrophic growth due to nutrient depletion, B. subtilis populations can persist in the VBNC state. This truly shows the versatility of dormant-like phenotypes in bacteria.
Besides, the observations made in this work draw attention to the typical methods of cell enumeration and their error-prone outcomes. Thus, it is extremely important to consider VBNC bacteria when testing new antibiotic compounds for clinical applications.
When testing new antibiotic compounds for clinical applications, it is extremely important to consider viable but non-culturable cells.
Gaining more insights into the physiology of VBNC bacteria and potential awakening signals could help develop new methods to fight reoccurring infections in antibiotic-treated patients. In future work, the robust RNA sequencing could shed a light on the transcriptional response and point to VBNC-inducing genetic targets.
- Read the article “Antibiotic tolerance in environmentally stressed Bacillus subtilis: physical barriers and induction of a viable but non-culturable state” in microLife by Morawska and Kuipers (2022).
Luiza Morawska is a molecular biologist currently working as a postdoctoral researcher at University College Cork, Ireland. Before moving to Ireland to work with gut-associated bacteria, she did her PhD at the University of Groningen, the Netherlands. During this time, she discovered her passion for microbiology and investigated the boundaries of horizontal gene transfer in members of Gram-positive bacteria, including Bacillus subtilis, Lactococcus lactis and Streptococcus thermophilus. Besides DNA transfer in bacteria, her research interests lie in exploring microbial adaptations to environmental stress conditions, including osmotic and antibiotic stress. In her work, she frequently employs fluorescent microscopy and microfluidics to show how single cells respond directly to conditions they typically meet in nature.
Oscar Kuipers is Professor in Molecular Genetics and heads the Department of Molecular Genetics at the University of Groningen since 1999. He was trained in molecular biology and biochemistry and moved to the direction of molecular genetics and synthetic biology after his PhD. Central topics in his research are the development of novel antimicrobials by synthetic biology approaches, studying population heterogeneity and bistability of clonal bacterial populations at the single-cell level, and bacterial gene regulation and genomics. He is a member of the Royal Netherlands Academy of Arts and Sciences and of the European Academy of Microbiology.
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The section #FascinatingMicrobes for the #FEMSmicroBlog explains the science behind a paper and highlights the significance and broader context of a recent finding. One of the main goals is to share the fascinating spectrum of microbes across all fields of microbiology.
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