Effective antibiotic use that minimizes treatment failures remains a challenge. A better understanding of how bacterial populations respond to antibiotics is necessary. Previous studies of large bacterial populations established the deterministic framework of pharmacodynamics. Here, characterizing the dynamics of population extinction, we demonstrated the stochastic nature of eradicating bacteria with antibiotics. Antibiotics known to kill bacteria (bactericidal) induced population fluctuations. Thus, at high antibiotic concentrations, the dynamics of bacterial clearance were heterogeneous. At low concentrations, clearance still occurred with a non-zero probability. These striking outcomes of population fluctuations were well captured by our probabilistic model. Our model further suggested a strategy to facilitate eradication by increasing extinction probability. We experimentally tested this prediction for antibiotic-susceptible and clinically-isolated resistant bacteria. This new knowledge exposes fundamental limits in our ability to predict bacterial eradication. Additionally, it demonstrates the potential of using antibiotic concentrations that were previously deemed inefficacious to eradicate bacteria.

Genetically identical microbial cells respond to stress heterogeneously, and this phenotypic heterogeneity contributes to population survival. Quantitative analysis of phenotypic heterogeneity can reveal dynamic features of stochastic mechanisms that generate heterogeneity. Additionally, it can enable a priori prediction of population dynamics, elucidating microbial survival strategies. Here, we quantitatively analyzed the persistence of an Escherichia coli population. When a population is confronted with antibiotics, a majority of cells is killed but a subpopulation called persisters survives the treatment. Previous studies have found that persisters survive antibiotic treatment by maintaining a long period of lag phase. When we quantified the lag time distribution of E. coli cells in a large dynamic range, we found that normal cells rejuvenated with a lag time distribution that is well captured by an exponential decay [exp(−kt)], agreeing with previous studies. This exponential decay indicates that their rejuvenation is governed by a single rate constant kinetics (i.e., k is constant). Interestingly, the lag time distribution of persisters exhibited a long tail captured by a power-law decay. Using a simple quantitative argument, we demonstrated that this power-law decay can be explained by a wide variation of the rate constant k. Additionally, by developing a mathematical model based on this biphasic lag time distribution, we quantitatively explained the complex population dynamics of persistence without any ad hoc parameters. The quantitative features of persistence demonstrated in our work shed insights into molecular mechanisms of persistence and advance our knowledge of how a microbial population evades antibiotic treatment.

Microorganisms adapt to frequent environmental changes through population diversification. Previous studies demonstrated phenotypic diversity in a clonal population and its important effects on microbial ecology. However, the dynamic changes of phenotypic composition have rarely been characterized. Also, cellular variations and environmental factors responsible for phenotypic diversity remain poorly understood. Here, we studied phenotypic diversity driven by metabolic heterogeneity. We characterized metabolic activities and growth kinetics of starved Escherichia coli cells subject to nutrient upshift at single-cell resolution. We observed three subpopulations with distinct metabolic activities and growth phenotypes. One subpopulation was metabolically active and immediately grew upon nutrient upshift. One subpopulation was metabolically inactive and non-viable. The other subpopulation was metabolically partially active, and did not grow upon nutrient upshift. The ratio of these subpopulations changed dynamically during starvation. A long-term observation of cells with partial metabolic activities indicated that their metabolism was later spontaneously restored, leading to growth recovery. Further investigations showed that oxidative stress can induce the emergence of a subpopulation with partial metabolic activities. Our findings reveal the emergence of metabolic heterogeneity and associated dynamic changes in phenotypic composition. In addition, the results shed new light on microbial dormancy, which has important implications in microbial ecology and biomedicine.

A key regulator of swarming in Proteus mirabilis is the Rcs phosphorelay, which represses flhDC, encoding the master flagellar regulator FlhD 4 C 2 . Mutants in rcsB, the response regulator in the Rcs phosphorelay, hyperswarm on solid agar and differentiate into swarmer cells in liquid, demonstrating that this system also influences the expression of genes central to differentiation. To gain a further understanding of RcsB-regulated genes involved in swarmer cell differentiation, transcriptome sequencing (RNASeq) was used to examine the RcsB regulon. Among the 133 genes identified, minC and minD, encoding cell division inhibitors, were identified as RcsB-activated genes. A third gene, minE, was shown to be part of an operon with minCD. To examine minCDE regulation, the min promoter was identified by 5= rapid amplification of cDNA ends (5=-RACE), and both transcriptional lacZ fusions and quantitative real-time reverse transcriptase (qRT) PCR were used to confirm that the minCDE operon was RcsB activated. Purified RcsB was capable of directly binding the minC promoter region. To determine the role of RcsB-mediated activation of minCDE in swarmer cell differentiation, a polar minC mutation was constructed. This mutant formed minicells during growth in liquid, produced shortened swarmer cells during differentiation, and exhibited decreased swarming motility. IMPORTANCEThis work describes the regulation and role of the MinCDE cell division system in P. mirabilis swarming and swarmer cell elongation. Prior to this study, the mechanisms that inhibit cell division and allow swarmer cell elongation were unknown. In addition, this work outlines for the first time the RcsB regulon in P. mirabilis. Taken together, the data presented in this study begin to address how P. mirabilis elongates upon contact with a solid surface. P roteus mirabilis, a Gram-negative member of the family Enterobacteriaceae, is a bacterium well known for its ability to swarm. Swarming is a specialized form of motility displayed by multicellular groups of flagellated bacteria across a solid or semisolid surface. In liquid culture, P. mirabilis exists as a peritrichously flagellated, rod-shaped cell. However, after coming into contact with a solid surface, the cells undergo differentiation into elongated, highly flagellated, multinucleate swarmer cells. Swarmer cells are 20-to 50-fold longer than vegetative cells and express thousands of flagella (1). Together, these swarmer cells form multicellular rafts, which they utilize to move across a solid surface (2). After a period of migration, the swarmer cells undergo consolidation (or dedifferentiation) and revert to vegetative rods. The repeated interchange from differentiation to consolidation is responsible for the characteristic bull's eye pattern that P. mirabilis forms on an agar plate (3, 4). Reviews on P. mirabilis swarming provide additional details on this process (5, 6).The switch from a rod-shaped cell to a swarmer cell is a complex process involving several global regulatory factors....

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