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

Bacterial persisters are able to tolerate high levels of antibiotics and give rise to new populations. Persister tolerance is generally attributed to minimally active cellular processes that prevent antibiotic-induced damage, which has led to the supposition that persister offspring give rise to antibiotic-resistant mutants at comparable rates to normal cells. Using time-lapse microscopy to monitor Escherichia coli populations following ofloxacin treatment, we find that persisters filament extensively and induce impressive SOS responses before returning to a normal appearance. Further, populations derived from fluoroquinolone persisters contain significantly greater quantities of antibiotic-resistant mutants than those from untreated controls. We confirm that resistance is heritable and that the enhancement requires RecA, SOS induction, an opportunity to recover from treatment, and the involvement of error-prone DNA polymerase V (UmuDC). These findings show that fluoroquinolones damage DNA in persisters and that the ensuing SOS response accelerates the development of antibiotic resistance from these survivors.

Enhanced antibiotic resistance development from fluoroquinolone persisters after a single exposure to antibiotic.

This study examined the microbicidal activity of 222-nm UV radiation (UV), which is potentially a safer alternative to the 254-nm UV radiation (UV) that is often used for surface decontamination. Spores and/or growing and stationary-phase cells of , , , , and  and a herpesvirus were all killed or inactivated by UV and at lower fluences than with UV spores and cells lacking the major DNA repair protein RecA were more sensitive to UV, as were spores lacking their DNA-protective proteins, the α/β-type small, acid-soluble spore proteins. The spore cores' large amount of Ca-dipicolinic acid (∼25% of the core dry weight) also protected  and  spores against UV, while spores' proteinaceous coat may have given some slight protection against UV Survivors among  spores treated with UV acquired a large number of mutations, and this radiation generated known mutagenic photoproducts in spore and cell DNA, primarily cyclobutane-type pyrimidine dimers in growing cells and an α-thyminyl-thymine adduct termed the spore photoproduct (SP) in spores. Notably, the loss of a key SP repair protein markedly decreased spore UV resistance. UV-treated  spores germinated relatively normally, and the generation of colonies from these germinated spores was not salt sensitive. The latter two findings suggest that UV does not kill spores by general protein damage, and thus, the new results are consistent with the notion that DNA damage is responsible for the killing of spores and cells by UV Spores of a variety of bacteria are resistant to common decontamination agents, and many of them are major causes of food spoilage and some serious human diseases, including anthrax caused by spores of  Consequently, there is an ongoing need for efficient methods for spore eradication, in particular methods that have minimal deleterious effects on people or the environment. UV radiation at 254 nm (UV) is sporicidal and commonly used for surface decontamination but can cause deleterious effects in humans. Recent work, however, suggests that 222-nm UV (UV) may be less harmful to people than UV yet may still kill bacteria and at lower fluences than UV The present work has identified the damage by UV that leads to the killing of growing cells and spores of some bacteria, many of which are human pathogens, and UV also inactivates a herpesvirus.

DNA Damage Kills Bacterial Spores and Cells Exposed to 222-Nanometer UV Radiation.

Antibiotics have vastly improved our quality of life since their discovery and introduction into modern medicine. Yet, widespread use and misuse have compromised the efficacy of these compounds and put our ability to cure infectious diseases in jeopardy. To defend themselves against antibiotics, bacteria have evolved an arsenal of survival strategies. In addition to acquiring mutations and genetic determinants that confer antibiotic resistance, bacteria can respond to environmental cues and adopt reversible phenotypic changes that transiently enhance their ability to survive adverse conditions, including those brought on by antibiotics. These antibiotic tolerant and persistent bacteria, which are prevalent in biofilms and can survive antimicrobial therapy without inheriting resistance, are thought to underlie treatment failure and infection relapse. At infection sites, bacteria encounter a range of signals originating from host immunity and the local microbiota that can induce transcriptomic and metabolic reprogramming. In this review, we will focus on the impact of host factors and microbial interactions on antibiotic tolerance and persistence. We will also outline current efforts in leveraging the knowledge of host-microbe and microbe-microbe interactions in designing therapies that potentiate antibiotic activity and reduce the burden caused by recurrent infections.

The social network: Impact of host and microbial interactions on bacterial antibiotic tolerance and persistence.

Spores of firmicute species contain 100s of mRNAs, whose major function in Bacillus subtilis is to provide ribonucleotides for new RNA synthesis when spores germinate. To determine if this is a general phenomenon, RNA was isolated from spores of multiple firmicute species and relative mRNA levels determined by transcriptome sequencing (RNA-seq). Determination of RNA levels in single spores allowed calculation of RNA nucleotides/spore, and assuming mRNA is 3% of spore RNA indicated that only ∼6% of spore mRNAs were present at >1/spore. Bacillus subtilis, Bacillus atrophaeus, and Clostridioides difficile spores had 49, 42, and 51 mRNAs at >1/spore, and numbers of mRNAs at ≥1/spore were ∼10 to 50% higher in Geobacillus stearothermophilus and Bacillus thuringiensis Al Hakam spores and ∼4-fold higher in Bacillus megaterium spores. In all species, some to many abundant spore mRNAs (i) were transcribed by RNA polymerase with forespore-specific σ factors, (ii) encoded proteins that were homologs of those encoded by abundant B. subtilis spore mRNAs and are proteins in dormant spores, and (iii) were likely transcribed in the mother cell compartment of the sporulating cell. Analysis of the coverage of RNA-seq reads on mRNAs from all species suggested that abundant spore mRNAs were fragmented, as was confirmed by reverse transcriptase quantitative PCR (RT-qPCR) analysis of abundant B. subtilis and C. difficile spore mRNAs. These data add to evidence indicating that the function of at least the great majority of mRNAs in all firmicute spores is to be degraded to generate ribonucleotides for new RNA synthesis when spores germinate.  Only ∼6% of mRNAs in spores of six firmicute species are at ≥1 molecule/spore, many abundant spore mRNAs encode proteins similar to B. subtilis spore proteins, and some abundant B. subtilis and C. difficile spore mRNAs were fragmented. Most of the abundant B. subtilis and other  spore mRNAs are transcribed under the control of the forespore-specific RNA polymerase σ factors, F or G, and these results may stimulate transcription analyses in developing spores of species other than B. subtilis. These findings, plus the absence of key nucleotide biosynthetic enzymes in spores, suggest that firmicute spores' abundant mRNAs are not translated when spores germinate but instead are degraded to generate ribonucleotides for new RNA synthesis by the germinated spore.

Levels and Characteristics of mRNAs in Spores of Firmicute Species.

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.

Antibiotic tolerant bacteria and persistent cells that remain alive after a course of antibiotic treatment can foster the chronicity of infections and the development of antibiotic resistance. Elucidating how bacteria overcome antibiotic action and devising strategies to bolster a new drug's activity can allow us to preserve our antibiotic arsenal. Here, we investigate strategies to potentiate the activities of topoisomerase inhibitors against nongrowing Escherichia coli that are often recalcitrant to existing antibiotics. We focus on sensitizing bacteria to the fluoroquinolone (FQ) levofloxacin (Levo) and to the spiropyrimidinetrione zoliflodacin (Zoli)-the first antibiotic in its class of compounds in clinical development. We found that metabolic stimulation either alone or in combination with inhibiting the AcrAB-TolC efflux pump sensitized stationary-phase E. coli to Levo and Zoli. We demonstrate that the added metabolites increased proton motive force generation and ATP production in stationary-phase cultures without restarting growth. Instead, the stimulated bacteria increased transcription and translation, which rendered the populations more susceptible to topoisomerase inhibitors. Our findings illuminate potential vulnerabilities of antibiotic-tolerant bacteria that can be leveraged to sensitize them to new and existing classes of topoisomerase inhibitors. These approaches enable us to stay one step ahead of adaptive bacteria and safeguard the efficacy of our existing antibiotics.

Stimulating Transcription in Antibiotic-Tolerant Escherichia coli Sensitizes It to Fluoroquinolone and Nonfluoroquinolone Topoisomerase Inhibitors.

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.

Metabolic and transcriptional activities underlie stationary-phase  sensitivity to Levofloxacin.

Staphylococcus aureus is an opportunistic pathogen whose treatment is further complicated by its ability to form biofilms. In this study, we examine the impact of growing S. aureus biofilms on different polymerizing surfaces, specifically agar and agarose, on the pathogen's tolerance to fluoroquinolones.

Agar and agarose used for Staphylococcus aureus biofilm cultivation impact fluoroquinolone tolerance.

If left unchecked, infections involving antibiotic-refractory bacteria are expected to cause millions of deaths per year in the coming decades. Beyond genetically resistant bacteria, persisters, which are genetically susceptible cells that survive antibiotic doses that kill the rest of the clonal population, can potentially contribute to treatment failure and infection relapse. Stationary-phase bacterial cultures are enriched with persisters, and it has been shown that stimulating these populations with exogenous nutrients can reduce persistence to different classes of antibiotics, including topoisomerase-targeting fluoroquinolones (FQs). In this study, we show that adding glucose and amino acids to nutrient-starved  cultures enhanced their sensitivity to FQs, including delafloxacin (Dela)-a drug that was recently approved for treating staphylococcal infections. We found that while the added nutrients increased nucleic acid synthesis, this increase was not required to sensitize  to FQs. We further demonstrate that addition of these nutrients increases membrane potential and the ability to generate harmful reactive oxygen species (ROS) during FQ treatment. Chelating iron, scavenging hydroxyl radicals, and limiting oxygenation during FQ treatment and during recovery following FQ treatment rescued nutrient-stimulated . In all, our data suggest that while nutrient stimulation increases the activity of FQ targets in stationary-phase , the resulting generation of ROS, presumably made possible through metabolic upregulation, is the primary driver of increased sensitivity to these drugs.IMPORTANCE causes many chronic and relapsing infections because of its ability to endure host immunity and antibiotic therapy. While several studies have focused on the nutrient requirements for the formation and maintenance of staphylococcal infections, the effects of the nutrient environment on bacterial responses to antibiotic treatment remain understudied. Here, we show that adding nutrients to starved  activates biosynthetic processes, including DNA synthesis, but it is the generation of harmful reactive oxidants that sensitizes  to DNA topoisomerase-targeting FQs. Our results suggest that the development of approaches aimed at perturbing metabolism and increasing oxidative stress can potentiate the bactericidal activity of FQs against antibiotic-tolerant .

Article	Year
Enhanced antibiotic resistance development from fluoroquinolone persisters after a single exposure to antibiotic. Nature communications\| PubMed ID: 30862812	2019
DNA Damage Kills Bacterial Spores and Cells Exposed to 222-Nanometer UV Radiation. Applied and environmental microbiology\| PubMed ID: 32033948	2020
The social network: Impact of host and microbial interactions on bacterial antibiotic tolerance and persistence. Cellular signalling\| PubMed ID: 32846197	2020
Levels and Characteristics of mRNAs in Spores of Firmicute Species. Journal of bacteriology\| PubMed ID: 33972352	2021
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
Stimulating Transcription in Antibiotic-Tolerant Escherichia coli Sensitizes It to Fluoroquinolone and Nonfluoroquinolone Topoisomerase Inhibitors. Antimicrobial agents and chemotherapy\| PubMed ID: 36951560	2023
Metabolic and transcriptional activities underlie stationary-phase sensitivity to Levofloxacin. Microbiology spectrum\| PubMed ID: 38078717	2024
Agar and agarose used for Staphylococcus aureus biofilm cultivation impact fluoroquinolone tolerance. Journal of applied microbiology\| PubMed ID: 39066496	2024
Metabolites augment oxidative stress to sensitize antibiotic-tolerant to fluoroquinolones. mBio\| PubMed ID: 39475229	2024

Wendy W. K. Mok

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