Antimicrobial Susceptibility of Persister Biofilm Cells of Bacillus cereus and Pseudomonas fluorescens

The present study evaluates the antimicrobial susceptibility of persister cells of Bacillus cereus and Pseudomonas fluorescens after their regrowth in suspension and as biofilms. Two conventional (benzalkonium chloride—BAC and peracetic acid—PAA) and two emerging biocides (glycolic acid—GA and glyoxal—GO) were selected for this study. Persister cells resulted from biofilms subjected to a critical treatment using the selected biocides. All biocide treatments developed B. cereus persister cells, except PAA that effectively reduced the levels of vegetative cells and endospores. P. fluorescens persister cells comprise viable and viable but non-culturable cells. Afterwards, persister cells were regrown in suspension and in biofilms and were subjected to a second biocide treatment. In general, planktonic cultures of regrown persister cells in suspension lost their antimicrobial tolerance, for both bacteria. Regrown biofilms of persister cells had antimicrobial susceptibility close to those regrown biofilms of biocide-untreated cells, except for regrown biofilms of persister P. fluorescens after BAC treatment, which demonstrated increased antimicrobial tolerance. The most active biocide against persister cells was PAA, which did not promote changes in susceptibility after their regrowth. In conclusion, persister cells are ubiquitous within biofilms and survive after critical biocide treatment. The descendant planktonic and biofilms populations showed similar properties as the original ones.

Persister control by leveraging dormancy associated reduction of antibiotic efflux

Persistent bacterial infections do not respond to current antibiotic treatment and thus present a great medical challenge. These conditions have been linked to the formation of dormant subpopulations of bacteria, known as persister cells, that are growth-arrested and highly tolerant to conventional antibiotics. Here, we report a new strategy of persister control and demonstrate that minocycline, an amphiphilic antibiotic that does not require active transport to penetrate bacterial membranes, is effective in killing Escherichia coli persister cells [by 70.8 ± 5.9% (0.53 log) at 100 μg/mL], while being ineffective in killing normal cells. Further mechanistic studies revealed that persister cells have reduced drug efflux and accumulate more minocycline than normal cells, leading to effective killing of this dormant subpopulation upon wake-up. Consistently, eravacycline, which also targets the ribosome but has a stronger binding affinity than minocycline, kills persister cells by 3 logs when treated at 100 μg/mL. In summary, the findings of this study reveal that while dormancy is a well-known cause of antibiotic tolerance, it also provides an Achilles’ heel for controlling persister cells by leveraging dormancy associated reduction of drug efflux.

Noise in a metabolic pathway leads to persister formation in Mycobacterium tuberculosis

Tuberculosis is difficult to treat due to dormant cells in hypoxic granulomas, and stochastically-formed persisters tolerant of antibiotics. Bactericidal antibiotics kill by corrupting their energy-dependent targets. We reasoned that noise in the expression of an energy-generating component will produce rare persister cells. In sorted low ATP M. tuberculosis grown on acetate there is considerable cell-to-cell variation in the level of mRNA coding for AckA, the acetate kinase. Quenching the noise by overexpressing ackA sharply decreases persisters, showing that it acts as the main persister gene under these conditions. This demonstrates that a low energy mechanism is responsible for the formation of M. tuberculosis persisters and suggests that the mechanism of their antibiotic tolerance is similar to that of dormant cells in a granuloma. Entrance into a low energy state driven by stochastic variation in expression of energy-producing enzymes is likely a general mechanism by which bacteria produce persisters.

Awakening sleeper cells: a narrative review on bacterial magic spot synthetases as potential drug targets to overcome persistence

Magic spot synthetases are emerging targets to overcome persistence caused by stringent response. The ‘stringent response’ is a bacterial stress survival mechanism, which results in the accumulation of alarmones (also called Magic spots) leading to the formation of dormant persister cells. These ‘sleeper cells’ evade antibiotic treatment and could result in relapse of infection. This review broadly investigates the phenomenon of stringent response and persistence, and specifically discusses the distribution, classification, and nomenclature of proteins such as Rel/SpoT homologs (RSH), responsible for alarmone synthesis. The authors further explain the relevance of RSH as potential drug targets to break the dormancy of persister cells commonly seen in biofilms. One of the significant factors that initiate alarmone synthesis is nutrient deficiency. In a starved condition, ribosome-associated RSH detects deacylated tRNA and initiates alarmone synthesis. Accumulation of alarmones has a considerable effect on bacterial physiology, virulence, biofilm formation, and persister cell formation. Preventing alarmone synthesis by inhibiting RSH responsible for alarmone synthesis will prevent or reduce persister cells’ formation. Magic spot synthetases are thus potential targets that could be explored to overcome persistence seen in biofilms.

BRAF-Inhibitor-Induced Metabolic Alterations in A375 Melanoma Cells

Acquired drug tolerance has been a major challenge in cancer therapy. Recent evidence has revealed the existence of slow-cycling persister cells that survive drug treatments and give rise to multi-drug-tolerant mutants in cancer. Cells in this dynamic persister state can escape drug treatment by undergoing various epigenetic changes, which may result in a transient metabolic rewiring. In this study, with the use of untargeted metabolomics and phenotype microarrays, we characterize the metabolic profiles of melanoma persister cells mediated by treatment with vemurafenib, a BRAF inhibitor. Our findings demonstrate that metabolites associated with phospholipid synthesis, pyrimidine, and one-carbon metabolism and branched-chain amino acid metabolism are significantly altered in vemurafenib persister cells when compared to the bulk cancer population. Our data also show that vemurafenib persisters have higher lactic acid consumption rates than control cells, further validating the existence of a unique metabolic reprogramming in these drug-tolerant cells. Determining the metabolic mechanisms underlying persister cell survival and maintenance will facilitate the development of novel treatment strategies that target persisters and enhance cancer therapy.

Persister cells: formation, resuscitation and combative therapies

Persister cells, or superfits, have been strongly implicated in the recalcitrance and recurrence of chronic bacterial infection through the dormant (metabolically reduced) phenotype they display and the tolerance to antimicrobial agents this dormancy grants them. The complex biochemical events that lead to the formation of persister cells are not completely understood, though much research has linked the degradation of type II toxin/antitoxin systems and reduced cellular ATP levels to the rise in stress response molecules (where (p)ppGpp is of particular interest), which induce this dormant state. The equally complex mechanism of resuscitation is initiated by the cells’ ability to sense nutrient availability via chemotaxis systems. Levels of secondary messenger proteins (i.e., cAMP) within the cell are reduced to allow the resuscitation of ribosomes, by ribosomal resuscitation factor HflX, to reinstate protein synthesis and, therefore, growth to re-populate. Techniques of superfit eradication utilise one, or more, of three approaches (i) direct killing, (ii) re-sensitising persister cells to conventional antimicrobials, or (iii) prevention of persister formation though few laboratory findings have been translated to clinical practice. This work will outline current findings in the field with a critical approach, where possible.