Advanced Methods for Enriching and Isolating Bacterial Persister Cells: A Comprehensive Guide for Researchers

Abstract

This article provides a detailed methodological guide for researchers and drug development professionals on the enrichment and isolation of bacterial persister cells, a dormant subpopulation responsible for chronic and relapsing infections. Covering foundational concepts, practical laboratory techniques, troubleshooting advice, and validation strategies, it synthesizes current research to address the significant challenge of obtaining these rare, transient cells for mechanistic studies and the development of more effective anti-infective therapies.

Understanding the Dormant State: Core Concepts and Characteristics of Persister Cells

The emergence of drug-tolerant persister (DTP) cells represents a fundamental challenge in oncology and infectious disease management, contributing significantly to treatment failure and disease recurrence. Unlike genetically resistant clones that acquire permanent, heritable resistance mutations, persister cells survive therapeutic stress through reversible, non-genetic adaptations [1] [2]. These cells constitute a reservoir within minimal residual disease that can seed relapse long after the visible tumor has regressed or the primary infection has cleared [1]. The clinical significance of DTPs is profound—they have been implicated in diverse malignancies including non-small cell lung cancer (NSCLC), melanoma, colorectal cancer, and breast cancer, as well as in chronic bacterial infections caused by pathogens such as Staphylococcus aureus, Pseudomonas aeruginosa, and Escherichia coli [1] [2] [3].

Critically, the reversible nature of the persister phenotype distinguishes it from permanent genetic resistance and offers unique therapeutic opportunities. When the selective pressure of anti-cancer or antimicrobial therapy is removed, persister cells can exit their drug-tolerant state and regenerate populations that remain sensitive to the original treatment [2] [4]. This biological plasticity underscores why patients may regain sensitivity to previously ineffective therapies after a "drug holiday," a clinical observation that cannot be explained by conventional genetic resistance models [4]. Understanding and targeting these transiently tolerant cells therefore represents a promising frontier for preventing relapse and improving long-term treatment outcomes.

Defining Characteristics: Tolerance Versus Genetic Resistance

Conceptual and Mechanistic Distinctions

Persister cells employ fundamentally different survival strategies compared to genetically resistant cells. The table below summarizes the key distinguishing features:

Table 1: Key Characteristics Distinguishing Persister Cells from Genetically Resistant Cells

Characteristic	Persister Cells (Non-Genetic Tolerance)	Genetically Resistant Cells
Heritability	Non-heritable, phenotypic plasticity	Heritable genetic mutations
MIC Change	No change in Minimum Inhibitory Concentration	Elevated Minimum Inhibitory Concentration
Prevalence	Rare subpopulation (typically <1% of total)	Can constitute majority of population
Stability	Reversible upon drug withdrawal	Permanent and stable
Mechanisms	Epigenetic reprogramming, metabolic shifts, dormancy	Target modification, efflux pumps, enzyme inactivation
Population Dynamics	Biphasic killing curves with slow second phase	Monophasic killing at elevated drug concentrations

The operational definition of a persister cell hinges on its ability to survive transient, high-dose drug exposure without stable genetic alterations [1] [2]. In both cancer and bacterial contexts, persisters demonstrate unchanged minimum inhibitory concentration (MIC) values compared to their drug-naïve counterparts, distinguishing them from resistant populations that exhibit elevated MICs [5]. When exposed to lethal drug concentrations, populations containing persisters exhibit characteristic biphasic killing curves, with rapid initial killing of the drug-sensitive majority population followed by a much slower decline representing the persister subpopulation [5] [6].

Molecular Mechanisms of Persistence

The molecular basis of persistence differs substantially from genetic resistance across multiple biological contexts:

• Epigenetic Reprogramming: Cancer DTPs frequently undergo chromatin remodeling mediated by histone-modifying enzymes such as KDM5A (a histone demethylase) and EZH2 (a histone methyltransferase) [2]. These reversible modifications create a transcriptionally repressive state that facilitates survival under drug pressure.

• Metabolic Adaptations: Both bacterial and cancer persisters shift toward quiescent or slow-cycling states with reduced metabolic activity. Cancer DTPs often increase dependence on oxidative phosphorylation and fatty acid oxidation while enhancing antioxidant defenses [2].

• Toxin-Antitoxin Systems: In bacterial persistence, toxin-antitoxin modules such as HipAB in E. coli induce dormancy by disrupting essential cellular processes when activated under stress conditions [5].

• Transcriptional Plasticity: Cancer DTPs activate alternative survival pathways including receptor tyrosine kinases (AXL, IGF-1R), developmental pathways (WNT/β-catenin, YAP/TEAD), and stress-response signaling [2].

Diagram 1: Transition from therapeutic stress to genetic resistance via persister state. The persister cell acts as a reversible intermediate that can facilitate the acquisition of permanent genetic resistance under continued drug pressure.

Enrichment and Isolation Methodologies

Core Principles for Persister Enrichment

The reliable enrichment and isolation of persister cells present significant technical challenges due to their low abundance, transient nature, and lack of universal surface markers. Successful methodologies typically exploit the fundamental biological properties that distinguish persisters: (1) their ability to survive lethal drug exposure while most cells die, and (2) their distinct physiological states such as dormancy or reduced metabolic activity. The enrichment process must carefully balance efficiency with preservation of the native persister phenotype, as extended antibiotic exposure or harsh processing conditions can artificially induce persistence or cause awakening [7].

Two broad strategic approaches have emerged for persister enrichment. Direct methods physically separate persisters based on survival after lethal treatment, while induction methods exploit physiological differences to increase the persister fraction before isolation. The choice between these approaches depends on the specific research questions, model system, and downstream applications. For cancer DTP studies, patient-derived models including organoids and xenografts that better recapitulate clinical complexity are increasingly favored over conventional cell lines [1].

Established Enrichment Protocols

Bacterial Persister Enrichment via Cephalexin-Induced Filamentation and Filtration

This highly efficient method leverages the differential response of susceptible cells versus persisters to cephalexin, a β-lactam antibiotic that inhibits cell division [7].

Table 2: Bacterial Persister Enrichment Protocol Using Cephalexin-Induced Filamentation

Step	Procedure	Parameters	Purpose
Culture Preparation	Grow bacterial culture to early exponential phase	OD~600~ ≈ 0.2-0.3; MHB medium, 37°C	Ensures optimal antibiotic activity and persister formation
Cephalexin Treatment	Add cephalexin to final concentration 40μg/mL	Incubate 1h with aeration, 37°C	Induces filamentation of susceptible cells while persisters remain unaffected
Filtration	Pass culture through membrane filter (5μm pore size)	Low protein-binding membrane	Retains filamented cells while persisters pass through
Collection	Collect flow-through containing persisters	Centrifuge at 5000×g, 10min	Concentrates persister cells for downstream applications
Validation	Assess antibiotic tolerance and regrowth capacity	Plate counts before/after antibiotic challenge	Confirms persister phenotype and enrichment efficiency

This protocol achieves approximately 28% enrichment efficiency while minimizing cellular debris and reducing antibiotic exposure time compared to alternative methods [7]. The resulting persister population demonstrates key persister characteristics: survival during extended cephalexin treatment, ability to reinitiate growth after treatment cessation, and multidrug tolerance to antibiotics with different cellular targets [7].

Cancer DTP Enrichment via Extended Drug Exposure

This method isolates cancer DTPs through their survival following prolonged exposure to chemotherapeutic or targeted agents.

Table 3: Cancer DTP Enrichment Protocol Using Extended Drug Exposure

Step	Procedure	Parameters	Purpose
Culture Establishment	Plate cancer cells at appropriate density	30-50% confluence; cell-type specific medium	Ensures logarithmic growth at treatment initiation
Drug Treatment	Add therapeutic agent at IC~90~ concentration	Incubate 72-144h with drug; refresh medium/drug every 48-72h	Eliminates drug-sensitive bulk population
DTP Recovery	Wash cells to remove drug; culture in drug-free medium	3× PBS washes; complete medium	Allows DTP recovery and proliferation
Validation	Functional and molecular characterization	Drug rechallenge, sphere formation, marker expression	Confirms DTP phenotype and reversible tolerance

This approach has successfully identified DTPs across diverse cancer types, including EGFR-mutant NSCLC treated with EGFR inhibitors, HER2+ breast cancer treated with lapatinib, and melanoma treated with BRAF/MEK inhibitors [1] [2]. The resulting DTPs typically exhibit characteristic features including slow-cycling phenotypes, epigenetic reprogramming, metabolic adaptations, and therapy-induced mutagenesis [1].

Diagram 2: Bacterial persister enrichment workflow using cephalexin-induced filamentation and filtration, with essential validation steps to confirm persister phenotype.

The Scientist's Toolkit: Essential Research Reagents and Materials

Successful persister research requires specialized reagents and tools designed to address the unique challenges of working with these rare, transient cell populations. The following table catalogues essential solutions for key experimental workflows in persister cell studies:

Table 4: Essential Research Reagent Solutions for Persister Cell Studies

Category	Specific Reagents/Tools	Application Notes
Selection Agents	Cephalexin, Osimertinib, Cisplatin, Enrofloxacin, Vancomycin	Use at optimized concentrations and exposure times specific to persister enrichment protocols
Detection Technologies	DTC-Flow panel (HER2/EpCAM/CD45), SCBC Mass Cytometry, Lineage tracing barcodes	Enables sensitive detection and molecular characterization of rare persister populations
Metabolic Probes	TMRE (membrane potential), CTC (respiratory activity), ALDEFLUOR assay	Assess metabolic state differences between persisters and normal cells
Epigenetic Modulators	HDAC inhibitors (Entinostat), KDM5A inhibitors, EZH2 inhibitors	Target epigenetic mechanisms maintaining persister state; used in combination therapies
Model Systems	Patient-derived organoids (PDOs), Patient-derived xenografts (PDXs), HipA7 mutant E. coli	Provide more physiologically relevant contexts for persister studies
Single-Cell Platforms	Mother machine microfluidics, Single-cell RNA-seq, Barcoding approaches	Enable analysis of persister heterogeneity and awakening dynamics

These tools facilitate the interrogation of persister biology across multiple dimensions, from initial isolation and characterization to mechanistic studies and therapeutic targeting. The selection of appropriate reagents should be guided by the specific experimental system (bacterial vs. cancer), the technical requirements of the enrichment protocol, and the downstream applications planned for the isolated persisters.

Future Directions and Concluding Perspectives

The study of persister cells continues to evolve rapidly, with several emerging technologies and conceptual frameworks promising to advance our understanding of these elusive populations. Single-cell analysis technologies are revealing unprecedented heterogeneity within persister populations, demonstrating that multiple molecular routes can lead to the shared phenotype of transient drug tolerance [1] [6]. Lineage tracing approaches have provided evidence that fate decisions leading to persistence may occur both before and after drug exposure, driven by inheritable cellular states that persist across multiple generations [6].

From a translational perspective, the reversible nature of persistence suggests unique therapeutic vulnerabilities. Rather than attempting to directly kill persisters—a challenge given their dormancy and multidrug tolerance—emerging strategies focus on manipulating their phenotypic state. Approaches include preventing persistence entry through epigenetic modulators, forcing persistence exit to sensitize cells to conventional therapies, and exploiting metabolic dependencies that become essential in the persister state [2] [4]. The development of these strategies will require increasingly sophisticated enrichment and characterization methodologies that preserve the native biology of persister cells while enabling functional and molecular analyses.

As persister research progresses from phenomenological observations to mechanistic understanding, the field must address key challenges including standardization of isolation protocols, validation of persister-specific markers, and development of models that better recapitulate clinical persistence. By distinguishing persistence from genetic resistance and developing targeted approaches to eliminate these transiently tolerant cells, researchers and clinicians may ultimately overcome a fundamental barrier to curative cancer and antimicrobial therapies.

Persister cells represent a rare subpopulation within bacterial and cancer cell communities that survive lethal stresses, such as antibiotic or chemotherapeutic treatment, through non-genetic, reversible mechanisms [8] [9]. These cells are clinically occult reservoirs that seed disease relapse long after the visible tumor or infection has regressed [8]. Understanding the physiological hallmarks of persistence—dormancy, metabolic downturn, and heterogeneity—is crucial for developing strategies to eradicate these resilient cells. This document details the core physiological features of persister cells and provides standardized protocols for their study, framed within the context of methods for enriching and isolating persister subpopulations.

Core Physiological Hallmarks

Persister cells are defined by three interconnected physiological states that enable their survival. The table below summarizes the key characteristics and functional implications of each hallmark.

Table 1: Core Physiological Hallmarks of Persister Cells

Hallmark	Key Characteristics	Functional Implications
Dormancy	Reversible entry into a non-proliferative, quiescent state (G0/G1 phase); temporary mitotic arrest [10] [8].	Enables survival by reducing vulnerability to treatments that target actively growing cells [10] [9].
Metabolic Downturn	Shift from anabolism to catabolism; reduced but not absent metabolic activity; reliance on oxidative phosphorylation in some models [11].	Conserves energy and resources under stress; maintains baseline ATP levels necessary for survival and reactivation [11].
Heterogeneity	Existence of multiple phenotypic states (e.g., mesenchymal-like, luminal-like) within a persister population; variable persistence levels (shallow to deep) [8] [9].	Allows a subset of cells to survive diverse and unpredictable stressors; complicates targeting with a single therapeutic approach [8].

Experimental Protocols for Persister Enrichment and Analysis

A primary challenge in persister research is their low natural abundance. The following protocols detail methods for enriching these rare cells to facilitate phenotypic and genomic studies.

Protocol 1: Antibiotic-Based Enrichment of Bacterial Persisters

This method enriches persisters by using antibiotics to lyse the majority of the non-persister population [12].

• Culture Preparation: Grow the bacterial strain of interest to the desired growth phase (e.g., mid-exponential or stationary phase) in an appropriate liquid medium.
• Antibiotic Challenge: Expose the culture to a high concentration of a bactericidal antibiotic. The concentration should be a multiple of the minimum inhibitory concentration (MIC) to ensure rapid killing of non-persisters.
  • Example: Treat a stationary-phase E. coli culture with 100x MIC of ampicillin or ofloxacin.
• Incubation: Incubate the culture with the antibiotic for a sufficient period to achieve several log reductions in viability (typically 3-5 hours, but duration is strain- and antibiotic-dependent).
• Harvesting: Centrifuge the antibiotic-treated culture to pellet the surviving cells.
• Washing: Wash the pellet 2-3 times with sterile phosphate-buffered saline (PBS) or fresh medium to remove all traces of the antibiotic.
• Resuspension: Resuspend the final pellet in a suitable buffer or medium. This enriched persister population is now ready for downstream applications, such as RNA extraction for transcriptomics or re-culturing to confirm regrowth.

Protocol 2: Culture Aging for Persister Enrichment

Nutrient limitation and culture aging induce a stress response that increases the frequency of persister cells [11] [12].

• Inoculation: Inoculate bacteria into a flask containing a rich liquid medium.
• Extended Incubation: Allow the culture to grow into stationary phase and continue incubating it without sub-culturing. The extended nutrient deprivation enriches for persisters.
  • Example: Incubate an E. coli culture for 24-48 hours (or longer) post-inoculation at 37°C with shaking [11].
• Sampling: Aseptically sample the aged culture. The persister frequency is typically highest in late stationary phase.
• Optional Antibiotic Confirmation: To confirm enrichment, a portion of the aged culture can be subjected to an antibiotic challenge as described in Protocol 1, and the survival rate can be compared to that of a mid-exponential phase culture.

Protocol 3: Flow Cytometry-Based Isolation of Bacterial Persisters

This method uses fluorescent staining to identify and sort persisters based on physiological activity [12].

• Staining: Take a sample from a bacterial culture and stain it with a viability or metabolic dye. The bis-oxonol dye DiBAC4(3) is suitable, as it enters cells with depolarized membranes, which can be associated with a non-growing state.
• Incubation: Incubate the stained cells in the dark according to dye-specific protocols.
• Flow Cytometry Setup: Configure the flow cytometer for cell sorting. Establish gating parameters based on forward and side scatter to identify the bacterial population.
• Cell Sorting: Sort the subpopulation of cells exhibiting low fluorescence intensity with DiBAC4(3), which may represent metabolically less active persister cells, into a sterile collection tube.
• Validation: Validate the sorted population by plating for viability counts and subjecting them to an antibiotic challenge to confirm a higher survival rate compared to the high-fluorescence population.

Protocol 4: Analysis of Cancer DTP Cells using Patient-Derived Organoids (PDOs)

For cancer Drug-Tolerant Persister (DTP) cells, 3D organoid models provide a physiologically relevant system [13] [8].

• Organoid Culture: Establish and maintain PDOs from patient tumor samples in a suitable 3D extracellular matrix with specialized growth medium.
• Drug Treatment: Expose the organoids to a relevant chemotherapeutic regimen at a defined concentration (e.g., IC50 or higher) for a sustained period.
  • Example: Treat colorectal cancer PDOs with FOLFOX (5-FU + oxaliplatin) or FOLFIRI (5-FU + SN38) regimens [13].
• Monitoring: Monitor cell survival over time (from 16 to 120 hours) using assays like CellTiter-Glo to measure ATP levels, which indicate viable cell mass.
• DTP Confirmation: Characterize the drug-tolerant response by assessing time-kill curves. An early monophasic survival curve indicates a tolerant population, which may later evolve into a biphasic curve characteristic of a small persister subpopulation [13].
• Functional Analysis: Harvest the surviving DTP cells for downstream molecular analysis (e.g., RNA-seq, proteomics) or to investigate mechanisms of reactivation upon drug withdrawal.

Visualization of Signaling and Metabolic Pathways

The following diagrams, generated using DOT language, illustrate key signaling pathways and metabolic states involved in regulating persistence.

Signaling Pathways in Cancer Cell Dormancy

This diagram illustrates the key signaling interactions that regulate the balance between proliferation and dormancy in cancer cells, particularly within the bone marrow microenvironment [10].

Metabolic State of Bacterial Persisters

This diagram visualizes the Crp/cAMP-mediated rewiring of energy metabolism observed in E. coli persister cells during the late stationary phase [11].

The Scientist's Toolkit: Research Reagent Solutions

The table below lists essential reagents and materials required for experiments focused on enriching and studying persister cells.

Table 2: Key Research Reagents and Materials for Persister Cell Studies

Reagent/Material	Function/Application	Example Usage
Bactericidal Antibiotics	Selective lysis of non-persister cells to enrich for the tolerant population [12].	Ampicillin, Ofloxacin, Ciprofloxacin used at high multiples of the MIC.
Fluorescent Viability Dyes (e.g., DiBAC4(3))	Staining based on membrane potential or metabolic activity to identify persister subpopulations via flow cytometry [12].	DiBAC4(3) enters cells with depolarized membranes, potentially marking a non-growing state.
Patient-Derived Organoids (PDOs)	Physiologically relevant 3D ex vivo models for studying cancer DTP cells and their microenvironment [13] [8].	Colorectal cancer PDOs treated with FOLFOX to model chemotherapy tolerance and relapse.
Compounds Inducing Metabolic Perturbation	Investigating the role of energy metabolism in persister survival and identifying potential synergies with antibiotics [11].	Carbon sources that modulate proton motive force and potentiate aminoglycoside uptake and killing.
Lysis Buffers & RNA Extraction Kits	Downstream molecular analysis (e.g., transcriptomics) of enriched persister populations to understand mechanisms of tolerance [12].	Extraction of high-quality RNA from a small number of sorted or antibiotic-selected persister cells for RNA-seq.

Bacterial persisters are a subpopulation of cells that are transiently tolerant to high concentrations of antibiotics without acquiring genetic resistance mutations [9] [5]. These phenotypic variants represent a significant challenge in clinical settings, underlying chronic and recurrent infections that are difficult to eradicate [14] [9]. The study of persister cells is fundamental to understanding treatment failures and developing more effective therapeutic strategies against persistent infections.

A primary obstacle in persister research lies in the inherent difficulties of isolating these cells for direct experimental analysis. This challenge stems from two interconnected fundamental characteristics: their remarkably low natural abundance within bacterial populations and their stochastic formation mechanisms. This application note details these core challenges and provides validated methodologies for enriching and isolating persister subpopulations to facilitate rigorous scientific investigation.

Core Challenges in Persister Isolation

The Problem of Low Natural Abundance

Persisters typically constitute a very small fraction of a bacterial population, a key trait noted since their discovery [5]. In most growing cultures, persisters represent less than 1% of the total population [5] [15]. This low proportion makes them difficult to detect, isolate, and study using standard microbiological techniques, as they are overshadowed by the vast majority of susceptible cells.

The Problem of Stochastic Formation

The formation of persisters is widely recognized as a stochastic process [16] [15]. These cells arise spontaneously within an isogenic population due to random, transient fluctuations in key cellular processes, rather than in a deterministic, programmed response [16]. Critical mechanisms include:

• Fluctuations in Metabolic States: Stochastic heterogeneity in the expression of energy-generating enzymes, such as those in the Krebs cycle (e.g., isocitrate dehydrogenase, Icd), leads to variations in cellular ATP levels [16]. Subpopulations of cells with low ATP are significantly more tolerant to antibiotic killing [16].
• Stochastic Toxin-Antitoxin System Activation: Random activation of toxin-antitoxin modules, such as HipAB and TisB/istR, can induce a dormant state by disrupting essential processes like ATP synthesis [5].

This non-deterministic nature means persister formation is unpredictable at the single-cell level, preventing researchers from simply inducing a synchronized, homogeneous persister state across an entire culture for easy harvest.

Quantitative Profile of Persister Subpopulations

The table below summarizes key characteristics of persister cells that directly impact isolation strategies.

Table 1: Key Quantitative and Qualitative Characteristics of Bacterial Persisters

Characteristic	Description	Experimental/Clinical Implication
Natural Abundance	Typically < 1% in planktonic cultures [5] [15]	Requires enrichment strategies prior to isolation.
ATP Level in Persisters	Significant reduction compared to normal cells [16] [3]	Can be exploited for sorting (e.g., via reporters like iATPSnFr1.0).
Formation Mechanism	Primarily stochastic [16]	Precludes deterministic, synchronized induction.
Phenotype Stability	Transient and reversible; non-heritable [5]	Isolated persisters can resuscitate, complicating analysis.
Metabolic Activity	Reduced or dormant, but heterogeneous [9] [17]	General metabolic inhibition can enrich persister fractions.

Established Methods for Persister Enrichment and Isolation

A cornerstone method for enriching persisters is antibiotic selection. This leverages the defining trait of persisters: survival after exposure to a lethal antibiotic dose that kills the majority of the population.

Protocol: Enrichment via Antibiotic Killing and Persister Isolation

This protocol is adapted from procedures used to study Staphylococcus aureus and Escherichia coli persisters [3].

Principle: A stationary-phase culture, which naturally contains a higher proportion of persisters, is treated with a high concentration of a bactericidal antibiotic. The surviving cells, enriched for persisters, are recovered by washing away the antibiotic.

Materials:

• Bacterial strain (e.g., S. aureus or E. coli)
• Appropriate rich broth medium (e.g., Lysogeny Broth, LB)
• Phosphate-Buffered Saline (PBS), sterile
• Bactericidal antibiotic (e.g., Ciprofloxacin, Vancomycin, Ampicillin)
• Centrifuge
• Cell culture incubator/shaker

Procedure:

• Culture Preparation: Inoculate the bacterial strain into 10-50 mL of broth and incubate with shaking for ~16-24 hours (overnight) to reach stationary phase. For some protocols, an aged stationary phase culture (e.g., 48 hours post-inoculation) is used to increase the initial persister frequency [16].
• Antibiotic Treatment:
  • Centrifuge the culture (e.g., 4000 x g, 10 min) and resuspend the pellet in fresh, pre-warmed broth or PBS containing antibiotic.
  • The antibiotic concentration should be significantly higher than the minimum inhibitory concentration (MIC). For example, use 5-10x MIC or a standardized high concentration (e.g., 100 µg/mL ciprofloxacin for E. coli, 50 µg/mL vancomycin for S. aureus).
  • Incubate the culture for a defined period (e.g., 3-5 hours) to ensure killing of the susceptible population.
• Recovery of Persisters:
  • Centrifuge the antibiotic-treated culture (e.g., 4000 x g, 10 min) to pellet the surviving cells.
  • Wash the pellet 2-3 times with sterile PBS to thoroughly remove the antibiotic.
  • The final pellet can be resuspended in a small volume of PBS or fresh medium for downstream applications, such as viability counting, molecular analysis, or further sorting.

Validation: The success of enrichment is typically confirmed by a biphasic killing curve, where the initial rapid death of susceptible cells is followed by a plateau representing the persister subpopulation [18] [5].

Diagram 1: Antibiotic-based persister enrichment workflow.

Protocol: Fluorescence-Activated Cell Sorting (FACS) Based on Metabolic Markers

For a more precise isolation of specific persister subtypes, FACS can be employed using fluorescent reporters for physiological states associated with persistence, such as low ATP.

Principle: A genetically encoded biosensor (e.g., iATPSnFr1.0 for ATP) is expressed in the bacterial population. After antibiotic treatment or in a heterogeneous culture, cells with low fluorescence intensity (indicating low ATP) can be sorted as the persister-enriched fraction [16].

Materials:

• Bacterial strain expressing a ratiometric ATP reporter (e.g., iATPSnFr1.0) [16]
• Microfluidics or standard culture equipment
• Fluorescence-Activated Cell Sorter (FACS)
• Sterile collection tubes

Procedure:

• Strain Preparation: Use a strain chromosomally expressing a constitutive ATP reporter like iATPSnFr1.0. This reporter is excited at 405 nm and 488 nm, with the emission ratio (488ex/405ex) correlating with ATP concentration [16].
• Sample Preparation: Grow the reporter strain to the desired phase (exponential or stationary). The culture can be treated with an antibiotic to enrich for survivors or used directly to capture stochastic persisters.
• Cell Sorting and Analysis:
  • Analyze the culture by FACS to measure fluorescence.
  • Gate and sort subpopulations based on fluorescence intensity (e.g., "Dim" cells with low ATP levels vs. "Bright" cells with high ATP) [16].
  • Sort the "Dim" population directly onto agar plates for survival assessment or into lysis buffer for molecular analysis.
• Validation: Confirm the sorted "Dim" population has a higher survival rate after a second antibiotic challenge compared to the "Bright" population [16].

Diagram 2: FACS strategy for isolating low-ATP persisters.

The Scientist's Toolkit: Key Research Reagents

The table below lists essential reagents and their applications in persister isolation research, as cited in the literature.

Table 2: Key Reagent Solutions for Persister Enrichment and Isolation Studies

Reagent / Tool	Function / Target	Application Example
iATPSnFr1.0 Reporter	Ratiometric fluorescent biosensor for ATP levels [16]	Single-cell identification and sorting of low-ATP persisters via FACS or microscopy [16].
Carbonyl Cyanide m-chlorophenylhydrazone (CCCP)	Protonophore that dissipates proton motive force and depletes ATP [17]	Chemical induction of a persister-like state for metabolic studies [17].
Ciprofloxacin	Fluoroquinolone antibiotic; inhibits DNA gyrase [16]	Killing of susceptible cells to enrich for persisters in cultures of E. coli and other Gram-negatives [16] [5].
Vancomycin	Glycopeptide antibiotic; inhibits cell wall synthesis [3]	Killing of susceptible cells to enrich for persisters in cultures of S. aureus [3].
Fluorescence-Activated Cell Sorter (FACS)	High-throughput cell sorting based on optical properties [16]	Isolation of persister subpopulations based on fluorescence from metabolic reporters or dye staining.
Microfluidics (Mother Machine)	Single-cell culture and long-term time-lapse microscopy [16]	Tracking persister formation, resuscitation, and heterogeneity in real-time at the single-cell level [16].

Key Microbial Species and Model Systems for Persister Research

Bacterial persisters are a subpopulation of genetically susceptible, non-growing, or slow-growing cells that exhibit remarkable tolerance to lethal doses of antibiotics and other environmental stresses [9] [19]. These phenotypically variant cells are now recognized as a primary culprit behind chronic, relapsing infections and the recalcitrance of biofilm-associated infections to antibiotic therapy [9] [20]. Research into persister cells faces a unique challenge: these cells are transient, metastable, and typically present at very low frequencies in bacterial populations [7] [21]. This application note, framed within a broader thesis on methods for enriching and isolating persister subpopulations, details the key microbial species and model systems that form the cornerstone of experimental persistence research. We provide a comparative analysis of organisms, standardized protocols for persister isolation, and essential reagent solutions to facilitate robust and reproducible research in this critical field.

Key Microbial Species in Persistence Research

The study of bacterial persistence spans a diverse range of microbial species, each offering unique advantages for investigating different aspects of the persister phenotype. The table below summarizes the primary model organisms and their specific relevance to persistence research.

Table 1: Key Microbial Species and Model Systems in Persister Research

Microbial Species	Gram Stain	Relevance to Persistence Research	Key Characteristics & Findings
Escherichia coli	Negative	The primary model organism for elucidating molecular mechanisms [16] [22] [21].	• Well-characterized genetics and extensive toolkit [22].• Existence of Type I (stationary phase-induced) and Type II (stochastic) persisters defined [9] [21].• Key pathways identified: Toxin-Antitoxin (TA) modules (e.g., HipA, TisB), SOS response, and reduced ATP levels [9] [16].
Mycobacterium tuberculosis	Acid-Fast	Model for studying persisters in chronic human infections [9].	• Natural persistence causes lengthy, multi-drug tuberculosis therapy [9].• Existence of viable but non-culturable (VBNC) states [9].• PZA (pyrazinamide) is a key clinical anti-persister drug [9].
Staphylococcus aureus	Positive	Model for Gram-positive pathogens and biofilm-associated infections [9].	• First species in which persisters were observed (Bigger, 1944) [9] [19].• High-persistence (hip) mutants isolated from clinical settings [9].• Studies link low ATP and Krebs cycle fluctuations to persistence [16].
Pseudomonas aeruginosa	Negative	Model for biofilm-associated chronic infections, particularly in cystic fibrosis (CF) [9] [20].	• High-persistence (hip) mutants frequently found in CF patients [9] [20].• Strong link between biofilms and persister cells [9] [20].• Mutations in mucA, mexT, lasR linked to persistence and resistance [20].
Other Clinically Relevant Species	Varies	Illustrates the broad relevance of persistence [9] [20].	• Salmonella enterica (typhoid fever), Borrelia burgdorferi (Lyme disease), Klebsiella pneumoniae, and Streptococcus pneumoniae are all known to form persister cells that contribute to persistent and relapsing infections [9] [20].

Experimental Protocols for Persister Enrichment and Isolation

A significant challenge in persistence research is the isolation of these rare cells without inducing the phenotype during the process itself. The following protocols represent established methods for enriching and isolating persister cells.

Protocol: Cephalexin-Induced Filamentation and Filtration for Persister Enrichment

This method leverages the specific killing dynamics of the β-lactam cephalexin to efficiently separate persisters from a population of susceptible, exponentially growing cells with minimal debris [7].

Application: Highly effective enrichment of persisters from exponential phase cultures for downstream single-cell analyses [7]. Principle: Cephalexin inhibits penicillin-binding protein 3 (PBP3/FtsI), halting cell division and causing susceptible cells to form long filaments before eventual lysis. Drug-tolerant persisters remain unaffected as short, non-filamented cells, allowing their physical separation by filtration [7].

Workflow Diagram: Cephalexin-Filtration Enrichment

Materials:

• Bacterial Strain: e.g., E. coli MG1655.
• Growth Medium: Mueller-Hinton Broth (MHB) or LB.
• Antibiotic: Cephalexin stock solution (e.g., 10 mg/mL in water).
• Filtration Unit: Sterile membrane filtration apparatus (e.g., 0.22 µm or 0.45 µm pore size).
• Centrifuge Tubes.

Procedure:

• Culture Growth: Grow bacteria to early exponential phase (OD~600nm~ ≈ 0.1-0.4) in an appropriate medium [7].
• Antibiotic Treatment: Add cephalexin to the culture at a concentration of 1-5 times the MIC. Mix thoroughly.
• Incubation: Incubate the culture with cephalexin for 1 hour at the growth temperature with shaking. Note: This duration is critical. Shorter treatments risk susceptible cell contamination; longer treatments generate more cell debris [7].
• Filtration: Pass the entire culture volume through the sterile membrane filter. The filamentous, susceptible cells will be retained on the filter.
• Collection: Collect the filtrate, which contains the non-filamented, persister cells.
• Concentration: Centrifuge the filtrate to pellet the persister cells. Discard the supernatant and resuspend the pellet in fresh medium or an appropriate buffer.
• Validation: Determine the concentration of viable cells by plating and colony counting. Validate persister phenotype by re-challenging an aliquot with cephalexin or other antibiotics; survival should remain high over several hours [7].

Protocol: Enzymatic Lysis for Rapid Persister Isolation

This antibiotic-free method uses a combination of alkaline and enzymatic lysis to rapidly kill growing cells, minimizing the risk of stress-induced persistence during isolation. It also allows for differentiation between Type I and Type II persisters [21].

Application: Rapid isolation of persisters from both exponential and stationary phase cultures without prolonged antibiotic exposure. Differentiation of Type I and Type II persisters [21]. Principle: The protocol disrupts the cell envelope of growing cells, which are more susceptible to lysis. Persister cells, often with altered membrane states or reduced metabolic activity, survive the brief lysis step. The intensity of the lysis treatment can be modulated to isolate total persisters or only the more robust Type I subpopulation [21].

Workflow Diagram: Enzymatic Lysis Isolation

Materials:

• Bacterial Strain: e.g., E. coli K-12 strains (WT, hipA7, hipQ).
• Lysis Solutions:
  • Osmotic Lysis Solution: Commercially available miniprep alkaline lysis solution (e.g., from Sigma) [21].
  • Enzymatic Lysis Solution: Lysozyme (45 mg/mL, ~485 U/mg) dissolved in TE buffer [21].
• Centrifuge Tubes.

Procedure:

• Sample Preparation: Take a 1 mL aliquot from a bacterial culture in the desired growth phase.
• Osmotic Lysis: Add 200 µL of the osmotic lysis solution to the 1 mL aliquot in a 15 mL tube. Vortex vigorously for 10 seconds. Incubate at room temperature for 10 minutes.
• Enzymatic Lysis: Add 200 µL of the enzymatic lysozyme solution. Mix by inverting the tube. Incubate at 37°C for 15 minutes with gentle shaking (200 rpm).
• Viability Count: Serially dilute the mixture and plate on LB agar plates to determine the frequency of persister cells (cfu/mL). Note: The surviving cells are the persisters isolated by this protocol [21].
• For Type I Persister Isolation: To selectively isolate the more robust Type I persisters, increase the volume of both the osmotic and enzymatic lysis solutions to 500 µL each. This harsher treatment kills both susceptible cells and the more fragile Type II persisters [21].

Quantitative Analysis of Persister Populations

Understanding the quantitative dynamics of persister formation and killing is essential for interpreting experimental results. The following table consolidates key quantitative findings from persistence research.

Table 2: Quantitative Survey of Persister Fractions and Dynamics

Parameter	Quantitative Findings	Experimental Context
Typical Persister Fraction	Ranges from <0.0001% to >70% of total population [23].	Varies massively by species, antibiotic, growth phase, and medium [22] [23].
Impact of Antibiotic Class	Membrane-active agents (e.g., colistin) yield lowest persister fractions (~0.001%). Protein synthesis inhibitors & antimetabolites yield highest (e.g., erythromycin ~63%) [23].	Survey of 54 antibiotics across 36 species [23].
Impact of Growth Phase	Exponentially growing cultures have lower persister fractions. Stationary phase cultures can have fractions 100-1000 times higher [9] [23].	Standard killing assays in rich media [9].
Killing Kinetics	Biphasic time-kill curve: rapid killing of normal cells followed by a plateau with a much slower death rate of persisters [7] [22].	Standard for defining and quantifying persistence [22].
Stochastic Awakening	Single-cell monitoring shows persister resuscitation occurs at a constant, stochastic rate after antibiotic removal [7].	Microfluidic "mother machine" studies with enriched E. coli persisters [7].
Energy (ATP) Levels	Persisters show significantly lower ATP levels. Subpopulations with low Krebs cycle enzyme expression have 2-10x higher survival upon antibiotic challenge [16].	FACS sorting and single-cell ATP reporting in E. coli and S. aureus [16].

The Scientist's Toolkit: Key Reagents and Materials

Table 3: Essential Research Reagent Solutions for Persistence Studies

Reagent / Material	Function / Application	Specific Examples & Notes
β-Lactam Antibiotics	Induce cell wall stress and lysis; used for enrichment and killing assays.	Cephalexin: PBP3 inhibitor for filtration-based enrichment [7].Ampicillin: Broad-target PBP inhibitor; classical persister studies [9] [21].
Fluoroquinolone Antibiotics	Induce DNA damage and the SOS response; used for killing assays.	Ciprofloxacin, Ofloxacin: Target DNA gyrase; used to study SOS-linked persistence [16] [21].
Lytic Enzymes	Rapidly disrupt cell wall of growing cells for antibiotic-free persister isolation.	Lysozyme: Digests peptidoglycan in Gram-positive and Gram-negative bacteria (with EDTA) [21].
ATP Reporters	Measure intracellular ATP levels at single-cell resolution to link metabolism to persistence.	iATPSnFr1.0: Ratiometric, genetically encoded ATP sensor [16].
Microfluidic Devices	Track growth, death, and resuscitation of individual cells over time.	Mother Machine: Ideal for studying stochastic awakening of persisters [16] [7].
Strains with High-Persistence (Hip) Mutations	Provide model systems with elevated persister fractions for mechanistic studies.	E. coli hipA7: Classic Type I persister mutant [9] [21].E. coli hipQ: Associated with Type II persistence [21].

Visualization of Core Persistence Mechanisms

The formation of persister cells is a systems-level property often emerging from fundamental trade-offs in cellular physiology. The following diagram integrates key molecular players into a coherent conceptual framework.

Conceptual Diagram: Integrated Network in Persister Formation

A Practical Toolkit: Proven Techniques for Persister Enrichment and Isolation

Bacterial persisters are a subpopulation of cells that exhibit transient, non-heritable tolerance to lethal concentrations of antibiotics without undergoing genetic mutation. These phenotypic variants enter a state of reduced metabolic activity or dormancy, enabling survival during antibiotic exposure and potentially leading to chronic, recurrent infections. The study of persister cells is complicated by their typically low abundance (approximately 0.01% in exponential-phase cultures), necessitating reliable methods for their enrichment. Chemical induction using stressors like carbonyl cyanide m-chlorophenyl hydrazone (CCCP) and specific antibiotics provides a synchronized, controllable approach to generate persister populations for downstream mechanistic studies and therapeutic screening [24] [12].

This protocol details established methodologies for inducing persister states in Escherichia coli and related bacterial species through chemical disruption of cellular energetics and antibiotic-mediated growth arrest, enabling reproducible enrichment of persister cells for subsequent analysis.

Key Mechanisms of Chemically-Induced Persistence

Chemical inducers trigger persistence by disrupting fundamental physiological processes, primarily cellular energetics and translation. The diagram below illustrates the core pathways targeted by CCCP and antibiotic inducers.

The induction of bacterial persistence by chemicals like CCCP and specific antibiotics converges on a core alarmone-GTP switch. The accumulation of the alarmone (p)ppGpp, triggered by various stressors, potently antagonizes intracellular GTP synthesis. A rapid, switch-like decrease in GTP levels beneath a critical threshold drives the transition from active growth to a dormant, persistent state in individual cells [25].

Comparative Analysis of Induction Methods

The table below summarizes the key parameters and outcomes for CCCP and antibiotic induction protocols.

Table 1: Quantitative Comparison of Chemical Induction Methods

Parameter	CCCP Induction	Rifampicin Induction	Aminoglycoside Tolerance
Primary Target	Membrane potential / Proton motive force [24]	RNA polymerase / Transcription [24]	Protein translation [25]
Typical Working Concentration	100 µg/mL [24]	Varies by MIC	Varies by MIC
Induction Duration	15 minutes [24]	30 minutes to 2 hours [24]	1 to 4 hours [25]
Metabolic State Post-Induction	Substantially reduced metabolism; delayed labeling in central pathways [24]	Growth arrested, dormant state [24]	Dormant, non- or slow-growing [9]
Key Metabolic Observations	More substantial metabolic shutdown with acetate vs. glucose carbon source [24]	N/A	N/A
Persistence Level Achieved	High (suitable for -omics) [24]	Can convert nearly 100% of population [24]	Subpopulation survival [25]

Experimental Protocols

CCCP Induction Protocol forE. coli

This method utilizes a protonophore to dissipate the membrane potential, inducing a persister state without permanent damage to essential cellular processes [24].

Materials and Reagents

• Bacterial Strain: Escherichia coli BW25113 (or other relevant strain)
• Growth Medium: M9 minimal medium supplemented with 2 g/L glucose (or other carbon source)
• Induction Reagent: Carbonyl cyanide m-chlorophenyl hydrazone (CCCP) stock solution (e.g., 10 mg/mL in DMSO or ethanol)
• Wash Buffer: M9 medium without a carbon source
• Equipment: Centrifuge, shaking incubator, spectrophotometer (for OD600 measurement)

Step-by-Step Procedure

• Culture Preparation:

  • Inoculate E. coli from an overnight culture into fresh M9 medium containing 2 g/L glucose.
  • Sub-culture to a starting OD600 of 0.05.
  • Incubate at 37°C with shaking at 200 rpm until the culture reaches mid-exponential phase (OD600 ≈ 0.5).

• Persister Induction:

  • Add CCCP from the stock solution to the culture to a final concentration of 100 µg/mL.
  • Incubate the culture for 15 minutes at 37°C with shaking at 200 rpm.

• Cell Harvesting and Washing:

  • Transfer the induced culture to centrifuge tubes.
  • Collect cells by centrifugation at 13,000 × g for 3 minutes at room temperature.
  • Carefully decant the supernatant.
  • Wash the cell pellet three times with 10 mL of M9 medium without any carbon source to remove residual CCCP.
  • Resuspend the final pellet in an appropriate volume of M9 medium to concentrate the cells (e.g., OD600 of 5) for downstream applications or analysis.

Antibiotic-Based Induction Protocol

Bacteriostatic antibiotics like rifampicin can induce a persister state by halting transcription, leading to growth arrest [24].

Procedure

• Culture Preparation: Grow the bacterial culture to the desired phase (exponential or stationary) as described in steps 1-2 of the CCCP protocol.

• Antibiotic Exposure:

  • Add the chosen bacteriostatic antibiotic (e.g., rifampicin) at a predetermined concentration. The concentration should be sufficient to inhibit growth.
  • Incubate for a defined period (e.g., 30 minutes to 2 hours) to allow for the induction of the persistent state.

• Cell Processing:

  • Following induction, wash the cells multiple times with a suitable buffer or antibiotic-free medium to remove the antibiotic.
  • The resulting cell population is enriched for persisters and can be used for subsequent experiments.

The Scientist's Toolkit: Essential Reagents

The table below lists key reagents and their applications in persister enrichment studies.

Table 2: Research Reagent Solutions for Persister Studies

Reagent / Material	Function / Application	Example Usage & Notes
CCCP (Protonophore)	Chemical inducer of persistence; disrupts the proton motive force and depletes ATP [24].	Used at 100 µg/mL for 15 min for synchronized induction in E. coli. Prepare fresh stock solution in DMSO.
Rifampicin	Antibiotic inducer of persistence; inhibits transcription by targeting RNA polymerase [24].	Can convert nearly all cells in a population to persisters. Concentration depends on the MIC of the strain.
Stable Isotope Tracers (¹³C-glucose, ¹³C-acetate)	Metabolic flux analysis; enables tracking of carbon source utilization in persisters vs. normal cells [24].	Use in tracer experiments post-induction to elucidate metabolic states via LC-MS/GC-MS.
DiBAC₄(3) Fluorescent Dye	Membrane potential staining; used in flow cytometry to sort and identify persister cells based on depolarized membranes [12].	Applied in flow sorting protocols for persister enrichment from heterogeneous populations.
M9 Minimal Medium	Defined growth medium; essential for controlling nutrient conditions and carbon source during induction and labeling studies [24].	Preferred over rich media like LB for its defined composition, especially in metabolic studies.

Downstream Applications and Validation

Following induction and enrichment, persister cells can be subjected to various downstream analyses. Stable Isotope Labeling (SIL) coupled with Mass Spectrometry (LC-MS/GC-MS) is a powerful approach to characterize the metabolic state of persisters. As demonstrated in foundational studies, persisters exhibit major differences in metabolic activities, including reduced metabolism and delayed labeling dynamics in central carbon pathways like the Pentose Phosphate Pathway and TCA cycle compared to normal cells [24].

Functional validation of the enriched persister population is critical. This typically involves performing time-kill assays with a relevant bactericidal antibiotic (e.g., a fluoroquinolone or aminoglycoside) to confirm the characteristic biphasic killing curve indicative of a subpopulation with enhanced survival [24] [25]. Furthermore, to confirm the non-genetic nature of the phenotype, regrown survivors should be tested to ensure they exhibit susceptibility profiles identical to the original, non-induced culture [25].

Bacterial persisters are a subpopulation of cells characterized by a transient, non-growing (or slow-growing) state that allows them to survive exposure to high concentrations of antibiotics. These genetically drug-susceptible cells are a significant culprit behind treatment failures, relapsing infections, and the development of antibiotic resistance, particularly in chronic and biofilm-associated infections [9]. A critical first step in persister research is their effective enrichment and isolation from a larger, susceptible population. This protocol details robust culture-based methods leveraging stationary phase growth and biofilm conditions to achieve this goal, providing a foundational technique for researchers and drug development professionals investigating persistent infections.

Theoretical Background: Linking Growth Conditions to Persistence

The connection between non-growing states and antibiotic tolerance is fundamental to persister biology. When bacterial cells enter the stationary phase due to nutrient depletion or are embedded in a biofilm, a subpopulation adopts a quiescent phenotype. This dormancy is key to their survival, as most bactericidal antibiotics target active cellular processes like cell wall synthesis, protein production, and DNA replication [9]. The following diagram illustrates the logical pathway from culture conditions to the formation and isolation of the persister subpopulation.

It is crucial to distinguish persister cells from resistant mutants. Antibiotic resistance involves genetic mutations that raise the minimum inhibitory concentration (MIC), allowing growth in the presence of the drug. In contrast, antibiotic tolerance/persistence involves survival without an increase in MIC, arising from a transient phenotypic switch [9] [26]. Stationary-phase cultures, for instance, often exhibit phenotypic tolerance, where the entire population survives antibiotic exposure but can be killed upon nutrient restoration. True persistence is a subpopulation phenomenon where a small fraction of cells survives even in a nutrient-replete, growing culture [26].

Experimental Protocols

Protocol 1: Enrichment from Stationary Phase Cultures

This protocol uses nutrient exhaustion to induce a dormant state in a portion of the bacterial population.

Materials:

• Bacterial Strain: e.g., Staphylococcus aureus ATCC 25923 or Escherichia coli K-12.
• Growth Medium: Tryptic Soy Broth (TSB) or Lysogeny Broth (LB).
• Antibiotic Stock: Levofloxacin or another bactericidal antibiotic relevant to the studied pathogen.
• Equipment: Shaking incubator, centrifuge, spectrophotometer (for OD measurement), cell culture plates or flasks.

Procedure:

• Inoculation and Growth: Inoculate 10 mL of fresh, pre-warmed medium with a single bacterial colony from an overnight agar plate.
• Prolonged Incubation: Incubate the culture at 37°C with vigorous shaking (e.g., 220 rpm) for an extended period (e.g., 72 hours). This ensures the culture reaches and maintains a deep stationary phase, enriching for quiescent cells [27].
• Antibiotic Challenge:
  • Harvest the stationary-phase culture.
  • Add a high concentration of a bactericidal antibiotic (e.g., 20x the MIC of levofloxacin) [26].
  • Incubate for a defined period (e.g., 24 hours) under the same conditions to kill the remaining susceptible, active cells.
• Persister Harvest and Washing:
  • Pellet the cells by centrifugation.
  • Wash the pellet twice with sterile phosphate-buffered saline (PBS) or an appropriate buffer to remove the antibiotic completely.
  • Resuspend the final pellet in PBS or fresh medium. This washed cell suspension is enriched with persister cells and can be used for downstream applications.

Protocol 2: Enrichment from Biofilm Cultures

Biofilms are natural reservoirs for persister cells due to their inherent heterogeneity and nutrient gradients [9] [27]. The physiological state of the inoculum used to initiate the biofilm significantly impacts the resulting persister population.

Materials:

• Materials from Protocol 1.
• Cell Culture-Treated Plates: 24-well or 96-well plates for biofilm formation.

Procedure:

• Inoculum Preparation: Prepare two types of inocula [27]:
  • Exponential Phase Inoculum (EDBF): Culture bacteria for ~2 hours to mid-exponential phase.
  • Stationary Phase Inoculum (SDBF): Culture bacteria for 72 hours to stationary phase.
• Biofilm Initiation: Transfer 1.5 mL of each inoculum into the wells of a cell culture-treated plate. Include multiple biological replicates.
• Biofilm Maturation: Allow biofilms to develop for a specified duration (e.g., 24 hours) at 37°C with or without shaking [27].
• Antibiotic Treatment and Harvest:
  • Gently wash the mature biofilms once with PBS to remove non-adherent planktonic cells.
  • Add fresh medium containing a high concentration of antibiotic (e.g., 400 µM levofloxacin) directly to the biofilm.
  • After incubation, harvest the biofilm cells by scraping. The surviving population is highly enriched for persisters.

The workflow for both protocols, highlighting the key decision points, is summarized below.

Data Presentation and Analysis

Quantitative Survival Data

The efficacy of persister enrichment is quantified by determining the survival rate after antibiotic challenge. Data should be presented as log reductions in Colony Forming Units (CFUs). The table below provides example data for S. aureus based on methodologies from the search results.

Table 1: Example survival data of S. aureus after levofloxacin treatment (400 µM for 24h) under different growth conditions. Data is presented as Mean log10 CFU ± SD.

Growth Condition	Pre-Treatment Viability (log10 CFU/mL)	Post-Treatment Viability (log10 CFU/mL)	Log Reduction	Approx. Survival %
Exponential Planktonic	9.0 ± 0.2	2.5 ± 0.5	6.5	~0.0003%
Stationary Planktonic (72h)	8.5 ± 0.3	7.5 ± 0.4	1.0	~10%
Biofilm (EDBF)	8.0 ± 0.2	6.0 ± 0.3	2.0	~1%
Biofilm (SDBF)	7.8 ± 0.3	6.5 ± 0.4	1.3	~5%

Key Characteristics of Enriched Populations

The persister populations enriched through these methods exhibit distinct features, as confirmed by surfaceomic and phenotypic analyses [27].

Table 2: Key characteristics of persister cells enriched via stationary phase and biofilm cultures.

Characteristic	Description	Research Implication
Metabolic State	Non-growing or slow-growing, but can be metabolically active and adapt their transcriptome [28].	Challenges the pure dormancy model; suggests active survival pathways.
Surfaceome Profile	Altered surface protein expression (e.g., reduced adhesins, increased immune evasion proteins like SpA in S. aureus SDBF) [27].	Impacts host-pathogen interactions; potential therapeutic target.
Tolerance Level	Can exhibit a continuum from "shallow" to "deep" persistence [9].	Different enrichment methods may select for persisters with varying resilience.
Inoculum Effect	Biofilms initiated from stationary phase cells (SDBF) show higher persistence and immune evasion traits than those from exponential phase cells (EDBF) [27].	The initial physiological state is a critical variable in experimental design.

The Scientist's Toolkit: Research Reagent Solutions

Table 3: Essential materials and reagents for culture-based persister enrichment.

Item	Function/Description	Example/Note
Tryptic Soy Broth (TSB)	A nutrient-rich, general-purpose medium for growing a wide range of bacteria, including Staphylococci.	Used for both planktonic and biofilm cultures of S. aureus [27].
Levofloxacin	A broad-spectrum fluoroquinolone bactericidal antibiotic. Induces DNA breakage.	Effective at 20x MIC; used at 400 µM for S. aureus biofilm treatment [27].
Cell Culture-Treated Plates	Polystyrene plates with treated surfaces to enhance cell adherence and biofilm formation.	Nunc plates are used in the cited protocol [27].
Triethylammonium Bicarbonate (TEAB)	A buffer used in sample preparation for proteomic analysis, specifically in "trypsin shaving."	Used to suspend cells for surfaceome analysis via mass spectrometry [27].
Sequencing Grade Trypsin	A high-purity protease used to digest surface-exposed proteins for LC-MS/MS identification.	Used at 55 ng/µL to shave surface proteins for surfaceomic studies [27].

{article title}

Advanced Sorting Strategies: Fluorescence-Activated Cell Sorting (FACS) Using Metabolic Reporters

A significant challenge in combating chronic and recurrent bacterial infections is the presence of bacterial persisters [29]. These cells are a transient, non- or slow-growing subpopulation that exhibits remarkable tolerance to high concentrations of antibiotics without acquired genetic resistance [30] [5]. Their ability to survive treatment and subsequently repopulate a biofilm is a major cause of therapeutic failure in infections ranging from tuberculosis to those caused by Escherichia coli and Pseudomonas aeruginosa [29] [31]. A critical hurdle in persister research has been the inability to isolate them to high purity. Persisters are rare, transient, and by all measures to date, extremely similar to the more abundant viable but non-culturable cells (VBNCs), as both exclude propidium iodide, harbor metabolic activity, and remain non-replicating during stress [30]. The defining characteristic that separates persisters is their capacity to resume growth on standard media after the antibiotic stress is removed, whereas VBNCs cannot [30].

In the absence of specific biomarkers for isolation, Fluorescence-Activated Cell Sorting (FACS) has emerged as the gold-standard technique for studying persister physiology [30]. This approach does not require prior isolation. Instead, it involves segregating a bacterial population into subpopulations (quantiles) based on a quantitative fluorescent characteristic—such as the activity of a metabolic enzyme or the expression of a fluorescent protein. Subsequent antibiotic tolerance assays on these sorted fractions quantify persister abundance across the physiological distribution, enabling the construction of a "persister phenotype distribution" that can be compared to the distribution of the entire population [30] [32]. This review details the application of FACS with metabolic and translational reporters as a core strategy for enriching and investigating persister subpopulations, providing detailed protocols and contextualizing these methods within the broader challenge of persister isolation.

Metabolic Basis for Persister Sorting

The physiological state that confers antibiotic tolerance is intimately linked to bacterial metabolism and energy levels. While persisters were historically characterized as dormant, recent studies reveal a more complex picture where their metabolism is rewired rather than simply shut down [11] [33]. A key insight is that persisters often constitute a subpopulation of low-energy cells. For instance, cells with diminished levels of Krebs cycle enzymes (e.g., isocitrate dehydrogenase Icd, citrate synthase GltA) show significant enrichment for persistence to ciprofloxacin [34]. Direct measurement of ATP in single cells using ratiometric sensors like iATPSnFr1.0 has confirmed that a subpopulation with low ATP levels is better able to survive antibiotic killing [34]. This metabolic downshift reduces the activity of antibiotic-targeted processes, thereby promoting tolerance.

Conversely, persister survival still relies on basal levels of energy metabolism. The global metabolic regulator Crp/cAMP plays a critical role in this metabolic rewiring, particularly in stationary-phase persisters. This complex redirects metabolism from anabolism to oxidative phosphorylation, sustaining the Tricarboxylic Acid (TCA) cycle, electron transport chain (ETC), and ATP synthase, which are all crucial for maintaining persister viability [11] [33]. This retained metabolic activity, albeit at a reduced rate, provides the foundation for using fluorescent metabolic reporters to distinguish persister cells from the general population. Furthermore, the link between translational inhibition and drug tolerance is well-established [31]. Various stress pathways lead to the repression of translation, making reporters that monitor the cell's capacity to synthesize protein powerful tools for identifying and isolating persister subpopulations.

The following diagram illustrates the core metabolic pathways and regulatory systems involved in persister formation that can be probed with FACS-based strategies.

Key Research Reagent Solutions

The successful implementation of FACS strategies for persister research relies on a suite of specialized reagents and tools. The table below catalogues the essential research reagent solutions, detailing their critical functions in staining, reporting, and isolating persister subpopulations.

Table 1: Key Research Reagents for FACS-Based Persister Analysis

Reagent / Tool Name	Function / Application	Key Characteristics
Redox Sensor Green (RSG) [30]	Fluorogenic metabolic stain for assessing metabolic activity via bacterial reductases.	Nontoxic; does not suppress cellular metabolism; yields green fluorescence upon reduction.
iATPSnFr1.0 [34]	Ratiometric, genetically encoded fluorescent reporter for single-cell ATP measurement.	Self-normalizing; uses 488 nm/405 nm excitation ratio to report ATP concentration.
Trans-mEos2 Reporter (PerSort) [31]	Fluorescent reporter system for isolating translationally dormant mycobacteria.	Genome-integrated; ATc-inducible transcription with strong Shine-Dalgarno sequence for translation reporting.
T5p-mCherry Reporter [30] [35]	Fluorescent protein reporter for monitoring cell division and gene expression.	Fluorescent protein is stable; dilution indicates cell division in inducer-free environments.
Crp/cAMP Reporter Systems [11] [33]	Tools for studying the role of the Crp/cAMP global metabolic regulator in persister metabolism.	Reveals metabolic rewiring towards oxidative phosphorylation in persistent cells.
Krebs Cycle Enzyme Reporters (e.g., Icd-mVenus) [34]	Translational fusions to key metabolic enzymes (e.g., Icd, GltA, SucA) for FACS.	Identifies subpopulations with low energy-generating enzyme levels, enriched for persisters.

Detailed FACS Protocols for Persister Analysis

The following protocols provide a framework for using FACS to assay the metabolic and growth states of bacterial persisters. These methods are adaptable but have been specifically used for E. coli.

Protocol 1: Assaying Metabolic State with Redox Sensor Green (RSG)

This protocol uses the metabolic stain RSG to segregate cells based on their metabolic activity before determining which subpopulations are enriched for persisters [30].

1. Sample Preparation and Staining:

• Grow the bacterial culture (e.g., E. coli MG1655) to the desired growth phase (exponential or stationary).
• Harvest 1 mL of the culture and wash the cells with phosphate-buffered saline (PBS).
• Resuspend the cell pellet in 1 mL of PBS containing a working concentration of RSG (e.g., 1-5 µM).
• Incubate the stained cells in the dark at room temperature for 15-60 minutes.

2. FACS Analysis and Sorting:

• Analyze the stained sample using a flow cytometer equipped with a 488-nm laser. Collect green fluorescence through a 525/50 nm bandpass filter.
• Establish sorting gates based on the fluorescence distribution of the population. For persister enrichment, often the dimmest 5-10% of the population (low metabolic activity) is sorted as one fraction, and the brightest 5-10% (high metabolic activity) as another.
• Sort a sufficient number of cells (e.g., 10,000 - 100,000) from each gated population into sterile microcentrifuge tubes containing PBS or a suitable recovery medium.

3. Persister Enumeration and Validation:

• Plate an aliquot of the sorted fractions onto LB agar to determine the total culturable cells (CFU) prior to antibiotic treatment.
• Treat the remaining sorted cells with a supra-lethal concentration of a bactericidal antibiotic (e.g., 5 µg/mL ofloxacin or 200 µg/mL ampicillin) for a duration sufficient to reach the second, slower killing phase (typically 3-5 hours).
• After antibiotic treatment, wash the cells to remove the antibiotic, serially dilute, and plate on LB agar to enumerate the surviving persisters.
• Calculate the persister frequency in each sorted fraction (CFU after antibiotic / CFU before antibiotic) and compare it to the frequency in the unsorted population.

Protocol 2: Monitoring Cell Division with a Fluorescent Protein (mCherry)

This protocol uses a stable fluorescent protein, mCherry, expressed from an inducible system to identify non-growing cells through the absence of fluorescence dilution [30] [35].

1. Strain Preparation and Reporter Induction:

• Use a strain harboring a chromosomally integrated, inducible mCherry system (e.g., T5p-mCherry induced by IPTG).
• Grow the reporter strain in medium containing 1 mM IPTG to fully induce mCherry expression during exponential growth.

2. Fluorescence Dilution and Sorting:

• Harvest the induced culture by centrifugation and wash thoroughly with PBS to remove IPTG.
• Resuspend the cells in fresh, inducer-free medium and allow them to grow for 1-2 generations. During this period, growing cells will dilute their mCherry fluorescence, while non-growing or slowly growing cells will retain high fluorescence.
• Analyze the cells by flow cytometry using a 560-nm laser and collect mCherry fluorescence through a 610/20 nm bandpass filter.
• Sort cells based on fluorescence intensity: the brightest cells (non-diluters, likely non-growing) and the dimmest cells (diluters, growing).

3. Persister Enumeration:

• Subject the sorted fractions to an antibiotic tolerance assay as described in Protocol 1, step 3.
• The high-fluorescence (non-diluting) fraction is typically enriched for persisters, as their lack of growth made them tolerant to antibiotics.

The workflow for these two core protocols, from cell preparation to data analysis, is summarized below.

Advanced Applications and Case Studies

The basic FACS protocols have been adapted and scaled into sophisticated methods to address specific challenges in persister research.

5.1. Persister-FACSeq for High-Throughput Physiology To overcome the throughput limitations of conventional FACS, Persister-FACSeq was developed to interrogate a library of fluorescent reporters simultaneously [32]. In this method:

• A library of strains, each with a different promoter fused to a fluorescent reporter, is grown and pooled.
• The pooled population is sorted by FACS into quantiles based on fluorescence intensity.
• Sorted cells from each quantile are plated, both with and without antibiotic treatment, to determine the promoter activity distribution of normal cells and persisters, respectively.
• Colonies from the plates are harvested, and their promoter regions are amplified with barcodes and quantified via next-generation sequencing.
• This approach allows for the massive parallelization of persister physiology studies, having revealed, for example, that persistence to ofloxacin is inversely correlated with the capacity of non-growing cells to synthesize protein [32].

5.2. PerSort for Isolation of Translationally Dormant Mycobacteria The PerSort method was specifically designed for mycobacteria to isolate translationally dormant persisters without applying antibiotic pressure, which can confound results [31].

• A reporter strain is created with a genome-integrated, ATc-inducible mEos2 fluorescent protein containing a strong translation initiation site.
• Under naive growth conditions, cells that are capable of transcription but not translation (i.e., translationally dormant) will show low fluorescence despite the presence of the inducer.
• FACS is used to isolate this low-fluorescing subpopulation, which has been demonstrated to exhibit multidrug tolerance and lag dormancy, consistent with persister phenotypes.
• Single-cell transcriptional profiling of these sorted cells can then elucidate the varied mechanisms (e.g., vapC30, mazF overexpression) leading to the persister state [31].

Data Interpretation and Technical Considerations

The quantitative data derived from FACS-based persister experiments provide a rich profile of persister heterogeneity. The following table summarizes typical findings from different experimental approaches.

Table 2: Representative Quantitative Findings from FACS-Based Persister Studies

Experimental Approach	Key Quantitative Finding	Bacterial System	Citation
Krebs Cycle Reporter FACS (Icd-mVenus)	The "Dim" population (low Icd) had ~10-fold higher survival after ciprofloxacin treatment compared to the "Bright" population.	E. coli	[34]
ATP Reporter (iATPSnFr1.0)	A subpopulation of cells with low ATP levels was enriched for survival after ampicillin treatment.	E. coli	[34]
Persister-FACSeq	Persistence to ofloxacin was inversely correlated with promoter activity from ribosomal and protein synthesis genes in non-growing cells.	E. coli (Stationary)	[32]
PerSort (Trans-mEos2)	The translationally dormant (low fluorescence) subpopulation exhibited multidrug tolerance and a significantly delayed mean time of colony appearance (41h vs 37h).	Mycobacterium smegmatis	[31]

When implementing these protocols, several technical considerations are paramount:

• VBNC Confounding: Always include controls to distinguish persisters (which regrow) from VBNCs (which do not). Persister quantification must be based on the ability to form colonies on standard media after antibiotic removal [30] [35].
• Fluorophore Impact: Validate that any fluorescent stain or protein used does not affect bacterial culturability or alter persister levels under experimental conditions [30].
• Gating Strategy: Persister enrichment is often found in the tails of fluorescence distributions (e.g., the dimmest 5% for metabolic activity or the brightest 5% for a non-diluting fluorescent protein). Pilot experiments are crucial for defining optimal gates.
• Antibiotic Selection: Use supra-lethal concentrations of bactericidal antibiotics (e.g., fluoroquinolones, β-lactams) and confirm biphasic killing kinetics to ensure the measured survivors are true persisters [30] [5].

Fluorescence-Activated Cell Sorting, empowered by metabolic and translational reporters, provides an indispensable and powerful strategy for enriching and characterizing bacterial persister subpopulations. By moving beyond the limitations of traditional isolation techniques, FACS allows researchers to probe the physiological heterogeneity of persisters within a population, revealing that these cells often occupy a low-energy state with rewired metabolism. Advanced methods like Persister-FACSeq and PerSort demonstrate how this core technology can be scaled and adapted to different bacterial species and research questions, from high-throughput genetic screens to mechanistic studies without antibiotic pre-selection. As these protocols continue to be refined and integrated with other 'omics' technologies, they will undoubtedly accelerate our understanding of persister biology and contribute to the development of novel therapeutic strategies aimed at eradicating these resilient cells to combat recalcitrant infections.

Bacterial persisters represent a small, transient subpopulation of cells that are metabolically dormant and can survive lethal antibiotic treatment without genetic resistance. These cells are a major contributor to chronic and recurrent infections, as they can repopulate once antibiotic pressure is removed [36]. Critically, this phenotypic heterogeneity exists within genetically identical populations, meaning traditional bulk measurement techniques are insufficient for isolating and studying these rare variants, which often constitute less than 1% of a population [16] [36]. Overcoming this technical challenge requires platforms capable of high-throughput single-cell analysis under precisely controlled conditions. Microfluidic technologies, particularly the mother machine platform, have emerged as powerful tools that meet this need, enabling researchers to isolate and monitor individual bacterial cells across multiple generations to unravel the mechanisms of persistence [16] [37].

The isolation and analysis of single cells are prerequisite steps for studying persister phenotypes. The performance of these technologies is typically evaluated by throughput, efficiency, spatial control, and cell viability [38]. The following table summarizes the primary techniques used for single-cell isolation.

Table 1: Key Single-Cell Isolation Techniques in Microbial Research

Technique	Throughput	Key Principle	Advantages for Persister Studies	Key Limitations
Mother Machine & SIFT [37]	High (10,000+ lineages)	Microfluidic trenches for cell lineage tracking with integrated optical trapping for retrieval.	Enables long-term, multigenerational imaging and isolation of live, unperturbed cells.	Requires specialized fabrication and setup.
Droplet Microfluidics [39]	Ultra-High (Thousands of droplets/sec)	Encapsulation of single cells in picoliter-volume droplets.	Ideal for high-throughput single-cell genomics/transcriptomics (e.g., Drop-seq).	Limited temporal monitoring of individual cells.
Fluorescence-Activated Cell Sorting (FACS) [40] [41]	High	Hydrodynamic focusing and electrostatic deflection of fluorescently-labeled cells.	Multi-parameter, high-speed sorting based on fluorescence markers.	Shear stress can damage cell viability; provides only a snapshot in time.
Magnetic-Activated Cell Sorting (MACS) [40] [41]	Medium	Labeling of cells with antibody-conjugated magnetic beads.	Simple, cost-effective for enriching cell populations.	Lower specificity and throughput compared to FACS; limited to surface markers.
Laser Capture Microdissection (LCM) [40] [41]	Low	Laser-based cutting and capture of specific cells from a solid sample.	Applicable to fixed tissues and biofilms.	Low throughput, potential for contamination, and requires high skill.
Micromanipulation [40] [41]	Low	Manual or robotic selection of single cells under a microscope.	High precision for selecting specific cells based on visual morphology.	Very low throughput and requires extensive skill.
Limiting Dilution [38] [41]	Medium	Serial dilution of a cell suspension to statistically achieve one cell per well.	Technically simple, low-cost, and reproducible.	Lack of direct control, requires downstream confirmation of clonality.

The Scientist's Toolkit: Essential Reagents and Materials

Successful execution of single-cell experiments, particularly in microfluidic platforms, relies on a suite of specialized reagents and tools.

Table 2: Key Research Reagent Solutions for Single-Cell Persister Studies

Item	Function/Application	Specific Examples & Notes
Fluorescent Biosensors	Reporters for tracking gene expression, protein localization, and metabolic states in live cells.	- iATPSnFR: A genetically-encoded ratiometric sensor for measuring intracellular ATP levels [16].- FRET-based Sensors: For studying protein-protein interactions and signaling dynamics [36].
Microfluidic Chips	The physical device that houses cells and enables precise fluidic control for imaging and isolation.	- Mother Machine Chips: Fabricated from PDMS, featuring arrays of dead-end growth trenches [16] [37].- Gel Encapsulation Chips: Used for immobilizing cells in thin agarose pads for antimicrobial exposure [42].
Barcoded Beads	For tracing the cellular origin of biomolecules in high-throughput sequencing.	- Drop-seq Beads: Microparticles with oligonucleotides containing cell barcodes and unique molecular identifiers (UMIs) for single-cell RNA-sequencing [39].
Viability Probes	Differentiate between live, dead, and viable-but-non-culturable (VBNC) cells.	- Nucleic Acid Stains (e.g., SYTOX Green, DAPI): Distinguish cells with compromised membranes [36].- Metabolic Probes: Report on enzymatic activity or membrane potential.
Cell Lysis Reagents	Release intracellular content for downstream molecular analysis within microfluidic compartments.	- Thermolabile Lysis Buffers: Activated by temperature shift within droplets or traps [39].- Enzymatic Lysis Mixes: Gentle on biomolecules but require optimized conditions.

Experimental Protocols for Persister Research

Protocol 1: Isoling Persisters Using the Mother Machine and SIFT

The Single-Cell Isolation Following Time-lapse Imaging (SIFT) method combines long-term imaging in a mother machine device with the retrieval of specific live cells for downstream analysis [37].

Workflow Overview:

Detailed Procedure:

• Device Preparation and Loading:

  • Fabricate a polydimethylsiloxane (PDMS) microfluidic device featuring a "mother machine" design. This includes a main growth lane, an array of dead-end growth trenches (typically ~1.5 µm x 1.5 µm x 25 µm), a separate collection lane, and integrated push-down valves [37].
  • Load an aged, stationary-phase bacterial culture (e.g., 48-hour post-inoculation) into the growth lane of the device. Cells are passively trapped in the growth trenches by flowing media.
  • The device is mounted on an inverted, automated microscope equipped with an environmental chamber maintained at 37°C.

• Time-Lapse Imaging and Phenotyping:

  • Flow fresh growth medium through the device to resuscitate cells and initiate growth. Image thousands of individual cell lineages every few minutes for multiple hours or days using phase-contrast and fluorescence microscopy (if reporters are used) [16] [37].
  • To induce persistence, switch the medium to one containing a lethal dose of an antibiotic (e.g., ampicillin). Continue time-lapse imaging to track cell responses (lysing, filamenting, or surviving without growth).
  • Use image analysis software to track lineages and identify persister cells—those that survive antibiotic exposure without lysing and remain intact.

• On-Chip Sterilization and Cell Retrieval:

  • Once target persister cells are identified, activate the push-down valves to seal the inlets and outlets of the growth lane.
  • Flow a bleach solution through the upstream tubing and the main growth lane inlet to sterilize and eliminate any potential contaminants. The sealed valves protect the cells in the growth trenches and the sterile collection lane [37].
  • Open the push-down valves separating the growth trenches from the collection lane. Use optical tweezers (a highly focused laser beam) to gently capture the target persister cell and transport it from its growth trench into the pristine collection lane. The typical transport time is under 15 seconds to minimize photodamage [37].

• Downstream Analysis:

  • Flush the contents of the collection lane to retrieve the isolated live persister cell. The cell can be plated for viability checks, inoculated into culture for propagation, or processed for whole-genome sequencing or transcriptomic analysis [37].

Protocol 2: High-Throughput Persister Screening via Microfluidic Gel Encapsulation

This protocol uses a micropatterned gel platform to rapidly analyze heterogeneous antimicrobial responses at the single-cell level, suitable for testing in physiological media like human urine [42].

Workflow Overview:

Detailed Procedure:

• Fabrication of Micropatterned Template:

  • Use a laser machining system to create an array of small wells (e.g., 0.5 mm diameter) on a thin double-sided tape. Attach this micropatterned tape to a glass coverslip to serve as the substrate [42].

• Cell Encapsulation in Gel Pads:

  • Prepare a 3% low-melting-point agarose solution in PBS or the desired physiological medium (e.g., urine). Heat to 65°C to dissolve completely, then cool to 37°C.
  • Mix the warm agarose solution with a concentrated bacterial suspension at a 1:1 ratio.
  • Immediately apply the mixture to the micropatterned template. Use a glass slide to scrape across the surface, creating an array of thin gel pads containing randomly distributed single cells [42].

• Antimicrobial Exposure and Time-Lapse Imaging:

  • Place the coverslip in a microscopy chamber. Flow antimicrobial solution at a physiologically relevant concentration over the gel pads.
  • Conduct time-lapse microscopy to monitor individual cells over time (e.g., 4-24 hours). Track phenotypic outcomes such as growth, death (lysis), or stasis for each cell.

• Regrowth Assay to Identify Persisters:

  • After a period of antimicrobial exposure, flow fresh, antibiotic-free medium over the gel pads to remove the drug.
  • Continue time-lapse imaging to monitor for regrowth. Persister cells are defined as those that survived the antibiotic exposure without dividing and then resumed growth upon antibiotic removal. In contrast, VBNC cells will survive but not regrow [42].

• Image and Data Analysis:

  • Use automated image analysis to quantify single-cell growth rates and killing kinetics. Generate time-kill curves for individual cells to characterize the heterogeneity of the population's response and identify distinct tolerant subpopulations.

Key Applications and Data Interpretation

Elucidating the Low-ATP Mechanism of Persistence

A pivotal application of the mother machine platform was the direct demonstration that bacterial persisters have low ATP levels. Researchers used an E. coli strain expressing the iATPSnFR ATP sensor and tracked cells in the mother machine during ampicillin treatment [16]. The quantitative data revealed that survival was not random; the small subpopulation of cells that survived antibiotic killing predominantly originated from cells with a low level of ATP before the antibiotic was even applied [16]. This provides strong evidence for a "low energy" mechanism of persister formation, driven by stochastic fluctuations in metabolic components.

Table 3: Quantitative Insights from Single-Cell Persistence Studies

Experimental Finding	Platform Used	Quantitative Result	Biological Insight
Link between Krebs Cycle and Persistence [16]	FACS & CFU Counting	Dim populations (low Krebs cycle enzyme levels) showed ~10-fold higher survival after ciprofloxacin treatment compared to Bright populations.	Diminished energy metabolism specifically enriches for persisters.
ATP Level of Persisters [16]	Mother Machine + iATPSnFR	The surviving cells after ampicillin treatment were overwhelmingly from the subpopulation with a pre-existing low ATP state.	Persister formation is linked to stochastic heterogeneity in ATP levels.
Physiological Medium Effects [42]	Bulk Time-Kill Curves in Urine	Pathogens in human urine showed highly heterogeneous time-kill kinetics, differing from standardized lab media.	The host environment significantly influences antimicrobial response heterogeneity.
Viability of SIFT-Isolated Cells [37]	SIFT Platform	Genomes of isolated cells showed no unique mutations, and growth dynamics were unperturbed post-isolation.	The SIFT process isolates live cells without genetic or physiological damage.

Microfluidic platforms, particularly the mother machine and its advanced derivatives like SIFT, have fundamentally transformed our approach to studying bacterial persistence. By enabling the long-term observation and precise isolation of rare persister cells from within a larger population, these technologies move research beyond population averages. The detailed protocols for single-cell isolation and analysis outlined here provide a roadmap for investigating the mechanisms of phenotypic heterogeneity and antibiotic tolerance. As these tools continue to evolve and become more accessible, they hold the promise of uncovering novel therapeutic targets to eliminate persister cells, thereby addressing a critical challenge in the management of chronic and recurrent bacterial infections.

The study of bacterial persisters—dormant phenotypic variants responsible for chronic infections and antibiotic treatment failure—is fundamentally constrained by the challenge of obtaining these cells in pure form. Their transient, non-hereditable nature and low frequency within isogenic populations make them notoriously difficult to isolate for mechanistic studies or drug screening [3] [5]. This protocol provides a validated, step-by-step workflow for enriching and isolating Staphylococcus aureus persisters, a major human pathogen. The methodologies presented herein are designed to facilitate a deeper mechanistic understanding of persistence and support the development of novel therapeutic strategies to combat recalcitrant infections [3]. The foundational principle of this isolation is the selective antibiotic killing of regular, susceptible cells, followed by the collection of the intact, tolerant persister subpopulation.

The following diagram outlines the core logical pathway and central biochemical mechanism underpinning persister formation and isolation in S. aureus. Critically, this process is independent of canonical toxin-antitoxin (TA) modules, which is a key distinction from persistence mechanisms in other bacteria like E. coli [43].

Step-by-Step Protocol

Step 1: Generation of S. aureus Persister Cells

The initial phase focuses on generating a culture enriched for persister cells via a extended stationary-phase incubation [44].

Materials

• Bacterial Strain: Staphylococcus aureus (e.g., strain MW2 or other relevant clinical isolates).
• Growth Medium: Tryptic Soy Broth (TSB).
• Equipment: 250 mL Erlenmeyer flasks, incubator with shaking capability, centrifuge, phosphate-buffered saline (PBS).

Procedure

• Inoculation: Dilute an overnight culture of S. aureus 1:10,000 into 25 mL of fresh, pre-warmed TSB within a 250 mL flask [44].
• Incubation: Incubate the culture at 37°C with shaking at 250 rpm for 24 hours. This extended incubation is critical for pushing the majority of the population into a stationary-phase state, thereby increasing the frequency of persisters [43] [44].
• Harvesting: After 24 hours, pellet the bacterial cells by centrifugation (e.g., 4,000 x g for 10 minutes).
• Washing: Carefully decant the supernatant and resuspend the cell pellet in sterile PBS. Repeat this wash step three times to ensure complete removal of the growth medium and metabolic waste [44].
• Final Resuspension: Resuspend the final, washed cell pellet in PBS to a standardized concentration of approximately 10^8 Colony Forming Units (CFU)/mL. The culture is now ready for antibiotic selection.

Step 2: Antibiotic Selection for Persister Enrichment

This step uses a high concentration of a bactericidal antibiotic to eliminate growing and susceptible cells, thereby selectively enriching for the tolerant persister subpopulation.

Materials

• Antibiotics: Ciprofloxacin, Vancomycin, or Oxacillin are commonly used and effective [43].
• Preparation: Prepare a stock solution of the chosen antibiotic in an appropriate solvent (e.g., ciprofloxacin in 0.1 N HCl) and then dilute it in PBS to the final working concentration [44].

Procedure

• Challenge: Add the antibiotic to the washed cell suspension from Step 1.4 to achieve a final concentration of 10x the Minimum Inhibitory Concentration (MIC) [43]. Note: The required MIC should be pre-determined for your specific strain.
• Incubation: Incubate the antibiotic-cell mixture for a defined period, typically 3 to 4 hours at 37°C [45] [44]. This duration is sufficient to achieve a biphasic kill curve, where the initial rapid killing of susceptible cells is followed by a plateau representing the persister population.
• Confirmation of Killing: The efficacy of the killing phase can be confirmed by plating an aliquot of the culture on a non-selective agar plate (e.g., TSB agar) before and after antibiotic exposure. A reduction of 2-3 logs in CFU is typically observed before the curve plateaus [45].

Step 3: Harvesting and Washing of Persister Cells

After antibiotic exposure, the surviving persister cells must be isolated from the antibiotic and cellular debris.

Procedure

• Centrifugation: Pellet the cells from the antibiotic-treated culture by centrifugation (e.g., 4,000 x g for 10 minutes).
• Washing: Carefully decant the supernatant, which contains the antibiotic and lysed cell material. Resuspend the pellet in fresh, sterile PBS. This wash step is crucial and should be repeated at least three times to ensure no residual antibiotic remains, which would interfere with downstream resuscitation or application experiments [44].
• Validation: The resulting cell population is a highly enriched preparation of S. aureus persisters. The success of the isolation can be validated by demonstrating that these cells:
  • Are non-growing during the antibiotic challenge.
  • Can resuscitate and proliferate upon removal of the antibiotic and transfer to fresh, nutrient-rich medium [45] [5].

Characterization and Validation of Isolated Persisters

Once isolated, persisters can be characterized using various techniques. The table below summarizes key quantitative findings from the literature regarding S. aureus persisters.

Table 1: Key Characteristics and Experimental Findings for S. aureus Persisters

Characteristic	Experimental Finding	Implication/Mechanistic Insight	Source
Frequency in Population	Bright cells (stationary markers) showed 100-1000x more survivors after ciprofloxacin.	Confirms enrichment and identifies a pre-existing, marker-positive subpopulation as persisters.	[43]
Morphology & Proteome	Distinct morphology and proteome/metabolome for vancomycin vs. enrofloxacin persisters.	Suggests antibiotic-specific persistence mechanisms; necessitates tailored study approaches.	[3]
Metabolic State	Persister formation linked to a stochastic drop in intracellular ATP.	ATP level is predictive of antibiotic efficacy; low energy state underlies tolerance.	[43]
Resuscitation Kinetics	Persisters resuscitate within 1 hour in fresh media, with a doubling time equal to normal cells (~23-24 min).	Indicates a rapid exit from dormancy and a return to full metabolic activity upon stress removal.	[45]

Analytical Methods for Validation

• Flow Cytometry & Cell Sorting (FACS): Using fluorescent reporters of stationary phase (e.g., Pcap5A-gfp or ParcA-gfp), cells expressing these markers can be sorted and shown to be significantly enriched for persisters (100-1000 fold) post-antibiotic treatment [43].
• ATP Assays: Direct measurement of intracellular ATP levels can confirm the low-energy state of the isolated persister population compared to exponentially growing cells [43].
• Proteomic and Metabolomic Analysis: As demonstrated in foundational studies, isolated persisters can be subjected to liquid chromatography-mass spectrometry (LC-MS/MS) analysis to profile their proteome and metabolome, revealing pathways critical for persistence [3] [46].

The Scientist's Toolkit: Essential Research Reagents

The following table lists key reagents and their critical functions in the persister isolation and study workflow.

Table 2: Key Research Reagent Solutions for S. aureus Persister Isolation

Research Reagent	Function/Application in Protocol
Tryptic Soy Broth (TSB)	Standard nutrient-rich medium for growing S. aureus cultures to stationary phase for persister enrichment.
Ciprofloxacin	Bactericidal antibiotic (fluoroquinolone) used for positive selection of persisters by killing susceptible cells.
Vancomycin / Oxacillin	Alternative bactericidal antibiotics (glycopeptide / β-lactam) for persister selection, allowing study of drug-class-specific persistence.
Phosphate-Buffered Saline (PBS)	Buffer for washing cells free of media, metabolites, and antibiotics before and after the selection step.
Pcap5A-gfp / ParcA-gfp Reporter	Plasmid-based fluorescent reporters for stationary phase; used to identify and sort the persister-prone subpopulation via FACS.
Bakuchiol	A plant-derived natural product identified as effective at killing S. aureus persisters (at 8 μg/mL), useful for testing anti-persister compounds.

Application Notes and Troubleshooting

Critical Parameters for Success

• Growth Phase and Time: The 24-hour stationary phase incubation is a key driver of enrichment. Do not shorten this step.
• Antibiotic Concentration and Viability: Always use a verified, lethal dose (e.g., 10x MIC) of a bactericidal antibiotic. Confirm killing via plating and biphasic kill curves.
• Thorough Washing: Incomplete removal of antibiotics during the post-selection wash will prevent persister resuscitation and lead to false negatives in downstream assays.

Downstream Applications

The isolated persisters obtained through this protocol are suitable for a wide range of applications, including:

• Mechanistic Studies: Proteomic [3] [46] and metabolomic profiling to identify molecular drivers of persistence.
• Drug Discovery: Screening for novel compounds like Bakuchiol [44] or adjuvant molecules like KL1 [47] that can kill persisters or sensitize them to conventional antibiotics.
• Single-Cell Analysis: Integration with microfluidic platforms [48] [42] or flow cytometry [45] to study heterogeneity and resuscitation dynamics at the single-cell level.

This comprehensive protocol provides a robust foundation for isolating and studying S. aureus persisters, thereby enabling advanced research into one of the most challenging aspects of antimicrobial therapy.

Navigating Technical Hurdles: Optimizing Yield and Purity in Persister Workflows

Within the broader scope of research on enriching and isolating persister subpopulations, the precise induction of the persister state represents a critical first step. Persister cells—non-growing or slow-growing cells that transiently survive lethal stressors—are a major contributor to chronic infections and therapy relapse in both bacteriology and oncology [9] [49]. Their induction is highly dependent on the specific stress conditions applied. This document provides detailed application notes and protocols for titrating stressor concentration and exposure time to maximize the efficiency of generating persister populations for downstream isolation and analysis. The principles outlined are applicable to bacterial persisters and cancerous Drug-Tolerant Persisters (DTPs), enabling researchers to systematically optimize this crucial initial phase of persister research.

Background and Significance

The Persister Phenotype and the Need for Precise Induction

The persister phenotype is characterized by transient, non-genetic tolerance to stressors like antibiotics and targeted therapies. Unlike resistant cells, persisters do not possess genetic mutations conferring tolerance; instead, they survive via reversible phenotypic adaptations, including metabolic quiescence, epigenetic remodeling, and transcriptional plasticity [2] [9]. A key challenge in their study is their low frequency and transient nature in naive populations. Therefore, reliable methods to induce this state in a reproducible, controlled manner are essential. Induction is not a simple on/off switch but a dynamic equilibrium between sensitive cells transitioning into and out of the persister state, a process that can be actively influenced by the stressor itself [50] [51].

Drug-Induced Plasticity and the Induction Trade-Off

Mounting evidence indicates that the stressors used to kill sensitive cells can simultaneously induce the persister state in a subset of the population—a phenomenon termed "drug-induced plasticity" [50] [49]. In cancer, targeted therapies can accelerate the adoption of a drug-tolerant state, thereby confounding traditional high-dose treatment strategies [50]. Similarly, in bacteria, antibiotic presence can influence switching rates between susceptible and persister states [52]. This creates a fundamental trade-off: while higher stressor concentrations maximize the killing of sensitive cells, they may also disproportionately increase the induction rate into the persister state or select for different types of persisters. Consequently, titrating concentration and exposure time is not merely about survival; it is about actively steering the population toward a desired equilibrium composition of sensitive and tolerant cells [50].

The following tables consolidate key quantitative findings from recent literature to guide the design of induction experiments. These parameters serve as a starting point for protocol optimization.

Table 1: Quantitative Parameters for Inducing Bacterial Persisters

Bacterial Species	Stressor	Effective Concentration	Exposure Time	Reported Persister Fraction	Key Findings
Staphylococcus aureus [53]	Vancomycin	Not Specified	24 hours	~0.1%	Generated stable persister population for isolation; proteomics revealed distinct response.
Staphylococcus aureus [53]	Enrofloxacin	Not Specified	24 hours	~0.1%	Generated stable persister population for isolation; proteomics distinct from vancomycin persisters.
E. coli (Environmental Isolates) [22]	Ciprofloxacin, Ampicillin, Nalidixic Acid	Not Specified	Varies (Model-based)	Highly variable between strains and drugs	Persister fractions were uncorrelated across antibiotics, even with similar modes of action.
E. coli (Model) [52]	Periodic Antibiotic Dosing	Tuned to dynamics	Periodic	N/A	Optimized periodic dosing reduced total antibiotic dose required for treatment by nearly 77%.

Table 2: Quantitative Parameters for Inducing Cancer Cell DTPs

Cancer Cell Model	Stressor (Therapy)	Experimental Context	Key Dynamic Parameters	Reported Persister Fraction/Behavior
Colorectal Cancer (DiFi, WiDr) [51]	Cetuximab (anti-EGFR) / BRAF Inhibitor	In vitro targeted therapy	Transition Rate (λ): Drug-induced.Death Rate (D): Drug-dependent.Persister Death Rate (Dp): >0, slow.	Biphasic killing curve observed. A fraction (0.2%-2.5%) of persisters slowly replicates during treatment.
NSCLC, Melanoma, etc. [50]	Targeted Therapies (e.g., EGFRi)	Mathematical modeling of dosing	Net Growth Rate (σ(c)): Function of dose.Equilibrium Composition (̄f₀(c)): Function of dose.	Optimal dosing strategy balances cell kill and tolerance induction, aiming for a fixed equilibrium composition.
Various Cancers [49]	TKIs, Immunotherapy	Review of DTP biology	Pre-existing and drug-induced subpopulations.	DTPs survive initial treatment, drive MRD, and can lead to relapse. Frequency can be low (e.g., ~0.3% initially).

Detailed Experimental Protocols

Protocol 1: Induction of Bacterial Persisters via Antibiotic Exposure

This protocol is adapted from methods used to isolate S. aureus persisters [53] and principles from optimization studies [52].

I. Materials

• Growth Medium: Appropriate rich broth (e.g., Tryptic Soy Broth for S. aureus).
• Antibiotic Stock Solutions: Prepare high-concentration stocks of the target antibiotic (e.g., Vancomycin, Enrofloxacin) in sterile water or appropriate solvent. Filter sterilize.
• Phosphate Buffered Saline (PBS), sterile.
• Culture Vessels: Erlenmeyer flasks or tissue culture tubes suitable for aerobic or anaerobic growth as required.
• Orbital Shaker Incubator.

II. Procedure

• Starter Culture:
  • Inoculate a single bacterial colony into 5-10 mL of growth medium.
  • Incubate overnight (12-16 hours) at the optimal temperature with shaking.

• Experimental Culture:

  • Dilute the overnight culture 1:100 into fresh, pre-warmed medium in a new flask.
  • Grow until the culture reaches the mid-exponential phase (OD₆₀₀ ~0.5). This ensures a uniform, actively growing population.

• Stressor Exposure and Titration:

  • Divide the Culture: Aseptically aliquot the exponential-phase culture into multiple separate vessels.
  • Apply Stressor:
    • To different aliquots, add the antibiotic stock solution to achieve a range of final concentrations. A typical range might be 1x, 10x, and 100x the Minimum Inhibitory Concentration (MIC).
    • Include a negative control (no antibiotic) to monitor normal growth.
  • Incubate: Return all cultures to the incubator for the defined exposure period. A common duration is 24 hours [53], but shorter (5-6h) and longer timepoints should be tested in a pilot experiment.
  • Sample for Viability: At predetermined timepoints (e.g., 0h, 3h, 6h, 24h), remove 1 mL aliquots from each treatment.

• Viability Assessment and Persister Enumeration:

  • Wash: Pellet the cells by centrifugation (e.g., 10,000 x g for 2 minutes). Carefully remove the supernatant and resuspend the pellet in an equal volume of sterile PBS to remove the antibiotic. Repeat this wash step twice.
  • Plate for CFU: Perform serial 10-fold dilutions of the washed cell suspension in PBS. Spot-plate or spread-plate appropriate dilutions onto antibiotic-free agar plates.
  • Incubate and Count: Incubate the plates until colonies form. Count the colonies to determine the number of Colony Forming Units (CFU) per mL remaining viable after antibiotic exposure.
  • The subpopulation that survives high-dose, prolonged antibiotic exposure but remains genetically susceptible is defined as the persister fraction.

III. Optimization Notes

• The optimal concentration and time are strain- and antibiotic-specific. The goal is to observe a biphasic killing curve: a rapid initial drop in viability (sensitive cells dying) followed by a stable, slower decline (persister population) [51].
• For some applications, periodic dosing rather than continuous exposure may be more effective for enrichment and can significantly reduce the total antibiotic used [52].

Protocol 2: Induction of Cancer DTPs via Targeted Therapy

This protocol is informed by work on colorectal cancer persisters [51] and reviews of DTP biology [2] [49].

I. Materials

• Cancer Cell Line: (e.g., DiFi or WiDr for CRC studies [51]).
• Cell Culture Medium: Appropriate medium (e.g., DMEM, RPMI) supplemented with serum and antibiotics.
• Drug Stock Solutions: Prepare high-concentration stocks of the targeted therapy (e.g., Cetuximab, Erlotinib, BRAF inhibitors) in sterile DMSO or PBS as required. Aliquot and store at -20°C.
• Trypsin-EDTA solution.
• PBS, sterile.
• Tissue Culture Flasks/Plates.
• Hemocytometer or Automated Cell Counter.

II. Procedure

• Cell Culture and Seeding:
  • Maintain cells in standard culture conditions. Ensure they are in the exponential growth phase at the start of the experiment.
  • Harvest and Count: Gently trypsinize, neutralize with medium, and centrifuge the cells. Resuspend in fresh medium and perform a cell count.
  • Seed Cells: Plate cells at a density optimized for the cell line and treatment duration (e.g., 50,000 - 100,000 cells/cm²). Ensure even distribution.

• Stressor Exposure and Titration:

  • Allow Attachment: Incubate seeded plates for 12-24 hours to allow cells to adhere and resume growth.
  • Apply Stressor:
    • Prepare a dilution series of the targeted therapy drug in fresh, pre-warmed medium. The range should span from sub-lethal to clinically relevant high doses.
    • Carefully remove the medium from the cells and replace it with the drug-containing medium.
    • Include a vehicle control (e.g., 0.1% DMSO) and a no-treatment control.
  • Incubate: Return cells to the incubator for the treatment period. This can range from 3 days to several weeks, with medium and drug replenished every 2-3 days.

• Monitoring and Viability Assessment:

  • Monitor Morphology: Observe cells daily using a phase-contrast microscope. DTPs often exhibit an elongated, mesenchymal-like morphology [49].
  • Quantify Viability:
    • At designated timepoints (e.g., day 3, 5, 7, 14), wash the cells with PBS and trypsinize to create a single-cell suspension.
    • Perform a cell count using a trypan blue exclusion assay or a similar viability stain to determine the number of live cells remaining.
    • The persister population is represented by the stable, low-level plateau of viable cells after the initial rapid decrease.

III. Optimization Notes

• The transition to the DTP state can be highly dynamic. Mathematical modeling suggests the optimal dosing strategy may be constant low-dose or intermittent high-dose, depending on whether the drug induces transitions into the persister state [50].
• Confirmation of the DTP state can involve assays for slow replication (e.g., CFSE staining, EdU incorporation [51]) and reversibility (i.e., regrowth upon drug withdrawal).

Signaling Pathways and Molecular Mechanisms of Induction

The induction of the persister state is governed by integrated intracellular stress responses. The following diagrams illustrate key pathways and the experimental workflow.

Key Signaling Pathways in Persister Induction

Diagram 1: Integrated stress response pathways in persister induction. Exposure to therapeutic stressors triggers a multi-faceted response. Intracellularly, this involves epigenetic, transcriptional, and metabolic reprogramming. In bacteria, toxin-antitoxin modules are key. Extracellularly, signals from the tumor microenvironment (TME) or external conditions reinforce the transition to the drug-tolerant persister phenotype [2] [9] [49].

Experimental Workflow for Induction Titration

Diagram 2: Workflow for titrating stressor parameters. A systematic approach involves synchronizing the starting population, applying a matrix of stressor concentrations and exposure times, and analyzing time-course viability data to identify conditions that generate a stable, analyzable persister fraction for downstream isolation [51] [53].

The Scientist's Toolkit: Essential Reagents and Materials

Table 3: Key Research Reagent Solutions for Persister Induction Studies

Reagent/Material	Function in Induction Protocol	Specific Examples & Notes
High-Purity Stressors	To apply a defined, selective pressure for persister enrichment.	Antibiotics: Vancomycin, Enrofloxacin, Ciprofloxacin [53].Targeted Therapies: Cetuximab (anti-EGFR), BRAF/MEK inhibitors, Erlotinib [51] [49].
Cell Line/Strain	The biological model system for studying persistence.	Bacteria: S. aureus, E. coli environmental isolates show variability [22] [53].Cancer: DiFi (CRC), PC9 (NSCLC), other cell lines with defined oncogenes [51] [49].
Defined Culture Media	To support robust growth prior to stress and maintain cells during extended exposure.	Tryptic Soy Broth (bacteria), RPMI/DMEM with serum (mammalian cells).
Viability Stains	To distinguish and quantify live, dead, and dormant cells.	Trypan Blue: Basic live/dead exclusion for mammalian cells.CFDA & Propidium Iodide (PI): Flow cytometry-based viability and metabolic activity staining for bacteria and mammalian cells [53].
Metabolic/Labeling Dyes	To track cell division and metabolic activity, confirming slow-cycling phenotype.	CFSE: Tracks dilution of fluorescence with each cell division [51].EdU: Click-chemistry detection of DNA synthesis in replicating cells [51].

Mitigating Contamination from Viable but Non-Culturable (VBNC) and Dead Cells

The viable but non-culturable (VBNC) state is a dormant survival strategy employed by many bacteria in response to environmental stress [54] [55]. Cells in this state are characterized by a loss of culturability on standard laboratory media that normally support their growth, while maintaining viability, metabolic activity, and the potential for resuscitation under favorable conditions [54] [56] [55]. This state poses significant challenges for researchers aiming to enrich and isolate persister subpopulations, as VBNC cells can contaminate preparations and lead to inaccurate conclusions about cellular viability and function. Distinguishing VBNC cells from dead cells and culturable persisters is therefore essential for rigorous research into bacterial persistence mechanisms [9] [57].

The VBNC state can be induced by a wide range of stressors commonly encountered in laboratory and natural environments, including nutrient starvation, extreme temperatures, osmotic pressure, oxidative stress, and exposure to antibiotics or disinfectants [54] [58] [55]. Over one hundred bacterial species are known to enter this state, including major pathogens such as Mycobacterium tuberculosis, Vibrio cholerae, Listeria monocytogenes, and Escherichia coli [56] [55]. Within the context of persister research, it is crucial to recognize that VBNC cells represent a distinct physiological state from both antibiotic-tolerant persisters and dead cells, each requiring specific detection and mitigation strategies [9] [57].

Distinguishing VBNC Cells from Persisters and Dead Cells

Accurate differentiation between VBNC cells, bacterial persisters, and dead cells is a fundamental prerequisite for successful persister enrichment protocols. Table 1 summarizes the key characteristics of these cell states, highlighting critical diagnostic features.

Table 1: Comparative Characteristics of VBNC Cells, Persister Cells, and Dead Cells

Feature	VBNC Cells	Persister Cells	Dead Cells
Culturability	Non-culturable on standard media	Culturable after antibiotic removal	Non-culturable
Metabolic Activity	Low but detectable	Very low to negligible	Absent
Membrane Integrity	Intact	Intact	Compromised
Resuscitation	Possible under specific conditions	Upon antibiotic removal	Not possible
Genetic Basis	Physiological response, typically no mutation	Phenotypic variant, no mutation	N/A
Response to Antibiotics	Tolerant (dormancy-mediated)	Tolerant (dormancy-mediated)	No response

The relationship between these states can be visualized as a continuum of metabolic activity and resuscitability. Active cells under stress can transition into persisters, which may further develop into VBNC cells under prolonged stress [57]. This continuum underscores the importance of using multiple complementary detection methods to accurately characterize bacterial subpopulations in experimental samples.

Detection and Quantification Methods

Relying on culturability alone significantly underestimates viable cell populations due to the presence of VBNC cells. A combination of direct viability assessment methods is therefore essential. Table 2 outlines the primary techniques used for detecting and quantifying VBNC cells in research settings.

Table 2: Analytical Methods for VBNC Cell Detection and Quantification

Method Category	Specific Technique	Principle	Key Advantages	Key Limitations
Molecular Methods	Reverse Transcription PCR (RT-PCR)	Detects short-lived mRNA molecules	High sensitivity, specific for viable cells	Does not confirm protein synthesis or cellular integrity
	Propidium Monoazide (PMA) qPCR	DNA-binding dye penetrates only compromised membranes, inhibiting PCR	Distinguishes intact from compromised cells	May not detect all VBNC cells with slight membrane alterations
Viability Staining	Live/Dead Staining (e.g., SYTO9/PI)	Dual staining based on membrane integrity	Rapid, allows microscopic enumeration	Can overestimate viability in some environmental conditions
	Fluorescent Diacetate (FDA) / CTC Staining	Detects esterase activity / respiratory activity	Confirms metabolic activity	Some VBNC cells may have very low metabolic rates
Advanced Techniques	Flow Cytometry / FACS	Multi-parameter analysis of stained cells	High-throughput, quantitative, enables cell sorting	Requires expensive instrumentation, expert interpretation
	Proteomic Analysis	Identifies protein expression profiles	Provides mechanistic insights into VBNC state	Complex sample preparation, data analysis

The following workflow diagram illustrates a recommended integrated approach for detecting and isolating VBNC cells in experimental samples:

Detailed Protocol: Flow Cytometry with Viability Staining for VBNC Cell Isolation

This protocol enables the differentiation and isolation of VBNC cells from mixed bacterial populations using fluorescence-activated cell sorting (FACS).

Materials:

• Bacterial culture in late exponential or stationary phase
• Appropriate stressor (e.g., antibiotic of interest)
• Phosphate-buffered saline (PBS)
• BacLight Live/Dead viability kit (SYTO9 and propidium iodide) or alternative viability stains (e.g., CTC for respiratory activity)
• Flow cytometer with cell sorter capability
• Appropriate culture media for viability confirmation

Procedure:

• Stress Induction: Expose bacterial culture to a predetermined stressor (e.g., 10× MIC of antibiotic for 24 hours) to induce VBNC state formation.
• Cell Harvesting: Centrifuge 1 mL of culture at 5,000 × g for 10 minutes. Wash cell pellet twice with sterile PBS to remove residual antibiotics or stressors.
• Viability Staining:
  • Resuspend cell pellet in 1 mL PBS.
  • Add SYTO9 (final concentration 5 µM) and propidium iodide (final concentration 30 µM).
  • Incubate in darkness for 15-30 minutes at room temperature.
• Flow Cytometry Analysis and Sorting:
  • Analyze stained cells using flow cytometer with 488 nm excitation.
  • Detect SYTO9 fluorescence at 500-550 nm (green channel).
  • Detect propidium iodide fluorescence at 600-650 nm (red channel).
  • Establish sorting gates based on the following populations:
    • Gate P1: SYTO9+/PI- (membrane-intact, potentially VBNC)
    • Gate P2: SYTO9+/PI+ (membrane-compromised, dead/dying)
  • Sort P1 population into sterile collection tubes containing resuscitation medium.
• Culturability Assessment:
  • Plate sorted P1 cells onto appropriate culture media.
  • Incubate under optimal growth conditions for 48-72 hours.
  • Compare colony counts with flow cytometry counts to determine percentage of non-culturable cells.
• VBNC Confirmation:
  • Cells that are SYTO9+/PI- (membrane-intact) but non-culturable are classified as VBNC.
  • Confirm with additional metabolic activity assays (e.g., CTC reduction) or molecular methods (e.g., RT-PCR for stress response genes).

Notes:

• Include appropriate controls (untreated cultures, heat-killed cells) for gating optimization.
• For enhanced resolution, combine membrane integrity staining with metabolic indicators like CTC (5-cyano-2,3-ditolyl tetrazolium chloride) to detect respiratory activity [54].
• Sorted VBNC cells can be used for downstream applications including transcriptomic, proteomic, or resuscitation studies.

Mitigation Strategies for VBNC Contamination in Persister Research

Effective mitigation of VBNC cell contamination requires strategic approaches throughout the experimental workflow. The following diagram illustrates the key pathways through which VBNC cells can interfere with persister research and potential intervention points:

Strategic Interventions

• Optimized Stress Induction Protocols

  • Carefully titrate antibiotic concentrations and exposure times to minimize VBNC induction while effectively selecting for persisters.
  • Consider combination approaches that utilize membrane-active compounds (e.g., SPR741, a polymyxin B derivative) to enhance antibiotic penetration without inducing dormancy [15].
  • Implement gradual stress application rather than acute high-dose exposure where experimentally feasible.

• Selective Elimination of VBNC Cells

  • Metabolic Activation: Utilize specific metabolites to resuscitate VBNC cells before antibiotic treatment. For example, lactate supplementation has been shown to aid resuscitation of VBNC Vibrio parahaemolyticus and extend the resuscitation window [59].
  • Membrane-Targeting Agents: Incorporate membrane-disrupting compounds such as thymol triphenylphosphine conjugates (TPP-Thy3) or synthetic cation transporters (SA-558) that target the intact membranes of VBNC cells [15].
  • Enzyme Targeting: Exploit metabolic vulnerabilities; for instance, targeting lactate dehydrogenase (LldD) in VBNC cells affects their ability to maintain the dormant state and can prevent VBNC formation [59].

• Validation of Persister Enrichment

  • Always combine culturability assays with direct viability assessment methods (as outlined in Section 3) to confirm that cells classified as persisters are truly culturable after stress removal.
  • Include VBNC-specific markers in analytical workflows, such as detection of stress response proteins (RpoS, OxyR) or unique membrane protein profiles [55] [59].
  • Perform periodic resuscitation attempts on supposedly "dead" cell fractions to check for VBNC cell presence.

The Scientist's Toolkit: Essential Reagents for VBNC Research

Table 3: Key Research Reagent Solutions for VBNC Cell Studies

Reagent/Category	Specific Examples	Function/Application	Key Considerations
Viability Stains	SYTO9/Propidium Iodide (BacLight Kit)	Membrane integrity assessment	Industry standard, works for most bacterial species
	CTC (5-cyano-2,3-ditolyl tetrazolium chloride)	Respiratory activity detection	Confirms metabolic activity in non-culturable cells
Molecular Assays	PMA (Propidium Monoazide) dye	Selective DNA amplification from intact cells	Requires optimization for different cell types
	RT-PCR reagents	Detection of gene expression in viable cells	Targets short-lived mRNA for viability confirmation
Membrane-Targeting Compounds	SA-558 (synthetic cation transporter)	Disrupts membrane potential in dormant cells	Effective against persisters and VBNC cells
	XF-70, XF-73	Porphyrin-based membrane disruptors	Light-activated for enhanced efficacy
Metabolic Modulators	Sodium lactate	Promotes resuscitation of VBNC cells	Concentration-dependent effect, requires optimization
	CSE inhibitors (for H₂S biogenesis)	Reduces persister and VBNC formation	Particularly effective in S. aureus and P. aeruginosa
Enzyme Targets	Pyrazinamide (for M. tuberculosis)	Disrupts membrane energetics via PanD targeting	Species-specific effectiveness
	ADEP4	Activates ClpP protease for protein degradation	Causes self-digestion in dormant cells

The presence of VBNC cells in bacterial populations represents a significant challenge in persister research, potentially compromising experimental outcomes and leading to misinterpretation of results. Successful mitigation requires a multifaceted approach that combines optimized stress induction protocols, rigorous detection methodologies employing multiple complementary techniques, and strategic interventions targeting VBNC-specific vulnerabilities. By implementing the protocols and strategies outlined in this application note, researchers can significantly improve the accuracy of persister enrichment and characterization, advancing our understanding of bacterial persistence mechanisms and supporting the development of novel therapeutic approaches against chronic and recurrent bacterial infections. Future directions should focus on developing more specific markers for distinguishing VBNC cells from other dormant states and creating standardized protocols for their detection and elimination across different bacterial species.

Drug-tolerant persister (DTP) cells represent a transient, non-genetic cellular state enabling survival under therapeutic stress, contributing to minimal residual disease and eventual tumor relapse [1] [60]. A core challenge in DTP research lies in accurately distinguishing these cells from other resistant or quiescent populations. This protocol details methods for the critical validation of two defining DTP characteristics: a dormant, slow-cycling phenotype during treatment and the reversibility of this state upon drug withdrawal [9] [60]. Proper confirmation of these traits is essential for any study focused on enriching or isolating DTP subpopulations, ensuring that the observed biology truly reflects the persister phenotype and not pre-existing genetic resistance or transient cytostasis.

Experimental Protocols for Validating Dormancy

A multi-faceted approach is required to confirm that putative DTPs have entered a dormant, slow-cycling state. Relying on a single metric is insufficient; proliferation, metabolic, and molecular markers must be assessed concurrently.

Protocol: Direct Proliferation Monitoring via Live-Cell Imaging

This protocol quantifies the replication dynamics of persister cells under continuous drug pressure, providing direct visual evidence of dormancy.

• Principle: Track individual cells over time to directly observe and quantify division and death events, confirming that a subpopulation remains viable but non-dividing [51].
• Procedure:
  • Cell Seeding: Seed cancer cells (e.g., DiFi or WiDr colorectal cancer clones) in multi-well glass-bottom plates at low density (e.g., 1,000-5,000 cells/cm²) to enable tracking of single cells.
  • Drug Treatment: After cell adhesion, introduce the targeted therapeutic agent at a clinically relevant concentration (e.g., cetuximab for DiFi cells). Include a vehicle-only control.
  • Image Acquisition: Place the plate in a live-cell imaging system maintained at 37°C and 5% CO₂. Acquire phase-contrast images from 10-20 non-overlapping fields per well every 30-60 minutes for a minimum of 72-96 hours.
  • Data Analysis: Use cell tracking software to generate lineage traces. Quantify the percentage of cells that do not divide over the entire imaging period, the time between divisions for those that do divide, and the frequency of cell death events [51].

Protocol: Metabolic Profiling of DTPs

This protocol assesses the metabolic shift toward quiescence, a hallmark of the DTP state.

• Principle: DTPs often undergo metabolic reprogramming, such as reduced glucose uptake and a shift toward mitochondrial oxidative phosphorylation (OXPHOS) [60].
• Procedure:
  • DTP Enrichment: Treat a large culture of cancer cells with a therapeutic agent for 5-7 days to enrich for persisters. Include a vehicle-treated control.
  • Staining: Harvest cells and stain with a fluorescent dye sensitive to metabolic activity (e.g., a tetrazolium salt-based dye for metabolic activity or a potentiometric dye for mitochondrial membrane potential).
  • Flow Cytometry: Analyze stained cells via flow cytometry. The DTP population is expected to show a distinct shift toward lower fluorescence intensity, indicating reduced metabolic activity compared to the vehicle-treated, proliferating population.
  • Seahorse Analysis: For a more detailed profile, isolate viable DTPs and analyze their bioenergetic profile using a Seahorse XF Analyzer to measure extracellular acidification rate (ECAR, a proxy for glycolysis) and oxygen consumption rate (OCR, a proxy for OXPHOS).

Experimental Protocols for Confirming Reversibility

The reversible nature of the DTP state is its cardinal feature. The following protocols test the ability of drug-surviving cells to re-enter the cell cycle and re-sensitize upon drug removal.

Protocol: Drug Withdrawal and Regrowth Assay

This is the fundamental test for DTP reversibility, confirming that survival is not due to genetic resistance.

• Principle: Upon removal of the drug pressure, reversible DTPs will resume proliferation and re-establish a population that remains sensitive to the original therapy [60].
• Procedure:
  • DTP Generation & Confirmation: Generate and validate a dormant DTP population using the methods above.
  • Drug Washout: Thoroughly wash the drug-treated cells and the vehicle-control cells to remove all traces of the therapeutic agent. Re-seed the cells into fresh drug-free medium at equal densities.
  • Regrowth Kinetics: Monitor cell number for 7-14 days using a live-cell imager or by performing daily cell counts. Compare the regrowth kinetics of the post-DTP population to the vehicle-treated control.
  • Re-challenge: Once the post-DTP population has regained logarithmic growth, re-treat it with the original drug. Perform a dose-response assay (e.g., CellTiter-Glo viability assay after 72 hours) to confirm that the cells have re-acquired sensitivity, with an IC50 comparable to the parental, drug-naïve cell line.

Protocol: Molecular Profiling of Epigenetic and Transcriptional States

This protocol validates reversibility at the molecular level by tracking dynamic changes in key regulators.

• Principle: The DTP state is maintained by reversible epigenetic and transcriptional adaptations, such as histone modifications and signaling pathway activation, which should reset upon drug withdrawal [1] [60].
• Procedure:
  • Sample Collection: Collect cell pellets at three critical time points:
    • T1: Vehicle-treated, proliferating cells.
    • T2: DTPs (after 5-7 days of drug exposure).
    • T3: Reverted cells (after 5-7 days of growth in drug-free media post-washout).
  • Western Blot Analysis: Probe lysates from each time point for key proteins associated with the DTP state. Expected results are summarized in Table 1 below.
  • RNA Sequencing: Perform transcriptomic analysis on samples from T1, T2, and T3. Expect to see upregulation of survival pathways (e.g., AXL, YAP/TEAD) in T2 that returns to baseline in T3 [60].

Table 1: Expected Molecular Marker Dynamics in a Reversible DTP Model

Target	Function	Expected Level in DTPs (T2)	Expected Level in Reverted Cells (T3)
KDM5A	Histone demethylase; represses differentiation genes [60]	Up	Returns to baseline
AXL	Receptor tyrosine kinase; promotes survival and EMT [60]	Up	Returns to baseline
Phospho-YAP	Transcriptional co-activator in Hippo pathway [60]	Up	Returns to baseline
Ki-67	Proliferation marker [1]	Down	Returns to baseline
p21	Cell cycle regulator; marker of stress/arrest [1]	Variable/Context-dependent	Returns to baseline

The following table consolidates key quantitative benchmarks from foundational studies, providing a reference for expected outcomes when validating DTP populations.

Table 2: Key Quantitative Parameters from DTP Studies

Parameter	Experimental System	Reported Value / Observation	Source
Replicating DTP Fraction	Colorectal Cancer (DiFi, WiDr)	0.2% to 2.5% of persisters showed slow replication during treatment [51]	Nature Genetics (2022)
Mutation Rate Increase	Colorectal Cancer (DiFi, WiDr)	7- to 50-fold increase in mutation rate during drug treatment [51]	Nature Genetics (2022)
Phenotype Origin	Colorectal Cancer (DiFi, WiDr)	Persister phenotype is predominantly drug-induced, not pre-existing [51]	Nature Genetics (2022)
Key Epigenetic Regulator	EGFR-mutant NSCLC	KDM5A essential for establishing reversible drug tolerance [60]	Frontiers in Pharmacology (2025)
Metabolic Shift	Various Cancers	Shift from glycolysis to Oxidative Phosphorylation (OXPHOS) [60]	Frontiers in Pharmacology (2025)

The Scientist's Toolkit: Essential Research Reagents

Table 3: Key Reagents for DTP Enrichment and Validation

Reagent / Material	Function in DTP Research	Example
Cell Tracker Dyes	Track cell division history and proliferation status via dye dilution in flow cytometry.	Carboxyfluorescein succinimidyl ester (CFSE) [51]
Nucleotide Analogs	Label cells actively synthesizing DNA; used to identify quiescent (label-negative) populations.	5-Ethynyl-2'-deoxyuridine (EdU) [51]
HDAC Inhibitors	Tool compounds to target epigenetic mechanisms of persistence and test combination strategies.	Entinostat [60]
Metabolic Inhibitors	Target the Oxidative Phosphorylation (OXPHOS) pathway to exploit metabolic vulnerabilities of DTPs.	IACS-010759 [60]
Patient-Derived Models	Study DTPs in a more physiologically relevant context that captures tumor heterogeneity.	Patient-Derived Organoids (PDOs) [1]
Live-Cell Imaging System	Essential for direct, long-term observation of cell division and death dynamics in DTP populations.	Incucyte or similar systems [51]

Experimental Workflow and Signaling Pathways

The following diagram illustrates the core experimental workflow for validating dormancy and reversibility, from initial treatment to final confirmation.

Experimental Workflow for DTP Validation

The signaling pathways that govern entry into and exit from the DTP state are complex and interconnected. The diagram below summarizes the key molecular players and their relationships.

Key Molecular Pathways in DTP State Transition

The systematic study of bacterial persisters through omics technologies (transcriptomics, proteomics, metabolomics) is fundamentally constrained by a critical biomass bottleneck. Persister cells are defined as a small subpopulation of genetically susceptible bacteria that enter a transient, non-growing or slow-growing state, enabling them to survive high-dose antibiotic treatment and subsequently regrow after stress removal [9] [5]. This phenotypic heterogeneity means persisters typically constitute less than 1% of a total bacterial population, making them exceptionally difficult to isolate in sufficient quantities for comprehensive multi-omics analyses [5] [61]. This application note details standardized protocols for overcoming this biomass limitation, enabling reliable generation, enrichment, and isolation of persister cells at scales necessary for transcriptomic, proteomic, and metabolomic profiling.

The core challenge stems from defining characteristics of persisters: their low abundance in typical cultures, their transient phenotype which reverts upon antibiotic removal, and their metabolic dormancy which complicates selective cultivation [5]. Without robust methods to overcome these limitations, omics studies risk analyzing contaminated samples or insufficient material, yielding unreliable data. The protocols outlined below provide standardized approaches for generating sufficient high-purity persister biomass for downstream omics applications.

Persister Enrichment Strategies: From Culture to Concentrated Biomass

Successful persister omics studies begin with strategic enrichment of persister cells prior to isolation. The following table summarizes the primary enrichment methods and their applications.

Table 1: Comparison of Persister Cell Enrichment Strategies

Enrichment Method	Underlying Principle	Typical Persister Yield	Compatible Downstream Omics	Key Considerations
High-Dose Antibiotic Treatment [61]	Kills vegetative cells while leaving dormant persisters intact	Varies by antibiotic and bacterial strain: ~0.001%-10% of initial population [26] [5]	Proteomics, Metabolomics	Risk of triggering stress responses; requires careful timing
Stationary Phase Culture [26] [5]	Nutrient limitation naturally induces dormancy in subpopulation	Up to 10% survival after antibiotic treatment [26]	All major omics approaches	Population heterogeneous; includes dying cells
Biofilm Culture [9] [5]	Structured communities contain naturally higher persister proportions	Significantly higher than planktonic cultures [9]	Transcriptomics, Proteomics	Technically challenging; complex matrix disruption
Enzymatic Lysis of Vegetative Cells [61]	Selective digestion of metabolically active cells while sparing persisters	Dependent on initial population density	Proteomics, Metabolomics	Potential persister loss; optimization required

Protocol: High-Dose Antibiotic Enrichment of Persister Cells

This protocol describes the enrichment of persister cells from Staphylococcus aureus and Escherichia coli cultures using high-dose antibiotic treatment, adapted from established methodologies [3] [26] [61].

Materials and Reagents

• Bacterial strains: Staphylococcus aureus (ATCC 25923), Escherichia coli (ATCC 25922)
• Antibiotics: Vancomycin (for Gram-positive), Enrofloxacin/Ciprofloxacin (for Gram-negative), prepared as stock solutions in appropriate solvents
• Growth media: Tryptic Soy Broth (TSB) for S. aureus; Luria-Bertani (LB) broth for E. coli
• Saline solution: 0.85% NaCl, sterile
• Equipment: shaking incubator, centrifuge, spectrophotometer, sterile culture tubes/flasks

Procedure

• Inoculum Preparation:

  • From frozen glycerol stocks, streak bacteria on appropriate agar plates and incubate overnight at 37°C.
  • Pick 3-5 colonies and inoculate into 10 mL of broth medium.
  • Incubate at 37°C with shaking (200 rpm) for 16-18 hours to reach stationary phase (OD600 ≈ 1.2-1.5).

• Antibiotic Treatment:

  • Dilute overnight culture 1:100 into fresh pre-warmed medium (50 mL in 250 mL flask).
  • Incubate with shaking at 37°C until mid-log phase (OD600 ≈ 0.5-0.6).
  • Add antibiotic at 100× MIC (Minimum Inhibitory Concentration):
    • For S. aureus: Vancomycin at 12.5 μg/mL or Enrofloxacin at 6.25 μg/mL [61]
    • For E. coli: Ciprofloxacin at 20× MIC [26]
  • Continue incubation for 3-5 hours to ensure complete killing of vegetative cells.

• Persister Collection:

  • Centrifuge culture at 4,000 × g for 10 minutes at 4°C.
  • Discard supernatant and resuspend cell pellet in 10 mL of cold saline solution.
  • Repeat washing step twice to remove residual antibiotics.
  • Resuspend final pellet in 1 mL of appropriate buffer for downstream processing.

• Viability Assessment:

  • Perform serial dilutions of the persister suspension in saline.
  • Spot 10 μL aliquots onto antibiotic-free agar plates in triplicate.
  • Incubate plates at 37°C for 24-48 hours and enumerate colony-forming units (CFUs).

The typical success criterion is a biphasic killing curve, with an initial rapid decline in viability followed by a stable plateau, indicating persister survival [5] [61]. For E. coli cultures, expect survival rates of approximately 0.001% for wild-type strains after ciprofloxacin treatment [26].

Advanced Persister Isolation and Purification Techniques

Following enrichment, precise isolation of persisters from residual debris and any remaining viable vegetative cells is crucial for high-quality omics data.

Protocol: Fluorescence-Activated Cell Sorting (FACS) of Persister Cells

This protocol describes the isolation of pure Bacillus subtilis persister populations using fluorescent staining and FACS, with adaptation for other bacterial species [61].

Materials and Reagents

• Fluorescent stains: 5-(and-6)-Carboxyfluorescein Diacetate (5(6)-CFDA), Propidium Iodide (PI)
• Buffers: Phosphate-Buffered Saline (PBS), filter-sterilized
• Equipment: Flow cytometer with cell sorting capability, centrifuge, vortex mixer, water bath

Procedure

• Sample Preparation:

  • Start with antibiotic-enriched persister population (from Protocol 2.1).
  • Adjust cell density to approximately 10^6 cells/mL in PBS.

• Fluorescent Staining:

  • Prepare 5(6)-CFDA working solution at 10 μM in PBS.
  • Add 10 μL of 5(6)-CFDA solution to 1 mL of cell suspension.
  • Incubate at 37°C for 30 minutes in the dark.
  • Add Propidium iodide to a final concentration of 5 μg/mL.
  • Incubate for an additional 5 minutes at room temperature in the dark.

• Flow Cytometry Gating and Sorting:

  • Use unstained and single-stained controls to establish fluorescence compensation.
  • Set up sorting gates to select for 5(6)-CFDA-positive/PI-negative populations (indicating metabolically active persisters) [61].
  • For B. subtilis, sort using a 100 μm nozzle at low pressure (≤ 20 psi) to maintain cell viability.
  • Collect sorted cells in sterile collection tubes containing appropriate culture medium or lysis buffer.

• Post-Sorting Processing:

  • Centrifuge sorted cells at 10,000 × g for 10 minutes.
  • For transcriptomics: resuspend in RNA stabilization reagent.
  • For proteomics: resuspend in protein lysis buffer.
  • For metabolomics: flash-freeze cell pellet in liquid nitrogen.

This method successfully isolates B. subtilis persisters with demonstrated viability and purity, enabling downstream molecular analyses [61]. The sorting approach effectively distinguishes persisters from both dead cells (PI-positive) and spores (5(6)-CFDA-negative), addressing a key challenge in persister isolation.

Figure 1: Comprehensive Workflow for Persister Cell Isolation and Omics Analysis. This diagram illustrates the integrated process from initial culture to pure persister preparation for omics studies, highlighting key purification steps.

Molecular Mechanisms of Persistence: Informing Omics Study Design

Understanding the biological basis of persistence is essential for designing appropriate omics experiments and interpreting results. The following diagram and table summarize key molecular pathways involved in persister formation.

Figure 2: Molecular Pathways Governing Persister Formation and Survival. Key mechanisms include toxin-antitoxin systems, stringent response, and suppression of reactive oxygen species (ROS) [9] [26] [5].

Table 2: Key Research Reagent Solutions for Persister Studies

Reagent/Category	Specific Examples	Function in Persister Research	Application Notes
Antibiotics for Enrichment	Vancomycin, Enrofloxacin, Ciprofloxacin	Selective killing of vegetative cells while sparing persisters	Use at 10-100× MIC; validate killing kinetics for each bacterial strain [3] [61]
Viability Stains	5(6)-CFDA, Propidium Iodide, SYTOX Green	Differentiation of metabolic states and membrane integrity	5(6)-CFDA indicates esterase activity; PI indicates membrane damage [61]
Cell Sorting Tools	Fluorescence-Activated Cell Sorter (FACS)	High-purity isolation of persister subpopulations	Enables collection of specific phenotypic subsets for omics analysis [61]
Metabolic Inhibitors	Carbonyl cyanide m-chlorophenyl hydrazone (CCCP)	Dissipation of proton motive force, induces persistence	Useful for studying energy metabolism in persisters [61]
Specialized Growth Media	Chemically defined minimal media	Controlled nutrient limitation to induce persistence	Enables study of specific nutrient effects on persistence frequency

Omics-Ready Biomass Preparation and Quality Control

The final critical step involves preparing isolated persisters for specific omics applications while maintaining sample integrity.

Biomass Requirements and Sample Preparation Guidelines

Table 3: Biomass Requirements and Processing Methods for Persister Omics

Omics Approach	Minimum Persister Cells Required	Optimal Processing Method	Key Quality Control Metrics
Transcriptomics	10^5 - 10^6 cells	Direct lysis in RNA stabilization reagent	RIN > 8.0 (RNA Integrity Number)
Proteomics	10^6 - 10^7 cells	Lysis in urea/thiourea buffer with protease inhibitors	Protein yield > 5 μg, identified proteins > 2000
Phosphoproteomics	10^7 - 10^8 cells	Immediate snap-freezing, phosphoprotein enrichment	Phosphosite identification > 1000 [62]
Metabolomics	10^6 - 10^7 cells	Quenching in cold methanol, rapid extraction	Detectable metabolites > 100, CV < 30% in QCs

Protocol: Quality Assessment of Persister Preparations

• Purity Validation:

  • Plate sorted persister samples on antibiotic-free agar to confirm culturability.
  • Verify susceptibility profile matches parental strain (no resistance development).
  • For S. aureus, confirm distinct morphology of persisters compared to vegetative cells [3].

• Viability Assessment:

  • Determine CFU/mL before and after sorting.
  • Calculate resuscitation ratio: (CFU after 24h)/(CFU immediately after sorting).
  • Expected ratio should be > 0.1 for healthy persister populations.

• Molecular Quality Controls:

  • For RNA: Assess RIN using microfluidic analysis.
  • For protein: Perform Bradford assay and SDS-PAGE.
  • For metabolomics: Monitor internal standards recovery rates.

The methodologies detailed in this application note provide a standardized framework for overcoming the fundamental biomass bottleneck in persister omics research. By implementing robust enrichment strategies, leveraging advanced sorting technologies, and applying rigorous quality control measures, researchers can generate high-quality multi-omics data from these elusive bacterial subpopulations. The integration of transcriptomic, proteomic, and metabolomic datasets from purified persisters will accelerate our understanding of persistence mechanisms and inform novel therapeutic strategies against chronic and recurrent infections [9] [5]. As these technologies evolve, particularly single-cell omics approaches, the biomass requirements will continue to decrease, further enhancing our ability to study persister biology at unprecedented resolution.

Confirming Phenotype and Function: Assays for Validating Isolated Persisters

Antibiotic killing and regrowth assays represent a cornerstone methodology in the burgeoning field of bacterial persistence research. Bacterial persisters are defined as a small, genetically susceptible subpopulation of cells that enter a transient, slow-growing or non-growing state, enabling them to survive exposure to high concentrations of bactericidal antibiotics [9]. These cells are not resistant; upon antibiotic removal and subsequent regrowth, they give rise to a population that remains fully susceptible to the same drug, distinguishing them from genetically resistant mutants [9] [42]. This phenomenon is a major contributor to chronic and recurrent infections, as it leads to treatment failure and relapse, complicating infections such as tuberculosis, recurrent urinary tract infections, and biofilm-associated conditions [9] [42].

The standard Antimicrobial Susceptibility Testing (AST), which determines the Minimum Inhibitory Concentration (MIC), is insufficient for studying persisters. While AST effectively identifies resistance, it fails to resolve the heterogeneity within a bacterial population and cannot detect the small, tolerant subpopulation that constitutes persisters [42]. Killing and regrowth assays address this gap by dynamically measuring a population's response to a lethal antibiotic challenge over time, typically generating a biphasic killing curve. This curve is characterized by an initial rapid decline in viable cells, representing the death of the majority, susceptible population, followed by a plateau phase where the persister subpopulation survives despite continued antibiotic exposure [63] [9]. The subsequent regrowth phase, assessed after antibiotic removal, confirms the viability and replicative potential of these persister cells, solidifying their identification.

Principles and Significance of the Assays

Distinguishing Persistence from Resistance and Tolerance

The killing and regrowth assay is fundamental for differentiating between three critical survival phenotypes: susceptibility, resistance, and persistence/tolerance. Table 1 outlines the core distinctions. Resistance is a stable, genetic trait that raises the MIC, allowing growth in the presence of an antibiotic. In contrast, tolerance or persistence is a non-genetic, phenotypic survival where the MIC remains unchanged, but the time required to kill the population is extended [9] [42]. The key operational difference between general tolerance and persistence lies in the survival curve: tolerance often describes the survival of the bulk population with a gradual kill rate, while persistence specifically refers to a biphasic curve with a distinct, surviving subpopulation [26] [9]. The regrowth assay confirms that these survivors are true persisters—viable but non-proliferating during antibiotic exposure—and not merely a pre-existing resistant subpopulation or cells in a transient, non-culturable state [42].

Table 1: Key Characteristics of Bacterial Survival Phenotypes

Phenotype	Minimum Inhibitory Concentration (MIC)	Survival under Lethal Antibiotic Exposure	Genetic Basis	Key Feature
Susceptible	Low	Killed rapidly	No	Killed by standard treatment [9]
Resistant	Elevated	Can grow	Yes, stable	Growth in presence of antibiotic [9]
Tolerant/Persistent	Unaffected	Survives (non-growing)	No, phenotypic	Biphasic killing curve; regrowth after removal [9] [42]

Quantitative Insights from Killing Kinetics

The time-kill curve provides rich, quantitative data beyond a simple biphasic pattern. The slope of the initial killing phase reveals the rate of killing of the main population, while the plateau level indicates the frequency of persisters within the total population [9]. This frequency is highly dynamic and can be influenced by numerous factors, including the bacterial growth phase, specific antibiotic mechanism, and environmental conditions. For instance, single-cell studies have demonstrated that the pre-treatment history of a cell significantly impacts its survival probability. Cells sampled from exponential growth phase and treated with ampicillin or ciprofloxacin showed that a majority of persisters were, surprisingly, actively growing before treatment [63] [48]. Conversely, stationary phase cultures generally contain a higher frequency of persisters, particularly for cell-wall active antibiotics like ampicillin [63]. Table 2 summarizes quantitative survival data for E. coli under different conditions, illustrating how persister frequency and the killing kinetics can vary.

Table 2: Quantitative Survival of E. coli in Killing Assays under Different Conditions

Strain / Condition	Antibiotic	Killing Profile	Key Quantitative Finding	Source Context
Wild-type (Exponential)	Ciprofloxacin (20x MIC)	Biphasic	Survival drops to ~0.001% [26]	Genetic vs. phenotypic tolerance
Wild-type (Stationary)	Ciprofloxacin (20x MIC)	Tolerance (Complete survival)	100% survival; killing to 0.001% upon nutrient restoration [26]	Phenotypic tolerance is reversible
hipA7 mutant (Stationary)	Ciprofloxacin (20x MIC)	Biphasic/Persistence	~10% survival even after nutrient restoration [26]	High-persistence mutant
Exponential Culture	Ampicillin (12.5x MIC)	Biphasic	Most persisters were growing before treatment [63]	Single-cell observation
Post-Stationary Culture	Ciprofloxacin (32x MIC)	Biphasic	All identified persisters were growing before treatment [48]	Single-cell observation

Experimental Protocols

Core Protocol: Bulk Killing and Regrowth Assay

This protocol is designed to characterize the persister subpopulation in a bacterial culture through a time-kill experiment followed by assessment of regrowth capacity.

Materials & Reagents

• Bacterial Strain: e.g., Escherichia coli MG1655 [63] [48] or relevant clinical isolate.
• Antibiotics: High-purity, laboratory-grade antibiotics (e.g., ciprofloxacin, ampicillin). Prepare stock solutions as per manufacturer's instructions and sterilize by filtration.
• Growth Media: Appropriate rich broth (e.g., Mueller-Hinton II (MH2), LB) and solid agar plates [42].
• Buffers: Phosphate-Buffered Saline (PBS), sterile.
• Equipment: Shaking incubator, water bath, centrifuge, spectrophotometer (for OD600 measurement), vortex mixer, colony counter.

Procedure

• Culture Preparation and Standardization:
  • Grow the bacterial strain overnight in appropriate broth to stationary phase.
  • Dilute the overnight culture 1:100 into fresh, pre-warmed broth and incubate with shaking until the desired growth phase is reached (typically mid-exponential phase, OD600 ~0.5).
  • Harvest cells by centrifugation (e.g., 5,000 x g for 10 min) and resuspend in fresh broth or PBS to standardize the cell density. A common starting density is ~1.5 x 10^7 CFU/mL [42].

• Antibiotic Exposure (Killing Phase):

  • Divide the standardized cell suspension into sterile tubes.
  • Add the antibiotic under test to achieve a lethal concentration, typically 10-100x the MIC of the strain [26] [63]. Include a no-antibiotic control.
  • Incubate the tubes under optimal growth conditions (e.g., 37°C with shaking).
  • At predetermined time points (e.g., 0, 2, 4, 6, 8, 24 hours), remove aliquots from each tube.

• Viable Cell Count (Plating):

  • Serially dilute the aliquots in sterile PBS to neutralize the antibiotic effect.
  • Plate appropriate dilutions onto antibiotic-free solid agar plates.
  • Incubate the plates for 16-24 hours until colonies are visible.
  • Count the colonies to determine the number of Colony-Forming Units (CFU) per mL at each time point. The survival curve is plotted as log(CFU/mL) versus time.

• Regrowth Assessment:

  • After an extended antibiotic exposure (e.g., 24 hours), take an aliquot from the treatment tube.
  • Pellet the cells by centrifugation and wash twice with sterile PBS to thoroughly remove the antibiotic.
  • Resuspend the cell pellet in fresh, pre-warmed, antibiotic-free broth.
  • Incubate the culture and monitor growth (e.g., by OD600) over time. The ability of the culture to regrow confirms the presence of viable persister cells.

Diagram 1: Workflow for Bulk Killing and Regrowth Assay

Advanced Single-Cell Analysis Using Microfluidics

Bulk assays average the behavior of millions of cells, masking the heterogeneity of persister formation and survival dynamics. Microfluidic devices allow for direct observation of individual cells before, during, and after antibiotic exposure [63] [48] [42].

Key Methodology (Based on MCMA Device [63] [48]):

• Device: A membrane-covered microchamber array (MCMA) is used. This consists of shallow (e.g., 0.8 µm deep) microchambers etched on a glass coverslip, sealed with a semi-permeable membrane.
• Cell Loading: Bacterial cells are loaded into the microchambers, forming a monolayer. The membrane allows rapid diffusion of media and antibiotics while physically trapping the cells for imaging.
• Antibiotic Treatment and Imaging: Fresh medium containing a lethal dose of antibiotic is perfused over the chambers. Time-lapse microscopy is used to track the morphological changes, division events, and death of individual cells in real-time.
• Regrowth Assessment: After a treatment period, the antibiotic-containing medium is replaced with fresh, drug-free medium. The chambers are monitored to identify which surviving cells resume growth and division, unequivocally classifying them as persisters.

This technique has revealed astonishing heterogeneity among persisters, including cells that continue to grow and divide with L-form-like morphologies under ampicillin pressure, or those that filament under ciprofloxacin [63].

The Scientist's Toolkit: Essential Research Reagents and Materials

Successful execution of persister assays relies on specific tools and reagents. This table details key solutions for the critical steps of the protocol.

Table 3: Key Research Reagent Solutions for Persister Assays

Item Name	Function/Application	Key Considerations
Lethal-Dose Antibiotic Solutions	To apply high-concentration antibiotic pressure for killing assays.	Use concentrations far above MIC (e.g., 20-100x MIC) [26]. Clinically relevant concentrations in specific body fluids (e.g., urine) can also be used [42].
Microfluidic Cell Culture Devices (e.g., MCMA)	To trap and image individual bacterial cells for single-cell analysis of persistence.	Enables tracking of cell lineage and fate before, during, and after antibiotic exposure [63] [48].
Physiological Culture Media (e.g., Human Urine)	To mimic the in vivo host environment during antimicrobial testing.	Host physiology (e.g., urine) can significantly alter bacterial growth and antimicrobial susceptibility compared to standard lab broth [42].
Synergistic Antibiotic Combinations (e.g., Aminoglycoside + Polymyxin)	To eradicate persister cells via simultaneous membrane disruption.	An ROS-independent combination shown to rapidly sterilize cultures of E. coli persister mutants [26].

Molecular Mechanisms and Signaling Pathways in Persistence

The ability to form persisters is tied to a network of intracellular signaling pathways that modulate bacterial growth and stress response. Key mechanisms include the Stringent Response, toxin-antitoxin (TA) systems, and suppression of reactive oxygen species (ROS) [26] [9].

Diagram 2: Core Pathways in Persister Cell Formation. The pathway shows how stress triggers a regulatory cascade leading to growth arrest, a key persister trait. ROS suppression is a correlated survival feature [26] [9].

The stringent response is triggered by nutrient limitation or other stresses, leading to accumulation of the alarmones (p)ppGpp. This directly suppresses growth-promoting processes like rRNA synthesis and stimulates the activation of TA modules [9]. In systems like HipBA, the toxin HipA inhibits growth by targeting essential processes, pushing the cell into a dormant state. A hallmark of many persister cells and tolerant populations is a global reduction in metabolic activity, which includes the active suppression of ROS accumulation, thereby avoiding a common death pathway triggered by bactericidal antibiotics [26]. This network creates a transient, non-growing state that is refractory to antibiotic killing.

Within the field of microbial research, particularly in the study of bacterial persister cells, confirming a state of metabolic dormancy is a complex challenge. Persisters are defined as genetically drug-susceptible but phenotypically tolerant cells that can survive antibiotic exposure, contributing to chronic and relapsing infections [9]. The longstanding conventional view that these cells are entirely metabolically dormant is being re-evaluated, with emerging evidence indicating that a spectrum of metabolic activities can persist even in a non-growing state [28] [9]. This application note details a consolidated experimental framework that combines direct ATP level measurement with 13C isotope tracing to provide a robust, multi-faceted profile of cellular metabolic activity. This methodology is essential for accurately characterizing the metabolic basis of dormancy during efforts to enrich and isolate persister subpopulations.

Theoretical Foundation

The Metabolic Spectrum of Bacterial Persisters

Bacterial persisters are not a uniform population but exhibit significant phenotypic heterogeneity. They are primarily characterized by their non-growing or slow-growing state, which allows them to tolerate bactericidal antibiotics that typically target active cellular processes [9]. This tolerance is distinct from genetic antibiotic resistance. The metabolic state of persisters can range from complete metabolic quiescence to slow or re-routed metabolism [9]. Some studies even challenge the traditional dormancy view, demonstrating that persisters can actively produce RNA and adapt their transcriptome to enhance survival despite not dividing [28]. This heterogeneity necessitates analytical methods that can both quantify overall metabolic capacity and trace specific pathway activities.

Analytical Principles

The two techniques described herein function on complementary principles to interrogate the metabolic state of cells:

• ATP Level Measurement: Adenosine triphosphate (ATP) is the universal energy currency of the cell. Its concentration provides a direct, real-time snapshot of the cell's energetic status [64]. Actively metabolizing cells maintain high ATP levels, while truly dormant cells exhibit drastically reduced ATP pools. This method offers a high-throughput, quantitative readout of the cell's metabolic capacity, defined here as its ability to generate energy.
• 13C Isotope Tracing: This technique involves supplying cells with nutrients containing 13C, a stable, heavy isotope of carbon. As the cells metabolize these labeled substrates, the 13C atoms are incorporated into downstream metabolic intermediates and products [65] [66] [67]. High-resolution mass spectrometry (MS) is then used to track the pattern and extent of this incorporation. This reveals the activity of specific metabolic pathways, such as glycolysis, the TCA cycle, or amino acid synthesis, and can identify which nutrients are being utilized [65] [67]. In persisters, the absence of 13C incorporation in key metabolites provides strong evidence for pathway inactivation or dormancy.

Integrated Methodologies

Protocol for ATP-Based Metabolic Capacity Assessment

This protocol, adapted for assessing bacterial persisters, enables the direct measurement of ATP levels to gauge metabolic activity [64].

• Step 1: Cell Preparation and Treatment. Enrich a persister population using a standard method (e.g., antibiotic treatment followed by washing). Seed the cells (persisters and a control growing population) into a 96-well plate at a density of ~10^5 - 10^6 cells per well. Treat experimental wells with selected metabolic inhibitors according to the experimental design.
• Step 2: Metabolic Inhibition. Systematically inhibit key pathways to dissect metabolic dependencies. A standard inhibitor set includes:
  • 2-Deoxy-D-glucose (50 mM): A glycolytic inhibitor.
  • Oligomycin A (10 µM): An ATP synthase inhibitor targeting oxidative phosphorylation (OXPHOS).
  • Combination (2-DG + Oligomycin A): To assess synergistic effects and total ATP production capacity.
• Step 3: ATP Measurement. Lyse the cells and use a commercial luminescent ATP detection assay kit. The assay relies on the luciferase enzyme, which produces light proportional to the ATP concentration. Measure luminescence using a plate reader.
• Step 4: Data Normalization and Analysis. Normalize the raw luminescence (Relative Light Units, RLU) to cell viability, typically assessed in parallel with a assay like XTT [64]. Calculate the dependency on specific pathways using normalized ATP values. For example:
  • Glycolytic Capacity = (ATP_Control - ATP_Oligomycin) / ATP_Control
  • Mitochondrial Dependency = (ATP_Control - ATP_2-DG) / ATP_Control

Table 1: Key Reagents for ATP Measurement Protocol

Reagent / Equipment	Function / Description	Example Source
2-Deoxy-D-glucose	Glycolysis inhibitor; competitive inhibitor of glucose	TCI (D0051)
Oligomycin A	ATP synthase inhibitor; targets oxidative phosphorylation	Sigma-Aldrich (75351)
Luminescent ATP Detection Assay Kit	Provides reagents for cell lysis and luciferase-based ATP quantification	Abcam (ab113849)
Cell Proliferation Kit II (XTT)	Measures cell viability and metabolic activity for normalization	Sigma-Aldrich (11465015001)
White 96-well plates	Optimum for luminescence assays, minimize well-to-well crosstalk	Thermo Fisher Scientific (136102)
Multimode Microplate Reader	Instrument to detect luminescence signals	BioTek Synergy HTX

Protocol for 13C Isotope Tracing to Determine Pathway Activity

This protocol outlines the use of deep 13C labeling to catalog active and inactive metabolic pathways in persister cells [65] [67].

• Step 1: Experimental Design and Tracer Selection. Design a custom 13C-labeled medium. For comprehensive coverage, use a medium where core carbon precursors like glucose and all amino acids are fully 13C-labeled, while other components (e.g., vitamins, serum) are unlabeled (12C) [65]. For targeted studies, use a single tracer like [1,2-13C] glucose.
• Step 2: Tracer Experiment and Sample Collection. Culture the enriched persister population in the 13C medium. Maintain the culture for a sufficient duration (e.g., ≥ 6 population doublings for actively growing controls) to allow for full 13C incorporation into metabolites. For slow-growing or non-growing persisters, longer incubation times may be required to detect any activity. Collect cells by rapid centrifugation and extract metabolites using a polar solvent (e.g., 80% methanol).
• Step 3: Metabolite Analysis via Mass Spectrometry. Analyze the polar extracts using Liquid Chromatography-High Resolution Mass Spectrometry (LC-HRMS). The high mass resolution is critical for distinguishing 13C-labeled ions and determining the exact mass of metabolites for sum formula assignment [65].
• Step 4: Data Processing and Interpretation. Process the raw MS data to identify metabolite ions and their mass isotopologue distributions (MIDs). The MID describes the pattern of 13C incorporation for a given metabolite. An endogenously synthesized metabolite will show a characteristic shift from its natural 13C abundance (all ~12C) to a distribution containing multiple 13C atoms [65]. Identify inactive pathways by the lack of 13C incorporation into their key intermediate and end-product metabolites.

Table 2: Key Reagents for 13C Isotope Tracing Protocol

Reagent / Equipment	Function / Description	Example Source / Specification
U-13C-Glucose	Uniformly labeled glucose tracer; core substrate for central carbon metabolism	Cambridge Isotope Laboratories
U-13C-Amino Acid Mix	Uniformly labeled mix; traces amino acid metabolism and anabolism	Cambridge Isotope Laboratories
Custom 13C Medium	Growth medium with defined 13C sources; enables hypothesis-free discovery	Formulated in-house per [65]
Liquid Chromatograph	Separates complex metabolite mixtures prior to MS analysis	e.g., Vanquish UHPLC
High-Resolution Mass Spectrometer	Precisely measures mass-to-charge (m/z) ratios for metabolite and 13C isotopologue identification	e.g., Orbitrap-based MS

The following diagram illustrates the logical workflow and integration points of the two methodologies for characterizing persister cell metabolism.

The Scientist's Toolkit: Essential Research Reagents

Successful implementation of these metabolic profiling techniques requires specific, high-quality reagents and tools. The following table catalogs the essential solutions for setting up these experiments.

Table 3: Research Reagent Solutions for Metabolic Profiling of Persisters

Category	Item	Critical Function & Application Notes
Stable Isotope Tracers	U-13C-Glucose ([1,2-13C] glucose recommended)	Core substrate for tracing central carbon metabolism; provides high flux resolution [67].
	U-13C-Amino Acid Mix	Enables profiling of amino acid utilization, protein synthesis, and specific anabolic pathways.
Metabolic Inhibitors	2-Deoxy-D-Glucose (2-DG)	Glycolytic inhibitor; used to dissect glycolytic capacity in ATP assays [64].
	Oligomycin A	ATP synthase inhibitor; used to assess mitochondrial dependency on OXPHOS [64].
Assay Kits	Luminescent ATP Detection Assay	Provides optimized lysis buffer and luciferase reagent for sensitive, high-throughput ATP quantitation [64].
	Cell Viability Assay (e.g., XTT)	Used for normalizing ATP data to viable cell count, crucial for accurate interpretation [64].
Analytical Tools	LC-HRMS System	The core platform for 13C-tracing; high mass resolution is essential for accurate isotopologue detection [65].
	Data Processing Software (e.g., Metran, OpenFLUX)	Computational tools for flux estimation and analysis of 13C-labeling data [67].

Data Interpretation and Integration

Quantitative Analysis and Meaning

The data generated from these protocols provides a multi-layered understanding of persister metabolism.

• ATP Assay Results: Normalized ATP levels should be interpreted as a direct measure of the cell's energy charge. A significant drop in ATP following inhibition of a specific pathway indicates that the cell is dependent on that pathway for energy maintenance. For example, if ATP levels plummet after Oligomycin A treatment, the persisters are reliant on OXPHOS, even in a non-growing state.
• 13C Isotope Tracing Results: The Mass Isotopologue Distribution (MID) is the key dataset. A metabolite that remains as mostly the M+0 isotopologue (all 12C) after incubation in 13C medium is not being synthesized de novo, indicating an inactive pathway. Conversely, a shift towards M+X isotopologues confirms active synthesis from the labeled precursor [65]. This can also reveal metabolic dependencies on exogenous serum components if 12C atoms are found in metabolites that should be fully labeled [65].

Correlating Findings for a Coherent Model

True confirmation of a dormant state is achieved when both techniques yield congruent results:

• Low ATP levels coupled with minimal to no 13C incorporation across a wide range of central metabolic metabolites (e.g., TCA cycle intermediates, nucleotides, lipids) provides strong evidence for a deep dormancy state.
• Moderate ATP levels with 13C incorporation in specific, re-routed pathways (e.g., gluconeogenesis but a silent TCA cycle) suggest a state of metabolic re-wiring rather than full dormancy. This integrated approach moves beyond a binary "active/dormant" classification and allows for the mapping of a metabolic phenotype continuum within persister populations [9].

The combination of ATP level measurement and 13C isotope tracing provides a powerful, orthogonal framework for confirming the metabolic state of bacterial persisters. The ATP assay offers a simple, high-throughput readout of the cell's energetic capacity, while 13C tracing delivers unparalleled insight into the specific pathways that are active or inactive. By applying these methods, researchers can move past simplistic classifications and begin to unravel the complex metabolic heterogeneity that defines persister subpopulations. This detailed metabolic profiling is a critical step in the broader research workflow of enriching and isolating persisters, ultimately enabling the discovery of novel therapeutic strategies to target these recalcitrant cells and eradicate persistent infections.

Persister cells are a transiently drug-tolerant subpopulation within an isogenic population of bacteria or cancer cells, characterized by a non-growing or slow-growing state that allows survival under lethal stress conditions such as antibiotic or chemotherapeutic treatment [9] [68]. Unlike resistant cells, persisters do not possess genetic mutations that confer resistance and exhibit normal minimum inhibitory concentration (MIC) values upon regrowth, making them a primary cause of chronic infections and therapy relapse [9] [69]. This Application Note details standardized methodologies for the enrichment, isolation, and subsequent molecular characterization of authentic persister cells, with a focus on proteomic and metabolomic profiling to uncover signatures of persistence.

The critical importance of persister cells in clinical settings is underscored by their role in recurrent infections and treatment failure across numerous pathogens, including Mycobacterium tuberculosis, Staphylococcus aureus, and Enterococcus faecium [9] [69]. Similarly, in oncology, cancer persister cells are recognized as a source of tumor recurrence following chemotherapeutic intervention [70] [71]. A comprehensive understanding of persister physiology is therefore essential for developing novel therapeutic strategies to eradicate these cells.

Experimental Protocols for Persister Enrichment and Isolation

Protocol 1: High-Dose Antibiotic Treatment for Bacterial Persister Enrichment

This protocol describes the induction of bacterial persisters using high concentrations of ciprofloxacin, as applied in Enterococcus faecium [69].

• Principle: A biphasic kill curve, where the majority of the population succumbs to antibiotic treatment, reveals a small, surviving subpopulation of persister cells.
• Materials:
  • Bacterial culture in mid-exponential growth phase (OD600 ≈ 0.3)
  • Ciprofloxacin stock solution
  • Mueller-Hinton (MH) broth and agar plates
• Procedure:
  • Determine MIC: Establish the Minimum Inhibitory Concentration (MIC) of ciprofloxacin for the bacterial strain using standard broth microdilution according to CLSI guidelines. For E. faecium strain AUS004, the MIC was determined to be 2 µg/mL [69].
  • Induce Persisters: Inoculate a culture of the test bacterium in MH broth and incubate until it reaches the mid-exponential phase (OD600 of 0.3). Add ciprofloxacin at a final concentration of 10x the MIC (e.g., 20 µg/mL). Continue incubation.
  • Monitor Kill Kinetics: At predetermined time points (e.g., 0, 3, 6, 12, 24, and 48 hours), remove aliquots, perform serial dilutions in sterile saline, and spot-plate onto MH agar plates. Count colony-forming units (CFU) after 24-48 hours of incubation.
  • Harvest Persisters: After 48 hours of antibiotic exposure, the surviving cells, which constitute the persister population, can be harvested for downstream analyses. Centrifuge the culture, and wash the pellet twice with phosphate-buffered saline (PBS) to remove the antibiotic.
• Validation: Confirm the persister phenotype by verifying that the MIC of the harvested survivors is unchanged from the original strain and that their growth kinetics in drug-free media are identical [69].

Protocol 2: PerSort – A Sorting-Based Method for Translational Dormancy

The PerSort method isolates translationally dormant mycobacterial persisters without prior antibiotic exposure, enabling the study of pre-existing persister subpopulations [31].

• Principle: A fluorescent reporter gene under the control of a strong translation initiation sequence is used to isolate cells with low translational activity via Fluorescence-Activated Cell Sorting (FACS).
• Materials:
  • pTrans-mEos2 reporter plasmid or equivalent
  • Mycobacterial culture (e.g., M. smegmatis or M. tuberculosis)
  • Anhydrotetracycline (ATc) for induction
  • FACS sorter
• Procedure:
  • Reporter Strain Construction: Transform the target mycobacterium with the pTrans-mEos2 plasmid, which carries a genome-integrating, ATc-inducible promoter driving the mEos2 fluorescent protein.
  • Induction and Staining: Grow the reporter strain under optimal conditions to mid-log phase. Induce reporter expression with ATc. Process the culture to minimize clumps for FACS analysis.
  • Cell Sorting: Perform FACS to isolate the subpopulation of cells exhibiting the lowest mEos2 fluorescence, representing the translationally dormant fraction.
  • Validation: Validate the sorted population by assessing its multidrug tolerance and regrowth capacity compared to the high-fluorescence population [31].

Proteomic and Metabolomic Characterization

The molecular characterization of persisters provides critical insights into the mechanisms underlying their dormant and drug-tolerant state.

Proteomic Workflow and Key Signatures

Proteomic analysis via mass spectrometry (MS) reveals proteins differentially abundant in persister cells, highlighting pathways critical for their survival.

• Sample Preparation: Harvest persister and control cells (e.g., exponential-phase). Lyse cells and extract proteins. Digest proteins into peptides using trypsin.
• Mass Spectrometry Analysis: Analyze peptides using LC-MS/MS (Liquid Chromatography with Tandem Mass Spectrometry). Use either label-free quantification or labeled approaches like SILAC (Stable Isotope Labeling by Amino Acids in Cell Culture) [68].
• Data Analysis: Identify and quantify proteins using bioinformatic tools. Perform statistical analysis to determine significantly differentially abundant proteins.

Table 1: Key Proteomic Signatures in Bacterial Persisters

Protein	Function	Change in Persisters	Organism	Inducing Stress
AhpF [68]	Oxidative stress response (Alkyl hydroperoxide reductase)	Increased during recovery	E. coli	Ampicillin (TisB-dependent)
OmpF [68]	Outer membrane porin	Increased during recovery	E. coli	Ampicillin (TisB-dependent)
CspA [69]	Cold shock protein, stress adaptation	Significantly different	E. faecium	Ciprofloxacin
ClpX [69]	ATP-dependent protease subunit	Significantly different	E. faecium	Ciprofloxacin
Proteins linked to oxidative stress [69]	Defense against reactive oxygen species	Significantly different	E. faecium	Ciprofloxacin

Metabolomic Workflow and Key Signatures

Metabolomics captures the immediate functional readout of cellular activity and is crucial for understanding persister physiology.

• Sample Preparation: For Intracellular (IC) metabolomics, rapidly quench metabolism (e.g., cold methanol), extract metabolites, and use a magnetic bead-based protocol to limit contamination from dead cells [69]. For Extracellular (EC) metabolomics, analyze the spent culture medium.
• Analysis by ¹H NMR: Conduct untargeted ¹H NMR spectroscopy on samples. Identify and quantify metabolites by comparing spectral profiles to reference libraries [72].
• Data Analysis: Use multivariate statistics (e.g., Principal Component Analysis - PCA) and hierarchical clustering to identify metabolites that are significantly altered in abundance.

Table 2: Key Metabolomic Alterations in Persister Cells and Cultures

Metabolite/Pathway	Change in Persisters	System	Implication
Trimethylamine metabolism [72]	Consistently altered	Bacterial pathogens under sub-MIC antibiotics	Alternative nitrogen and carbon utilization
Krebs Cycle (TCA cycle) [71]	Upregulated	Melanoma persisters to chemotherapy	Increased mitochondrial energy metabolism
Fatty Acid Oxidation [70]	Upregulated	Cycling cancer persisters	Supports survival and proliferation under drug pressure
D-alanine metabolism [72]	Suppressed	S. aureus under vancomycin	General suppression of metabolism
Glutathione metabolism [70]	Upregulated	Cycling cancer persisters	Enhanced antioxidant defense

Visualization of Core Persister Physiology and Workflows

The following diagrams summarize the key physiological traits of persister cells and the experimental workflow for their molecular validation.

The Scientist's Toolkit: Essential Research Reagents

Table 3: Key Reagents for Persister Cell Research

Reagent / Material	Function / Application	Example / Specification
pTrans-mEos2 Plasmid	Fluorescent reporter for isolating translationally dormant cells via FACS. Integrated into the genome for single-copy stability [31].	Available through addgene or constructed in-house with an ATc-inducible promoter.
Anhydrotetracycline (ATc)	Inducer for the pTrans-mEos2 and similar inducible reporter systems.	High-purity grade, dissolved in a suitable solvent (e.g., DMSO or ethanol).
Ciprofloxacin	Fluoroquinolone antibiotic used for enriching persisters via high-dose treatment.	Clinical-grade powder for preparation of stock solutions. Store at -20°C.
Magnetic Beads coupled with Propidium Iodide (PI)	Sample "cleaning" to remove dead or membrane-compromised cells prior to omics analysis, improving signal-to-noise ratio [69].	Commercial kits for live cell isolation.
SILAC Amino Acids (¹³C, ¹⁵N labeled)	Metabolic labeling for quantitative pulsed-SILAC proteomics to measure de novo protein synthesis in persisters [68].	L-Arginine and L-Lysine, heavy isotope-labeled.
PBS with 0.05% Tween 80	Nutrient-starvation medium for inducing a multidrug-tolerant state in mycobacteria and for washing cell pellets [31].	Filter sterilized.

Concluding Remarks

The methodologies detailed in this Application Note provide a robust framework for the enrichment, isolation, and molecular validation of authentic persister cells. The integration of proteomic and metabolomic data is paramount, as it reveals that persisters are not simply inert entities but undergo active metabolic reprogramming and stress response to achieve a transiently tolerant state. Key conserved signatures, such as upregulation of antioxidant systems and a shift toward specific energy pathways like the Krebs cycle and fatty acid oxidation, present a compelling set of potential therapeutic targets.

Moving forward, leveraging these molecular signatures to screen for compounds that selectively eliminate persister cells holds immense promise for combating chronic recurrent infections and preventing cancer relapse. The continued refinement of isolation protocols like PerSort, coupled with multi-omics integration, will accelerate the discovery of novel anti-persister therapies and improve patient outcomes.

Within the field of microbiology, the study of bacterial persisters—a transiently drug-tolerant subpopulation of cells—presents a significant technical challenge. Their low abundance and non-heritable, phenotypically diverse nature make their isolation and study particularly difficult [5]. A critical first step in this research is the effective enrichment of these elusive cells from a larger, susceptible population. This application note provides a comparative analysis of established methodologies for enriching persister cells, framing them within the context of a broader thesis on persister isolation. We summarize quantitative data for direct comparison, detail standardized experimental protocols, and provide visual workflows to guide researchers, scientists, and drug development professionals in selecting and implementing the most appropriate enrichment strategy for their specific research goals.

Comparative Analysis of Enrichment Methods

The choice of enrichment strategy is paramount, as it dictates the physiological state of the persisters under investigation and influences subsequent experimental findings. The following table summarizes the key characteristics, outputs, and considerations of three primary enrichment approaches.

Table 1: Strengths and Limitations of Primary Persister Enrichment Methods

Enrichment Method	Theoretical Basis	Typical Persister Yield	Key Strengths	Major Limitations
Stationary Phase Enrichment	Nutrient depletion in late-stage cultures induces a slow-growing or non-growing state [26] [73].	~10% survival post-treatment [26].	Simple, high-yield, reproducible; models chronic infections where nutrients are limited [9].	Highly heterogeneous population; may include viable but non-culturable (VBNC) cells [73] [5].
Nutrient Starvation	Abrupt removal of nutrients suspends metabolic activity, promoting tolerance [26].	Varies with starvation duration and cell density.	Rapid induction of tolerance; useful for studying stochastic formation.	Phenotypic tolerance is readily reversible upon nutrient restoration, unlike genetic persistence [26].
Antibiotic Treatment (e.g., Ciprofloxacin)	Kills growing, susceptible cells, leaving a surviving persister subpopulation [26].	Wild-type: ~0.001% [26]. hipA7 mutant: ~10% [26].	Directly isolates the phenotypically defined subpopulation of interest.	Very low yield in wild-type strains; requires high antibiotic concentrations and precise timing.

Detailed Experimental Protocols

Protocol 1: Enrichment from Late Stationary Phase Culture

This protocol is designed to obtain a high yield of type I persisters, which are formed in response to nutrient limitation and are prevalent in biofilm-related infections [9] [73].

Research Reagent Solutions

• LB Broth: Standard culture medium for growing E. coli.
• Phosphate-Buffered Saline (PBS): Salt solution for washing and resuspending cells.
• Ciprofloxacin Stock Solution: 10 mg/mL in water or DMSO; used for positive selection of persisters.
• Agar Plates: For viable cell counting.

Methodology

• Inoculation and Growth: Inoculate 5 mL of LB broth with a single bacterial colony and incubate overnight at 37°C with shaking (200 rpm).
• Extended Incubation: Dilute the overnight culture 1:1000 into 50 mL of fresh LB in a 250 mL flask. Incubate at 37°C with shaking for 72 hours to ensure entry into the late stationary phase.
• Cell Harvesting: Centrifuge 10 mL of the culture at 5,000 × g for 10 minutes at room temperature.
• Washing: Gently resuspend the cell pellet in 10 mL of sterile PBS to remove residual nutrients.
• Persister Selection: Resuspend the final pellet in 10 mL of PBS containing ciprofloxacin at a concentration of 10x the MIC (e.g., 20 µg/mL for E. coli). Incubate for 5 hours at 37°C.
• Assessment of Enrichment: Serially dilute the culture in PBS at the end of the antibiotic exposure and plate on LB agar plates. Incubate overnight at 37°C and count the resulting colonies to determine the persister titer [26] [73].

Diagram 1: Stationary phase persister enrichment workflow.

Protocol 2: Induction via Nutrient Starvation for Phenotypic Tolerance

This method induces a reversible, phenotypic tolerance, useful for studying the response to acute environmental stress [26].

Research Reagent Solutions

• Saline Solution: 0.85% NaCl for nutrient starvation.
• LB Broth & Agar Plates: For culture and enumeration.

Methodology

• Culture Preparation: Grow a bacterial culture to mid-exponential phase (OD₆₀₀ ≈ 0.5).
• Starvation Induction: Pellet cells by centrifugation and resuspend in an equal volume of pre-warmed saline solution.
• Starvation Incubation: Incubate the cell suspension in saline for 2-4 hours at 37°C with shaking.
• Challenge and Analysis: Treat the starved culture with a lethal concentration of antibiotic (e.g., 20x MIC ciprofloxacin) for 3 hours. Serially dilute and plate to quantify the surviving tolerant population [26].

Protocol 3: Direct Isolation Using High-Dose Antibiotic Selection

This protocol is the definitive method for isolating the persister subpopulation from an otherwise susceptible culture.

Methodology

• Standard Culture: Grow a bacterial culture to the desired growth phase (exponential or stationary).
• Lethal Antibiotic Exposure: Add a high concentration of a bactericidal antibiotic (e.g., 20x MIC of ciprofloxacin, ampicillin, or kanamycin) directly to the culture.
• Incubation and Sampling: Incubate the culture under standard growth conditions. Sample at multiple time points (e.g., 0, 2, 4, 6 hours).
• Persister Quantification: At each time point, wash the sampled cells with PBS to remove the antibiotic, perform serial dilutions, and plate on antibiotic-free agar to determine the number of surviving persister cells. The killing curve will typically show a biphasic pattern, with a sharp initial drop followed by a plateau representing the persister fraction [26] [5].

Associated Signaling Pathways in Persister Formation

Understanding the molecular mechanisms behind persistence is crucial. The following diagram integrates key pathways, such as the stringent response and toxin-antitoxin systems, which are triggered by enrichment methods and lead to the dormant, tolerant state.

Diagram 2: Core molecular pathways of persister formation.

The Scientist's Toolkit: Essential Research Reagents

The following table lists key reagents used in the protocols above, along with their critical functions in persister cell research.

Table 2: Key Research Reagent Solutions for Persister Studies

Reagent / Material	Function / Application	Example Usage in Protocol
Ciprofloxacin	Fluoroquinolone antibiotic; induces DNA breaks and a toxic metabolic response.	Positive selection of persisters in all protocols [26].
Aminoglycoside-Polymyxin B Combination	Synergistic, ROS-independent membrane disruption.	Rapid eradication of persister cells in validation studies [26].
Crp/cAMP System	Global metabolic regulator; shifts metabolism to oxidative phosphorylation.	Studying the role of energy metabolism in persister survival [73].
hipA7 Mutant Strains	High-persistence mutant with elevated (p)ppGpp levels.	Positive control for high persister yields in Method 3 [26] [5].

Conclusion

The successful enrichment and isolation of persister cells are pivotal for deconstructing the mechanisms underlying antibiotic tolerance and developing therapies against chronic infections. This guide has synthesized key methodologies, from chemical induction and FACS to microfluidics, each with distinct applications and trade-offs between throughput and single-cell resolution. Future directions must focus on standardizing these protocols across different bacterial species, improving the viability of sorted persisters for downstream functional studies, and leveraging isolated populations for high-throughput drug screening. Mastering these techniques will directly accelerate the discovery of anti-persister compounds, ultimately bridging a critical gap in our ability to treat persistent biofilm and relapsing infections.

References

• 1. Drug-tolerant persister cells in cancer: bridging the gaps ... [https://www.nature.com/articles/s41467-025-66376-6]
• 2. Drug-tolerant persister cell in cancer - PubMed Central - NIH [https://pmc.ncbi.nlm.nih.gov/articles/PMC12391116/]
• 3. Isolation and characterization of persisters of the ... [https://www.sciencedirect.com/science/article/pii/S2589004224012276]
• 4. Targeting Cancer Drug-Tolerant Persister Cells in Minimal ... [https://www.sciltp.com/journals/ijddp/articles/2505000693]
• 5. Molecular mechanism and application of emerging ... [https://bmcmicrobiol.biomedcentral.com/articles/10.1186/s12866-024-03628-3]
• 6. Inheritable cell-states shape drug-persister correlations and ... [https://journals.plos.org/ploscompbiol/article?id=10.1371/journal.pcbi.1013446]
• 7. Enrichment of persisters enabled by a ß-lactam-induced ... [https://www.nature.com/articles/s42003-019-0672-3]
• 8. Drug-tolerant persister cells in cancer: bridging the gaps ... [https://pmc.ncbi.nlm.nih.gov/articles/PMC12623778/]
• 9. Bacterial persisters: molecular mechanisms and ... [https://www.nature.com/articles/s41392-024-01866-5]
• 10. Cancer cell dormancy: An update to 2025 [https://bmrat.org/index.php/BMRAT/article/view/994]
• 11. Unraveling CRP/cAMP-mediated metabolic regulation in ... [https://elifesciences.org/articles/99735]
• 12. Methods for Enrichment of Bacterial Persister Populations ... [https://pubmed.ncbi.nlm.nih.gov/34590252/]
• 13. Early tolerance and late persistence as alternative drug ... [https://www.nature.com/articles/s41467-024-54728-7]
• 14. Treatment strategies targeting persister cell formation in ... [https://pubmed.ncbi.nlm.nih.gov/33022189/]
• 15. Mini review: Persister cell control strategies [https://www.frontiersin.org/journals/pharmacology/articles/10.3389/fphar.2025.1706115/full]
• 16. Bacterial persisters are a stochastically formed subpopulation ... [https://pmc.ncbi.nlm.nih.gov/articles/PMC8084331/]
• 17. Differential carbon source utilization drives metabolic state ... [https://www.frontiersin.org/journals/pharmacology/articles/10.3389/fphar.2025.1634627/full]
• 18. Cancer drug-tolerant persister cells: from biological questions ... [https://pmc.ncbi.nlm.nih.gov/articles/PMC12622869/]
• 19. Integrative biology of persister cell formation - PubMed Central [https://pmc.ncbi.nlm.nih.gov/articles/PMC9470271/]
• 20. Bacterial Persister Cells and Development of Antibiotic ... [https://www.frontierspartnerships.org/journals/british-journal-of-biomedical-science/articles/10.3389/bjbs.2024.12958/full]
• 21. Novel Protocol for Persister Cells Isolation - PMC [https://pmc.ncbi.nlm.nih.gov/articles/PMC3931647/]
• 22. Quantitative analysis of persister fractions suggests different ... [https://pmc.ncbi.nlm.nih.gov/articles/PMC3682893/]
• 23. A Quantitative Survey of Bacterial Persistence in the ... [https://www.mdpi.com/2079-6382/9/8/508]
• 24. Differential carbon source utilization drives metabolic state ... [https://pmc.ncbi.nlm.nih.gov/articles/PMC12440864/]
• 25. A shared alarmone–GTP switch controls persister ... [https://www.nature.com/articles/s41564-025-02015-6]
• 26. Antibiotic-persistent bacterial cells exhibiting low-level ROS ... [https://pmc.ncbi.nlm.nih.gov/articles/PMC12345145/]
• 27. Growth Mode and Physiological State of Cells Prior to Biofilm ... [https://pmc.ncbi.nlm.nih.gov/articles/PMC7023439/]
• 28. Antibiotic persisters are metabolically active, non-growing ... [https://www.sciencedirect.com/science/article/pii/S0924857924003029]
• 29. Antibiotic Persistence as a Metabolic Adaptation [https://pmc.ncbi.nlm.nih.gov/articles/PMC5874710/]
• 30. Analyzing persister physiology with fluorescence activated ... [https://pmc.ncbi.nlm.nih.gov/articles/PMC4908830/]
• 31. PerSort Facilitates Characterization and Elimination of ... [https://pmc.ncbi.nlm.nih.gov/articles/PMC7716392/]
• 32. Development of Persister-FACSeq: a method to massively ... [https://www.nature.com/articles/srep25100]
• 33. Unraveling CRP/cAMP-Mediated Metabolic Regulation In ... [https://elifesciences.org/reviewed-preprints/99735v1]
• 34. Bacterial persisters are a stochastically formed subpopulation ... [https://journals.plos.org/plosbiology/article?id=10.1371/journal.pbio.3001194]
• 35. Establishment of a Method To Rapidly Assay Bacterial ... [https://pmc.ncbi.nlm.nih.gov/articles/PMC3754326/]
• 36. Single-Cell Technologies to Study Phenotypic ... [https://www.mdpi.com/2076-2607/9/11/2277]
• 37. Isolating live cells after high-throughput, long-term, time-lapse ... [https://pmc.ncbi.nlm.nih.gov/articles/PMC7525750/]
• 38. Microfluidic Biochips for Single‐Cell Isolation and ... [https://pmc.ncbi.nlm.nih.gov/articles/PMC11267386/]
• 39. Microfluidic Chips for Single-cell Analysis and Isolation [https://www.ufluidix.com/microfluidics-applications/single-cell-analysis/]
• 40. Single Cell Isolation and Analysis - PMC - PubMed Central [https://pmc.ncbi.nlm.nih.gov/articles/PMC5078503/]
• 41. The 7 Single-Cell Isolation Techniques [https://www.atlantisbioscience.com/blog/the-7-single-cell-isolation-techniques/?srsltid=AfmBOoqWJmL3z07w0mCiJvAA4SiZv8dWTmV1y-FPkqqme7omJqCJpmfu]
• 42. Precision single cell analysis to characterize host dependent ... [https://pubs.rsc.org/en/content/articlehtml/2025/lc/d4lc00765d]
• 43. Persister formation in Staphylococcus aureus is associated ... [https://pmc.ncbi.nlm.nih.gov/articles/PMC4932909/]
• 44. Bakuchiol kills Staphylococcus aureus persisters and ... [https://www.frontiersin.org/journals/pharmacology/articles/10.3389/fphar.2025.1592183/full]
• 45. Flow-cytometry analysis reveals persister resuscitation ... [https://pmc.ncbi.nlm.nih.gov/articles/PMC7346475/]
• 46. A Simplified Method for Comprehensive Capture of the ... [https://www.nature.com/articles/s41597-025-05361-6]
• 47. A host-directed adjuvant sensitizes intracellular bacterial ... [https://www.nature.com/articles/s41564-025-02124-2]
• 48. Observation of persister cell histories reveals diverse ... [https://pmc.ncbi.nlm.nih.gov/articles/PMC12074638/]
• 49. Drug-induced tolerant persisters in tumor - Molecular Cancer [https://molecular-cancer.biomedcentral.com/articles/10.1186/s12943-025-02323-9]
• 50. Optimal dosing of anti-cancer treatment under drug- ... [https://www.nature.com/articles/s41540-025-00571-5]
• 51. A modified fluctuation-test framework characterizes the ... [https://www.nature.com/articles/s41588-022-01105-z]
• 52. Optimization of periodic treatment strategies for bacterial ... [https://pmc.ncbi.nlm.nih.gov/articles/PMC11003776/]
• 53. Isolation and characterization of persisters of the pathogenic ... [https://pmc.ncbi.nlm.nih.gov/articles/PMC11166702/]
• 54. Viable but Nonculturable - an overview [https://www.sciencedirect.com/topics/immunology-and-microbiology/viable-but-nonculturable]
• 55. Viable but Nonculturable Bacteria: Food Safety and Public ... [https://pmc.ncbi.nlm.nih.gov/articles/PMC3804398/]
• 56. Viable but nonculturable [https://en.wikipedia.org/wiki/Viable_but_nonculturable]
• 57. Viable but non-cultivable state in oral microbiota [https://www.frontiersin.org/journals/cellular-and-infection-microbiology/articles/10.3389/fcimb.2025.1533768/full]
• 58. Latest Developments of Research on the Viable Non ... [https://www.mdpi.com/2076-3417/15/3/1454]
• 59. Bacterial dormancy: A subpopulation of viable but non ... [https://pmc.ncbi.nlm.nih.gov/articles/PMC7837498/]
• 60. Drug-tolerant persister cell in cancer: reversibility ... [https://www.frontiersin.org/journals/pharmacology/articles/10.3389/fphar.2025.1612089/full]
• 61. Isolation of Persister Cells of Bacillus subtilis and ... [https://www.mdpi.com/1422-0067/22/18/10059]
• 62. Integration of multi-omics data accelerates molecular ... [https://www.nature.com/articles/s41467-025-57550-x]
• 63. Observation of persister cell histories reveals diverse ... [https://elifesciences.org/articles/79517]
• 64. Protocol for analyzing energy metabolic pathway ... [https://pmc.ncbi.nlm.nih.gov/articles/PMC10960080/]
• 65. Profiling the metabolism of human cells by deep 13C labeling [https://pmc.ncbi.nlm.nih.gov/articles/PMC6239935/]
• 66. Metabolomics and Isotope Tracing [https://www.sciencedirect.com/science/article/pii/S0092867418303878]
• 67. Overview of 13c Metabolic Flux Analysis [https://www.creative-proteomics.com/resource/13c-metabolic-flux-analysis.htm]
• 68. High-Throughput Proteomics Identifies Proteins With ... [https://pmc.ncbi.nlm.nih.gov/articles/PMC6414554/]
• 69. Proteomic characterization of persisters in Enterococcus ... [https://pmc.ncbi.nlm.nih.gov/articles/PMC10765921/]
• 70. Cycling cancer persister cells arise from lineages with ... [https://pmc.ncbi.nlm.nih.gov/articles/PMC9209846/]
• 71. A Transient Metabolic State in Melanoma Persister Cells ... [https://www.frontiersin.org/journals/molecular-biosciences/articles/10.3389/fmolb.2021.780192/full]
• 72. Proteomic and metabolomic responses of priority bacterial ... [https://www.nature.com/articles/s44259-025-00147-7]
• 73. Unraveling CRP/cAMP-mediated metabolic regulation in ... [https://pmc.ncbi.nlm.nih.gov/articles/PMC12237405/]