time-lapse-epifluorescence-microscopy-imaging-pseudomonas-aeruginosa

Time-Lapse Epifluorescence Microscopy Imaging of <i>Pseudomonas aeruginosa</i> and <i>Staphylococcus aureus</i> Heterogeneous Phenotypes

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

Uconn Health

Bacteria have a repertoire of strategies to overcome antibiotics in clinical use, complicating our ability to treat and cure infectious diseases. In addition to evolving resistance, bacteria within genetically clonal cultures can undergo transient phenotypic changes and tolerate high doses of antibiotics. These cells, termed persisters, exhibit heterogeneous phenotypes; the strategies that a bacterial population deploys to overcome one class of antibiotics can be distinct from those needed to survive treatment with drugs with another mode of action. It was previously reported that fluoroquinolones, which target DNA topoisomerases, retain the capacity to kill nongrowing bacteria that tolerate other classes of antibiotics. Here, we show that in Escherichia coli stationary-phase cultures and colony biofilms, persisters that survive treatment with the anionic fluoroquinolone delafloxacin depend on the AcrAB-TolC efflux pump. In contrast, we did not detect this dependence on AcrAB-TolC in E. coli persisters that survive treatment with three other fluoroquinolone compounds. We found that the loss of AcrAB-TolC activity via genetic mutations or chemical inhibition not only reduces delafloxacin persistence in nongrowing E. coli MG1655 or EDL933 (an E. coli O157:H7 strain), but it limits resistance development in progenies derived from delafloxacin persisters that were given the opportunity to recover in nutritive medium following antibiotic treatment. Our findings highlight the heterogeneity in defense mechanisms that persisters use to overcome different compounds within the same class of antibiotics. They further indicate that efflux pump inhibitors can potentiate the activity of delafloxacin against stationary-phase E. coli and block resistance development in delafloxacin persister progenies.

The AcrAB-TolC Efflux Pump Impacts Persistence and Resistance Development in Stationary-Phase Escherichia coli following Delafloxacin Treatment.

Antibiotic persistence is a phenomenon in which rare cells of a clonal bacterial population can survive antibiotic doses that kill their kin, even though the entire population is genetically susceptible. With antibiotic treatment failure on the rise, there is growing interest in understanding the molecular mechanisms underlying bacterial phenotypic heterogeneity and antibiotic persistence. However, elucidating these rare cell states can be technically challenging. The advent of single-cell techniques has enabled us to observe and quantitatively investigate individual cells in complex, phenotypically heterogeneous populations. In this review, we will discuss current technologies for studying persister phenotypes, including fluorescent tags and biosensors used to elucidate cellular processes; advances in flow cytometry, mass spectrometry, Raman spectroscopy, and microfluidics that contribute high-throughput and high-content information; and next-generation sequencing for powerful insights into genetic and transcriptomic programs. We will further discuss existing knowledge gaps, cutting-edge technologies that can address them, and how advances in single-cell microbiology can potentially improve infectious disease treatment outcomes.

Single-Cell Technologies to Study Phenotypic Heterogeneity and Bacterial Persisters.

Bacterial persisters are rare phenotypic variants in clonal bacterial cultures that can endure antimicrobial therapy and potentially contribute to infection relapse. Here, we investigate the potential of leveraging microbial interactions to disrupt persisters as they resuscitate during the post-antibiotic treatment recovery period.

Probiotic Escherichia coli Nissle 1917 inhibits bacterial persisters that survive fluoroquinolone treatment.

Antibiotic resistance is a major danger to public health that threatens to claim the lives of millions of people per year within the next few decades. Years of necessary administration and excessive application of antibiotics have selected for strains that are resistant to many of our currently available treatments. Due to the high costs and difficulty of developing new antibiotics, the emergence of resistant bacteria is outpacing the introduction of new drugs to fight them. To overcome this problem, many researchers are focusing on developing antibacterial therapeutic strategies that are "resistance-resistant"-regimens that slow or stall resistance development in the targeted pathogens. In this mini review, we outline major examples of novel resistance-resistant therapeutic strategies. We discuss the use of compounds that reduce mutagenesis and thereby decrease the likelihood of resistance emergence. Then, we examine the effectiveness of antibiotic cycling and evolutionary steering, in which a bacterial population is forced by one antibiotic toward susceptibility to another antibiotic. We also consider combination therapies that aim to sabotage defensive mechanisms and eliminate potentially resistant pathogens by combining two antibiotics or combining an antibiotic with other therapeutics, such as antibodies or phages. Finally, we highlight promising future directions in this field, including the potential of applying machine learning and personalized medicine to fight antibiotic resistance emergence and out-maneuver adaptive pathogens.

Resistance-resistant antibacterial treatment strategies.

The bacterial pathogen  is responsible for a variety of chronic human infections. Even in the absence of identifiable resistance mutations, this pathogen can tolerate lethal antibiotic doses through phenotypic strategies like biofilm formation and metabolic quiescence. In this study, we determined that  maintains greater metabolic activity in the stationary phase compared to the model organism, , which has traditionally been used to study fluoroquinolone antibiotic tolerance. We demonstrate that hallmarks of  fluoroquinolone tolerance are not conserved in , including the timing of cell death and necessity of the SOS DNA damage response for survival. The heightened sensitivity of stationary-phase  to fluoroquinolones is attributed to maintained transcriptional and reductase activity. Our data suggest that perturbations that suppress transcription and respiration in  may actually protect the pathogen against this important class of antibiotics.

Article	Year
The AcrAB-TolC Efflux Pump Impacts Persistence and Resistance Development in Stationary-Phase Escherichia coli following Delafloxacin Treatment. Antimicrobial agents and chemotherapy\| PubMed ID: 34097492	2021
Single-Cell Technologies to Study Phenotypic Heterogeneity and Bacterial Persisters. Microorganisms\| PubMed ID: 34835403	2021
Probiotic Escherichia coli Nissle 1917 inhibits bacterial persisters that survive fluoroquinolone treatment. Journal of applied microbiology\| PubMed ID: 35332984	2022
Resistance-resistant antibacterial treatment strategies. Frontiers in antibiotics\| PubMed ID: 36845830	2023
Metabolic and transcriptional activities underlie stationary-phase sensitivity to Levofloxacin. Microbiology spectrum\| PubMed ID: 38078717	2024

Patricia J. Hare

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