Work in the Van Melderen lab is supported by the ARC actions 2018-2023, the Fonds National de la Recherche Scientifique (FNRS CDR J.0182.21F). T.O. is supported by a ULB fellowship. T.S. is supported by an FRIA fellowship (FNRS). J.C. is supported by a post-doctoral fellowship &#34;charg&#233; de recherches&#34; (FNRS).

<ol>
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The authors declare no competing interests.

The protocol presented in this paper allows the analysis of the persistence phenotype observed in response to antibiotic treatment at the population and single-cell levels. The experiments were performed with the E. coli MG1655 strain, which was grown in a chemically defined medium (MOPS glycerol 0.4%). Time-kill assays and microscopy experiments were carried out on exponential-phase cultures. We used OFX, a fluoroquinolone, at a concentration of 5 &#956;g&#183;mL&#8722;1 to reveal the persister cells. The approaches described here can be applied to other bactericidal antibiotics, such as &#946;-lactams, aminoglycosides, or antimicrobial compounds31. Accordingly, other bacterial strains, media, or growth conditions can be used. Monitoring different fluorescent fusions in a similar setup to that described here can be useful for following cellular processes such as DNA replication32, DNA repair25,33, and cell division34 before, during, and after the antibiotic treatment. Similarly, fluorescent reporters can be exploited to investigate distinct aspects of cell physiology, such as intracellular pH35, ATP36, or ROS37 levels. Alternatively to fluorescent fusions, chemical dyes could also be applied. For example, the hupA-mCherry fusion could be replaced by 4&#39;,6-diamidino-2-phenylindole (DAPI), a fluorescent dye that stains DNA38. Performing time-lapse microscopy coupled with such fluorescent dyes should, however, be avoided, as these staining techniques can perturb the dynamics of the cell cycle during time-lapse experiments. Alternatively, such experiments can be replaced by time-course analyses of snap-shot imaging at relevant timepoints.
While such fluorescent reporters are helpful, the amount of information that can be extracted through the analysis of phase-contrast images should not be neglected. Here, we monitored cell length evolution throughout the growth, OFX treatment, and recovery stages. Other parameters based on phase-contrast images, such as cell width, phase-contrast intensity, and curvatures of the bacterial cells, can also be extracted with ease by using adequate software, such as MicrobeJ27.
In summary, the procedure described here can be applied to other conditions and bacterial species to monitor cellular responses to changing environments or stressors18,19. By using other fluorescent reporters (transcriptional and translational reporters, chemical dye) in combination with a population analysis, such as flow cytometry/FACS, interesting questions can be addressed in a multi-scale framework.

This protocol aims to analyze the bacterial persistence phenotype in response to specific antibiotic treatment at the single-cell and population levels. Persistence describes the capacity of small subpopulations within an isogenic population to endure high doses of bactericidal antibiotics (fluoroquinolones, aminoglycosides, &#946;-lactams, etc.), with the minimal inhibitory concentration (MIC) of the so-called persister cells being identical to that of the bulk of the population. Biphasic killing dynamics, when measuring bacterial survival over time in the presence of an antibiotic, reveal the presence of transiently drug-tolerant cells, with an initial rapid eradication of the non-persister cells, followed by a much slower killing rate of the persister cells. Upon antibiotic removal, these cells give rise to a genetically identical population that displays similar killing dynamics when treated with the same antibiotic1,2. In contrast to persistence, antibiotic resistance is defined at the population level and is generally a consequence of either de novo mutations or the horizontal gene transfer of a resistance-conferring plasmid3. While the mutations responsible for resistance are mostly located in the target of the drug or in the promotor regions of the drug efflux pumps, genes altering the persistence frequency identified by genome-wide and targeted mutant analysis approaches have proven to be numerous and diverse2,3,4,5,6,7,8. Therefore, it is likely that bacterial cells can enter the persister state through multiple pathways9,10,11, and approaches to investigate the persistence phenomenon at the single-cell level are needed to characterize the physiology of these persister cells.
The recent development of microfluidic tools used in combination with fluorescence microscopy has paved the way for the characterization of the persistence phenotype and highlighted the role of key cellular processes, such as chromosome replication12, DNA repair13, and cell division14, in persister cell formation. In this paper, we describe an integrated approach combining classical microbiology assays with single-cell live imaging to characterize persister cells generated in exponentially growing Escherichia coli cultures treated with a high dose of ofloxacin. The protocol described here can be applied to study the antibiotic persistence phenomenon in other bacterial species, such as Bacillus subtilis15, or conditions (e.g., antibiotic persistence following &#946;-lactam treatment16) and can easily be modified to investigate the many phenomena involving phenotypic heterogeneity17,18,19. Furthermore, the setup described in this paper can be combined with other fluorescent reporters to investigate distinct cellular parameters of interest, such as intracellular levels of pH20 or ATP21 at the single-cell level, which may potentially produce novel insights into the antibiotic persistence phenomenon.

<table><tbody><tr><td>Axio Observer</td><td>Zeiss</td><td></td><td>Inverted fluorescence microscope</td></tr><tr><td>CaCl&lt;sub&gt;2&lt;/sub&gt;.2H&lt;sub&gt;2&lt;/sub&gt;O&amp;nbsp;</td><td>Merck</td><td>1.02382.0250</td><td>For MOPS glucose 0.4% and MOPS glycerol 0.4% growth medium</td></tr><tr><td>CellASIC ONIX Microfluidic System&amp;nbsp;</td><td>Merck</td><td>CAX2-S0000&amp;nbsp;</td><td>Microfluidic system</td></tr><tr><td>CellASIC ONIX2 FG</td><td>Merck</td><td>ONIX2 1.0.1&amp;nbsp;</td><td>Microfluidic software</td></tr><tr><td>CellASIC ONIX2 Manifold Basic&amp;nbsp;</td><td>Merck</td><td>CAX2-MBC20</td><td>Manifold system</td></tr><tr><td>CoCl&lt;sub&gt;2&lt;/sub&gt;.6H&lt;sub&gt;2&lt;/sub&gt;O</td><td>Merck</td><td>1.02539.0100</td><td>For MOPS glucose 0.4% and MOPS glycerol 0.4% growth medium</td></tr><tr><td>CuSO&lt;sub&gt;4&lt;/sub&gt;.5H&lt;sub&gt;2&lt;/sub&gt;O</td><td>Merck</td><td>1.02790.0250</td><td>For MOPS glucose 0.4% and MOPS glycerol 0.4% growth medium</td></tr><tr><td>D-(+)-glucose</td><td>Sigma Aldrich</td><td>G7021-1KG</td><td>Carbon source for MOPS glucose 0.4% growth medium</td></tr><tr><td>&lt;em&gt;E. coli&lt;/em&gt; K-12 MG1655 CF1648 (&lt;em&gt;fnr+&lt;/em&gt;)</td><td></td><td>BE10</td><td>wt reference strain, lab strain</td></tr><tr><td>&lt;em&gt;E. coli&lt;/em&gt; K-12 MG1655 CF1648 (&lt;em&gt;fnr+&lt;/em&gt;) &lt;em&gt;hupA-mCherry::FRT-kan-FRT&lt;/em&gt;</td><td></td><td>BE16</td><td>HU-mCherry fusion integrated via P1 transduction at the native locus, lab strain</td></tr><tr><td>FeSO4.7H&lt;sub&gt;2&lt;/sub&gt;O&amp;nbsp;</td><td>VWR Chemicals</td><td>24244.232</td><td>For MOPS glucose 0.4% and MOPS glycerol 0.4% growth medium</td></tr><tr><td>Fiji</td><td>ImageJ</td><td>https://fiji.sc/&amp;nbsp;</td><td>Image software; Schindelin et al. if used in publication</td></tr><tr><td>Glycerol</td><td>Merck</td><td>56815</td><td>Carbon source for MOPS glycerol 0.4% growth medium</td></tr><tr><td>Greiner CELLSTAR&amp;reg; multiwell culture plate</td><td>Merck</td><td>M8812-100EA</td><td>24-well clear flat bottom 2ml volume culture plate for automated plate reader</td></tr><tr><td>H&lt;sub&gt;3&lt;/sub&gt;BO&lt;sub&gt;3&amp;nbsp;&lt;/sub&gt;</td><td>Sigma-Aldrich</td><td>B6768-1KG</td><td>For MOPS glucose 0.4% and MOPS glycerol 0.4% growth medium</td></tr><tr><td>Hamamatsu ORCA Flash 4.0 digital camera</td><td>Hamamatsu</td><td>C13440-20CU</td><td>Digital Image Acqusition</td></tr><tr><td>K2HPO4</td><td>Merck</td><td>1.05099.1000</td><td>For MOPS glucose 0.4% and MOPS glycerol 0.4% growth medium</td></tr><tr><td>KOH</td><td>Merck</td><td>1.05029.1000</td><td>For MOPS glucose 0.4% and MOPS glycerol 0.4% growth medium</td></tr><tr><td>K&lt;sub&gt;2&lt;/sub&gt;SO&lt;sub&gt;4&lt;/sub&gt;&amp;nbsp;</td><td>Merck</td><td>1.05153.0500</td><td>For MOPS glucose 0.4% and MOPS glycerol 0.4% growth medium</td></tr><tr><td>Luria-Broth agar medium</td><td>Invitrogen</td><td>22700041</td><td>Growth medium for plating assay</td></tr><tr><td>Luria-Broth medium</td><td>Invitrogen</td><td>12780029</td><td>Growth medium for MIC determination</td></tr><tr><td>MgCl&lt;sub&gt;2&lt;/sub&gt;.6H&lt;sub&gt;2&lt;/sub&gt;O&amp;nbsp;</td><td>Merck</td><td>1.05832.1000</td><td>For MOPS glucose 0.4% and MOPS glycerol 0.4% growth medium</td></tr><tr><td>MgSO4</td><td>Merck</td><td>7487-88-9</td><td>Dilution solution for bacterial survival assay used at 10mM</td></tr><tr><td>MicrobeJ&amp;nbsp;</td><td>Imagej/Fiji plugin</td><td>https://www.microbej.com/</td><td>Microscopy image analysis plugin. Ducret et al.for in publication; Detection settings: For bacteria : Area (&amp;mu;m2): 0,1-max; Length (&amp;mu;m): 0,5-max; Width (&amp;mu;m): 0,6-max; Range (&amp;mu;m): 0,5-max; Angularity (rad): 0-0,3; 0-max for all other parameters.&amp;nbsp;</td></tr><tr><td>Microfluidic Plates CellASIC ONIX</td><td>Merck</td><td>B04A-03-5PK&amp;nbsp;</td><td>Plate for microfluidic system</td></tr><tr><td>MnCl&lt;sub&gt;2&lt;/sub&gt;.4H&lt;sub&gt;2&lt;/sub&gt;O</td><td>Merck</td><td>1.05927.0100</td><td>For MOPS glucose 0.4% and MOPS glycerol 0.4% growth medium</td></tr><tr><td>MOPS, Free Acid ULTROL&amp;reg; Grade</td><td>Merck</td><td>475898-500GM</td><td>For MOPS glucose 0.4% and MOPS glycerol 0.4% growth medium</td></tr><tr><td>NaCl</td><td>SIgma-Aldrich</td><td>S5886-1KG</td><td>For MOPS glucose 0.4% and MOPS glycerol 0.4% growth medium</td></tr><tr><td>(NH4)6Mo7O24.4H&lt;sub&gt;2&lt;/sub&gt;O</td><td>Merck</td><td>1.01180.0250</td><td>For MOPS glucose 0.4% and MOPS glycerol 0.4% growth medium</td></tr><tr><td>NH&lt;sub&gt;4&lt;/sub&gt;Cl&amp;nbsp;</td><td>Merck</td><td>1.01145.0500</td><td>For MOPS glucose 0.4% and MOPS glycerol 0.4% growth medium</td></tr><tr><td>Ofloxacin</td><td>Merck</td><td>82419-36-1</td><td>Fluoroquinolone antibiotic used to treat the bacterial cells</td></tr><tr><td>Phosphate Buffered Saline (PBS) pH 7.4, 1x</td><td>Merck</td><td>P3813</td><td>Dilution buffer</td></tr><tr><td>GENESYS 10S UV-Visible Spectrophotometer</td><td>Thermofisher Scientific</td><td>&amp;nbsp;840-208200</td><td>UV-Visible Spectrophotometer, Single/Six Cell Holder with PrinterMeasurement of optical density OD600nm</td></tr><tr><td>SoftMax Pro</td><td>Molecular Devices</td><td></td><td>Microplate reader software</td></tr><tr><td>SpectraMax i3x</td><td>Molecular Devices</td><td></td><td>Microplate reader</td></tr><tr><td>N-[Tris(hydroxym&amp;eacute;thyl)-m&amp;eacute;thyl]-glycine</td><td>Merck</td><td>1.08602.0250</td><td>For MOPS glucose 0.4% and MOPS glycerol 0.4% growth medium</td></tr><tr><td>Zeiss&amp;reg; immersion Oil 518F</td><td>Zeiss</td><td></td><td>Immersion oil to increase resolution of microscope</td></tr><tr><td>Zen3.2 Pro</td><td>Zeiss</td><td></td><td>Microscopic image acquisition and processing software</td></tr><tr><td>ZnSO4.7H&lt;sub&gt;2&lt;/sub&gt;O&amp;nbsp;</td><td>Merck</td><td>1.08883.0500</td><td>For MOPS glucose 0.4% and MOPS glycerol 0.4% growth medium</td></tr></tbody></table>

population and single-cell analysis of antibiotic persistence in escherichia coli

Antibiotic persistence refers to the capacity of small bacterial subpopulations to transiently tolerate high doses of bactericidal antibiotics. Upon bactericidal antibiotic treatment, the bulk of the bacterial population is rapidly killed. This first rapid phase of killing is followed by a substantial decrease in the rate of killing as the persister cells remain viable. Classically, persistence is determined at the population level by time/kill assays performed with high doses of antibiotics and for defined exposure times. While this method provides information about the level of persister cells and the killing kinetics, it fails to reflect the intrinsic cell-to-cell heterogeneity underlying the persistence phenomenon. The protocol described here combines classical time/kill assays with single-cell analysis using real-time fluorescence microscopy. By using appropriate fluorescent reporters, the microscopy imaging of live cells can provide information regarding the effects of the antibiotic on cellular processes, such as chromosome replication and segregation, cell elongation, and cell division. Combining population and single-cell analysis allows for the molecular and cellular characterization of the persistence phenotype.

Watch this Scientific Journal Video about Population and Single-Cell Analysis of Antibiotic Persistence in Escherichia coli at JoVE.com

Population and Single-Cell Analysis of Antibiotic Persistence in Escherichia coli

Antibiotic persistence describes the ability of small subpopulations within a sensitive isogenic population to transiently tolerate high doses of bactericidal antibiotics. The present protocol combines approaches to characterize the antibiotic persistence phenotype at the molecular and cellular levels after exposing Escherichia coli to lethal doses of ofloxacin.

Population and Single-Cell Analysis of Antibiotic Persistence in Escherichia coli

Antibiotic persistence refers to the capacity of small bacterial subpopulations to transiently tolerate high doses of bactericidal antibiotics. Upon ...

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Research

JoVE Journal

Biology

1.7K Views.  Université Libre de Bruxelles (ULB). Antibiotic persistence describes the ability of small subpopulations within a sensitive isogenic population to transiently tolerate high doses of bactericidal antibiotics. The present protocol combines approaches to characterize the antibiotic persistence phenotype at the molecular and cellular levels after exposing Escherichia coli to lethal doses of ofloxacin.

Video: Population and Single-Cell Analysis of Antibiotic Persistence in Escherichia coli

Testing the Role of Multicopy Plasmids in the Evolution of Antibiotic Resistance

Multicopy plasmids are extremely abundant in prokaryotes but their role in bacterial evolution remains poorly understood. We recently showed that the increase in gene copy number per cell provided by multicopy plasmids could accelerate the evolution of plasmid-encoded genes. In this work, we present an experimental system to test the ability of multicopy plasmids to promote gene evolution. Using simple molecular biology methods, we constructed a model system where an antibiotic resistance gene can be inserted into Escherichia coli MG1655, either in the chromosome or on a multicopy plasmid. We use an experimental evolution approach to propagate the different strains under increasing concentrations of antibiotics and we measure survival of bacterial populations over time. The choice of the antibiotic molecule and the resistance gene is so that the gene can only confer resistance through the acquisition of mutations. This &#34;evolutionary rescue&#34; approach provides a simple method to test the potential of multicopy plasmids to promote the acquisition of antibiotic resistance. In the next step of the experimental system, the molecular bases of antibiotic resistance are characterized. To identify mutations responsible for the acquisition of antibiotic resistance we use deep DNA sequencing of samples obtained from whole populations and clones. Finally, to confirm the role of the mutations in the gene under study, we reconstruct them in the parental background and test the resistance phenotype of the resulting strains.

Here we present an experimental method to test the role of multicopy plasmids in the evolution of antibiotic resistance.

Multicopy plasmids are extremely abundant in prokaryotes but their role in bacterial evolution remains poorly understood. We recently showed that the ...

Genetics

Quantification of Plasmid-Mediated Antibiotic Resistance in an Experimental Evolution Approach

Plasmids play a major role in microbial ecology and evolution as vehicles of lateral gene transfer and reservoirs of accessory gene functions in microbial populations. This is especially the case under rapidly changing environments such as fluctuating antibiotics exposure. We recently showed that plasmids maintain antibiotic resistance genes in Escherichia coli without positive selection for the plasmid presence. Here we describe an experimental system that allows following both the plasmid genotype and phenotype in long-term evolution experiments. We use molecular techniques to design a model plasmid that is subsequently introduced to an experimental evolution batch system approach in an E. coli host. We follow the plasmid frequency over time by applying replica plating of the E. coli populations while quantifying the antibiotic resistance persistence. In addition, we monitor the conformation of plasmids in host cells by analyzing the extent of plasmid multimer formation by plasmid nicking and agarose gel electrophoresis. Such an approach allows us to visualize not only the genome size of evolving plasmids but also their topological conformation-a factor highly important for plasmid inheritance. Our system combines molecular strategies with traditional microbiology approaches and provides a set-up to follow plasmids in bacterial populations over a long time. The presented approach can be applied to study a wide range of mobile genetic elements in the future.

Our experimental approach provides a strategy to follow plasmid abundance and antibiotic resistance over time in bacterial populations.

Plasmids play a major role in microbial ecology and evolution as vehicles of lateral gene transfer and reservoirs of accessory gene functions in microbial ...

ScanLag: High-throughput Quantification of Colony Growth and Lag Time

Growth dynamics are fundamental characteristics of microorganisms. Quantifying growth precisely is an important goal in microbiology. Growth dynamics are affected both by the doubling time of the microorganism and by any delay in growth upon transfer from one condition to another, the lag. The ScanLag method enables the characterization of these two independent properties at the level of colonies originating each from a single cell, generating a two-dimensional distribution of the lag time and of the growth time. In ScanLag, measurement of the time it takes for colonies on conventional nutrient agar plates to be detected is automated on an array of commercial scanners controlled by an in house application. Petri dishes are placed on the scanners, and the application acquires images periodically. Automated analysis of colony growth is then done by an application that returns the appearance time and growth rate of each colony. Other parameters, such as the shape, texture and color of the colony, can be extracted for multidimensional mapping of sub-populations of cells. Finally, the method enables the retrieval of rare variants with specific growth phenotypes for further characterization. The technique could be applied in bacteriology for the identification of long lag that can cause persistence to antibiotics, as well as a general low cost technique for phenotypic screens.

ScanLag is a high-throughput method for measuring the delay in growth, namely lag time, as well as the growth rate of colonies for thousands of cells in parallel. Screening using ScanLag enables the discrimination between long lag-time and slow growth at the level of a single variant.

Growth dynamics are fundamental characteristics of microorganisms. Quantifying growth precisely is an important goal in microbiology. Growth dynamics are ...

Immunology and Infection

Isolation and Identification of Waterborne Antibiotic-Resistant Bacteria and Molecular Characterization of their Antibiotic Resistance Genes

The development and spread of antibiotic resistance (AR) through microbiota associated with freshwater bodies is a major global health concern. In the present study, freshwater samples were collected and analyzed with respect to the total bacterial diversity and AR genes (ARGs) using both conventional culture-based techniques and a high-throughput culture-independent metagenomic approach. This paper presents a systematic protocol for the enumeration of the total and antibiotic-resistant culturable bacteria from freshwater samples and the determination of phenotypic and genotypic resistance in the culturable isolates. Further, we report the use of whole metagenomic analysis of the total metagenomic DNA extracted from the freshwater sample for the identification of the overall bacterial diversity, including non-culturable bacteria, and the identification of the total pool of different ARGs (resistome) in the water body. Following these detailed protocols, we observed a high antibiotic-resistant bacteria load in the range of 9.6 &#215; 105-1.2 &#215; 109 CFU/mL. Most isolates were resistant to the multiple tested antibiotics, including cefotaxime, ampicillin, levofloxacin, chloramphenicol, ceftriaxone, gentamicin, neomycin, trimethoprim, and ciprofloxacin, with multiple antibiotic resistance (MAR) indexes of &#8805;0.2, indicating high levels of resistance in the isolates. The 16S rRNA sequencing identified potential human pathogens, such as Klebsiella pneumoniae,&#160;and opportunistic bacteria, such as Comamonas spp., Micrococcus spp., Arthrobacter spp., and Aeromonas spp. The molecular characterization of the isolates showed the presence of various ARGs, such as blaTEM, blaCTX-M (&#946;-lactams), aadA, aac (6&#39;)-Ib (aminoglycosides), and dfr1 (trimethoprims), which was also confirmed by the whole metagenomic DNA analysis. A high prevalence of other ARGs encoding for antibiotic efflux pumps-mtrA, macB, mdtA, acrD, &#946;-lactamases-SMB-1, VIM-20, ccrA, ampC, blaZ, the&#160;chloramphenicol acetyltransferase gene catB10, and&#160;the rifampicin resistance gene rphB-was also detected in the metagenomic DNA. With the help of the protocols discussed in this study, we confirmed the presence of waterborne MAR bacteria with diverse AR phenotypic and genotypic traits. Thus, whole metagenomic DNA analysis can be used as a complementary technique to conventional culture-based techniques to determine the overall AR status of a water body.

Here, we present a detailed protocol for the isolation and identification of antibiotic-resistant bacteria from water and the molecular characterization of their antibiotic resistance genes (ARGs). The use of culture-based and non-culture-based (metagenomic analysis) techniques provides complete information about the total bacterial diversity and the total pool of different ARGs present in freshwaters from Mumbai, India.

The development and spread of antibiotic resistance (AR) through microbiota associated with freshwater bodies is a major global health concern. In the ...

Environment

Continuous Measurement of Biological Noise in Escherichia Coli Using Time-lapse Microscopy

The protocol developed here offers a tool to enable computer tracking of Escherichia coli division and fluorescent levels over several hours. The process starts by screening for colonies that survive on minimal media, assuming that only Escherichia coli harboring the correct plasmid will be able to thrive in the specific conditions. Since the process of building large genetic circuits, requiring the assembly of many DNA parts, is challenging, circuit components are often distributed between multiple plasmids at different copy numbers requiring the use of several antibiotics. Mutations in the plasmid can destroy transcription of the antibiotic resistance genes and interject with resources management in the cell leading to necrosis. The selected colony is set on a glass-bottom Petri dish and a few focus planes are selected for microscopy tracking in both bright field and fluorescent domains. The protocol maintains the image focus for more than 12 hours under initial conditions that cannot be regulated, creating a few difficulties. For example, dead cells start to accumulate in the lenses' field of focus after a few hours of imaging, which causes toxins to buildup and the signal to blur and decay. Depletion of nutrients introduces new metabolic processes and hinder the desired response of the circuit. The experiment's temperature lowers the effectivity of inducers and antibiotics, which can further damage the reliability of the signal. The minimal media gel shrinks and dries, and as a result the optical focus changes over time. We developed this method to overcome these challenges in Escherichia coli, similar to previous works developing analogous methods for other micro-organisms. In addition, this method offers an algorithm to quantify the total stochastic noise in unaltered and altered cells, finding that the results are consistent with flow analyzer predictions as shown by a similar coefficient of variation (CV).

Continuous Measurement of Biological Noise in Escherichia Coli Using Time-lapse Microscopy

This work presents a microscopy method that allows live imaging of a single cell of Escherichia coli for analysis and quantification of the stochastic behavior of synthetic gene circuits.

The protocol developed here offers a tool to enable computer tracking of Escherichia coli division and fluorescent levels over several hours. The process ...

Bioengineering

Multi-scale Analysis of Bacterial Growth Under Stress Treatments

Analysis of the bacterial ability to grow and survive under stress conditions is essential for a wide range of microbiology studies. It is relevant to characterize the response of bacterial cells to stress-inducing treatments such as exposure to antibiotics or other antimicrobial compounds, irradiation, non-physiological pH, temperature, or salt concentration. Different stress treatments might disturb different cellular processes, including cell division, DNA replication, protein synthesis, membrane integrity, or cell cycle regulation. These effects are usually associated with specific phenotypes at the cellular scale. Therefore, understanding the extent and causality of stress-induced growth or viability deficiencies requires a careful analysis of several parameters, both at the single-cell and at the population levels. The experimental strategy presented here combines traditional optical density monitoring and plating assays with single-cell analysis techniques such as flow cytometry and real time microscopy imaging in live cells. This multiscale framework allows a time-resolved description of the impact of stress conditions on the fate of a bacterial population.

This protocol allows a time-resolved description of bacterial growth under stress conditions at the single-cell and the cell population levels.

Analysis of the bacterial ability to grow and survive under stress conditions is essential for a wide range of microbiology studies. It is relevant to ...

Visualization of Bacterial Resistance using Fluorescent Antibiotic Probes

Fluorescent antibiotics are multipurpose research tools that are readily used for the study of antimicrobial resistance, due to their significant advantage over other methods. To prepare these probes, azide derivatives of antibiotics are synthesized, then coupled with alkyne-fluorophores using azide-alkyne dipolar cycloaddition by click chemistry. Following purification, the antibiotic activity of the fluorescent antibiotic is tested by minimum inhibitory concentration assessment. In order to study bacterial accumulation, either spectrophotometry or flow cytometry may be used, allowing for much simpler analysis than methods relying on radioactive antibiotic derivatives. Furthermore, confocal microscopy can be used to examine localization within the bacteria, affording valuable information about mode of action and changes that occur in resistant species. The use of fluorescent antibiotic probes in the study of antimicrobial resistance is a powerful method with much potential for future expansion.

Fluorescently tagged antibiotics are powerful tools that can be used to study multiple aspects of antimicrobial resistance. This article describes the preparation of fluorescently tagged antibiotics and their application to studying antibiotic resistance in bacteria. Probes can be used to study mechanisms of bacterial resistance (e.g., efflux) by spectrophotometry, flow cytometry, and microscopy.

Fluorescent antibiotics are multipurpose research tools that are readily used for the study of antimicrobial resistance, due to their significant ...

High-throughput Screening of Chemical Compounds to Elucidate Their Effects on Bacterial Persistence

Bacterial persisters are defined as a small subpopulation of phenotypic variants with the capability of tolerating high concentrations of antibiotics. They are an important health concern as they have been associated with recurrent chronic infections. Although stochastic and deterministic dynamics of stress-related mechanisms are known to play a significant role in persistence, mechanisms underlying the phenotypic switch to/from the persistence state are not completely understood. While persistence factors triggered by environmental signals (e.g., depletion of carbon, nitrogen and oxygen sources) have been extensively studied, the impacts of osmolytes on persistence are yet to be determined. Using microarrays (i.e., 96 well plates containing various chemicals), we have designed an approach to elucidate the effects of various osmolytes on Escherichia coli persistence in a high throughput manner. This approach is transformative as it can be readily adapted for other screening arrays, such as drug panels and gene knockout libraries.

In this method paper, we present a high-throughput screening strategy to identify chemical compounds, such as osmolytes, that have a significant impact on bacterial persistence.

Bacterial persisters are defined as a small subpopulation of phenotypic variants with the capability of tolerating high concentrations of antibiotics. ...

Visualizing Antibiotic Persistence in Bacterial Pathogens

Time-Lapse Epifluorescence Microscopy Imaging of Pseudomonas aeruginosa and Staphylococcus aureus Heterogeneous Phenotypes

Antibiotic persistence is a phenomenon in which a small number of bacterial cells in a genetically susceptible population survive antibiotic treatment that kills the other genetically identical cells. Bacterial persisters can resume replication once antibiotic treatment ends and are commonly thought to&#160;underlie clinical treatment failure.&#160;Recent work harnessing the power of time-lapse fluorescence microscopy, in which bacteria are labeled with fluorescent transcriptional reporters, translational reporters, and/or dyes for a variety of cellular features, has advanced our understanding of Escherichia coli persisters beyond what could be learned from population-level antibiotic survival assays. Such single-cell approaches, rather than bulk population assays, are essential for delineating the mechanisms of persister formation, damage response, and survival. However, methods for studying persisters in other important pathogenic species at this level of detail remain limited.
This study provides an adaptable approach for time-lapse imaging of Pseudomonas aeruginosa (a gram-negative rod) and Staphylococcus aureus (a gram-positive coccus)&#160;during antibiotic treatment and recovery. We discuss molecular genetic approaches to introduce fluorescent reporters into these bacteria. Using these reporters, as well as dyes, we can track the phenotypic changes, morphological features, and fates of individual cells in response to antibiotic treatment. Additionally, we are able to observe the phenotypes of individual persisters as they resuscitate following treatment. In all, this work serves as a resource for those interested in tracking the survival and gene expression of individual antibiotic-treated cells, including persisters, both during and after treatment, in clinically important pathogens.

In this manuscript, we provide a comprehensive protocol for assessing antibiotic survival for Pseudomonas aeruginosa and Staphylococcus aureus, transforming plasmids into P. aeruginosa and S. aureus to create reporter strains and visualize&#160;phenotypic variants, such as persisters, by time-lapse epifluorescence&#160;microscopy.

Antibiotic persistence is a phenomenon in which a small number of bacterial cells in a genetically susceptible population survive antibiotic treatment ...

In Vitro Persister Assay: A Method to Evaluate the Effect of Osmolytes on Bacterial Persistence

Source: Karki, P. et al. <a href="https://app.jove.com/v/61597">High-throughput Screening of Chemical Compounds to Elucidate Their Effects on Bacterial Persistence.</a> J. Vis. Exp. (2021)
In this video, we demonstrate a method to evaluate the effect of osmolytes on the formation of bacterial persisters. The bacterial cells are first exposed to osmolytes to induce osmotic stress, triggering persister formation, then treated with antibiotics to enumerate the persisters.

Test próbny in vitro: metoda oceny wpływu osmolitów na trwałość bakterii

Source: Karki, P. et al. High-throughput Screening of Chemical Compounds to Elucidate Their Effects on Bacterial Persistence. J. Vis. Exp. (2021)
In this ...

Population and Single-Cell Analysis of Antibiotic Persistence in Escherichia coli

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Testing the Role of Multicopy Plasmids in the Evolution of Antibiotic Resistance

Quantification of Plasmid-Mediated Antibiotic Resistance in an Experimental Evolution Approach

ScanLag: High-throughput Quantification of Colony Growth and Lag Time

Isolation and Identification of Waterborne Antibiotic-Resistant Bacteria and Molecular Characterization of their Antibiotic Resistance Genes

Continuous Measurement of Biological Noise in Escherichia Coli Using Time-lapse Microscopy

Multi-scale Analysis of Bacterial Growth Under Stress Treatments

Visualization of Bacterial Resistance using Fluorescent Antibiotic Probes

High-throughput Screening of Chemical Compounds to Elucidate Their Effects on Bacterial Persistence

Time-Lapse Epifluorescence Microscopy Imaging of Pseudomonas aeruginosa and Staphylococcus aureus Heterogeneous Phenotypes

Test próbny in vitro: metoda oceny wpływu osmolitów na trwałość bakterii